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homozygous or heterozygous for the structural Lepore comigrates with HbS on cellulose acetate and agarose hemoglobin variant and coexpress any of the possible at an alkaline pH and with HbA on citrate agar at an acid pH. a@gene combinations. In the heterozygous state, hematologic findings are The severity of the combination disorder is directly pro- similar to thalassemia minor (Figure 14-12): Hemoglobin portional to the total number of affected genes and ranges electrophoresis reveals a mean Hb Lepore concentration of from moderate to asymptomatic. Coexistent a@thalassemia 10%; HbA2 is decreased with a mean of 2%; HbF is usually decreases synthesis of a@chains resulting in fewer a@globin slightly elevated to 2–3%; and HbA makes up the remainder chains available to combine with the structurally abnormal (Table 14-6). b@chain. Thus, the concentration of the structural variant The severely anemic cases of Hb Lepore require a regular (in heterozygotes) is usually decreased because the limited transfusion protocol from early childhood. Splenectomy can number of a@chains preferentially combines with the nor- be performed in an attempt to lessen the degree of anemia. mal b@, d@, or g@ chains. The percentage of HbA, HbF, and 312 Chapter 14 Table 14.8 Characteristics of Combination Thalassemia and Hemoglobinopathy Disorders Most Common Disease Genetic Defect CBC/Diff Hb Electrophoresis Other Data Ancestry Hb S/b@thal Hb S b@allele African Moderate Micro/Hypo No Hb A Severity like Hb SS with b@thal allele Mediterranean Hb 5-8 g/dL c Hb A2 (+ ) Hb solubility test Hb S/b0@Type 1 N. American Sickle cells c Hb F Middle Eastern Hb S Hb S/b+@Type 1 Slight Micro/Hypo Same but less severe Severity like Hb AS (+ ) Hb8-19g/dl Hb solubility test Sickle cells Hb S/b+@Type 2 Normal RBCs and Hb Same but less severe Asymptomatic; Sickle cells (+ ) Hb solubility test Hb C/b@thal Hb C b@allele Same as Same as Hb S/b@thal; Same as Severity like with b@thal allele Hb S/b@thal Hb C crystals Hb S/b@thal Hb S/b@thal Hb C instead of Hb S (- ) Hb solubility test Hb S/a@thal Hb S b@allele(s) African Moderate Micro/Hypo Hb Bart's at Birth a@thal decreases Hb S with a@thal allelc(s) Mediterranean Sickle cells T Hb A levels and symptoms N. American ? Hb A 4a and 2b = 8 2 Middle Eastern genotypes (+ ) Hb solubility test cTHbF HbS Hb S/HPFH Hb S b@allele African No anemia No Hb A (+ ) Hb solubility test with HPFH allele Mild anisocytosis c Hb F Rare sickle cells T Hb A2 Hb S Hb E/a@thal Hb E b@allele SE Asian Mild Micro/Hypo Hb Bart's at Birth Not as severe as Hb with a@thal allele(s) African Moderate/severe anemia T Hb A SS ? Hb A2 4a and 2b = 8 c Hb F possible Hb S genotypes (- ) Hb solubility test thai = thalassemia, HPFH = hereditary persistence of fetal hemoglobin, c = slight increase, T = slight decrease, Micro/Hypo = microcytic and hypochromic, Hb = hemoglobin, N = normal HbA2 increase relative to a typical heterozygote, reducing sometimes show HbC crystals. Hemoglobin electrophore- the abnormal pathophysiology associated with the particu- sis is helpful in resolving the structural hemoglobin variant lar structural hemoglobin variant inherited. and shows the quantitative changes in normal and abnor- In the case of a homozygous bSbS individual with an mal hemoglobins associated with a@ and b@thalassemias. In a@thalassemia syndrome, the clinical severity of sickling is HbS@a@thalassemia, the concentration of HbS is inversely often reduced because of a net decrease in MCHC, which proportional to the number of a@gene deletions. HbS con- reduces the tendency of HbS to produce cell sickling. Thus, centration in heterozygotes is about 35% with one a@gene cell hemolysis and the clinical symptoms associated with deletion, about 28% with two a@deletions, and about 20% occlusion of the microvasculature decrease (Chapter 13). with three a@gene deletions.60 HbF has also been shown to decrease the sickling process.8 When less common structural variants (HbE, HbO, The wide variety of clinical severities seen in sickle cell ane- HbD, etc.) are coexpressed with thalassemias, more test- mia can be partially related to the high incidence of a@ and ing may be necessary because they comigrate with HbS or b@thalassemia in the same population (Figure 14-13). HbC on hemoglobin electrophoresis (Figure 14-2). Molecu- Laboratory diagnosis is accomplished by applying the lar techniques including automated sequencing, dot-blot techniques used to identify each of the disorders individu- analysis, or allele-specific amplification can be used in these ally. Patients present with a mild microcytic, hypochromic cases (Chapter 43).61 anemia with target cells and the poikilocytes associated The incidence of double heterozygotes expressing with the inherited structural hemoglobin variant. Patients both HbE and b@thalassemia is increasing in Southeast who inherit HbS can have sickle cells, and those with HbC Asia, and many patients are presenting with symptoms Thalassemia 313 Figure 14.14 Peripheral blood smear from a patient with hemoglobin E and a@thalassemia. The RBCs are microcytic, Figure 14.13 Peripheral blood smear from a patient with hypochromic, and target cells are present (Wright-Giemsa stain; hemoglobin S and a@thalassemia. The cells are microcytic. There are 1000* magnification). acanthocytes and cells with pointed ends present (Wright-Giemsa stain; 1000* magnification). cell anemia. Only HbS, HbF, and HbA2 are present with HbF levels of 15–35%. HbA2 is normal or reduced. Family studies that rival b@thalassemia major in severity.62 Approximately are helpful in identifying the double heterozygous state. 3000 people with both mutations are born each year in Due to the significant variations in clinical expres- Thailand, and many others are diagnosed in other parts of sion of the various hemoglobin structural variants and Southeast Asia, India, and Burma. In Thailand, Laos, and b@thalassemia alleles, it is suggested that identification of Cambodia, HbE penetrance is approximately 50%. HbE these conditions should go beyond hematologic analysis heterozygotes usually have relatively mild disease; there- and hemoglobin electrophoresis.63 A diagnosis defined at fore, HbE/b@thal double heterozygotes might be expected the molecular level to identify the genetic mutation can lead to have a moderately severe disease.63 However, the HbE to better clinical management of the disease. A summary mutation at the 26th position of the b@globin gene activates of the differentiating characteristics of combination thalas- a cryptic splice site at codon 25, resulting in significantly semia and hemoglobinopathy disorders can be found in reduced b@chain production. Therefore, in double hetero- Table 14-8. zygotes of HbE and b0@thal, few to no bE@ or bA@chains are produced. This results in the accumulation of a@chains that Checkpoint 14.9 precipitate within the erythrocytes, producing hemolysis In combination disorders of structural Hb variants and thalas- as in b@thalassemia major.63,64 A more moderate anemia semia, why is a@thalassemia inherited with sickle cell trait less can result if the b+@thalassemia gene is inherited with HbE. severe than b@thalassemia coexpressed with sickle cell trait? In this form of the disease, there are HbE, HbA, HbF, and HbA2. The HbA, however, is less than would be expected in HbE trait, and the anemia is more severe. When the b+@thalassemia gene is inherited with the bE@gene, the result Differential Diagnosis is microcytic, hypochromic anemia with significant poikilo- cytosis, and nucleated erythrocytes. of Thalassemia Hemoglobin E can also be found in combination with Clinical signs, symptoms, and CBC results are strikingly a@thalassemia (Figure 14-14). This combination produces similar in microcytic, hypochromic anemias regardless of a more severe anemia than does HbE alone. The amount the etiology of the anemia, making the clinical diagnosis of HbE depends on whether the patient is heterozygous difficult. Differentiating the various thalassemias is even or homozygous for HbE and the a@thalassemia genotype more difficult because they are all inherited and occur in inherited (Chapter 13). similar nationalities. Additional laboratory tests are there- Double heterozygotes for sickle cell and HPFH (deletion fore crucial in making the differential diagnosis.25 Table 14-9 variant) exhibit a mild form of sickle cell trait with no occur- summarizes the tests used to differentiate thalassemia from rence of crises or anemia. It has been suggested that the sur- other anemias. prisingly favorable clinical picture is related to the distribution of HbF in erythrocytes. The peripheral blood smear shows Checkpoint 14.10 anisocytosis and target cells, and the sodium metabisulfite Which laboratory tests should be performed first to differentiate and solubility tests are positive. Hemoglobin electrophoresis thalassemia and iron deficiency? produces a pattern that is easily confused with that of sickle 314 Chapter 14 Table 14.9 Typical Hematological Parameters Helpful in Differentiating Well-Developed Iron Deficiency from a@Thalassemia, b@Thalassemia, and HPFH Disease RBC RDW MCV MCH MCHC Hb Elect. Serum Iron Serum Ferritin TIBC Sat(%) FEP Hb H disease Relative c c c T T T T T T T T T T T Hb A2 T T Hb F Normal can be c Normal Can be c Nornal Can be T Normal Can Normal Hb H Hb Bart's from transfusions from transfusions from transfusions be c from and/or increased and/or increased transfusions absorption of iron absorption of iron a@thal minor c Normal T T Normal Normal Hb A2 Normal Normal Normal Normal Normal Normal Hb F Hb Bart's at birth b@thal major Relative c c T T T 1–9% Hb A2 20–90% Normal Can be c Normal Can be c Normal Can be T Normal Can Normal Hb F from transfusions from transfusions from transfusions be c from and/or increased and/or increased transfusions absorption of iron absoprtion of iron b@thal minor c Normal T T T 4–8% Hb A2 1–2% Normal Normal Normal Normal Normal Hb F IDA T c T T T Normal Hb A2 Normal T T c T c Hb F HPFH homo c Normal T T Normal No Hb A2 100% Hb F Normal Normal Normal Normal Normal HPFH hetero c Normal T T Normal 1–2% Hb A2 10–30% Normal Normal Normal Normal Normal Hb F RBC = red blood cell, RDW = red cell distribution width, MCV = mean corpuscular volume, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, TIBC = total iron binding capacity, % Sat = , transferrin saturation, FEP = free erythroid protoporphyrin, c = slight increase, c c = moderate increase, T = slight decrease, T T = moderate decrease, T T T = marked decrease, thal = thalassemia, IDA = iron@deficiency anemia, HPFH = hereditary persistence of fetal hemoglo- bin, Hb Elect. = hemoglobin electrophoresis SOURCE: Tim R. Randolph. Thalassemia 315 Summary Thalassemias result from genetic defects that affect the produc- The thalassemias generally produce a microcytic, tion of globin chains. Any of the globin chains can be affected, hypochromic anemia with changes in the concentrations but the most clinically significant are a@ and b@chain defects. of HbF, HbA2, and HbA. HbF and HbA2 are elevated The clinical severity of the disease is related to the number of in b@thalassemia and decreased in a@thalassemia with mutated genes and the type of genetic defect. The human dip- decreased concentrations of HbA in both. In the more loid genome normally has four a@genes, so in a@thalassemia, severe a@thalassemias, HbH (b4) and Hb Bart (g4) can be from one to four of the a@genes can be deleted. If only one detected. Erythrocyte morphology is similar in both major gene is affected, the condition is not clinically or hematologi- forms of thalassemia and in iron-deficiency anemia; how- cally apparent, but if two or three are affected, both clinical ever, hemoglobin electrophoresis and iron studies assist in and hematological abnormalities of mild or moderate sever- differentiating these two entities. Some structural hemoglo- ity, respectively, occur. Deletion of all four a@genes is incom- bin variants are synthesized in decreased quantities (i.e., Hb patible with life. There are two b@alleles and three categories Lepore, Hb Constant Spring, HbE) and have clinical and of b@thalassemia gene defects; one causes a complete absence morphologic similarities to thalassemias. Molecular tech- of b@chain production (b0@thalassemia), one causes variably niques (Chapter 43) are now available to identify the genetic decreased synthesis of b@chain production (b+@thalassemia), mutation in the globin gene but are not always necessary and the third minimally affects b@chain production (silent for diagnostic purposes. carrier or bSC). This results in seven potential b@thalassemia Current therapies are improving, and medical access in genotypes (b0b0, b0b+, b+b+, b0b, b+b, bSCbSC, bSCb) that are underdeveloped countries is expanding. As a result of these categorized into four clinical severities (b@thalassemia major, advances, the

general health and quality of life are improv- intermedia, minor, and silent carrier) ranging from severe to ing for patients with thalassemia. asymptomatic. Review Questions Level I c. Elevations in embryonic and fetal hemoglobins can be observed. 1. The statement that best defines thalassemia is: (Objective 1) d. The amino acid sequence of the globin chains of the abnormal hemoglobins is normal. a. qualitative disorder of hemoglobin synthesis derived primarily from a genetic point mutation in 3. What is the typical morphologic classification of one or more globin genes erythrocytes in thalassemia? (Objective 3) b. disorder of inappropriate iron metabolism due to a. Macrocytic, normochromic abnormal transferrin b. Normocytic, normochromic c. quantitative disorder of hemoglobin synthesis c. Microcytic, hyperchromic resulting from deletional and nondeletional muta- tions of globin genes d. Microcytic, hypochromic d. single amino acid substitution in a globin chain 4. Select the disorder that is an a@thalassemia. affecting the function of hemoglobin (Objectives 5a, 5b) 2. Which of the following statements is false for a patient a. HbH disease with thalassemia but true in certain hemoglobinopa- b. Cooley’s anemia thies? (Objective 2) c. HPFH a. Abnormal hemoglobin will polymerize inside d. Hb Lepore erythrocytes, altering red cell shape. b. Novel hemoglobins composed of abnormal combi- 5. a@Thalassemia is characterized by: (Objective 5c) nations of normal globin chains can be detected on a. deletion of b@genes hemoglobin electrophoresis. b. amino acid substitutions in the a@chain 316 Chapter 14 c. excess a@chain production 2. Why is hydrops fetalis incompatible with life? d. deletion of a@genes (Objective 4b) 6. Which nationality is most likely to be affected by a. Life cannot exist without HbA. thalassemia? (Objectives 5b, 6a) b. Lack of embryonic hemoglobins precludes fetal development. a. Chinese c. All three normal adult hemoglobins contain b. South American Indians a@chains. c. Southeast Asians d. Fetal hemoglobin is essential to sustain life after d. Europeans birth. 7. Which of the following laboratory results would be 3. Which pathophysiologic event is involved in the expected in a patient with a@thalassemia? pathogenesis of HbH disease? (Objective 4b) (Objective 5e) a. HbH has a higher affinity for oxygen that hampers a. MCH = 32 pg oxygen release. b. MCV = 70 fL b. HbH is an embryonic hemoglobin that is not c. Stomatocytes present at birth. d. Increased HbA c. HbH cannot bind and transport oxygen. d. Polymerization of HbH alters erythrocyte shape. 8. The pathogenesis of b@thalassemia includes: (Objective 6b) 4. The genetic designation heterozygous a. decreased production of b@chains a0@thal@1/normal refers to: (Objective 4a) b. abnormal structure of a@chains a. a@thalassemia minor c. bone marrow hypoproliferation b. Cooley’s anemia d. decreased synthesis of erythropoietin c. HbH disease 9. In b@thalassemia major, hemoglobin electrophoresis d. silent carrier will show: (Objective 6d) 5. The single best laboratory test to distinguish a. reduced HbF b@thalassemia minor from a@thalassemia, iron- b. reduced HbA deficiency anemia, HPFH, and hemoglobinopathies 2 is: (Objective 8) c. reduced HbA d. increased HbH a. hemoglobin solubility b. serum iron 10. Select the thalassemia type in which the patient c. Heinz body stain survives and presents with an abnormal h emoglobin that is sensitive to oxidation and precipitates in d. HbA2 level red cells after incubation with brilliant cresyl blue. 6. Hemoglobin Constant Spring can best be described (Objective 6d) as: (Objective 7c) a. hydrops fetalis a. deletion of three a@genes b. HbH disease b. two normal b@chains and two elongated a@chains c. b@Thalassemia minor c. two normal a@chains and two b/g@fusion chains d. Silent carrier d. continued synthesis of g@chains throughout adult Level II life 1. a@Thalassemia most commonly results from which of 7. Select the statement that best describes hereditary the following genetic lesions? (Objective 1) persistence of fetal hemoglobin. (Objective 7d) a. Gene deletion a. The homozygous state is incompatible with life. b. Promoter mutation b. HbF is elevated in adults. c. Termination codon mutation c. It results from the deletion of the g@gene. d. Splice site mutation d. It is a form of b@thalassemia. Thalassemia 317 8. A 4-year-old male patient has a microcytic, hypochro- c. HPFH and b@thalassemia minor mic anemia. Hemoglobin electrophoresis shows 46% d. HbS and HPFH HbS, 49% HbA, 3.5% HbA2, 1.5% HbF. His parents have no symptoms of anemia. What are his parents’ 10. A 28-year-old female from Laos who had a hemoglo- most likely phenotypes? (Objectives 5, 7) bin of 11.2 g/dL was diagnosed with iron-deficiency anemia. She was given iron supplements. Her reticu- a. Sickle cell trait and b@thalassemia major locyte count increased from 4% to 5% after 6 days of b. Sickle cell anemia and a@thalassemia treatment. Six months later, she returned for a follow- c. Sickle cell anemia and heterozygous b@thalassemia up CBC. Her hemoglobin was 11.5 g/dL, and the red cells were microcytic (75 fL), normochromic. What d. Sickle cell trait and normal reflex test should be done? (Objective 8) 9. Which of the following combination disorders would a. Hemoglobin electrophoresis exhibit more severe symptoms? (Objective 7f) b. Serum iron a. HbS and b@thalassemia minor c. Bone marrow b. HbC trait and a@thalassemia minor d. Serum ferritin References 1. The Human Gene Mutation Database at the Institute of Medical with hemoglobinopathies given either cord blood or bone Genetics in Cardiff. Retrieved from www.hgmd.cf.ac.uk. marrow transplantation from an HLA-identical sibling. Blood, 2. Cooley, T. B., & Lee, P. (1925). 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The diagnosis of thalas- related conditions: therapeutic goals and response to chelation semia trait by starch block electrophoresis of the hemoglobin. therapies. Hematology and Oncology Clinics of North America, 24(6), Blood, 13(1), 61–69. 1109–1130. 58. Wood, W. G., Weatherall, D. J., Hart, G. H., Bennett, M., & Marsh, 40. Porter, J. B. (2010). Deferasirox—current knowledge and future G. W. (1982). Hematologic changes and hemoglobin analysis challenges. Annal of the New York Academy of Science, 1202, 87–93. in beta thalassemia heterozygotes during the first year of life. 41. Phrommintikul, A., Sukonthasarn, A., Kanjanavanit, R., & Pediatric Research, 16, 286–289. Nawarawong, W. (2006). Splenectomy: A strong risk factor for 59. Bunn, H. F., & Forget, B. G. (1986). Sickle cell disease: Clinical pulmonary hypertension in patients with thalassaemia. Heart, and epidemiological aspects. In: O. Platt & D. G. Nathan, eds. 92(10), 1467–1472. Hemoglobin: Molecular, genetic, and clinical aspects (pp. 502–554). 42. Singer, S. T., Kuypers, F. A., Styles, L., Vichinsky, E. P., Foote, D., & Philadelphia: W.B. Saunders. Rosenfeld, H. (2006). Pulmonary hypertension in thalassemia: 60. Embury, S. H., Dozy, A. M., Miller, J., Davis, J. R. Jr., Kleman, K. M., association with platelet activation and hypercoagulable state. Preisler, H., . . . Mentzer, W. C. (1982). Concurrent sickle-cell American Journal of Hematology, 81(9), 670–675. anemia and alpha-thalassemia: Effect on severity of anemia. 43. Krishnamurti, L., Bunn, H. F., Williams, A. M., & Tolar, J. (2008). New England Journal of Medicine, 306(5), 270–274. Hematopoietic cell transplantation for hemoglobinopathies. 61. Huisman, T. H. (1997). Combinations of beta-chain abnormal Current Problems in Pediatric and Adolescent Health Care, 38(1), 6–18. hemoglobins with each other or with beta-thalassemia 44. Lucarelli, G., Isgro, A., Sodani, P., & Gaziev, J. (2012). Hematopoi- determinants with known mutations: Influence on phenotype. etic stem cell transplantation in thalassemia and sickle cell ane- Clinical Chemistry, 43(10), 1850–1856. mia. Cold Spring Harbor Perspectives on Medicine, 2(5). doi: 10.1101/ 62. Weatherall, D. J. (2000). Introduction to the problem of hemoglo- cshperspect.a011825. bin E-beta thalassemia. Journal of Pediatric Hematology and Oncol- 45. Lucarelli, G., & Gaziev, J. (2008). Advances in the allogeneic ogy, 22(6), 551. transplantation for thalassemia. Blood Review, 22(2), 53–63. 63. Winichogoon, P., Fucharoen, S., Chen, P., & Wasi, P. (2000). Genetic 46. Pace, B. S., & Zein, S. (2006). Understanding mechanisms of factors affecting clinical severity of beta-thalassemia syndromes. g@globin gene regulation to develop strategies for p harmacological Journal of Pediatric Hematology and Oncology, 22(6), 573–580. fetal hemoglobin induction. Developmental Dynamics, 235(7), 64. Fucharoen, S., Ketvichit, P., Pootrakul, P., Siritanaratkul, N., 1727–1737. Piankijagum, A., & Wasi, P. (2000). Clinical manifestations of 47. Cavazzana-Calvo, M., Payen, E., Negre, O., Wang, G., Hehir, K., beta-thalassemia/hemoglobin E disease. Journal of Pediatric Fusil, F., . . . Leboulch, P. (2010). Transfusion independence and Hematology and Oncology, 22(6), 552–557. Chapter 15 Megaloblastic and Nonmegaloblastic Macrocytic Anemias Joel Hubbard, PhD Stacey Robinson, MS Objectives—Level I At the end of this unit of study, the student should be able to: 1. Explain the cause and process of megalo- 7. Describe the etiology and pathophysiology blastic maturation in the bone marrow. of pernicious anemia, including clinical 2. Describe the body’s requirements for symptoms and clinical subtypes. vitamin B12 (cobalamin) and folate and their 8. Name three causes of a folate deficiency physiologic roles. and give two distinguishing clinical or 3. List the laboratory tests used to confirm a laboratory characteristics of each. diagnosis of cobalamin deficiency and give 9. Differentiate the pathophysiology and expected results. peripheral blood findings of nonmegalo- 4. List the laboratory tests used to confirm a blastic macrocytic anemia from those of diagnosis of folic acid deficiency and give megaloblastic anemias. expected results. 10. Summarize the typical blood picture seen 5. Recognize the six most common disorders with a folate or cobalamin deficiency. associated with a macrocytic anemia. 6. Name four causes of a cobalamin deficiency and give two distinguishing clinical or laboratory characteristics of each. 319 320 Chapter 15 Objectives—Level II At the end of this unit of study, the student should be able to: 1. Summarize the process of cobalamin and 6. Categorize the causes and clinical variations folic acid metabolism and explain how a of pernicious anemia. deficiency can result in megaloblastosis. 7. Compare and contrast the various clinical 2. Compare macrocytosis associated with a forms and causes of a folic acid deficiency. normoblastic marrow and macrocytosis 8. Choose and briefly explain four l aboratory associated with a megaloblastic marrow tests used to identify the cause of a on the basis of physiological defect, and macrocytic anemia; give the expected differentiate them based on the laboratory results of these four tests in a patient with blood picture. an a utoantibody directed against intrinsic 3. Distinguish between the metabolic pathway factor. of folate and cobalamin (B12) based on 9. Compare and contrast the causes of how they complement each other and how macrocytosis that have a normoblastic a deficiency of either vitamin can affect marrow. hematopoiesis in the marrow. 10. Construct an algorithm of laboratory t esting 4. Summarize the mechanism of maturation to distinguish between a megaloblastic defects that lead to megaloblastosis and anemia and a macrocytic, normoblastic recognize the morphologic blood cell anemia. abnormalities. 11. Evaluate a case study from a patient with 5. Compare and contrast the various clinical anemia. Determine the most probable forms and causes of a cobalamin and folate diagnosis from the medical history and deficiency on the basis of clinical symptoms laboratory results. and laboratory results. Chapter Outline Objectives—Level I and Level II 319 Megaloblastic Anemia 323 Key Terms 320 Macrocytic Anemia Without Megaloblastosis 340 Background Basics 321 Summary 342 Case Study 321 Review Questions 343 Overview 321 References 345 Introduction 321 Key Terms Achlorhydria Folate Megaloblastic Cobalamin Folic acid Nuclear-cytoplasmic asynchrony Demyelination Glossitis Pernicious anemia (PA) Dyspepsia Intrinsic factor (IF) Megaloblastic and Nonmegaloblastic Macrocytic Anemias 321 Background Basics The information in this chapter builds on the concepts • Outline the functional and morphologic classification learned in previous chapters. To maximize your learning of anemia and list the basic laboratory tests to experience, you should review these concepts before diagnose anemia. (Chapter 11) starting this unit of study: Level II Level I • Summarize the concepts of cell development, regula- • Describe the maturation process of erythrocytes in tion, and the process of cell division. (Chapter 2) the marrow. (Chapter 5) • List and describe the laboratory tests used in differential diagnosis of anemia. (Chapter 11) CASE STUDY Basophilic stippling 1+ We refer to this case study throughout the chapter. Occasional Howell-Jolly bodies Kathy, a 36-year-old female, experienced a recent Consider the reflex tests that might be impor- 35-lb weight loss. Her tongue was red and fissured. tant in identifying the etiology of this anemia. She also complained of chronic fatigue and short- ness of breath upon exertion. Physical examination suggested signs of jaundice and increased numb- ness and a tingling sensation of fingers and toes. Overview She was hospitalized with the general diagnosis of moderate anemia, jaundice, and neurological This chapter is a study of the macrocytic anemias, which can symptoms. Her admitting CBC demonstrated the be megaloblastic or nonmegaloblastic. The first part of the following laboratory results: chapter discusses the megaloblastic anemias beginning with a description of the clinical and laboratory findings. Because Differential megaloblastic anemia is most often due to deficiencies or WBC 4.5 * 103/mcL Lymphs 36.0% abnormal metabolism of folate or cobalamin (vitamin B12), RBC 2.50 * 106/mcL Monos 3.6% the metabolism of these vitamins is discussed in detail. The latter part of the chapter reviews the causes of nonmegalo- Hb 10.0 g/dL Neutrophils 59.4% blastic macrocytic anemia and compares the laboratory test (100.0 g/L) results in nonmegaloblastic and megaloblastic anemia. The Hct 31% (0.31 L/L) Eosinophils 1.0% laboratory professional can often identify diagnostic clues MCV 124 fL Basophils 0.0% of megaloblastic anemia on review of a blood smear. MCH 40.5 pg NRBCs/100 5 WBCs MCHC 32.7 g/dL Moderate Introduction hyperseg- Macrocytic anemias are characterized by large erythrocytes mented (mean MCV more than 100 fL) with an increased MCH and RDW 21.2 neutrophils a normal hemoglobin content (MCHC). This is an important PLT 155 * 103/mcL group of anemias because macrocytosis is frequently a sign of a disease process that can result in significant morbidity The following abnormal erythrocyte morphol- if left untreated. ogy was reported: Macrocytosis is found in 2.5–4.0% of adults who have Macrocytes 2+ a routine complete blood count.1 In up to 60% of cases, Anisocytosis 3+ macrocytosis is not accompanied by anemia,2 but isolated Poikilocytosis 2+ macrocytosis should always be investigated. Macrocyto- sis without anemia can indicate early folate or cobalamin Ovalocytes 1+ (vitamin B12) deficiency because macrocytosis precedes the development of anemia in these disorders. 322 Chapter 15 Macrocytosis detected by automated cell counters is not laboratory analysis to help distinguish causes of macrocytic always apparent microscopically on stained blood smears. anemia is shown in Figure 15-2. In some cases, the erythrocyte size on automated counters is falsely elevated due to hyperglycemia, cold agglutinins, and extreme leukocytosis. These causes of false macrocyto- Megaloblastic Anemia sis need to be differentiated from true macrocytosis. Although very little was known about the function or origin The most common cause of true macrocytosis is alco- of blood cells before the twentieth century, some perceptive holism. Other causes include folate and cobalamin deficien- individuals began to make associations between anemia cies, drugs including chemotherapy, reticulocytosis due to and other clinical signs in patients. In 1822, J. S. Coombe, hemolysis or bleeding, myelodysplasia, liver disease, and a Scottish physician, made the initial clinical description of hypothyroidism.2 a patient who appeared to have megaloblastic anemia. He Macrocytic anemias are generally classified as megalo- was the first to suggest that this anemia might be related to blastic or nonmegaloblastic (normoblastic), depending on dyspepsia.3 In 1855, Thomas Addison reported his descrip- morphologic characteristics of erythroid precursors in the bone tion of a macrocytic anemia, but he made no reference to marrow (Table 15-1). The megaloblastic anemias are the result the typical microscopic blood findings.4 The discovery and of a defect in DNA synthesis. Frequently there is an arrest in description of the abnormal erythroid precursors in the the S phase of the cell cycle and to a lesser extent during other bone marrow associated with this anemia were made pos- phases of the cell cycle due to delayed nuclear development. sible by the advent of triacid stains. Paul Ehrlich is credited RNA and protein synthesis, however, are relatively unim- with coining the term megaloblast in 1891 to describe the paired. The result is unbalanced cell growth and impaired cell large abnormal precursors in megaloblastic anemia.5 division

characterized by erythroblasts with distinct abnormal Megaloblastic anemia is classified as a nuclear matura- morphologic features. The nucleus appears immature with a tion defect. Anemia is attributed primarily to a large degree fine particulate chromatin pattern, whereas the cytoplasm is of ineffective erythropoiesis resulting from disrupted DNA increased and relatively more mature (referred to as nuclear- synthesis. The anemia was called megaloblastic in an attempt cytoplasmic asynchrony). These cells are referred to morpho- to describe the giant, abnormal-appearing erythroid precur- logically as megaloblastic. All proliferating cells are affected. sors (megaloblasts) in the bone marrow. The generic word The basis for the nonmegaloblastic anemias is not megaloblast describes any maturation stage of the megaloblas- always as well defined but is often related to an increase tic erythroid series (i.e., polychromatic megaloblast). Other in membrane lipids. The macrocytes in nonmegaloblastic nucleated cells of the marrow are also typically abnormal. macrocytic anemia are usually round, but in megaloblas- About 95% of megaloblastic anemias are caused by tic anemia, they are oval (Figure 15-1a, b). A flow chart for deficiencies of either vitamin B12 (cobalamin) or folic Table 15.1 Conditions Associated with Megaloblastic and Nonmegaloblastic (Normoblastic) Macrocytic Anemias Megaloblastic Normoblastic Folate deficiency Alcoholism Nutritional deficiency Liver disease Increased requirement (pregnancy) Shift reticulocytosis in hemolytic anemia or hemorrhage Intestinal malabsorption Hypothyroidism Drug inhibition Aplastic anemia Cobalamin deficiency Obstructive jaundice Pernicious anemia Splenectomy Small bowel resection Pregnancy Gastrectomy Artifactual: Intestinal malabsorption hyperglycemia Nutritional deficiency cold agglutinins Increased requirement (pregnancy) leukocytosis Transcobalamin deficiency Nitrous oxide abuse Other causes Chemotherapy with metabolic inhibitors Orotic aciduria Congenital dyserythropoietic anemia (CDA) Megaloblastic and Nonmegaloblastic Macrocytic Anemias 323 a b Figure 15.1 (a) Peripheral blood film from a patient with pernicious anemia. Note the anisocytosis with oval macrocytes and the nucleated red blood cell with a Howell-Jolly body. (Wright-Giemsa stain; 1000* magnification) (b) Peripheral blood film from a patient with normoblastic, macrocytic anemia. Compare the size of the RBCs with the lymphocyte. The RBCs are primarily round macrocytes (Wright- Giemsa stain, 1000* magnification). Hb & Hct MCV .100 fL Increased Bleeding, hemolysis, or Reticulocyte count response to therapy for anemia Normal, low Blood smear morphology: Macrocytes absent Consider false increase in MCV Macro-ovalocytes No due to cold agglutinins or Dacryocytes hyperglycemia Howell-Jolly bodies Hypersegmentation Round macrocytes Suspect normoblastic Yes macrocytic anemia Serum cobalamin assay; go to Figure 5 Liver disease; alcoholism; aplastic anemia Figure 15.2 Algorithm for the differential diagnosis of the megaloblastic anemias from other macrocytic anemias. Hb, hemoglobin; Hct, hematocrit; MCV, mean cell volume. acid, vitamins necessary as coenzymes for nucleic acid Clinical Presentation synthesis. In the majority of cases, cobalamin deficiency is secondary to a deficiency of intrinsic factor (IF), a The onset of megaloblastic anemia is usually insidious; protein necessary for absorption of cobalamin, rather because the anemia develops slowly, it produces few symp- than to a nutritional deficiency of the vitamin. Folic acid toms until the hemoglobin and hematocrit are significantly deficiency, on the other hand, is most often due to an depressed. Patients can present with typical anemic symp- inadequate dietary intake. Inherited disorders affecting toms of lethargy, weakness, and a yellow or waxy pallor. DNA synthesis or vitamin metabolism are rare causes of Dyspeptic symptoms are common. Glossitis with a beefy megaloblastosis. red tongue, or more commonly a smooth pale tongue, is characteristic. Loss of weight and loss of appetite are common complaints. In pernicious anemia (see “Perni- Checkpoint 15.1 cious Anemia”), atrophy of the gastric parietal cells causes Explain why patients with cobalamin or folate deficiency have decreased secretion of intrinsic factor and hydrochloric acid. megaloblastic maturation. Bouts of diarrhea can result from epithelial changes in the gastrointestinal tract. 324 Chapter 15 Neurological disturbances occur only in cobalamin thalassemia, chronic renal insufficiency, and chronic inflam- deficiency, not in folic acid deficiency. They are the most mation or infection.9 Sometimes these coexisting causes serious and dangerous clinical signs because neurological of anemia are not recognized until after the megaloblas- damage can be permanent if the deficiency is not treated tic anemia has been treated. If coexisting iron deficiency, promptly. The patient’s initial complaints occasionally are thalassemia, or chronic disease is suspected, patient medi- related to neurological dysfunction rather than to anemia. cal history, racial/ethnic background, and previous MCV Neurological damage has been reported to occur even should be considered.10 before anemia or macrocytosis in some cases, particularly in Hematologic parameters vary considerably (Table 15-2). elderly people. The bone marrow, however, usually reveals The hemoglobin and erythrocyte count range from normal megaloblastic changes even in the absence of anemia. Tin- to very low. The erythrocyte count is occasionally less than gling, numbness, and weakness of the extremities reflect 1.0 * 106/mcL. However, anemia is not always evident. In peripheral neuropathy. Loss of vibratory and position (pro- one study of 100 patients with confirmed cobalamin defi- prioceptive) sensations in the lower extremities can cause ciency, only 29% had a hemoglobin of less than 12 g/dL.11 the patient to have an abnormal gait. The patient’s rela- This is significant because neurologic symptoms can be tives sometimes note mental disturbances such as loss of present even if the MCV and/or hematocrit are normal.12 memory, depression, and irritability. Megaloblastic madness Because the abnormality is a nuclear maturation defect, the is a term that was used to describe severe psychotic mani- megaloblastic anemias affect all three blood cell lineages: festations of cobalamin deficiency. A patient with severe erythrocytes, leukocytes, and platelets. This is unlike most anemia occasionally is asymptomatic, which is probably a other anemias that typically involve only erythrocytes. The reflection of a very slowly developing anemia. It has been leukocyte count can be decreased due to an absolute neu- suggested that cobalamin deficiency should be suspected tropenia. Platelets can also be decreased but do not usually in all patients who have an unexplained anemia and/or fall below 100 * 103/mcL. The relative reticulocyte count neurological disturbances or in individuals who are at risk (percentage) is usually normal; however, because of the of developing a deficiency such as elderly people or those severe anemia, the corrected reticulocyte count is less than with intestinal diseases.6 2%, the absolute reticulocyte count is low, and RPI is less than 2 (Chapter 11). The distinguishing features of megaloblastic ane- Checkpoint 15.2 mia on the stained blood smear include the triad of oval Patients with megaloblastic anemia often present with a yellow macrocytes (macro-ovalocytes), Howell-Jolly bodies, and or waxy pallor. What is the diagnostic significance of this clini- cal symptom? hypersegmented neutrophils (Figure 15-1a). Anisocytosis is moderate to marked with normocytes and a few micro- cytes in addition to the macrocytes. Poikilocytosis can be Laboratory Evaluation striking and is usually more so when the anemia is severe. Polychromatophilia and megaloblastic erythroblasts can be Laboratory tests are critical to a diagnosis of megaloblastic seen, especially when the anemia is severe, indicating the anemia. The routine CBC with a review of the blood smear futile attempt of the bone marrow to increase peripheral gives important diagnostic clues and helps in selecting erythrocyte mass. Cabot rings occasionally can be seen in reflex tests. erythrocytes. PERIPHERAL BLOOD Granulocytes and platelets can also show changes evi- Megaloblastic anemia is typically a macrocytic, normo- dent of abnormal hematopoiesis. Hypersegmented neu- chromic anemia. The MCV is usually more than 100 fL trophils can be found in megaloblastic anemia even in the and can reach a volume of 140 fL. However, an increased absence of macrocytosis (Figure 15-3). Finding 5% or more MCV is not specific for megaloblastic anemia. The MCH is neutrophils with five lobes or one neutrophil with six or increased because of the large cell volume, but the MCHC more lobes is considered hypersegmentation. This find- is normal. In cobalamin deficiency, a macrocytosis can pre- ing of hypersegmented neutrophils is considered highly cede the development of anemia by months to years.7–8 On sensitive and specific for megaloblastic anemia. Therefore, the other hand, the MCV can remain within the reference hypersegmented neutrophils offer an important clue to interval. Epithelial changes in the gastrointestinal tract can megaloblastic anemia in the face of a coexisting disease cause impaired iron absorption. If an iron deficiency (which that tends to keep erythrocyte volume less than 100 fL. One characteristically produces a microcytic, hypochromic ane- study showed that in patients with renal disease, iron defi- mia) coexists with megaloblastic anemia, macrocytosis can ciency, or chronic disease with a normal or decreased MCV be masked, and the MCV can be in the normal range.9 Other and 1% hypersegmented neutrophils, 94% had vitamin B12 conditions that have been shown to coexist with megalo- or folic acid deficiency.8 If 5% hypersegmented neutrophils blastic anemia in the absence of an increased MCV include were counted, the incidence of the vitamin B12 or folic acid Megaloblastic and Nonmegaloblastic Macrocytic Anemias 325 Table 15.2 Comparison of Common Laboratory Values in Megaloblastic and Nonmegaloblastic Macrocytosis Laboratory Value Megaloblastic Macrocytosis Nonmegaloblastic Macrocytosis WBC count Decreased Normal Platelet count Decreased Normal RBC count Decreased Decreased Hemoglobin Decreased Decreased Hematocrit Decreased Decreased MCV Usually more than 110 fL More than 110 fL RBC morphology Ovalocytes, Howell-Jolly bodies, polychromasia Polychromasia, target cells, and stomatocytes (liver disease), schistocytes (hemolytic anemias) Hypersegmentation of neutrophils Present Absent Reticulocyte count Normal to decreased Normal, decreased, or increased Serum cobalamin Decreased in cobalamin deficiency Usually normal Serum folate Decreased in folate deficiency Normal (except in alcoholism when it can be decreased) FIGLU Increased in folate deficiency Normal MMA Increased in B12 deficiency Normal Homocysteine Increased Normal Serum bilirubin Increased Normal to increased LD Increased Normal to increased MCV, mean corpuscular volume; FIGLU, formiminoglutamic acid; MMA, methylmalonic acid; LD, lactic dehydrogenase deficiency increased to 98%. Hypersegmented neutrophils In megaloblastic states, the bone marrow is hypercellular tend to be larger than normal neutrophils. A mild shift to with megaloblastic erythroid precursors and a decreased the left with large hypogranular bands may also be noted. M:E ratio. In a long-standing anemia, red marrow can Platelets may be large, especially when the platelet count expand into the long bones. About half the erythroid is decreased. precursors typically show megaloblastic changes. Meg- aloblasts are large nucleated erythroid precursors that display nuclear-cytoplasmic asynchrony with nuclear Checkpoint 15.3 maturation lagging behind cytoplasmic maturation Why are abnormalities of leukocytes and platelets present in (Figure 15-4). The nucleus of the megaloblast contains megaloblastic anemia? loose, open chromatin that stains poorly; cytoplasmic development continues in a normal fashion. At each stage BONE MARROW of development, the cells contain more cytoplasm with a If physical examination, patient history, and peripheral more mature appearance relative to the size and maturity of blood findings suggest megaloblastic anemia, a bone mar- the nucleus (resulting in a decreased nuclear:cytoplasmic row examination can help establish a definitive diagnosis. [N:C] ratio). CASE STUDY (continued from page 321) Based on the initial CBC results, further testing was ordered with the following results: B12 (cobalamin) 50 pg/mL Low Folate 10.3 ng/mL Normal Total billirubin 2.5 mg/dL High Direct billirubin 0.8 mg/dL Normal AST 35 U/mL Normal ALT 30 U/mL Normal Figure 15.3 Hypersegmented neutrophils from the peripheral blood of a patient with pernicious anemia (Wright-Giemsa stain; 1000* magnification). (Continued) 326 Chapter 15 OTHER LABORATORY FINDINGS CASE STUDY (continued from page 325) If CBC results suggest megaloblastic anemia, further testing Examination of a bone marrow aspirate is necessary to distinguish the cause. Although no major revealed an erythroblastic hyperplasia with mega- medical organization has published guidelines for reflex loblastic erythroblasts. testing, the most common next step is to measure serum cobalamin and serum, or red cell, folate. Laboratories use 1. What is the morphologic classification of the different methods (chemiluminescence, radioassay) to mea- patient’s anemia? sure cobalamin, so there is no “gold standard” to use as a 2. Based on the information obtained so far, what is reference interval. Generally, values less than 150 pg/mL the most likely defect? are consistent with cobalamin deficiency, whereas levels more than 400 pg/mL suggest adequate cobalamin. Border- 3. What is the significance of the AST/ALT results? line levels (150–400 pg/mL) can be associated with cobala- 4. What further testing can be done to obtain a min deficiency. definitive diagnosis? Measurement of erythrocyte folate is not influenced as much by recent dietary changes as is serum folate and gives an accurate estimate of the average folate levels over the The megaloblastic features are

more easily noted in later preceding several months.13,14 On the other hand, if there stages of erythroid development, especially at the polychro- is a cobalamin deficiency, folate will leak out of the cells, matophilic stage in which the presence of hemoglobin mixed which will give a false low red cell folate and false increased with RNA gives the cytoplasm the gray-blue color typical serum folate. In addition, red cell folate is measured by of this erythroid precursor. The polychromatophilic mega- folate-binding protein assays that rely on chemilumines- loblast nucleus, however, still has an open (lacy) chromatin cence methodology. These methods show considerable ana- pattern more typical of an earlier stage of development. lytic variability. Therefore, the less expensive serum folate Leukocytes and platelets also show typical features measurement is preferred for initial testing. If serum folate of a nuclear maturation defect as well as ineffective leu- is more than 4 ng/mL, folate deficiency can be ruled out. kopoiesis and thrombopoiesis. Giant metamyelocytes and Early megaloblastic changes can be detected by testing bands with loose, open chromatin in the nuclei are diag- for methylmalonic acid (MMA) and homocysteine levels in nostic (Figure 15-4a). The myelocytes can show poor granu- the blood. These metabolites are intermediates in folate and lation as do more mature stages. Megakaryocytes may be cobalamin metabolism and are elevated early in functional decreased, normal, or increased. Maturation, however, is vitamin deficiencies. Tests for these metabolites are more distinctly abnormal. Larger than normal megakaryocytes sensitive than serum cobalamin levels and increase earlier may be found with separation of nuclear lobes and nuclear than a drop in the cobalamin level. By performing tests for fragments. both MMA and homocysteine, it is possible to differentiate a b Figure 15.4 (a) Basophilic and orthochromatic megaloblasts in the bone marrow from a patient with pernicious anemia. Note the large size of the cells, the open chromatin network in the nuclei, and the presence of Howell-Jolly bodies in the orthochromatic megaloblasts. Note the nuclear-cytoplasmic asynchrony. There is a large band neutrophil and a segmented neutrophil with at least five nuclear lobes. (Bone marrow; Wright-Giemsa stain; 1000* magnification) (b) Nuclear-cytoplasmic asynchrony in the bone marrow. Note the large size of the cells (bone marrow; Wright-Giemsa stain; 1000* magnification). Megaloblastic and Nonmegaloblastic Macrocytic Anemias 327 cobalamin deficiency from folate deficiency. Homocysteine uric acid, and alkaline phosphatase are decreased. Addi- is elevated in folate deficiency, whereas MMA is usually tional tests are discussed in the following sections. normal. On the other hand, both homocysteine and MMA are elevated in cobalamin deficiency. An increase in both MMA and homocysteine is also found in combined cobala- Checkpoint 15.4 min and folate deficiencies. In these cases, clinical informa- What abnormal morphological findings on a stained blood tion is important to help establish a differential diagnosis. A smear compose the triad in megaloblastic anemia? block in the metabolism of histidine to glutamic acid occurs in folic acid deficiency and causes increased urinary excre- tion of formiminoglutamic acid (FIGLU), an intermediate Folate metabolite, after the administration of histidine. These A folate deficiency must be considered in the differential metabolites return to normal levels when the appropri- diagnosis of macrocytosis associated with megaloblastic ate vitamin is given to the patient. It is recommended that anemia. clinicians first use the lower cost tests of serum cobalamin and serum folate to diagnose cobalamin and folate defi- STRUCTURE AND FUNCTION ciencies and use the higher cost MMA and homocysteine Folic acid is the parent substance of a large group of com- tests if cobalamin and folate test results are not definitive15 pounds known as folates. Chemically, folic acid is known as (Figure 15-5). pteroylmonoglutamate (PteGlu). Structurally, folic acid is com- The large degree of ineffective erythropoiesis results in posed of three parts: (1) pteridine, a nitrogen-containing hemolysis in the marrow and an increase in plasma iron ring; (2) a ring of p-amino-benzoic acid; and (3) a glutamic turnover, serum iron, indirect bilirubin, and urobilinogen. acid residue (Figure 15-6). The term folic acid refers to this The characteristic marked increase in fractions 1 and 2 of base structure (inert form) and to the commercially avail- serum lactic dehydrogenase (LD) is partially caused by able synthetic form used for the fortification of food sup- the destruction of megaloblasts rich in LD. The increase is plies and for dietary supplements. Folic acid itself does not roughly proportional to the degree of anemia. Haptoglobin, occur naturally. Most dietary folates vary in their oxidation Megaloblastic hematologic changes and/or neurologic abnormalities Cobalamin assay <150 pg/mL 150–400 pg/mL >400 pg/mL Serum MMA assay Serum RBC or serum folate assay Homocysteine assay Both MMA & MMA elevated, Both MMA & MMA normal, homocysteine homocysteine homocysteine homocysteine Decreased Normal are elevated normal are normal elevated Cobalamin deficiency Consider further evaluation with bone marrow Folate deficiency Consider further evaluation with bone marrow Figure 15.5 Algorithm that can be used to determine the cause of megaloblastic anemia using laboratory tests. Analysis can begin with a serum cobalamin assay. If the results are in the low normal range (150–400 pg/mL) or the patient has unexplained neurologic symptoms, measurement of cobalamin metabolic intermediates, methylmalonic acid (MMA), and homocysteine is suggested. If both are elevated, cobalamin deficiency is considered. If MMA is normal and homocysteine is elevated, folate deficiency is probable. RBC or serum folate also can be measured, especially if cobalamin is within the reference interval. If these test results conflict with the clinical diagnosis, therapeutic trials with cobalamin can be used. Bone marrow is rarely performed for diagnostic purposes but can be necessary in some cases. The bone marrow shows megaloblastic features in both folate and cobalamin deficiencies. 328 Chapter 15 N O N 4 5 3 6 CH2 NH C NH CH CH2 CH2 COOH 9 10 2 7 1 8 COOH NH2 N N Pteridine p-Amino-benzoic acid Glutamic acid Figure 15.6 Molecular structure of the folic acid molecule. status, their one-carbon substituent unit, and in their num- The function of THF is to transfer one-carbon com- ber of glutamic acid residues (they exist as polyglutamates). pounds from donor molecules to acceptor molecules in Tetrahydrofolate (THF), the active form, is produced by a intermediary metabolism. In this capacity, folate serves a four-hydrogen reduction of the pteridine ring. Dihydrofo- vital role in the metabolism of nucleotides and amino acids late (DHF) is an intermediate in this reaction. (Figure 15-7a ; Table 15-3). Deconjugation, reduction, methylation a Dietary folate N5-methyl THF Homocysteine MeCbl1 Methionine synthase Methionine SAM THF Serine NADPH1H1 Dihydrofolate reductase Glycine Serine hydroxymethyl transferase N5,N10-methylene THF DHF duMP dTMP dTTP DNA THF b Histidine Urocanic acid FIGLU Glutamic acid 1 N5-formimino-THF Hydrolase AdoCbl c Homocysteine Propionyl CoA MMA Methylmalonyl CoA Succinyl CoA Mutase Figure 15.7 Biochemical reactions using folic acid, cobalamin, and derivatives. (a) The role of dietary folate and cobalamin in the synthesis of DNA and methionine. The carbon transfer reaction involved in the de novo synthesis of DNA is initiated when the carbon side chain of serine is transferred to THF to form N5, N10@methylene THF. This carbon is then transferred to deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP), a pyrimidine of DNA. In this reaction, THF is oxidized to DHF. The DHF is reduced back to THF by DHF reductase. A deficiency of folate or cobalamin blocks the synthesis of the nucleotide deoxythymidine triphosphate (dTTP). The synthesis of methionine from homocysteine requires the donation of a methyl group from N5@methyl THF and the action of methylcobalamin (MeCbl) as a cofactor, resulting in THF and methionine that can be used to generate S-adenosylmethionine (SAM). A deficiency of either folate or cobalamin (methylcobalamin) blocks this reaction, resulting in an increase in homocysteine levels. (b) The role of folate in the catabolism of histidine. The intermediate metabolite of this reaction is formiminoglutamic acid (FIGLU), which requires THF for conversion to glutamic acid. A deficiency of folate blocks this reaction, resulting in an increase in FIGLU excretion. (c) The role of cobalamin (in the form of adenosyl- cobalamin [AdoCbl]) in the conversion of methylmalonic acid (MMA) to succinyl CoA. A decrease in cobalamin (AdoCbl) results in urinary excretion of MMA. Megaloblastic and Nonmegaloblastic Macrocytic Anemias 329 Table 15.3 Metabolic Reactions Requiring Folic Acid Coenzymes System Reaction Serine 4 Glycine Ser + THF 4 N5, N10@methylene THF + Gly dTTP synthesis dUMP + N5, N10@methylene THF S DHF + dTMP Histidine catabolism Formininoglutamate + THF S N5@formimino THF + glutamic acid Methionine synthesis Homocysteine + N5@methyl THF S THF + Methionine Ser, serine; THF, tetrahydrofolate; Gly, Glycine; dTTP, deoxythymidine triphosphate; dUMP, deoxyuridine monophosphate; deoxythymidine monophosphate; DHF, dyhydrofolate METABOLISM heat; thus, when food is overcooked, much of the folate is Folate is present in food and is synthesized by microorgan- destroyed. Ascorbate protects folate from oxidation and, isms. Most folate in food is in the conjugated polyglutamate when present, can protect folate to some extent from heat form. It is deconjugated in the intestine to a monoglutamate degradation. The recommended daily dietary allowance of prior to absorption. Absorption can take place throughout food folic acid for adults is about 400 mcg of which about the small intestine but is especially significant in the proxi- 50–80% is absorbed in the intestine. This is adequate to pro- mal jejunum. Once taken up by the intestinal epithelial cell, vide the minimum daily requirement of about 50 mcg/day the folate is reduced to N5@methyl THF, the primary circulat- needed to sustain normal metabolism. The liver stores from ing form of THF in the blood. N5@methyl THF is distributed 5 to 10 mg of folate, which is sufficient to provide the daily throughout the body via the blood and attaches to cells by requirement for three to six months if folate is omitted from means of specific receptors called cell surface folate receptor@a the diet. (a glycosyl-phosphatidylinositol [GPI]–anchored receptor; Folate plays an important role in normal embryogen- Chapter 17). This receptor is upregulated through transcrip- esis. The folate receptor@a is activated early in embryonic tional, translational, and post-translational mechanisms stem cells and increases as the need for folate increases. when intracellular and extracellular folate is decreased. Observations have revealed a high incidence of low folate Translational receptor upregulation involves the covalent levels in women who give birth to babies with neural tube binding of accumulated homocysteine (which occurs when defects (NTD) compared with women who give birth to there is a folate deficiency) with a protein called heteroge- normal babies. Experimental studies suggest perturbations neous nuclear ribonucleoprotein-E1 (hnRNP-E1).16 in folate receptor@a may be involved in these neural tube Once inside the cell, N5@methyl THF must be demeth- closure abnormalities.17 Several studies have shown that ylated and reconjugated by the addition of seven or eight supplementation of folate during pregnancy can reduce glutamic acid residues to keep it from leaking out again. the rate of occurrence of these birth defects by as much as Demethylation is a reaction that requires cobalamin and one-half. In an effort to reduce NTDs, the U.S. government methionine synthase (Figure 15-7a). Thus, a deficiency of required the fortifications of grain products with folic acid cobalamin traps folate in its methylated form and blocks the beginning in the fall of 1997.17 The goal was to increase the formation of other forms of THF and the synthesis of dTTP, daily dietary folate to 100 mcg per person. Pregnant women a nucleotide of DNA. This is commonly referred to as the need an intake of about 800 mcg/day. folate trap or methyl trap. Although free THF is easily conju- Folate can become depleted quickly in conditions with gated within cells, methyl-THF is not; consequently, much rapid cell turnover such as sickle cell anemia and other of the methyl-THF taken up by a cobalamin-deficient cell hemolytic anemias, during growth, pregnancy, and lacta- leaks out before additional glutamates can be added.14 The tion. Thus, the daily requirement in patients with these con- cells in cobalamin deficiency are unable to retain their folate, ditions is increased. leading to tissue folate depletion. In the demethylation of N5@methyl THF by cobalamin, homocysteine is methylated to methionine, a precursor of S-adenosylmethionine (SAM). Checkpoint 15.5 This reaction requires methionine synthase and cobalamin. Hal had small bowel resection due to carcinoma. Explain why SAM is thought to be critical to nervous system function. he is at high risk for

folate deficiency. REQUIREMENTS Folate is present in most foods including eggs, milk, yeast, PATHOPHYSIOLOGY OF FOLATE DEFICIENCY mushrooms, and liver but is especially abundant in green Folate deficiency results in decreased synthesis of leafy vegetables (from which it gets its name). It is also syn- N5, N10@methylene THF, which is needed as a cofactor in thesized by microorganisms. The vitamin is destroyed by DNA synthesis. Consequently, there is a marked slowing 330 Chapter 15 of DNA synthesis, and the S phase of the cell cycle is pro- to obtain enough of the appropriate foods to maintain longed. The impairment of DNA synthesis is due to the adequate folic acid intake. People who are alcoholics inability to convert deoxyuridine monophosphate (dUMP) whose diet consists mainly of large quantities of ethanol to deoxythymidine monophosphate (dTMP), the precur- have a deficiency of many vitamins in addition to folic sor of dTTP. Subsequently, the dUMP is phosphorylated to acid. Complicating the folate deficiency in alcoholic indi- the triphosphate form (dUTP). DNA polymerase does not viduals, the ethanol appears to impair release of folate effectively distinguish dUTP from dTTP, and dUTP is erro- from the liver and can be toxic to erythroid precursors. neously incorporated into the DNA of folate-deficient cells. Erythroid precursors in alcoholism are frequently vacu- The DNA “proofreading” function of the cells recognizes olated. In alcoholic individuals who have liver disease but the mistake and tries to repair the DNA by replacing uridine have an adequate diet, the anemia is macrocytic but not with thymidine, but the repair attempt fails due to the lack megaloblastic. of available dTTP. The result is ultimately DNA fragmen- Increased Requirement In individuals with increased cell tation and cell death by apoptosis.14 All rapidly dividing replication, the normal daily intake of folic acid may not cells—including erythrocyte, leukocyte, and platelet pre- be sufficient to maintain normal DNA synthesis. Without cursors and intestinal epithelium—are affected by a folate folate supplements, folate stores can be rapidly depleted. deficiency. Surviving hematopoietic cells show characteris- This occurs in hemolytic anemias such as sickle cell anemia tic megaloblastic changes. and thalassemia, in myeloproliferative diseases such as leu- The bone marrow can exhibit a three-fold increase in kemia, and in metastatic cancers. Anemia in pregnancy is erythropoiesis, but the peripheral blood reticulocyte count common and can be caused by deficiencies of iron and/or is low, indicating a large degree of ineffective erythropoi- folic acid. The deficiency of folic acid is related to the lim- esis. Increased apoptosis of the blood cell precursors leads ited reserves of this nutrient and a 5- to 10-fold increased to increased heme catabolism and iron turnover, signs of demand for its use created by the growing fetus. Prophy- hemolysis, jaundice, and pancytopenia. Extramedullary lactic folic acid supplements are usually prescribed during hemolysis also occurs, and circulating red cell survival pregnancy.18 can be decreased by 30–50%. The compounds requiring folic acid as a cofactor for metabolism (homocysteine and Malabsorption Intestinal diseases affecting the upper small FIGLU) accumulate. Mildly elevated levels of homocysteine intestine, which interfere with the absorption of nutrients, are considered a major risk factor for atherosclerosis and can cause a folate deficiency. The most common conditions venous thrombosis (Chapter 35). of this type include ileitis, tropical sprue, and nontropical The clinical findings of folate deficiency develop sprue. The blind loop syndrome associated with an over- sequentially. Serum folate decreases within 1–2 weeks of growth of bacteria can cause a folate deficiency because the onset of a folate deficiency. Hypersegmented neutrophils bacteria preferentially utilize the folate. are the first morphologic change and occur at about 2 weeks. Drug Inhibition Megaloblastic anemia has also been asso- The urinary excretion of FIGLU increases next at about ciated with certain drugs including oral contraceptives, 13 weeks, and anemia appears last at about 19–20 weeks. long-term anticoagulant drugs, phenobarbital, primidone, CAUSES OF FOLATE DEFICIENCY and phenytoin. Anemia occasionally is not present even Folate deficiency can occur as the result of an inadequate though serum and erythrocyte folate are depressed. dietary intake, an increased requirement, malabsorption in the small intestine, or drug inhibition (Table 15-4). Checkpoint 15.6 Inadequate Diet The most common cause of folate defi- What is the most common cause of folate deficiency, and in ciency is an inadequate dietary intake of folic acid. This what groups of individuals is it usually found? is seen most often in poor and elderly people who fail Table 15.4 Causes of Folate Deficiency Cause Examples Inadequate diet Low income, elderly with limited function/income, alcoholics Increased requirement Diseases/conditions associated with rapid cell turnover (sickle cell anemia, thalassemia, leukemias, other malignancies, pregnancy, infancy) Malabsorption Ileitis, tropical sprue, nontropical sprue, blind loop syndrome Drug inhibition Oral contraceptives, long-term anticoagulant therapy, phenobarbital, primidone, phenytoin, antimetabolite chemotherapy Biologic competition Bacterial overgrowth in small intestine Megaloblastic and Nonmegaloblastic Macrocytic Anemias 331 LABORATORY EVALUATION OF FOLATE DEFICIENCY cobalt. The more accurate terminology when referring to Both serum and erythrocyte folate levels are decreased in this family of vitamins is cobalamin. Vitamin B12 refers spe- folate deficiency. Serum folate reflects the folic acid intake cifically to the therapeutic form of crystalline cobalamin that over the last several days, whereas erythrocyte folate contains the ligand cyanide (cyanocobalamin), a form not reflects the folate available when the red cell was maturing naturally found in the body but used for treating cobalamin in the bone marrow and reflects the net folate level over deficiency. In hematology literature, the terms cobalamin and the preceding several months. Serum folate can be falsely vitamin B12 are often used interchangeably. In this text we increased with even slight hemolysis of the sample. Low use the term cobalamin. serum folate can indicate an imminent folic acid deficiency Cobalamin is structurally classified as a corrinoid, a and precedes erythrocyte folate deficiency.15 family of compounds with a corrin ring. The molecule has Care must be taken in interpreting folate results three portions: (1) a corrin ring composed of four reduced because both serum and erythrocyte folate can be pyrrole groups with a cobalt at the center (cobalamin gets falsely increased or decreased in a variety of conditions its name from the central cobalt), (2) a nucleotide that lies (Table 15-5). Neither serum (methyl-THF) nor erythrocyte almost perpendicular to the ring attached to the cobalt, and folate (heterogeneous folate mixture with various polyglu- (3) various ligands (a b@group) attached to the cobalt on the tamate chain lengths) is a good indicator of folate stores if opposite side of the ring from the nucleotide (Figure 15-8). there is a deficiency of cobalamin. A cobalamin deficiency The b@group in cobalamin is cyanide, methyl, adenosyl, leads to the accumulation of methyl-THF because the dona- or hydroxyl. Adenosylcobalamin (AdoCbl) and methylco- tion of the methyl group to homocysteine and the release of balamin (MeCbl) act as coenzymes in biological reactions. free THF is impaired. In addition, cobalamin is required for Hydroxycobalamin (OHCbl) and cyanocobalamin (CnCbl) normal transfer of methyl-THF to the cells and for keeping are not metabolically active forms of cobalamin but can be the folate in the cell as conjugated folate. Thus, serum folate converted to the active methyl and adenosyl forms by tis- can be falsely increased (by 20–30%) and erythrocyte folate sue enzymes. falsely decreased in cobalamin deficiency due to folate trap- As mentioned previously, methionine is formed ping as methyl-THF. Epithelial changes in the GI tract that from homocysteine when a methyl group is transferred accompany cobalamin deficiency can lead to malabsorption from N5@methyl THF to homocysteine in a cobalamin- of folic acid in which case both serum and erythrocyte folate dependent reaction catalyzed by methionine synthase levels are decreased. (Figure 15-7a). For this reaction, the vitamin must be in Competitive folate-binding assays (indirect immuno- the MeCbl form. A cobalamin deficiency traps newly assays) using chemiluminescence methods have replaced acquired folate in the N5@methyl THF form (the form microbiological assays for both serum and RBC folate. in which the cell acquires folate from the plasma). N5@methyl THF is not efficiently conjugated with glu- Cobalamin (Vitamin B12) tamic acids and thus leaks back out of cells (see the discussion of folate metabolism). The result is an intra- Cobalamin is required for DNA synthesis and neurologic cellular functional deficiency of folate and a block of function. Specific diagnosis of cobalamin deficiency is DNA synthesis. critical because associated neurologic damage can be irre- AdoCbl is required for only one mammalian reaction: versible. Pernicious anemia is a specific form of cobala- the conversion of methylmalonyl CoA to succinyl CoA min deficiency and will be discussed in detail later in this (Figure 15-7c). AdoCbl acts as a coenzyme with methyl- chapter. malonyl CoA mutase in this reaction. Increased urinary STRUCTURE AND FUNCTION excretion of methylmalonic acid (MMA), a precursor of Vitamin B12 is a commonly used generic term for a family methylmalonyl-CoA, is a diagnostic aid in cobalamin of cobalamin vitamins in which ligands can be chelated to deficiency. Table 15.5 Causes of False Increases and Decreases in Folate Levels Erythrocyte Folate Serum Folate False increase Early folate deficiency Recent increase in dietary folate Reticulocytosis Hemolysis of blood sample Recent RBC transfusion Coexisting cobalamin deficiency False decrease Cobalamin deficiency Recent low dietary intake Recent alcohol consumption Gallium or technetium administration 332 Chapter 15 CN, CH3, OH–, or deoxyadenosyl METABOLISM Defects in any of the steps of cobalamin metabolism can lead to a cobalamin deficiency and megaloblastic anemia. NH2OCH2CH CH 2 3 CH3 CH2OCNH The laboratory plays a critical role in both assessing the 2 NH2COCH2 C level of the vitamin and determining the cause of low lev- CH2CH2CONH2 CH els. Thus, it is important to understand the metabolism of 3 N N CH this important nutrient. 3 Co CH N N CH Absorption Cobalamin is present in most foods of animal 3 origin including milk, eggs, and meat. The vitamin complex C CH OC CH H 3 2C 2 CH3 is released from food by peptic digestion at the low pH in CH3 CH2CH2CONH2 the stomach and binds tightly to a haptocorrin (HC)–like protein, a cobalamin binding protein secreted in the saliva and in the stomach (Figure 15-9). This binding protein Nucleotide was previously known as R-protein, named because of its (dimethylbenzimidazole derivative) rapid electrophoretic mobility compared with the mobility Figure 15.8 of intrinsic factor. HC is the preferred binding protein for Molecular structure of the cobalamin molecule. cobalamin released from food. The HC–cobalamin complex protects the cobalamin from degradation by the hydro- chloric acid in the stomach. In the duodenum, pancreatic Checkpoint 15.7 proteases degrade HC, releasing cobalamin. The released A patient has the following results: cobalamin 50 pg/mL, serum cobalamin quickly binds to intrinsic factor (IF), which resists folate 4 ng/dL, RBC folate 100 ng/mL. Interpret these results. pancreatic degradation. Intrinsic factor is a glycoprotein secreted by parietal cells of the gastric mucosa in response Cbl ? food complex IF Stomach (secretes HC and IF) HC 1 Cbl HC ? Cbl Duodenum HC (haptocorrin degraded) Cbl Cbl 1 IF Cbl ? IF Cbl ? IF Cbl ? IF Ileum receptors IF Cbl ? IF on cells Cbl Cbl ? TC Cbl ? TC leaves the cell to enter the plasma Mucosal lining cells Figure 15.9 Absorption of cobalamin in the gastrointestinal tract. HC, haptocorrin; IF, intrinsic factor; TC, transcobalamin; Cbl, cobalamin. Megaloblastic and Nonmegaloblastic Macrocytic Anemias 333 to the presence of food, vagal stimulation, histamine, and the bile where it binds to haptocorrin. This HC–-cobala- gastrin. IF binds cobalamin with a 1:1 stoichiometry and min complex enters the intestine where the cobalamin is is required for the intestinal absorption of cobalamin. The released from HC by pancreatic proteases and bound by IF IF–cobalamin complex resists digestion and passes through in the same manner as dietary cobalamin. the jejunum into the ileum where it binds the specific IF HC binds ligands less specifically and more tightly receptor (cubulin) on the microvilli of ileal mucosal cells.14 than TC. It binds a wide variety of corrinoids in addition to Binding requires a pH of 5.4 or higher as well as calcium. cobalamin. These analogues are transported by HC to the Following attachment of the IF–cobalamin complex to liver, metabolized, and excreted in the bile. The analogues cubulin, the entire complex is taken into the mucosal cell bind poorly to IF and therefore are not absorbed but

are by endocytosis. Cobalamin is released from IF and enters excreted in the feces. Thus, HC can serve to clear the body the portal blood while IF is degraded.14 The IF-receptors are of nonphysiologic cobalamin analogues. recycled to the microvilli of the ileum where they partici- In plasma, HC is largely produced by granulocytes. pate in absorption of more IF–cobalamin complexes. Large Haptocorrins are increased in myeloproliferative disorders, oral doses of cobalamin can be absorbed by simple diffusion presumably due to excess proliferation of granulocytes. without IF (see the section “Therapy”). REQUIREMENTS Transport Proteins that transport cobalamin in the blood About 3–5 mcg of cobalamin per day is needed to main- are transcobalamin (previously known as transcobalamin II) tain normal biochemical functions. It is estimated that only and haptocorrins (previously known as transcobalamin I about 70% of cobalamin intake is absorbed, which sug- and transcobalamin III or R-proteins). These plasma hapto- gests that the diet should include 5–7 mcg of the vitamin corrins belong to the same family of proteins as the gastric per day. This amount is available in a regular “balanced” haptocorrin. diet but not in a strict vegetarian diet. Cobalamin stores Transcobalamin (TC) is produced in many types of (about 5,000 mcg) are sufficient to provide the normal daily cells, including hepatocytes, enterocytes, macrophages, and requirement for about 1,000 days. Therefore, it takes several hematopoietic precursors in the bone marrow. Although years to develop a deficiency if no cobalamin is absorbed TC carries only a small fraction of the total cobalamin in from the diet. About half of the vitamin is stored in the liver, and the rest is located in the heart and kidneys.19 the plasma, it binds 90% of the newly absorbed cobalamin and is the primary plasma protein that mediates transfer of cobalamin into the tissues. TC also binds corrins that Checkpoint 15.8 are chemically similar to cobalamin but have no known Explain why there is a megaloblastic anemia in transcobalamin function in mammalians (cobalamin analogues). The deficiency when the serum cobalamin concentration is normal. TC–cobalamin complex is thought to be formed within the ileal mucosal cells and released to the blood.14 This transport complex disappears rapidly from blood (T1/2 of PATHOPHYSIOLOGY OF COBALAMIN DEFICIENCY 6–9 minutes) as it is taken up by cells in the liver, bone mar- Deficiency of cobalamin is reflected by (1) impaired DNA row, and other dividing cells that have specific receptors for synthesis (megaloblastic anemia) and (2) defective fatty acid TC (TCblR, CD320). The TC–cobalamin complex is internal- degradation (neurologic symptoms). ized by receptor-mediated endocytosis. Once inside the cell, cobalamin is released from TC and utilized, and the TC is Impaired DNA Synthesis A deficiency in either cobalamin degraded. Congenital deficiency of TC produces a severe or folic acid results in impaired production of methylene-THF, megaloblastic anemia in infancy. However, serum cobala- a defect in dTTP synthesis, and ultimately a defect in DNA min concentration in this condition is normal. synthesis (Figure 15-7a). This produces megaloblastic anemia and epithelial cell abnormalities. All dividing cells including The functions of the haptocorrins, a group of immuno- the hematopoietic cells in the bone marrow are affected. logically related proteins, are less well understood. They are synthesized by the mucosal cells of the organs that secrete Defective Fatty Acid Degradation AdoCbl is a cofactor in them and by phagocytes. They are found in body fluids the conversion of methylmalonyl CoA to succinyl CoA. In including plasma, saliva, amniotic fluid, milk, and gastric cobalamin deficiency, there is a defect in degradation of juice. Haptocorrins (HC) bind 70–90% of circulating cobal- propionyl CoA to methylmalonyl CoA and, finally, to suc- amin forming the cobalamin–HC complex. Cobalamins cinyl CoA. As propionyl CoA accumulates, it is used as a undergo a significant amount of enterohepatic recirculation. primer for fatty acid synthesis, replacing the usual primer The cobalamin–HC complex attaches to an asialoglycopro- acetyl CoA. This results in fatty acids with an odd number tein receptor on hepatocytes. Clearance of HC from plasma of carbons. These odd-chain fatty acids are incorporated is much slower (T1/2 9–10 days) than that of TC. Within the into neuronal membranes, causing disruption of membrane hepatocyte, HC is degraded and cobalamin is excreted into function. Demyelination (destruction, removal, or loss of 334 Chapter 15 the lipid substance that forms a myelin sheath around the CAUSES OF COBALAMIN DEFICIENCY axons of nerve fibers), a characteristic finding in cobalamin Cobalamin deficiency has many causes, including lack deficiency, is a result of this erroneous fatty acid synthesis. of intrinsic factor (pernicious anemia), malabsorption, A critical feature of demyelination in cobalamin defi- nutritional deficiency, and impaired utilization by tissues ciency is neurological disease. Peripheral nerves are most due to defective or absent transport proteins or enzymes often affected, presenting initially as motor and sensory (Table 15-6). neuropathy. The brain and spinal cord can also be affected leading to dementia, spastic paralysis, and other serious Pernicious Anemia Pernicious anemia (PA) is a specific neurological disturbances. Neurologic damage has been term used to define the megaloblastic anemia caused by an known to occur occasionally without any sign of anemia absence of IF secondary to gastric atrophy. An absence of IF or macrocytosis, making accurate diagnosis difficult but leads to cobalamin deficiency because the vitamin cannot critical.20 The bone marrow, however, always shows mega- be absorbed in its absence. PA is the most common cause of loblastic hematopoiesis. Neurological disease might not cobalamin deficiency, accounting for 85% of all deficiencies. be totally reversible but, if treated early, can be partially Atrophy of gastric parietal cells is demonstrated by find- resolved. Neurological disease does not occur in folate ing achlorhydria of gastric juice after histamine stimula- deficiency. Administration of synthetic folic acid corrects tion (these cells produce HCl as well as IF). PA is generally the anemia of cobalamin deficiency but does not halt or a disease of older adults, usually occurring after 40 years reverse neurological disease because synthetic folic acid, of age. This anemia is seen more commonly among people unlike dietary folate, is reduced directly to THF without the of northern European background, especially Great Britain requirement of cobalamin as a cofactor. The THF can correct and Scandinavia, but can be found in all ethnic groups. the megaloblastosis, but because there is a bypass of the More women than men are affected, and some patients have cobalamin-dependent reaction of conversion of homocyste- prematurely graying or whitening hair. Although no par- ine to methionine, SAM—a metabolite considered critical to ticular genetic abnormality has been identified, a positive nervous system function—is not formed. Thus, it is essential family history of PA increases the risk of developing it by to differentiate folate deficiency and cobalamin deficiency 20-fold. The incidence of gastric carcinoma in patients with PA is increased.21,22 so that appropriate treatment can be given. Gastritis and abnormalities of the gastrointestinal epithe- PA is an autoimmune disease. The gastric atrophy is lium secondary to cobalamin deficiency can interfere with the thought to result from immune destruction of the acid- absorption of folic acid and iron, complicating the anemia. secreting portion of the gastric mucosa.14 Up to 90% of PA patients have antibodies against parietal cells.23 However, these antibodies are not specific for pernicious anemia and are also found in patients with gastritis, thyroid disease, Checkpoint 15.9 and Addison’s disease. On the other hand, serum antibod- Explain why severe cobalamin deficiency sometimes presents with neurological disease. ies against intrinsic factor are found in about 75% of PA patients and are highly specific for PA. These IF antibodies Table 15.6 Causes of Cobalamin Deficiency and Associated Conditions Causes Malabsorption Biologic competition Nutritional deficiency Impaired utilization Associated Conditions Pernicious anemia (lack of IF) Intestinal parasite Strict vegetarian diets Transcobalamin deficiency (i.e., Diphyllobothrium latum) Gastrectomy or gastric bypass Leishmaniasis Pregnant women on a poor Nitrous oxide inhalation diet Crohn’s disease Bacterial overgrowth Malnutrition Tropical sprue Celiac disease Surgical resection of the ileum Imerslund-Grääsbeck disease Pancreatic insufficiency Drugs (colchicine, neomycin, p-aminosalicylic acid, or omeprazole) Blind loop syndrome Diverticulitis Megaloblastic and Nonmegaloblastic Macrocytic Anemias 335 are of two types: blocking and binding. Type I, or blocking tapeworm Diphyllobothrium latum can cause a deficiency antibodies, are antibodies to IF and prevent formation of the since the worm accumulates the vitamin avidly. IF–cobalamin complex. Type II, or binding antibodies, are Nutritional Deficiency Dietary deficiency of cobalamin is directed against the IF–cobalamin complex and prevent the rare in the United States. Food from animal sources, espe- IF–cobalamin complex from binding to ileal receptors. Bind- cially liver, is rich in cobalamin. Strict vegetarian diets, how- ing antibodies are found in about half the sera that contain ever, do not supply cobalamin and individuals following blocking antibodies. A number of findings suggest that the these diets can develop a deficiency over a period of years. immune destruction of the gastric mucosa is not antibody Occasionally, pregnant women with a poor diet can develop mediated but more likely T-cell mediated. Patients with a deficiency presumably due to an increased demand by the agammaglobulinemia have a higher than expected inci- developing fetus. However, folic acid deficiency is a more dence of PA. Also, lymphocytes from PA patients have been common cause of megaloblastic anemia in pregnancy due shown to be hyperresponsive to gastric antigens.14 to the lower preexisting stores of this nutrient. Pernicious anemia frequently occurs with other auto- immune diseases such as Graves’ disease and Hashimoto’s Other Causes Transcobalamin deficiency produces mega- thyroiditis, type I diabetes, and Addison’s disease. In addi- loblastic anemia because it is the major transport protein tion, a predisposition to PA can be inherited. Relatives of responsible for delivering the vitamin to the tissues. In TC patients with PA have a higher incidence of antiparietal cell deficiency, cobalamin is absorbed normally, and serum antibodies and anti-intrinsic factor antibodies than the gen- cobalamin levels are normal because of the relatively high eral population, even in the absence of overt PA.14 amount bound to HC. Tissue cobalamin deficiency, includ- Juvenile pernicious anemia is rare in children. It can occur ing megaloblastic anemia, develops, however, because the secondary to a variety of conditions, including a congenital cellular receptors recognize only TC-bound, not the hapto- deficiency or abnormality of IF (the more common type, corrin-bound cobalamin.14 with lack of IF but otherwise normal gastric secretion), or Nitrous oxide, N2O (“laughing gas”), abuse has been more rarely, true PA of childhood. True PA of childhood has reported to result in a cobalamin deficiency and megalo- an absence of intrinsic factor, gastric atrophy, decreased gas- blastic anemias. N2O rapidly inactivates methionine synthe- tric secretion, and antibodies against IF and parietal cells. tase, for which cobalamin is a coenzyme. Cobalamin cleaves Megaloblastic anemia in childhood from malabsorption of N2O and at the same time is oxidized to an inert form. This cobalamin can also be due to a congenital deficiency of TC, leads to a rapid deficiency. a congenital gastric haptocorrin deficiency, or selective mal- absorption of cobalamin (Imerslund-Gräsbeck disease). The Checkpoint 15.10 latter can be due to abnormal cubulin receptors in the ileum. Why is pernicious anemia considered an autoimmune disorder? Other Causes of Malabsorption Pernicious anemia is only one specific cause of cobalamin malabsorption, LABORATORY EVALUATION OF COBALAMIN which also can be caused by a loss of IF secondary to gas- DEFICIENCY trectomy or secondary to diseases that prevent binding In 2014, the British Committee for Standards in Haematol- of the IF–cobalamin complex in the ileum. An iron defi- ogy published a set of guidelines for diagnosis and treat- ciency usually precedes cobalamin deficiency in patients ment of cobalamin and folate disorders.25 Since there is no who have had a gastrectomy. Diseases that can affect the gold standard test that defines a cobalamin deficiency, it is absorption of the IF–cobalamin complex in the ileum recommended that the clinical picture be used to assess the include Crohn’s disease, tropical sprue, celiac disease, and significance of test results. surgical resection of the ileum. In Imerslund-Gräsbeck Laboratory diagnosis of pernicious anemia and/or disease, the IF receptors (cubulin) are missing or abnor- cobalamin deficiency usually begins with a serum cobala- mal, causing a form of juvenile megaloblastic anemia. min assay. Serum cobalamin measures both haptocorrin- Patients with severe pancreatic insufficiency experience a bound cobalamin (holo-HC) and transcobalamin-bound lack of absorption of cobalamin because the vitamin

can- cobalamin (holo-TC), the form of cobalamin taken up by not be released from HC and transferred to IF. Normally, cells. Both serum cobalamin and holo-TC are now highly pancreatic proteases are responsible for degrading HC. automated using competitive binding luminescence tech- Certain medications can interfere with intestinal absorp- nologies, which are suitable for large-scale screening of tion. In addition, conditions that allow a buildup of bac- cobalamin status. However, this technology can give spuri- teria in the small bowel can cause a cobalamin deficiency ous results in patients with high-titer anti-IF antibodies.26 as the bacteria preferentially take up the vitamin before it Holo-TC is also available as an automated monoclonal reaches the ileum. This situation occurs in the blind loop antibody assay with a reference interval of approximately syndrome and in diverticulitis. Infestation with the fish 20–125 pmol/L. 336 Chapter 15 Measurement of holo-TC can be an early marker for are the most sensitive and specific indicators of defi- cobalamin deficiency. Because cellular receptors specifically ciency.24–33 Some studies have shown that in addition to mediate the uptake of holo-TC but not holo-HC, holo-TC being a good indicator of cobalamin deficiency, increased could provide a more accurate view of patients’ cobalamin homocysteine levels are associated with a three-fold increase status than serum cobalamin levels that include both holo- risk in myocardial infarction as well as venous thrombosis.32 TC and holo-HC.27 Low holo-TC levels, however, may only Increased concentrations of both analytes can be found in be temporary or may even remain at the subclinical level many patients with normal serum cobalamin concentra- rather than progress to pathologic levels. The interpreta- tions.32 Some clinicians recommend initial testing for serum tion of holo-TC values is complicated by the fact that it is cobalamin levels and following up low or borderline nor- unclear whether low results reflect insufficient availability mal results with MMA and homocysteine measurements.33 to tissues or some impairment in the ability of the holo- Others have found that MMA and homocysteine measure- TC complex to leave the ileal mucosal cells.28 If the cobala- ments are more sensitive tests of early pernicious anemia, min level is decreased and there are clinical symptoms of preceding hematologic abnormalities and decreased serum a deficiency, no further testing is warranted before therapy cobalamin levels, and recommend them as a superior test- is initiated.29 However, studies have shown that serum ing regimen (Figure 15-7). cobalamin does not always detect a cobalamin deficiency, Homocysteine and MMA tests also are helpful in and further testing could be necessary if a deficiency is sus- determining a patient’s response to treatment with cobala- pected. Furthermore, serum cobalamin can appear falsely min and folate. The serum MMA level remains increased decreased or falsely normal/increased in some conditions in cobalamin-deficient patients treated inappropriately (Table 15-7). Cobalamin deficiency can be masked by folate with folate. Folate-deficient patients treated inappropri- therapy. ately with cobalamin have increased serum homocysteine Addition of second line tests to help clarify underly- level. See Table 15-8 for a list of the advantages and disad- ing biochemical and functional deficiencies include tests for vantages of laboratory tests used to diagnose cobalamin methylmalonic acid (MMA) and homocysteine. Increased deficiency.6 excretion of methylmalonic acid (MMA) in the urine indi- Although not used routinely due to cost associated rectly indicates a decrease in cobalamin concentration. Up with multiple tests, a formula called “Fedosov’s well- to 40% of patients may have increased MMA levels in the ness score,” transforms the results of four variables into urine but normal cobalamin levels.22 These patients, how- a single variable based on age.26 The variables include ever, show laboratory and clinical evidence of cobalamin holo-TC, serum cobalamin, MMA, and total homocyste- deficiency. The only condition in which MMA is increased ine. The result is a combined indicator of cobalamin status in addition to cobalamin deficiency is congenital meth- expressed as cB12. In an effort to decrease costs, three-vari- ylmalonic aciduria. This condition is caused by complete able and two-variable analyses may also be calculated. The or partial deficiencies of enzymes and cofactors involved three-variable analysis includes serum cobalamin, holo-TC, in the conversion of methlmalonyl CoA to succinyl CoA. and MMA. The two-variable analysis includes holo-TC and There is no associated hyperhomocysteinemia or megalo- MMA. Results are expressed as degrees of high, normal, blastic anemia. Determination of MMA concentration is and deficient. also useful in distinguishing a cobalamin deficiency and The serum cobalamin and MMA assays establish the folate deficiency. existence of a cobalamin deficiency but do not provide a Homocysteine is increased in the plasma of patients distinction between PA and other causes of cobalamin defi- with cobalamin or folate deficiency.30 Monitoring serum ciency. Gastric analysis may be more useful in establish- levels can be an early detector of cobalamin deficiency, and ing the specific diagnosis of PA but is rarely performed. A recent studies have concluded that MMA and homocysteine recommended testing algorithm for laboratory diagnosis of PA is shown in Figure 15-10.34 The order of testing can improve the efficiency of proper diagnosis by first mea- Table 15.7 Causes of False Decrease and False Normal/ suring serum cobalamin and MMA. If serum cobalamin Increase in Serum Cobalamin Levels is borderline or low and MMA is increased, the diagnosis False Decrease False Normal/Increase of PA should be confirmed with testing for intrinsic fac- tor blocking antibody (IFBA) and serum gastrin levels. Folate deficiency Cobalamin treatment Gastrin is usually markedly increased in PA due to gastric Pregnancy (last trimester) Liver disease atrophy.33–35 Oral contraceptive use Nitrous oxide exposure Multiple myeloma Chronic myelogenous leukemia Gastric Analysis Because atrophy of the parietal cells is Elderly Polycythemia vera a universal feature of PA, an absence of free HCl in gastric Haptocorrin deficiency Transcobalamin deficiency juice after histamine stimulation is indicative of PA. Parietal Megaloblastic and Nonmegaloblastic Macrocytic Anemias 337 Table 15.8 Tests Used to Diagnose Cobalamin Deficiencya Test Advantages Disadvantages Cobalamin/serum High sensitivity; widely available; variation in reference Only holo-TC is available for cells; falsely decreased in intervals according to method of analysis; measures folate and HC deficiency both HC and TC Methylmalonic acid (MMA)/urine High sensitivity and specificity; increased concentra- Falsely increased in reduced renal function; not read- tion helps differentiate cobalamin from folate defi- ily available; expensive ciency; increased level may precede hematologic abnormalities; helpful in determining patient response to cobalamin treatment Homocysteine/plasma High sensitivity; increased level may precede hema- Low specificity; does not differentiate cobalamin tologic abnormalities; helpful in determining patient deficiency from folate deficiency; falsely increased in response to cobalamin treatment reduced renal function, hypothyroidism, hypovolemia Holo-TC Sensitivity and specificity similar to cobalamin; mea- Falsely increased in liver disease and in reduced renal sure of cobalamin available to tissue function; complicated interpretation (see the section “Laboratory Evaluation of Cobalamin Deficiency”); not a readily available test Complete blood count with differential High sensitivity of macrocytosis with megaloblastic Specificity of macrocytosis without megaloblastic changes changes is low; not readily recognizable in subclinical cobalamin deficiency a Sensitivity and specificity of these tests may be lower in subclinical cobalamin deficiency; subclinical deficiency has no known clinical expression and diagnosis depends on biochemical markers; recommended diagnostic test combination for cobalamin deficiency is serum cobalamin and MMA. Both should be abnormal for a diagnosis of cobalamin deficiency. cells secrete both HCl and intrinsic factor; thus an absence CobaSorb Test Once a cobalamin deficiency has been of HCl is indirect evidence for lack of IF. After histamine defined, the capacity to absorb cobalamin should be stimulation in patients with pernicious anemia, the pH assessed before a treatment choice is made. The CobaSorb fails to fall below 3.5, and gastric volume, pepsin, and ren- test is a test designed to measure the absorption of cobal- nin are decreased. Approximately 80% of cases of PA have amin.6,36,37 There are three parts to this test in which the increased gastrin. Testing for anti-intrinsic factor antibodies, patient is given nonradioactive cobalamin either alone or serum gastrin, and serum pepsinogin A and C together as a with recombinant IF or haptocorrin. Blood levels of holo- group provides a sensitive indication of gastric atrophy and TC and holo-HC are measured before and after administra- pernicious anemia.35 tion of the cobalamin. An increase in the concentrations of Cobalamin Assay, Serum < 150 ng/L > 400 ng/L Intrinsic blocking antibody 150–400 ng/L Neg Intermediate Pos > 0.4 nmol/mL Methylmalonic acid ≤ 0.4 nmol/mL Result consistent Result does not imply a Result does not imply with pernicious deficiency of cobalamin cobalamin deficiency. Gastrin anemia. at the cellular level. No further testing No further testing suggested. suggested. ≤ 200 pg/mL > 200 pg/mL Result does not Results consistent suggest with pernicious pernicious anemia. anemia. Figure 15.10 Testing algorithm for the diagnosis of pernicious anemia. 338 Chapter 15 these plasma transport proteins saturated with cobalamin (OHCbl) because of these patients’ inability to absorb oral after administration of the cobalamin is reflective of active cobalamin. Large doses of cobalamin therapy (usually cobalamin absorption. Refer to Table 15-9 for a list of tests 1,000–2,000 mcg/day) administered orally could be feasi- used to investigate the cause of a cobalamin deficiency. ble if the patient is followed carefully.24 The oral treatment can be better tolerated and less expensive.38 The rationale behind oral therapy using large doses of vitamin is that a Checkpoint 15.11 small amount (from 1-3%) of the vitamin is absorbed by What two lab tests are the most specific indicators of cobalamin diffusion without IF. deficiency? THERAPY CASE STUDY (continued from page 326) Therapeutic trials in megaloblastic anemia using physiologic Antibody testing had the following result: doses of either vitamin B12 or folic acid produce a reticulo- Intrinsic-factor-blocking antibodies positive to cyte response only if the specific vitamin that is deficient a titer of 1:6400 is being administered. For instance, small doses (1 mcg) of 5. What is this patient’s definitive diagnosis? vitamin B12 given daily produce a reticulocyte response in cobalamin deficiency but not in folic acid deficiency. On the 6. What would you predict this patient’s reticulo- other hand, large therapeutic doses of cobalamin or folic cyte count to be? acid can induce a partial response to the other vitamin defi- ciency as well as the specific deficiency. Generally, it is best to determine which deficiency exists and to treat the patient with the specific deficient vitamin. Other Megaloblastic Anemias Large doses of folic acid will correct the anemia in cobal- A megaloblastic anemia is occasionally associated with amin deficiency but do not correct or halt demyelination drugs, congenital enzyme deficiencies, or other hemato- and neurologic disease. This makes diagnosis and specific poietic diseases. therapy in cobalamin deficiency critical. Specific therapy DRUGS causes a rise in the reticulocyte count after the fourth day of A large number of drugs that act as metabolic inhibitors can therapy. Reticulocytosis peaks at about 5–8 days and returns cause megaloblastosis (Table 15-10). Some of these drugs to normal after 2 weeks. The degree of reticulocytosis is pro- are used in chemotherapy for malignancy. Although aimed portional to the severity of the anemia with more striking at eliminating rapidly proliferating malignant cells, these reticulocytosis in patients with severe anemia. The hemo- drugs are not selective. Any normal proliferating cells, globin rises about 2–3 g/dL every 2 weeks until normal lev- including hematopoietic cells, are also affected.39 els are reached. The marrow responds quickly to therapy, as evidenced by normal pronormoblasts appearing within 4–6 ENZYME DEFICIENCIES hours and nearly complete recovery of erythroid morpho- Methionine synthase reductase (MSR) deficiency is a rare logic abnormalities within 2–4 days. Granulocyte abnormal- autosomal recessive disorder. A deficiency of this enzyme ities disappear more slowly. Hypersegmented neutrophils leads to a dysfunction of folate/cobalamin metabolism and can usually be found for 12–14 days after therapy begins. results in hyperhomocysteinemia, hypomethioninemia, Specific therapy can reverse the peripheral neuropathy and megaloblastic anemia. MSR is necessary for the reduc- of cobalamin deficiency, but spinal cord damage is usu- tive activation of methionine synthase and the resultant ally irreversible. Pernicious anemia must be treated with folate-cobalamin-dependent conversion of homocysteine lifelong monthly parenteral doses of hydroxycobalamin to methionine. Table 15.9 Tests Used to Investigate the Cause of Cobalamin Deficiencya Test Advantages Disadvantages IF blocking antibodies High specificity for lack of IF; characteristic for PA Low sensitivity Pepsinogen and gastrin High sensitivity; mirrors gastric function Low specificity for PA; increased

in gastric atrophy Parietal cell antibodies May be present in PA Low specificity CobaSorb Reflects absorption of cobalamin Use in routine practice needs further evaluation a These tests are used primarily to evaluate cobalamin deficiency due to pernicious anemia. Additional testing may be necessary to evaluate other causes of cobalamin deficiency. Patient history is also important in diagnosis. IF, intrinsic factor; PA, pernicious anemia Megaloblastic and Nonmegaloblastic Macrocytic Anemias 339 Table 15.10 Drugs That Can Cause Megaloblastosis DNA Base Inhibitors Pyrimidine Purine Antimetabolites Other Azauridine Acyclovir Cytosine arabinoside Azacytidine Adenosine arabinoside Fluorocytidine Cyclophosphamide Azathioprine Fluorouracil Zidovudine (AZT) Gancyclovir Hydroxyurea Mercaptopurine Methotrexate Thioguanine Vidarabine CONGENITAL DEFICIENCIES (Figure 15-11). Type II is distinguished by a positive A congenital deficiency is a physiological aberration pres- acidified serum test (Ham test; Chapter 17) but a nega- ent at birth. The following congenital deficiencies result in tive sucrose hemolysis test. In the Ham test, only about megaloblastosis. 30% of normal sera are effective in lysing CDA II cells. This type has also been termed hereditary erythroblastic Orotic Aciduria Inborn defects in enzymes required for pyrimidine synthesis or folate metabolism can result in multinuclearity with positive acidified serum test (HEMPAS). megaloblastic anemia. Orotic aciduria is a rare autosomal CDA II is the most common of the three types of CDA. recessive disorder in which there is a failure to convert • CDA III This type of CDA is morphologically distinct orotic acid to uridylic acid. The result is excessive excretion from Types I and II because of the presence of giant of orotic acid. Children with this disorder also fail to grow erythroblasts (up to 50 mcM) containing up to 16 nuclei. and develop normally. The condition responds to treatment Sometimes the erythrocytes are agglutinated by anti-I with oral uridine. and anti-i antibodies. Congenital Dyserythropoietic Anemia Congenital dys- erythropoietic anemia (CDA) is actually a heterogeneous group of refractory, congenital anemias characterized by both abnormal erythropoiesis and ineffective erythropoi- esis (Table 15-11). There are three types: CDA I, CDA II, and CDA III. Types I and II are inherited as autosomal recessive disorders, and Type III is inherited in an autosomal domi- nant fashion. Red cell multinuclearity in the bone marrow and secondary siderosis are recognized in all types; how- ever, megaloblastic erythroid precursors are present only in Type I and Type III. • CDA I Bone marrow erythroblasts are megaloblas- tic and often binucleate with incomplete division of Figure 15.11 Peripheral blood from a case of congenital nuclear segments. The incomplete nuclear division is dyserythropoietic anemia type II (CDA-II). There is anisocytosis characterized by internuclear chromatin bridges. with microcytic, hypochromic cells as well as macrocytes and normocytes. The nucleated cell is an orthochromic normoblast • CDA II Bone marrow precursors are not megaloblastic showing lobulation of the nucleus (Wright-Giemsa stain; 1000* but are typically multinucleated with up to seven nuclei magnification). Table 15.11 Comparison of Congenital Dyserythropoietic Anemia (CDA) Types Characteristics CDA I CDA II CDA III Inheritance Autosomal recessive Autosomal recessive Autosomal dominant RBC multinuclearity Present Present Present Number of nuclei 2 Up to 7 Up to 16 Siderosis Present Present Present Megaloblastosis Present Absent Present Other characteristics Incomplete nuclear division Positive Ham test RBC agglutination by anti-I and anti-i antibodies 340 Chapter 15 OTHER HEMATOPOIETIC DISEASES with macrocytosis should be questioned about their alcohol The myelodysplastic syndromes are a group of stem cell consumption.40 Macrocytosis associated with alcoholism is disorders characterized by peripheral blood cytopenias and usually multifactorial and can be megaloblastic. Macrocy- dyshematopoiesis. Erythroid precursors in the bone mar- tosis is probably the result of one or more of four causes: row frequently exhibit megaloblastic-like changes. There is (l) folate deficiency due to decreased dietary intake, (2) occasionally a nonmegaloblastic macrocytic anemia. These reticulocytosis associated with hemolysis or gastrointestinal diseases are discussed in Chapter 25. bleeding, (3) associated liver disease, and (4) alcohol toxicity. Folate deficiency associated with a megaloblastic ane- mia is the most common cause of the macrocytosis found Checkpoint 15.12 in hospitalized alcoholic patients. The deficiency probably Which clinical type of CDA gives a positive Ham test result and results from poor dietary habits, although ethanol also presents with a normoblastic marrow? appears to interfere with folate metabolism. The reduced erythrocyte survival with a corresponding reticulocytosis has been associated with chronic gastrointes- Macrocytic Anemia tinal bleeding secondary to hepatic dysfunction (decreased coagulation proteins) or thrombocytopenia, hypersplenism Without Megaloblastosis from increased portal and splenic vein pressure, pooling of cells in splenomegaly, and altered erythrocyte mem- The typical findings of megaloblastic anemia are not branes caused by abnormal blood lipid content in liver dis- evident in other macrocytic anemias. The macrocytes in ease (Chapter 17). Stomatocytes are associated with acute macrocytic anemias without megaloblastosis are usually alcoholism, but there appears to be no abnormal cation not as pronounced and are usually round rather than oval permeability, and hemolysis of these cells is not significant. as seen in megaloblastic anemia (Figure 15-1a, b). Hyper- Liver disease is common in alcoholic individuals; typi- segmented neutrophils are not present, and leukocytes and cal hematologic findings associated with this disease are platelets are quantitatively normal. Jaundice, glossitis, and discussed in the following section. Even when anemia is neuropathy, the typical clinical findings in megaloblastosis, absent, most alcoholic individuals have a mild macrocytosis are absent. The cause of the macrocytosis without mega- (100–110 fL) unrelated to liver disease or folate deficiency. loblastosis is unknown in many cases. In some cases the This can be caused by a direct toxic effect of ethanol on macrocytes can be due to an increase in membrane lipids or developing erythroblasts. Vacuolization of red cell precur- to a delay in erythroblast maturation. Some diseases associ- sors, similar to that seen in patients taking chloramphenicol, ated with nonmegaloblastic macrocytic anemia are listed in is a common finding after prolonged alcohol ingestion. If Table 15-12. Three of the most common—alcoholism, liver alcohol intake is eliminated, the cells gradually assume their disease, and reticulocytosis (stimulated erythropoiesis)— normal size, and the bone marrow changes disappear. The are discussed in this section. association of a sideroblastic anemia and alcoholism is dis- cussed in Chapter 12. Alcoholism The multiple pathologies of this type of anemia result Alcohol abuse is one of the most common causes of non- in the possibility of a variety of abnormal hematologic find- anemic macrocytosis. It has been suggested that all patients ings. Thus, it is possible to have a blood picture resembling Table 15.12 Conditions Associated with Nonmegaloblastic Macrocytosis Condition Cause of Macrocytosis Alcoholism Direct toxic effect of alcohol on erythroid precursors Reticulocytosis associated with hemolysis or GI bleeding Liver disease (abnormal RBC membrane lipid composition) Can also be megaloblastic from folate deficiency Liver disease Increased RBC membrane lipids Hemolysis or posthemorrhagic anemia Reticulocytosis associated with stimulated erythropoiesis Hypothyroidism Unknown Aplastic anemia Unknown Artifactual Cold agglutinin disease Severe hyperglycemia RBC clumping Swelling of RBCs Megaloblastic and Nonmegaloblastic Macrocytic Anemias 341 that of megaloblastic anemia, chronic hemolysis, chronic or patients also show abnormal liver function and have mark- acute blood loss, liver disease, or (more than likely) a com- edly increased levels of plasma triglycerides. bination of these conditions. Alcohol can also cause disor- Abnormalities in erythrocyte membrane lipid compo- dered heme synthesis, as discussed in Chapter 12. sition are common in hepatitis, cirrhosis, and obstructive jaundice. Both cholesterol and phospholipid are increased, Liver Disease resulting in cells with an increased surface area–to–volume ratio. This abnormality is not thought to cause decreased cell The most common condition associated with a nonmegalo- survival. In contrast, in severe hepatocellular disease, eryth- blastic macrocytic anemia is liver disease (including alco- holic cirrhosis). The causes of this anemia are multifactorial rocyte membranes have an excess of cholesterol relative to and include hemolysis, impaired bone marrow response, phospholipid, which decreases the erythrocyte deformabil- folate deficiency, and blood loss (Table 15-13). Although ity. This membrane lipid imbalance is associated with the macrocytic anemia is the most common form of anemia in formation of spur cells in which the erythrocyte exhibits liver disease, occurring in more than 50% of the patients, spike-like projections. These cells have a pronounced short- normocytic or microcytic anemia can also be found depend- ened life span leading to an anemia termed spur cell anemia ing on the predominant pathologic mechanism. (Chapter 17). Erythrocyte survival appears to be significantly short- Kinetic iron studies have revealed that the bone mar- ened in alcoholic liver disease, infectious hepatitis, biliary row response in liver disease can be impaired. It has been cirrhosis, and obstructive jaundice. The reason for this is proposed that liver disease can affect the production of unknown. Cross-transfusion studies in which patient cells erythropoietin because this organ has been shown to be an are infused into normal individuals demonstrate an increase important extrarenal source of the hormone.41 In alcoholic in patient cell survival. This suggests that an extracellular cirrhosis, the alcohol may have a direct suppressive effect factor is probably responsible for cell hemolysis. The spleen on the bone marrow. is thought to play an important role in sequestration and Clinical findings and symptoms in liver disease are sec- hemolysis in individuals with splenomegaly or hypersplen- ondary to the abnormalities in liver function. The liver is ism. In some cases, hemolysis is well compensated for by involved in many essential metabolic reactions and in the an increase in erythropoiesis and there is no anemia. In synthesis of many proteins and lipids. Therefore, the ane- some patients with alcoholic liver disease, a heavy drink- mia is a minor finding among the abnormalities associated ing spree produces a brisk but transient hemolysis. These with this organ’s dysfunction. Table 15.13 Causes and Characteristics of Anemia in Liver Disease Causes Anemia Type Characteristics Abnormal liver function Macrocytic MCV normal to increased Increased RBC membrane cholesterol resulting in target cells and acanthocytes Normal to slightly increased reticulocytes Folic acid deficiency Macrocytic MCV increased Pancytopenia Ovalocytes and teardrop cells Normal to decreased reticulocytes Functional folate deficiency Hemolysis Normocytic or macrocytic MCV normal to increased Spur cells and schistocytes Increased reticulocytes Hypersplenism Normocytic MCV normal Pancytopenia Increased reticulocytes Portal hypertension Marrow hypoproliferation Normocytic MCV normal Pancytopenia Decreased reticulocytes Associated with renal disease or alcohol suppression Chronic blood loss Microcytic MCV decreased Iron deficiency Leukocyte and platelet counts slightly increased Reticulocytes increased Commonly associated with gastrointestinal bleeding 342 Chapter 15 The anemia is usually mild with an average hemoglo- bone marrow. These cells are larger than normal with an bin concentration of about 12 g/dL. With complications, MCV as high as 130 fL. A reticulocyte count and examina- the anemia can be severe. The erythrocytes can appear tion of the blood smear allow distinction of this macro- normocytic, macrocytic (usually not more than 115 fL cytic entity from megaloblastic anemia. In the presence MCV), or microcytic. A discrepancy between the MCV and of large numbers of shift reticulocytes, polychromasia is the appearance of the cells microscopically often occurs. markedly increased. The WBC count is slightly increased, In these cases, thin, round macrocytes (as determined by and the platelet count is normal. In addition, the oval mac- diameter) with target cell formation are found on the blood rocytes typical of megaloblastic anemia are not present smear, but the MCV is within normal limits. The reticu- in conditions associated with increased erythropoietin locyte count can be increased, but the RPI is usually less stimulation. than 2 unless hemolysis is a significant factor. Thrombo- cytopenia is a frequent finding, and platelet function can Hypothyroidism be abnormal. Various nonspecific leukocyte abnormalities have been described including neutropenia, neutrophilia, Anemia of hypothyroidism presents as a mild to moderate and lymphopenia. The bone marrow is either normocellular anemia with a normal reticulocyte count. Thyroid hormone or hypercellular, often with erythroid hyperplasia. The pre- regulates cellular metabolic rate and therefore tissue oxy- cursors are qualitatively normal unless folic acid deficiency gen requirement. With a decrease in thyroid hormone (i.e., is present. In this case, megaloblastosis is apparent with the hypothyroidism), tissue oxygen requirement is reduced typical associated blood abnormalities. and the kidneys adapt appropriately. The net result is a Other laboratory tests of liver function are variably abnor- decrease in the production of EPO and, correspondingly, mal including increased serum bilirubin and increased hepatic erythrocytes. Thus, although the hemoglobin concentration enzymes. Tests for carbohydrate and lipid metabolites are fre- is reduced,

the tissue oxygenation is adequate. This type quently abnormal depending on the degree of liver disease. of “anemia” often presents as a macrocytic, normochromic anemia but can also be a normocytic, normochromic ane- mia. The anemia can be complicated by iron, folic acid, or Checkpoint 15.13 cobalamin deficiency, and the blood picture can reflect these What are the causes of macrocytosis seen in alcoholism? forms of anemia. Stimulated Erythropoiesis Checkpoint 15.14 What are three clinical or laboratory findings (in addition to Increased erythropoietin (stimulation) in the presence of assessing the bone marrow) that can distinguish a nonmegalo- an adequate iron supply (e.g., autoimmune hemolytic ane- blastic macrocytic anemia from a megaloblastic anemia? mia) can result in the release of shift reticulocytes from the Summary Macrocytosis due to megaloblastic anemia must be differen- Clinically, symptoms from inadequate dietary folate can tiated from macrocytosis with a normoblastic marrow. The occur within months as compared with years after onset of laboratory test profile of a patient with megaloblastic anemia a cobalamin deficiency. commonly indicates pancytopenia. The blood smear reveals Vitamin B12 (i.e., cyanocobalamin) absorption occurs macro-ovalocytes (i.e., large ovalocytes), poikilocytosis, tear- in the small intestine. Dietary cobalamin is released from drop cells, Howell-Jolly bodies, and neutrophil hyperseg- digestion of animal proteins in meats and bound by gastric mentation. The marrow is characterized by megaloblastosis haptocorrins and subsequently intrinsic factor (IF). Once of precursor cells due to a block in thymidine production. absorbed, cobalamin is bound to specific plasma proteins Because thymidine is one of the four DNA bases, the defi- known as transcobalamin and haptocorrins. Normal serum ciency leads to a diminished capacity for DNA synthesis and cobalamin values are highly variable based on age and sex. a block in mitosis. The marrow is hypercellular, but erythro- Cobalamin function is related to DNA synthesis because poiesis is ineffective. Causes of megaloblastosis are nearly cobalamin is a vital cofactor in the conversion of methyl tet- always due to cobalamin or folic acid deficiencies. rahydrofolate to tetrahydrofolate. This product is an impor- Folate is primarily acquired from the diet. The liver is tant cofactor needed for the production of DNA thymidine. the main storage site of folic acid. Folate deficiency results Defective production of intrinsic factor is the most in decreased synthesis of N5, N10@methylene THF, a cofac- common cause of cobalamin deficiency (pernicious ane- tor in DNA synthesis. Consequently, DNA synthesis slows. mia [PA]), which is caused by failure of the gastric mucosa Megaloblastic and Nonmegaloblastic Macrocytic Anemias 343 to secrete IF. PA most commonly occurs in people after stores in the presence of cobalamin deficiency. Cobalamin 40 years of age. Central nervous system symptoms can be levels can be directly assessed by measuring serum cobala- present in advanced cases. Laboratory tests used to diag- min or indirectly by measuring MMA in the urine/serum/ nose PA include serum cobalamin, MMA, IFBA, and serum plasma or homocysteine in plasma. gastrin. Other causes of cobalamin deficiency include gas- Macrocytosis due to megaloblastic anemia must be dif- trectomy, malabsorption diseases such as Crohn’s disease, ferentiated from macrocytosis with a normoblastic marrow. and drugs. Megaloblastic anemia rarely results from che- Normoblastic, macrocytic anemias can result from acute motherapeutic drugs or congenital enzyme deficiencies blood loss or hemolysis due to shift reticulocytosis from the or with other hematopoietic diseases such as congenital marrow. Alcohol abuse is one of the most common causes dyserythropoietic anemia. of normoblastic macrocytosis. Liver disease, often result- Serum and RBC folate can be measured to diagnose ing from alcohol abuse, is also commonly associated with folate deficiency, but the tests do not reliably indicate folate macrocytosis without a megaloblastic marrow. Review Questions Level I c. 2 years 1. The most common cause of macrocytosis is: d. 3–6 months (Objective 5) 6. Which of the following conditions increases the daily a. folate deficiency requirement for cobalamin? (Objective 2) b. alcoholism a. Pregnancy c. liver disease b. Aplastic anemia d. pernicious anemia c. Hypothyroidism 2. In the majority of cases, cobalamin deficiency is due d. Splenectomy to a deficiency of: (Objective 6) 7. A deficiency of cobalamin leads to impaired: a. intrinsic factor (Objective 1) b. vitamin B6 a. folic acid synthesis c. folate b. DNA synthesis d. methylmalonic acid c. intrinsic factor secretion 3. Which of the following is the best clue in diagnosing d. absorption of folate megaloblastic anemia? (Objective 3) 8. Laboratory diagnosis of pernicious anemia can a. Decreased hemoglobin and hematocrit include which of the following? (Objective 3) b. Leukocytosis a. Urinary FIGLU c. Hypersegmented neutrophils b. WBC count d. Poikilocytosis c. Gastric analysis 4. Increases in urinary excretion of f ormiminoglutamic d. LDH acid (FIGLU) most likely indicate which of the 9. Alcoholic individuals commonly develop a macro- following? (Objective 4) cytic anemia due to: (Objective 8) a. Cobalamin deficiency a. folate deficiency b. Autoantibodies to intrinsic factor b. increased blood cholesterol levels c. Folic-acid deficiency c. development of autoantibodies against intrinsic d. Hemolysis factor d. intestinal malabsorption of cobalamin 5. The liver stores enough folate to meet daily require- ment needs for how long? (Objective 2) a. 1 month b. 6–8 weeks 344 Chapter 15 10. Anemia due to liver disease is often associated with c. folate deficiency which of the following RBC morphological forms? d. large bowel resection (Objective 9) 7. Which of the following is more typical of nonmegalo- a. Ovalocytes blastic than megaloblastic anemia? (Objectives 2, 3) b. Microcytes a. Oval macrocytes c. Spur cells b. Round macrocytes d. Teardrop cells c. Howell-Jolly bodies Level II d. Hypersegmented neutrophil 1. If a patient presents with anemia, macrocytosis, pan- 8. Which of the following tests will help determine if cytopenia, and malnutrition, which of the following there is a problem with cobalamin absorption? should be investigated first as a possible cause of the (Objective 7) anemia? (Objectives 4, 11) a. Holo-TC assay a. Pernicious anemia b. Homocysteine assay b. Folic-acid deficiency c. Serum TC assay c. Cobalamin deficiency d. CobaSorb test d. Celiac disease 9. Which type of congenital dyserythropoietic anemia 2. Which laboratory test is a first-line test to help deter- (CDA) presents with giant multinucleated erythro- mine a cobalamin deficiency? (Objective 8) cytes in the marrow? (Objective 10) a. Red cell folate a. CDA I b. Serum folate b. CDA II c. Homocysteine c. CDA III d. Serum cobalamin assay d. CDA IV 3. Which of the following can be found in a patient with 10. A 48-year-old Caucasian female experiencing fatigue, megaloblastic anemia? (Objectives 2, 4, 10) loss of appetite, and weight loss over a period of three a. Giant metamyelocytes and hypolobulated months was seen by her physician. A medical history neutrophils revealed she had a history of alcohol abuse. An initial laboratory workup demonstrated that she was ane- b. Howell-Jolly bodies and Pappenheimer bodies mic, had a leukocyte count of 3 * 106/mcL and had c. Hypersegmented neutrophils and oval macrocytes an MCV of 119 fL. Macro-ovalocytes and neutrophil d. Hypochromic macrocytes and thrombocytosis hypersegmentation were noted on her blood smear evaluation. Based on the initial laboratory test results, 4. The metabolic function of tetrahydrofolate is to: her physician obtained a serum cobalamin and folate (Objective 1) workup. Results were as follows: a. synthesize methionine Serum cobalamin 550 pg/mL b. transfer carbon units from donors to receptors Serum folate 4.0 ng/dL c. serve as a cofactor with cobalamin in the synthesis of dTTP RBC folate 105 ng/mL d. synthesize intrinsic factor Based on her clinical history and laboratory results, the best possible diagnosis is which of the following? 5. Folic acid deficiency can be caused by: (Objective 7) (Objective 11) a. alcoholism a. Pernicious anemia b. chronic blood loss b. Folate deficiency c. strict vegetarian diet c. Primary cobalamin deficiency d. vitamin B6 deficiency d. Anemia of liver disease 6. A lack of intrinsic factor could be due to: (Objective 8) a. gastrectomy b. cobalamin deficiency Megaloblastic and Nonmegaloblastic Macrocytic Anemias 345 References 1. Hoggarth, K. (1993). Macrocytic anaemias. Practitioner, 237(1525), and subsequent cancer: A population-based cohort study. Cancer, 331–335. 71(3), 745–750. 2. 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ity. American Journal of Clinical Nutrition, 94(1), 359S–365S. ment of methylmalonic acid and homocysteine vs vitamin B12 36. Havas, A. M., Morkbak, A. L., & Nexo, E. (2007). Plasma holo- and folate. Clincial Chemistry, 46(8, Pt. 2), 1277–1283. transcobalamin compared with plasma cobalamins for assess- 16. Schrier, S. L. (2013). Diagnosis and treatment of vitamin B12 and folate ment of vitamin B12 absorption; optimization of a non-radioactive deficiency. Retrieved July 22, 2013, from www.uptodate.com. vitamin B12 absorption test (CobaSorb). Clinica Chimica Acta, 17. Antony, A. C. (2013). Megaloblastic anemias. In: R. Hoffman, E. 376(1–2), 150–154. J. Benz Jr, L. E. Silberstein, H. Heslop, J. Weitz, J. Anaztasi, eds. 37. Bhat, D. S., Thuse, N. V., Lubree, H. G., Joglekar, C. V., Naik, S. S., Hematology Basic Principles and Practice (6th ed., pp. 473–504). Ramdas, L. V., . . . Yajnik, C. S. (2009). Increases in plasma holo- Philadelphia: Elsevier. transcobalamin can be used to assess vitamin B-12 absorption in 18. Crider, K. S., Bailey, L. B., & Berry, R. F. J. (2011). Folic acid food individuals with low plasma vitamin B-12. Journal of Nutrition, fortification: Its history, effect, concerns, and future directions. 139(11), 2119–2123. Nutrients, 3(3), 370–384. 38. Bolaman, Z., Kadikoylu, G., Yukselen, V., Yavasoglu, I., Barutca, 19. MRC Vitamin Study Research Group (1991). Prevention of neural S., & Senturk, T. (2003). Oral versus intramuscular cobalamin tube defects: Results of the Medical Research Council Vitamin treatment in megaloblastic anemia: a single-center, prospec- Study. Lancet, 338(8760), 131–137. tive, randomized, open-label study. Clinical Therapeutics, 25(12), 20. Chanarin, I. (1987). Megaloblastic anaemia, cobalamin, and 3124–3134. folate. Journal of Clinical Pathology, 40(9), 978–984. 39. Hesdorffer, C. S., & Longo, D. L. (2015). Drug-induced mega- 21. Elin, R. J., & Winter, W. E. (2001). Methylmalonic acid: A test loblastic anemia. New England Journal of Medicine, 373(17), whose time has come. Archives of Pathology Laboratory Medicine, 1649–1658. 125(6), 824–827. 40. Seppa, K., Sillanaukee, P., & Saarni, M. (1993). Blood count and hema- 22. Hsing, A. W., Hansson, L. E., McLaughlin, J. K., Nyren, O., Blot, tologic morphology in nonanemic macrocytosis: Differences between W. J., Ekborn, A., & Fraumeni, J. F. Jr. (1993). Pernicious anemia alcohol abuse and pernicious anemia. Alcohol, 10(5), 343–347. Chapter 16 Hypoproliferative Anemias Grace B. Athas, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define hypoproliferative anemia. 6. List the major clinical and laboratory charac- 2. Cite the diagnostic criteria for aplastic teristics of aplastic anemia. anemia. 7. Identify environmental factors associated 3. Describe the epidemiology and etiology of with the development of aplastic anemia. aplastic anemia. 8. Describe the etiology, bone marrow, and 4. Explain the pathophysiology of aplastic peripheral blood in pure red cell aplasia. anemia. 9. Identify peripheral blood findings associated 5. Compare and contrast acquired and inher- with the following: aplastic anemia, pure red ited aplastic anemia. cell aplasia, and anemia due to renal disease. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Propose and explain possible causes of findings and peripheral blood and bone aplastic anemia. marrow findings. 2. Discuss prognosis in aplastic anemia. 5. Compare and contrast pure red cell aplasia 3. Compare methods of treatment and with aplastic anemia and other causes of management of patients with aplastic erythroid hypoproliferation. anemia. 6. Compare and contrast major characteristics 4. Contrast aplastic anemia with other causes of Diamond-Blackfan syndrome and of pancytopenia on the basis of clinical transient erythroblastopenia of childhood. 346 Hypoproliferative Anemias 347 7. Explain the pathophysiology of anemia due hypoproliferative anemia, suggest addi- to renal disease. tional laboratory tests if appropriate, and a 8. Evaluate laboratory test results and medical possible diagnosis. history of a clinical case for a patient with Chapter Outline Objectives—Level I and Level II 346 Aplastic Anemia 348 Key Terms 347 Pure Red Cell Aplasia 356 Background Basics 347 Other Hypoproliferative Anemias 358 Case Study 347 Summary 359 Overview 348 Review Questions 360 Introduction 348 References 362 Key Terms Aplasia Fanconi anemia (FA) Idiopathic Aplastic anemia Hypocellularity Myelophthisis Diamond-Blackfan anemia (DBA) Hypoplasia Pancytopenia Dyshematopoiesis Hypoproliferative Pure red cell aplasia Background Basics The information in this chapter builds on the concepts • Outline the classification of anemias. (Chapter 11) learned in previous chapters. To maximize your learning • Explain the process of hematopoiesis. (Chapter 4) experience, you should review these concepts before start- ing this unit of study: Level II • Explain the concepts of stem cell renewal and dif- Level I ferentiation. (Chapter 4) • Describe basic laboratory procedures used to screen • Describe the normal bone marrow structure and for and assess anemia. (Chapters 10, 11) cellularity and the process of bone marrow examina- • Identify abnormal values and results for basic hema- tion. (Chapters 3, 38) tologic laboratory procedures. (Chapter 10) CASE STUDY and in no acute distress. There was no lymphade- We refer to this case study throughout the chapter. nopathy or organomegaly. Many petechial hemor- Rachael, a 13-year-old female, was admitted to the rhages covered her chest and legs. Several bruises hospital with complaints of progressive weakness were found on her legs and thighs. Laboratory and shortness of breath with minimal physical tests were ordered upon admission. effort. She has experienced recurrent fevers reach- Consider the diagnostic possibilities in this case ing 38.9 °C. Physical examination revealed a well- and how laboratory tests can be used to assist in developed adolescent with good nutritional status differential diagnosis. 348 Chapter 16 Overview in aplastic anemia (AA) usually appear normal. Aplasia of the bone marrow is only one of several possible causes of Finding cytopenias in the peripheral blood often suggests peripheral blood pancytopenia, but pancytopenia due to the presence of a serious medical condition. Although there causes other than AA can result in morphologically abnor- are a variety of causes, bone marrow hypoproliferation is mal blood or bone marrow cells. Whereas AA is usually one of the most serious. This chapter discusses the acquired characterized by pancytopenia, granulocyte, platelet, and and inherited anemias that are associated with bone marrow erythrocyte, levels may not be depressed uniformly. hypoproliferation. The first section describes the classifica- Diagnostic criteria for severe aplastic anemia are tion of hypoproliferative anemias followed by discussions included in Table 16-1. Additional diagnostic criteria are of aplastic anemia and pure red cell aplasia. These anemias based on disease severity.1 With disease progression, con- are contrasted to other causes of cytopenias. centrations of cells in all three cell lineages eventually become further depleted. This reflects an impaired prolif- erative capacity of the marrow stem cells, which lose their Introduction ability for normal cellular renewal. The hypoproliferative anemias are a heterogeneous group of acquired and inherited disorders in which there is a nor- CASE STUDY (continued from page 347) mocytic or macrocytic, normochromic anemia associated with chronic bone marrow hypocellularity. Much of the 1. Select laboratory tests appropriate for screening area in the bone marrow normally occupied by hemato- for aplastic anemia. poietic tissue is replaced by fat. The terms aplastic, apla- 2. Justify the selection of laboratory screening tests sia, and hypoplastic refer to a bone marrow with an overall based on Rachael’s clinical signs and symptoms. decrease in hematopoietic cellularity. If there is hypoplasia of only one of the hematopoietic lineages, the terms ery- throid, myeloid, or megakaryocytic hypoplasia should be used to define the specific entity. Checkpoint 16.1 The hematopoietic defect is due to depletion, damage, An anemic patient has a (corrected) reticulocyte count of 1.5%, or inhibition of hematopoietic stem cells (HSCs) and/or hemoglobin of 10.0 g/dL (100 g/L), hematocrit of 30% (0.30 hematopoietic progenitor cells (HPCs). Either the unipo- L/L), total neutrophil count of 0.4 * 103/mcL and a platelet tent erythroid progenitor cell or a multilineage hematopoi- count of 30 * 103/mcL. Is it likely that this patient has aplastic etic progenitor cell can be affected. The peripheral blood anemia? findings provide important clues to help identify the bone marrow abnormality. If only the erythroid progenitor cells Epidemiology (BFU-E, CFU-E) are affected, platelets and leukocytes remain normal, and the diagnosis is pure red cell aplasia. The anemias described in this chapter are rare, occurring at More commonly, the multilineage hematopoietic precur- a frequency of two cases per million. The actual prevalence sor cells (HSC, MPP, CMP, CFU-GEMM, etc.) are affected, of aplastic anemia may be somewhat higher due to underdi- resulting in pancytopenia (decreases of all three cell lin- agnosis and imprecision in applying diagnostic standards.1 eages), and the diagnosis is aplastic anemia. A geographic variation in incidence occurs with 2–3 times more cases in Asia than in western countries and more often in developing countries than in developed countries.2 It Aplastic Anemia is believed that this variation is related to environmental and occupational factors.3 Individual susceptibility could The term aplastic anemia is used to describe the condition also play a role. Aplastic anemia is most commonly seen in of pancytopenia that is associated with a hypocellular 15- to 25-year-olds with two smaller affected groups consist- bone marrow. The mature blood cells that are produced ing of 2-5-year-olds and adults older than 60. The occur- rence of AA in young children is mostly due to inherited forms, whereas the occurrence in adults is most frequently Table 16.1 Diagnostic Criteria for Aplastic Anemia due to acquired forms, although there are exceptions.1,4 Bone marrow cellularity less than 25% plus two of the following: Granulocyte count less than 0.5 * 103/mcL Pathophysiology Platelet count less than 20 * 103/mcL The pathophysiology of aplastic anemia is complex. It Anemia with corrected reticulocyte count 61, (absolute concentration may involve an abnormal microenvironment for hemato- less than 40 less * 109/L) poietic cells, deficiencies of hematopoietic stem cells and Hypoproliferative Anemias 349 progenitor cells, and/or cytokine inhibition of cell growth cells, thus lending further support to the immune basis of and development. It is estimated that the number of HSCs in aplastic anemia.5 severe acquired AA is decreased to less than 1% of normal. Defective telomere maintenance in HSCs and HPCs Response of marrow failure to immunosuppressive therapy has been observed in some patients with AA and may con- has led to the characterization of most cases of acquired AA tribute to its pathophysiology. Telomeres are the ends of as an immune-mediated disease.5,6 The immune mechanism chromosomes consisting of 500–2,000 tandem repeats of responsible is thought to involve the suppression of HSC the hexanucleotide TTAGGG. The telomere ends of chro- growth and differentiation by abnormal T-lymphocytes. mosomes shorten with each cell division (Chapter 2). Main- This is supported by finding that up to 80% of AA patients tenance of telomere length occurs through the action of the will respond to treatment with immunosuppressive drugs, enzyme telomerase. Mutations in telomerase-related genes, including antithymocyte globulin (ATG) and cyclosporine.7 including telomerase reverse transcriptase (TERT), telom- Additional evidence supporting the immunosuppression erase RNA template (TERC), and shelterin (TINF) result theory comes from the finding that in tissue culture, the in shortened telomeres, reduced proliferation of hemato- addition of lymphocytes taken from patients with AA poietic precursors, and increased apoptosis. Furthermore, decreases hematopoietic cell production, and their removal shortened telomeres may predispose to malignant transfor- improves the production of hematopoietic cells.8 However, mation, which is observed in some patients with AA. Addi- the auto-antigens that trigger the abnormal immune attack tional environmental or genetic factors can also contribute on HSC remain to be identified. to telomere shortening. Shortened telomeres and the muta- There are a large number of dysregulated genes in tions associated with them have been observed in patients aplastic anemia patients’ CD4+ T-cells and cytotoxic with both acquired and inherited forms of aplastic anemia.13 CD8+ T-cells.9 A consistent finding is abnormal expan- sion of subsets of CD4+ T-helper cells including Th1 Classification and Etiology (interferon-producing cells), Th2 (interleukin-4-produc- ing cells), and Th17 (IL-17-producing cells). On the other Aplastic anemia can be classified as either acquired or inher- hand, there is a decreased or skewed number of Treg ited (Table 16-2). Historically, much attention has focused cells (CD4+ , CD25+ , FoxP3+ ).9 Treg cells are important on an association between acquired AA and environmental in inducing immune tolerance and suppressing autore- exposures. Drugs, chemicals, radiation, infectious agents, active effector T-cell proliferation.10 Impairment of Treg and other factors have been linked to the

development cells plays a critical role in the pathophysiology of aplastic of acquired AA, which can be temporary or persistent. In anemia.10,11 most cases, no environmental link can be identified, and Flow cytometric analysis of lymphocyte subpopula- the cause is said to be idiopathic. The immune pathophysi- tions in acquired aplastic anemia has revealed a marked ologic model (discussed in the previous section) provides a increase in activated CD8+ lymphocytes. The cause of acti- unifying basis for understanding the disorder regardless of vation of T-lymphocytes in AA remains unclear, although the presence or absence of environmental factors. Although it may be associated with viral infections or medical drugs acquired AA is more common in adults, it is also an infre- in a small number of cases. A genetic basis of aberrant cyto- quent cause of aplasia in children. Both idiopathic and other toxic T-lymphocyte activation may also exist. In any case, acquired forms of AA are discussed in the next sections. activated cytotoxic T-lymphocytes produce interferon Inherited forms of AA result in chronic failure of the @g (IFN bone marrow, which may or may not be present at birth. A @g) and tumor necrosis factor (TNF), substances known to inhibit hematopoiesis.5 Both IFN@g and TNF induce over- congenital condition is one that is manifest early in life, often expression of Fas (a cell membrane receptor) on HSCs and at birth, but that is not necessarily inherited; it can be caused HPCs. When the Fas receptor is activated by binding with by acquired factors (e.g., maternal exposure to an environ- its ligand, apoptosis is initiated (Chapter 2). mental toxin while pregnant). Inherited AA is quite rare and An increased incidence of AA is associated with the is often associated with other congenital anomalies. presence of the class II histocompatibility antigen DR2, which is also linked to other autoimmune disorders. Aplas- tic anemia patients positive for this antigen respond well to CASE STUDY (continued from page 348) immunosuppressive therapy.12 3. Evaluate the relationship between Rachael’s age The success of hematopoietic stem cell transplants and the likelihood that she has aplastic anemia. (SCT) in many patients with AA indicates that the pancy- topenia can be corrected by repopulation of the marrow 4. If aplastic anemia is present, would you expect with normal HSCs and immune cells. The aberrant clone of her to have an idiopathic or secondary form? T-lymphocytes is eliminated through a pretransplant con- Explain your answer. ditioning regimen and subsequently replaced with normal 350 Chapter 16 Table 16.2 Classification of Hypoproliferative Anemias Aplastic Anemia Pure Red Cell Aplasia Other Hypoproliferative Anemias Acquired Transitory infections Anemia of chronic renal disease Idiopathic Acquired pure red cell aplasia Anemia associated with endocrine abnormalities Drugs: chloramphenicol, phenylbutazone, gold Acute: infections, transient erythroblastopenia of compounds, sulfa drugs, antihistamines, antithyroid, childhood (TEC), drugs tetracyclines, penicillin, methylphenylethylhydantoin, Chronic: thymoma, autoimmune disorders indomethacin, ibuprofen, quinacrine, chloraquine Chemical agents: benzene, insecticides, carbon tet- rachloride, chemotherapeutics (vincristine, busulfan, etc.), arsenic Ionizing radiation Biological agents: parovirus, infectious mononucleo- sis, infectious hepatitis, measles, influenza, errors of amino acid metabolism, starvation Pregnancy Paroxysmal nocturnal hemoglobinuria Inherited Fanconi anemia Familial aplastic anemia Dyskeratosis congenita Congenital amegakarocytic thrombocytopenia Shwachman-Diamond syndrome Diamond-Blackfan anemia ACQUIRED FORMS OF APLASTIC ANEMIA increase susceptibility to HSC damage.15 In cases associated Acquired forms of aplastic anemia may be idiopathic or with drug exposure, the pathophysiology is thought to associated with exposure to drugs/chemicals, ionizing involve an abnormal immune response to the HSC. Unlike radiation, and infectious agents. Various metabolic disor- agranulocytosis and drug-induced thrombocytopenia, stop- ders may also be associated with aplastic anemia. ping the putative drug does not usually lead to hematopoi- etic recovery, with a few exceptions.1 Idiopathic The majority of cases of aplastic anemia cannot The widespread use of toxic chemical agents in indus- be linked to an environmental factor and are referred to as try and agriculture is probably responsible for some cases idiopathic. It is possible that previous exposure to an unrec- of bone marrow aplasia. Benzene derivatives are well estab- ognized agent or event could be responsible for stimulating lished as a cause of bone marrow suppression. Although the immune system. most cases develop within a few weeks after exposure, some Drugs And Chemical Agents Recent research has shown occur after months or years of chronic exposure. Although that exposure to drugs or chemical agents is rarely associ- stem cells can be damaged, the main toxic effect of ben- ated with aplastic anemia, although historically these asso- zene is usually expressed on transient stages of committed ciations were given prominent attention. A study conducted proliferating precursor cells. Elevated risk of AA was docu- in Thailand, where the incidence of AA is 2–3 times higher mented in the Thai study for persons exposed to benzene than in the United States, indicated that an elevated risk and other solvents as well as agricultural pesticides such of developing AA was associated with exposure to only as organophosphates and DDT and for persons who drink a small number of substances, including sulfonamides, nonbottled water in rural regions.14 thiazide diuretics, and mebendazole. In the Thai study, an Most of the cytotoxic drugs used in chemotherapy of increased risk was not found for chloramphenicol, a drug malignant diseases kill rapidly proliferating cells. How- frequently implicated in case reports of AA.14 Other drugs ever, the drugs do not distinguish between malignant and that have been implicated include gold, anticonvulsants, normal cells. Therefore, all proliferating cells are damaged, nonsteroidal analgesics, antiprotozoals, and antithyroid including normal cells of the hematopoietic compartment. medications.1 Most individuals taking such medications, Although quiescent (G0) stem cells are spared from immedi- however, do not develop AA. One possible explanation ate drug exposure, repeated doses of the drug over a long is that persons with diminished P-glycoprotein, an efflux period of time can eventually deplete the remaining HSCs pump that is the product of the multi-drug resistance gene as they enter the proliferating pool. In most cases, timely MDR-1, may have excessive accumulation of drugs that can withdrawal of the drug leads to hematopoietic recovery. Hypoproliferative Anemias 351 Ionizing Radiation Aplastic anemia has been encountered abortion. Aplasia may be related to estrogen inhibition of in persons exposed to ionizing radiation in industrial acci- stem cell proliferation.17 dents, military nuclear tests, and therapeutic regimens for ASSOCIATION WITH CLONAL DISORDERS malignancy. Ionizing radiation is directly toxic to HSCs Even prior to the widespread use of immunosuppressive and HPCs, and the effects are dose dependent. Small therapy for AA, patients occasionally developed clonal dis- doses affect all cells but are especially destructive to rap- orders, including paroxysmal nocturnal hemoglobinuria (PNH) idly proliferating cells. The bone marrow can recover from and myelodysplastic syndromes (MDS). Thus, the develop- sublethal doses of irradiation because quiescent stem cells ment of these disorders in AA patients is not necessarily are induced to begin proliferating after exposure-induced related to the treatment regimens currently used. As many depletion of more differentiated progeny. Therapy in these as 15% of patients treated with immunosuppressive therapy cases is mostly supportive and needed only until hemato- for aplastic anemia may develop MDS.1 poietic function is restored. With high doses (more than PNH is an acquired stem cell disease in which a blood 4,000 rads) bone marrow aplasia and peripheral blood pan- cell membrane abnormality increases the cells’ susceptibil- cytopenia are usually permanent. ity to in vivo complement-mediated hemolysis. PNH is Infectious Agents Viral and bacterial infections can be fol- caused by mutations in the PIGA gene resulting in the lowed by a transient cytopenia. The aplasia can be limited absence of a glycolipid that serves to attach and anchor pro- to the erythroid elements (red cell aplasia) or can include all teins to the cell membrane. Although considerable variation three cell lineages. Aplasia has been described in patients in clinical manifestations occurs, the typical picture in severe after recovery from infectious mononucleosis, tuberculosis, cases is pancytopenia and marrow hypoplasia. Between 40 and hepatitis but is typically transient. Hepatitis-associated and 50% of patients with an initial diagnosis of AA develop AA occurs most frequently in males and does not appear an abnormal erythrocyte population similar to that seen in to be caused by any of the known hepatitis viruses. It is PNH, but they do not usually develop the full clinical mani- often fatal if untreated.16 The pathophysiology of hepatitis- festations of PNH (intravascular hemolysis and venous associated AA is thought to be immune mediated because thrombosis).18 PNH is a prominent, late complication in it responds to immunosuppressive therapy.5,16 An aplastic many AA patients who have received immunosuppressive crisis in patients with hereditary hemolytic anemias is com- ATG therapy.19 PNH is discussed in detail in Chapter 17. monly associated with human parvovirus infection. This aplasia, however, is limited to the erythroid lineage. In Epstein-Barr viral infections as well as other viral CASE STUDY (continued from page 349) infections, the virus can infect the HSC, triggering an For the past 3 months, Rachael’s family physician immune response. Cytotoxic lymphocytes then destroy has been following her recovery from viral hepati- the virus-infected stem cell. Other mechanisms of stem cell tis. Her recovery was uneventful; her liver enzyme damage in viral infections have been postulated, including levels returned to normal within 2 months. She has direct cytotoxicity of the virus and inhibition of cellular pro- no other past medical history. There is no family liferation and differentiation.5 history of hematologic disorders. Metabolic The rare inborn errors of amino acid metabo- 5. What aspect of this patient’s history could lism, which result in accumulation of ketones and glycine, be associated with the occurrence of aplastic have been associated with aplastic anemia. anemia? Starvation or protein deficiency results in hypoprolif- erative anemia after about 3 months of deprivation. Starva- tion that is not self-induced usually occurs in areas where other endemic pathologies are also present, such as parasitic INHERITED APLASTIC ANEMIA A number of disorders have a congenital predisposition for infection and blood loss. Thus, the causes of this anemia can developing AA, as described below. be multifactorial. Decreased hormonal stimulation of hematopoietic Fanconi Anemia Fanconi anemia (FA) is an autosomal precursor cells is important primarily as a factor in ery- recessive (or rarely X-linked recessive) disorder resulting throid hypoplasia. Renal disease and endocrine diseases from a variety of molecular defects and is characterized by are examples of hypoproliferation caused by a decrease in childhood-onset aplastic anemia, abnormal chromosomal erythropoietin. fragility, and an increased predisposition to developing A life-threatening pancytopenia rarely can occur dur- leukemia and cancer. FA has a prevalence of about 1–5 ing pregnancy. The mother is at risk for hemorrhage and per million persons in North America with a frequency of sepsis. The fetus may show growth restriction and could the heterozygous carrier state of about 1 in 300.20 Patients die in utero. The condition usually remits after delivery or can have a complex assortment of congenital anomalies 352 Chapter 16 in addition to progressive bone marrow hypoplasia. The FA proteins are thus responsible for the genetic instability congenital defects include dysplasia of bones, renal abnor- associated with DNA damage, resulting in increased apop- malities, and other organ malformations as well as mental tosis of hematopoietic stem cells and AA.1,4 In addition to retardation, dwarfism, microcephaly, hypogonadism, and genetic defects leading to defective DNA repair and DNA skin hyperpigmentation. Some patients lack congenital instability, TNFa is elevated in most patients. The telo- defects and may not be diagnosed until AA develops, which meres of the HSC and HPC are shortened, suggesting an occasionally may not occur until adulthood.21 abnormally high proliferative rate, probably induced by Aplastic anemia eventually develops in about 90% of the marrow’s attempt to replace damaged HSCs/HPCs and FA patients. The hematological manifestations are gener- ultimately lead to HSC/HPC premature senescence.13 ally slowly progressive from birth and need to be moni- Other Causes of Inherited Aplastic Anemia Several other tored closely. Clinical signs of pancytopenia usually appear rare disorders have a predisposition to developing AA. between the ages of 5 and 10 years with the median age Dyskeratosis congenita (DC) is an inherited bone marrow at diagnosis of 6.5 years.4 Anemia is usually macrocytic failure syndrome, resulting in AA in about 50% of cases. (although it can be normocytic) with macrocytosis often The physical abnormalities and mutations in patients preceding anemia. Erythrocytes often

show increased levels with DC differ from those seen in patients with FA. Most of Hemoglobin F and the i antigen (reflecting hematologic DC patients have dystrophic nails and leukoplakia of stress and the development of erythrocytes from earlier the oral mucosa, conditions not seen in FA. Mutations in erythroid progenitor cells). Leukopenia primarily involves several genes are associated with DC. Markedly reduced the granulocytes. Thrombocytopenia often precedes anemia telomerase activity, telomere shortening, early HSC/HPC and leukopenia. Androgen therapy can reverse the pancyto- senescence, and a reduced stem cell compartment are char- penia for several years in about 50% of FA patients. G-CSF acteristic. There are multiple subtypes of DC with varying therapy can help increase neutrophil counts. clinical features.4 HSCT offers the only possible curative treatment Congenital amegakaryocytic thrombocytopenia (CAT) for bone marrow failure in FA patients. In patients with presents in the neonatal period with isolated thrombocy- HLA-matched sibling donors, 5-year survival is close to topenia due to reduced or absent marrow megakaryocytes. 75%. Stem cells from HLA-matched unrelated donors have Aplastic anemia subsequently develops in 45% of patients, also been used. Because FA patients are acutely sensitive usually in the first years of life. The defect is a mutation in to chemotherapeutic agents and radiation, pretransplant the MPL gene, which codes for the thrombopoietin (TPO) conditioning protocols must be altered to reduce toxicity receptor,22 is required for maintenance of HSC viability that could increase the risk of developing a clonal disor- (Chapter 4). der. Although HSCT can cure the marrow failure in FA Patients with Shwachman-Diamond syndrome (SDS) can patients, the increased risk of nonhematologic malignan- present with some of the same congenital anomalies, includ- cies remains. ing AA as is seen in FA patients. However, they also have The risk for developing cancer in FA is high and exocrine pancreatic dysfunction and malabsorption syn- increases with age. Acute nonlymphocytic leukemia and drome and do not display increased chromosomal fragility solid tumors are common complications. Leukemia is dif- and defective DNA repair processes. This disorder is due to ficult to treat, and survival is poor. With improved treat- mutations of the SBDS gene, although the exact pathogen- ment options, the median survival for FA patients is now esis of the disorder is still unknown. Diagnosis is confirmed 29 years of age.4 by genetic testing.22 Karyotyping of FA cells shows increased spontaneous chromosomal breakage, gaps, rearrangements, exchanges, and duplications. Diagnosis of FA involves exposing blood lymphocytes or skin fibroblasts to DNA crosslinking agents such as mitomycin C, diepoxybutane (DEB), or cisplatin, CASE STUDY (continued from page 351) which amplify chromosomal breakage.1,4 Molecular tech- 6. Is it likely that Rachael has an inherited form of niques can be used to identify specific mutations. Testing aplastic anemia? Explain your answer. for FA is performed when warranted based on clinical find- ings and/or abnormal blood counts, usually in infants and children. The molecular defects in FA are heterogeneous. To date, mutations in 15 genes that are associated with the FA pheno- Clinical Presentation type have been identified.22 Several of the FA-identified pro- The onset of symptoms in AA is usually insidious and teins function in a common cellular DNA repair mechanism, related to the cytopenias. Common initial signs are bleeding which is activated in response to DNA damage. Defective accompanied by petechial and mucosal hemorrhages and Hypoproliferative Anemias 353 infection. Pallor, fatigue, and cardiopulmonary complica- tions can be present as the anemia progresses. RBC 2.42 * 106/mcL Hepatosplenomegaly and lymphadenopathy are Hb 7.1 g/dL (71 g/L) absent. Splenomegaly has occasionally been noted in later stages of the disease, but if found in the early stages, the Hct 24% (0.24 L/L) diagnosis of aplastic anemia should be questioned. PLT 8.0 * 103/mcL WBC 1.2 * 103/mcL Laboratory Evaluation Differential Laboratory studies of peripheral blood and bone marrow Segmented neutrophils 2% are essential if a diagnosis of aplastic anemia is suspected. Lymphocytes 94% PERIPHERAL BLOOD Monocytes 4% Pancytopenia is typical. Although the degree of sever- Uncorrected reticulocyte 0.7% ity can vary, the diagnosis of AA should be questioned count unless the leukocyte count, erythrocyte count, and platelet 8. Evaluate each of Rachael’s laboratory results by count are all below the reference intervals. Hemoglobin is comparing them to reference intervals. usually less than 7.0 g/dL (70 g/L). Erythrocytes appear normocytic and normochromic, or they can be slightly 9. Which of Rachael’s routine laboratory results macrocytic. The presence of nucleated erythrocytes and are consistent with those expected for aplastic teardrops is not typical of AA but suggests marrow anemia? replacement (myelophthisic anemia). Myelodysplastic 10. Classify the morphologic type of anemia. syndrome, rather than AA, is suggested by the presence of dysplastic neutrophils and other abnormal cells. The rela- 11. Calculate the absolute lymphocyte count. Are tive reticulocyte count (%) can be misleading due to the Rachael’s lymphocytes truly elevated as sug- severe anemia. Therefore, the reticulocyte count should gested by the relative lymphocyte count? always be determined in absolute concentration and/or 12. Correct the reticulocyte count. Why is this step be corrected for anemia before interpretation. The abso- important? lute reticulocyte count is usually less than 25 * 109/L. The corrected reticulocyte count is less than 1%. Most often, 13. Calculate the absolute reticulocyte count. thrombocytopenia is present at the time of diagnosis. Neutropenia precedes leukopenia; initially, lymphocyte and monocyte counts are normal. Because of the neutro- penia, the differential count reflects a relative lymphocy- BONE MARROW tosis. When the leukocyte count is below 1.5 * 103/mcL, Examination of the bone marrow is necessary to differen- an absolute lymphocytopenia is also present. The band to tiate aplastic anemia from other diseases accompanied by segmented neutrophil ratio is increased, and occasionally pancytopenia. In AA, the bone marrow is hypocellular with more immature forms are found. Neutrophil granules are more than 70% fat (Figure 16-1). Thus, it is often difficult to frequently larger than normal and stain a dark red; these obtain an adequate sample. Bone marrow infiltration with granules should be distinguished from toxic granules, granulomas or cancer cells can lead to fibrosis, also result- which are bluish black. Flow cytometry of the peripheral ing in a hypocellular dry tap on aspiration. Both aspiration blood can be ordered to detect CD59+ cells when PNH is and biopsy are needed for a correct diagnosis.23 It is rec- suspected23 (Chapter 20). ommended that several different sites be aspirated because focal sampling of the marrow can be misleading. Some areas of acellular stroma and fat can be infiltrated with clusters of lymphocytes, plasma cells, and reticulum cells. Areas of residual hematopoietic tissue termed hot spots may CASE STUDY (continued from page 352) be found primarily early in the disease but may occasionally 7. Correlate Rachael’s clinical findings of w eakness be found in severe refractory cases. Iron staining reveals and shortness of breath as well as petechial many iron granules in macrophages, but granules are rarely hemorrhages and bruises with her laboratory seen in normoblasts. Flow cytometry should be performed; screening results, which follow. the percentage of CD34+ cells in the bone marrow in AA is typically less than 0.3%. Bone marrow karyotyping is use- Admission laboratory data for patient: ful for differentiating hypocellular forms of myelodysplastic syndromes from aplastic anemia.1 354 Chapter 16 Prognosis and Therapy Recent advances in treatment have tempered the previ- ously grim prognosis of patients diagnosed with aplastic anemia. HSCT and immunosuppressive therapy (IST) have greatly improved survival. Presently, the 5-year survival rate is 79%.24 Choice of definitive therapy for severe acquired AA depends on the age of the patient and availability of a matched donor. HSCT is recommended for patients up to age 45 who have a matched sibling donor, although some recommendations extend the age limit to age 55. HSCT is also recommended for patients up to age 21 with a fully com- Figure 16.1 Bone marrow preparation from a patient with patible HLA-matched unrelated donor. IST is recommended aplastic anemia shows marked hypocellularity (10%). The patches for patients without a matched related donor, the situation of cells remaining are primarily lymphocytes (Wright-Giemsa stain; 100* magnification). faced by the majority of patients with aplastic anemia.3 Before beginning HSCT or IST, putative causative drugs should be withdrawn or the patient removed from a hazardous environment. The immediate treatment is CASE STUDY (continued from page 353) often supportive with the administration of erythrocytes, platelets, and antibiotics. Granulocyte transfusions can be Rachael was referred to a hematologist who given to severely neutropenic patients with life-threatening ordered a bone marrow examination. The aspirate sepsis.23 obtained was inadequate for evaluation due to lack To avoid alloimmunization, transfusion should of marrow spicules. Only a single site was aspi- be minimized if HSCT is anticipated. Irradiated and/ rated. Touch preps made from the biopsy showed or leukocyte-reduced blood products may be ordered a markedly hypocellular marrow with very few (Chapter 1). Hematopoietic growth factors such as G-CSF hematopoietic cells. Cells present consisted of generally do not have a beneficial effect on patients with AA and may increase the risk of MDS.5lymphocytes, plasma cells, and stromal cells. There were no malignant cells present. HSCT using cells collected from bone marrow has become a relatively common procedure and is curative in 14. Compare these results with those expected for a many patients with aplastic anemia. Bone marrow is pre- person with aplastic anemia. ferred over peripheral blood stem cells due to the increased 15. Interpret the significance of the lack of malignant risk of graft-versus-host disease with the latter.25 Antithy- cells and hematopoietic blasts. mocyte globulin (ATG) and cyclosporine are typically used as preconditioning regimens to suppress the immune 16. Suggest a way to improve the validity of bone response of the recipient. Preconditioning is nonmyeloab- marrow examination results for this patient. lative.26 The current 5-year survival rate is 77% for HLA- matched sibling donors. Patients who are transfused with bone marrow from donors who have cells with longer pre- transplant telomere length have a higher 5-year survival OTHER LABORATORY FINDINGS probability.27 Survival rates are highest for children and Other abnormal findings are not specific for aplastic ane- for patients who have been minimally transfused.5 Treat- mia but are frequently found associated with the disease. ment complications and post-transplant mortality remain Hemoglobin F can be increased, especially in children. high, especially in older patients; therefore, the probabil- Erythropoietin is often increased, particularly when com- ity for long-term cure must be weighed against the inher- pared with the erythropoietin levels in patients with similar ent risks of complications, including graft-vs-host disease degrees of anemia. Serum iron is increased with more than and early and late toxicities of the conditioning regimen. 50% saturation of transferrin, reflecting erythroid hypo- Although engraftment of HSCs is successful in many cases, plasia. The clearance rate of iron (Fe59) from the plasma some transplants, even when performed between identi- is decreased because of the decrease in iron utilization by cal twins, do not correct the AA. These unsuccessful trans- a hypoactive marrow. Patients who are younger than age plants suggest that the donor HSC growth is suppressed 50 should be screened for FA using tests for chromosomal by the same immune mechanism that induced the original breakage.23 Results of these tests will be normal in other aplasia (Chapter 29). An additional constraint to HSCT is forms of inherited AA and in acquired/idiopathic forms that matched sibling donors are available for only 20–30% of AA. of patients with aplastic anemia. Hypoproliferative Anemias 355 Combined, intensive immunosuppressive therapy common. The bone marrow, however, is usually normocel- (IST) using ATG in combination with cyclosporine has lular or hypercellular with various degrees of qualitative become standard treatment for those patients with abnormalities of one or more cell lineages (dyshematopoi- acquired severe aplastic anemia who lack a suitable bone esis). Signs of dyshematopoiesis are also reflected in mor- marrow donor. IST is effective in restoring hematopoiesis phologic abnormalities of one or more cell lineages in the in 60–90% of patients who are over 45 or who lack an HLA- peripheral blood. These qualitative abnormalities of periph- matched sibling donor.13,22 More favorable outcomes are eral blood cells are useful in differentiating cytopenias due observed in children. Relapse requiring additional IST to true hypoproliferation of stem cells (AA) from cytopenias occurs in 30–40% of patients. It is estimated that 15 to 20% due

to ineffective erythropoiesis. of patients with aplastic anemia who are treated with IST A subgroup of patients with MDS may have a hypo- develop clonal disorders such as PNH, leukemia, and cellular marrow. This condition is referred to as hypoplastic myelodysplastic syndromes.28 HSCT may be curative in MDS (HMDS). In these cases, differentiation of MDS and these patients.28 AA may be complicated if there are few bone marrow cells from a biopsy or aspirate to examine. If granulocytic and megakaryocytic dysplasias are seen and/or if an abnormal karyotype consistent with MDS is present, HMDS may be CASE STUDY (continued from page 354) diagnosed. However, if only erythroid dysplasia is present, 17. Appraise the prognosis for Rachael. the evidence is insufficient to differentiate HMDS and AA because erythroid dysplasia may be found in some cases 18. Predict a treatment regimen. of AA.18 CONGENITAL DYSERYTHROPOIETIC ANEMIA Congenital dyserythropoietic anemia (CDA) is a very Differentiation of Aplastic Anemia rare familial refractory anemia characterized by both from other Causes of Pancytopenia abnormal and ineffective erythropoiesis. The bone mar- row is normocellular or hypercellular, but the peripheral Pancytopenia can be associated with disorders other than blood is pancytopenic. In contrast to the normal ery- AA, but these disorders differ in that the pancytopenia is throid precursors found in aplastic anemia, erythrocyte not the result of a defect in stem cell proliferation. Rather, precursors in the bone marrow of CDA patients exhibit the bone marrow can be normocellular, hypercellular, or multinuclearity, and myeloblasts and promyelocytes are infiltrated with abnormal cellular elements. increased. The anemia may be normocytic, but most often MYELOPHTHISIC ANEMIA it is macrocytic. The three types of CDA are discussed in Myelophthisis signifies marrow replacement or infil- Chapter 15. tration by fibrotic, granulomatous, or neoplastic cells (Chapter 21). The abnormal replacement cells reduce HYPERSPLENISM normal hematopoiesis and disrupt the normal bone Hypersplenism due to a variety of causes can result in marrow architecture, allowing the release of immature a decrease of one or more cellular elements of the blood cells into the peripheral blood. Anemia may be accom- as these elements become pooled and sequestered in the panied by normal, increased, or decreased leukocyte and spleen. In this condition, the bone marrow is hyperplastic, platelet counts. The most characteristic findings are a corresponding to the peripheral blood cytopenia. Anemia is leukoerythroblastic reaction and a moderate to marked accompanied by a reticulocytosis as opposed to the reticu- poikilocytosis. Dacryocytes are common, as are large locytopenia found in the true hypoproliferative anemias. bizarre platelets. By contrast, nucleated erythrocytes and Granulocytopenia may be accompanied by a shift to the significant morphologic changes are almost never found left in these cells. Splenomegaly and other findings of the in the peripheral blood in AA. Myelophthisic anemia is underlying disease are important in diagnosing this disor- associated with diffuse cancer of the prostate, breast, and der. Splenectomy, although not always advisable, corrects stomach and is typical of myelofibrosis and lipid storage the cytopenias. disorders. OTHER MYELODYSPLASTIC SYNDROMES Deficiency of cobalamin or folic acid may be accompanied Myelodysplastic syndromes (MDS) are a group of hema- by pancytopenia (Chapter 15). The bone marrow in these tological disorders that have a propensity to terminate cases, however, reveals normocellularity or hypercellularity in acute leukemia (Chapter 25). The principal peripheral with megaloblastic changes. In the peripheral blood, hyper- blood findings are pancytopenia, bicytopenia, or isolated segmented neutrophils and Howell-Jolly bodies are typical cytopenias with reticulocytopenia. A macrocytic anemia is but are not found in pancytopenia of aplastic anemia. 356 Chapter 16 Inherited forms of PRCA, including Diamond-Blackfan Checkpoint 16.2 anemia, are discussed in the section “Diamond-Blackfan A pancytopenic patient has a presumptive diagnosis of aplas- Anemia.” The underlying pathophysiology of PRCA is tic anemia. While performing a smear review, you observe the variable and depends on associated clinical conditions. presence of several types of poikilocytes, dysmorphic neutro- Treatment of the underlying condition can resolve the phils, and large agranular platelets. Do these findings support the presumptive diagnosis? If not, which disorder(s) would be anemia, but unresponsive cases are considered to have an more likely for this patient? immune basis.30 Refer to Table 16-3 for results of labora- tory tests in PRCA. Acute Acquired Pure Red Cell CASE STUDY (continued from page 355) Aplasia 19. What other hematologic conditions must be Viral (parvovirus, Epstein-Barr virus, and viral hepatitis) ruled out for Rachael? and/or bacterial infections can be associated with a tempo- 20. What laboratory test is most beneficial in differ- rary suppression of erythropoiesis. B19 parvovirus infects entiating aplastic anemia from these other disor- erythroid progenitor cells, lyses the target cell, and blocks ders? Compare the expected results of AA with erythropoiesis. Infection, as well as the block to erythro- those of the other disorders. poiesis, is terminated by the production of neutralizing antibodies to the virus. In individuals who have a normal erythrocyte life span and hemoglobin level, temporary erythroid hypoproliferation is not noticed. However, if the Pure Red Cell Aplasia erythrocyte life span is decreased due to an underlying con- dition, the complication of sudden erythroid hypoprolifera- Pure red cell aplasia (PRCA) is a heterogeneous group tion (aplastic crisis) can be life threatening. These aplastic of disorders characterized by a selective decrease in ery- crises are most frequently noted in patients with hemolytic throid precursor cells in the marrow and by peripheral anemias including sickle cell anemia, paroxysmal nocturnal blood anemia. The anemia can be severe and is typically hemoglobinuria, and autoimmune hemolytic anemias. normocytic, normochromic. PRCA is thought to occur The aplastic crisis cases brought to the attention of a due to selective hypoproliferation of the committed ery- physician probably represent only a minor fraction of the throid progenitor cell. The term aplastic anemia should be actual occurrence of temporary erythroid aplasia. The aplas- avoided in describing PRCA because there is no distur- tic crises are often preceded by fever with upper respira- bance of granulopoiesis or thrombopoiesis. Reticulocytes tory and intestinal complaints. Several members of the same can be present but are less than 1% when corrected for the family are frequently affected with the illness. Patients with degree of anemia. PRCA can be acquired (acute or chronic) concurrent hemolytic anemia have a rapid onset of lethargy or inherited and can affect any age group. Acquired PRCA and pallor. Patients without hemolytic anemia may seek can develop in patients with thymoma, hematopoietic medical attention because of the primary illness, and ane- neoplasms, other cancers, autoimmune disorders, infec- mia is only an incidental finding. tion following administration of certain drugs, immuno- If the anemia is severe, supportive therapy of packed suppressive therapy, and post-transplantation.29 Transient erythrocyte transfusions may be necessary until spontane- erythroblastopenia of childhood (TEC) is an acquired, self- ous recovery occurs. PRCA associated with viral hepatitis limiting form of erythroid hypoplasia found in children. has a poor prognosis. Table 16.3 Laboratory Findings in Pure Red Cell Aplasia Test Findings Erythrocyte count, hematocrit, and hemoglobin Decreased Reticulocyte count Severely decreased Leukocyte count Normal or possibly increased Platelet count Normal or possibly increased Bilirubin Low or normal Bone marrow Absence of erythroid cells if examined early in disease; if examined later in disease progres- sion, increase in young erythroid cells, which can be mistaken for an erythroid maturation arrest. If the patient is followed, however, these cells show normal maturation and differentia- tion. There is normal myelopoiesis and granulopoiesis. Hypoproliferative Anemias 357 Acute erythroid hypoplasia also occurs with the admin- DBA is actually a diverse family of diseases with a istration of some drugs and chemicals. After removal of the common hematologic phenotype. Evidence exists for both inciting agent, normal erythropoiesis usually resumes. autosomal dominant and autosomal recessive modes of Transient erythroblastopenia of childhood (TEC) is a inheritance. About 20–25% of cases have a mutation of the form of acquired acute pure red cell aplasia, but because of RPS19 gene (which encodes for a protein involved in ribo- the importance of distinguishing this pediatric anemia from some assembly). Other genes mutated in patients with DBA Diamond-Blackfan anemia (DBA), TEC is discussed with have been identified and although the basic defect in most DBA in the section “Diamond-Blackfan Anemia.” cases appears to be defective ribosome biogenesis, the exact functional defect in all mutations is not clear. A wide range Chronic Acquired Pure Red Cell of physical abnormalities can be observed in approximately 50% of DBA patients, and patients are predisposed to occur- Aplasia rences of cancer.29 Many cases have no familial pattern, sug- An acquired selective depression of erythroid precursors gesting spontaneous mutations or acquired disease. is a rare disorder encountered in middle-aged adults. This The anemia in DBA is not due to a deficiency of erythro- disease usually occurs in association with thymoma, auto- poietin (EPO) because EPO levels are consistently increased immune hemolytic anemia, systemic lupus erythematosus, and actually higher than expected for the degree of ane- rheumatoid arthritis, or hematologic neoplasms (MDS, mia. The EPO is active, and no antibodies directed against large granular lymphocytic leukemia, chronic lymphoid this cytokine are found, nor are there apparent mutations leukemia, Hodgkin disease). The high incidence of autoim- in either EPO or the EPO receptor. The probable defect in mune disorders associated with this disorder suggests that DBA is an intrinsic defect of erythroid progenitor cells, an immunologic mechanism could be responsible for the resulting in their inability to respond normally to inducers red cell aplasia. Cytotoxic antibodies to erythropoietin-sen- of proliferation and differentiation. Hence, there is typically sitive cells in the marrow and to erythropoietin have been a deficit of erythroid precursors in the marrow of patients demonstrated in some cases. More commonly, the mecha- with DBA.29 Physical findings and symptoms are those nism appears to be a T-cell-mediated immunosuppression associated with anemia or related to the congenital defects. of erythropoiesis. In some cases, PRCA may be the initial Anemia is severe with erythroid hypoplasia in an otherwise presenting sign of the underlying disorder.29 normocellular bone marrow. Clinical presentation is nonspecific. Pallor is usually Diagnostic criteria for DBA are included in (Table 16-4).29 the only physical finding. Therapies include transfusion Testing for RPS19 mutations is helpful in only about 20–25% with packed erythrocytes, thymectomy if the thymus is of cases. Testing for the chromosomal breakage associ- enlarged, and immunosuppression. Various drugs, includ- ated with FA is negative. Cobalamin and folate levels are ing corticosteroids, cyclosporine, cytotoxic agents, ATG, and normal. Fetal-like erythrocytes are present; hemoglobin F monoclonal antibodies against B lymphocytes (rituximab/ is increased to 5–25%, and the i antigen is increased. The anti-CD20, alemtuzumab/anti-CD52), have been used to presence of fetal-like erythrocytes is not particularly useful suppress the immune response. Androgens or erythropoi- in diagnosing DBA in children younger than 1 year of age etin to stimulate erythrocyte production is rarely used. In because children in this age group normally possess eryth- drug-induced red cell aplasia, withdrawal of the putative rocytes with fetal characteristics. Serum iron and serum fer- agent is indicated. Approximately 80% of patients with ritin are increased, and transferrin is 100% saturated. acquired PRCA have a spontaneous remission or remission Therapy includes erythrocyte transfusions and adminis- induced by immunosuppression. About half will relapse, tration of adrenal corticosteroids. Up to half of DBA patients but with additional immunosuppression, 80% will enter develop prolonged remission, but most eventually require a second remission. By retreating relapsing patients or additional therapy. Transfusion dependence requires iron- continuing maintenance immunosuppression, many can chelation therapy (deferroximine). Most deaths are due to be maintained transfusion-free for years.30 HSCT is rarely complications of therapy such as hemosiderosis. indicated for acquired pure red cell aplasia. Table 16.4 Diagnostic Criteria for Diamond-Blackfan Diamond-Blackfan Syndrome Anemia Macrocytic (or normocytic) normochromic anemia in first year of life Diamond-Blackfan anemia (DBA) is a rare congenital Reticulocytopenia progressive erythrocyte aplasia that occurs in very young children. Anemia is present at birth in 25% of cases, and in Normocellular marrow with marked erythroid hypoplasia 98% of cases, diagnosis is made within the first year of life.29 Increased serum erythropoietin (EPO) Fanconi anemia, there is no leukopenia or thrombocytope- Normal or slightly decreased leukocyte count nia. The anemia is typically severe. Normal or increased platelet count 358 Chapter 16 Table 16.5 Comparison of the Features of Diamond-Blackfan Anemia and Transient Erythroblastopenia of Childhood (TEC) Feature Diamond-Blackfan TEC Pure red cell aplasia (PRCA) Present Present

Fetal erythrocyte characteristics (i antigen, HbF Present Absent (if child is more than 1 year old) increased) Etiology Inherited Acquired following viral infection (mechanism unknown) MCV Can be increased Normal Age at onset Birth to 1 year More than 1 year Physical abnormalities Present in 50% Absent or unrelated to anemia Therapy Corticosteroids, transfusions None, spontaneous recovery, or one or two transfusions DBA must be distinguished from transient erythroblas- PATHOPHYSIOLOGY topenia of childhood (TEC), a temporary suppression of Due to the complexity of the clinical settings in uremia, ane- erythropoiesis that frequently occurs after a viral infection mia is frequently the result of several different pathophysi- in otherwise normal children (Table 16-5). The age of onset ologies (Table 16-6): of TEC ranges from 1 month to 10 years with most cases 1. The most important and consistent factor is bone occurring in children over the age of 2. Progressive pallor marrow erythroid hypoproliferation attributed to a in a previously healthy child is the primary clinical finding. decrease in erythropoietin (EPO) production by the Fetal characteristics of erythrocytes (HbF, i antigen) seen in diseased kidney. DBA are not found in TEC. The pathophysiology of TEC is 2. In some cases, the EPO level is normal, but the bone thought to be either a virus-associated, antibody-mediated, marrow does not respond. The unresponsiveness can or a T-cell-mediated suppression/inhibition of erythroid be caused by the presence of a low molecular weight precursors. Although a preceding viral infection is associ- dialyzable inhibitor of erythropoiesis present in the ated with TEC, parvovirus B19 is not the etiologic agent. serum of uremic patients. In these cases, improvement It is important that the distinction be made between DBA in hemoglobin levels is seen after dialysis. and TEC because DBA requires treatment that is unneces- sary and potentially harmful to children with TEC. Patients 3. In addition to hypoproliferation, decreased eryth- with TEC recover within 2 months of diagnosis. Therapy, if rocyte survival compounds the anemia. One fac- needed, usually involves one or two red cell transfusions.29 tor responsible for the shortened survival is related to an unknown extracorpuscular cause, perhaps an unfavorable metabolic environment or mechanical Other Hypoproliferative trauma. Another cause of hemolysis can be related to an acquired abnormality in erythrocyte metabo- Anemias lism that involves the pentose phosphate shunt. This abnormality causes impaired generation of NADPH Other hypoproliferative anemias due primarily to defective and reduced glutathione.31 Thus, when exposed to oxi- hormonal stimulation of erythroid progenitor cells include dants, the erythrocytes develop Heinz bodies, induc- the anemia associated with chronic renal disease and the ing acute hemolysis. Hemolysis can also be related to anemias associated with endocrine disorders. In most cases, a reversible defect in erythrocyte membrane sodium- these anemias can be traced to a decrease in erythropoietin potassium ATPase. production. 4. The anemia can be related to blood loss from the gastro- intestinal tract because of a decrease in platelets and/or Renal Disease platelet dysfunction. Blood is also lost during priming Chronic renal disease is a common cause of anemia. The for dialysis. Patients receiving dialysis lose about 5–6 patient usually seeks medical attention for symptoms mg of iron daily. Thus, an anemia complicated by iron related to renal failure, and the anemia is discovered dur- deficiency is common (Chapter 12). ing the initial workup. The hemoglobin begins to decrease 5. Patients on dialysis can become folate deficient when the blood urea nitrogen level increases to more than because folate is dialyzable. Without folate supple- 30 mg/dL. Anemia develops slowly, and most patients tol- ments, the patient can develop a megaloblastic anemia erate the low hemoglobin levels well. (Chapter 15). Hypoproliferative Anemias 359 THERAPY Table 16.6 Possible Causes of Anemia in Chronic Renal Therapy for chronic renal disease includes hemodialy- Disease sis, continuous ambulatory peritoneal dialysis, and renal Decreased erythropoietin production transplantation. All treatments tend to ameliorate the ane- Presence of an inhibitor of erythropoiesis mia, but hemodialysis exposes the patient to additional Decreased erythrocyte survival causes of anemia including blood loss, iron and folate Blood loss deficiency, and hemolysis. Thus, iron and folic acid supple- Iron deficiency ments are frequently given in conjunction with hemodi- Folate deficiency alysis. Intermittent doses of EPO three times a week cause improvement in 1–2 weeks. In some cases, a normal hemo- globin is achieved, and in all cases, the patients remain LABORATORY EVALUATION transfusion-independent. A normocytic and normochromic anemia is typical in renal disease except when the patient is deficient in folate or iron; then a macrocytic anemia or microcytic Endocrine Abnormalities anemia prevails. Moderate anisocytosis with some degree Disorders of the thyroid, adrenals, parathyroid, pituitary, of microcytosis may be present. Hemoglobin levels are or gonads can result in a mild to moderate anemia.32 Endo- reduced to 5.0-8.0 g/dL (50–80 g/L), and the reticulocyte crine deficiencies are sometimes associated with a decrease production index is approximately 1. There is moderate to in EPO. The resulting anemia is usually normocytic, nor- severe poikilocytosis with echinocytes and schistocytes. mochromic with normal erythrocyte morphology. The The number of echinocytes correlates roughly with the bone marrow findings suggest erythroid hypoproliferation. severity of azotemia. Spherocytes are associated with Treatment of the endocrine disorder can resolve the anemia. hypersplenism. Nucleated erythrocytes are noted in the A slowly developing normocytic, normochromic ane- peripheral blood. Leukocytes and platelets are usually mia is characteristic of hypothyroidism. Erythrocyte sur- normal. The bone marrow reveals erythroid hypopro- vival is normal, and reticulocytosis is absent. The anemia liferation, especially when compared with the degree of is most likely a physiologic response to a decrease in tissue anemia.31 demands for oxygen. With hormone replacement therapy, Other laboratory findings vary depending on the the anemia slowly remits. severity of renal impairment. Typically blood urea Hypopituitarism is associated with an anemia more nitrogen is more than 30 mg/dL, serum creatinine is severe than that of hypothyroidism, and the leukocyte count increased, and electrolytes are abnormal. Hemostatic can be decreased. However, anemia is a minor component of abnormalities can be present. Serum ferritin levels are the other manifestations of hypopituitarism. The pituitary has higher than normal in chronic renal failure even if iron an effect on multiple endocrine glands including the thyroid deficiency is present. Therefore, it has been suggested and adrenals. In males, a decrease in androgens (gonadal dys- that if the serum ferritin level is less than 40 ng/mL, iron function) can be partly responsible for the anemia because they deficiency should be considered. Increased iron-binding stimulate erythropoiesis. In addition, a decrease in growth capacity can be a useful predictor of iron deficiency in hormone can have a trophic effect on the bone marrow. Mild these cases. anemia has also been associated with hyperparathyroidism. Summary The hypoproliferative anemias include a group of acquired anemia is a form of inherited aplastic anemia with progressive and inherited disorders in which a chronic marrow fail- bone marrow hypoplasia and other congenital defects. The ure of erythropoiesis occurs. If only the erythrocytes are disorder is characterized by chromosomal instability and fra- affected, the term pure red cell aplasia is appropriate. More gility, secondary to defective DNA repair mechanisms. commonly, hypocellularity involves all cell lineages, and The laboratory findings in AA reveal pancytopenia. The the diagnosis is aplastic anemia. erythrocytes are usually normocytic and normochromic but Immune suppression has been shown to frequently can be macrocytic. The reticulocyte count is low, and the underlie the hypocellularity in acquired aplastic anemia. corrected reticulocyte count is less than 1%. The bone mar- Acquired AA can be idiopathic or secondary to drugs, chemi- row is less than 25% cellular. cal agents, ionizing radiation, or infectious agents. Inherited Pure red cell aplasia is characterized by a selective AA can be associated with other congenital anomalies. Fanconi decrease in erythroid cells. This disorder can be acquired 360 Chapter 16 or inherited. The acquired forms are seen in thymoma, with with endocrinopathies. The laboratory findings reflect not administration of certain drugs, autoimmune disorders, and only anemia but also pathologies of the primary disorder. infection, especially viral infections. DBA is an inherited Immunosuppressive therapy using antithymocyte progressive erythrocyte aplasia occurring in young chil- globulin, cyclosporine, or monoclonal antibodies against dren. This inherited form of aplasia must be differentiated B-cell antigens is the treatment of choice for the majority from TEC, a temporary aplasia occurring after viral infec- of older patients who are not candidates for hematopoietic tion in otherwise healthy children. stem cell transplants (HSCT). HSCT is potentially curative Other hypoproliferative anemias are due primarily but not without risks. Transplantation still remains unavail- to defective hormonal stimulation of erythroid stem cells. able for many patients due to the inability to find matched These include anemia associated with renal disease and donors. Review Questions Level I c. Fanconi anemia 1. Which statement best explains current theory on the d. Acquired aplastic anemia etiology of aplastic anemia? (Objective 3) 4. Which laboratory test result is inconsistent with a diag- a. An immune mechanism, possibly involving abnor- nosis of aplastic anemia? (Objective 9) mal T-lymphocytes, leads to suppression of hema- topoietic stem cells. a. Bone marrow cellularity = 50, b. Granulocyte count = 0.2 * 103 b. A deficiency of cytokines, such as erythropoietin, /mcL results in hematopoietic stem cells which are inca- c. Platelet count = 15 * 103/mcL pable of mitosis. d. Reticulocyte count = 0.3, c. Hematopoietic stem cells are crowded out by neo- plastic cells invading the bone marrow. 5. Which statement best describes current research on d. The bone marrow microenvironment is damaged potential exposure to environmental agents and the and rendered nonsupportive of the growth of development of aplastic anemia? (Objective 7) hematopoietic stem cells. a. There is no documented association between any envi- ronmental factors and development of aplastic anemia. 2. A 60-year-old patient with a history of severe anemia b. The majority of aplastic anemia cases are not linked was found to have a marked decrease in erythrocyte to environmental exposures, but links to benzene precursors in the bone marrow with no dysplasia. and radiation exposure and some medications Leukocyte and platelet precursors were normal. have been reported. Her hemoglobin was 7.4 g/dL, reticulocyte count was decreased, and red cell indices were normal. c. Environmental factors associated with develop- Erythrocyte morphology was normal. These test ment of most cases of aplastic anemia include results and patient presentation are consistent with a wide array of infectious and chemical agents, which disorder? (Objectives 8, 9) drugs, and radiation. d. Only the antibiotic chloramphenicol and radiation a. Aplastic anemia exposure are tied to aplastic anemia. b. Iron-deficiency anemia c. Sickle cell disease 6. What is the typical morphologic classification of erythrocytes in aplastic anemia? (Objective 9) d. Pure red cell aplasia a. Hypochromic, microcytic 3. A 6-year-old child has CBC and bone marrow results b. Normochromic, normocytic consistent with a diagnosis of aplastic anemia. c. Normochromic, microcytic Chromosome fragility was present, and genetic mutations were demonstrated by molecular analysis. d. Hypochromic, macrocytic Which disorder is most consistent with this scenario? (Objectives 5, 6) 7. The bone marrow in aplastic anemia is typically: (Objective 8) a. Congenital dyserythropoietic anemia a. Hypocellular b. Diamond-Blackfan anemia b. Hypercellular Hypoproliferative Anemias 361 c. Dysplastic c. Because current theory emphasizes the role of envi- d. Normal ronmental factors such as toxin and drug exposure, IST enhances immune function and helps the indi- 8. Which of the following is (are) considered a cause of vidual with AA mount an immune response. hypoproliferation in aplastic anemia? (Objective 3) d. There is no evidence of a relationship between the 1. Damage to stem cells cause of AA and IST; we know only empirically that it is effective. 2. Depletion of stem cells 3. Inhibition of stem cells 3. A 70-year-old patient with a recent history of severe a. 1 only anemia, thrombocytopenia, and neutropenia was referred to a hematologist. Further testing revealed a b. 1 and 2 only marked decrease in all hematopoietic precursors in c. 2 and 3 only the bone marrow with no dysplasia. Pancytopenia d. 1, 2, and 3 and severely low hemoglobin was observed in the peripheral blood. Red cell indices and erythrocyte 9. What term best describes the peripheral blood find- morphology was normal. These test results and ings of a person with aplastic anemia? (Objective 9) patient presentation are consistent with which

disor- a. Pancytopenia der? (Objective 8) b. Bicytopenia a. Aplastic anemia c. Granulocytopenia only b. Iron-deficiency anemia d. Anemia only c. Anemia of chronic disease d. Pure red cell aplasia 10. Diagnostic criteria for aplastic anemia include: (Objec- tive 2) 4. Mr. Garcia is a 68-year-old man who is being a. Corrected reticulocyte count of more than 1% evaluated for kidney disease. His blood urea nitrogen (BUN) is 15 mmol/L (reference b. Platelet count less than 100 * 103/mcL interval = 2.1 - 7.1 mmol/L). His hemoglobin c. Granulocyte count less than 0.5 * 103/mcL concentration was found to be 10.9 g/dL. Which d. Bone marrow less than 50% cellular statement is the best possible explanation for his hemoglobin concentration? (Objectives 5, 7, 8) Level II a. He most likely has experienced severe blood loss 1. What next line of testing would you recommend be (hemorrhage) as result of kidney disease. ordered when a previously healthy adult presents b. His kidney disease is causing decreases in erythro- with pancytopenia on a routine automated CBC? poietin, red cell survival, iron, and folate, resulting (Objective 8) in anemia. a. Molecular assays for FA mutations c. Mr. Garcia’s hemoglobin concentration is nor- b. Chromosome breakage assay mal for a male his age and is unrelated to kidney disease. c. Review of RBC morphology on the peripheral blood smear d. Nothing in the clinical or laboratory data presented explains the hemoglobin and additional testing is d. Bone marrow examination required. 2. Immunosuppressive therapy (IST) can be used when 5. What are drawbacks to treatment of aplastic a patient has a diagnosis of aplastic anemia. What anemia with hematopoietic stem cell transplants? is the relationship between this treatment and a (Objective 3) prevalent theory on pathophysiology of the disorder? (Objectives 1, 3) a. This is considered merely supportive therapy and must be continuously administered. a. Current theory holds that the cause of AA is due to hematopoietic stem cells suppression by abnormal b. Not all patients will respond to this therapy, which cytotoxic T-lymphocytes, which are sensitive to is most successful with older patients. IST. c. Treatment complications and post-transplant mor- b. Current theory is that AA is due to neoplastic tality are high. changes in hematopoietic stem cells, which must d. This treatment is recommended only for very be eliminated by IST. young children and is not useful for adults. 362 Chapter 16 6. What confirmatory test should be performed in sus- normal numbers of other cell lineages is most pected cases of AA? (Objective 4) consistent with a diagnosis of: (Objective 5) a. Serum iron and TIBC a. Fanconi anemia b. Hemoglobin electrophoresis b. aplastic anemia c. Bone marrow examination c. pure red cell aplasia d. Direct antiglobulin test d. myelophthisic anemia 7. A 3-year-old patient presents with severe n ormocytic, 9. A male patient with previously diagnosed infectious normochromic anemia. Platelet counts and leukocyte mononucleosis infection has become suddenly ane- counts are normal. The mother reported that the mic. A possible cause of the anemia is: (Objective 1) child has been healthy since birth but recently had a a. iron deficiency cold. Which of the following laboratory test results would support a diagosis of TEC? b. folic acid deficiency (Objectives 1, 5, 6) c. anemia of chronic disease a. Decreased serum erythropoietin (EPO) d. aplastic anemia b. Normal hemoglobin F level on hemoglobin 10. What is the standard treatment for patients with electrophoresis acquired aplastic anemia? (Objective 3) c. Abnormal karyotype a. Immunosuppressive therapy d. I antigen on the patient’s erythrocytes b. Bone marrow transplant 8. A bone marrow from an anemic patient that c. Administration of growth factors demonstrates a marked erythroid hypoplasia but d. None; treatment typically not needed References 1. Brodsky, R. A. (2014). Acquired aplastic anemia. In: J. P. Greer, 11. Shi, J., Ge, M., Lu, S., Li, X., Shao, Y., Huang, J., . . . Zheng, Y. D. A. Arber, B. Glader, A. F. List, R. T. Means, F. Paraskevas, . . . (2012). Intrinsic impairment of CD4+ CD25+ regulatory T cells J. Forester, eds. Wintrobe’s clinical hematology (13th ed., pp. in acquired aplastic anemia. Blood, 120(8), 1624–1632. 965–974). Baltimore: Lippincott Williams & Wilkins. 12. Young, N. S., & Kaufman, D. W. (2008). The epidemiology of 2. Raj, R. (2015). Aplastic anemia in children. Journal of the Associa- acquired aplastic anemia. Haematologica, 93(4), 489–492. tion of Physicians of India, 63(Suppl. 3), 13–15. 13. Calado, R. T., & Young, N. S. (2008). Telomere maintenance and 3. Issaragrisil, S., Leaverton, P. E., Chansung, K., Thamprasit, T., human bone marrow failure. Blood, 111(9), 4446–4455. Porapakham, Y., Vannasaeng, S., . . . Young, N. S. (1999). Regional 14. 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Arber, B. Glader, A. F. List, anemia. Journal of the American Medical Association, 313(6), 594–602. R. T. Means, F. Paraskevas . . . J. Foerster, eds. Wintrobe’s clinical 28. Yoshimi, A., Strahm, B., Baumann, I., Furlan, I., Schwarz, hematology (13th ed., pp. 975–989). Baltimore: Lippincott Williams & S., Teigler-Schlegel, A., . . . Niemeyer, C. M. (2014). Hematopoietic Wilkins. Chapter 17 Hemolytic Anemia: Membrane Defects Diana Cochran-Black, DRPH Objectives—Level I At the end of this unit of study, the student should be able to: 1. List the hereditary membrane disorders 4. Discuss the principle of the osmotic fragility involved with erythrocyte skeletal protein test and interpret the results. abnormalities. 5. Describe the etiology, pathophysiology, and 2. List the hereditary erythrocyte membrane laboratory features of paroxysmal nocturnal disorders involved with abnormal hemoglobinuria (PNH). membrane permeability. 6. Explain the principle of the sugar water 3. Describe the pathophysiology and (sucrose hemolysis) test and interpret its recognize laboratory features associated results. with hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis, and hereditary stomatocytosis. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Differentiate the protein defects associated stomatocytosis, and paroxysmal nocturnal with each hereditary membrane defect hemoglobinuria. discussed in the chapter. 3. Explain the role of decay-accelerating factor 2. Create a flow chart using laboratory tests (DAF) and membrane inhibitor of reactive to differentiate hereditary spherocytosis, lysis (MIRL) in PNH. elliptocytosis, pyropoikilocytosis, 364 Hemolytic Anemia: Membrane Defects 365 4. Explain the results of the sucrose hemolysis 5. Evaluate a clinical case study and determine test and immunophenotyping to determine the type of membrane disorder present by a diagnosis of paroxysmal nocturnal correlating clinical history and laboratory hemoglobinuria (PNH). features. Chapter Outline Objectives—Level I and Level II 364 Hereditary Pyropoikilocytosis (HPP) 373 Key Terms 365 Hereditary Stomatocytosis Syndromes 374 Background Basics 365 Abnormal Membrane Lipid Composition: Case Study 366 Acanthocytosis 375 Overview 366 Paroxysmal Nocturnal Hemoglobinuria (PNH) 377 Introduction 366 Summary 379 Skeletal Protein Abnormalities 366 Review Questions 380 Hereditary Spherocytosis 367 References 382 Hereditary Elliptocytosis 371 Key Terms Abetalipoproteinemia Hereditary elliptocytosis (HE) Overhydrated hereditary Cholecystitis Hereditary pyropoikilocytosis stomatocytosis (OHS) Cholelithiasis (HPP) Paroxysmal nocturnal Decay-accelerating factor (DAF) Hereditary spherocytosis (HS) hemoglobinuria (PNH) Dehydrated hereditary Horizontal interaction Rh null disease stomatocytosis (DHS) Membrane inhibitor of reactive Spur cell anemia Exchange transfusion lysis (MIRL) Vertical interaction Background Basics The information in this chapter builds on concepts • Describe the general clinical and laboratory features learned in previous chapters. To maximize your learn- associated with hemolytic anemias. (Chapter 11) ing experience, you should review these concepts before • Calculate erythrocyte indices, recognize abnormal starting this unit of study: erythrocyte morphology, and list reference intervals for common hematology parameters. (Chapters 10, 11, 37) Level I • Describe the function, composition, and metabolism Level II of the erythrocyte membrane. (Chapter 5) • List the erythrocyte membrane proteins associ- • Identify the differences between extravascular and ated with cell deformability and permeability, and intravascular erythrocyte destruction. (Chapters 6, 11) describe their involvement in horizontal and vertical interactions. (Chapter 5) 366 Chapter 17 CASE STUDY Skeletal Protein We refer to this case study throughout the chapter. Abnormalities Jack, a 12-year-old male,

was brought to his family The membrane protein and lipid interactions associated physician for evaluation of right-upper-quadrant with abnormal erythrocyte membranes can be divided pain. He has a lifelong history of hemolytic and into two categories, vertical and horizontal interactions1,2,3 aplastic crises. (Figure 17-1 ; Table 17-1). Consider why this patient history is important when selecting and evaluating laboratory tests. Vertical Interactions Vertical interactions are perpendicular to the plane of the erythrocyte membrane and include interactions between Overview the skeletal lattice on the cytoplasmic side of the membrane and its attachment to the integral proteins and lipids of the This chapter focuses on a group of hemolytic anemias that membrane. These interactions stabilize the lipid bilayer of the result from defects in the erythrocyte membrane. These membrane. Defects in vertical contacts between the skeletal defects include hereditary spherocytosis, hereditary elliptocy- lattice proteins and the membrane’s integral proteins and lip- tosis, hereditary pyropoikilocytosis, and hereditary stomato- ids cause uncoupling of the lipid bilayer from the underlying cytosis; membrane lipid disorders; and paroxysmal nocturnal skeletal lattice, allowing a selective loss of portions of the lipid hemoglobinuria. The format for presentation of each of these bilayer. The net loss of cell membrane results in a decrease in disorders is pathophysiology, clinical presentation, labora- the surface-area-to-volume ratio, the formation of a sphero- tory evaluation, and therapy. Because these anemias result cyte, and the eventual hemolysis of the cell. The skeletal lat- from defects in the erythrocyte membrane, the reader should tice, however, is not disrupted, and the cell is mechanically have a good understanding of the normal erythrocyte mem- stable. See Table 17-1 for the genetic defects associated with brane structure (Chapter 5) before beginning. the vertical protein interactions of the red cell membrane. Introduction Horizontal Interactions Horizontal interactions are parallel to the plane of the Erythrocyte life span can be significantly shortened if the membrane and are important in the formation of the stress- cell is intrinsically defective (intracellular defect). Hemo- supporting skeletal protein lattice that provides mechanical lytic anemia has been associated with defective erythrocyte stability to the membrane. Horizontal interactions include membranes, structurally abnormal hemoglobins (hemo- spectrin heterodimer head-to-head association to form tet- globinopathies), defective globin synthesis (thalassemias), ramers as well as skeletal protein interactions in the junc- and deficiencies of erythrocyte enzymes. Almost all of these tional complexes at the distal ends of spectrin tetramers defects are hereditary. The hemoglobinopathies and thalas- (spectrin, actin, protein 4.1R, glycophorin C, and adducin semias, which have a significant hemolytic component, are contacts). Horizontal defects characterized by defects of the discussed in Chapters 13 and 14. skeletal protein interactions beneath the lipid bilayer lead An erythrocyte membrane that is normal in both to disruption of the skeletal lattice and, consequently, mem- structure and function is essential to the survival of the brane destabilization. This causes cell fragmentation with cell (Chapter 5). Composed of proteins and lipids, the mem- formation of poikilocytes. brane is responsible for maintaining stability and the nor- mal discoid shape of the cell, preserving cell deformability, and retaining selective permeability. Lipid Composition Abnormalities Hemolytic anemia can result from abnormalities in Disorders that affect the composition of the membrane lipid constituent membrane proteins or lipids, both of which can bilayer lead to the formation of acanthocytes or stomatocytes. alter the membrane’s stability, shape, deformability, and/ The erythrocyte membrane normally contains equal amounts or permeability. The abnormal cells are particularly sus- of free cholesterol and phospholipids. Excess free plasma ceptible to entrapment in the splenic cords. Anemia results cholesterol in patients can accumulate in the outer bilayer of when the rate of hemolysis is increased to the point that the the erythrocyte. Preferential expansion of the outer face of the bone marrow cannot adequately compensate. Most hemo- lipid bilayer in comparison to the inner face leads to forma- lysis associated with abnormal membranes is extravascular, tion of acanthocytes.4 Acanthocytes are more spheroidal cells occurring primarily in the spleen. with irregular projections and an absence of central pallor. Hemolytic Anemia: Membrane Defects 367 Lipid bilayer-skeleton uncoupling: Sp-lipid Sp-2.1 -3 Sp-lipid Sp-4.1R -GPC A defect of vertical interactions interaction interaction interaction interaction Deficiency of spectrin, ankyrin Band GPC lipid or band 3 protein 3 bilayer 2.1 b a 4.1R actin a b spectrin SpD-D interaction Sp-4.1R -actin interaction Lipid bilayer - adducin protein skeleton uncoupling Horizontal interactions Gross membrane fragmentation: A defect of horizontal protein interactions Normal skeleton Disrupted skeleton Whole cell fragmentation Membrane loss in the form of microvesicles Surface area deficiency leading to spherocytosis Figure 17.1 Pathobiology of the red cell lesions in hereditary red blood cell membrane defects. Vertical interactions of membrane lipids and proteins are perpendicular to the plane of the membrane. These interactions stabilize the lipid bilayer. Deficiencies or defects of spectrin, ankyrin (2.1), or band 3 protein cause the lipid bilayer to decouple from the underlying skeletal lattice and subsequent membrane loss in the form of microvesicles. This leads to the formation of spherocytes (hereditary spherocytosis). Horizontal interactions are parallel to the plane of the membrane. These interactions provide mechanical stability to the membrane. Horizontal defects include abnormal spectrin heterodimer association to form tetramers and defective skeletal protein interactions of junctional complexes at the end of the spectrin tetramers (spectrin, actin, protein 4.1R, previously referred to as protein 4.1). Horizontal defects result in fragmentation of the red blood cell (hereditary elliptocytosis, hereditary pyropoikilocytosis). Sp, spectrin; GP, glycophorin; SpD-D, spectrin dimer dimer. These cells are poorly deformable and readily trapped in the Table 17.1 Protein Mutations in the Erythrocyte spleen. A variety of conditions can lead to acanthocytosis. Membrane That Result in Vertical or Horizontal Interaction Hemolytic anemias associated with the various Defects and Cause Hemolytic Anemia membrane defects are listed in Table 17-2. In general, Interaction Defect Causes Gene vertical interaction defects are characterized by the pres- Vertical Ankyrin ANK1 ence of spherocytes; horizontal defects are characterized by the presence of other types of poikilocytes; and lipid Band 3 SLC4A1 composition defects are characterized by the presence of Protein 4.2 EPB42 acanthocytes. a@Spectrin SPTA1 b@Spectrin SPTB Horizontal Protein 4.1R EPB41 Hereditary Spherocytosis Glycophorin C GYPC Hereditary spherocytosis (HS) is the most common inher- a@Spectrin SPTA1 ited hemolytic disorder seen in individuals of northern Euro- b@Spectrin SPTB pean descent. The estimated prevalence of this membrane Actin ACTB disorder ranges from 1 in 2500 to 1 in 5000 Caucasians.3,5,6,7 Vertical interactions 368 Chapter 17 Table 17.2 Hemolytic Anemias Associated with Erythrocyte Membrane Defects Abnormal Membrane Erythrocyte Disorder Inheritance Pattern Membrane Defect Function Morphology Hereditary Usually autosomal dominant; Combined deficiencies Defective vertical protein Spherocytes spherocytosis (HS) autosomal recessive; de novo of ankyrin and spectrin interaction between RBC mutations or isolated deficiencies skeleton and membrane; loss of spectrin, band 3, of lipid bilayer and subsequent or protein 4.2 formation of a spherocyte with decreased deformability; abnormal permeability to Na+ Hereditary Autosomal dominant or rare Defective spectrin Defect in horizontal protein Elliptocytes elliptocytosis (HE) de novo mutation; Melanesian Deficiency or defect in interactions resulting in variant is autosomal recessive band 4.1R membrane instability; also can Abnormal integral proteins be a defect in permeability Hereditary Autosomal recessive Two defects: Partial spectrin Defect in horizontal protein Schistocytes, pyropoikilocytosis (HPP) deficiency and presence interactions resulting in elliptocytes, and of a mutant spectrin membrane instability microspherocytes Overhydrated hereditary Autosomal dominant Unknown Abnormally permeable to Stomatocytes stomatocytosis Na+ and K+; overhydration and swelling; decreased deformability Dehydrated hereditary Autosomal dominant Unknown Abnormally permeable Target cells stomatocytosis resulting in loss of K+; water loss; decreased deformability Acanthocytosis Autosomal recessive Increased sphingomyelin, Expansion of outer lipid layer Acanthocytes (abetalipoproteinemia) which can be secondary causes abnormal shape; to abnormal plasma lipid increased membrane viscosity composition and decreased fluidity/ deformability Paroxysmal nocturnal Acquired Deficiency of DAF or MIRL Increased sensitivity to Normocytic or hemoglobinuria (PNH) complement lysis macrocytic; microcytic, hypochromic if iron deficient DAF, decay-accelerating factor; MIRL, membrane inhibitor of reactive lysis. with both deformability and permeability. The intrinsic red CASE STUDY (continued from page 366) cell defect is compounded by the function of the spleen, which A CBC was ordered on Jack, and the results follow: retains and further damages the abnormal HS erythrocytes. The membrane defect in HS is a disorder of vertical WBC 8.0 * 109>L protein interactions most often characterized by a combined RBC 4.0 * 1012>L deficiency of spectrin and ankyrin, although there is con- Hb 10.8 g/dL siderable genetic heterogeneity at the molecular level. The Hct 0.292 L/L (29.2%) spectrin deficiency can be a primary deficiency of spectrin or a secondary deficiency due to defective attachment of the Platelets 504 * 109>L skeleton to the lipid bilayer. Defective attachment can occur 1. Calculate the erythrocyte indices. as a result of mutations of ankyrin, a@ or b@spectrin, protein 4.2, or band 3 protein.8 Identified mutations can be found at 2. Based on the calculated indices, describe the www.hgmd.cf.ac.uk/ac/index.php. patient’s red blood cells. These defects in spectrin and ankyrin and their interac- tions with other skeletal proteins result in a weakening of the vertical connections between the skeletal proteins and Pathophysiology lipid bilayer of the membrane. The uncoupling between the inner membrane skeleton and outer lipid bilayer leads to the Hereditary spherocytosis is a clinically heterogeneous disor- shedding of the lipid bilayer in the form of lipid microvesi- der characterized by mild to moderate hemolysis. The eryth- cles.3 The cell has a decreased surface-area-to-volume ratio, rocytes in this disorder are deficient in certain membrane secondary to membrane loss, changing the morphology of proteins and are abnormally permeable to monovalent cat- the cell from a discocyte to a spherocyte. The most sphe- ions. These features lead to erythrocytes that have problems roidal cells have a greatly increased cytoplasmic viscosity. Hemolytic Anemia: Membrane Defects 369 The spheroidal shape and increased viscosity result in varying directly with the individual’s age upon presentation reduced cellular flexibility. Reticulocytes and young eryth- of symptoms. Infants have the lowest values, 8–11 g/dL. rocytes in HS are normal in shape, emphasizing the fact that Older children usually have concentrations above erythrocytes lose their membrane vesicles after encounter- 10 g/dL. The reticulocyte count is usually more than 8% ing the stress of the circulation. and often is disproportionately increased relative to the In addition to the abnormal cytoskeleton of HS erythro- degree of anemia.5 The diagnosis of hereditary spherocy- cytes, other membrane abnormalities can be present. Total tosis is suspected when many densely stained spherocytic lipid content in the HS erythrocyte membrane is decreased cells with a decreased diameter and increased polychroma- both before and after splenectomy. Although the organiza- sia on the blood smear are found (Figure 17-2). Small, dense tion of lipids in the membrane is known to affect membrane microspherocytes with a decreased MCV and increased fluidity, an association between abnormal fluidity and HS MCHC also can be found. The number of microspherocytes erythrocytes has not been established. The HS erythrocytes varies considerably, and these cells may not be prominent also are abnormally permeable to sodium causing an influx in 20–25% of patients. In mild forms of HS, the changes in of Na+ at a much higher rate than normal.9 An increase in erythrocyte morphology may be too subtle to detect even the activity of the cation pump can compensate for the leak by experienced hematologists. Nucleated erythrocytes can if adequate glucose is available for ATP production. The be found in children with severe anemia. increased permeability is probably related to a functional When the inheritance pattern of HS cannot be estab- abnormality of the membrane proteins. lished, HS must be distinguished from other conditions The spherocytic shape, increased cytoplasmic viscos- causing spherocytosis. Spherocytes also can be found in ity, and increased membrane permeability account for the acquired autoimmune hemolytic anemia (AIHA), but in HS, eventual destruction of HS cells in the spleen. The nonde- the spherocytes are more uniform in size and shape than formable spherocytes lack the flexibility of normal cells and in AIHA. Automated reticulocyte analyzers may be use- become trapped in the splenic cords. In this acidic, hypoxic, ful in differentiating AIHA from HS. HS will demonstrate hypoglycemic environment, the cell quickly runs out of the a higher reticulocyte to immature retic fraction (RET/IRF) ATP needed to pump out the excess Na+ resulting from the ratio as compared to AIHA.10 increased membrane permeability. As energy production Erythrocyte indices are helpful

in diagnosing HS. The decreases, splenic macrophages destroy the metabolically MCV usually is normal or only slightly decreased (77–87 fL). stressed cells. If reticulocytosis is marked, the MCV can be increased. The MCH is normal, but the MCHC is generally more than Clinical Presentation 36 g/dL.3 Modern blood analyzers utilizing laser or aper- ture impedance methodology are extremely sensitive in The clinical severity of HS varies among families and even detecting erythrocytes with an MCHC of more than 41 g/dL among patients in the same family. About 25% of the patients (hyperhemoglobin).11 These erythrocytes are typically sphe- have compensated hemolytic disease, no anemia, little or no rocytes. This technology can detect even the mild forms of jaundice, and only slight splenomegaly. In contrast, the dis- HS. Importantly, the indices can vary, depending on iron and ease can be lethal for some patients, especially those homo- folate stores. Folate frequently becomes depleted in chronic zygous for dominant HS. Most patients, however, develop hemolytic states, resulting in macrocytosis (Chapter 15). a partially compensated hemolytic anemia in childhood and appear asymptomatic. HS in some asymptomatic individu- als can be detected only when family studies are conducted on patients with more severe forms of the disease. Intermit- tent jaundice can occur and is especially apparent during viral infections. Splenomegaly is present in about 50% of affected infants increasing to 75–95% in older children and adults. Aplastic crisis is a life-threatening complication that can occur in childhood during or following a viral infec- tion. Untreated older patients commonly develop pigment bile stones from excess bilirubin catabolism (cholelithiasis). These patients also are predisposed to cholecystitis. Laboratory Evaluation Figure 17.2 Peripheral blood from a patient with hereditary Hemoglobin levels in patients with HS can be normal or spherocytosis shows the presence of many densely staining decreased, (about two-thirds to three-fourths have compen- spherocytes (arrows) (peripheral blood, Wright-Giemsa stain, sated or partially compensated mild to moderate anemia), 1000* magnification). 370 Chapter 17 Other markers of ongoing hemolysis may be present including increased serum bilirubin, increased lactic dehydrogenase (LD), and increased urine and fecal urobi- linogen. Variable components of extravascular and intra- vascular hemolysis may be present including decreased haptoglobin. As in other hemolytic states, the bone marrow demon- strates normoblastic erythroid hyperplasia with an increase in storage iron. Checkpoint 17.1 List various factors related to changes in the erythrocyte that can lead to a decrease or increase in the MCV in hereditary spherocytosis. OSMOTIC FRAGILITY TEST In many laboratories, the principal screening test in the diagnosis of HS is the osmotic fragility test. This test mea- sures the erythrocyte’s resistance to hemolysis by osmotic stress, which depends primarily on the cell’s volume, sur- face area, and its membrane function. Erythrocytes are incubated in varying concentrations of hypotonic sodium chloride (NaCl) solution. During the process of osmotic equilibration in hypotonic solutions, spherocytes are unable to expand as much as normal discoid shaped cells because of their decreased surface-area-to-volume ratio. Very little Figure 17.3 Graph depicting the osmotic fragility of normal fluid needs to be absorbed before the cells hemolyze. The cells, spherocytes, and cells from a patient with thalassemia. spherocytes also can have increased membrane perme- Spherocytes show an increased fragility with a shift to the left in the ability, contributing to their increased fragility. Lysis of HS osmotic fragility curve, and the thalassemia cells show a decreased fragility with a shift to the right in the osmotic fragility curve. erythrocytes, therefore, begins at higher NaCl concentra- The decreased fragility in thalassemia is caused primarily by the tions than normal cells. These HS cells are said to exhibit presence of target cells. increased osmotic fragility. The osmotic fragility test is not abnormal unless sphero- cytes constitute at least 1–2% of the erythrocyte population; cells) have a decreased osmotic fragility, which is found in thus, patients with mild HS may have a normal osmotic fra- a variety of conditions including thalassemia and sickle cell gility. These cells, however, show marked abnormal hemo- anemia. lysis if the blood is incubated overnight (24 hours) at 37°C AUTOHEMOLYSIS TEST before it is added to the NaCl solution. Incubation increases The autohemolysis test is another screening test used in the loss of erythrocyte surface area in HS cells compared diagnosing HS but it does not have an advantage over the with normal erythrocytes due to their leaky and unstable osmotic fragility test. It is more valuable in differentiat- membrane. The difference in osmotic fragility between HS ing various types of congenital, nonspherocytic hemolytic cells and normal cells is particularly apparent at the 100% anemias. This test measures the degree of spontaneous red cell lysis point, which occurs at ∼0.43 { 0.05 g NaCl/ hemolysis of blood incubated at 37°C. The degree of dL with HS cells and at ∼0.23 { 0.07 g NaCl/dL with nor- hemolysis depends on the integrity of the cell membrane mal cells.12 Because of its increased sensitivity, this incu- and the adequacy of cell enzymes involved in glycolysis. bated osmotic fragility test is the most reliable diagnostic Incubation of blood in vitro probably causes an alteration test for hereditary spherocytosis. The osmotic fragility test results are graphed to depict the degree of fragility in comparison to the normal state Table 17.3 Example of Increased Osmotic Fragility (Figure 17-3). A shift to the left of normal in the curve indi- Patient Normal Control cates increased osmotic fragility, whereas a shift to the right indicates decreased osmotic fragility (Table 17-3). Cells Initial hemolysis 0.65% NaCl 0.45% NaCl with an increased surface area–to–volume ratio (e.g., target Complete hemolysis 0.45% NaCl 0.30% NaCl Hemolytic Anemia: Membrane Defects 371 of membrane lipids, which leads to a change in cell perme- corrects the anemia and hemolysis, but the basic membrane ability and an increase in the utilization of both glucose defect remains, and spherocytes can still be found in the and ATP. peripheral blood. Fragments of the unstable membrane are In HS, autohemolysis is increased to between 5 and probably removed in the liver. 25% at 24 hours (normal is 0.2–2.0%) and can increase to 75% at 48 hours. If glucose is added to the blood before CASE STUDY (continued from page 368) incubation, hemolysis significantly decreases. If large num- bers of microspherocytes are present, hemolysis might not The patient’s peripheral smear revealed numer- be corrected with glucose. Autohemolysis also increases in ous elliptocytes, spherocytes, teardrop cells, and immune spherocytic anemias, but glucose does not usually micropoikilocytes. affect the test results in these conditions. 3. What additional lab tests should be ordered? ANTIHUMAN GLOBULIN TEST The antihuman globulin (AHG) test is negative, a find- ing helpful in distinguishing HS from immune hemolytic anemias in which large numbers of spherocytes also are found (Chapter 19). The AHG test detects antibodies or Hereditary Elliptocytosis complement bound to erythrocytes in vivo (direct anti- Hereditary elliptocytosis (HE) is inherited as an autoso- globulin test [DAT]) or free antibodies in the serum (indi- mal-dominant trait except for a rare Melanesian type that rect antiglobulin test [IAT]). Immune hemolytic anemias are is inherited as a recessive trait. Worldwide, the incidence usually associated with a positive DAT whereas the DAT in is one case per 2000–4000 individuals, although the true HS is negative. incidence is unknown because many patients with HE are asymptomatic.5 The disease is heterogeneous in the degree of hemolysis and in clinical severity. As its name indicates, Checkpoint 17.2 the most prominent peripheral blood finding is an increase Explain why a patient with hereditary spherocytosis will dem- onstrate increased hemolysis in an autohemolysis test and why in oval and elongated erythrocytes (elliptocytes). adding glucose prior to incubation will lead to a normal rate of Three classifications of HE can be distinguished based hemolysis in these patients. on erythrocyte morphology3,16: (1) common HE, (2) sphero- cytic HE (hemolytic ovalocytosis), and (3) stomatocytic HE, Melanesian ovalocytosis, or Southeast Asian ovalocytosis Identification of Deficient/Defective (SAO) (Table 17-4). Membrane Protein Pathophysiology To determine which erythrocyte membrane protein(s) is/ are involved, initial studies usually include sodium dodecyl The abnormal erythrocyte shape in hereditary elliptocyto- sulfate polyacrylamide gel electrophoresis (SDS-PAGE). sis is the result of a defect of one of the skeletal proteins. Genetic linkage analysis using PCR-based techniques or Similar to HS, reticulocytes and young erythrocytes in this cDNA and genomic DNA analyses can be done if a molecu- disorder are normal in shape; therefore, the elliptical eryth- lar diagnosis is needed (Chapter 42). The eosin-5-maleimide rocyte shape is acquired progressively as they circulate. (EMA) binding test uses flow cytometry analysis to identify Erythrocytes must deform (elongate) to enter capillaries various erythrocyte membrane defects. This test utilizes a in the microcirculation (vessels with diameters as small as dye that binds to the integral protein band 3 and Rh-related ∼3 mcM3mM4 ) Also, erythrocytes are subjected to shear proteins. Flow cytometry reveals a decrease in fluorescence stress as they circulate, which also contributes to their in HS erythrocytes reflecting the relative amount of band 3. This test has high specificity and sensitivity for HS and Table 17.4 Classification of Hereditary Elliptocytosis (HE) is useful in screening. The EMA test also can detect other Based on Erythrocyte Morphology membrane abnormalities associated with band 3 including Type of HE Hemolysis Erythrocyte Shape abnormalities of erythrocyte hydration, variants of dyser- thropoietic anemia, and sickle cell disease.5,11,13 Common HE Variable—minimal Biconcave elliptocytes to severe Spherocytic HE Present Spherocytes and fat Therapy (hemolytic ovalocytosis) elliptocytes Mild forms of HS do not require therapeutic intervention. Southeast Asian Mild or absent Roundish elliptocytes ovalocytosis (SAO) that are also Total or partial splenectomy is the standard treatment in (stomatocytic HE; stomatocytic patients with symptomatic hemolysis.14,15 This therapy Melanesian ovalocytosis) 372 Chapter 17 acquiring an elliptical shape. Normal erythrocytes revert an increase in bone marrow erythropoiesis (compensated to a biconcave shape once they re-enter larger vessels and hemolytic disease). Anemia is not characteristic. experience less circulatory stress, whereas HE erythrocytes Common HE is rare in the Western populations but more remain in the elliptical shape. It is possible that the stress in common in blacks, particularly in equatorial Africa. The the microcirculation causes disruption of the skeletal pro- severity ranges from asymptomatic to severe clinical dis- tein contacts in the membrane of HE erythrocytes and leads ease. There can be minimal hemolysis and only mild ellip- to the formation of new protein contacts that prevent HE tocytosis (15%) or severe hemolysis with cell fragmentation erythrocytes from resuming the normal biconcave shape. and formation of poikilocytes. The principal defect involves horizontal membrane pro- A variant of common HE noted in black infants is tein interactions. Evidence indicates that several different associated with a moderately severe anemia at birth and membrane protein defects can be linked to this disease5,17: neonatal jaundice. The peripheral blood smear exhibits erythrocytes similar to those seen in hereditary pyropoi- 1. Decreased association of spectrin dimers to form tetra- kilocytosis with budding and fragile bizarre poikilocytes. mers because of defective spectrin chains Variable numbers of elliptocytes are noted. Between 6 and 2. Deficiency or defect in band 4.1R that aids in binding 12 months of age, the hemolysis decreases, and the num- spectrin to actin ber of elliptocytes increases. One of the parents of affected 3. Abnormalities of the integral proteins including defi- infants has mild hereditary elliptocytosis. ciency of glycophorin C and abnormal anion transport The spherocytic HE variant constitutes a relatively protein (band 3) with increased affinity for ankyrin rare form of HE characterized by the presence of hemolysis (vertical interaction defect). despite minimal changes in erythrocyte morphology. The Each of these defects can lead to skeletal disruptions erythrocytes have characteristics of both HS and HE cells: that can cause the cell to become elliptical in shape and/ Some are spherocytic and others are fat elliptocytes. or fragment under the stresses of the circulation, depend- The Southeast Asian variant of HE (SAO, stomato- ing on the extent of the defect. Cells become more ellipti- cytic HE) is characterized by a mild or absent hemolytic cal as they age.12 Mildly dysfunctional proteins cause only component. Erythrocyte cation permeability appears to be elliptocytosis, whereas severely dysfunctional proteins increased, and the expression of blood group antigens is cause membrane fragmentation in addition to elliptocyto- muted. The elliptocytes are roundish and stomatocytic with sis. Alteration in shape only does not appear to affect

cell one or two transverse bars or a longitudinal slit. Evidence deformability and viability, and the cells have a nearly nor- indicates that these cells can have more stable cytoskeletons than normal.3mal life span. Elliptocytosis with membrane fragmentation, however, causes a decrease in cell surface area and reduced The high prevalence of the SAO variant of HE in some cell deformability. The life span of these cells is severely parts of the world is related to the resistance of the SAO shortened. erythrocytes to invasion by malarial parasites. The resis- In addition to membrane instability, HE erythrocytes tance may result from the abnormal rigidity of the eryth- are abnormally permeable to cations. This altered perme- rocyte membrane. This protection against malaria has led ability demands an increase in ATP to run the cation pump to natural selection of individuals with HE in areas of the world where malaria is endemic.3and maintain osmotic equilibrium. Cells detained in the spleen can quickly deplete their ATP and become osmoti- HE cells are poorly agglutinable with antisera against cally fragile. erythrocyte antigens. This is presumably the result of defec- The defect in the SAO variant of HE is an abnormal tive lateral movement and clustering of surface antigens band 3 protein rather than a defect in the cytoskeletal pro- associated with the abnormal band 3 protein. The labora- teins under the lipid bilayer. There is a nine-amino acid tory scientist should be aware of this problem because it deletion near the boundary of the membrane and cytoplas- can interfere with testing of patients’ cells in the blood bank. mic domains of the protein. This is associated with a tighter binding of band 3 to ankyrin, a lack of transport of sulfate Laboratory Evaluation anions, and a restriction in the lateral and rotational mobil- ity of band 3 protein within the membrane. As a result, these The most consistent and characteristic laboratory finding erythrocytes are very rigid.3,16 in all variants is prominent elliptocytosis (Figure 17-4). Elliptocytes also can be found in association with other dis- eases, but elliptocytes in these conditions usually constitute Clinical Presentation less than 25% of the erythrocytes. Acquired elliptocytosis Ninety percent of patients with HE show no overt signs of is seen in megaloblastic anemias and iron-deficiency ane- hemolysis. Although erythrocyte survival can be decreased, mia. In contrast, the elliptocytes in HE make up more than the hemolysis is usually mild and well compensated for by 25% of the erythrocytes and usually more than 60%. In the Hemolytic Anemia: Membrane Defects 373 The membrane defect, however, remains, and elliptocytes are still present. The asymptomatic variants require no therapy. Checkpoint 17.3 Why do the elliptocytes in HE demonstrate normal osmotic fragility? CASE STUDY (continued from page 371) Jack’s osmotic fragility test revealed the following: Patient Control Figure 17.4 Peripheral blood from a patient with nonhemolytic Initial hemolysis 0.65% NaCl 0.45% NaCl HE reveals almost 100% elliptocytes (peripheral blood, Wright- Complete hemolysis 0.40% NaCl 0.30% NaCl Giemsa stain, 1000* magnification). 4. Interpret the results of the osmotic fragility test. asymptomatic variety of HE, elliptocytes could be the only morphologic clue to the disease. Hemoglobin levels are usu- ally higher than 12 g/dL. Reticulocytes are mildly elevated, up to about 4%. Hereditary In the hemolytic HE variants, hemoglobin concentra- tion is 9–10 g/dL, and reticulocytes are elevated to as high Pyropoikilocytosis (HPP) as 20%. Microelliptocytes, bizarre poikilocytes, schistocytes, Hereditary pyropoikilocytosis (HPP), a rare autosomal reces- and spherocytes are usually evident (Figure 17-5). The sive disorder, is closely related to HE. Based on genetic and bone marrow shows erythroid hyperplasia with normal biochemical data, it has been established that HPP is a severe maturation. subtype of HE.3,17 It occurs primarily in blacks.5 The disease The incubated and unincubated osmotic fragility tests presents in infancy or early childhood as a severe hemolytic and autohemolysis tests are usually abnormally increased anemia with extreme poikilocytosis. The morphologic simi- in the overt hemolytic variants. However, the obvious larities of erythrocytes in HPP and that of erythrocytes asso- blood picture suggests that there is no need to perform ciated with thermal injury led investigators to examine the these tests. thermal stability of HPP cells. In contrast to normal eryth- rocytes that fragment at 49–50°C, HPP cell membranes frag- Therapy ment when heated to 45–46°C. In addition, pyropoikilocytes disintegrate when incubated at 37°C for 76 hours. The hemolytic variants of HE respond well to splenec- tomy. As in HS, splenectomy reduces hemolysis and pro- tects the patient from complications of chronic hemolysis. Pathophysiology The HPP cells have two defects with one inherited from each parent. One is related to a deficiency of a@spectrin and the other to the presence of a mutant spectrin that prevents self- association of heterodimers to tetradimers.3 The parent carry- ing the mutant spectrin either has mild HE or is asymptomatic. The parent with the deficiency of spectrin, is usually hemato- logically normal. The HPP phenotype also is found in patients who are homozygous or doubly heterozygous for one or two spectrin mutations characteristically found in HE trait.3 These defects lead to a disruption of the membrane skeletal lattice and cell destabilization followed by erythrocyte fragmentation and poikilocytosis. Poikilocytes are removed in the spleen. Clinical Presentation Figure 17.5 Peripheral blood from a patient with hemolytic hereditary elliptocytosis. Schistocytes as well as elliptocytes are Clinical features consistent with a hemolytic anemia are present (Wright-Giemsa stain, 1000* magnification). present at birth. Hyperbilirubinemia requiring exchange 374 Chapter 17 transfusion (a procedure involving simultaneous with- Therapy drawal of blood and infusion with compatible blood) or phototherapy (therapeutic exposure to sunlight or artificial Patients show improvement after splenectomy, but the light) is present.5 Laboratory serologic studies for hemolytic membrane defect remains, and fragmented erythrocytes disease of the newborn, however, are negative. are still present. Laboratory Evaluation Checkpoint 17.4 Interpret the results of the following thermal sensitivity test: Stained blood smears exhibit striking erythrocyte mor- phologic abnormalities including budding, fragments, Patient’s erythrocytes Marked erythrocyte fragmentation microspherocytes (2–4 fL), elliptocytes, triangulocytes after 10-minute incubation at 46°C (fragmented erythrocytes that are triangle shaped), and Normal control No significant change in erythro- other bizarre erythrocyte shapes (Figure 17-6). The MCV cyte morphology after 10-minute is decreased (25–55 fL), most likely as a result of the eryth- incubation at 46°C. rocyte fragmentation. The osmotic fragility is abnormal, especially after incubation, and the thermal sensitivity test demonstrates an increase in erythrocyte fragmentation (Figure 17-7). Autohemolysis is increased, and the hemoly- sis is not corrected with glucose. Hereditary Stomatocytosis Syndromes The term hereditary stomatocytosis includes a group of rare autosomal dominant hemolytic anemias in which the erythrocyte membrane exhibits abnormalities in cation permeability.9 These syndromes include overhydrated hereditary stomatocytosis (OHS) (also known as hereditary hydrocytosis) and dehydrated hereditary stomatocytosis (DHS) (also known as hereditary xerocytosis). The red cell membrane in OHS is abnormally permeable to both Na+ and K+. The net gain of Na+ ions is greater than the net loss of K+ ions as the capacity of the cation pump (fueled by ATP derived from glycolysis) to maintain normal intracellular osmolality is exceeded. Because the pump exchanges 3 Na+ Figure 17.6 Peripheral blood from a patient with hereditary (outward) for 2 K+ (inward), as the pump fails, the intra- pyropoikilocytosis (peripheral blood, Wright-Giemsa stain, 1000* cellular concentration of cations increases, water enters the magnification). cell, and the overhydrated cells take on the appearance of stomatocytes. Stomatocytes (hydrocytes) on dried, stained blood films are erythrocytes with a slitlike (mouthlike) area of pallor (Figure 17-8). These cells are uniconcave and appear to be bowl shaped on wet preparations. The primary defect in DHS is a net loss of intracellular K+ that exceeds the passive Na+ influx, and net intracellular cation and water content are thus decreased. Consequently, the cell dehydrates and the cells appear targeted or con- tracted and spiculated. CASE STUDY (continued from page 373) A thermal sensitivity test demonstrated that Jack’s erythrocytes fragment when incubated for 10 min- utes at 46°C. Figure 17.7 Peripheral blood from a patient (same as in 5. What do the results of the thermal sensitivity test Figure 17-6) with hereditary pyropoikilocytosis. Smear made after reveal about Jack’s red blood cells? incubation of blood for 10 minutes at 46°C (peripheral blood, Wright-Giemsa stain, 1000* magnification). Hemolytic Anemia: Membrane Defects 375 alcoholism, liver disease, and cardiovascular disease. How- ever, these acquired conditions lack abnormal cation perme- ability, and little hemolysis is present. The cells are dehydrated in DHS, which is reflected by an increased MCHC. As the MCHC of the cell increases beyond 37 g/dL, the cytoplasmic viscosity increases, and cellular deformability decreases. The rigid cells become trapped in the spleen. Laboratory Evaluation Anemia is usually mild to moderate with a hemoglobin con- centration of 8–10 g/dL. Bilirubin is increased and reticulo- Figure 17.8 This peripheral blood picture from a patient with cytosis is moderate. The MCHC of the stomatocytes seen in hereditary overhydrated stomatocytosis reveals erythrocytes with OHS is decreased, and the MCV can be increased. The blood slitlike or mouthlike 1stoma = mouth2 areas of pallor (peripheral smear is remarkable for 10–50% stomatocytes. Osmotic fra- blood, Wright-Giemsa stain, 1000* magnification). gility and autohemolysis are increased, and autohemolysis is partially corrected with glucose and ATP. Pathophysiology Target cells and erythrocytes that have hemoglobin Specific membrane defects for OHS and DHS have recently puddled at the periphery are observed in DHS. The cells been identified. Patients with OHS demonstrate a defi- demonstrate a slightly increased MCV, increased MCHC, ciency of stomatin (band 7.2b), an integral red cell mem- and decreased osmotic fragility. brane protein. The role of stomatin in cation permeability has not been established.9 Abnormalities of erythrocyte lip- Checkpoint 17.5 ids have been demonstrated to induce stomatocytosis with Explain why the osmotic fragility is decreased in DHS. impaired sodium transport. However, stomatocytosis also occurs when membrane lipids are normal. In these cases, membrane proteins may be abnormal. Although membrane Therapy proteins are usually electrophoretically normal, their con- Splenectomy is not a required therapeutic measure for OHS formation may be altered. and DHS. In fact, this procedure is usually contraindicated From the variability of clinical findings, labora- because some patients have experienced catastrophic tory results, and response to splenectomy, these disor- thrombotic episodes after splenectomy.9 Most patients are ders appear to be caused by several different membrane able to maintain an adequate hemoglobin level without this defects. Some patients have marked stomatocytosis but no procedure. abnormal sodium transport and, thus, no overt hemolysis. Rh null disease (a disorder associated with the lack of all Rh antigens on erythrocytes) also is associated with the Checkpoint 17.6 presence of stomatocytes. Rh proteins are normally nonco- Determine the type of erythrocyte membrane disorder pres- valently linked with Rh-associated glycoproteins (RhAG) ent based on the following lab results: reticulocyte count: 4%; and together they form the Rh–RhAG complex. A lack of osmotic fragility: increased; autohemolysis: increased; bilirubin: this complex alters the cation regulation of the erythrocyte. increased; peripheral smear: 30% stomatocytes present. Molecular defects associated with OHS include mutations in SLC4A1, RHAG, and GLUT1 genes.9,18,19 Mutations in the PIEZ01 protein have been linked to DHS.20,21 All of these mutations play a role in the disturbances of erythrocyte vol- Abnormal Membrane ume homeostasis. The stomatocytic cells in OHS are osmotically fragile Lipid Composition: and less deformable than normal cells, and, as a result, the cells are sequestered in the spleen where glucose supplies Acanthocytosis are readily exhausted. As the ATP levels fall, the cation Acanthocytosis is most often associated with acquired pump is unable to maintain osmotic equilibrium, causing or inherited abnormalities of the membrane lipids. This cell lysis or phagocytosis. occurs in liver disease and in a rare inherited condition, OHS must be differentiated from acquired stomatocy- abetalipoproteinemia. Although the mature erythrocyte has tosis. Stomatocytes are seen as an acquired defect in acute no capacity for de novo synthesis of lipids or proteins, the 376 Chapter 17 lipids of the membrane continually exchange with plasma and keratocytes. Spherocytes and echinocytes can also be lipids. Thus, erythrocytes can acquire excess lipids when found. An increase in unconjugated bilirubin and liver the concentration of plasma lipids increases. Excess mem- enzymes and a decrease in serum albumin reflect evidence brane lipids expand the membrane surface area and cause of liver disease. the cell to acquire abnormal shapes, including target cells

Studies with transfused cells have shown that spur (codocytes), leptocytes, or acanthocytes. If portions of cells acquire their shape as innocent bystanders and that the membrane are lost because of “grooming” of excess when normal cells are transfused into the patient, they lipids in the spleen or if the membrane’s lipid viscosity acquire the abnormal shape and are hemolyzed at the increases, the cells lose their ability to deform and are same rate as the patient’s cells. This suggests that the dis- sequestered in the spleen. On the other hand, a signifi- eased liver and associated plasma lipid abnormalities are cant decrease in one or more lipids in the cell membrane responsible for the transformation of normal red cells into can also lead to increased destruction. The rare hereditary spur cells.22 forms of acanthocytosis also can result from abnormal Biliary obstruction is often associated with the pres- proteins. ence of normocytic or slightly macrocytic target cells that result from an acquired excess of lipids in the cell mem- Spur Cell Anemia brane. However, in contrast to the acanthocytes found in severe hepatocellular disease, the excess lipid in target cells Spur cell anemia is an acquired hemolytic condition asso- associated with biliary obstruction includes an increase in ciated with severe hepatocellular disease such as cirrhosis both cholesterol and phospholipids in a ratio similar to that in which serum lipoproteins increase, leading to an excess of normal cells. As a result, lipid viscosity and membrane of erythrocyte membrane cholesterol. The total phospho- deformability are normal. These target cells have a normal lipid content of the membrane, however, is normal. As the survival. membrane ratio of cholesterol to phospholipid increases, the cell becomes flattened (leptocyte) with a scalloped edge. The increased cholesterol-to-phospholipid ratio also causes Abetalipoproteinemia (Hereditary a decrease in membrane fluidity and an associated decrease Acanthocytosis) in cell deformability. During repeated splenic passage or Abetalipoproteinemia is a rare autosomal recessive dis- conditioning, membrane fragments are lost, and the cell order characterized by the absence of serum b@lipoprotein, acquires irregular spikelike projections typical of acan- low serum cholesterol, low triglyceride, and low phospho- thocytes (spur cells). Eventually, the cell is hemolyzed. In lipid and an increase in the ratio of cholesterol to phospho- patients with cirrhosis, this hemolysis is enhanced by con- lipid. The primary abnormality is the defective processing gestive splenomegaly. and secretion of apolipoprotein B, a microsomal triglyc- The peripheral blood shows a moderate to severe nor- eride transfer protein. This defect is due to mutations in mocytic, normochromic anemia with a hemoglobin concen- the microsomal triglyceride transfer protein (MTP) gene, tration of 5–10 g/dL. Reticulocytes are increased to 5–15%. which encodes for the MTP protein.23,24 Acanthocytes are Approximately 20–80% of the erythrocytes are acanthocytes typically found, but hemolysis is minimal with little or (Figure 17-9). On the peripheral blood smear, acantho- no anemia. Reticulocytes are usually normal but can be cytes must be distinguished from echinocytes (burr cells) slightly increased. The acanthocytes have normal cho- lesterol levels, but lecithin is decreased and sphingomy- elin is increased. This is in contrast to spur cells found in severe liver disease that have increased membrane cholesterol. Membrane fluidity is decreased, presumably because of the increase in sphingomyelin, which is less fluid than other phospholipids. The degree of distortion 1 3 of erythrocytes increases with cell age. The acanthocytes have normal membrane permeability, normal glucose metabolism, and normal osmotic fragility. Autohemoly- sis at 48 hours is increased and only partially corrected 2 by glucose. In addition to hypolipidemia, the disorder is characterized by steatorrhea, retinitis pigmentosa, and Figure 17.9 neurological abnormalities. Transport of fat-soluble vita- Spur cell anemia in a patient with alcoholic cirrhosis. Note the echinocytes (1) and acanthocytes (spur cells; 2). mins (A, D, E, K) is impaired, and the prothrombin time Spherocytes (3) are also present (peripheral blood, Wright-Giemsa can be increased because of decreased vitamin K stores stain, 1000* magnification). (Chapters 32 and 36). Hemolytic Anemia: Membrane Defects 377 Lecithin-Cholesterol Acyl Transferase abnormal clone of differentiated hematopoietic cells. The (LCAT) Deficiency abnormal stem cell clone produces erythrocytes, platelets, and neutrophils that bind abnormally large amounts of This rare autosomal recessive disorder affects metabolism complement and that are abnormally sensitive to comple- of high-density lipoproteins. Onset is usually during young ment lysis (Chapter 19). Complement is composed of at adulthood. The disorder is characterized by a deficiency of least 20 proteins responsible for a variety of biologic activi- LCAT, the enzyme that catalyzes the formation of choles- ties. These proteins are abbreviated as C1 through C9. They terol esters from cholesterol. As a result, patients with this normally circulate in an inactive form and are activated as deficiency demonstrate low levels of high-density lipopro- part of the body’s defense system (Chapter 7). If activated tein (HDL), serum apolipoprotein AI and AII, and elevated complement components attach to the erythrocyte mem- levels of apo E.25 Because erythrocyte membrane choles- brane, the cell can be hemolyzed. terol is in a relatively rapid equilibrium with unesterified The susceptibility of PNH cells to complement-induced plasma cholesterol, the activity of LCAT indirectly regulates lysis is related to deficient regulation of complement activa- the amount of free cholesterol in the cell. The most charac- tion. At least two regulatory proteins, decay-accelerating teristic hematologic findings include a mild hemolytic ane- factor (DAF) (CD55) and membrane inhibitor of reactive mia marked by the presence of numerous macrocodocytes lysis (MIRL) (CD59), found on normal cell membranes are (target cells). The target cells are loaded with cholesterol. responsible for preventing amplification of complement LCAT activity decreases in most patients with spur cells and activation. DAF is a complement regulatory protein that target cells, but the relationship to lipid abnormalities of the enhances the decay of C3 convertase, accelerates decay (dis- membrane is not known. sociation) of membrane-bound C3bBb, and thus prevents amplification of C3 convertase and activation of other com- Rare Forms plement components. The major role of MIRL is to prevent the interaction between C8 and C9; thus, it interferes with The rare forms of acanthocytosis associated with abnormali- the formation of the membrane attack complex (MAC) (the ties of membrane proteins include the McLeod phenotype final steps of complement activation). These regulatory fac- with a deficiency of Kx protein and the Kell system anti- tors help normal cells avoid lysis by autologous comple- gens, chorea-acanthocytosis syndrome, and acanthocytosis ment. Lack of DAF and MIRL on PNH cells results in the with band 3 protein abnormalities. excessive sensitivity of these cells to complement.26 Deficiency of DAF and MIRL in PNH is not due to Checkpoint 17.7 the lack of production of these proteins but to the absence Compare the membrane lipid abnormalities seen in LCAT defi- of a membrane glycolipid that serves as an anchor that ciency, spur cell anemia, and abetalipoproteinemia, and explain attaches these proteins to the cell membrane. Other proteins how they result in hemolysis of the cell. anchored to the cell membrane in a similar manner also are deficient on hematopoietic cells in PNH. The common link in the deficiencies of these membrane proteins is the lack Paroxysmal Nocturnal of the glycolipid anchoring structure, glycosyl-phosphatidyl inositol (GPI). GPI embedded in the cell membrane is Hemoglobinuria (PNH) important for the covalent linkage of a wide variety of pro- All erythrocyte membrane disorders discussed so far are teins to the cell membrane. These GPI-linked proteins vary hereditary except spur cell anemia. Paroxysmal noctur- in structure and function, and include adhesion molecules, hydrolases, and receptors.27 nal hemoglobinuria (PNH) is a rare acquired disorder of the erythrocyte membrane. The membrane defect makes The GPI-anchoring deficiency is because of a somatic the cell abnormally sensitive to lysis by complement. The mutation of the PIGA gene, which encodes the enzyme disease derives its name from the classic pattern of inter- phosphatidyl inositol glycan Class A, essential for synthesis mittent bouts of intravascular hemolysis and nocturnal of the GPI anchor. A large number of PIGA mutations have hemoglobinuria. The condition is exacerbated during sleep been identified. Analysis of bone marrow cells indicates that and remits during the day. However, many patients have the mutation occurs in the hematopoietic stem cell (HSC). chronic hemolysis that is not associated with sleep and with Although not understood, the mutated HSC has a prolif- no obvious hemoglobinuria. erative advantage. All blood cell lineages derived from the mutant HSC are deficient in DAF activity, confirming PNH Pathophysiology as a clonal disorder of hematopoiesis. Thus, many patients with PNH are not only anemic but also granulocytopenic An intrinsic erythrocyte disorder, PNH results from an and thrombocytopenic. The abnormal clone can appear acquired stem cell somatic mutation that leads to an after damage to the marrow or spontaneously (idiopathic). 378 Chapter 17 A significant number of patients with PNH have or eventu- acetylcholinesterase decrease (Chapter 23). Although hemo- ally develop another clonal blood disorder such as acute globinuria can be intermittent or even mild, hemosiderin- myeloid leukemia (Chapter 26) or a myelodysplastic syn- uria is a constant finding, indicating chronic intravascular drome (Chapter 25).28 hemolysis. The increased sensitivity of PNH cells to complement The bone marrow usually exhibits normoblastic hyper- can be demonstrated in vitro by activation of either the plasia but can be hypocellular. In some cases, marrow classic or the alternate pathway of complement activation. failure develops during the course of the disease. Interest- In vivo, however, activation is probably primarily via the ingly, aplastic anemia may be the initial diagnosis with an alternate pathway. abnormal clone of PNH cells developing during the course of the disease (in up to 50% of patients with aplastic ane- Clinical Presentation mia; Chapter 16). PNH rarely precedes aplastic anemia and should be considered in the differential diagnosis when PNH occurs most often in adults but is occasionally hypoplastic anemia is found in association with hemoly- found in children. The three basic disease manifesta- sis.29 Bone marrow iron is decreased or absent. tions are hyperhemolysis, venous thrombosis, and bone The osmotic fragility is normal. Autohemolysis is marrow hypoplasia.27 PNH begins insidiously with increased after 48 hours, and when glucose is added, the irregular brisk episodes of acute intravascular hemolysis hemolysis can increase even more. The DAT is negative accompanied by hemoglobinuria. The patient usually for immunoglobulin (Ig) but can be positive for comple- seeks medical attention when reddish-brown urine is ment given the fact that PNH cells have a propensity to noted. In some patients, the irregular exacerbations of bind C3b. hemolysis are associated with sleep, hence the name The sucrose hemolysis (sugar-water) test is useful in paroxysmal nocturnal hemoglobinuria. In other patients, identifying erythrocytes that are abnormally sensitive to these hemolytic episodes can follow infections, transfu- complement lysis. The patient’s blood is incubated in a sions, vaccinations, surgery, or ingestion of iron salts. In sucrose solution. The sucrose provides a low-ionic-strength a large number of patients, hemolysis is unrelated to any medium that promotes the binding of complement specific event. Iron-deficiency anemia can occur because to the erythrocytes. PNH cells show hemolysis in this of the chronic intravascular hemolysis and urinary loss medium. of iron-containing degradation products. Renal function The Ham (acidified-serum lysis) test is a more spe- can become abnormal as the result of chronic iron deposi- cific test for PNH cells (Chapter 37), but laboratories have tion in the renal tubules. Folic acid deficiency can occur replaced it with immunophenotyping for confirmation of because of increased demand for this nutrient. Abdominal PNH (Chapter 40). Immunophenotyping uses monoclonal and lower back pain, eye pain, and headaches can occur antibodies directed against the GPI anchored molecules. during hemolytic episodes. When the GPI link is missing, these molecules are also miss- In spite of the moderate thrombocytopenia, venous ing. This technology can detect three types of cells in PNH thrombosis is a prominent and severe complication and is a related to the degree of deficiency of GPI-linked proteins common cause of death. Thrombotic events may be related on cell membranes: to abnormal platelet or neutrophil function because of the lack of GPI-anchored proteins. • Type I Little or no hemolysis by complement; nearly When leukopenia is present, infections are common. normal GPI-linked protein expression Propensity for infection also can be related to the absence of • Type II Moderately sensitive to complement lysis; granulocyte glycoproteins and altered functional responses intermediate

GPI-linked protein expression of granulocytes. Immunological abnormalities can be present. • Type III Highly sensitive to complement lysis; no expression of GPI-linked proteins Laboratory Evaluation Because different hematopoietic cells display differ- Most patients with PNH experience anemia with a hemo- ent types of GPI-anchored proteins, it is recommended globin concentration of 8–10 g/dL. The erythrocytes are that at least two different antibodies be used for a diagno- normocytic or macrocytic but can appear microcytic and sis of PNH (CD55, CD59, CD14). CD55 (DAF) and CD59 hypochromic if iron deficiency develops. Reticulocytes are (MIRL) antibodies are used most frequently and are attrac- increased (5–10%) but not to the extent expected for a hemo- tive because they are involved in the complement pathway lytic anemia. Nucleated red blood cells can be found on the that directly leads to the hemolysis in PNH. PNH cells blood smear. Isolated development of leukopenia and/or show low-intensity staining for these molecules. CD55 and thrombocytopenia often occurs during the course of the CD59 are both normally found on all hematopoietic cells. disease. Neutrophil alkaline phosphatase and erythrocyte CD14 is normally on monocytes, and some laboratories Hemolytic Anemia: Membrane Defects 379 use it to verify PNH. The analysis of molecules that can be Therapy expressed on the membrane in a GPI-anchored form as well as a transmembrane form is not as useful because it can give Treatment is primarily supportive in the form of transfu- false normal results. sions, antibiotics, and anticoagulants. A humanized mono- The most accurate measurement of the PNH defect clonal antibody against C5 (eculizumab) has been shown to with immunophenotyping is performed using nucleated relieve the hemoglobinuria in PNH patients by inhibiting cells rather than erythrocytes because the patient with the formation of the membrane attack complex (MAC).30 In PNH often receives red blood cell transfusions. In addi- patients with PNH-induced marrow aplasia, bone marrow tion, results on erythrocytes may not be accurate because transplantation may be indicated. PNH type III erythrocytes have a shorter life span than do type I or II, thus underestimating type III erythrocyte concentration. CASE STUDY (continued from page 374) The fluorescent-labeled inactive toxin aerolysin This case study depicts a young man who pre- (FLAER) test is another method used to diagnose PNH.26,27 sented with right-upper-quadrant pain. The CBC This technique utilizes toxin from the bacterium Aeromonas disclosed anemia with a decreased MCV and hydrophila bound to a fluorochrome. The toxin binds directly increased MCHC. The peripheral blood smear to the GPI anchor itself, not to an attached protein, which revealed numerous elliptocytes, teardrop cells, and makes it a direct measurement of GPI-deficient cells. micropoikilocytes. The patient’s erythrocytes dem- onstrated increased osmotic fragility and thermal sensitivity. Checkpoint 17.8 Explain why immunophenotyping with CD14, CD55, and CD59 6. What disorder do the patient’s lab findings is used to establish a diagnosis of PNH. suggest? Summary Intrinsic defects of the erythrocyte such as an abnormal hereditary hemolytic anemias including hereditary sphero- membrane, structurally abnormal hemoglobin, defec- cytosis, hereditary elliptocytosis, and hereditary pyropoi- tive globin synthesis, or deficient erythrocyte enzymes kilocytosis. Abnormally permeable membranes can cause can lead to a shortened erythrocyte life span because of hereditary overhydrated and dehydrated stomatocytosis. increased hemolysis. An anemia will result if the bone Abnormalities in the lipid portion of the membrane can be marrow is unable to compensate for the erythrocyte loss. inherited or acquired. These disorders result in the forma- These intrinsic abnormalities are almost always inherited tion of acanthocytes. defects. Paroxysmal nocturnal hemoglobinuria (PNH), a Erythrocyte membrane defects can be caused by abnor- stem cell disorder, is an acquired membrane abnormal- malities of membrane proteins or lipids, which can affect ity in which complement-mediated destruction of the cell cell deformability, stability, shape, and/or permeability. occurs. The defect is due to a lack of decay-accelerating fac- The abnormal erythrocytes become trapped in splenic tor (DAF) and membrane inhibitor of reactive lysis (MIRL) cords and are removed from the circulation. Interactions on the erythrocytes, secondary to the absence of a mem- between the skeletal lattice proteins and integral proteins brane glycolipid, glycosyl-phosphatidyl inositol (GPI) to and lipids of the membrane are vertical interactions that anchor these proteins to the cell membrane. Hemolysis is when disrupted cause a reduced density of spectrin and intravascular, resulting in hemoglobinuria and decreased uncoupling of the lipid bilayer. This causes selective loss haptoglobin. Leukopenia and thrombocytopenia are com- of the lipid bilayer and formation of spherocytes. Skeletal mon because these cells also are susceptible to complement lattice proteins interact horizontally to form the stress-sup- destruction. The sucrose hemolysis test is a screening test porting skeletal protein lattice. Defects in these interactions for PNH. Although the Ham test was used in the past, result in mechanical instability and fragmentation of the immunophenotyping and the FLAER test are now the stan- cell. Inherited defects in membrane proteins produce the dard technologies for confirming a diagnosis of PNH. 380 Chapter 17 Review Questions Level I 7. The red blood cells in paroxysmal nocturnal hemoglo- binuria (PNH) demonstrate a(n) ___________________ 1. What is the most prevalent erythrocyte morphology osmotic fragility test. (Objectives 4, 5) observed in hereditary spherocytosis? (Objectives 1, 3) a. decreased a. Small, spherical erythrocytes with little or no cen- b. increased tral pallor c. normal b. Spheroidal erythrocytes with sharp irregular d. unpredictable projections 8. Which of the following statements describes the basic c. Fragmented erythrocytes principle behind the sucrose hemolysis test? This test d. Oval-shaped erythrocytes determines whether: (Objective 6) 2. What erythrocyte membrane disorder has a. a patient’s erythrocytes incubated in their own erythrocytes that are thermally unstable and serum will demonstrate increased hemolysis. fragment when heated to 45–46°C? (Objectives 1, 3) b. a patient’s erythrocytes are sensitive to comple- a. Hereditary spherocytosis ment lysis when exposed to acidified serum. b. Hereditary elliptocytosis c. the patient’s erythrocytes incubated in a low ionic medium such as sucrose will demonstrate c. Hereditary pyropoikilocytosis increased hemolysis. d. PNH d. the patient’s erythrocytes will resist acid elution. 3. Choose the principal screening test for a diagnosis of 9. The laboratory features associated with asymptomatic hereditary spherocytosis. (Objective 3) common hereditary elliptocytosis include: a. Autohemolysis test (Objective 3) b. Sucrose hemolysis test a. fragmented erythrocytes on the peripheral smear c. Thermal stability test b. increased osmotic fragility d. Osmotic fragility test c. positive autohemolysis test 4. Erythrocyte membrane disorders associated with d. mild reticulocytosis known skeletal protein defects include all of the 10. The osmotic fragility test determines whether a following except: (Objective 1) patient’s erythrocytes are osmotically fragile by a. hereditary spherocytosis measuring the amount of hemolysis that occurs: b. hereditary overhydrated stomatocytosis (Objective 4) c. hereditary elliptocytosis a. after a patient’s erythrocytes have been incubated d. hereditary pyropoikilocytosis for 24 hours in acidified serum b. when a patient’s erythrocytes are incubated in 5. Which of the following erythrocyte disorders is various concentrations of hypotonic saline associated with abnormal membrane permeability? (Objective 2) c. when the patient’s erythrocytes are incubated in a sucrose solution a. Hereditary elliptocytosis d. hemolysis when the patient’s erythrocytes are b. Hereditary dehydrated stomatocytosis incubated in their own serum for 48 hours c. PNH Level II d. HPP 1. Which of the following is the red blood cell mem- 6. Laboratory features associated with hereditary brane protein defect associated with hereditary pyro- spherocytosis include: (Objective 3) poikilocytosis? (Objective 1) a. spherocytes on the peripheral smear a. Deficiency of band 3 b. MCHC more than 36% b. Defective ankyrin protein c. increased osmotic fragility c. Mutant spectrin d. all of the above d. Excess cholesterol Hemolytic Anemia: Membrane Defects 381 2. Which of the following erythrocyte disorders will c. CD33 and CD34 demonstrate an increased osmotic fragility pattern? d. CD56 and CD10 (Objective 2) 6. The RBC membrane permeability in hereditary over- a. Hereditary elliptocytosis and paroxysmal hydrated stomatocytosis is ___, and the cells have a(n) nocturnal hemoglobinuria ___________________ osmotic fragility. (Objective 2) b. Hereditary overhydrated stomatocytosis and hereditary spherocytosis a. increased; decreased c. Paroxysmal nocturnal hemoglobinuria and b. decreased; increased hereditary xerocytosis c. increased; increased d. Sickle cell anemia and thalassemia d. decreased; decreased 7. The following results were obtained on an osmotic Use this case study to answer questions 3 and 4. fragility test. A 5-year-old white male was admitted with the Normal Control Patient diagnosis of a fractured tibia following a playground Initial hemolysis 0.50% 0.65% NaCl accident. His admission laboratory results follow: Complete hemolysis 0.30% 0.45% NaCl WBC 125 * 109>L Differential These results most closely relate to which of the RBC 3.6 * 1012>L Segmented following statements? (Objective 2) Hb 10.2 g/dL neutrophils 70% a. The patient’s peripheral smear will reveal Hct 27% Lymphocytes 22% spherocytes. MCV 96.3 fL Monocytes 5% b. The patient’s peripheral smear will reveal target cells. MCH 28.3 pg Eosinophils 2% c. The patient’s peripheral smear will reveal sickle MCHC 38 g/dL Basophils 1% cells. RBC morphology: Slight polychromasia and sphe- d. The test is out of control and should be repeated. rocytes present 8. A decrease in the level of which erythrocyte enzyme Osmotic fragility test: Initial hemolysis: 0.65% NaCl occurs in PNH? (Objective 3) Complete hemolysis: 0.45% NaCl a. Leukocyte alkaline phosphatase (LAP) b. Acid phosphatase c. C5 convertase 3. Which erythrocyte index differentiates this membrane d. Acetylcholinesterase disorder from most of the other e rythrocyte membrane disorders discussed in this chapter? 9. Which of the following statements best describes (Objectives 2, 5) the role of the decay-accelerating factor (DAF)? (Objective 3) a. MCV b. MCH a. 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American Journal of Hematology, 91(4), nectomy for hereditary spherocytosis: A multi-institutional 366–370. doi: 10.1002/ajh.24278 Chapter 18 Hemolytic Anemia: Enzyme Deficiencies Jean Sparks PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Identify the two main pathways by which 5. Explain how the diagnosis of G6PD defi- erythrocytes catabolize glucose. ciency is made. 2. Explain the role of erythrocyte enzymes in 6. List the tests used to detect G6PD deficiency maintaining the cell’s integrity, and describe and describe their principles. how deficiencies in these enzymes can lead 7. Recognize the erythrocyte morphology in a to anemia. Romanowsky-stained blood smear associ- 3. Identify the most common erythrocyte ated with G6PD deficiency. enzyme deficiency. 8. Identify common compounds that induce 4. Describe the inheritance pattern for glucose- anemia in G6PD deficiency. 6-phosphate dehydrogenase (G6PD). Objectives—Level II At the end of this unit of study, the student should be able to: 1. Recommend appropriate laboratory testing 4. Correlate clinical and laboratory findings and interpret results for suspected G6PD with the common G6PD variants. deficiency following a hemolytic episode. 5. Diagram the reaction catalyzed by pyruvate 2. Explain the function of glutathione in main- kinase, and explain how a defect of this taining cellular integrity. enzyme can cause hemolysis. 3. Associate the mechanisms of hemolysis with 6. Recognize erythrocyte morphology associ- defects in the glycolytic and hexose mono- ated with pyruvate kinase deficiency. phosphate shunt pathways. 7. Review and interpret laboratory findings in a case study presentation of G6PD deficiency. 383 384 Chapter 18 Chapter Outline Objectives—Level I and Level II 383 Pyruvate Kinase (PK) Deficiency 393 Key Terms 384 Other Enzyme Deficiencies in the Glycolytic Background Basics 384 Pathway 394 Case Study 384 Abnormal Erythrocyte Nucleotide Metabolism 395 Overview 385 Summary 395 Introduction 385 Review Questions 396 Glucose-6-Phosphate Dehydrogenase Deficiency 387 References 397 Other Defects and Deficiencies of the HMP Shunt and GSH Metabolism 393 Key Terms Bite cell Chronic nonspherocytic hemolytic anemia Hypoxia Blister cell Favism Lyonization Background Basics The information in this chapter builds on concepts learned • Recognize RBC morphology as it relates to hemolytic in previous chapters. To maximize your learning experi- disease processes. (Chapters 10, 11) ence, you should review the following concepts from pre- • Understand basic enzymology and techniques to vious chapters and other resources: measure enzyme activity. (Other resources) Level I and Level II • Describe the normal erythrocyte metabolic pro- cesses. (Chapter 5) CASE STUDY CBC We refer to this case study throughout the chapter. WBC 12 * 103>mcL The patient, Henry, is a 20-year-old African Ameri- Hb 9.1 g/dL (91 g/L) can male who was in the process of being assigned Hct 27% (0.27 L/L) to West Africa for a 12-month period with the MCV 90 fL Peace Corps. Because of the high prevalence of PLT 423 * 103>mcL malaria in the area, he was started on antimalarial prophylaxis (primaquine) 3 days prior to his flight Total bilirubin 5.0 mg/dL to Africa. Twenty-four hours after starting the Conjugated bilirubin 0.2 mg/dL medication, he developed fever, chills, and general Unconjugated 4.8 mg/dL malaise. He subsequently reported to the emer- bilirubin gency department and was admitted to the hospi- Haptoglobin 39 mg/Dl tal for observation and additional testing. The physical exam revealed a normal appear- Based on the clinical history and these laboratory ing, well-nourished adult male in no acute distress. results, consider what could have precipitated this His family history was noncontributory, and patient’s condition. he had no known drug allergies. Laboratory analy- sis yielded the following: Hemolytic Anemia: Enzyme Deficiencies 385 Overview Table 18.1 Erythrocyte Enzyme Deficiencies Associated Defects within the erythrocyte can shorten the red cell life with Congenital (Chronic) Nonspherocytic Hemolytic Anemia span, resulting in hemolytic anemia. These defects are intrinsic Metabolic Pathway Enzyme Deficiency to the cell and can involve the erythrocyte membrane (Chap- Hexose monophosphate Glucose-6-phosphate dehydroge- ter 17), hemoglobin (Chapter 13), or enzymes. The two most nase (G6PD) common enzyme defects within the erythrocyte are glucose- Glutathione synthetase 6-phosphate dehydrogenase (G6PD) and pyruvate kinase Glutamylcysteine synthetase (PK). Of these two enzyme deficiencies, G6PD defects are Glutathione reductase found more frequently and affect the hexose monophosphate Glycolytic (Embden-Meyerhof) Pyruvate kinase (PK) shunt. PK defects are the second most common enzyme defi- Glucose phosphate isomerase ciency and affect the glycolytic pathway. Thus, the chapter (GPI) begins with a description of the role of these two pathways in Hexokinase (HK) erythrocyte metabolism and a general overview of the clini- Phosphoglycerokinase (PGK) cal and laboratory findings in enzyme deficiencies associated Phosphofructokinase (PFK) with these pathways. G6PD and PK enzyme deficiencies are Triosephosphate isomerase (TPI) discussed in detail in the format of pathophysiology, clinical Nucleotide Pyrimidine 5′@nucleotidase (P5N) and laboratory findings, and therapy. Introduction and is catabolized via the glycolytic pathway or the hex- ose monophosphate (HMP) shunt. The HMP shunt catab- Even though the mature erythrocyte lacks a nucleus, mito- olizes approximately 10% of the glucose, and is essential chondria and ribosomes, the cell is still metabolically active for maintaining adequate concentrations of reduced gluta- (Chapter 5). Metabolism provides energy to keep hemo- thione (GSH). GSH protects the erythrocyte from oxidant globin iron in the reduced state, pump ions across the cell damage: it maintains hemoglobin in the reduced functional membrane, keep the sulfhydryl groups in enzymes and state and safeguards vital cellular enzymes from oxida- other proteins in the reduced state, and maintain the cell’s tion. GSH is maintained at high levels by conversion of shape. These activities are essential for red cell survival. The NADPH to NADP; NADP is reduced back to NADPH in erythrocyte life span can be significantly shortened if the a reaction catalyzed by glucose-6-phosphate dehydroge- cell is intrinsically abnormal. Lacking the ability to synthe- nase (G6PD) (Figure 18-1). When the cell is exposed to an size proteins and undergo oxidative phosphorylation for oxidizing agent, more NADPH is consumed as part of the ATP production, the mature erythrocyte depends entirely cell’s protective mechanism to maintain sufficient levels of on anaerobic metabolism for its energy needs. Maturation GSH, and G6PD is required to restore levels of NADPH. of the reticulocyte is associated with a decline in the activity Oxidized GSH (GSSG) is also actively transported out of of some enzymes, but the activity usually is relatively stable the erythrocyte. If enzymes in the HMP shunt are deficient, in the mature erythrocyte.1 Because the erythrocyte cannot the cell’s reducing power is compromised, and oxidized synthesize new enzymes, the amount of enzyme present for hemoglobin accumulates, subsequently denaturing in the normal cell function is limited. form of Heinz bodies. Heinz bodies aggregate at the cell An inherited deficiency in one of the erythrocyte membrane, causing membrane damage. As the cells pass enzymes can disrupt cellular metabolism and compromise through the spleen, the macrophages attempt to remove the the integrity of the cell membrane or hemoglobin and cause Heinz bodies, leading to premature extravascular hemoly- hemolysis. Refer to Table 18-1 for the more common heredi- sis. The most common enzyme deficiency of the HMP shunt tary enzyme deficiencies known to cause hemolytic anemia. is G6PD deficiency. Most are associated with two erythrocyte metabolic path- ways: the hexose monophosphate (HMP) shunt and the gly- Glycolytic Pathway colytic (Embden-Meyerhof) pathway. To understand how Most of the cell’s energy is produced via glycolysis in the defects in these two pathways can result in hemolysis, they glycolytic pathway. About 90% of the glucose is metabo- will be reviewed. For a more thorough discussion of each, lized by this pathway as one mole of glucose is catabolized review Chapter 5. to lactic acid with a net production of two moles of ATP. Hexose Monophosphate Shunt ATP is needed for active cation transport of Na+, K+, and Ca++, maintaining membrane deformability and the normal Glucose, the cell’s primary metabolic substrate, enters the erythrocyte biconcave disc shape.1 The erythrocyte’s abil- cell in a carrier-mediated, energy-free transport process ity to deform is an important determinant of its survival. 386 Chapter 18 Oxidant Stress H2O2 H2O Glutathione peroxidase GSH GSSG Glutathione reductase Glucose Hexokinase NADP NADPH Glucose-6-phosphate 6-phosphogluconate G6PD Glycolytic Pathway 2 ATP 2 Pyruvate Figure 18.1 G6PD is needed for maintaining adequate quantities of glutathione (GSH), an important buffer to oxidants within the erythro- cyte. As GSH reduces H2O2 to H2O, it is oxidized (GSSG). G6PD generates NADPH in the conversion of glucose-6-phosphate to 6-phosphoglu- conate. NADPH, in turn, regenerates

reduced glutathione from oxidized glutathione. Deficiencies in enzymes of the glycolytic pathway decrease hemolysis/anemia, to intermittent hemolysis, to a chronic ATP production and lead to hemolysis. The mechanism of hemolytic state. Affected individuals generally have a nor- hemolysis is related to decreased ATP, leading to impaired mocytic, normochromic anemia, reticulocytosis, hyperbili- cation pumping. Membrane integrity is compromised and rubinemia, and neonatal jaundice. The direct antiglobulin increased osmotic fragility results. The osmotically fragile test (DAT) is negative, indicating an absence of antibodies cells are trapped in the hostile splenic environment and coating the erythrocytes, and there is no evidence sug- phagocytized. Heinz bodies are not formed because the gesting a defect in either the erythrocyte membrane or cell’s reducing power is primarily linked to the HMP shunt, hemoglobin. which is not affected. The anemias associated with inherited defects of eryth- The Rapoport-Luebering shunt of the glycolytic path- rocyte metabolism can be acute or chronic, depending on way provides the erythrocyte with 2,3-bisphosphoglycer- the mutant enzyme inherited. Some mutations cause low ate (2,3-BPG). When 1,3-BPG is shunted to 2,3-BPG instead enzyme activity or instability, whereas other mutations are of directly to 3-PG, a critical reaction producing ATP is associated with enzymes that have moderately to mildly bypassed and there is no net gain of ATP from glycolysis. decreased activity. Some mild types are associated with The activity of this shunt is stimulated during hypoxia to hemolysis only under stressful conditions such as admin- facilitate oxygen delivery to the tissues. 2,3-BPG binds to istration of oxidant drugs, whereas severely deficient or hemoglobin, decreasing hemoglobin’s oxygen affinity and unstable types cause chronic hemolysis in the absence of making more oxygen available to the tissues. stress. The chronic hemolytic types are often collectively referred to as hereditary nonspherocytic hemolytic anemia. Clinical and Laboratory Evaluation Crosby introduced this term in 1950 to describe hemolytic in Erythrocyte Enzyme Deficiencies anemia that appeared at an early age but in whom test- ing revealed a normal osmotic fragility,1 and it was used Most erythrocyte enzyme deficiencies are inherited as to differentiate a hereditary hemolytic anemia that was autosomal recessive traits. However, the most common not hereditary spherocytosis. This heterogeneous group of enzyme deficiency, G6PD, is inherited as an X-linked anemias does not have significant poikilocytosis. Although recessive (sex-linked) disorder. Individuals homozygous the anemias might not be life threatening, they can be dis- for the autosomal recessive enzyme deficiencies and males abling and lead to debilitating complications. Although with X-linked G6PD deficiency are most likely to be symp- anemias other than those caused by enzyme mutations can tomatic. The clinical presentation is variable, depending also be chronic, hemolytic, and nonspherocytic, they are not on the specific mutation inherited and can range from no included in this group (e.g., sickle cell disease, thalassemia). Hemolytic Anemia: Enzyme Deficiencies 387 Diagnosis earlier stages of parasite maturation (ring stage) may be more readily phagocytized.6,7 The exact mechanism of this Definitive diagnosis of enzyme deficiencies requires spec- hypothesized protection is unknown, however, and epide- trophotometric measurement of the suspected deficient miological and clinical studies have not consistently shown enzyme and/or molecular testing for the specific gene. The a clear connection between protection against malaria and timing of enzymatic testing and interpretation of the results G6PD deficiency.8,9,10 Nevertheless, the geographic coinci- is important for accurate diagnosis. With some enzyme defi- dence is compelling. ciencies, enzyme activity is nearly normal in reticulocytes but decreases as the cell ages (e.g., G6PD@A- described in “G6PD Variants,” below). Thus, depending on the age dis- Etiology tribution of the circulating cells and the degree of reticulo- G6PD deficiency is a sex-linked recessive disorder (the cytosis, the enzyme content may appear normal. For this cytogenetic location of the gene, G6PD, is Xq28). It is fully reason, the patient’s blood should not be collected for test- expressed in males with a genetic mutation and in females ing immediately after a hemolytic episode when most of the homozygous or double heterozygous for a mutant allele. enzyme-deficient cells have been hemolyzed and there is a More than 400 G6PD variants have been identified.1 compensatory reticulocytosis. Diagnosis during or shortly G6PD deficiency is heterogeneous with differences in after a hemolytic episode can be made if the blood is centri- severity among races, sexes, and the mutant variant inher- fuged and the older dense erythrocytes at the bottom of the ited. The majority of people with a G6PD variant allele have column of blood are tested.2,3 If a patient has been recently no clinical expression of the deficiency unless they are transfused, testing should be delayed until the transfused exposed to oxidative chemicals or drugs, or have severe cells are no longer present.1 infections. See Table 18-2 for compounds that have been associated with precipitating hemolytic anemia in G6PD deficiency. It is now considered good practice to test for Checkpoint 18.1 G6PD deficiency before giving drugs known to cause hemo- Transfusion of red blood cells in a patient with chronic nons- lysis, especially in populations in whom G6PD deficiency is pherocytic, hemolytic anemia as the result of an erythrocyte common. This may become a public health issue as more enzyme deficiency does not reverse or prevent the recipi- countries are attempting to eliminate malaria in a process ent’s condition. However, a transfusion does help to raise the that may involve mass administration of primaquine.11 patient’s hemoglobin. If tests are performed to quantitate the enzyme after a transfusion, the results can be within the normal reference intervals. Explain. Checkpoint 18.2 Oxidant compounds are harmful because they result in the pro- duction of toxic peroxides or other oxygen radicals that over- whelm the body’s natural mechanisms to scavenge them. Why Glucose-6-Phosphate is the protection against oxidants easily compromised in G6PD deficiency? Dehydrogenase Deficiency G6PD deficiency is the most common erythrocyte enzyme disorder. It was first recognized during the Korean War Pathophysiology when 10% of African American soldiers who were given G6PD is necessary for maintaining adequate levels of GSH the antimalarial drug primaquine developed a self-limited for reducing cellular oxidants (Figure 18-1). In G6PD defi- hemolytic anemia. Currently, the U.S. Army, Navy, Air ciency, the generation of NADPH, and subsequently GSH, Force, and Marine Corps screen for G6PD deficiency in all is impaired and cellular oxidants accumulate. The buildup personnel entering the military services and those service of cellular oxidants leads to erythrocyte injury and both members on active duty.4 G6PD deficiency is found world- intravascular and extravascular hemolysis. Oxidants cause wide but occurs most frequently in people from the Medi- the oxidation of free –SH groups in hemoglobin and other terranean area, Africa, and China. intracellular proteins, forming disulfide bridges. Oxidized The geographic distribution coincides with that of hemoglobin has decreased solubility and precipitates as malaria, suggesting that G6PD deficiency may provide pro- Heinz bodies. Heinz bodies attach to the erythrocyte mem- tection against this disease. One mechanism for this protec- brane, causing increased membrane permeability to cations, tion could be oxidant injury to the parasite. Due to the cell’s osmotic fragility, and cell rigidity. Heinz bodies are removed inability to restore NADPH and GSH, the parasite may from the erythrocytes by splenic macrophages, producing be more vulnerable to the reactive intermediates formed bite cells and blister cells. With progressive membrane loss, when the parasite breaks down hemoglobin.5 Evidence spherocytes can be formed (Figure 18-2). Spherocytes are also suggests that G6PD-deficient erythrocytes containing less deformable than normal cells and become trapped and 388 Chapter 18 Table 18.2 Compounds Associated with Hemolysis in G6PD Deficiency Antimalarials Sulfonamides Sulfones Nitrofurans Analgesics Miscellaneous Primaquine Sulfanilamide Thiazosulfone Nitrofurantoin Acetanilid Fava beans (Furadantin) Pamaquine Sulfacetamide Diaphenylsulfone Hydroxylamines (DDS, Dapsone) Pentaquine Sulfapyridine Sulfoxone (Diasone) Methylene blue Quinacrine (Atabrine) Sulfamethoxazole Naphthalene (moth (Gantonol) balls) Sulfasalazine Naxidlic acid (Azulfidine) Niridazole Phenylhydrazine Rasburicase Toluidine blue Trinitrotoluene (TNT) Antimalarials Sulfonamides Sulfones Nitrofurans Analgesics Miscellaneous Primaquine Sulfanilamide Thiazosulfone Nitrofurantoin Acetanilid Fava beans (Furadantin) Pamaquine Sulfacetamide Diaphenylsulfone Hydroxylamines (DDS, Dapsone) Pentaquine Sulfapyridine Sulfoxone (Diasone) Methylene blue Quinacrine (Atabrine) Sulfamethoxazole Naphthalene (moth (Gantonol) balls) Sulfasalazine Naxidlic acid (Azulfidine) Niridazole Phenylhydrazine Rasburicase Toluidine blue Trinitrotoluene (TNT) hemolyzed in the spleen (extravascular hemolysis). Heinz results in removal of the cell from the circulation by splenic bodies require a supravital stain to be visualized because macrophages. Cell membrane damage can occasionally be they are not evident on Romanowsky-stained blood smears. severe enough for the cell to hemolyze in the circulation. Oxidant stress also can oxidize membrane lipids and pro- This intravascular hemolysis can be acute and accompanied teins. This disruption of the membrane structural integrity by hemoglobinemia and hemoglobinuria. G6PD activity is highest in young cells and decreases rapidly as cells age. Reticulocytes have approximately 5 times higher enzyme activity than the oldest circulating erythrocytes. However, normal erythrocytes use only 0.1% of their maximum G6PD enzyme capacity.12 Thus, under normal conditions, even older cells retain enough G6PD activity to maintain adequate GSH levels. This explains why most G6PD-variant cells can maintain normal function and hemolysis is sporadic. When erythrocytes are overwhelmed by excessive oxidant stress, the G6PD activity becomes inad- equate to maintain normal metabolic function in individuals who have inherited a G6PD variant. Those cells that are most deficient (i.e., the oldest) undergo oxidative damage and are Figure 18.2 Peripheral blood from a patient with G6PD rapidly removed from circulation. With most G6PD variants, deficiency during a hemolytic episode. The erythrocytes with a hemolysis is self-limited (e.g., hemolysis stops after a time portion of the cell missing are known as bite cells. The spleen even if the oxidant stress continues). Self-limited hemolysis pits the Heinz bodies with a portion of the cell producing these misshapen erythrocytes. Some of the cells reseal and become occurs because the older, most G6PD-deficient erythrocytes spherocytes (Wright-Giemsa stain, 1000* magnification). initially are destroyed, but the younger cells remain because Hemolytic Anemia: Enzyme Deficiencies 389 they have sufficient enzyme activity to avoid hemolysis. The according to the degree of deficiency and hemolysis13 (Table reticulocytes released from the bone marrow in response to 18-3). Most of the identified variants actually have normal the hemolytic episode also have enough enzyme activity to enzyme activity. Deficient enzyme variants tend to have maintain metabolic activity even under oxidant stress. mutations clustered in domains responsible for stable However, it is important to recognize that under the stress dimerization of the active form of the protein, or the NADP of severe oxidants (drugs, chemicals), even normal cells can binding site.14 Except for G6PD-B, G6PD@A+, and G6PD@A-, experience oxidant damage and hemolysis. variants are given geographic or other types of names. Checkpoint 18.3 Females with G6PD Deficiency Erythrocyte morphology should always be examined carefully. Female heterozygotes for G6PD deficiency always contain The ability to pick up subtle clues regarding the cause of a dis- two populations of cells, one normal and one G6PD deficient. ease process can be acquired from a comprehensive evaluation In contrast, all cells in affected males are G6PD deficient. of abnormal erythrocyte morphology. How is this likely to aid the The dual population in females is caused by lyonization, diagnosis of G6PD deficiency? the random inactivation of one|multi|chromosome in each hematopoietic stem cell. Depending on the proportion of G6PD Variants abnormal erythrocytes and the nature of the inherited vari- ant, females might not have clinical expression of the defi- More than 400 variants of the G6PD enzyme have been ciency or be affected as severely as males. Although rare, identified.1 Many of them differ in activity, stability, and/ there are case reports of homozygous or double heterozy- or electrophoretic mobility. The World Health Organization gous-deficient females.15 Figure 18-3 illustrates the expected (WHO) has categorized the variant enzymes into five classes progeny from G6PD-deficient males or females. Table 18.3 WHO Classification of Mutant G6PD Classes Class G6PD Activity Hemolysis Important Variants I Severely deficient Chronic nonspherocytic hemolytic Minnesota, Iowa anemia, not self-limited II Severely deficient (less than 10%) Acute, episodic, can be chronic and G6PD-Mediterranean, common not self-limited severe oriental variants III Moderately to mildly deficient Acute, episodic G6PD@A-, G6PD-Canton (10–60%) IV Normal (60–150%) Absent G6PD-B, G6PD@A+ V Increased activity Absent X Y X X X Y X X X X X X X Y X Y X X X X X Y X Y Female carrier Female carrier Normal male Normal male Normal female Female carrier Normal male Affected male X chromosome carrying G-6PD deficiency gene Normal X chromosome Figure

18.3 G6PD deficiency is a sex-linked disorder that is carried by a gene on the X chromosome. The disease is fully expressed in males who carry the affected X chromosome. Females who carry one affected X chromosome (female carrier) and one normal X chromosome have two populations of cells, one deficient in G6PD and one with normal G6PD. This happens because of random inactivation of one X chromosome in each cell of the female embryo. The chromosome remains inactive throughout subsequent divisions of the cell. This figure illustrates the expected progeny from G6PD-deficient males or females. 390 Chapter 18 Clinical Presentation Even though favism occurs more often in males due to the X-linked nature of the disease, heterozygous females can A spectrum of clinical presentations is associated with this also be affected depending on the proportion of enzyme- disorder: (1) acute, acquired hemolytic anemia (episodic), deficient erythrocytes present. including favism; (2) hereditary (congenital) nonsphero- cytic hemolytic anemia (chronic); and (3) neonatal hyperbil- HEREDITARY NONSPHEROCYTIC HEMOLYTIC ANEMIA irubinemia with jaundice. Homozygotes and heterozygotes Hereditary nonspherocytic hemolytic anemia syndrome can be symptomatic, depending on the severity of the defi- is associated with G6PD variants (WHO class I) that have ciency. G6PD deficiency is most common in those of Afri- low in vitro activity or are markedly unstable. Hemolysis is can, Asian, Mediterranean, and Middle Eastern heritage. chronic and not associated with ingestion of drugs or infec- tions, although drugs and infections can exacerbate the ACUTE ACQUIRED HEMOLYTIC ANEMIA hemolysis. The hemolysis is usually compensated so ane- Most persons with G6PD deficiency have no clinical symp- mia can be mild. Reticulocytosis is in the range of 4–35%. toms, and they are not anemic. Diagnosis usually occurs This type of G6PD deficiency is often referred to as chronic during or after infectious illnesses or following exposure to nonspherocytic hemolytic anemia. certain drugs because these conditions commonly precipi- Leukocyte and platelet G6PD are controlled by the tate hemolytic episodes. Hemolysis is variable and depends same gene locus as erythrocyte G6PD. Thus, some patients on the degree of oxidant stress, the G6PD variant, and sex of with G6PD-deficient red cells also have leukocyte G6PD the patient. The symptoms are those of an acute intravascu- deficiency. An increase in pyogenic infections has been lar hemolytic anemia (Chapter 11). Drug-induced hemolysis reported in those with less than 5% normal activity.5,19,20,21 usually occurs within 1 to 3 days after exposure to the drug. However, a group of Israeli patients with severe G6PD defi- Sudden anemia develops with a 3–4 g/dL drop in hemoglo- ciency without an increased incidence of infections had bac- bin. Jaundice is not prominent. The patient may experience tericidal activity in their G6PD-deficient neutrophils within abdominal and low back pain, as well as dark or black urine the range of healthy controls.22 Considerable fluctuation of due to hemoglobinuria. In one study of 35 G6PD-deficient enzyme activity in leukocytes was found to depend on the children in India, the most common significant complica- time of day it was measured. This fluctuation can produce tion, occurring in more than 50% of the cases, was renal enough NADPH to initiate the respiratory burst and pre- failure.16 Hemoglobinemia is prominent. Often, however, vent infection. Neutrophils also are relatively short-lived hemolysis is less striking and is not accompanied by hemo- cells, so those with unstable variants might not show func- globinuria or conspicuous symptoms. tional impairment. Favism refers to the sudden severe hemolytic episode that develops in some individuals with G6PD deficiency NEONATAL HYPERBILIRUBINEMIA after the ingestion of fava beans (broad beans). Hemolytic Some neonates with G6PD deficiency have severe hyper- episodes occur in much the same way as drug-induced bilirubinemia with the potential of kernicterus if untreated. hemolytic episodes. The most likely components of the bean The prevalence of this severe jaundice occurs twice as often responsible for the sensitivity are isouramil and divicine.17 in G6PD-deficient male neonates compared with neonates These acute hemolytic episodes were thought to be asso- from the general population. The mechanism is unknown. ciated with severe G6PD deficiency, especially the G6PD- Although hemolysis may be present, other factors, includ- Mediterranean variant. It is now known that other forms of ing inability to conjugate and clear bilirubin, seem to play G6PD deficiency are also associated with favism, including a more important role.23 In one study, G6PD deficiency was G6PD@A- and G6PD-Aures, a variant identified in Algerian identified in at least 21% of infants who were readmitted subjects.18 with kernicterus.24 Treatment can include phototherapy and The hemolysis associated with favism is similar to exchange transfusion. the acute hemolytic episodes that occur after primaquine In an effort to diminish kernicterus associated with administration in individuals with the G6PD@A- variant. G6PD, The WHO Working Group in 1989 recommended Consumption of fava beans is widespread in the Mediter- screening programs in areas with a G6PD deficiency inci- ranean area and the Middle East. The first signs of favism dence in males of greater than 3–5% in combination with are malaise, severe lethargy, nausea, vomiting, abdominal parental education. The effectiveness of the recommenda- pain, chills, tremor, and fever.1 Hemoglobinuria occurs a tions has not been validated but evidence currently avail- few hours after ingestion of the beans. Persistent hemo- able suggests the Working Group recommendations should globinuria usually prompts the individual to seek medical continue to be supported.25 attention. Jaundice can be intense. Severe favism usually Healthy neonates and preterm infants (29–32 weeks’ affects children between the ages of 2 and 5 years. The inci- gestation) have higher G6PD activity than do adults. How- dence in young children is changing in some countries, ever, the higher activity does not affect the diagnosis of however, due to neonatal screening and parental education. G6PD because neonates and preterm infants with G6PD Hemolytic Anemia: Enzyme Deficiencies 391 deficiency have lower activity than do normal neonates.26 episode. In G6PD-Mediterranean, however, even young Neonates who need transfusions should not be given blood cells have gross deficiencies of G6PD, and enzyme activity from G6PD deficient donors because their immature liver appears abnormal even with reticulocytosis. Both severe function makes them less able to metabolize an increased and mild types of G6PD deficiency are detected by measur- bilirubin load.27 ing the enzyme if the patient is not undergoing hemolysis. Laboratory Evaluation CASE STUDY (continued from page 384) Anemia is absent and peripheral blood findings are nor- Because of the low hemoglobin, Henry was trans- mal for most of the common G6PD variants associated with fused with 2 units of packed red cells. Examination episodic disease except during hemolytic episodes. Patients of the peripheral blood smear was remarkable for with the hereditary nonspherocytic hemolytic anemia form occasional spherocytes and bite cells. This blood may exhibit chronic hemolysis. During or immediately fol- smear finding, presentation of anemia, and onset of lowing a severe hemolytic episode, polychromasia, occa- illness coinciding with the initiation of primaquine sional spherocytes, small hypochromic cells, erythrocyte suggested shortened RBC life span due to oxida- fragments, blister cells, and bite cells may be seen on the tive damage. blood smear. Bite cells (degmacytes) have a chunk of the cell removed from one side and are thought to be formed 1. What test should be considered after finding bite when phagocytes in the spleen remove the denatured hemo- cells on a blood smear? globin (Heinz bodies) bound to the cell membrane. They are frequently thought to be typical of G6PD deficiency (Figure 18-2). However, bite cells are more characteristic of drug-induced oxidant hemolysis in individuals with nor- QUALITATIVE FLUORESCENT SPOT TEST mal hemoglobin and enzyme activity.28 The fluorescent spot test (Beutler fluorescent spot test) is a rapid, reliable, and sensitive screening test for G6PD A peculiar cell, the blister cell, referred to by a variety deficiency. Whole blood is added to a mixture of glucose- of descriptive terms (irregularly contracted cell, eccentro- 6-phosphate (G6P), NADP, and saponin. A drop of this cyte, erythrocyte hemighost, double-colored erythrocyte, mixture is placed on a piece of filter paper and examined and cross-bonded erythrocyte) may be seen in G6PD defi- under ultraviolet light for fluorescence. The G6PD enzyme ciency after oxidant-related hemolysis (Figure 18-4). The red present in erythrocytes normally metabolizes G6P, produc- cell membrane is oxidized, and the hemoglobin is confined ing NADPH, which fluoresces. NADP does not fluoresce, to one side of the cell, whereas the other side is transpar- so lack of fluorescence indicates G6PD deficiency. Although ent. The transparent side often contains Heinz bodies and this test often produces false negative results, especially has flattened membranes in which the opposing membrane after a hemolytic episode, it is useful in identifying severe sides are juxtaposed. This cross-bonding of the membrane deficiencies. A recent modification of the fluorescent spot appears to decrease deformability and destine the cell for test is to report “intermediate” fluorescence along with phagocytosis by macrophages. These cells have a decreased “absent” and “present” fluorescence. This modification is volume and increased MCHC. A variety of abnormal laboratory findings related to hemolysis can be found during or after a hemolytic episode. Reticulocytosis is characteristic following a hemolytic epi- sode.29 Leukocytes may increase, but platelets are usually normal. Unconjugated bilirubin and serum lactate dehy- drogenase (LD) may be increased. Haptoglobin commonly decreases during the acute hemolytic phase. Absence of hap- toglobin in the recovery stage indicates chronic hemolysis. Definitive diagnosis depends on the demonstration of a decrease in erythrocyte G6PD activity. In affected indi- viduals, the enzyme activity may appear normal during and for a time after a hemolytic episode because older cells with less G6PD are preferentially destroyed and the newly released reticulocytes have higher activity. A reticulocytosis Figure 18.4 The arrows are pointing to blister cells, which have various names (see the chapter text). Hemoglobin is condensed of more than 7% is generally associated with a normal to one side of the cell, leaving a transparent area (blister) on the enzyme screen after hemolysis.30 For this reason, assays for other. These cells may be found in G6PD-deficient individuals after a G6PD should be performed 2–3 months after a hemolytic hemolytic attack (Wright-Giemsa stain, 1000* magnification). 392 Chapter 18 recommended to improve the diagnosis of G6PD deficiency sample can be submitted to a reference laboratory that can in heterozygous females and individuals with moderate perform the quantitative erythrocyte enzyme assays. G6PD deficiencies.31 In a recent clinical trial, the fluorescent spot enzyme levels can be quantitated by incubating an eryth- test had 26% false negatives and 2% false positives.32 rocyte hemolysate with G6P and NADP, and measuring the rate of reduction of NADP to NADPH at 340 nm in a DYE REDUCTION TEST spectrophotometer. The dye reduction screening test incubates a hemolysate of patient’s blood with G6P, NADP, and the dye brilliant MOLECULAR METHODS cresyl blue. If the hemolysate contains G6PD, the NADP is In the case of heterozygous females or when the quantita- reduced to NADPH, which in turn reduces the blue dye to tion of the enzyme can yield misleading results such as dur- its colorless form. The time it takes for this change to occur ing or after a hemolytic episode, it is best to perform is inversely proportional to the amount of G6PD present. polymerase chain reaction (PCR) tests to reveal the genetic Normal blood is also tested as a control. The test is specific abnormality. In a DNA-based screening test, DNA is and is available as a commercial kit. A recent study of a extracted from spots of dried blood and then submitted for point-of-care dye reduction test kit reported a significant PCR. Fluorescent-labeled probes are used to detect mutant problem with false negatives.33 Another commercial kit uses G6PD alleles. a lateral flow test strip and is read visually. This kit had a positive predictive value of 72% with heparinized blood CASE STUDY (continued from page 391) and 65% with EDTA-preserved blood.34 A quantitative ver- sion of this test measures the rate of reduction of NADP to A spectrophotometric assay for G6PD was per- NADPH at 340 nm in a spectrophotometer. formed on Henry’s peripheral blood. The result was borderline normal, and a preliminary diag- ASCORBATE CYANIDE TEST nosis of G6PD deficiency was made. Primaquine The ascorbate cyanide test is the most sensitive screening usage was discontinued, and the patient recov- test for detecting heterozygotes and G6PD deficiency dur- ered without complications.

Upon follow-up, the ing hemolytic episodes. The test is not specific for G6PD patient was retested for G6PD deficiency and was deficiency, since it also detects other defects or deficiencies found to be abnormal, confirming the diagnosis. in the HMP shunt. The test is also positive in paroxysmal nocturnal hemoglobinuria (PNH), PK deficiency, and unsta- 2. Why was the initial G6PD test result normal but ble hemoglobin disorders. The test’s principle is that G6PD- the repeat test abnormal? deficient cells fail to reduce hydrogen peroxide. A patient’s blood sample is incubated with sodium ascorbate, sodium cyanide, and glucose. Hydrogen peroxide is generated by Differential Diagnosis the interaction of ascorbate with hemoglobin. The sodium G6PD deficiency has similar clinical features and some cyanide inhibits the activity of normal erythrocyte catalase, overlapping laboratory test results that are similar to which catalyzes the degradation of hydrogen peroxide. drug-induced hemolysis associated with unstable hemo- Erythrocytes deficient in G6PD cannot reduce the perox- globins.1 Abnormalities in hemoglobin electrophoresis ide, and hemoglobin is oxidized to methemoglobin, which and the hemoglobin stability test help identify the unsta- imparts a brown tint to the solution. ble hemoglobins but are normal in G6PD deficiency. The CYTOCHEMICAL STAINING ascorbate cyanide test may be abnormal in both unstable The enzyme activity in individual cells is detected by the hemoglobins and G6PD deficiency, but the G6PD assay and cytochemical staining method. The G6PD in the red cells fluorescent screening test are positive only in G6PD defi- reacts with a sensitive tetrazolium salt to ultimately form ciency. Exclusion of anemias associated with defects of the formazan. The level of formazan is directly related to the erythrocyte membrane can be made as these anemias have G6PD activity. The activity in the cells can be scored by characteristic poikilocytosis (e.g., spherocytes, ovalocytes, hand, but automated methods utilizing flow cytometry are stomatocytes). available.35 QUANTITATION OF G6PD Therapy Screening tests for the enzyme are recommended before Most of the patients with G6PD deficiency are asymptom- performing more expensive quantitative tests. Semiquan- atic and do not experience chronic hemolysis; thus, no ther- titative tests that use a low cutoff of 2.10 U/g Hb fail to apy is indicated. However, patients should avoid exposure detect most heterozygous female neonates. Sensitivity of to the oxidant drugs and foods that can precipitate hemo- the test can be increased if the cutoff is raised to 2.55 U/g lytic episodes. In acute hemolytic episodes, supportive ther- Hb for females and to 2.35 U/g Hb for males.36 A blood apy including blood transfusions, treatment of infections, Hemolytic Anemia: Enzyme Deficiencies 393 and removal of the precipitating agent are used. Exchange associated with the genetic mutations appears to reflect transfusion may be necessary in cases of severe neonatal not only the aberrant properties of the mutant protein but jaundice. Dialysis may be indicated in patients with oliguria also interactions of the genotype with physiological and and severe azotemia.10 environmental factors including epigenetic modifications, ineffective erythropoiesis, splenic function, and coexisting polymorphisms of other enzymes.39 Simple heterozygotes CASE STUDY (continued from page 392) are usually asymptomatic. Acquired PK deficiency is seen in 3. What was the precipitating cause of Henry’s some leukemias and myelodysplastic disorders. This type is anemia? more common and milder than the hereditary type. Pathophysiology More than 220 different mutations in the PK gene, 178 of Other Defects and which result in PK deficiency (PKLR gene on chromosome 1q21) have been identified.40 Most are missense mutations, Deficiencies of the Hmp but nonsense mutations, deletions, and insertions are also Shunt and Gsh Metabolism found. PK catalyzes the conversion of phosphoenolpyru- vate (PEP) to pyruvate, concurrent with the conversion of Erythrocytes synthesize about 50% of their total glutathi- ADP to ATP (Figure 18-5). PK deficiency compromises this one every 4 days. Congenital deficiencies of the enzymes energy-producing reaction, resulting in a decrease of ATP needed for glutathione synthesis (glutathione synthetase, production. The cell’s inability to maintain normal ATP glutamylcysteine synthetase) have been reported to be levels results in alterations of the erythrocyte membrane: associated with a decrease in GSH and a hereditary nons- failure of the cation pumps causing potassium loss as well pherocytic hemolytic anemia. Hemolysis increases during as sodium and calcium gain and dehydration (echinocytes). administration of certain drugs. Deficiencies of glutathione The echinocytes are sequestered in splenic cords and phago- reductase, an enzyme that catalyzes the reduction of GSSG cytized by macrophages. to GSH, although rare, have been reported.37,38 Glutathione peroxidase catalyzes the detoxification of hydrogen perox- Clinical Presentation ide by GSH. Deficiencies of this enzyme, although common, Clinical symptoms vary depending on the degree of ane- are not a cause of hemolysis. This might be explained by the mia, which can vary from mild to severe. Individuals tol- fact that peroxide reduction by GSH occurs nonenzymati- erate the anemia relatively well because of the increase in cally at a significant rate. 2,3-BPG that accompanies this distal block in glycolysis. The two to three times normal increase in 2,3-BPG enhances the release of oxygen to the tissues. Jaundice may occur with Pyruvate Kinase (Pk) intermittent hemoglobinuria. Gallstones are a common Deficiency complication due to high levels of bilirubin excreted from the liver in the bile. Pyruvate kinase deficiency is the most common enzyme deficiency in the glycolytic pathway and the second most Glucose common erythrocyte enzyme deficiency. Although PK deficiency is less common than G6PD deficiency, it is the most common inherited enzyme defect associated with a chronic hemolytic anemia.5 Many pyruvate kinase enzyme 2 Phosphoenolpyruvate mutations account for the disorder’s variability of clini- cal manifestations. The more severe types are noted in ADP Pyruvate infancy, whereas the milder types may not be detected until kinase adulthood. ATP 2 Pyruvate Etiology Inheritance is autosomal recessive. Clinically significant Figure 18.5 Glucose is metabolized to pyruvate in the glycolytic pathway. ATP is generated as phosphoenolpyruvate and hemolytic anemias due to PK deficiency are associated is converted to pyruvate with a net gain of 2 ATP. Two molecules of with the homozygous state or double heterozygosity for pyruvate are formed from 1 glucose molecule. In PK deficiency, this two mutant enzymes. The variation in clinical phenotype reaction is slowed, resulting in deficient ATP production. 394 Chapter 18 PK deficiency can be life threatening in neonates. The first locus produces PK active in muscle, leukocytes, When anemia is present at birth, PK deficiency should be platelets, and various other tissues. The second locus pro- considered and differentiated from other etiologies associ- duces the PK active in erythrocytes. Thus, in erythrocyte PK ated with anemia in newborns (ABO/Rh incompatibility, deficiency (mutations of the second locus), only the eryth- thalassemia, G6PD deficiency, and hereditary spherocyto- rocytes are deficient; leukocytes are normal. The screening sis).41 Severe PK deficiency can cause pronounced jaundice procedure is based on the disappearance of fluorescence in neonates that may require exchange transfusions, and as erythrocytes are incubated with phosphoenol-pyruvate some cases have been associated with hydrops fetalis.41,42 (PEP), LD, ADP, and NADH.43 Splenectomy in older children may result in stabilization PEP + ADP ¡PK Pyruvate + ATP of the hemoglobin and decrease the need for transfusions. + Patients with milder forms of PK deficiency are commonly Pyruvate + NADH + ¡LD + H Lac.tate + NAD Fluorescence No Fluorescence diagnosed in early adulthood although they may have had neonatal jaundice.22 Some mutant PK enzymes have normal activity at high substrate concentrations and abnormal activity at low Laboratory Evaluation substrate concentrations. A modification of this procedure has been developed to improve the interpretation of the Patients with PK deficiency have a normocytic, normochro- endpoint.44 In this modification, patient blood is frozen mic anemia with hemoglobin levels of 6–12 g/dL. Reticulo- and thawed to ensure complete hemolysis of the specimen cytosis ranges from 2–15% and increases after splenectomy, before testing. often more than 40%. The degree of reticulocytosis before Fluorescence in normal erythrocytes disappears in splenectomy is not proportional to the degree of anemia as it 30 minutes. In PK-deficient erythrocytes, fluorescence is in most other hemolytic anemias, because the spleen pref- persists for 45–60 minutes. The quantitative test for PK erentially sequesters and removes the younger PK deficient deficiency is performed in the same manner as the screening erythrocytes. The blood smear exhibits irregularly contracted test except that the rate of disappearance of fluorescence cells and occasional echinocytes before splenectomy; more is measured in a spectrophotometer at 340 nm. A rapid are found following splenectomy (Figure 18-6). In contrast potentiometric method has also been developed to mea- to G6PD deficiency, Heinz bodies and spherocytes are not sure enzymatic activity by monitoring the change in pH in found in PK deficiency. Serum unconjugated indirect bili- a reaction buffer during the conversion of the substrate to rubin and LD are increased, and haptoglobin is decreased pyruvate. Because of the large number of mutations and the or absent. Osmotic fragility is normal, but cells demonstrate low incidence of PK deficiency, molecular methods were increased hemolysis when incubated at 37 °C. Autohemol- initially not widely used to detect the disease45 but more ysis is increased at 48 hours and is not corrected with the recently have largely replaced enzymatic assays.5 addition of glucose but is corrected with the addition of ATP. In performing enzyme tests for PK, the erythrocytes must be separated from leukocytes because leukocytes Therapy contain more PK than erythrocytes. Two genes located on There is no specific therapy for PK deficiency. Transfusions chromosomes 15q22 and 1q21 encode for pyruvate kinase. help maintain the hemoglobin above 8–10 g/dL. Splenec- tomy can improve the hemoglobin level and decrease the need for transfusions in some affected individuals; how- ever, hemolysis continues. Checkpoint 18.4 What are the differentiating characteristics of PK and G6PD defi- ciencies found on the peripheral blood smear? Other Enzyme Deficiencies in the Glycolytic Pathway Figure 18.6 Other enzyme deficiencies in the glycolytic pathway, Blood smear from a patient with PK deficiency. when associated with anemia, have clinical manifesta- Note the echinocyte, acanthocyte, target cells, and irregularly contracted cells. Howell-Jolly bodies are also present (Wright- tions and laboratory findings that resemble those of PK Giemsa stain, 1000* magnification). deficiency. Hemolytic Anemia: Enzyme Deficiencies 395 • Glucose phosphate isomerase deficiency (GPI) This • Triosephosphateisomerase (TPI) deficiency This is the second most common disorder of the glycolytic causes severe abnormalities in erythrocytes resulting pathway. Almost all GPI mutants are unstable, causing in severe hemolysis. Abnormalities are also noted in hemolytic anemia. Affected individuals show a partial striated muscle and the central nervous system. Death response to splenectomy. in infancy is common. • Hexokinase (HK) deficiency As the first enzyme in the glycolytic pathway HK is responsible for prim- ing the glycolytic pump. Of the two types of HK defi- ciency, one is associated with hemolytic anemia that Abnormal Erythrocyte responds to splenectomy. The other is associated not Nucleotide Metabolism only with hemolytic anemia but also with an array of other abnormalities. Because the metabolic defect Pyrimidine 5′@nucleotidase (P5N) contributes to the deg- occurs before the generation of 2,3-BPG, production radation of nucleic acids by cleaving pyrimidine nucleo- of 2,3-BPG is reduced and patients tolerate the anemia tides into smaller nucleosides that can diffuse out of the poorly. cell. The buildup of pyrimidine nucleotides decreases the • Phosphoglycerokinase (PGK) deficiency A sex-linked adenine nucleotide pool needed for normal function. P5N disorder, PGK deficiency causes hemolytic anemia and deficiency is an autosomal recessive disorder that leads to intellectual disability in males, but females have a a severe hemolytic anemia unresponsive to splenectomy. milder form of the disorder. Partially degraded mRNA and rRNA accumulate within the cell and are visualized as basophilic stippling in stained • Phosphofructokinase (PFK) deficiency This is indi- smears. Lead inhibits this enzyme, which may explain the cated when subunits of the PFK enzyme are found in similar coarse basophilic stippling seen in lead poisoning various tissues. Deficiency of this enzyme can appear (Chapter 12). as myopathy, hemolytic anemia, or both.45 Summary The erythrocyte life span may be significantly shortened infections or with the administration of certain drugs, if the erythrocyte has intrinsic defects such as deficient and is self-limited. In these variants, the younger cells metabolic machinery. Maintaining a balance of intra- have adequate enzyme activity, but the older cells are cellular constituents is compromised when enzymes severely deficient and selectively hemolyzed. Testing for responsible for

various metabolic pathways are defi- the enzyme should be delayed until 2 months after the cient or fail to function properly. These abnormalities hemolytic episode when reticulocytes are at a steady state are almost always inherited defects. Erythrocytes with (normal) and the erythrocytes produced after the hemo- intrinsic defects are susceptible to early destruction, and lytic episode have aged. Screening tests for the enzyme when this destruction exceeds the marrow capacity to include the fluorescent dye test, cytochemical staining, replace cells, hemolytic anemia results. These hemolytic dye reduction test, and ascorbate cyanide test. Definitive anemias are known as chronic or hereditary nonspherocytic testing requires quantitation of the enzyme or genetic hemolytic anemias. testing for mutations. Erythrocyte enzyme deficiencies can compromise the Deficiencies of enzymes in the glycolytic pathway integrity of the cell membrane or hemoglobin and lead decrease ATP production and lead to hemolysis. Cation to hemolysis. The HMP shunt provides the cell-reducing pump activity may be impaired and osmotic fragility may power, protecting it from oxidant damage. Defects in be increased. Pyruvate kinase (PK) is the most common this shunt allow hemoglobin to be oxidized and dena- enzyme abnormality in this pathway. Many PK enzyme tured to Heinz bodies. The Heinz bodies damage the mutants exist, resulting in a diverse array of clinical and cell membrane. The finding of bite cells (degmacytes) laboratory findings. Reticulocytosis may be significant. on the blood smear is evidence that Heinz bodies have The blood film is remarkable for the presence of irregu- been pitted from the cells. The most common deficiency larly contracted cells and echinocytes. Heinz bodies and in this pathway is G6PD deficiency, a sex-linked disor- bite cells are not found. Screening and definitive tests der. This enzyme has many different variants, some of for the enzyme are based on fluorescence. Several other which cause severe hemolysis and others mild hemolysis. rare enzyme defects that also lead to hemolysis have been In most cases, hemolysis is sporadic, occurring during identified. 396 Chapter 18 Review Questions Level I 7. Following a hemolytic episode, which of the following is a common finding in G6PD deficiency? (Objective 5) 1. What are the two main metabolic pathways that erythrocytes use for glucose metabolism? (Objective 1) a. Reticulocytosis a. Krebs cycle and glycolytic pathway b. Appearance of burr cells on the blood smear b. Hexokinase and Krebs cycle c. Increased haptoglobin c. Oxidative phosphorylation and glycolytic pathway d. Decreased unconjugated bilirubin d. Hexosemonophosphate shunt and glycolytic 8. An abnormal erythrocyte resulting from splenic removal pathway of Heinz bodies in erythrocytes is called: (Objective 7) 2. The main protective functions of erythrocyte enzymes a. macrocyte result from which of the following? (Objective 2) b. target cell a. Electron transport and cation pumping using ATP c. bite cell b. Cation pumping using ATP and protection of d. dacryocyte hemoglobin by reduced glutathione c. Protection of hemoglobin by reduced glutathione 9. Following a hemolytic episode in a G6PD-deficient and electron transport individual, a characteristic finding on a blood smear d. Cation pumping and bilirubin production is: (Objective 7) a. increased polychromasia 3. In G6PD deficiency, anemia ultimately results from: b. echinocytes (Objective 2) c. Howell-jolly bodies a. buildup of 2,3-BPG and poor iron binding d. Pappenheimer bodies b. inability to maintain enough ATP to pump cations c. oxidative damage to hemoglobin and splenic 10. What compound can induce anemia in G6PD defi- removal of erythrocytes ciency? (Objective 8) d. membrane protein defects and loss of erythrocyte a. Aspirin flexibility b. Vitamin C 4. Which is the most common erythrocyte enzyme defi- c. Iron ciency? (Objective 3) d. Primaquine a. Pyruvate kinase Level II b. Hexokinase 1. Why should G6PD testing be delayed for an individ- c. Glucose phosphate isomerase ual following a hemolytic episode? (Objective 1) d. Glucose-6-phosphate dehydrogenase a. The level of glucose must have time to replenish. 5. Which is true for the inheritance pattern for G6PD? b. Heinz bodies can interfere with the test method. (Objective 4) c. Deficient cells have been selectively destroyed. a. It is X-linked and only found in males. d. The patient needs time to build up iron stores. b. It is autosomal dominant and affects all offspring. c. It is X-linked; however, females can be affected. 2. An 18-year-old black male was suspected of having G6PD deficiency when he experienced hemolytic ane- d. It is autosomal, and males are affected; females are mia after administration of primaquine. An erythro- carriers. cyte G6PD analysis performed on blood taken 2 days after symptoms appeared was normal. A reticulocyte 6. Which of the following is a quantitative test for count revealed 12% reticulocytes at this time. These G6PD? (Objective 6) results suggest that: (Objective 7) a. Rate reduction test a. the patient definitely does not have G6PD defi- b. Fluorescent spot test ciency but could have pyruvate kinase deficiency c. Dye reduction test b. another G6PD test should be done in several months d. Ascorbate cyanide test when the reticulocyte count returns to normal Hemolytic Anemia: Enzyme Deficiencies 397 c. leukocytes could be contaminating the sample, giv- c. Heinz bodies are not formed in pyruvate kinase ing a false result deficiency. d. the patient probably has the G6PD-Mediterranean d. Pyruvate kinase deficiency does not cause abnor- variant mal erythrocyte morphology. 3. The main consequence of enzyme defects in the glyco- 7. Hereditary nonspherocytic hemolytic anemia syn- lytic pathway is: (Objective 3) drome is associated with which G6PD variants? (Objective 4) a. decreased ATP production b. Heinz body formation a. Class I c. decreased formation of reduced glutathione b. Class II d. decreased formation of 2,3-BPG c. Class III d. Class IV 4. Which of the following G6PD isoenzyme variants will not result in hemolysis? (Objective 4) 8. Harmful peroxides in the erythrocyte are neutralized by which of the following? (Objective 2) a. G6PD-Mediterranean b. G6PD-B a. ATP production c. G6PD-Canton b. Heinz bodies d. G6PD@A- c. Bilirubin d. Glutathione 5. The blood smear of a patient with chronic nonsphero- cytic hemolytic anemia reveals echinocytes, acantho- 9. A patient is suspected of having a form of G6PD defi- cytes, target cells, and irregularly contracted cells. ciency but is not affected by ingestion of fava beans. Which follow-up test could help define the cause of How can this be explained? (Objective 4) this anemia? (Objective 6) a. The patient has the G6PD-Mediterranean type. a. PEP fluorescent test b. The G6PD-B type of G6PD is present. b. Ascorbate cyanide test c. The patient does not have G6PD deficiency. c. Fluorescent spot test d. A mild form of G6PD is present. d. Quantitation of G6PD 10. Which lab test is most useful in screening for G6PD 6. Explain why bite cells are not characteristic of pyru- deficiency? (Objective 1) vate kinase deficiency. (Objectives 3, 5, 6) a. Fluorescent spot test a. The spleen removes them as they are formed. b. CBC b. The erythrocyte forms a spherocyte as inclusions c. 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Vora, S., Corash, L., Engel, W. K., Durham, S., Seaman, C., & Disease in Childhood. Fetal and Neonatal Edition, 89(6), F555–557. Piomelli, S. (1980). The molecular mechanism of the inherited 27. Renzaho, A. M., Husser, E., & Polonsky, M. (2014). Should phosphofructokinase deficiency associated with hemolysis and blood donors be routinely screened for glucose-6-phosphate myopathy. Blood, 55(4), 629–635. Chapter 19 Hemolytic Anemia: Immune Anemias Linda A. Smith, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. List the antibody systems or specificities a. Cold agglutinin syndrome usually involved in: b. Warm autoimmune hemolytic anemia a. Cold agglutinin syndrome (WAIHA) b. Paroxysmal cold hemoglobinuria (PCH) 4. Describe and recognize the typical hema- c. Warm autoimmune hemolytic anemias tologic laboratory findings in ABO and Rh (WAIHA) hemolytic disease of the fetus and newborn 2. Describe the purpose and general procedure (HDFN). for the direct antiglobulin test (DAT) and 5. Contrast alloimmune and autoimmune identify the typical DAT profile (polyspecific hemolytic anemias including stimulus for AHG, anti-IgG, anti-C3) found in patients antibody production, site of hemolysis, type with cold agglutinin syndrome and warm of antibody involved, and direct antiglobu- autoimmune hemolytic anemia. lin test (DAT) and indirect antiglobulin test 3. Describe and recognize the characteristic (IAT) reactions. hematologic findings associated with the following conditions: Objectives—Level II At the end of this unit of study, the student should be able to: 1. Compare the pathophysiology of extra- 2. Given a set of laboratory data, determine vascular and intravascular hemolysis in the underlying mechanism of hemolysis and immune hemolytic anemia (IHA) including suggest confirmatory tests. site of destruction, immunoglobulin class of antibody, and underlying mechanism. 399 400 Chapter 19 3. Contrast the different mechanisms of drug- 6. Interpret the results of laboratory tests for induced immune hemolytic anemia and HDFN, and determine whether evidence of tests used for confirmation, and identify the hemolysis is present. drugs commonly involved. 7. Compare and contrast the pathophysiol- 4. Describe the mechanism of hemolysis, select ogy and clinical findings of an immediate the confirmatory tests for paroxysmal cold transfusion reaction with those of a delayed hemoglobinuria (PCH), and evaluate the reaction. results. 8. List the causes of the secondary types of 5. Compare the prenatal and postnatal patho- cold agglutinin syndrome and warm auto- physiology of hemolytic disease of the fetus immune hemolytic anemia and identify key and newborn (HDFN). laboratory results linking the cause and the autoimmune condition. Chapter Outline Objectives—Level I and Level II 399 Mechanisms of Hemolysis 404 Key Terms 400 Laboratory Identification of Sensitized Red Background Basics 400 Cells 405 Case Study 401 Autoimmune Hemolytic Anemias (AIHA) 407 Overview 401 Alloimmune Hemolytic Anemia 417 Introduction 401 Summary 423 Classification of Immune Hemolytic Anemias 401 Review Questions 423 Sites and Factors that Affect Hemolysis 403 References 425 Key Terms Alloimmune hemolytic anemia Donath-Landsteiner (D-L) antibody Immune hemolytic anemia (IHA) Antihuman globulin (AHG) Drug-induced hemolysis Immune tolerance Autoimmune hemolytic anemia Erythroblastosis fetalis Indirect antiglobulin test (IAT) (AIHA) Hemolytic disease of the fetus and Kernicterus Cold agglutinin syndrome (CAS) newborn (HDFN) Paroxysmal cold hemoglobinuria Direct antiglobulin test (DAT) Hemolytic transfusion reaction (PCH) Background Basics The information in this chapter builds on the concepts Level I learned in previous chapters. Additionally, a basic under- • Summarize the normal production, life span, and standing of immunology principles and an introduction destruction of erythrocytes. (Chapter 5) to immunohematology will be helpful. To maximize your • List the reference interval for the hematology param- learning experience, you should review these concepts eters: hemoglobin, hematocrit, erythrocyte count, before starting this unit of study: and reticulocytes. (Chapters 10, 11) Hemolytic Anemia: Immune Anemias 401 • List the different intrinsic and extrinsic factors that destruction to laboratory parameters in the d iagnosis can result in hemolytic anemia. (Chapter 11) of abnormal hemolysis. (Chapters 5, 11) • Describe the basic structure of immunoglobulin and • Choose laboratory tests to assist in diagnosing its normal physiologic role. (Chapter 8) anemia in a cost-efficient and effective manner. (Chapter 11) Level II • Identify clinical signs of hemolytic anemia and • Describe the role of erythropoietin and regulation of changes that occur in laboratory tests that signal pos- its production. (Chapter 5) sible hemolytic anemia. (Chapter 11) • Differentiate intravascular and extravascular destruction of erythrocytes and relate these types of CASE STUDY Introduction We refer to this case study throughout the chapter. When an immune-mediated process (antibody and/or com- plement) destroys erythrocytes prematurely, the disorder Nancy, a 28-year-old female, made an appointment is referred to as an immune hemolytic anemia (IHA). The with her physician because she was feeling tired all individual, however, might or might not become anemic, the time and was short of breath with minor exer- depending on the severity of hemolysis and the ability of tion. She indicated that the symptoms had been the bone marrow to compensate for erythrocyte loss. Diag- ongoing for about 3 weeks. She has no known his- nosis of anemia (and underlying hemolysis) is determined tory of chronic diseases. by laboratory findings such as a decrease in hemoglobin Consider the initial laboratory tests that should and hematocrit, an increase in reticulocytes and/or uncon- be performed to evaluate this patient’s condition jugated bilirubin, and a decrease in serum haptoglobin. based on clinical history and symptoms. Initial confirmation of the underlying immune mechanism is accomplished by demonstrating attachment of antibody or complement to the patient’s erythrocytes. Table 11-9 (Chapter 11) summarizes some common laboratory values Overview characteristically seen in hemolytic anemia. This chapter compares the different types of immune- mediated hemolytic anemias: autoimmune, alloimmune, Classification Of Immune and drug induced. The underlying mechanism for each of these anemias involves the reaction of an antibody Hemolytic Anemias with erythrocyte antigens and subsequent cell destruc- tion by either intravascular or extravascular processes. Determining the underlying process of immune hemolysis The chapter also compares mechanisms of intravascular is important because each type requires a specific treatment and extravascular hemolysis and describes the tests nec- regimen. Initially, IHA can be classified into three broad cat- essary to detect erythrocyte sensitization and identify the egories based on the stimulus for antibody production1,2 causative antibody. (Table 19-1). These are: The chapter discusses pathophysiology, clinical pre- • Autoimmune hemolytic anemia sentation and laboratory evaluation, differential diagnosis, • Drug-induced hemolytic anemia and therapy for each of the major types of autoimmune • Alloimmune hemolytic anemia hemolytic anemia. Hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN) are Autoimmune hemolytic anemia (AIHA) is a com- included as examples of alloimmune hemolytic anemia. plex and incompletely understood process characterized The causative antibody and clinical presentation for acute by an immune reaction against self-antigens and increased and delayed transfusion reactions are compared and the destruction of erythrocytes. Destruction may be mediated laboratory tests required to confirm hemolytic transfusion by anti-erythrocyte antibodies. Antibody alone, antibody reactions are described. The section on HDFN compares with complement, or complement alone. Individuals pro- ABO-HDFN and Rh-HDFN. The treatment of a fetus and a duce antibodies against their own erythrocyte antigens newborn with this condition as well as preventative mea- (autoantibodies), which are usually directed against high- sures are included. incidence antigens (antigens present on the erythrocytes of 402 Chapter 19 react best at body temperature (37°C); the anemia they pro- Table 19.1 Classification of Immune Hemolytic Anemias duce is termed warm autoimmune hemolytic anemia (WAIHA). Classification Causes About 70% of the AIHAs are of the warm type. Most warm Autoimmune • Warm-reactive antibodies (37°C) autoantibodies are of the IgG class (most frequently IgG1) Primary or idiopathic and cause extravascular hemolysis of the erythrocyte. A few Secondary warm-reacting autoantibodies of either the IgM or IgA class • Autoimmune disorders (systemic lupus erythe- have been identified. Cold hemolytic anemias, on the other matosus, rheumatoid arthritis, and others) hand, are usually due to the presence of an IgM antibody • Chronic lymphocytic leukemia and other immu- noproliferative diseases with an optimal thermal reactivity below 30°C. Hemolysis • Viral infections with cold-reacting antibodies results from IgM binding to • Neoplastic disorders and activating complement. The IgM antibody attaches to • Chronic inflammatory diseases erythrocytes in the cold and fixes complement. After warm- • Cold-reactive antibodies (less than 30°C) ing, the antibody dissociates from the cell, but the comple- Primary or idiopathic (cold hemagglutinin disease) ment remains, either causing direct cell lysis or initiating Secondary extravascular destruction. Included in the cold hemolytic • Infectious diseases (Mycoplasma pneumoniae, anemia classification is a special condition, paroxysmal cold Epstein-Barr virus, other organisms) hemoglobinuria (PCH), which is characterized by a cold- • Lymphoproliferative disorders Paroxysmal cold hemoglobinuria reacting IgG antibody capable of fixing complement. • Idiopathic A third category, mixed-type autoimmune hemolytic ane- • Secondary mia, demonstrates both warm-reacting (IgG) autoantibodies Viral syndromes and cold-reacting (IgM) autoantibodies. • Syphilis (tertiary) Drugs that attach to the erythrocyte membrane or alter • Mixed type it in some way can cause drug-induced hemolysis. Histori- • DAT negative cally, several

different mechanisms of hemolysis have been Drug induced • Drug dependent hypothesized based on whether the drug binds directly to • Drug independent the cell, reacts with an antibody in the circulation to form an • Nonimmunologic protein adsorption (NIPA) immune complex that binds to the cell, or alters the eryth- Alloimmune • Hemolytic transfusion reaction rocyte antigens to stimulate formation of autoantibodies. • Hemolytic disease of the fetus and newborn Now, however, these antibodies are broadly classified as either drug dependent or drug independent based on reac- tions of patient’s erythrocytes and the drug in the in vitro most people). The autoantibodies characteristically react not test systems. These mechanisms are discussed in detail later only with the individual’s own erythrocytes but also with in this chapter. the erythrocytes of other individuals carrying that antigen. AIHA occurs because of antibody development to an Autoimmune hemolytic anemias are further classified erythrocyte antigen that the individual lacks. When an indi- as warm or cold hemolytic anemia based on clinical symp- vidual is exposed to erythrocytes from another person, there toms and on the optimal temperature at which the antibody could be antigens on the transfused cells that are not pres- reacts in vivo and in vitro (Table 19-2). Some antibodies ent on the recipient’s erythrocytes. Therefore, the recipient’s Table 19.2 Characteristics of Agglutinins in Hemolytic Anemia Warm-Reacting Antibodies Cold-Reacting Antibodies Immunoglobulin (Ig) class IgG IgM IgM (rare) IgG (PCH only) IgA (usually with IgG) Optimal reactivity 37°C Less than 30°C, usually less than 10°C Mechanism of hemolysis Extravascular Intravascular: complement-mediated lysis Attachment of membrane-bound IgG or C3b to macro- Extravascular: attachment of membrane-bound C3b to phage receptors macrophage receptors Specificity Usually broad specificity anti-Rh Usually autoanti-I Occasionally autoanti-i PCH: autoanti-P PCH, paroxysmal cold hemoglobinuria. Hemolytic Anemia: Immune Anemias 403 lymphocytes recognize antigens on the transfused cells as foreign and stimulate the production of antibodies (alloan- Sites And Factors That tibodies). In contrast to autoantibodies, these alloantibodies Affect Hemolysis react only with the antigens on the transfused cells or cells from individuals who possess the antigen. The alloanti- Regardless of whether it is caused by alloantibodies or bodies do not react with the individual’s own erythrocytes. autoantibodies, hemolysis can be intravascular or extra- Examples of alloimmune hemolytic anemia are: vascular, depending on the class of antibody involved and whether the complement cascade has been completely • HDFN in which the mother makes antibodies against activated. Most immune-mediated hemolysis is extravas- antigens on the fetal erythrocytes cular. Erythrocytes sensitized (coated) with antibody • Transfusion reactions in which the recipient makes anti- (IgG) or complement components (e.g., C3b) attach to bodies to antigens on the transfused (donor) cells macrophages in the spleen or liver via macrophage recep- The presence of alloantibodies can be detected in vitro tors for the Fc portion of IgG or the C3b component of by performing an antibody screen in which the patient’s complement. These cells are then phagocytized serum reacts with commercial erythrocytes containing most (Figure 19-1). Intravascular hemolysis occurs if the com- of the clinically significant antigens. An autocontrol consist- plement cascade is activated through C9 (the membrane ing of the patient’s serum and erythrocytes can also be set attack complex), resulting in lysis of the cell. The rate at up. When only alloantibodies are present, the autocontrol which hemolysis occurs in hemolytic anemia is related to shows no hemolysis or agglutination, whereas the mixture several factors.1 Table 19-3 summarizes the major factors of patient’s serum and the commercial cells produce agglu- and their effects. tination and, in rare cases, hemolysis. Checkpoint 19.2 Checkpoint 19.1 Explain how the class of immunoglobulin, amount of antibody What are the three major categories of immune hemolytic ane- bound, and thermal reactivity of the antibody affect hemolysis. mia, and how is antibody production stimulated in each type? CASE STUDY (continued from page 401) Macrophage Nancy’s initial complete blood count (CBC) shows Fc receptor C3b receptor a hemoglobin value of 7.0 g/dL and a hematocrit of 21%. Her white blood cell (WBC) count and RBC RBC C3b platelet count are within the reference interval. Antibody 1. What are some reasons for her to have a low hemoglobin value? Figure 19.1 Immune-mediated extravascular hemolysis. Erythrocytes sensitized with antibody or complement (C3b) attach to macrophages via specific cell receptors for these immune proteins. Table 19.3 Factors Affecting the Rate of Hemolysis in Immune Hemolytic Anemias Factor Effect Class of immunoglobulin coating the erythrocytes For IgG subclasses, the affinity of macrophage receptors and rate of hemolysis is greatest for IgG3 and IgG1 Quantity of Ig molecules per erythrocyte Immunoglobulins coat a cell with a high density (large number) of corresponding antigens more heavily than a cell with low density (fewer) antigens Titer of the antibody Higher titer is more likely to cause increased hemolysis Ability of the Ig to activate complement IgM and IgG can activate complement; for IgG, the ability to activate complement is IgG3 7 IgG1 7 IgG2 Thermal amplitude of the antibody Warm-reacti ng (37°C) antibodies can cause hemolysis but cold-reacting (0–4°C) antibodies usually do not The activity level of macrophages Suppression of Fc receptors decreases rate of hemolysis Complement components on the membrane Macrophages have receptors for C3b but no effective receptor for C3d 404 Chapter 19 Mechanisms Of Hemolysis and destruction of cells and microorganisms. The major roles of complement in immune hemolytic anemias are The mechanism for hemolysis is based on whether IgM, sensitization and lysis of erythrocytes. Sensitization occurs IgG, and/or complement are/is present on the erythro- when only a portion of the cascade is activated and depos- cyte. Specific phagocytic cells in the spleen or liver initiate ited on the erythrocyte membrane; lysis results when the extravascular hemolysis of cells coated with IgG or comple- entire system is activated. ment. Complete activation (through C9) of the complement The complement proteins are designated numerically cascade results in intravascular hemolysis. The type of lysis (e.g., C1, C2, C3) or by letters or historical names. Comple- (intravascular or extravascular) can affect rate of hemolysis ment proteins normally circulate in an inactive state but and number of erythrocytes destroyed. Intravascular hemo- under certain circumstances become activated in a cascade- lysis may affect up to 200 mL erythrocytes/hour versus only like fashion. An activated component is identified by plac- 20 cells/hour with extravascular hemolysis.2 ing a bar over the component (C5). The complement cascade can be initiated by at least IgG-Mediated Hemolysis three separate mechanisms: classic, alternate, and lec- IgG mediates erythrocyte destruction by first attaching to tin. The usual and most important activation pathway in the erythrocyte membrane antigens through the Fab por- IHA is the classic, although on some occasions the alter- tion of the Ig molecule. The Fc portion of the bound IgG is nate pathway can be activated (Figure 19-2). The classic exposed and binds to Fc receptors (FcgR@I, -II, -III) on mac- complement pathway is initiated by an antigen–antibody rophages in the red pulp of the spleen. After binding, the reaction involving IgM as well as IgG1 and IgG3 antibod- macrophage pits the antigen–antibody (Ag/Ab) complex, ies (IgG2 antibodies activate but less efficiently). The first fragmenting the cell membrane, which then reseals itself. complement component (C1q) must attach to two antibody With repeated splenic passage, the erythrocyte continues to Fc regions, requiring that two antibody-binding sites be lose membrane and gradually assumes a spherocytic shape. near. Thus, the attachment of complement depends on the As the cell becomes more spherocytic, it becomes rigid and density or concentration of antibody molecules and their less deformable and is eventually phagocytized by splenic spatial arrangement when attached to antigens on the cell macrophages. Alternatively, the antibody-sensitized cell can surface. Only one IgM molecule (a pentameric structure) be entirely engulfed by the macrophages (phagocytosis). is required to activate the complement pathway, whereas Natural killer (NK) cells and neutrophils also have Fc two IgG molecules (monomeric) are required; as a result, gR. Neutrophils are capable of phagocytosis (FcgR@I,@III). Inter- IgM is much more likely to activate complement than IgG. action of antibody-coated cells with NK cells usually results Therefore, the nature of the antibody involved is an impor- in the recognition and lysis of cells by NK cells, which is tant determinant of the extent of erythrocyte destruction by referred to as antibody-dependent cell-mediated cytotox- complement. icity (ADCC). This occurs through binding of IgG by the Activation of the complement cascade is initiated FcgRIII (CD16) on NK cells. when the first complement component, C1, binds to two As the spleen becomes saturated with sensitized red Fc regions of IgG or IgM antibody molecules. This attach- blood cells (RBCs) (RBCs coated with antibody and/or ment initiates activation of the other complement compo- complement), the liver assists in removing the cells. Lightly nents (C4, C2, C3). The complex containing C3b activates coated cells are more efficiently removed in the spleen due the terminal components, C5 to C9. Activation of these to the sluggish splenic blood flow. The liver can be of some terminal complement components, known as the membrane importance in removing heavily sensitized cells. The splenic attack complex (MAC), is responsible for the lytic attack on tissue proliferates in response to an increase in erythrocyte the erythrocyte membrane. Membrane leakage begins at the sequestration and can be responsible for splenic enlarge- C8-activation stage when a transmembrane pore is formed. ment (splenomegaly) in chronic warm-immune hemolytic C9 prevents the pore from resealing, resulting in osmotic anemias (Chapter 3). In immune hemolytic anemias in lysis. The alternate pathway of complement activation, which complement is activated, it, as well as IgG, is present which bypasses C1, C2, and C4 and activates C3 directly, on the erythrocyte membrane, which enhances phagocyto- can be initiated by aggregated IgG, IgA, and IgE as well as sis by increasing the likelihood of the cell binding to either by several polysaccharides and liposaccharides. the Fc and/or C3b receptors of macrophages. If complement activation on the erythrocyte membrane is complete (C1 S C9), intravascular hemolysis occurs. However, activation of complement does not always go Complement-Mediated Hemolysis through C9 and thus does not always lead to direct cell The complement system consists of more than 20 serum lysis. More commonly, activation proceeds only through proteins responsible for several diverse biological activities C3 on the erythrocyte membrane (in which case the cell is including the mediation of acute inflammatory responses said to be sensitized). As C3 is activated, it is broken down Hemolytic Anemia: Immune Anemias 405 into two fragments: C3a, which is released into the plasma, encounters macrophages. C3c dissociates from the mem- and C3b, which remains attached to the cell membrane. brane, but C3d remains attached. Erythrocytes coated with The sensitized cell with attached C3b is totally or partially C3d have a normal survival because macrophages have no engulfed by binding to the C3b receptors (CR1, CR3) of receptors for this complement component. Thus, the bal- macrophages in the liver (most complement-coated cells are ance between C3b deposition on the membrane and C3b removed in this organ). Because of the enzymatic action of inactivation determines the susceptibility of erythrocytes to the protein C3b inactivator in plasma, C3b on erythrocytes phagocytosis by macrophages via the C3b receptors. can be further cleaved to form C3c and C3d before the cell In addition to C3b inactivator, other inhibitors of com- plement activation exist. Classic Pathway Alternate Pathway IgM-Mediated Hemolysis C1q,r,s Ag/Ab, polysaccharides In cold agglutinin syndrome, IgM molecules attach to the Ag/Ab Ca++ erythrocyte membrane, but these sensitized cells are not C1q,r,s removed from circulation in the same manner as those sensi- attaches to Fc of Ag/Ab Factor D tized with IgG because macrophages do not have receptors for the Fc portion of IgM. However, IgM is an efficient acti- C1q,r,s,-Ag/Ab C3b+Factor B C3b,Bb Mg++ vator of complement, and cells can be lysed intravascularly if complement activation through C9 is complete. If acti- Properdin (P) C1q,r,s,-Ag/Ab vation is incomplete and only C3b coats the erythrocytes, C4 they can be destroyed extravascularly via adherence to CR1 C4a C3b,P,Bb and CR3 receptors on macrophages. Adherence of the cell C1q,r,s,-Ag/Ab-C4b to macrophages via complement receptors and subsequent C2 C3 C2b phagocytosis, however, is less efficient than immune adher- C1q,r,s,-Ag/Ab-C4b,2a ence (adherence mediated via immunoglobulin) and phago- C3 C3b inactivator cytosis via macrophage Fcg receptors. It has been estimated

C3a Ag/Ab-4b,2a,3b that more than 100,000 molecules of the complement com- ponent C3b are required on the cell surface to induce effec- tive macrophage phagocytosis via complement alone. C3b C5 Inhibition C5a is also inefficient in promoting adherence to macrophages Activation C5b because much of the C3b is inactivated to C3d. Thus, extra- C6 vascular hemolysis of cells sensitized with complement is C5b,6 not as severe as hemolysis of cells sensitized with IgG. C7 In addition to activating complement, IgM antibodies C5b,6,7 agglutinate cells. In vitro, agglutination is a useful phenom- C8 enon for detecting the presence of cold agglutinins. C5b,6,7,8 C9 C5b,6,7,8,9 Checkpoint 19.3 Compare the mechanisms of IgG-mediated hemolysis with Figure 19.2 The complement cascade. The central event in those of IgM-mediated hemolysis. complement activation is the activation of C3 by C3 convertases. This can occur by two separate but interrelated mechanisms, the classic and alternate pathways. The classic complement pathway is initiated by an antigen–antibody reaction. The antigen–antibody Laboratory Identification of complex activates the C1q, r, s complex, which in turn activates C4 by proteolytic cleavage to C4a and C4b. C2 binds to C4b and Sensitized Red Cells is proteolytically cleaved by C1s to form C2a and C2b. The C4b2a complex serves as C3 convertase. In the alternate pathway, C3b Suspicion of immune hemolytic anemia indicates specific serves as the cofactor of the C3-cleaving enzyme complex (C3b, tests to detect and identify the causative antibody. In gen- P, Bb), also known as C3 convertase. Thus, C3b serves to prime its eral, two distinct techniques are used: own activation. The C3b formed through the classic pathway can directly initiate the assembly of the alternate pathway C3 convertase. • Agglutination in saline, which will detect IgM C3 can also be activated by spontaneous hydrolysis. The C3b antibodies complexes formed by the classic and alternate pathways activate C5 to C5a and C5b. Membrane damage is initiated by the assembly of • Antihuman globulin (AHG) test, which will detect IgG C5b with C6, 7, 8, 9. antibodies and/or complement Spontaneous ©UTHSCSA 1993 406 Chapter 19 IgM antibodies can be detected by agglutination reac- are used to specifically identify what component is on the tions using test sera (antibody) and appropriate erythro- erythrocyte. cytes suspended in saline, but IgG antibodies cannot. The The two types of tests using AHG are: difference in the ability of IgG and IgM to cause aggluti- • Direct antiglobulin test detects erythrocytes coated nation in saline can be explained by the difference in size with antibody or complement in vivo of the two antibodies in relation to the zeta potential. The erythrocyte zeta potential is an electrostatic potential cre- • Indirect antiglobulin test detects antibodies in the ated by the ionic cloud surrounding erythrocytes when plasma or serum they are suspended in saline. This force tends to keep the erythrocytes about 25 nm apart in solution. Thus, an anti- Direct Antiglobulin Test body must be large enough to span the 25 nm gap between The direct antiglobulin test (DAT) detects erythrocytes that cells to agglutinate saline- or plasma-suspended cells. The have been sensitized with antibody and/or complement in IgM pentamer has a possible span of 35 nm; therefore, it vivo. This test should always be performed in suspected can overcome the electrostatic forces separating the cells cases of AIHA because it differentiates AIHA from all other and cause agglutination (Figure 19-3). However, the maxi- types of hemolytic anemia, confirming the immunologic mum span of an IgG molecule is about 14 nm, and it cannot basis of erythrocyte destruction. Results must be evaluated bridge antigens on two separate cells to cause agglutina- in conjunction with clinical symptoms and other laboratory tion. Thus, detection of IgG antibodies requires a different data.3 Specimens collected in tubes with ethylenediamine- technique using AHG, which is an antibody to human IgG. tetraacetate (EDTA) are preferred to clotted specimens for AHG can connect the antibody molecules on separate cells the DAT procedure. EDTA chelates Ca++ and Mg++, pre- and cause agglutination. venting the in vitro binding of complement to red cells The AHG test (historically referred to as the Coombs that can be mediated by naturally occurring cold-reactive test) is a laboratory procedure designed to detect erythro- antibodies (e.g., autoanti-I). However, complement that has cytes sensitized with IgG and/or complement. Polyspecific been bound in vivo will be detected. Polyspecific AHG is AHG is composed of antibodies to both IgG and C3, capa- added to saline-washed patient cells, and agglutination is ble of attaching to the Fc region of IgG immunoglobulins considered positive evidence for the presence of IgG and/or or complement components on two separate cells, bridg- complement components on the cells due to in vivo coating. ing the distance between them and leading to the lattice A positive test with polyspecific AHG should be fol- formation known as agglutination. Monoclonal antiserum lowed by a DAT with monospecific AHG antiserum that against IgG only (anti-IgG AHG) or C3 only (anti-C3 AHG) reacts specifically with either IgG or complement to deter- mine the type of proteins bound to the erythrocyte. Either or both monospecific IgG-AHG and C3b-AHG can be positive. If the anti-IgG test is positive, the antibody can be removed from the cell by an elution process, and the resulting elu- IgG ate (solution containing the antibody) tested to identify the specificity of the antibody. 14nm If an autoantibody is IgM, only complement usually is detected on the erythrocytes as the IgM tends to dissociate RBC RBC from the cells in the warmer part of the circulation. The poly- 25 nm specific and the anti-C3 monospecific DAT test will be positive; the anti-IgG will be negative. An elution procedure is not used if only complement is detected on the erythrocyte because no antibody would be recovered for identification. In general, if the antibody coating the cells is IgA, the DAT will be negative because the AHG reagent cannot detect that immunoglobulin class. IgA-specific or IgM-specific AHG is not routinely avail- 35 nm able in most clinical laboratories but may be used in reference IgM pentamer laboratories for workup of difficult-to-diagnose cases. Figure 19.3 The zeta potential of erythrocytes keeps the cells about 25 nm apart when suspended in saline. IgG antibodies have Indirect Antiglobulin Test a span of about 14 nm, not enough to bridge the gap between cells The indirect antiglobulin test (IAT), sometimes referred to and cause agglutination. IgM antibodies, however, are pentamers with a span of about 35 nm, a distance sufficient to bridge the space as an antibody screen, is used to detect antibodies in the between cells and cause agglutination. patient’s serum. A positive IAT indicates alloimmunization Hemolytic Anemia: Immune Anemias 407 (immunization to antigens from another individual) and/ Positive DAT in Normal Individuals or the presence of autoantibody in the patient’s serum. In the IAT, free antibody is detected by incubating the patient’s Some healthy blood donors and hospitalized patients can serum with reagent erythrocytes of known antigenic have a positive DAT but not shortened erythrocyte survival. makeup (commonly referred to as screening cells). An auto- The reason for this observation is not clear,1 but factors that control consisting of the patient’s serum and erythrocytes could be responsible include.21,22 may also be included. After a specified incubation period at 1. The individual’s macrophages may not be as active in 37°C, the cells are washed free of excess serum, and AHG removing sensitized cells as the macrophages in indi- antiserum is added. If the patient’s antibody has attached viduals with hemolytic disease. to the corresponding erythrocyte antigen during the incuba- 2. The amount of antibody bound to cells might not be tion period, the reagent cells will agglutinate with AHG. sufficient to cause decreased erythrocyte life span. Further testing to identify the specificity of the antibody is 3. Macrophages might not recognize the subclass of anti- then carried out. body sensitizing the cell. Macrophage Fc receptors have low affinity for the IgG2 and IgG4 subclasses. Eryth- rocytes coated with these immunoglobulins will give Checkpoint 19.4 a positive direct DAT, but in vivo, survival of the cells Compare the purpose of the DAT and the IAT, and state the type will be normal. of specimen used for each test. 4. The thermal amplitude of the antibody may be less than 37°C 5. The positive DAT can be due to the presence of cer- Negative DAT in AIHA tain complement fragments on erythrocytes. Increased amounts of C3d can be found on the erythrocytes, but In some cases of AIHA, antibody cannot be detected because this component is not detected by macrophage on the patient’s cells or in the serum.4,5,6,7,8,9,10 This can receptors, erythrocytes will not have decreased survival. result from an insufficient number of IgG molecules on 6. Patients with hypergammaglobulinemia or receiving the erythrocyte for detection, autoantibodies of the IgA high-dose intravenous immunoglobulin (IVIG) or anti- or IgM class, or the presence of autoantibodies with a low lymphocyte globulin could have a positive DAT affinity for the erythrocyte.1,7,11 The DAT can detect as because of nonspecific binding of immunoglobulins to few as 100–500 molecules of IgG per cell or 400–1100 mol- the erythrocytes. ecules of complement per cell. However, in vivo removal of sensitized cells by macrophages can occur when cells are coated with fewer IgG molecules. Thus, the in vivo CASE STUDY (continued from page 403) life span of the sensitized cell can be significantly short- ened, as evidenced by the clinical findings of a typical When examining Nancy’s peripheral blood smear, DAT-positive hemolytic anemia, but the concentration the laboratory professional noted that spherocytes of antibodies on the cell could be insufficient to give a were present. The reticulocyte count was elevated. positive DAT.4,8 Newer, more sensitive techniques, such The laboratory professional called the blood bank as enzyme-linked DAT, gel tests, dual DAT procedure, and found that the DAT on Nancy was positive Polybrene tests, immunoblotting, and flow cytometry, can with polyspecific AHG, anti-IgG, but negative with detect lower concentrations of antibodies than the conven- anticomplement. tional DAT technique.9,12,13,14,15,16,17 The dual DAT test is 2. What is the significance of the spherocytes? able to identify IgM bound to erythrocytes in WAIHA.18 IgA or IgM autoantibodies are not detected using polyspe- 3. Based on these results, what do you suspect is cific AHG. However, if complement has been activated, going on in Nancy’s blood? Explain. it can be detected if sufficient molecules are present.1,11,19 In other cases the polyclonal activation in WAIHA may result in increased levels of reactive IgA.20 Specialized antihuman globulin reagents with anti-IgA or anti-IgM Autoimmune Hemolytic specificity is used in some reference laboratories to detect erythrocyte sensitization by immunoglobulins of these Anemias (AIHA) classes. In general, these cases of DAT-negative hemolysis Usually a person’s immune system recognizes self-antigens respond to steroid therapy in a similar manner to DAT- (those that appear on the individual’s own cells) and does positive cases.4 not mount an immune response to these antigens. These 408 Chapter 19 antigens can, however, stimulate an immune response if rate of hemolysis and hemoglobin levels (LAI). The type of injected into another individual because the recipient’s lysis may affect hemoglobin levels, since intravascular lysis immune system recognizes them as being “foreign.” The may destroy more cells per hour than extravascular lysis.2 immune regulatory mechanisms that govern response to Although WAIHA can occur at any age, the incidence self-antigens are collectively known as immune tolerance. increases after age 40. Childhood incidence peaks in the first It is generally accepted that autoimmune diseases occur 4 years of life, often after acute infections, and may pres- as the result of several factors including genetic predisposi- ent with more severe symptoms than adult onset.11,20,30,31 tion, exposure to infectious agents that can induce antibody About 60% of the cases of WAIHA are idiopathic and can be production due to molecular mimicry, and defects in the acute or chronic. In acute idiopathic WAIHA, severe anemia mechanism regulating immune tolerance including altered develops over 2 to 3 days, but the hemolysis is self-limited levels of CR1, and lack of effective T-regulatory cells (TReg). with a duration of several weeks to several years. In other T-regulatory cells are a subpopulation of T-cells that have instances, the hemolysis is chronic and does not abate. a role in immune tolerance and maintaining self-tolerance.

The underlying disorders most frequently associated The cells are known to inhibit T-cell proliferation and cyto- with secondary WAIHA are: kine production and help prevent autoimmunity.23 They • Lymphoproliferative disease, including chronic lym- also inhibit self-reactive T-cells. Decreased numbers of TRegs phocytic leukemia (CLL) and Hodgkin’s disease have been found in patients with autoimmune hemolytic (Chapter 28). CLL, found most frequently in older anemia.23,24,25 In addition, there is evidence for the role of adults, is the disease most frequently associated with Th17 cells in autoimmunity, including diseases such as rheu- the development of secondary WAIHA. WAIHA often matoid arthritis and systemic lupus erythematosus.26,27,28 develops late in the disease when the immune dysregu- The Th17 cells belong to a novel class of CD4+ T-cells that lation is greatest. Many children with idiopathic WAIHA secrete several cytokines including IL-17, IL-21, and IL-22, eventually develop a lymphoproliferative disease. which may play a role in development of autoimmune dis- ease. The level of IL-17, which is pro-inflammatory cytokine • Neoplastic diseases that amplifies inflammation, is increased in patients with • Other autoimmune disorders, including systemic lupus autoimmune hemolytic anemia. Significantly elevated lev- erythematosus and rheumatoid arthritis, ulcerative els of Th 17 cells correlate with disease activity.26,27 Another colitis, and Crohn’s disease. Those patients with SLE factor that may influence development of autoimmune dis- and WAIHA often also have increased risk for immune eases, including warm autoimmune hemolytic anemia, is an thrombocytopenia, which may or may not reflect levels imbalance of specific cytokines. IL-10 helps to promote anti- necessary for diagnosis of Evan’s syndrome.32 body production by B-cells while IL-12 suppresses humoral • Certain viral and bacterial infections, especially in immune response and promotes cell-mediated responses.24 infants and young children In one study patients with warm autoimmune hemolytic • Vaccinations anemia showed an imbalance in the levels of these cyto- kines.23 Warm or cold AIHA is also categorized based on • Solid organ transplant whether there is an underlying disease associated with it. Presence of the underlying disorder can complicate The two categories are: initial diagnosis and treatment of hemolytic anemia. In • Primary or idiopathic: no underlying disease identified many cases, the AIHA is resolved by treating the underly- ing disease. • Secondary: underlying disease present PATHOPHYSIOLOGY Warm Autoimmune Hemolytic The autoantibody in WAIHA is reactive with antigens on Anemia the patient’s erythrocytes. Most often the specificity of the antibody is directed against polypeptide antigens of the Rh Warm autoimmune hemolytic anemia (WAIHA) is the most system, although other antigen systems can be involved. common form of AIHA (up to 70% of cases).29 It is most When directed against the Rh system, the antibody can be often mediated by IgG antibodies (usually IgG1 or IgG3) specific for a single antigen (such as autoanti-e) or more whose optimal in vivo reactivity is at 37°C. In a majority commonly, the antibody reacts with a complex Rh antigen (90%) of WAIHA cases, erythrocytes are sensitized with IgG found on all erythrocytes except Rh null or Rh-deleted and complement or IgG alone. About 13% of cases are sensi- cells. The epitope against which these latter antibodies are tized with complement alone.29 WAIHA is rarely associated directed has not been defined. Recent research suggests that with IgM-only or IgA-only sensitization. In cases where the antibody can be against a component of the erythrocyte IgM is involved, the presence of complement alone on the membrane such as glycophorins, band 3 protein, or protein erythrocyte may be a clue.10 The subclass of IgG may affect band 4.1. Hemolytic Anemia: Immune Anemias 409 Most hemolysis in WAIHA is extravascular via splenic macrophages. Subclass of IgG present may affect the degree Table 19.4 Laboratory Findings Associated with WAIHA of hemolysis, with IgG3 associated with lower hemoglo- Common findings • Positive DAT bin levels.22 Although complement is not needed for cell • Normocytic, normochromic anemia destruction, if both antibody and complement are on the • Increased reticulocytes cell membrane, phagocytosis is enhanced. If erythrocyte • Spherocytes and other erythrocyte abnormalities destruction exceeds the compensatory capacity of the bone • Presence of autoantibody in the serum marrow to produce new cells, anemia develops. Direct • Increased serum bilirubin (total and complement-mediated intravascular hemolysis associated unconjugated) with IgM antibodies in WAIHA is rare. • Decreased serum haptoglobin • Elevated lactate dehydrogenase (LD) CLINICAL PRESENTATION • Positive antibody screen with all cells The most common presenting symptoms in idiopathic including autocontrol WAIHA are related to anemia. Progressive weakness, dizzi- • Incompatible crossmatches with all donors ness, dyspnea on exertion, back or abdominal pain, and jaundice are common. In secondary WAIHA, signs and Other laboratory findings • Increased osmotic fragility that can be associated with • Increased urine and fecal urobilinogen symptoms of the underlying disorder can obscure the fea- hemolysis in WAIHA • Hemoglobinemia,a hemoglobinuria, tures of the hemolytic anemia. The patient can present with methemoglobinemia, hemosiderinuria signs and symptoms of both the underlying disease and a Seen only in acute hemolytic episode. hemolysis or just the disease. The signs of WAIHA in a few patients can precede development of the underlying dis- ease. Although mild to moderate splenomegaly is present other poikilocytes are characteristic (Figure 19-4). Sphero- in more than 50% of patients with WAIHA, massive splenic cytes found in AIHA are usually more heterogeneous than enlargement suggests an underlying lymphoproliferative are the spherocytes associated with hereditary spherocytosis. disorder. Hepatomegaly is found in about one-third of This anisocytosis is readily noted upon examining the blood patients with primary WAIHA. smear and is indicated by an increase in red cell distribution width (RDW) on automated hematology analyzers. Eryth- rophagocytosis by monocytes is rarely seen. The mono- Checkpoint 19.5 cyte-engulfed erythrocyte is readily detected if the cell still What are the clinical findings and the immune stimuli for contains its pink-staining hemoglobin. If the hemoglobin has WAIHA? leaked out of the cell, however, only colorless vacuoles are seen. Leukocyte counts are normal or increased with neutro- philia in idiopathic WAIHA but can vary in the secondary Laboratory Evaluation form based on the underlying disease. Platelet counts are Refer to Table 19-4 for the most common laboratory findings associated with WAIHA. Findings such as a positive DAT, autoantibody in the serum, and presence of spherocytes on the peripheral blood smear reflect the immune-mediated destruction of the erythrocyte. Peripheral Blood Moderate to severe normocytic, normo- chromic anemia is typical and decreased hemoglobin is the most direct indicator of clinical severity.33 In well-compen- sated hemolytic disease, anemia can be mild or absent, and the only abnormal parameters may be a positive DAT and an increase in reticulocytes. Reticulocytes are invariably increased in uncomplicated hemolytic disease. The reticu- locyte production index can be as high as 6 or 7. Depending on the degree of reticulocytosis, macrocytosis can be present. Presence of reticulocytopenia may represent an autoimmune reaction against erythrocyte precursors and presents a more severe presentation with clinical necessity for transfusion.25,34 Figure 19.4 A blood smear from a patient with warm autoimmune hemolytic anemia (WAIHA). The marked anisocytosis The blood smear frequently shows erythrocyte abnor- is due to the presence of spherocytes and large polychromatophilic malities that suggest a hemolytic process. Polychromasia, erythrocytes. The nucleated cells are orthochromic normoblasts nucleated red blood cells, spherocytes, schistocytes, and (PB; Wright stain, 1000* original magnification). 410 Chapter 19 usually normal or slightly increased. Evan’s syndrome results the cross-match. In addition, approximately 30% of patients when severe thrombocytopenia accompanies WAIHA. with WAIHA have underlying alloantibodies, which can Bone Marrow Bone marrow examination is not necessary present an additional challenge to finding compatible blood because the autoantibody can mask their presence.35 for the diagnosis of WAIHA. The bone marrow can show erythroid hyperplasia, depending on the degree of hemoly- If the patient has an alloantibody, blood negative for the sis. Erythrophagocytosis by macrophages can be seen. Com- specific antigen must be administered. If serologically com- pensatory bone marrow response can be less than expected patible blood cannot be found because of the presence of the with concomitant folic acid deficiency. In chronic hemolysis, autoantibody, donor cells demonstrating the least incom- the folic acid requirement increases two to three times nor- patibility are usually chosen. These transfusions appear to mal; without folic acid supplements, the stores are quickly be safe and have little risk of increased hemolysis.36 In some depleted. If the patient contracts viral infections associated cases, the blood transfused is matched only for ABO and Rh with bone marrow suppression, life-threatening anemia type. The clinical problems with this approach, however, (aplastic crisis) can occur. are two-fold: donor cells (1) are often destroyed as rapidly as the patient’s own erythrocytes and (2) can also stimulate Other Laboratory Tests The DAT is a useful test to distin- the production of alloantibodies. The precautions of delay- guish the immune nature of this hemolytic anemia from ing or denying transfusion to patients who require it due nonimmune-mediated hemolytic anemias. The test is usu- to lack of serologically compatible blood may jeopardize ally positive with polyspecific AHG and anti-IgG mono- their survival.37 In some cases, the use of flow cytometry specific AHG antiserum. Only about 30% of the cases of can help evaluate the survival of donor cells in patients WAIHA have a reaction with anti-C3, either with or without with WAIHA. In other cases, such as individuals who must the concurrent presence of IgG. Complement only, IgM- or receive serial transfusions, finding phenotypically matched IgA-mediated lysis in WAIHA is rare.11,19 donor blood can decrease the risk of stimulating alloanti- Reaction of patients’ serum with commercial screening bodies. In patients who require serial transfusion, genotyp- cells typically shows agglutination with all cells when AHG ing of the patient may be an option that permits selection of is added. The autocontrol (patient serum and patient eryth- antigen-matched units without interference from autoanti- rocytes) shows similar reactions. Other laboratory findings body-coated cells and may provide cells with better in vivo are nonspecific but reflect the hemolytic component of the survival.38,39 If the autoantibody has an identifiable single condition (Table 19-4). specificity (such as autoanti-e), the donor blood chosen for DIFFERENTIAL DIAGNOSIS the compatibility test should be negative for the antigen. The diagnosis of WAIHA requires clinical features of hemo- Therapies for WAIHA focus on decreasing the produc- lysis as well as serologic evidence. WAIHA with the pres- tion of autoantibodies and slowing down the destruction of ence of spherocytes can be differentiated from hereditary erythrocytes. These therapies include:35,37,40 spherocytosis (HS) by the DAT. Antibodies are not respon- • Corticosteroids The standard initial or first-line ther- sible for forming spherocytes in HS; therefore, the DAT is apy for patients with WAIHA is often a course of negative. The autohemolysis test is abnormal in both HS immunosuppressive drugs such as corticosteroids. and in WAIHA with spherocytes. The addition of glucose Corticosteroids are used to produce immunosuppres- significantly corrects autohemolysis in HS, but not in sion by decreasing lymphocyte proliferation and sup- WAIHA. The peripheral blood smear also gives clues to pressing macrophage sequestration of sensitized cells diagnosis. The spherocytes in HS appear as a rather homo- by affecting the Fc receptors. Although many patients geneous population, but WAIHA has a mixture of sphero- show a good initial response with a decrease in eryth- cytes, microspherocytes, and normocytes. rocyte destruction, few patients experience complete remission with this therapy. A decrease in production of Checkpoint 19.6 autoantibodies is often demonstrated by a decrease in What is the DAT pattern in WAIHA? Explain why spherocytes the strength of the DAT reaction and indirect antiglob- are commonly seen in WAIHA. ulin test. Response usually begins within the second week of treatments, and many patients require long- term low-dose maintenance to prevent relapse. Patients THERAPY who do not respond are often placed on second-line Transfusion therapy is not necessary in self-limiting hemo- therapy. Patients with underlying lymphoproliferative lytic disorders without life-threatening hypoxemic anemia. diseases or SLE often show a reduced response to cor- When transfusion is indicated (more often associated with ticosteroids, necessitating second-line therapy.35,40,41,42,43 acute onset), finding a suitable donor is difficult because the Standard second-line treatments with the most efficacy patient’s autoantibody usually reacts with all donor cells in for individuals who do not respond to corticosteroids Hemolytic Anemia: Immune Anemias 411 includes splenectomy or the use of monoclonal anti- some refractory patients as adjunct therapy.44

There is body treatment (rituximab). no recommended therapy for secondary WAIHA.35,40 • Splenectomy Splenectomy has been a standard second- Treatment of the underlying disease in patients with line therapy for years and provides long-term remission secondary WAIHA is important. Often resolution for some patients.35,40 Removal of the spleen decreases of the disease leads to decreased production of the the destruction of IgG-coated erythrocytes that would autoantibody. normally be removed by splenic macrophages. If, however, the antibody concentration remains high, the destruction of sensitized erythrocytes can continue in Cold Autoimmune Hemolytic the liver. Some evidence suggests that splenectomy can Anemia be more beneficial in patients with idiopathic WAIHA Cold AIHA, also termed cold agglutinin disease (CAD) or than in those with the secondary type. The primary cold agglutinin syndrome (CAS), is associated with an IgM complication is sepsis related to infections, especially antibody that fixes complement and is reactive below 37°C. with encapsulated bacteria. This disorder, which comprises 16–30% of the AIHAs, is less • Rituximab (monoclonal anti-CD20) Rituximab has common in children than adults. IgA and IgG antibodies been used in the treatment of clonal B-cell malignan- rarely have been implicated in hemolysis in CAS. cies, including chronic lymphocytic leukemia, in CAS, like WAIHA, is either idiopathic or second- cases of autoimmune collagen vascular disease and ary (Table 19-5). Unlike the autoantibody associated with in patients with cold agglutinin syndrome. It is rec- WAIHA, however, this autoantibody is routinely present ommended as a second-line therapy in patients with in the plasma and is often not of clinical significance. Idio- idiopathic and secondary WAIHA that are refractory to pathic CAS is usually chronic, occurring after age 50 with other treatments and is being used in place of splenec- a peak onset after age 70. The antibody involved is usually tomy in many patients.18 Children appear to respond a monoclonal IgM/k light chain with autoanti-I specificity. better than adults.40 Rituximab depletes the B cells that The secondary type can occur after an infectious disease produce autoantibodies but does not apparently affect or be associated with lymphoproliferative disease. The sec- plasma cells or hematopoietic stem cells. ondary type associated with infectious disease is usually • Danazol This drug is a synthetic anabolic steroid that has an acute, self-limiting form that has an onset 1–3 weeks been used as a first-line treatment with corticosteroids. after infection and resolves within 4–6 weeks.45 Most of • Cytotoxic drugs Various alternative therapies are avail- these autoantibodies are polyclonal and have a specificity able for patients who do not respond to first- or second- for antigens of the Ii system. Anti-I is usually associated line therapy or who have recurrent disease. Cytotoxic with Mycoplasma pneumoniae infections but may also be drugs such as cyclophosphamide, cyclosporine A, vin- associated with viral infections including HIV. Anti-i can cristine-loaded platelets, mycophenolate mofetil, and be seen in infectious mononucleosis. Up to 75% of chronic azathioprine cause general suppression of the immune CAS have an underlying bone marrow lymphoprolifera- system, which decreases synthesis of autoantibody. tive B cell disorder.46,47,48,49,50 The clonality can be demon- strated via flow cytometry of immunohistochemistry. The • High-dose intravenous immunoglobulin (IVIG) This secondary chronic form of CAS associated with lymphop- treatment blocks Fc receptors on macrophages and roliferative disorders, such as lymphoma or Waldenstrom’s affects T- and B-cell function by increasing TReg cells or macroglobulinemia, is typically found in older individuals. reducing B-cell function. It has a variable success rate The antibody in these cases is usually a monoclonal IgMk and is most often used as an adjunct therapy with cor- ticosteroids or in selected patients with severe anemia who are refractory to drugs. Table 19.5 Autoimmune Hemolytic Anemia Caused by • Therapeutic plasma exchange and plasmapheresis Cold-Reacting Antibodies This procedure can dilute or temporarily remove the Cold agglutinin syndrome Primary (idiopathic) autoantibody from the patient’s circulation. It has been (CAS) Secondary successful in reducing antibody load for a short time in • Infection associated some cases but is not a satisfactory long-term therapy. It • Mycoplasma pneumonia infections, infectious mononucleosis is now recommended as a “last ditch” effort for patients • Lymphoproliferative disease associated with severe hemolysis.35,40 Paroxysmal cold Primary (idiopathic) • Hematopoietic stem cell transplants are not recom- hemoglobinuria Secondary mended as their effectiveness has not been proven. Use • Viral infections of erythropoiesis-stimulating agents has been used in • Tertiary syphilis 412 Chapter 19 protein. A more severe type of cold AIHA, paroxysmal cold cell volume (MCV) is falsely elevated (Table 19-6). These hemoglobinuria (PCH), is associated with a biphasic cold- erroneous values occur when erythrocyte agglutinates are reacting IgG antibody and is discussed in the next section. sized and counted as single cells. The hematocrit, calcu- lated from the erroneous erythrocyte count and MCV, is PATHOPHYSIOLOGY falsely low. The mean cell hemoglobin (MCH) and mean The severity of CAS is related to the thermal range of the cell hemoglobin concentration (MCHC) calculated from the antibody. Cold-reacting antibodies with a wide range of erythrocyte count and hematocrit are falsely elevated. The thermal activity (up to 32°C) can cause problems when the hemoglobin assay is accurate because the cells are lysed peripheral circulation cools to this temperature. Comple- to determine this parameter. Accurate cell counts can be ment-mediated lysis accounts for most of the erythrocyte obtained by warming the blood and diluting reagents to destruction. 37°C before performing the test. Visible autoagglutination The cold-reacting antibody is usually directed against in tubes of anticoagulated blood can be observed as the the I antigen, which is expressed on erythrocytes of almost blood cools to room temperature. all adults. I antigen specificity of the antibody can be defined When blood counts are performed at 37°C, the results by reactivity of the patient’s serum with all adult erythro- indicate a mild to moderate normocytic, normochromic cytes but minimal or no reactivity with cord cells (which anemia. The blood film shows polychromasia, some sphe- lack the I antigen). In CAS associated with infectious mono- rocytes, rouleaux, or clumps of erythrocytes, and some- nucleosis however, the antibody may have anti-i specific- times nucleated red cells (Figure 19-5). Erythrophagocytosis ity. These antibodies react strongly with cord cells because can be seen but is more typical on smears made from buffy the i antigen is generally expressed strongly on cord cells coats after the blood has incubated at room temperature. and erythrocytes of children younger than 2 years old and Leukocyte and platelet counts are usually normal. Leuko- weakly or not at all with adult erythrocytes. The second cytosis can occur during acute hemolysis as the result of a most common specificity for cold auto agglutinins is anti-Pr, bone marrow stress response. The bone marrow exhibits which can be stimulated by infections with rubella virus or normoblastic hyperplasia often with an increase in lym- the varicella virus. The Pr antigens are expressed on both phoid cells. Patients with chronic CAS may show decreased adult and infant erythrocytes. C3 and/or C4 levels. Patients also can have a decreased CLINICAL PRESENTATION value for the CH50 assay, a functional hemolytic assay In some instances, CAS is associated with a chronic hemo- that measures the integrity of the entire complement cas- lytic anemia with or without jaundice. In others, hemoly- cade. Bilirubin and LD may be increased depending on the sis is episodic and associated only with chilling. degree of hemolysis. Erythrocyte agglutination occurs in areas of the body that cool to the thermal range of the antibody and cause sludg- ing of the blood flow within capillaries. Vascular changes including acrocyanosis in which the hands and/or feet Table 19.6 Criteria for Clinical Diagnosis of Cold turn blue and cold can occur. In Raynaud’s phenomenon, Agglutinin Syndrome (CAS) pain can be accompanied by a characteristic pattern of Clinical history • Acrocyanosis color changes in the skin from white (due to spasm of the • Hemoglobinuria on exposure to cold vessels) to blue (caused by cyanosis) to red (which indi- Laboratory evaluation • Serological cates a return of blood flow to the area). These conditions DAT: Positive with polyspecific AHG primarily affect the extremities, especially the tip of the Negative with anti-IgG; Positive with nose, fingers, toes, and ears. Hemoglobinuria accompa- anti-C3 IAT: Antibody showing characteristic reac- nies the acute hemolytic attacks. Splenomegaly can be tions at less than 25°C present (Table 19-6). Cold agglutinin titer more than 1000 at 4°C • False increase in MCV, MCH, and MCHC • False decrease in erythrocyte count Checkpoint 19.7 • Normocytic, normochromic anemia Describe the mechanism of cell destruction in CAS. • Reticulocytosis • Spherocytes, agglutinated RBCs, rouleaux, nucleated RBCs on blood smear • Increased bilirubin (total and unconjugated) LABORATORY EVALUATION • Increased LD The first indication of the presence of unsuspected cold • Decreased haptoglobin agglutinins often is from blood counts performed on elec- • Hemoglobinemia, hemoglobinuria in acute tronic cell counters. The erythrocyte count is inappropri- hemolysis ately decreased for the hemoglobin content, and the mean • Hemosiderinuria in chronic hemolysis Hemolytic Anemia: Immune Anemias 413 or only weakly positive with benign cold agglutinins. The DAT, utilizing monospecific anti-IgG, is negative both in CAS and benign cold agglutinins. Historically, the cold agglutinin test was performed when CAS was suspected. This test demonstrates the ability of the pathologic antibody to agglutinate the patient’s cells at temperatures from 0°C to 20°C in saline. The reaction is reversible with agglutinates dispersing at 37°C. With benign cold antibodies, agglutination occurs at 0–4°C and can occur up to 20°C. Titers of benign cold agglutinins reach 1:64 in normal individuals; in cold agglutinin disease, the titer is usually more than 1:1000. Titers of 1:256 or more with a positive DAT using monospecific anti-C3 antisera and a negative DAT using monospecific anti-IgG antisera are Figure 19.5 Cold autoimmune hemolytic anemia from a highly suggestive of cold agglutinin disease. patient with chronic lymphocytic leukemia. Some of the erythrocytes are in small clumps. Spherocytes are also present (peripheral blood, Wright stain, 1000* original magnification). Checkpoint 19.9 Compare the DAT findings and the antibody specificity in WAIHA and CAS. Checkpoint 19.8 Explain why the MCV, MCH, and MCHC can be falsely increased when blood from someone with CAS is tested using an auto- THERAPY mated cell counter. The most effective therapy in primary CAS is usually achieved by keeping the individual’s extremities warm. Corticosteroids are not usually effective. Chemotherapy DIFFERENTIATION OF CAS AGGLUTININS FROM using rituximab has been somewhat effective in primary BENIGN COLD AGGLUTININS CAS and CAS associated with an underlying lympho- The serum of most normal individuals exhibits the pres- proliferative disease and is recommended as a first-line ence of cold autoantibodies with anti-I specificity when the therapy.35,40 When used with fludarabine, the response rate serum and cells are incubated at 4°C. These antibodies are for patients increases. Recently, Eculizumab, a monoclonal termed benign cold autoagglutinins because their thermal antibody that binds to C5, has been used to treat patients amplitude and concentration (titer) are not high enough refractory to rituximab. This complement inhibitor binds to to cause clinical problems. When pathologic cold aggluti- C5 and inhibits formation of the membrane attack complex nins (increased titer and increased thermal amplitude) are (MAC), thus inhibiting hemolysis.51 Another new drug tar- suspected as the cause of anemia, laboratory tests should gets the serine protease C1s, thus preventing attachment be performed to differentiate pathologic cold agglutinins of the C1 molecule to antibody and preventing intravas- from the benign ones (Table 19-7). The DAT with polyspe- cular hemolysis.46,52,53 Plasma exchange can be used in cific AHG and monospecific anticomplement antiserum is acute hemolytic episodes because most IgM is distributed positive in pathologic cold agglutinin disease but negative in the intravascular spaces. However, plasma exchange is Table 19.7 Comparison of Characteristics of Pathologic Cold Agglutinins Found in CAS with Those of Benign Cold Agglutinins Found in Normal Individuals Pathologic Agglutinins Benign Agglutinins Antibody class IgM IgM Antibody specificity Usually anti-I but in secondary CAS can be anti-i anti-I Antibody clonality Monoclonal in idiopathic type and secondary type due to lympho- Polyclonal proliferative disease; polyclonal in secondary type due to infec- tious disease Thermal amplitude 0–30°C 0–4°C Agglutination at room temperature Significant Not present Titer Usually more than 1:1000 Less than 1:64 DAT Positive with polyspecific AHG and monospecific anticomplement Negative 414 Chapter 19 effective for only a short time because

protein regeneration CLINICAL PRESENTATION half-life is ∼5 days. Splenectomy, which can be effective in Hemoglobinuria is the most common clinical symptom. WAIHA, is not effective in CAS because the C3b-coated cells The patient can also experience jaundice, pallor, and are destroyed primarily by Kupffer cells in the liver. Theo- hepatosplenomegaly. Raynaud’s phenomenon can occur retically, patients with strongly reacting cold agglutinins during acute episodes followed by jaundice. Rare cases may have complications if surgical procedures requiring of acute renal failure have been associated with severe hypothermia are undertaken. There is little evidence this hemolysis.61 is a great risk.51,54,55 Like secondary WAIHA, resolution of LABORATORY EVALUATION secondary CAS usually occurs when the underlying condi- The degree of anemia depends on the frequency and sever- tion has been treated. ity of hemolytic attacks. During the attack, hemoglobin con- centration drops sharply accompanied by hemoglobinemia, Paroxysmal Cold Hemoglobinuria methemalbuminemia, and hemoglobinuria. Hemoglobin Paroxysmal cold hemoglobinuria (PCH) is a rare but acute values can decrease to as low as 5 g/dL. Neutropenia, a and rapidly progressive autoimmune hemolytic disorder neutrophilic shift to the left, reticulocytopenia, and sphe- that is characterized by massive intermittent acute hemoly- rocytes can accompany erythrocyte lysis. Other abnormal sis and hemoglobinuria. Although it is the cause of about 2% erythrocyte morphology including anisocytosis and poi- of autoimmune hemolytic anemias overall, it is the cause of kilocytosis as well as erythrocyte fragments may be seen.61 30–40% of AIHA in children and is most frequent in children Serum bilirubin, blood urea nitrogen, and lactic dehydro- under the age of 5 years. Historically, PCH was associated genase (LD) are elevated, whereas serum complement and with congenital or tertiary syphilis in adults. It is now most haptoglobin are decreased. Erythrophagocytosis occurs often seen in children with viral and bacterial infections, more commonly in PCH than in other types of AIHA, and although it has been rarely reported in adults due to various the phagocytic cells usually involved are segmented neu- clinical presentations.56 The infections linked to PCH include trophils, not monocytes. Epstein-Barr virus, cytomegalovirus, measles, mumps, Hae- Antibodies on the cells are not usually detected by the mophilus influenzae, and Klebsiella pneumoniae. Infections have DAT because the D-L antibody elutes at warm tempera- been linked with Parvovirus 19 as the P antigen serves as a tures. A weakly positive DAT with anticomplement AHG receptor for this virus. Post-vaccination PCH has also been can appear and persist for several days after the hemolytic reported.57,58 PCH is usually a transient disorder that appears episode. The IAT can be positive if performed in the cold. 5 days to 3 weeks after infection onset but can persist up to 3 Normal erythrocytes incubated with patient serum react months. It generally resolves spontaneously, but transfusion more positively in the IAT than will patient cells. can be required in cases of severe, life-threatening anemia. D-L antibodies are usually present in low titers (less Treatment with plasma exchange or immunosuppressive than 1:32) and express a low thermal amplitude, but their drugs may be used in cases of severe hemolysis. presence can be verified by the D-L test, which employs a biphasic reaction (Table 19-8). In this test, the patient’s PATHOPHYSIOLOGY blood is collected in two clot tubes (serum, not plasma, is PCH was the first hemolytic anemia for which a mechanism required); one is incubated at 4°C for 30 minutes and the of hemolysis was established. This hemolytic anemia is dis- other at 37°C for 30 minutes. Both tubes are then incubated tinct from the other cold AIHAs because of the nature of the at 37°C. If the D-L antibody is present, it causes hemolysis in antibody involved. It is caused by a biphasic complement- the tube initially incubated at 4°C and then warmed to 37°C. fixing IgG antibody, the Donath-Landsteiner (D-L) anti- No hemolysis is present in the tube kept at 37°C. Hemoly- body, which has an auto-anti-P specificity. Although IgG sis in this test can also occur in cold agglutinin syndrome is the most common antibody class involved in PCH, there (CAS), but the hemolysis occurs very slowly. See Table 19-9 have been reports of IgM and IgA.58,59,60 In rare cases of IgM for a comparison of PCH and CAS. class antibody, the specificity was biphasic anti-I, while oth- ers were anti-P specificity.58,60 Biphasic refers to the two tem- THERAPY peratures necessary for optimal lysis of the erythrocytes. The PCH associated with acute infections terminates upon antibody reacts with erythrocytes in the capillaries at temper- recovery from the infection. Steroids are not usually atures less than 20°C and avidly binds the early acting com- helpful. Transfusion can be required if the hemolysis is plement components. Upon warming to 37°C, the antibody severe. In rare cases when the hemolysis persists, plas- molecule detaches from the cell, but the membrane attack mapheresis can be used. Rituximab has also been used complement components are activated on the cell membrane as therapy in rare adult cases. The chronic form of the causing cell lysis. Because the antibody repeatedly attaches disease is best treated by avoiding exposure to the cold. and detaches from erythrocytes with subsequent comple- Since hemolysis is primarily intravascular, splenectomy ment activation, it can cause significant hemolysis. is not indicated.59 Hemolytic Anemia: Immune Anemias 415 Both C3 and IgG can be detected on the erythrocyte in Table 19.8 Donath-Landsteiner (D-L) Test for Detecting the Presence of D-L Antibodiesa the DAT. The cold-reacting antibody often has specificity for the Ii system antigens. The warm-reacting autoantibody is Patient’s Whole Blooda Control Test like those found in classic WAIHA and often has a complex Incubate for 30 min at 37°C 4°C Rh specificity. Incubate for 30 min at 37°C 37°C Patients usually respond well to treatment with cortico- Centrifuge: observe plasma steroids and do not require transfusion. Rituximab has been for presence of hemolysis used in cases with underlying lymphoproliferative disease.5 Interpretation D-L antibodies present No hemolysis Hemolysis Drug-Induced Hemolytic Anemias No D-L antibodies present No hemolysis No hemolysis Although the occurrence is rare, certain drugs can cause a Two tubes of patient’s whole blood are used; one tube serves as the control and the immune cytopenias that involve one or more cell lineages other as the test. including neutrophils, platelets, and erythrocytes. Anemia, thrombocytopenia, and agranulocytosis can occur together or separately. It has been proposed that a drug’s ability to Checkpoint 19.10 induce production of antibodies against different cell lin- Compare the antibody specificity and the confirmatory test for eages is related to its affinity for the cells. The greater the PCH and CAS. affinity, the more likely sensitization against the drug–cell complex is to occur. Drug-induced immune hemolytic anemia is a relatively uncommon acquired condition pre- Mixed-Type AIHA cipitated by certain drugs. The drug itself does not cause erythrocyte injury, and not all individuals taking the drug Mixed-type AIHA is characterized by the presence of a develop this immune reaction. warm-reacting IgG autoantibody and a cold-reacting IgM More than 135 drugs have been found to induce a autoantibody that has both high titer and increased ther- positive DAT or an immune-mediated hemolytic anemia.65 mal amplitude (reacts at more than 30°C). It is less common The classes of drugs implicated include antimicrobials, non- in children than adults.62 About 50% of the cases are idio- steroidal anti-inflammatory drugs, antineoplastic drugs, pathic; most of the remainder are associated with diseases diuretics, and antidiabetic drugs. Note that second- and such as systemic lupus erythematosus or lymphoprolifera- third-generation cephalosporins such as cefotetan and tive diseases.63,64 Patients may have symptoms that are con- ceftriaxone constitute the majority of cases of drug- sistent with each type and frequently present with an acute, induced immune hemolytic anemia and are responsible for more severe anemia and can have a more chronic course most fatalities with ceftriaxone causing more than 50% of the with intermittent exacerbations.34,62 Reticulocytopenia has fatalities. Piperacillin is now the most common drug asso- also been noted in several cases of mixed WAIHA and DAT- ciated with DIHA.66 Chemotherapeutic drugs, especially negative hemolysis.34 The IgG antibody mediates extravas- purine analogs such as fludarabine that are used to treat cular hemolysis, and the IgM is responsible for complement patients with leukemia and some types of lymphoma, are fixation and intravascular hemolysis. In some cases, the another class of drugs associated with DIHA. Drug-induced cold-reacting antibody triggers increased hemolysis and immune hemolysis must be distinguished from both drug- hemoglobinuria when the patient is exposed to the cold. induced nonimmune hemolysis that occurs secondarily to Table 19.9 Comparison of Cold Agglutinin Syndrome (CAS) and Paroxysmal Cold Hemoglobinuria (PCH) CAS PCH Patient Usually adults more than 50 years of age Usually children after viral infection Clinical findings Acrocyanosis Chills, fever, hemoglobinuria DAT Positive with polyspecific AHG and monospecific C3 Positive with polyspecific AHG and monospecific C3 Donath-Landsteiner test Negative Positive Antibody class IgM Biphasic IgG (D-L) Antibody specificity Anti-I Anti-P Thermal amplitude of antibody Up to 30°C Under 20°C Hemolysis Chronic extravascular/intravascular Acute intravascular Therapy Avoid the cold Supportive; treatment of underlying illness 416 Chapter 19 erythrocyte metabolic defects such as G6PD deficiency and MECHANISM OF IN VIVO ACTION FOR DRUG-INDE- from spontaneous autoimmune disorders. This is important PENDENT ANTIBODIES because drug-induced, immune hemolytic anemias are the In the drug-independent mechanism, the drug binds to the result of an immune response to drug-induced alteration of erythrocyte; however, the antibody is produced against the erythrocyte. The resolution for this immune hemolysis primarily erythrocyte epitopes. This can be due to altera- involves withdrawal of the offending drug and support- tion of the erythrocyte membrane by the drug or molecu- ive treatment, not the use of immunosuppressives or other lar mimicry. This mechanism mimics warm autoimmune therapy. hemolytic anemia in that the patient’s serum reacts with all Although historically there were four mechanisms used erythrocytes against which it is tested. It is important that to describe the mechanisms of drug-induced hemolysis these be distinguished so that appropriate treatment can be (drug adsorption, immune complex, membrane modifica- initiated.69,70 The mechanism by which antibody produc- tion, and autoantibody-like), with our current understand- tion is induced is unknown; however, evidence indicates ing of the process a “unifying hypothesis” has been that the drug alters normal erythrocyte antigens so they proposed to explain antibody formation in drug-induced are no longer recognized as self. Historically this mecha- hemolytic anemias. The hypothesis proposes that once a nism was linked to the antihypertensive drug Aldomet drug binds to the erythrocyte membrane, antibodies can be (a@methyldopa). In approximately 20% of patients on the produced to react with epitopes specific to the drug, with drug, a positive DAT develops after about 3–6 months. epitopes that represent a combination of drug and erythro- However, hemolytic anemia develops in only 1% of these cyte proteins, or with epitopes primarily on the erythrocyte patients. Fludarabine now is the drug most commonly asso- membrane. This helps to explain how patients can develop ciated with this type of drug-independent mechanism for more than one type of drug-induced antibody. antibody formation, although other drugs have been impli- Currently, two major types of drug-induced antibodies cated when there is an underlying condition.5,65,71 Eryth- have been described: drug dependent and drug indepen- rocyte destruction is extravascular, and anemia develops dent. They are named based on the type of in vitro testing gradually. If the drug is withdrawn, the antibody produc- required for identification. Drug-dependent antibodies are tion gradually stops, but the DAT can remain positive for those that require the presence of the drug during in vitro years. The DAT is dose dependent. The larger the dose testing. Drug-independent antibodies are those that react of the drug, the more likely the patient is to have a posi- in vitro without the presence of the drug. A third category, tive DAT. The DAT using anti-IgG is positive but because nonimmunological protein adsorption (NIPA), causes posi- complement is rarely activated, the DAT using anti-C3 is tive DAT but is rarely linked with hemolysis.66 usually negative. Workups for investigating drug-induced hemolytic In nonimmunological protein adsorption (NIPA) some anemia may be quite extensive. They can require DAT, drugs, b@lactamase inhibitors, cefotetan, and platinum- reaction of patient’s serum with drug-coated cells, and rou- based chemotherapeutic agents such as oxaliplatin can tine antibody investigations. This is important to be able to alter the erythrocyte membrane so that

immunoglobu- distinguish these reactions from transfusion reactions, true lins and other serum proteins bind nonspecifically to the autoantibody formation, or other clinical conditions.65,67 membrane.66,70 No drug antibody is involved. DAT using polyspecific AHG is positive; the anti-IgG or anti-C3 can MECHANISM OF IN VIVO ACTION FOR be positive or negative. This mechanism is known to cause DRUG-DEPENDENT ANTIBODIES hemolysis in some patients. The coated cells are removed The mechanism of action for the drug-dependent type anti- via macrophage receptors.65,70 bodies can occur in two ways. In one, the drug binds cova- Table 19-10 contains a summary of the characteris- lently to the erythrocyte and antibodies are produced only tics of each mechanism. Regardless of the underlying against the drug epitopes. Antibody forms and attaches mechanism, erythrocytes sensitized with either anti- to the drug-coated erythrocyte, which is then removed body and/or complement have a shortened life span. by splenic macrophages. Drugs often associated with this Each of these mechanisms causes a positive DAT with mechanism are intravenous penicillin, piperacillin, and polyspecific AHG and with either anti-IgG and/or anti- cephotetan. The second mechanism for drug-dependent C3 AHG. antibody occurs when the drug binds only weakly to the erythrocyte and antibody is formed to epitopes that consist of both drug and erythrocyte membrane. The antibody can Checkpoint 19.11 fix complement and cause intravascular lysis. Ceftriaxone is Compare the different types of drug-induced hemolysis includ- one of the drugs commonly associated with this mechanism ing the type of hemolysis, the drug usually associated with the and is linked to severe intravascular hemolysis often within mechanism, and the DAT profile. hours of administration.66,68 Hemolytic Anemia: Immune Anemias 417 Table 19.10 Summary of Classic Mechanisms of Drug-Induced Immune Hemolytic Anemia Type (Prototypic Drug) Action Direct Antiglobulin Test Mechanism of Cell Destruction Drug dependent Drug bound to cell S antibody forms primarily Polyspecific AHG positive, anti-IgG Extravascular adhesion to to drug epitopes and binds to drug positive, anti-C3 can be positive macrophages via FcgR and phagocytosis Drug binds loosely to erythrocyte S antibody Polyspecific AHG positive, anti-IgG Intravascular complement- forms to epitopes of drug and cell S activates negative, anti-C3 positive mediated lysis complement cascade S antibody leaves cell Drug independent Drug adheres to cell membrane S antibody Polyspecific AHG positive, anti-IgG Extravascular adhesion to Autoantibody-like forms primarily against epitopes on erythrocyte positive, anti-C3 positive or negative macrophages via FcgR and membrane S antibody reacts with erythrocyte phagocytosis Nonimmune protein Modification of cell membrane that results in Polyspecific AHG positive, mono- Hemolysis can result adsorption nonimmunologically absorbed IgG, IgA, IgM, C3 specific can be positive or negative autoimmunity, or passenger lymphocyte syndrome. Pas- CASE STUDY (continued from page 407) senger lymphocyte syndrome is an immune hemolytic process Two days later, Nancy’s hemoglobin dropped to that develops often within the first week post-transplant. 50 g/L. The physician ordered several more tests. It may occur following solid organ (heart, lung, kidney), She had a positive IAT, and the antibody reacted bone marrow, or stem cell transplant.75 The donor B lym- with all cells including her own. Other test results phocytes and/or plasma cells transplanted with the organ indicated that this patient had systemic lupus or bone marrow produce antibodies against the recipient’s erythematosus. blood group antigens. Although ABO incompatibility is most frequently implicated, other blood group system anti- 4. What type of antibody appears to be present in bodies (anti-D, anti-K, or other Rh system antibodies) may Nancy? Explain. be involved.75,76 Development of autoantibodies after solid 5. What is the relationship of Nancy’s primary organ transplant is rare but is more common after bone mar- disease, systemic lupus erythematosus, and her row or stem cell transplants.34,35 Hemostatic expansion post- anemia? HSCT may trigger loss of self-tolerance, leading to increases in autoreactive cells.73,77 Post-HSCT infections may also trigger development of antibodies that have the ability to cross react with human erythrocyte antigens, stimulating Alloimmune Hemolytic an autoimmune process.77 Blood transfusions can, in some patients, lead to the development of autoantibodies. In most Anemia cases, the hemolysis from passenger lymphocyte syndrome is usually self-limiting as the donor B lymphocytes gener- Hemolytic anemia induced by an individual’s immuni- ally do not survive.74 zation with erythrocyte antigens on the infused cells of Another type of immune-mediated hemolysis is recog- another individual is known as alloimmune hemolytic nized in patients receiving high-dose intravenous immu- anemia. The patient’s erythrocytes lack the antigen(s) pres- noglobulin (IVIG) therapy (more than 2 g/kg body weight ent on infused cells. These transfused antigens are recog- or 100 g IVIG in 2–4 days). IVIG, made from the plasma nized as foreign and induce the recipient to form antibodies of thousands of donors, is used in treating a variety of that, in turn, react with the transfused cells. This type of autoimmune and immunodeficiency syndromes including immunologic destruction of erythrocytes is characteristic Guillian-Barre syndrome, Kawasaki disease, immune-medi- of transfusion reactions and hemolytic disease of the fetus ated thrombocytopenia, and antibody-mediated transplant and newborn (HDFN). Factors such as the immunogenicity rejection. of the antigen, number of transfusions, and function of the A two-hit theory is proposed to explain the hemolysis recipient’s immune system can influence the development seen with IVIG. The first factor is that the IVIG recipient is a of alloantibodies. non–Group O individual (Group A, Group B, or Group AB). There is evidence of the production not only of alloan- Most cases are seen with individuals with Group A blood. tibodies in patients undergoing solid organ and allogeneic The second factor is the presence of an underlying inflam- bone marrow/stem cell transplants but also of autoanti- matory condition.78,79 The non-immune-stimulated anti-A bodies.72,73,74 Post-transplant immune-mediated hemolysis and anti-B in IVIG may attach to erythrocytes. The under- can be linked to major blood group incompatibility, pas- lying condition activates splenic macrophages to increase sive transfer of antibody from the donor, development of lysis of antibody or complement coated cells. Despite the 418 Chapter 19 relatively common positive DAT and hemolysis caused by complications of the reaction (e.g., shock, renal failure, dis- anti-A or anti-B, there is no evidence that anti-D in IVIG seminated intravascular coagulation [DIC]). Laboratory causes lysis. investigation of the reaction must be performed. Lysis develops in about one-third of cases as early as The delayed hemolytic transfusion reaction is usually 24 hours after treatment. In other cases, it may take as the result of an anamnestic response in which the donor long as 2 weeks post-treatment.79 The lysis is usually erythrocytes contain an antigen to which the patient has self-limiting and primarily extravascular and may result been previously sensitized. In these cases, the antibody was in a slight decrease in hemoglobin, a positive DAT.78,80,81 not detectable prior to transfusion, but the infused eryth- Patients receiving high dose IVIG should have hemoglobin rocytes restimulate antibody production, destroying the levels followed at 36 and 96 hours post-infusion.81 donor cells containing the antigen. In a delayed transfu- sion reaction, some patients show no clinical signs, and the Hemolytic Transfusion Reactions reaction is detected only when laboratory tests demonstrate that the expected increase in hemoglobin has not occurred Transfusion of blood can cause a hemolytic transfusion or when, upon subsequent testing, demonstrate a positive reaction as the result of the interaction of foreign (nonself) DAT or presence of a previously unidentified alloantibody. antigens on transfused erythrocytes and alloantibodies Other patients can present with dark urine or vague symp- in the patient’s plasma. In contrast to AIHA, the antibod- toms such as fever and fatigue post-transfusion, which will ies produced in transfusion reactions cause immunologic trigger an investigation. Patients at highest risk for delayed destruction of donor cells but do not react with the eryth- reactions are those who receive multiple transfusions over rocytes of the person making the antibody. However, many their lifetime (e.g., patients with aplastic anemia, sickle cell of the same factors involved in autoimmune hemolytic ane- anemia). Patients with sickle cell anemia or other hemo- mia (e.g., antibody class/subclass, complement activation, globinopathies may undergo hyperhemolysis in which not antigen density, antibody titer, efficiency of Fc receptors) only the donor cells but also the patient’s own erythrocytes are also involved in this immune response.82 The two types are lysed.82,84 of transfusion reactions involving alloantibodies to eryth- rocyte antigens are acute (occurring within 24 hours) and PATHOPHYSIOLOGY delayed (occurring 2–14 days after transfusion) (Table 19-11). An acute hemolytic transfusion reaction is characterized by An acute hemolytic transfusion reaction results when the intravascular hemolysis with hemoglobinuria because of infused erythrocytes react with antibodies that already exist complete activation of the complement cascade. IgM anti- in the recipient, usually ABO system antibodies, although bodies usually mediate this type of hemolysis of donor cells, other system antibodies have been implicated.83 This type although IgG antibodies are rarely involved. Acute hemo- of reaction usually is caused by clerical or other human lytic transfusion reaction is typical of an ABO incompatibil- error that results in an ABO mismatch between donor and ity and begins very shortly after the infusion of the donor recipient. For example, the patient is given the wrong unit unit has begun. As little as 10 mL of ABO-incompatible of blood or is misidentified by the phlebotomist, nurse, or blood can trigger symptoms of an acute hemolytic laboratory personnel. Most errors occur in patient care areas, reaction.29 As cells are lysed, the release of erythrocyte mem- not in the transfusion service area of the laboratory. Patients brane phospholipids can activate the intrinsic and extrinsic given the wrong blood exhibit classic clinical signs includ- coagulation cascade (Chapter 32). The resulting consump- ing increased pulse rate, hypotension, chills and fever, pain tive coagulopathy (disseminated intravascular coagulation) (chest, flank, or at the infusion site), or difficulty breathing. can damage the kidney by deposition of fibrin in the micro- When an acute transfusion reaction is suspected, the transfu- vasculature (Chapters 32, 34). The intravascular hemolysis sion must be stopped immediately because of the potential may induce a “cytokine storm” with increased production of Table 19.11 Comparison of Acute and Delayed Hemolytic Transfusion Reactions Acute Delayed Timing Immediate (within 24 hours) 2–14 days Underlying cause Usually ABO antibodies Other antibodies: often Kidd system (anamnestic response) Hemolysis Intravascular Extravascular; rare, slower intravascular if antibody capable of fixing complement Symptoms Fever, chills, back pain, hypotension, pain at site of infusion Uncommon (fever, hemoglobinuria) Laboratory findings Hemoglobinemia Positive DAT May see hemoglobinuria Positive DAT (possible) Antibody in eluate Hemolytic Anemia: Immune Anemias 419 pro-inflammatory cytokines such as increased tumor necro- LABORATORY EVALUATION sis factor@a (TNF@a) and other interleukins that can mediate The laboratory findings vary, depending on whether the some of the clinical symptoms.29,82 Mortality rates from ABO transfusion reaction is acute or delayed. Intravascular hemo- acute hemolytic transfusion reactions range from 10–50%. lysis usually accompanies the acute reaction and typically Extravascular hemolysis is typical of a delayed hemo- can be associated with hemoglobinemia, hemoglobinuria, lytic transfusion reaction occurring when erythrocytes methemoglobinemia, and decreased haptoglobin and hemo- are coated with IgG antibodies and removed via macro- pexin. Extravascular hemolysis usually accompanies the phage Fc receptors in the spleen. The speed of the removal delayed reaction. The DAT might or might not be positive depends on the amount of antibody on the cell. Comple- in acute hemolytic transfusion reactions, depending on the ment is not usually involved but when present can enhance extent of erythrocyte destruction. The DAT is usually posi- phagocytosis. tive in a delayed hemolytic transfusion reaction but might Delayed transfusion reactions occur 2–14 days after a not be detected until days after transfusion. A patient who is transfusion. Although the antibody cannot be detected in suspected of experiencing either an acute or delayed trans- pretransfusion testing because its concentration is lower fusion reaction should have a transfusion reaction workup. than the sensitivity level of the test, antigens on infused donor cells induce a secondary (anamnestic) antigenic response. The antibody produced is usually IgG, and Checkpoint 19.13 hemolysis is extravascular. The first indication of a delayed Compare the characteristic laboratory findings in acute hemo- lytic transfusion reactions and in delayed transfusion reactions. reaction is a drop in the hemoglobin concentration several days after the transfusion with no signs of bleeding. Intra- vascular hemolysis can also occur but is less pronounced THERAPY than in acute reactions. Laboratory investigation reveals a The most important immediate action to take when an acute

positive DAT because of antibody-coated donor cells in the transfusion reaction occurs is to terminate the transfusion. patient’s circulation. Antibodies characteristically associ- A major effort should be made to maintain urine flow to ated with a delayed transfusion reaction are in the Kidd prevent renal damage. Shock and bleeding require immedi- system (anti@Jka, anti@Jkb), although antibodies to other anti- ate attention. In a delayed reaction, treatment is generally gens have also caused delayed reactions. not required, although in potentially severe cases red blood CLINICAL PRESENTATION exchange may be required.86 Future units of blood given to Symptoms of an immediate transfusion reaction begin within the patient, however, must lack the antigen to which the minutes to hours after beginning the transfusion. A variety of patient has made an antibody. Cases of hyperhemolysis nonspecific symptoms, including fever, chills, low back pain, treatment with monoclonal antibodies that affect the sensations of chest compression or burning at the site of infu- immune response (Rituximab or Eculizumab) has shown sion, hypotension, and nausea and vomiting, can occur. Unless variable effectiveness.84,87 the transfusion is immediately stopped, shock can occur. Anuria due to tubular necrosis secondary to inadequate renal blood flow and bleeding due to DIC are common complica- CASE STUDY (continued from page 417) tions. The severity of the reaction and extent of organ damage The clinician wants to start Nancy’s therapy and are directly proportional to the amount of blood infused. give her a transfusion. Although some types of non-erythrocyte-related trans- 6. How would knowing that Nancy had not been fusion reaction (e.g., allergic, febrile, nonhemolytic) are transfused in the last several months help you common in the pediatric population, delayed hemolytic decide on the underlying cause of the antibody? transfusion reactions are uncommon in the pediatric popu- lation compared with the adult population.85 Most delayed 7. What would you tell the clinician about giving transfusion reactions cause few signs or symptoms. The a transfusion? most common signs are malaise and unexplained fever sev- 8. What kind of therapy could be used? eral days after the transfusion. Some patients notice the presence of hemoglobin in the urine. Hemolytic Disease of the Fetus and Checkpoint 19.12 Newborn (HDFN) Compare the underlying mechanisms, pathophysiology, and clinical symptoms of an acute hemolytic transfusion reaction Hemolytic disease of the fetus and newborn (HDFN) is an with a delayed one. alloimmune disease associated with increased erythrocyte destruction during fetal and neonatal life and is caused by 420 Chapter 19 fetomaternal blood group incompatibility. Maternal anti- or transfusion. Normally, the placenta does not allow free bodies are transported across the placenta and react with passage of erythrocytes from fetal to maternal circulation, antigens of paternal origin on the fetal erythrocytes.29 The but small numbers of erythrocytes can enter the maternal three categories of HDFN are: ABO caused by anti-A, anti- circulation during gestation. Additionally, small amounts B, and/or anti-A, B; Rh(D) caused by anti-D; and “other” of fetal blood also can enter the mother’s circulation during caused by alloantibodies to other Rh system antigens (C, delivery. The risk of sensitization increases as the volume of c, E, e) or antibodies to other blood group system antigens the fetal bleed increases. If the fetal-maternal bleed is suf- (e.g., Kell, Kidd, Duffy). More than 95% of HDFN cases are ficient to stimulate the production of maternal antibodies, due to either anti-D or ABO system antibodies. Although subsequent pregnancies could be at risk for HDFN. HDFN caused by ABO antibodies is more common than Although three classes of immunoglobulins can be HDFN caused by anti-D, Rh(D) incompatibility causes more produced during the mother’s immunization—IgG, IgM, severe disease (Table 19-12). The antibodies most commonly IgA—only IgG can cross the placenta and cause HDFN. The associated with the remaining HDFN cases include anti-K, IgG antibody is actively transported across the placenta and anti-c, anti-C, and anti-E, although any IgG antibody can causes destruction of fetal erythrocytes. The fetus/newborn be implicated. HDFN caused by anti-D and anti-K are the has anemia and bilirubinemia of varying degrees of severity most severe. HDFN caused by anti-K is unique in that the based on the strength of the immune response and degree anti-K suppresses erythropoiesis in the fetus and is respon- of hemolysis. sible for the early destruction of erythroid progenitor cells, In ABO-HDFN, the mother already has the naturally leading to a more severe anemia and increased numbers of occurring ABO system antibodies, generally a mixture of IgM intrauterine transfusions.88,89,90 and IgG anti-A, anti-B, and/or anti-A,B. The pre-existing IgG antibodies can cross the placenta to destroy fetal cells, PATHOPHYSIOLOGY potentially affecting all pregnancies including the first. The pathophysiology of HDFN involves initial sensitiza- Approximately 15–20% of the group A or group B babies tion and antibody production, in utero effects, and postnatal born to group O mothers develop serologic evidence of effects. Four conditions must be met for HDFN to occur: ABO-HDFN (positive DAT).3 In HDFN caused by all other • The mother must be exposed (sensitized) to an erythro- antibodies including anti-D, the mother generally lacks cyte antigen that she lacks. preformed antibodies capable of recognizing fetal antigens. • The fetus must possess the antigen to which the mother The firstborn is not usually affected because the first preg- has been sensitized. nancy serves as the sensitizing event. In each subsequent • The mother must produce antibodies to the foreign pregnancy with an antigen-positive fetus, the risk for sever- antigens. ity of HDFN increases as the antibody response increases. • The mother’s antibody must be able to cross the pla- Prenatal Period If destruction of fetal erythrocytes is severe centa and enter the fetal circulation enough in utero, the fetus becomes severely anemic and can develop complications as a result. Extramedullary hemato- Sensitization The mother may have been exposed to for- poiesis occurs in the liver and spleen, causing their enlarge- eign (nonself) erythrocyte antigens by previous pregnancy ment. Because of hemolysis, the unconjugated (indirect) Table 19.12 Comparison of Hemolytic Disease of the Fetus and Newborn Caused by ABO and Rh(D) Feature Rh ABO Other Antibody Immune IgG Nonimmune IgG Immune IgG Blood group Mother Rh negative Mother, group O; newborn, group A or B Mother lacks antigen that is on fetal cell Baby Rh positive Obstetric history Only pregnancies after the first are usually First pregnancy and subsequent preg- Pregnancy can be first if mother previ- affected nancies can be affected ously sensitized by transfusion. If preg- nancy is sensitizing event, usually second and subsequent pregnancies affected Clinical findings Moderate to severe anemia and Mild anemia if present; mild to moderate Mild to severe anemia and bilirubinemia bilirubinemia bilirubinemia with a peak 24–48 hours after birth Laboratory findings DAT positive DAT weakly positive or negative DAT positive No spherocytes Spherocytes present Therapy Exchange transfusion if severe Phototherapy Phototherapy and/or exchange transfu- sion, if severe Hemolytic Anemia: Immune Anemias 421 bilirubin concentration increases. In the fetus, this biliru- the greatest risk thereafter. In Rh(D) incompatibility, the bin crosses the placenta and is conjugated and excreted by cord blood hemoglobin can be low normal at birth (nor- the mother. With procedures such as amniocentesis, the mal hemoglobin at birth is 14–20 g/dL), and the newborn amount of bilirubin in amniotic fluid can be measured to may not appear jaundiced. However, significant hemoly- help determine the relative severity of hemolysis. Cordo- sis occurring in the first 24 hours of life outside the womb centesis in which a sample of the fetal blood is taken via a results in anemia with pallor and jaundice. In severe cases, needle inserted into the umbilical vein allows testing of the hepatosplenomegaly can be present. Severe anemia can be fetal blood for blood type, DAT, hemoglobin, and bilirubin. accompanied by heart failure and edema. As the level of Noninvasive procedures such as Doppler ultrasound can unconjugated bilirubin rises, kernicterus can occur and can help predict anemia based on blood flow in the fetus. In be fatal in severe cases. The risk of hyperbilirubinemia in recent years, methods to obtain fetal cells or cell-free DNA premature infants is even greater because of the inability of from the maternal circulation have been introduced. These the premature liver to excrete the excess bilirubin. methods help in predicting Rh-HDFN and eliminating the ABO incompatibility is not as severe as Rh incompati- need for invasive procedures such as amniocentesis. Pre- bility. The clinical course is usually benign, and hemolysis natal fetal genotyping diagnostic procedures have been is minimal. Within 24–48 hours after birth, the infant developed. Although they were initially designed to detect appears jaundiced, but kernicterus is extremely rare. Ane- the D antigen, they have been expanded to detect other Rh mia is mild and pallor is uncommon. Hepatosplenomegaly, system antigens (C, c, E, e) and K, Fy, and JK genotypes as if present, is mild. well and to predict paternal zygosity.91,92 HDFN is signifi- cantly more severe when women have multiple antibodies that include anti-D than those with anti-D alone or when Checkpoint 19.14 specificities of the antibodies do not include anti-D.93,94 This Compare the pathophysiology and clinical findings of ABO- may be because more antigen sites are coated with antibody, HDFN and Rh-HDFN. leading to a more aggressive immune response. The most serious complication of HDFN is cardiac failure and hydrops fetalis, which occurs when the fetus is LABORATORY EVALUATION unable to produce sufficient erythrocytes. Hydrops fetalis Laboratory tests are essential to identify the etiology is characterized by edema and accumulation of fluid in the of HDFN, determine prognosis, and select appropriate peritoneal, pericardial, or pleural cavities. treatment. Classic prenatal testing on a pregnant woman Erythroblastosis fetalis, a term also used to describe includes ABO and Rh typing of the erythrocytes as well as characteristics of HDFN, reflects the presence of large an antibody screen (IAT) on her serum. If the IAT is positive, numbers of nucleated erythrocytes found in the newborn’s the antibody is identified so that an assessment of HDFN peripheral blood in very severe cases. risk can be performed. The postnatal HDFN workup will Postnatal Period Erythrocyte destruction persists after involve laboratory tests on both the mother and the infant. birth because of maternal antibodies in the newborn’s Rh Incompatibility The DAT in Rh incompatibility is circulation. After birth, the newborn must conjugate and usually positive, reflecting antibody coating of the new- excrete the bilirubin on its own. In the neonate, albumin born’s erythrocytes. About 50% of affected infants have a levels for bilirubin transport are limited, and liver glucuro- cord blood hemoglobin concentration of less than 14 g/dL. nyl transferase for bilirubin conjugation is low; therefore, Because the capillary blood hemoglobin can be up to 4 g/dL considerable amounts of toxic unconjugated bilirubin can higher due to placental transfer of blood at birth, the cord accumulate in the newborn after delivery. In the unconju- blood hemoglobin concentration is most useful as an indica- gated state, bilirubin is toxic because it is lipid soluble and tor of anemia at birth and a baseline to follow destruction of can easily cross cell membranes. This form of bilirubin has erythrocytes after birth. A direct relationship exists between a high affinity for basal ganglia of the central nervous sys- the initial cord blood hemoglobin level and the severity of tem. Thus, the excess unconjugated bilirubin can lead to the disease. Lower cord hemoglobin levels at birth are asso- kernicterus, an irreversible form of brain damage. The con- ciated with a more severe clinical course. After birth, hemo- jugated form of bilirubin cannot cause this problem because globin levels can fall at the rate of 3 g/dL/day. The lowest it is water soluble and lipid insoluble and cannot cross cell hemoglobin values are present at 3–4 days. The erythro- membranes. cytes are macrocytic and normochromic. Reticulocytes are CLINICAL PRESENTATION markedly increased, sometimes reaching 60%. Nucleated Anemia resulting from increased cell destruction is the red cells are markedly increased in the peripheral blood greatest risk to both the fetus in utero and to the newborn (10@100 * 103/mcL) reflecting the rapid production of cells with HDFN in the first 24 hours of life. Bilirubinemia is in response to erythrocyte destruction. Normal infants also 422 Chapter 19 have nucleated red cells in the peripheral blood, but their successful delivery. In severe cases of HDFN the mother values are

much lower (0.2@2.0 * 103/mcL). may also undergo routine plasmapheresis to remove circu- A blood smear shows marked polychromasia, mild or lating antibody until the fetus can be delivered. IVIG may absent poikilocytosis, and few, if any, spherocytes. In some be used in conjunction with plasmapheresis to suppress an cases, the leukocyte count is increased to 30 * 103/mcL immune response.95,96 or more due to a rise in neutrophils reflecting the marrow In mild cases postnatally, the newborn is treated with response to stress (the normal leukocyte count at birth is phototherapy, which slowly lowers the toxic bilirubin 15920 * 103/mcL). However, recent studies have shown level. Although toxic levels of bilirubin are 19–20 mg/dL, that a neutropenia is often present, regardless of the type exchange transfusion is usually performed before that level of HDFN.4 A significant neutrophilic shift to the left often is reached. Exchange transfusions can also be indicated if occurs. The platelet count is usually normal, but thrombo- the bilirubin is rising more than 1 mg/dL/hour or if there cytopenia can develop in severe cases. is significant anemia. The transfusion has several beneficial Cord blood bilirubin is elevated in Rh-HDFN but is effects: usually less than 5.5 mg/dL. However, the elevated bili- • Removes plasma containing maternal antibodies and rubin does not accurately reflect the severity of hemolysis dilutes the concentration of remaining antibodies because bilirubin produced before birth readily crosses the placenta and is metabolized by the mother. The newborn’s • Removes some of the antibody-coated erythrocytes serum bilirubin peaks on the third or fourth day and can • Lowers the level of bilirubin reach 40–50 mg/dL if not treated. Most bilirubin is in the • Treats the anemia toxic unconjugated form. Full-term infants with bilirubin concentrations more than 10 mg/dL are at increased risk RH IMMUNE GLOBULIN (RHIG) for kernicterus, and premature infants can develop it with The passive injection of Rh immunoglobulin (RhIG) levels as low as 8–10 mg/dL. that contains anti-D prevents maternal immunization. ABO Incompatibility In the case of ABO incompatibility, a About 7–8% of Rh-negative women develop antibodies weakly positive DAT is found in the cord blood, but it often to Rh-positive cells after the birth of an Rh-positive ABO- becomes negative within 12 hours. The weak reaction is due compatible infant. The routine use of prophylactic RhIG in to the small number of anti-A or anti-B antibodies attached Rh-negative women during gestation (at 28 weeks) and fol- to the erythrocyte. Bilirubin is not usually significantly ele- lowing the birth of an Rh-positive child has decreased the vated, but the bilirubin level 6 hours after birth can be used incidence of HDFN considerably, although other changes to predict development of hyperbilirubinemia in severe in prenatal care and number of pregnancies also may have cases.5 The peripheral blood smear in severe cases can show affected the incidence. Most women (92%) who develop increased numbers of nucleated erythrocytes and the pres- anti-D during pregnancy do so at 28 weeks or later. There- ence of schistocytes, spherocytes, and polychromasia. fore, antepartum administration of RhIG is given between weeks 28 and 30 of gestation to any Rh-negative woman who has not developed anti-D. The RhIG acts as an immu- Checkpoint 19.15 nosuppressant, depressing the production of immune IgG. Compare the laboratory findings including the peripheral blood However, this prenatal anti-D binds to fetal cells and can smear and the DAT for newborns with ABO-HDFN and those result in a positive DAT and must be evaluated for clinical with Rh-HDFN. and serological significance. Postnatally, a dose of RhIG given within 72 hours of birth protects against the consequences of a fetal-maternal THERAPY bleed. Rh-positive erythrocytes of a fetus that enter the The major efforts of therapy are to prevent hyperbilirubi- mother’s circulation at birth can stimulate the mother’s nemia and anemia. If the destruction of erythrocytes and immune system to make antibodies. As in the antepar- degree of anemia appear to affect the viability of the fetus, tum administration of RhIG, postnatal RhIG can bind fetal an intrauterine transfusion can be given. Non-invasive cells and mediate their removal in the spleen. The dose methods such as Doppler velocity have, in many cases, of RhIG should be determined based on the number of replaced amniocentesis and cordocentesis to determine fetal cells in the maternal circulation. Several tests can be the degree of anemia. These techniques, in conjunction used to estimate the volume of fetal maternal hemorrhage with molecular methods that identify fetal DNA for detect- (FMH), including the qualitative rosette and the quantita- ing in utero hemolysis have allowed decisions to be made tive K leihauer-Betke tests, and, more recently, immuno- on whether an intrauterine transfusion should be given. fluorescent flow cytometry using a monoclonal antibody Intrauterine transfusions often must be given on a routine against fetal hemoglobin to determine the number of fetal basis until the fetus reaches a gestational age that allows cells present. Hemolytic Anemia: Immune Anemias 423 Summary Immune hemolytic anemia (IHA) is mediated by antibodies appears to cause a change in the erythrocyte membrane that and/or complement and can be classified as autoimmune, causes the body to recognize it as foreign and produce an alloimmune, or drug induced, depending on the underly- antibody against the cell. ing process. Patients with IHA have a positive DAT due Alloimmune hemolytic anemia has two presentations: to immunoglobulin and/or complement on the cell. The transfusion reaction and hemolytic disease of the newborn. autoimmune hemolytic anemias (AIHA) occur when the In both conditions, infusion of erythrocytes containing for- offending antibody reacts with an antigen on the patient’s eign (nonself) antigens stimulates the antibody. This sensiti- erythrocytes (self-antigen). These antibodies can also react zation causes the production of alloantibodies to the foreign with erythrocytes of most other persons. AIHA are either antigens, resulting in the cell’s destruction. The antibody idiopathic or secondary to an underlying disease and are can be detected in the serum and cause a positive DAT. further classified as warm or cold, depending on the ther- Transfusion reactions can be acute (immediate) or mal reactivity of the causative antibody. WAIHA is most delayed (2–14 days after transfusion). Acute reactions most often caused by IgG antibodies that react at body tempera- often involve an ABO mismatch between donor and recipient ture (37°C) and cause extravascular hemolysis. Cold hem- and result in intravascular hemolysis because of complement agglutinins are IgM antibodies that generally react at less activation. Delayed transfusion reactions are due to non-ABO than 20°C but are efficient complement activators and can antibodies. These reactions result in extravascular lysis and cause intravascular hemolysis. In both cases, the DAT is are characterized by a positive DAT. The causative antibody positive, and the IAT procedure can detect the antibody. An must be identified so that future transfused erythrocytes lack IgG biphasic antibody that binds complement in the cool the antigen corresponding to the patient’s antibody. peripheral circulation mediates PCH, another type of cold ABO, Rh(D) or other blood group system antibod- autoimmune hemolytic condition. As the coated cells reach ies that react with fetal erythrocytes can cause hemolytic warmer portions of the circulation, the complement cascade disease of the fetus and newborn (HDFN). ABO-HDFN is is activated and intravascular hemolysis occurs. generally milder than Rh-HDFN and can occur in any preg- In drug-induced immune hemolytic anemia, the drug nancy because prior antigenic sensitization is not required. binds to the erythrocyte membrane and induces antibody Rh and other types of HDFN require prior sensitization. formation, which occurs by several mechanisms, depending Maternal antibodies that coat fetal cells can cause anemia on the causative drug. The antibodies can be drug depen- and/or elevated bilirubin in the newborn. Maternal and dent, and the drug must be present for the antibody to react. newborn ABO and Rh group, maternal antibody, and new- In autoantibody formation (drug independent), the drug born DAT are used to evaluate HDFN. Review Questions Level I 3. Which of the following parameters on an automated hematology instrument could be seen in cases of cold 1. The characteristic erythrocyte seen in a peripheral agglutinin syndrome (CAS)? (Objective 3) blood smear in WAIHA is a(n): (Objective 3) a. Falsely elevated MCV a. macrocyte b. Falsely elevated RBC count b. spherocyte c. Falsely decreased MCHC c. dacrocyte d. Falsely decreased hemoglobin d. elliptocyte 4. One purpose of the DAT is to: (Objective 2) 2. Which of the following might be observed on a peripheral blood smear in cases of cold autoimmune a. detect erythrocytes coated with immunoglobulin hemolytic anemia? (Objective 3) in vivo a. Helmet cells b. detect antibodies in the serum b. Macrocytes c. neutralize serum complement c. Agglutination d. prevent agglutination by IgM antibodies d. Spherocytes 424 Chapter 19 5. Intravascular hemolysis is characteristic of which of c. penicillin the following alloantibody situations? (Objective 5) d. rituximab a. Delayed hemolytic transfusion reaction 2. Because a newborn shows evidence of jaundice, a b. ABO-HDFN workup for HDFN is started. The newborn has a c. Rh-HDFN weakly positive DAT with anti-IgG. The mother is d. Acute hemolytic transfusion reaction group O, Rh negative, and the newborn is group A, Rh negative. The blood smear shows evidence 6. A patient with WAIHA would most likely have serum of spherocytes. What is the most likely cause? that reacts in which of these patterns? (Objective 5) (Objective 6) Serum + Own Serum + Erythrocytes a. Rh-HDFN Erythrocytes of Others b. ABO-HDFN a. Negative Negative c. Combined ABO- and Rh-HDFN b. Negative Positive d. Cold agglutinins c. Positive Positive 3. Which autoimmune syndrome is characterized by the d. Positive Negative presence of a biphasic complement-fixing IgG antibody? (Objective 4) 7. Which of the following is not considered to be a con- dition caused by autoantibodies? (Objective 5) a. PCH b. WAIHA a. PCH c. Cold agglutinin syndrome b. CAS d. Immune complex drug induced c. Delayed transfusion reaction d. Drug-induced hemolytic anemia 4. Intravenous administration of what drug is char- acteristically associated with covalent attachment 8. The Donath-Landsteiner antibody is found in which of the drug to the erythrocyte membrane and type of hemolytic anemia? (Objective 1) drug-dependent antibody formation? (Objective 3) a. Warm autoimmune hemolytic anemia a. Penicillin b. Paroxysmal cold hemoglobinuria b. Aldomet c. Cold autoimmune hemolytic anemia c. Quinidine d. Drug-induced hemolytic anemia d. Third-generation cephalosporins 9. Which of the following is not part of the typical 5. Based on maternal and fetal testing, a newborn is sus- peripheral blood picture in Rh-HDFN? (Objective 4) pected of having Rh-HDFN. Which of the following a. Macrocytes peripheral blood smear morphologies might be seen? (Objective 6) b. Polychromasia c. Spherocytes a. Microcytic, hypochromic erythrocytes, decreased reticulocytes d. Increased nucleated erythrocytes b. Microcytic, normochromic erythrocytes, increased 10. The antibody found in PCH is: (Objective 1) nucleated red blood cells c. Macrocytic, hypochromic erythrocytes, decreased a. IgM reticulocytes b. IgG d. Macrocytic, normochromic erythrocytes, increased c. directed against the Rh antigens nucleated red blood cells d. directed against the ABO antigens 6. What is the most likely mechanism of hemolysis in Level II WAIHA? (Objective 1) 1. The drug(s) most often associated with drug-induced a. Increased sensitivity to complement hemolytic anemia is/are: (Objective 3) b. Fixing of complement by IgM antibody a. aldomet c. Biphasic reactions by IgG antibodies b. second- and third-generation cephalosporins d. Phagocytosis of IgG coated erythrocytes Hemolytic Anemia: Immune Anemias 425 7. A patient who received a transfusion 6 days ago is Spherocytes are present. The physician suspects suspected of having a delayed transfusion reaction. hemolytic anemia, and laboratory tests suggest a Which of the following would not be a characteristic hemolytic process with an increase in reticulocytes finding? (Objective 7) and bilirubin. 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B. (2015). Hemolytic events associated with intravenous immune Pediatric Blood Cancer, 59, 953–955. globulin therapy: A qualitative analysis of 263 cases reported 61. Slemp, S. N., Davisson S. M., Layten, J., Cipkala, D. A. & to four manufacturers between 2003 and 2012. Transfusion, 55, Waxman, D. A. (2014). Two case studies and a review of paroxys- S36–S46. mal cold hemoglobinuria. Lab Medicine, 46(3), 253–258. 80. Padmore, R. (2015). Possible mechanisms for intravenous 62. Bass, G. F., Tuscano, E. T., & Tuscanom, J. M. (2014). Diagnosis immunoglobulin-associate hemolysis: Clues obtained from and classification of autoimmune hemolytic anemia. Autoimmu- review of clinical case reports. Transfusion, 55, S59–S64. nity Reviews, 13, 560–564. 81. Scott, D. E., & Epstein, J. S. (2015). Safeguarding immune globulin 63. Hirano, Y., Itonaga, T., Yasudo, H., Isojima, T., Miura, K., recipients against hemolysis: What do we know and where do we Harita, Y., . . . Oka, A. (2016). 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Life-threatening delayed cause drug-induced immune hemolytic anemia or positive direct hyperhemolytic transfusion reaction in a patient with sickle cell antiglobulin tests: Some interesting findings since 2007. Immuno- disease: Effective treatment with eculizumab followed by ritux- hematology, 30, 66–79. imab. Transfusion, 55, 2398–2403. 66. Arndt, P. A. (2014). Drug-induced immune hemolytic anemia: 85. Oakley, F. D., Woods, M., Arnold, S., & Young, P. P. (2015). Trans- The last 30 years of changes. Immunohematology, 30, 44–54. fusion reactions in pediatric compared with adult patients: A 67. Leger, R. M., Arndt, P. A., & Garratty, G. (2014). How we investi- look at rate, reaction type, and associated products. Transfusion, gate drug-induced immune hemolytic anemia. Immunohematol- 55, 563–570. ogy, 30, 85–94. 86. Tormey, C. A., & Stack, G. (2013). Limiting the extent of a delayed 68. Arndt, P. A., Leger, R., & Garratty, G. (2012). Serologic character- hemolytic transfusion reaction with automated red blood cell istics of ceftriaxone antibodies in 25 patients with drug-induced exchange. Archives of Pathology and Laboratory Medicine, 137, immune hemolytic anemia. Transfusion, 52, 602–612. 861–864. 69. Mayer, B., Bartolmas, T., Yurek, S., & Salama, A. (2015). Variabil- 87. Gupta, S., Fenves, A., Nance, S. T., Sykes, D. B., & Dzik, W. (2015). ity of findings in drug-induced immune haemolytic anaemia: Hyperhemolysis syndrome in a patient without a hemoglobin- Experience over 20 years in a single centre. Transfusion Medicine opathy, unresponsive to treatment with eculizumab. Transfusion, and Hemotherapy, 42, 333–339. 55, 623–628. 70. Garratty, G. (2010). Immune hemolytic anemia associated with 88. de Haas, M., Thurik, F. F., Koelewijn, J. M., & Van der Schoot, C. drug therapy. Blood Reviews, 24, 143–150. E. (2015). Haemolytic disease of the fetus and newborn. 71. Satori, A., Staley, B., & Skipper, A. (2014). Drug-induced auto- Vox Sanguinis, 109, 99–113. immune hemolytic anemia in a 78-year-old African-American 89. Rath, M. E., Smits-Wintjens, V. E., Lindenburg, I. T., Brand, A., man with chronic lymphocytic leukemia. Lab Medicine, 45(3), van Kamp, I. L., Oepkes, D., . . . Lopriore, E. (2011). Exchange e105–e108. transfusions and top-up transfusions in neonates with Kell hae- 72. Holbro, A., & Passweg, J. R. (2015). Management of hemolytic molytic disease compared to Rh D haemolytic disease. anemia following allogeneic stem cell transplantation. Vox Sanguinis, 100, 312–316. Hematology, 2015, 376–384. 90. Denomme, G. A. (2015). Kell and Kx blood systems. Immunohema- 73. Rovira, J., Cid, J., Gutierrez-Garcia, G., Pereira, A., Fernandez- tology, 31, 14–19. Aviles, F., Rosinol, L, . . . Lozano, M. (2013). Fatal immune 91. Fasano, R. M. (2016). Hemolytic disease of the fetus and newborn in hemolytic anemia following allogeneic stem cell transplantation: the molecular era. Seminars in Fetal & Neonatal Medicine, 21, 28–34. Report of 2 cases and review of literature. Transfusion Medicine 92. Pirelli, K. L., Pietz, B. C., Johnson, S. T., Pinder, H. I., & Bellissimo, Reviews, 27, 168–170. D. B. (2010). Molecular determination of RHD zygosity: Predict- 74. Nadarajah, L., Ashman, N., Thuraisingham, R., Barber, C., ing risk of hemolytic disease of the fetus and newborn related to Allard, S., & Green, L. (2013). Literature review of passenger anti-D. Prenatal Diagnosis, 30, 1207–1212. lymphocyte syndrome following renal transplantation and two 93. Smith, H. M., Shirey, R. S., Thoman, S. K., & Jackson, J. B. (2013). case reports. American Journal of Transplantation, 13, 1594–1600. Prevalence of clinically significant red blood cell alloantibodies in 75. Hurtarte-Sandoval, A. R., Navarro-Cabello, M. D., Alvarez-Rivas, pregnant women at a large tertiary-care facility. Immuohematology, M. A., Robles-Lopez, A. I., Salmeron-Rodrguez, M. D., 29, 127–130. 428 Chapter 19 94. Markham, K. B., Rossi, K. Q., Nagaraja, H. N., & O’Shaughnessy, 96. Houston, B. L., Govia, R., Abou-Setta, A. M., Reid, G. J., Hadfield, R. W. (2015). Hemolytic disease of the fetus and newborn due M., Menard, C, . . . Zarychanski, R. (2015). Severe Rh alloim- to multiple maternal antibodies. American Journal of Obstetrics & munization and hemolytic disease of the fetus managed with Gynecology, 213, e1–e5. plasmapheresis, intravenous immunoglobulin and intrauterine 95. Fernandez-Alba, J. J., Leon, R., Gonzalez-Macias, C., Paz, A., transfusion: A case report. Transfusion and Apheresis Science, 53, Prado, F., Moreno, L. J., . . . Torrejon, R. (2014). Treatment of D 399–402. alloimmunization in pregnancy with plasmapheresis and intra- venous immune globulin: Case report. Transfusion and Apheresis Science, 51, 70–72. Chapter 20 Hemolytic Anemia: Nonimmune Defects Linda A. Smith, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define microangiopathic hemolytic anemia thrombocytopenic purpura (TTP), and (MAHA) and list several associated disorders hemolytic uremic syndrome (HUS). and the age group most commonly affected. 3. Recognize the characteristic erythrocyte 2. Describe the general morphology and morphology of MAHA on a stained blood hematologic values associated with MAHA film. and criteria that distinguish disseminated 4. List organisms that can cause erythrocyte intravascular coagulation (DIC), thrombotic hemolysis. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Summarize the general pathophysiology for 4. Given a set of data and clinical history, MAHA. determine whether MAHA is a probable 2. Compare and contrast the clinical findings, diagnosis, identify the possible etiology, underlying cause, treatment, and character- and propose follow-up tests that should be istic findings for erythrocytes, platelet count, performed. and coagulation tests for each of the follow- 5. Compare the underlying mechanism of ing types of MAHA: hemolysis by the following infectious a. Hemolytic uremic syndrome (HUS) agents: b. Thrombotic thrombocytopenic purpura a. Plasmodium parasites (malaria) (TTP) b. Babesia parasites c. Disseminated intravascular coagulation c. Bartonella bacteria (DIC) d. Clostridium bacteria 3. Define exercise-induced hemoglobinuria. 429 430 Chapter 20 Chapter Outline Objectives—Level I and Level II 429 Hemolytic Anemia Caused by Physical Key Terms 430 Injury to the Erythrocyte 431 Background Basics 430 Hemolytic Anemias Caused by Antagonists in the Blood 440 Case Study 430 Summary 443 Overview 431 Review Questions 443 Introduction 431 References 445 Key Terms Cryosupernatant Hemolytic uremic syndrome (HUS) Thrombotic thrombocytopenic Disseminated intravascular Microangiopathic hemolytic purpura (TTP) coagulation (DIC) anemia (MAHA) von Willebrand factor (VWF) Fresh frozen plasma (FFP) Plasma exchange Hemolysis, elevated liver enzymes and low platelet (HELLP) syndrome Background Basics The information in this chapter builds on concepts • Identify the intrinsic hemolytic anemias, and describe learned in previous chapters. To maximize your learn- how they differ from extrinsic hemolytic anemias. ing experience, you should review the following (Chapter 11) concepts: Level II Level I • Describe the structure and function of the major • Describe the normal production, life span, and proteins of the erythrocyte membrane. (Chapter 5, 17) destruction of the erythrocyte. (Chapter 5) • Identify the key tests that can be used in diagnosis • List the reference intervals for basic adult hematology of anemia, and identify clinical signs of anemia. parameters. (Chapters 1, 10, inside book covers) (Chapter 11) • Identify sources of defects that lead to hemolytic • Describe the different nonimmune mechanisms of anemia. (Chapter 11) hemolysis and how they are detected. (Chapters 5, • Review the immune hemolytic anemias, and describe 11, 17, 18) how they differ from other hemolytic anemias. • Identify the laboratory tests that differentiate immune (Chapter 19) from nonimmune anemia. (Chapters 11, 19) CASE STUDY contraceptives but was not taking any other drugs. We refer to this case study throughout the chapter. Her initial laboratory tests showed: Mai, a 35-year-old woman, saw her physician. Hb 6 g/dL (60g/L) She complained of weakness, low-grade fever, Hct 18% (0.18 L/L) and periods of forgetfulness, and memory loss for WBC 8.9 * 103>mcL the last week or so. She denied any viral illness before the onset of symptoms. She was on oral Consider reflex testing that could be helpful in identifying the cause of the anemia. Hemolytic Anemia: Nonimmune Defects 431 Overview or with foreign surfaces such as artificial heart valves com- monly induce such damage. There are other causes of injury, This chapter deals with the mechanisms of hemolysis not including Shiga toxin from organisms such as Esherichia coli included in the chapters on membrane defects (Chap- (E. coli) 0157:H7. Infectious agents such as Plasmodium sp. ter 17), metabolic deficiencies (Chapter 18), or immune and Babesia sp. can cause injury to the erythrocytes during mechanisms (Chapter 19) but includes microangiopathic their intracellular life cycle. Some drugs and chemicals can hemolytic anemias (MAHAs). Hemolytic uremic syndrome cause membrane oxidant injury, leading to intravascular (HUS), an MAHA, is discussed in detail including disease hemolysis or removal of the damaged cell by the spleen association, pathophysiology, and clinical presentation and (Table 20-1). laboratory evaluation. It also provides a brief overview of thrombotic thrombocytopenic purpura (TTP), which is dis- cussed in detail in Chapter 35. Uncommon causes of hemo- lysis such as hypertension, mechanical heart devices, burns, Hemolytic Anemia Caused exercise, and infectious agents are also briefly discussed. by Physical Injury to the Erythrocyte Introduction Intravascular and/or extravascular hemolysis and striking Erythrocytes that have normal hemoglobin structure, abnormal shapes of the circulating peripheral blood eryth- enzymes, and membranes can be prematurely destroyed by rocytes, including fragments (schistocytes) and helmet cells factors extrinsic to the cell. This destruction can be immune characterize hemolytic anemia caused by traumatic physi- mediated via antibodies and/or complement (Chapter 19). cal injury to the erythrocytes in the vascular circulation. However nonimmune factors also can cause either extravas- cular or intravascular hemolysis, depending on the type and Thrombotic Microangiopathic extent of injury to the erythrocyte. This chapter discusses nonimmune causes that lead to premature erythrocyte Anemia (TMA) destruction. Erythrocytes can undergo traumatic physical Thrombotic microangiopathic anemia (TMA) is an inclusive injury in the peripheral circulation, resulting in the pres- term referring to several conditions in which a hemolytic ence of schistocytes in the peripheral blood. Contact with process is caused by microcirculatory lesions. The condi- fibrin strands or platelet aggregates in the microcirculation tions described by this term are sometimes referred to as Table 20.1 Hemolytic Anemias Caused by Nonimmune Antagonists in the Erythrocyte Environment Category Antagonist Mode of Hemolysis Thrombotic microangiopathic anemia Thrombi in microcirculation Physical damage to erythrocytes by microthrombi Malignant hypertension Unknown Physical damage to erythrocytes Other physical trauma Exercise-induced hemoglobinuria External force Fragmentation of erythrocytes from exces- Oxidative stress sive external force as they pass through microcapillaries Thermal injury Heat Thermal damage to erythrocyte membrane proteins Traumatic cardiac Physical stress Erythrocyte fragmentation Infectious agents Plasmodium sp. Direct parasitization of erythrocyte; hypersplen- ism; acute intravascular hemolysis (P. falciparum infection) Babesia sp. Invasion of the erythrocyte and cell lysis Bartonella sp. Invasion of the erythrocyte and cell lysis Clostridium sp. Hemolytic toxins Animal venoms Snake bites Mechanical cell damage due to DIC Spider bites Venom Chemicals and drugs Water Osmotic lysis Oxidants Hemoglobin denaturation Lead Erythrocyte membrane

damage 432 Chapter 20 the microangiopathic hemolytic anemias (MAHA) and are characterized by the presence of schistocytes, thrombocyto- CASE STUDY (continued from page 430) penia, and ischemic damage to organs.1,2,3,4,5 In these condi- Mai’s peripheral blood smear showed moderate tions, damage to the endothelial lining of the small vessels schistocytes and polychromasia. results in deposits of fibrin (which sometimes includes 1. What are some conditions that result in the pres- platelets) within the vessel. In addition, neutrophils’ role ence of schistocytes? in inflammation and thrombosis through neutrophil extra- cellular traps (NETs) has been explored6 (Chapter 7). As the erythrocytes are forced through the fibrin strands, the membrane can be sliced open. Sometimes the erythrocyte HEMOLYTIC UREMIC SYNDROME membrane reseals itself, leading to abnormal erythrocyte Hemolytic uremic syndrome (HUS) is a multisystem dis- shapes that are noted as schistocytes and keratocytes on order first described in the mid-1950s. It is characterized by the peripheral blood smear (Chapter 10). These damaged a triad of clinical findings: cells are often prematurely removed in the spleen (extra- • Hemolytic anemia with erythrocyte fragmentation vascular hemolysis). Severely damaged erythrocytes can be destroyed intravascularly. Depending on the underly- • Thrombocytopenia ing pathology, leukocytes can be increased and platelets • Acute nephropathy, which can include acute renal can be decreased. Evidence of intravascular coagulation failure (formation of fibrin within the blood vessels) and fibrino- Most individuals recover renal function.1,4,7 In some cases, lysis (breakdown of fibrin) could be present. The plasma there can also be evidence of mild neurologic problems, concentration of markers of hemolysis (bilirubin and hap- although only a small percentage develop severe symptoms. toglobin) varies depending on the type and extent of hemo- HUS can be subdivided into several categories. In this lysis (Chapter 11). text, classic STEC-HUS, secondary HUS (due to other infec- The underlying diseases and conditions responsible tious and noninfectious causes), and atypical HUS are dis- for TMA include classic Shiga toxin–producing E. coli hemo- cussed (Table 20-3). A previous categorization of two groups lytic uremic syndrome (STEC-HUS), atypical hemolytic uremic (D+ HUS and D- HUS) based on the presence or absence syndrome (aHUS), and thrombotic thrombocytopenic purpura of a bloody diarrheal prodrome is no longer recommended. (TTP). Other conditions that are characterized by microan- giopathic hemolytic anemia include malignant hyperten- Shiga Toxin–Producing E. Coli Hemolytic Uremic Syndrome sion, autoimmune disorders, sepsis, disseminated cancer, (Stec-Hus) Classic STEC-HUS is related to the presence and pregnancy (eclampsia, preeclampsia). A severe form of an infectious organism that produces Shiga-like toxin of preeclampsia characterized by hemolysis, elevated (other organisms that do not produce Shiga-like toxin may liver enzymes and low platelet counts (HELLP) syndrome also cause HUS). STEC-HUS is the most common form and can also cause MAHA. HUS and TTP can present with represents about 90% of cases. Onset tends to occur in chil- similar initial clinical symptoms, but the underlying etiol- dren between the ages of 6 months and 10 years; however, ogy, the age group affected, and the target organ(s) differ most cases are seen in children up to the age of 5 years. It (Table 20-2). is also considered the most common cause of acute renal failure in children and can lead to chronic renal insuffi- ciency (Table 20-2). Adult onset of this type is infrequent Checkpoint 20.1 and occurs in those over 16 years of age. The elderly are at What abnormal erythrocytes characterize microangiopathic highest risk for severe disease. hemolytic anemia? How are these cells formed? Classic STEC-HUS is characterized by bloody diarrhea (or hemorrhagic colitis) with more than 90% of the cases Table 20.2 Comparison of Characteristics Associated with STEC-HUS and TTP TTP HUS Adults ages 20–50 Children less than 5 years old Hemolytic anemia with red cell fragmentation Hemolytic anemia with red cell fragmentation Renal dysfunction (mild to moderate) Acute renal failure Thrombocytopenia Thrombocytopenia Severe CNS symptoms Mild CNS symptoms Fever Hemolytic Anemia: Nonimmune Defects 433 associated with gastrointestinal infections by specific sero- the individual had an invasive infection such as pneumo- types of Escherichia coli, which produce Shiga toxin (Stx). nia or bacteremia and the causative organism belonged to Most other cases are associated with infection by Shigella a serotype not included in pneumococcal vaccines. Occa- dysenteriae Type 1.7 The incubation period from infection sional cases have been linked to viral infections such as to onset of diarrhea is usually 5–6 days but can range from Epstein-Barr virus, human immunodeficiency virus, and 1–14 days. However, only about 15% of individuals who are cytomegalovirus. infected with Shiga toxin-producing E. coli develop HUS. Secondary HUS that is due to noninfectious include In the United States, the most common serotype of connective tissue diseases such as systemic lupus erythe- Shiga toxin–producing E. coli (STEC), or enterohemorrhagic matosus and lupus anticoagulant, as well as some types of E. coli (EHEC), is E. coli O157:H7. Other enterohemorrhagic cancer (especially stomach, colon, and breast) and diabe- serotypes including 026, 011, 0104, and 0145 have been iden- tes.2,3 The disease has been reported in young women with tified.8,9,10 The causative organism is not part of the normal complications of pregnancy, after normal childbirth, or with flora of humans but is found in the gastrointestinal tract of the use of oral contraceptives. Many drugs including anti- a small percentage of cattle. Most human infections have platelet drugs and antineoplastic agents have been reported been traced to the ingestion of incompletely cooked beef to cause rare cases of HUS. HUS secondary to other diseases contaminated with the organism, but the organism can also has a higher risk of recurrence and a lower survival rate be transmitted by the fecal-oral route (inadequate hand than cases that are associated with colitis or have no identi- washing). fiable trigger (primary). Other factors such as elevated neutrophil counts, Atypical HUS The term atypical HUS (aHUS) is used to increased C-reactive protein (CRP), and fever are associated describe cases of HUS caused by mutations in complement with an increased risk for developing HUS. Those under regulatory proteins or autoantibodies to these proteins.13,14,15 In 5 years of age and over 75 years of age are also at greater many cases these are inherited or familial. Episodes of aHUS risk.11 The use of antibiotics or antimotility drugs for treat- often recur.16 Almost 50% of cases are mutations related to three ing individuals with diarrhea from E. coli O157:H7 has also regulatory proteins associated with the alternate complement been associated with increased risk for developing HUS.11,12 pathway. These proteins, which function to control ampli- Secondary HUS The term, secondary HUS is often used fication of the pathway by C3bBb, are factor H, membrane for post-infectious HUS not caused by STEC or caused cofactor protein (MCP, CD46), and factor I. Other mutations in complement factor B and C3 have been identified.14,17,18,19,20,21 by other noninfectious causes (Table 20-3). Streptococcus pneumoniae (S. pneumoniae) is the organism most com- In many cases, these mutations do not manifest until a trigger- ing event occurs.18,19 monly linked to the post-infectious type. In most cases, An autoantibody against factor H has also been implicated in up to 15% of cases.14 The impaired regula- tion of the complement cascade leads to increased endothelial Table 20.3 deposition of C3, increased C5a, activation of the membrane Types of HUS and Associated Conditions attack complex (MAC), glomerular endothelial cell destruc- That Can Precipitate HUS tion, and microvascular thrombosis.20 Type of HUS Associated Condition CLASSIC STEC-HUS Diarrhea related (classic) Post-infection with: Checkpoint 20.2 • Escherichia coli O157:H7 What are the two types of HUS, and what organisms or dis- • other serotypes of E. coli eases are most commonly associated with each type? • Shigella dysenteriae serotype SECONDARY Post-infection with: • S. pneumoniae Pathophysiology More than 70% of the cases of HUS • Viral infections have been associated with damage to the renal glomeru- Immunosuppression related lar capillary endothelium by the Shiga toxin produced Chemotherapy/cytotoxic drugs by E. coli O157:H7 and S. dysenteriae Type 1.8,9 Once the ATYPICAL Mutations in the genes for comple- organism enters the human gastrointestinal tract, it colo- ment regulatory proteins nizes and forms an attachment lesion and begins to secrete Autoantibodies to complement regulatory proteins virulence factors including Stx. The organism’s toxin is Pregnancy or oral contraceptive absorbed into the circulation through damaged gastroin- related testinal tissue and onto the surface of neutrophils (Figure OTHER Renal and bone marrow transplantation 20-1). Neutrophils then deliver the Stx to the kidney. This Pregnancy or oral contraceptive toxin has a predilection for endothelial cells of the micro- related vasculature of the glomerulus and exerts a direct toxic 434 Chapter 20 Shiga toxin produced by organism Catabolic enzymes (especially leukocyte elastase) and oxi- dative products released from the granules of activated neutrophils have been implicated in causing additional Local damage to colon mucosa endothelial damage. Toxin enters circulation Checkpoint 20.3 Explain how infection with E. coli O157:H7 results in intravas- cular hemolysis. Toxin damages endothelial cells of capillaries in glomeruli Clinical Presentation STEC-HUS occurs in previously Platelet activation/fibrin microthrombi form Renal failure healthy children with the highest incidence in the first year of life. The onset is acute with sudden pallor, abdominal pain, vomiting, foul-smelling and bloody diarrhea, and mac- Erythrocytes damaged as trapped in thrombi roscopic hematuria. Other symptoms include a low-grade fever, hypertension, petechiae, bruising, and jaundice. The most important and/or serious complication of HUS is acute Schistocytes renal failure, which can lead to chronic renal insufficiency in some children. The duration of oliguria and anuria is vari- able. Organs other than the kidney can be affected as well. Intravasular hemolysis Splenic sequestration Regardless the organ affected, the pathology is the same (i.e., thrombosis of the microcirculation). Central ner- Figure 20.1 A possible mechanism for damage by Shiga-like vous system symptoms can result directly from microan- toxin of Escherichia coli O157:H7. giopathy of the central nervous system or from resulting hypertension. Lethargy and minor seizures are the most effect when bound to the endothelial cells. The toxin’s B common symptoms. Hepatomegaly can be present; sple- subunits bind to plasma membrane Gb3 receptors while nomegaly is less common. Hyperglycemia is common in the A subunit inhibits ribosomal protein synthesis, leading children because of pancreatic damage secondary to HUS. to cell death. Infiltrates of inflammatory cells and lipo- aHUS may occur at any age and clinical findings are like polysaccharides and the production of pro-inflammatory those of STEC-HUS.19 Most cases have an abrupt onset. cytokines such as IL-8 and tumor necrosis factor@a and chemokines contribute to the cytotoxic damage in glo- Laboratory Evaluation A moderate to severe normocytic, merular and renal tubular cells.7,9,10,11 Endothelial damage normochromic anemia is typical with hemoglobin levels as leads to the release of prothrombotic-, vasoactive-, and low as 3–4 g/dL (median values 7–9 g/dL) (Table 20-4). The platelet-aggregating substances that cause platelet activa- peripheral blood smear shows fragmented and deformed tion with the subsequent formation of thrombi. Although damage primarily occurs in the renal microvasculature, other organ systems (central nervous system, heart, liver) Table 20.4 Laboratory Findings in HUS and TTP can be affected. The resulting thrombotic microangiopa- Evidence of hemolysis Decreased hemoglobin/hematocrit thy that traps erythrocytes and causes fragmentation is Increased reticulocytes/ polychromasia responsible for the schistocytes commonly seen in HUS. Thrombocytopenia In addition, Shiga toxin has been shown to transiently Leukocytosis with shift to the left activate the alternative complement pathway and inhibit Presence of schistocytes regulatory proteins.11,21,22,23 Evidence of intravascular Hemoglobinemia hemolysis Hemoglobinuria In S. pneumoniae infections, capillary damage is medi- Decreased haptoglobin ated by the bacterial enzyme neuraminidase.12,24 Neuramin- Increased total and unconjugated serum bilirubin idase cleaves cell membrane glycoproteins and glycolipids, Increased lactic dehydrogenase facilitating tissue invasion by the bacteria and exposing the (LD) normally hidden T-antigen (Thomsen-Friedenreich antigen) Evidence of thrombotic Thrombocytopenia microangiopathy Fibrin degradation products (nor- on capillary walls, platelets, and erythrocytes. Naturally mal to slightly increased); D-dimer occurring anti-T antibodies cause agglutination of cells and increased platelets leading to thrombosis in the small vessels. Some PT and APTT (normal to slightly abnormal) evidence suggests that increased C3b deposition on cells Factors I, V, VIII (normal to may lead to cell destruction via the complement pathway. increased) Hemolytic Anemia: Nonimmune Defects 435 cells (schistocytes, burr cells, helmet cells, spherocytes) with later section “Disseminated Intravascular Coagulation”), the degree of anemia correlated directly with the degree coagulation screening tests are abnormal. The

direct and of morphologic change (Figure 20-2). Polychromasia and indirect antiglobulin tests are usually negative, reflecting a an occasional nucleated erythrocyte can be seen. A leuko- nonimmune pathology. cytosis with a shift to the left is common. Platelet counts Urinalysis results show moderate to massive amounts vary from low normal to markedly decreased with a median of protein (1–2 g/24 hours to 10 g/24 hours), gross or value of 50 * 103>mcL. The duration of thrombocytopenia microscopic hematuria, increased numbers of neutrophils is 1–2 weeks. (pyuria), and casts (hyaline, granular, renal epithelial), Hemoglobinemia with an increase in total serum bili- reflecting the damage to the glomerulus. The presence of rubin (2–3 mg/dL) and a decrease in serum haptoglobin hemosiderin in the urine sediment reflects chronic intravas- reflects chronic intravascular hemolysis. Serum lactate cular hemolysis (Chapter 11). dehydrogenase (LD) is markedly elevated, and cardiac enzymes can be elevated because of myocardial dam- age. Blood urea nitrogen (BUN), and creatinine levels are Checkpoint 20.4 What are the typical erythrocyte morphology and coagulation increased, and the glomerular filtration rate is decreased, test results in children with HUS? reflecting renal damage. Metabolic acidosis, hyponatremia, and hypokalemia are common. Screening for complement abnormalities (CH50 assay, C3 and C4 levels, complement Therapy Mild to moderately severe cases of STEC-HUS factor H [CFH] and complement factor I [CFI] levels) can have the best prognosis for recovery (more than 80%). be indicated in patients with symptoms of aHUS.22,23 The With improvement in early diagnosis and supportive care, CH50 is a test of total complement activity and is reported especially during oliguric or anuric phases, the mortal- as the reciprocal of the serum dilution that lyses 50% of a ity of the disease has been reduced to 5–15%. Supportive red cell suspension. In addition, tests for antibodies to these care includes close observation, blood transfusion if neces- factors, flow cytometry for MCP on white blood cells, and sary, control of electrolyte and water imbalances, control of molecular studies to identify mutations in specific genes hypertension, and peritoneal dialysis in anuria.7,15,25 Plate- may be indicated.15 In HUS caused by S. pneumoniae, the let transfusions are not recommended because they can direct antiglobulin test (DAT) may be positive.7 exacerbate the thrombotic process but may be required in Screening tests for coagulation abnormalities include some patients with excessive bleeding. Antibiotic therapy the prothrombin time (PT) and activated partial throm- in STEC-HUS is not recommended.12 The beneficial use of boplastin time (APTT) (Chapter 32). Many cases of HUS fresh frozen plasma exchange (removal of patient plasma exhibit no detectable consumption of coagulation factors. and replacement with donor plasma) has not been shown The PT can be normal or slightly prolonged, but the APTT to be efficacious in patients with STEC-HUS. In contrast, is usually normal. Although fibrin-degradation products patients with aHUS, especially that caused by complement (fragments of fibrin produced by plasmin degradation of dysregulation, can benefit from plasma exchange or plasma fibrin) and D-dimer are elevated, disseminated intravascu- infusions. Plasma infusions are contraindicated in patients lar coagulation (DIC) is rare (Chapter 34). In DIC (see the with HUS who have a positive direct antiglobulin test or who are infected with S. pneumoniae because of the presence of naturally occurring anti-T in the infused plasma. Potentially promising preventative measures in indi- viduals infected with Shiga toxin-producing E. coli is the use of monoclonal antibodies against Shiga toxin to provide passive immunity or the use of Shiga toxin receptor ana- logs.12,15,25 There is only a small window of time (3–5 days) from onset of diarrhea in which treatment can be effective, so detection of the toxin in stool or blood by flow cytometry must be performed. Although plasma exchange has been the primary therapy for aHUS, the understanding of the etiology has changed treatment recommendations. The drug eculizamab is a recombinant, monoclonal anti-C5 antibody. Originally Figure 20.2 used to treat paroxysmal nocturnal hemoglobinuria, it has A peripheral blood smear from a patient with hemolytic uremic syndrome. The platelets are markedly decreased. proved to be effective in aHUS.14,15,19 The drug suppresses Schistocytes and spherocytes are present (Wright-Giemsa stain, complement activation by binding to C5 and preventing 1000* magnification). cleavage into C5a and C5b, thus preventing progression to 436 Chapter 20 the membrane attack complex (MAC). This drug is now rec- capillaries and arterioles in several organs including the ommended as a first-line therapy in aHUS.14,25,26 Treatment kidneys, heart, brain, and pancreas (VWF is a plasma pro- should be initiated as soon as STEC-HUS and ADAMST13 tein needed for platelets to adhere to collagen). Although deficiency have been ruled out and may be required for these thrombi contain platelets and sometimes immunoglo- life.14 Kidney transplant in patients with aHUS has poor bin and complement, there is little fibrin, inflammation, or outcome because of the recurring nature of the condition.19 subendothelial exposure as in disseminated intravascular coagulation (DIC). 27 THROMBOTIC THROMBOCYTOPENIC PURPURA Thrombotic thrombocytopenic purpura (TTP) is another Ultra large multimers of VWF normally are cleaved relatively uncommon disorder in which platelet aggrega- into smaller forms by the protease ADAMTS13 (a dis- tion on the microvascular endothelium results in serious integrin and metalloprotease with thrombospondin complications. In most cases, it is an acute disorder that type 1 motif) (Chapter 35). It is now known that a defi- affects young adults (ages 20–50 with a peak incidence in ciency in ADAMTS13 is the cause of TTP. Because of an the third decade). TTP occurs more frequently in females ADAMTS13 deficiency, the ultra large VWF multimers than males. TTP can be congenital or acquired (nonidio- remain attached to the endothelial cells and adhere to pathic or idiopathic). Genetic mutations in ADAMTS13 platelets, inducing platelet aggregation and formation of Chapter 32 may be associated with a single gene or combi- platelet thrombi. As erythrocytes are forced through the nations of mutations in multiple genes.17,27 thrombi, fragmentation occurs. There is some evidence Various clinical events have been identified as possible that these ULVWF may also activate the alternative com- precipitating factors in acquired TTP (Table 20-5). Infections plement pathway.5,6,27 The familial form of TTP occurs are the most common precipitating factor (40%) followed because of a mutation in the ADAMTS13 gene, resulting by pregnancy (10–25%). Without treatment, TTP has a mor- tality rate more than 90% because of multiorgan failure. in a deficient/dysfunctional enzyme. The acquired type The disorder is discussed in detail in Chapter 35, but an is caused by autoantibodies against ADAMTS13, which overview comparison with HUS in this chapter highlights block its activity. several key findings. Clinical Presentation The clinical aspects of TTP are like Pathophysiology TTP is characterized by microthrombi those of HUS except that TTP occurs most often in young composed of platelets and unusually large forms of von adults and involves more organ systems. Neurologic symp- Willebrand factor (VWF) (Chapters 31, 32) that occlude toms are more prominent, renal dysfunction is less severe, and the mortality rate is higher than in HUS (Table 20-2). Symptoms can be eliminated with early treatment, although some patients recovering from TTP can have permanent Table 20.5 Some Reported Clinical Conditions That Can manifestations of renal damage and require dialysis. Be Precipitating Factors in TTP Laboratory Evaluation Typical laboratory results are Conditions Examples shown in Table 20-4. The hemoglobin is usually less than Infections Bacterial—enteric organisms (E. 10.5 g/dL (average 8.0–9.0 g/dL). The mean cell volume coli, Shigella, Salmonella, Campy- lobacter, or Yersinia species) (MCV) is variable, either normal or decreased if there is marked erythrocyte fragmentation or increased in the Bacterial—other (S. pneumoniae, Legionella, Mycoplasma species) presence of reticulocytosis. The mean corpuscular hemo- Viral (HIV, EBV, influenza, herpes globin (MCH) and mean cell hemoglobin concentration simplex) (MCHC) are normal. Reticulocytes and nucleated eryth- Drugs Antimicrobials—penicillin rocytes can be found in the peripheral blood, reflecting Ticlopidine, Chemotherapeutic the bone marrow response to hemolysis. The most striking agents blood finding is the abundance of schistocytes (generally Connective tissue diseases Systemic lupus erythematosus more than 1%). Leukocytosis with counts of more than Rheumatoid arthritis 20 * 103>mcL occurs in 50% of patients and is usually Ankylosing spondylitis accompanied by a shift to the left. Thrombocytopenia is Sjögren’s syndrome often severe (8@44 * 103>mcL) because of consumption of Miscellaneous Bee sting platelets in the formation of microthrombi. Megakaryo- Dog bite cytes are abundant in the bone marrow, reflecting an Carbon monoxide poisoning increased hematopoietic response to the consumption of Pregnancy or oral-contraceptive platelets.28 related Coagulation tests are usually normal or only slightly Lymphomas and carcinomas abnormal in TTP; this helps to differentiate TTP from DIC Hemolytic Anemia: Nonimmune Defects 437 in which an increase in D-dimer as well as a prolonged PT, APTT, and thrombin time occur (Chapter 34). ADAMTS13 Table 20.6 Causes of Disseminated Intravascular Coagulation (DIC) activity level is the primary test to distinguish TTP from other TMas; the activity level is usually 610, and may be Bacterial sepsis Endotoxins as low as 5%.27,28 Exotoxins Hemoglobinemia, hemoglobinuria, decreased hapto- Neoplasm Solid tumors globin levels, and increased total and unconjugated serum Myeloproliferative disorders bilirubin are direct evidence of intravascular hemolysis. The Serious trauma —— direct antiglobulin test (DAT) is negative. Obstetrical complications Therapy Studies have shown that plasma exchange with Immunologic disorders Hemolytic transfusion reactions fresh frozen plasma (FFP) can be effective in providing the Transplant rejection needed ADAMTS13 protease.29,30 It is also effective in Miscellaneous Venom—snake or insect removing autoantibody (in acquired TTP) and is usually Drugs continued until the platelet count has recovered.31 Cryosu- pernatant, which lacks the large VWF multimers present in FFP, yet still contains the needed VWF cleaving protease, activation of plasma procoagulant proteins lead to deposi- can be used.32 Treatment options for those with autoanti- tion of fibrin and formation of widespread microthrombi in bodies include corticosteroids, the monoclonal antibody the microvasculature. As erythrocytes become entangled in rituximab (anti-CD20), antiplatelet or platelet-inhibiting the fibrin meshwork in the capillaries (clothesline effect), agents, intravenous administration of steroids, or combina- they fragment to form schistocytes. Complications that result tions of corticosteroids and plasma. Targeted therapies include thrombotic occlusion of vessels, bleeding because of including use of recombinant ADAMTS13 may be a future consumption of coagulation proteins, and ultimately organ option.31,33 failure. Hemolysis is not usually severe, but the effects of the consumptive coagulopathy can cause severe thrombocyto- penia and serious bleeding complications. Checkpoint 20.5 The typical findings on the blood smear include How does the clinical presentation of TTP differ from that of the presence of schistocytes and thrombocytopenia HUS? How is it similar? (Figure 20-3). The presence of schistocytes is not specific for DIC and they may only be present in low numbers.37 However, the abnormal coagulation tests help distinguish CASE STUDY this condition from others (TTP and HUS) that give a similar (continued from page 432) picture on a peripheral blood smear and increase diagnostic As Mai was questioned further, she indicated that accuracy.28,37 Abnormal coagulation tests include: she had noticed many bruises on her extremities. Her platelet count was 31 * 103>mcL. She had a • Prolonged prothrombin time (PT), activated partial 2.5% reticulocyte count. thromboplastin time (APTT), and thrombin time (TT) 2. What is the significance of these results? • Elevated D-dimer test 3. Why might the clinician order coagulation tests? DISSEMINATED INTRAVASCULAR COAGULATION Disseminated intravascular coagulation (DIC) is a complex thrombohemorrhagic condition in which the normal coagu- lation process (coagulation and fibrinolysis) is altered by an underlying condition. The more common conditions that precipitate DIC include bacterial sepsis, neoplasms, immu- nologic disorders, or trauma.34,35 It may also be observed as an obstetrical complication associated with severe pre- eclampsia, intrauterine fetal death, or post-partum hemor- rhage36 (Table 20-6). DIC can be initiated by damage to the endothelial lining of vessels, which causes release of throm- Figure 20.3 Peripheral blood from patient with disseminated boplastic substances that activate the coagulation mecha- intravascular coagulation. Notice the schistocytes, spherocytes, and nism. As a result, platelet activation and aggregation and thrombocytopenia (Wright stain, 1000* magnification). 438 Chapter 20 • Decreased platelet count of pregnancies with eclampsia develop HELLP syndrome • Increased fibrin degradation products (FDP) with a mortality rate of about 1%. The peripheral blood findings are like those found in • Decreased fibrinogen (often less than 150 mg/dL) TTP, HUS, and other microangiopathic conditions. Over- • Decreased antithrombin (AT) all, however, the hemolysis and thrombocytopenia are

less In some cases, specific tests such as procalcitonin, an inflam- severe than those associated with TTP or HUS. Liver dam- matory biomarker, can be useful if septic DIC is suspected. age is due primarily to obstruction of hepatic sinusoids and In these cases, there may also be decreased protein C and can lead to subsequent hepatic hemorrhage or necrosis. decreased antithrombin.34,38 Microvascular fibrin-like deposits that resemble those in Treatment can include erythrocyte and platelet trans- TTP/HUS are responsible for the presence of schistocytes. fusions as well as infusion of fresh frozen plasma or factor Laboratory markers are used to determine the presence concentrates to replace coagulation factors. Some patients of HELLP. The liver enzyme most frequently measured may require specific fibrinogen replacement therapy.36 Most is aspartate aminotransferase (AST), and concentrations important, however, are the treatment and resolution of the more than 70 IU/L are common. Increased total (more than underlying disorder responsible for the DIC. The etiology, 1.2 mg/dL) and/or unconjugated bilirubin, increased LD diagnosis, and treatment of DIC is discussed further in (generally more than 600 IU/L), and decreased haptoglo- Chapter 34. bin can also be seen in HELLP. Coagulation tests such as the PT and APTT are usually normal until late in the dis- ease course. The platelet count decreases (usually less than Checkpoint 20.6 100 * 103>mcL) as a result of platelet aggregation and con- Explain how DIC can be differentiated from TTP and HUS based sumption at the site of endothelial damage.43 Although DIC on coagulation tests. infrequently occurs as a complication of HELLP, patients with lower platelet counts (less than 50 * 103>uL) can be at increased risk. There is evidence that some patients HELLP SYNDROME have a compensated form of DIC based on changes in The HELLP syndrome is an obstetric complication char- D-dimer values. Acute tubular necrosis with renal failure, acterized by hemolysis, elevated liver enzymes, and a low hepatic rupture, and pulmonary edema can also occur as platelet count that usually develops prior to childbirth, often complications.40 between the 26th and 28th week of gestation.39,40 Up to 30% Corticosteroid therapy can be useful in controlling cell of cases develop within 48 hours post-delivery.40 A partial destruction and decreasing liver enzymes if the fetus can- or incomplete form in which only 1 or 2 of the characteristic not be delivered immediately. Plasma exchange is rarely findings is present has also been described. HELLP must used as a treatment except in those whose values do not be distinguished from preeclampsia, TTP, and complement- normalize within 48 hours post-delivery or who progress induced thrombotic microangiopathy.41,42 The etiology and to post-partum thrombotic syndrome. The use of plasma pathogenesis are not well understood, but an association exchange will remove procoagulant factors.40,45 The use of with pregnancy-induced hypertension and DIC is sug- cortisone dexamethasone has eliminated the need for plate- gested.43 As with TTP and HUS, the precipitating factor is let transfusions in most patients with platelet counts less often unknown, but the clinical aspects are characterized than 50 * 103>mcL. by capillary endothelial damage and intravascular platelet activation as well as microangiopathic hemolytic anemia.39 Several gene variations including ones in the FAS and coag- CASE STUDY (continued from page 437) ulation Factor V Leiden have shown to increase risk. Placen- Mai’s PT and APTT were slightly prolonged. The tal factors may induce an inflammatory response that leads fibrinogen levels were slightly decreased. to activation of the complement and/or coagulation cas- cade.43 Although a significant prevalence of HELLP occurs 4. What do these findings indicate about the under- in patients with antiphospholipid syndrome, there seems lying problem? to be no direct association with the presence of IgM or IgG anticardiolipin antibodies or anti-b2-glycoprotein-I antibod- ies.44 Some experts consider HELLP to be a severe form of preeclampsia or eclampsia, which shares some of their char- MALIGNANT HYPERTENSION acteristics such as hypertension, proteinuria, and thrombo- Malignant hypertension can present with a low platelet cytopenia, but it is distinguished from them by the presence count, erythrocyte fragmentation, and renal failure. In addi- of hemolysis and elevated liver enzymes. Severe cases can tion, the presence of schistocytes, low platelet count, and compromise fetal growth and survival. Approximately 10% increased LD has been used to predict renal insufficiency as Hemolytic Anemia: Nonimmune Defects 439 well as recovery.The mechanism of hemolysis is unknown. depends on the percentage of body surface area burned. It may be caused by endothelial injury, fibrinoid necrosis Hemolysis probably results from the direct effect of heat- of arterioles, or deposition of fibrin fed by thromboplas- causing protein denaturation of spectrin in the eryth- tic substances released from membranes of lysed erythro- rocyte membrane. If erythrocytes are heated to 48 °C in cytes.15,46,47 Some individuals with malignant hypertension vitro, spectrin degradation causes a loss of elasticity and have decreased levels of ADAMTS13 activity; this may be deformability. In addition, the fatty acid and lipoprotein caused by an increased release of VWF that occurs because metabolism in both plasma and erythrocytes are altered of endothelial cell stimulation. after burn injuries, which can contribute to abnormal erythrocyte morphology. Peripheral blood smears show OTHER CONDITIONS ASSOCIATED WITH MAHA erythrocyte budding, schistocytes, and spherocytes. After Hematopoietic stem cell transplant recipients may show 48 hours, signs of hemolysis such as hemoglobinuria and transplant-associated thrombotic microangiopathy (TA- hemoglobinemia decrease. Thermal injury to erythrocytes TMA). TA-TMA may represent a form of graft-versus- also has occurred during hemodialysis when the dialysate host disease (GVHD) and result from endothelial damage is overheated. induced by donor cytotoxic T-cells or by treatment-related endothelial cell injury.48,49 It is characterized by many of EXERCISE-INDUCED HEMOGLOBINURIA the same diagnostic criteria as other TMAs including schis- Exercise-induced hemoglobinuria (sometimes described tocytes, decreased platelet counts, decreased hemoglobin, as march or runner’s hemoglobinuria) describes a transient and increased LD. The alternative and classical complement hemolysis occurring after strenuous exercise and often pathways are involved.49 Although the condition can resem- involves contact with a hard surface (e.g., running, tennis, ble TTP, ADAMTS13 activity is more than 5%. The kidney marching). The hemolysis is probably because of physical is most commonly affected but lungs, gastrointestinal tract, injury to erythrocytes as they pass through the microvas- and brain may also be affected. Treatment may include use of plasma exchange and eculizamab. 48,49 culature. Plasma hemoglobin and haptoglobin levels indi- cate that the primary cause of the transient hemolysis is Cancer-related MAHA (CR-MAHA) is uncommon but intravascular lysis.57,58 However, it is not seen in all indi- may be related to destruction of erythrocytes and plate- viduals participating in these activities and is occasion- lets in small vessels in cancerous tissue. Patients have a ally seen in other physical activities such as swimming, DAT-negative hemolytic anemia with schistocytes and thrombocytopenia.50 cycling, and rowing in which contact with a hard surface is limited. Hand drumming or percussion hemolysis has Drugs may also be linked to TMA in a few circum- a similar pathogenesis, only the damage is initiated in the stances. The process must be distinguished from immune- palms of the hands, not the soles of the feet.59 In recent mediated lysis (drug dependent antibodies or drug independent antibodies).51,52 years, the role of exercise-induced oxidative stress and red cell age have been recognized as potential additional causes of lysis, especially in normally sedentary indi- Other Erythrocyte Physical Trauma viduals who participate in strenuous exercise. Increased Resulting in Hemolytic Anemia osmotic fragility and decreased deformability leading to TRAUMATIC CARDIAC HEMOLYTIC ANEMIA intravascular hemolysis were noted in these individuals. Hemolytic anemia is a recognized complication following In addition, changes in erythrocyte membrane proteins surgical insertion of prosthetic heart valves or with aortic such as spectrin, especially in older cells, can increase sus- valve stenosis.53,54,55 However, massive hemolysis is rarely ceptibility to extravascular hemolysis during strenuous a complication.53 Unlike the microangiopathic anemia seen exercise. with TTP or DIC, the platelet count usually does not signifi- In contrast to the other hemolytic conditions dis- cantly decrease.53,56 Excessive acceleration or turbulence of cussed so far in this chapter, no erythrocyte fragments blood flow around the valve can fragment the erythrocytes appear on the peripheral blood smear, but the hallmarks as a result of “shear stress.” Many erythrocyte fragments of intravascular hemolysis—hemoglobinemia and hemo- are apparent on the blood smear. The spleen removes some globinuria—can be present. The passage of reddish urine of the severely traumatized cells, but many cells undergo immediately after exercise and for several hours thereaf- intravascular hemolysis. Newly designed prosthetic valves ter is usually the only complaint from affected individu- have helped decrease the shear stress and resulting eryth- als. Anemia and decrease in total erythrocyte volume are rocyte fragmentation. uncommon because less than 1% of the erythrocytes are hemolyzed during an episode.60 Individuals can present THERMAL INJURY with a slightly increased MCV because of increased reticu- Hemolytic anemia occurs within the first 24–46 hours locytes. Iron deficiency can occur if exercise and hemolysis after extensive thermal burns, and the degree of hemolysis are frequent.60 440 Chapter 20 methods, including invasion of the erythrocyte by parasites, CASE STUDY (continued from page 438) damage to the cell membrane, production of hemolysins Mai’s symptoms continued to worsen with that lyse the erythrocyte, or stimulation of antibodies or f requent seizures, headaches, and dizziness. immune complex deposits that result in increased phago- Her urinalysis results showed a 2+ protein and cytosis of the cell.61 moderate blood. However, she had normal urinary volume. Infectious Agents 5. Based on these results, what is the most likely Parasites and bacteria can infect erythrocytes and lead condition associated with these clinical and directly to their destruction. Alternatively, toxins produced laboratory results? Explain. by infectious agents can cause hemolysis. 6. What therapy might be used? MALARIAL PARASITES The anemia accompanying malaria is due directly and indirectly to the intracellular malarial parasites that spend part of their life cycle in the erythrocyte (Figure 20-4). The Hemolytic Anemias anemia resulting from this infection is usually a mild, nor- Caused by Antagonists in mocytic normochromic anemia but can be severe in infec- tion with Plasmodium falciparum because of the high levels of The Blood parasitemia. Thrombocytopenia can also be present. There are some kits available to detect parasite-specific antigens Antagonists such as drugs or venoms and infectious in the blood, but definitive diagnosis involves finding the organisms can cause premature erythrocyte destruction life cycle stage within the erythrocyte on a peripheral blood (Table 20-1). This hemolysis is precipitated by multiple smear. Infection with P. falciparum, a cause of severe anemia a b c Figure 20.4 Peripheral blood smears from patients with malaria. (a) A ring form of malaria in the erythrocyte. (b) An immature schizont form of malaria in the erythrocyte. (c) A gametocyte of Plasmodium falciparum in the erythrocyte (Wright stain, 1000* magnification). Hemolytic Anemia: Nonimmune Defects 441 in children, can be accompanied by ineffective erythropoi- esis and decreased reticulocytes. The hemoglobin in these cases can reach levels as low as 5 g/dL. In addition, poor diet, malnutrition, and decreased iron and folate stores contribute to the severity of the anemia. Exchange transfu- sions can be used in the severest cases to remove infected erythrocytes. The release of the intraerythrocytic parasite from the cell results in the cell destruction. Also, the spleen can remove the entire parasitized cell, or splenic macrophages can pit the parasite from the erythrocyte, damaging the cell membrane. The decreased deformability of the erythrocyte is due to changes in the cell membrane by splenic pitting, remodeling of the erythrocyte cytoskeleton by parasite proteins, as well as the presence of the large intracellular organism itself.62,63,64 The resulting decreased deformability can lead to removal of the cell by the spleen. Anemia can also result from an immune-mediated process. Antimalarial Figure 20.5 Peripheral blood smear of a patient with antibodies react with malarial antigens on the erythrocyte babesiosis. Several infected erythrocytes are in the field (Wright membrane, resulting in removal of the sensitized cell by the stain, 1000* magnification). splenic macrophages. In some cases, the concentration of complement regulatory proteins decrease, which can facili- doubles or tetrads in the form of a Maltese cross. Travel tate complement-mediated hemolysis. history, the absence of the characteristic banana-shaped Blackwater fever, an uncommon complication of infec- gametocytes, and the lack of pigment help distinguish it tion with P. falciparum, is characterized by massive acute from P.

falciparum. Most infections are asymptomatic, but intravascular hemolysis with hemoglobinemia, hemoglo- some cases present with a flulike syndrome. Generally, binuria, methemalbuminemia, and hyperbilirubinemia. there is 1–10% parasitemia. Extravascular hemolysis can However, the parasitemia level is often low. The mechanism occur in a manner like that seen with malaria. A mild to that precipitates this is unclear. One possible mechanism is moderate normocytic anemia as well as thrombocytopenia the development of an autoantibody to the infected eryth- can be present. Other possible laboratory findings include rocyte. Another is a direct reaction to the drug quinine or to increased reticulocyte count, liver enzymes, and bilirubin. repeated incomplete treatment with the drug. Quinine can In a rare fulminating infection, severe anemia, intravascular act as a hapten to stimulate formation of a drug-dependent hemolysis, and hemoglobinuria are seen.67 Complications antibody that has complement-fixing ability. In some cases, associated with intravascular hemolysis include renal fail- the direct antiglobulin test (DAT) can be positive with either ure and DIC. Individuals at greatest risk of complications monospecific anticomplement or anti-IgG. Use of synthetic are the elderly and those who are splenectomized or immu- quinine drugs has considerably decreased the frequency of nosuppressed.68 Patients who are splenectomized generally this complication. It is possible that other antimalarial drugs have a more severe clinical presentation and higher levels of including mefloquine could trigger this response. parasitemia. In severe cases of hemolysis or renal complica- tions, exchange transfusion may be indicated. BABESIOSIS Babesiosis, a protozoan infection of rodents and cattle, is most commonly transmitted to humans by the bite of a hard Checkpoint 20.7 tick. However, the organism can be acquired transplacen- Why do malaria and babesiosis result in anemia? tally and through packed red blood cell transfusions. In the United States, it occurs most frequently in the New England area but cases can be found in Virginia and the Midwest. BARTONELLOSIS The most common organism for insect and transfusion Gram-negative bacterial organisms in the genus Barton- transmission is Babesia microti; however, other species such ella are zoonotic pathogens transmitted by blood-sucking as B. divergensa and B. duncani have been linked to trans- arthropods or by direct inoculation by the scratch or bite of a mission.65,66,67 On the peripheral blood smear, the parasites mammal. The organisms infect erythrocytes and endothelial appear as intracellular, pleomorphic, 1 - 5 mcM 1mm2 cells. Infection by Bartonella bacilliformis is associated with ring-like structures resembling ring-form trophozoites of Carrion’s disease, which is currently restricted to Columbia, Plasmodium falciparum (Figure 20-5). Some can appear as Peru, and Ecuador. Travelers to these areas are at risk for 442 Chapter 20 acquiring infections, which may manifest months to years Thomsen-Friedenrich (T-antigen), which can react with non- after leaving the endemic area.69 Other Bartonella species immune autoantibody. The peripheral blood smear shows infect erythrocytes but are not associated with hemolytic many microspherocytes and few erythrocyte fragments. manifestations. The disease is transmitted by the bite of a female sand-fly and is biphasic with the acute phase char- acterized by an often fatal syndrome consisting of myalgia, Animal Venoms high fever, and an acute, severe hemolytic anemia (Oroya Venoms injected by bees, wasps, spiders, and scorpions may fever) with onset in approximately 21 days after inocula- cause hemolysis in some susceptible individuals. One of the tion. Hepatosplenomegaly and jaundice can also be present. more common bites that can result in hemolysis is that of the Changes in T-lymphocyte counts and levels of cytokines brown recluse spider (Loxosceles reclusa). The characteristic such as IL-10 can also be associated with acute infection. symptom of envenomation is a localized lesion that shows Some patients may suffer temporary immunosuppression.70 signs of inflammation and a central thrombosis surrounded The disease can progress to coma and death within weeks by ischemic areas. However, up to 15% of individuals, and has a high mortality in untreated cases, especially if especially children, the elderly, and immunocompromised other comorbidities exist.71 If the patient survives, a chronic patients, develop systemic symptoms including nausea and phase characterized by an inflammatory reaction presents vomiting, fever, jaundice, and intravascular hemolysis that weeks to months later. In this phase of the disease the can be severe.77,78 Patients have leukocytosis, anemia, hema- organisms invade endothelial cells. The disease is charac- turia, thrombocytopenia, and increased levels of creatine terized by a granulomatous reaction (verruga peruana) with phosphokinase (CK). There are some reports of a positive the appearance of cutaneous hemangioma-like lesions that DAT with complement on the erythrocyte after bites.79 The can contain bacteria, neutrophils, macrophages, endothelial mechanism of venom damage appears to involve sphingo- cells, and deposits of immunoglobulins.72 The pleomorphic myelinase D2, which cleaves glycophorin from the erythro- coccobacillary, organisms are readily visualized as single, cyte membrane.80 Hyaluronidase and other esterases may paired, or chained organisms on or within erythrocytes on also be involved in cell damage.81 This decreases the struc- Wright- or Giemsa-stained peripheral blood smears during tural integrity of the membrane and increases its sensitivity the course of the disease. The organism releases several pro- to complement-mediated lysis. Systemic loxoscelism may teins including deformin that are responsible for inducing be treated with plasma exchange.82 the pitting or invagination of the erythrocyte membrane. Although snake bites rarely cause hemolysis directly, These structures, as well as other proteins such as hemo- hemolysis can be the result of DIC or a venom-induced lysins, can serve as entry portals for the bacteria and help consumption coagulopathy (VICC) resulting from activa- explain the mechanism of cell destruction.72,73 tion of the coagulation pathway. Snake venom may contain a mixture of biologically active proteins (including proco- CLOSTRIDIUM PERFRINGENS agulant toxins, anticoagulant toxins, protein C activators, C. perfringens is part of the normal flora of the gastrointes- and platelet aggregation inducers and inhibitors that affect tinal tract. Infection may present as a transient bacteremia hemostasis.83 Phospholipid A2 can degrade lecithin in the or a life-threatening condition. It is one of the few organ- cell membrane.84,85 VICC has no systemic microthrombi, but isms (along with C. septicum and C. novyi) that can cause the D-dimer levels and activated PTT are elevated.85 a rapid, massive intravascular hemolysis.73,74 Although this complication is very rare, it carries a high mortality risk unless aggressively treated with antimicrobials and Chemicals and Drugs surgical drainage.75 Patients with neoplasms such as colon Various chemicals and drugs have been identified as possi- cancer or invasive tumors of the genitourinary tract are at ble causes of erythrocyte hemolysis; many of these are dose highest risk. The bacteria produce several potent exotoxins dependent. In addition to erythrocyte hemolysis, chemicals that affect host cell membranes. The major hemolytic toxin and drugs can also produce methemoglobinemia and cya- 1a@toxin2 is a phospholipase C that hydrolyzes sphingomy- nosis, or in some instances bone marrow aplasia. elin, phospholipids, and lecithin present in the erythrocyte Hemoglobinemia and hemoglobinuria can occur membrane and leads to changes in membrane integrity and because of osmotic lysis of erythrocytes when water enters the presence of spherocytes. Streptolysin O and perfringo- the vascular system during transurethral resection or when lysin have been implicated in development of DIC in these inappropriate solutions are used during a blood transfusion. patients.76 Fever, thrombocytopenia, neutrophilia, hemoglo- Some drugs known to cause hemolysis in G6PD-defi- binemia, and hemoglobinuria are present. Anuria or acute cient persons can also cause hemolysis in normal persons renal failure can develop as a result of the hemoglobinuria. if the dose is sufficiently high. The hemolysis mechanism is Lysis of the erythrocyte or other cells can cause DIC. Bac- like that in G6PD deficiency with hemoglobin denaturation terial neuraminidase may expose cryptic antigens such as and Heinz body formation because of strong oxidants. Hemolytic Anemia: Nonimmune Defects 443 Anemia associated with lead poisoning is usually iron within mitochondria (Chapter 12). However, lead classified with sideroblastic anemias because the patho- also damages the erythrocyte membrane, which is mani- physiologic and hematologic findings are similar. Lead fested by an increase in osmotic fragility and mechanical inhibits heme synthesis, causing an accumulation of fragility. Summary Mechanisms of nonimmune damage to the erythrocyte disorder in which platelet aggregation and unusually large are varied. Hemolytic anemia caused by traumatic physi- forms of von Willebrand factor (VWF) on the microvascu- cal injury to the erythrocytes in the vascular circulation lar endothelium leads to formation of platelet thrombi that can be characterized by extravascular or intravascular occlude capillaries and arterioles in multiple organs. The hemolysis. Microangiopathic hemolytic anemia refers to underlying cause of TTP is a deficiency in the ADAMTS13 a group of anemias caused by microcirculatory lesions that protease that cleaves the ultralarge multimers of VWF injure the erythrocytes, producing schistocytes. Of those into smaller forms. HELLP syndrome, another MAHA, is discussed in this chapter, HUS and TTP are the more com- thought to be a severe form of preeclampsia. HELLP is monly encountered. Classic HUS is the most common and characterized by hemolysis, elevated liver enzymes, and is mediated by the Shiga-like toxin of E. coli O157:H7. The a low platelet count. Fibrin-like deposits are responsible toxin enters the gastrointestinal tract and damages the for the presence of schistocytes. Antagonists in the blood mucosa. Once it enters the circulation, it has a predilection such as venoms and infectious organisms can also cause for the endothelial cells of the glomerular microvascula- hemolytic anemia. Intraerythrocytic parasitic infections ture and exerts a toxic effect. Endothelial damage leads with organisms such as Plasmodium sp. or Babesia sp. can to release of prothrombotic substances that cause plate- cause hemolysis and anemia without the presence of schis- let activation and formation of thrombi. The thrombi trap tocytes. In susceptible individuals, drugs or chemicals can erythrocytes and cause fragmentation of the cells. TTP is a also lead to hemolysis. Review Questions Level I c. spur cell anemia d. immune hemolytic anemia 1. Which of the following results is associated with HUS and TTP? (Objective 2) 4. Which of the following organisms does not cause damage of the erythrocyte because of an intraerythro- a. Increased haptoglobin cytic life cycle? (Objective 4) b. Thrombocytopenia a. Plasmodium falciparum c. Reticulocytopenia b. Babesia sp. d. Decreased LD c. Bartonella sp. 2. One of the major criteria that distinguishes DIC from d. Clostridium perfringens other causes of microangiopathic hemolytic anemia is: (Objective 2) 5. A characteristic finding on a blood smear in MAHA is the presence of: (Objective 3) a. the presence of schistocytes b. thrombocytopenia a. target cells c. decreased hemoglobin b. spur cells d. an abnormal coagulation test c. schistocytes d. echinocytes 3. A patient who has anemia with an increased reticulo- cyte count, increased bilirubin, and many schistocytes 6. All the following are characterized as causes of on the blood smear could have: (Objective 2) MAHA except: (Objective 1) a. MAHA a. TTP b. high cholesterol in the blood b. DIC 444 Chapter 20 c. HUS 2. A 34-year-old woman is brought into the ER after fall- d. March hemoglobinuria ing off a ladder while painting her house. Selected lab results include: 7. All the following are associated with HUS except: (Objective 2) Hb 8.0 g/dL PT 36 seconds (80 g/L) a. thrombocytosis Hct 25% APTT 775 seconds b. nucleated RBCs in peripheral blood (0.25 L/L) c. schistocytes Platelet 20 * 103>mcL Fibrinogen 100 mg/Dl d. reticulocytosis count 8. MAHA is most frequently caused by: (Objective 1) Peripheral blood smear shows schistocytes. Given these results, what is the most likely diagnosis? a. microcirculatory lesions (Objectives 2, 4) b. immune destruction a. HELLP syndrome c. antagonists in the blood b. TTP d. plasma lipid abnormalities c. DIC 9. Intravascular hemolysis in MAHA would be d. Traumatic hemolytic anemia associated with which of the following parameters? (Objective 2) 3. Laboratory results for a child who had easy bruis- ing, tiredness, difficulty breathing, and decreased Bilirubin Haptoglobin urinary output were hemoglobin: 6.5 g/dL; platelets: A. decreased decreased 41 * 103>mcL; PT and APTT within normal reference B. decreased increased intervals. His mother indicated he had an episode of bloody diarrhea about 2 weeks earlier but it had not C. increased decreased recurred. Based on the clinical and limited laboratory D. increased increased findings, what is the most likely condition? (Objectives 2, 4) 10. MAHA caused by HUS is usually seen in which age group? (Objective 1) a. TTP a. Children younger than 5 years of age b. Bartonellosis b.

Females between 20 and 50 years of age c. Clostridium sp. infection c. Either sex younger than 50 years of age d. HUS d. Males older than 16 years of age 4. The most likely age group for developing TTP is: (Objective 2) Level II a. female children younger than 1 year 1. A 43-year-old woman presents to her physician with a 3-week history of fatigue, constant headache, and b. women between 20 and 50 years low-grade fever. Selected laboratory results include: c. either gender younger than 5 years d. men older than 16 years Hb 7.5 g/dL Platelet count 16 * 103>mcL (75 g/L) 5. Which of the following disorders is not characterized Hct 23% Reticulocytes 11% by the presence of schistocytes? (Objective 3) (0.23 L/L) a. March hemoglobinuria RDW 15 b. Insertion of a prosthetic valve Peripheral blood smear showed schistocytes. Which c. Third-degree burns of the following drugs that the patient was taking d. Malignant hypertension could cause these symptoms and lab values? (Objectives 2, 4) 6. A patient with a deficiency in the VWF protease a. Ticlopidine ADAMTS13 would be at risk to develop which condi- tion? (Objective 2) b. Aspirin c. Estrogens a. HUS d. Quinine b. Spur cell anemia Hemolytic Anemia: Nonimmune Defects 445 c. TTP Her platelet count was less than 60 * 103>mcL and d. Hereditary acanthocytosis her hemoglobin was 7.5 g/dL. She had no history of chronic disease. What laboratory test(s) might give a 7. The formation of schistocytes in MAHA is primarily clue to the underlying cause? (Objective 4) due to: (Objective 1) a. Haptoglobin a. pitting by splenic macrophages b. Reticulocyte count b. defective cell membranes c. Liver enzymes c. increased membrane phospholipids d. APTT and PT d. shearing of erythrocytes by fibrin threads 10. Hemolytic toxins are the major cause of intravascular 8. Plasma exchange is used as a primary treatment for hemolysis in diseases or conditions caused by which which of the following? (Objective 2) of the following organisms? 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Huarcaya, E., Best, I., Rodriguez-Tafur, J., Maguina, C., S olorzano, 82. Said, A., Hmiel, P., Goldsmith, M., Dietzen, D., & Hartman, M. E. N., Menacho, J., . . . Ventosilla, P. (2011). Cytokines and (2014). Successful use of plasma exchange for profound hemoly- T-lymphocyte count in patients in the acute and chronic phases sis in a child with loxoscelism. Pediatrics, 134, e1464–e1467. of Bartonella bacilliformis infection in an endemic area in Peru: A doi: 10.1542/peds.2013-3338 pilot study. Revista do Instituto de Medicina Tropical de Sao Paulo, 53, 83. McCleary, R. J. R., & Kini, R. M. (2013). Snake bites and hemosta- 149–154. doi: http://dx.doi.org/10.1590/S0036-46652011000300006 sis/thrombosis. Thrombosis Research, 132, 642–646. 71. Angelakis E., & Raoult, D. (2014). Pathogenicity and treatment of 84. Bhagwat, K., & Amar, L. (2013). Blood hemoglobin, lactate dehy- Bartonella infections. International Journal of Antimicrobial Agents, drogenase and total creatine kinase combinely as markers of 44, 16–25. hemolysis and rhabdomyolysis associated with snakebite. Inter- 72. Pulliainen, A. T., & Dehio, C. (2012). Persistence of Bartonella sp. national Journal of Toxicology and Pharmacological Research, 5(1), 5–8. Stealth pathogens: From subclinical infections to vasoprolifera- 85. Berling, I., & Isbister, G. K. (2015). Hematologic effects and com- tive tumor formation. FEMS Microbiology Reviews, 36, 563–599. plications of snake envenomation. Transfusion Medicine Reviews, doi: 10.1111/j.1574-6976.2012.00324.x 29, 82–89. This page intentionally left blank Section Four Nonmalignant Disorders of Leukocytes 449 Chapter 21 Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes Kristin Landis-Piwowar, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Recognize neutrophilia from hematologic 6. Identify neutropenia from hematologic data data and name the common disorders and list the common disorders associated associated with neutrophilia. with neutropenia. 2. Explain the quantitative and qualitative 7. Recognize the conditions associated with neutrophil response to acute bacterial false or pseudo-neutropenia. infections. 8. Identify neutrophil nuclear alterations 3. Identify immature granulocytes and including Pelger-Huët, hypersegmentation, morphologic changes (toxic granulation, and pyknotic forms on stained blood films Döhle bodies, intracellular organisms, and and microscopic pictures. vacuoles) often seen in reactive neutrophilia. 9. Recognize morulae, Alder-Reilly granules, 4. Define and recognize leukemoid reaction, and Chédiak-Higashi inclusions on stained leukoerythroblastosis, and pyknotic nuclei blood films and microscopic pictures. on stained blood films and microscopic 10. State the common conditions associated pictures. with abnormal eosinophil, basophil, and 5. Distinguish leukemoid reaction from monocyte counts. chronic myeloid leukemia based on 11. Define Gaucher and Niemann-Pick diseases. laboratory data. 450 Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 451 Objectives—Level II At the end of this unit of study, the student should be able to: 1. Assess the etiology, associated conditions, functional abnormalities (Chédiak-Higashi, and peripheral blood findings for immediate, Alder-Reilly, May-Hegglin, and chronic acute, chronic, and reactive neutrophilia. granulomatous diseases) and differentiate 2. Compare and contrast the hematologic and their cellular abnormalities. clinical features for leukemoid reaction and 8. Evaluate alterations in the relative and/or chronic myeloid leukemia (CML). absolute numbers of eosinophils, basophils, 3. Organize neutropenia to include etiology and monocytes and associate them with the and associated conditions as well as blood clinical condition of the patient. and bone marrow findings. 9. Evaluate the etiology, laboratory findings, 4. Recognize, evaluate, and select appropriate and clinical features of the lysosomal storage corrective action for false neutropenia. disorders. 5. Appraise the nuclear abnormalities of 10. Identify and differentiate the abnormal neutrophils including Pelger-Huët, pseudo- macrophages seen in Gaucher disease, Pelger-Huët, hypersegmentation, and Niemann-Pick disease, and sea-blue pyknotic nuclei, and reconcile them with the histiocytosis syndrome. appropriate clinical conditions of the patient. 11. Construct an efficient and cost-effective 6. Appraise the cytoplasmic abnormalities of reflex testing pathway for following-up neutrophils including toxic granulation, neutrophilia, neutropenia, and qualitative Döhle bodies, vacuoles, intracellular organ- granulocyte abnormalities. isms, and morulae and reconcile them with 12. Evaluate a case study from a patient with a the patient’s appropriate clinical condition. nonmalignant granulocyte disorder. 7. Recognize and summarize the clinical features of the inherited granulocyte Chapter Outline Objectives—Level I and Level II 450, 451 Eosinophil Disorders 464 Key Terms 451 Basophil and Mast Cell Disorders 466 Background Basics 452 Monocyte/Macrophage Disorders 466 Case Study 452 Summary 468 Overview 452 Review Questions 469 Introduction 452 References 471 Neutrophil Disorders 453 Key Terms Agranulocytosis Döhle body Hypereosinophilic syndrome Basophilia Egress (HES) Demargination Hypereosinophilia Leukemoid reaction 452 Chapter 21 Leukocytosis Monocytosis Pseudo-neutrophilia Leukoerythroblastic reaction Morulae Reactive neutrophilia Leukopenia Myelophthisis Sea-blue histiocytosis syndrome Lysosomal storage disorders Neutropenia Shift to the left/left shift Mastocytosis Neutrophilia Toxic granules Monocytopenia Pelger-Huët anomaly (PHA) Background Basics The information in this chapter builds on concepts Level II learned in previous chapters. To maximize your learning • Describe the role of specific neutrophil granules and experience, you should review before starting this unit enzymes in antimicrobial systems. (Chapter 7) of study: • Identify normal macrophages and discuss their role in the bone marrow and the rest of the monocyte- Level I macrophage system. (Chapter 7) • Summarize the production, kinetics, distribution, life • Describe leukocyte maturation and proliferation pools span, and basic function of neutrophils and mono- in the bone marrow; describe the role of cytokines in cytes. (Chapter 7) bone marrow release of leukocytes; describe the pro- • Describe how leukocytes are counted and differ- cess of leukocyte egress to tissue. (Chapters 3, 4, 7) entiated; recognize normal and immature granulo- • Correlate the function of the hematopoietic organs to cytes. (Chapters 7, 10, 37) leukocyte distribution and demise. (Chapter 3) acquired and inherited physiologic states. The laboratory CASE STUDY professional must recognize these abnormalities and cor- We refer to this case throughout the chapter. relate them with the patient’s clinical condition. Dennis, a 24-year-old man, was taken to emer- gency surgery to repair several bone fractures sustained in an automobile accident. His previ- Introduction ous medical history was unremarkable. He was in Changes in leukocyte concentration and morphology excellent health prior to the accident. are often the body’s normal responses to various disease Consider why Dennis’s condition could result processes and toxic challenges. Most often, one class of in abnormal hematologic test results and the possi- leukocyte is affected more than the others, providing ble complications that could occur during Dennis’s an important clue to diagnosis. The type of cell affected treatment and recovery. depends in a large part on its function (e.g., bacterial infec- tion commonly results in an absolute neutrophilia, viral infections are characterized by an absolute lymphocytosis, Overview and certain parasitic infections cause an eosinophilia). Thus, the absolute concentrations of each leukocyte class aids in This chapter discusses benign changes in granulocytes and the differential diagnosis of a patient, especially when the monocytes as a response to various nonmalignant disease total leukocyte concentration is abnormal. states and toxic challenges. These changes include both Leukocytosis refers to a condition in which the total quantitative and qualitative variations that can be detected leukocyte count is more than 11.0 * 103/mcL in an adult. by laboratory professionals. The chapter is organized by See Appendix D, Table B for leukocyte and differential class of leukocyte (neutrophils, eosinophils, basophils, reference intervals. Although leukocytosis usually occurs and monocytes) and by the changes in quantity and qual- because of an increase in neutrophils, it can also be related to ity (morphology) of those leukocytes. These variations an increase in lymphocytes or (rarely) in monocytes, eosino- in leukocyte number and appearance are correlated with phils, or basophils.1 Quantitative variations of leukocytes Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 453 are evaluated by performing a total leukocyte count and a Automated hematology analyzers detect neutrophil counts differential count. The absolute concentration of each type outside the reference interval, but they do not detect quali- of leukocyte can be calculated from these two values as tative changes in the neutrophils. Detection of these changes follows (Chapter 7): requires careful microscopic examination of stained blood films and provides information for differential diagnosis, Differential count (in decimal form) * WBC count especially in patients with recurring bacterial infections. (* 103/mcL) = Absolute cell count (* 103/mcL) Quantitative Disorders Checkpoint 21.1 Quantitative neutrophil abnormalities can result from An adult patient’s total leukocyte count is 5.0 * 103/mcL. There malignant or benign disorders. Malignant disorders are are 60% segmented neutrophils, 35% lymphocytes, and 5% caused by neoplastic transformation of hematopoietic stem monocytes on the differential. Calculate the absolute number of cells and are discussed in Chapters 23–28. Benign disorders each cell type. Is each of these relative and absolute cell counts normal or abnormal? are usually acquired and most often occur as a consequence of a physiologic insult (stressful stimulus that affects nor- mal function and can result in morbidity). Three interre- Leukopenia refers to a condition in which the total leu- lated mechanisms affecting neutrophil concentration in the kocyte count falls to less than 4.5 * 103/mcL. This condi- peripheral

blood include: (1) bone marrow production and tion is usually the result of a decrease in neutrophils, but release of neutrophils, (2) rate of neutrophil egress to tissue lymphocytes and other leukocytes may also contribute. or survival time in blood, and (3) ratio of marginating to Morphologic or qualitative variations in leukocytes circulating neutrophils in peripheral blood. are noted by examination of the stained blood smear NEUTROPHILIA (Chapters 10, 37). Some qualitative changes affect cell func- The reference interval for neutrophil concentration varies tion, whereas others do not. Variations in the appearance of with age and race, which emphasizes the importance of eval- the cell together with its concentration can provide specific uating the count for each demographic group. Neutrophilia clues to the pathologic process. refers to an increase in the total circulating absolute neu- trophil concentration (ANC). In adults, neutrophilia occurs when the ANC exceeds 7.0 * 103/mcL. See Appendix D, Neutrophil Disorders Table B for age- and race-specific reference intervals. Because neutrophils are the most numerous type of leuko- Neutrophilia that is not caused by malignancy most cyte that circulates in the peripheral blood in adults, quan- often occurs as a response to a physiologic or pathologic titative disorders of neutrophils are often accompanied by process and is termed reactive neutrophilia; it can be imme- changes in the total leukocyte count. Although neutrophilia diate, acute, or chronic and can involve any or all of the (increase in neutrophils) is more common than neutropenia three mechanisms listed in Table 21-1. (decrease in neutrophils), the consequences to the health of Immediate Neutrophilia Of the neutrophils inside a a patient are more severe when the neutrophil count is low. blood vessel, approximately 50% freely circulate, and the Table 21.1 Conditions Associated with Neutrophilia Immediate Neutrophilia Acute Neutrophilia Chronic Neutrophilia Strenuous exercise Acute bacterial, fungal, and some viral infections Persistence of infections that cause acute neutrophilia Stress Inflammatory processes Persistence of inflammation and chronic inflammatory • Tissue damage from burns, trauma, or surgery disorders • Collagen, vascular, and autoimmune disorders • Hypersensitivity reactions Pain Drugs, hormones, and toxins Drugs, hormones, and toxins Extreme temperatures Colony-stimulating factors, vaccines, venoms Metabolic and endocrine disorders • Eclampsia • Adrenocorticotropic hormone Childbirth Acute hemorrhage or hemolysis Hematologic neoplasms Epinephrine Hereditary and congenital disorders Anesthesia Various cancers Emotional stimuli (panic, severe stress) 454 Chapter 21 remaining 50% are loosely attached to the endothelial cells neutrophil count may be normal, but toxic morphologi- of the blood vessel (marginated; Chapter 7). Immediate neu- cal changes may be observed including toxic granulation, trophilia can occur without pathologic stimulus and is prob- Döhle bodies, and cytoplasmic vacuoles (discussed later in ably a simple redistribution of the marginating pool to the this chapter and illustrated in Figure 21-1). circulating pool. Routine laboratory analysis of a peripheral Bacterial Infection Bacterial infection is the most com- blood sample detects only those neutrophils in the circulat- mon cause of neutrophilia, especially infection caused by ing pool. pyogenic organisms such as staphylococci and streptococci Although the increase in circulating neutrophils occurs (Table 21-1). Depending on the virulence of the microorgan- rapidly in immediate neutrophilia, it is transient (lasting ism, extent of infection, and response of the host, the neu- only 20–30 minutes). Furthermore, immediate neutrophila trophil count can range from 7.0- 70 * 103/mcL, although is independent of bone marrow output and tissue egress the count is usually in the range of 10- 25 * 103/mcL. As (movement of neutrophils out of the circulation and into the the demand for neutrophils at the site of infection increases, tissues). Therefore, immediate neutrophilia is also referred the early response of the bone marrow is to increase out- to as pseudo-neutrophilia or demargination because no put of storage neutrophils to the peripheral blood, causing actual change in the number of neutrophils within the vas- a left shift. The inflow of neutrophils from the bone mar- culature occurs. The increased circulating neutrophils are row to the blood continues until it exceeds the neutrophil typically mature, normal cells. This redistribution of neutro- outflow to the tissues, causing an absolute neutrophilia. phils causes the physiologic neutrophilia that accompanies In very severe infections, the storage pool of neutrophils active exercise, epinephrine administration, anesthesia, and can become exhausted, the mitotic pool may be unable anxiety and can increase the neutrophil count by as much to keep up with the demand, and neutropenia develops.4 as twofold.2 Neutropenia in overwhelming infection is a poor prognos- Acute Neutrophilia Acute neutrophilia occurs when neu- tic indicator. Chronic bacterial infection can lead to chronic trophils egress from the bone marrow storage pool into stimulation of the marrow, whereby the production of neu- the peripheral blood. Within hours following a pathologic trophils remains high and a new steady state of production stimulus (e.g., bacterial infection, toxin) the circulating pool develops. of neutrophils can increase by as much as 10-fold.3 Neutrophilia is neither a unique nor an absolute finding The neutrophilia is far more pronounced than in in bacterial infections. Infections with other organisms such pseudo-neutrophilia, and the proportion of immature neu- as fungi, rickettsia, spirochetes, and parasites also can cause trophils can be increased. More bands appear if the tissue a neutrophilia. Certain bacterial infections are characterized demand for neutrophils creates an acute shortage of seg- by neutropenia rather than neutrophilia.4 Rarely, bacterial mented neutrophils in the bone marrow storage pool. Con- infections lead to lymphocytosis rather than neutrophilia, tinued demand in extreme circumstances can result in the release of metamyelocytes.2 As bone marrow production increases and the storage pool is replenished, the leukocyte differential returns to normal. Chronic Neutrophilia Chronic neutrophilia generally follows acute neutrophilia and occurs if the stimulus for neutrophils continues beyond a few days. This results in a depleted storage pool and increased production of the mitotic pool in an attempt to meet the demand for neutro- phils. In this state, the marrow shows increased numbers of early neutrophil precursors including myeloblasts, pro- myelocytes, and myelocytes. The blood contains increased numbers of bands, metamyelocytes, myelocytes, and (rarely) promyelocytes. An increase in the concentration of immature forms of leukocytes in the circulation is termed a shift to the left or left shift. Conditions Associated with Neutrophilia Chronic neutro- philia caused by benign or toxic conditions is usually charac- Figure 21.1 A leukemoid reaction. There is an increased leukocyte count and a left shift. The cells have heavy toxic terized by a total leukocyte count of less than 50 * 103/mcL granulation and Döhle bodies (arrows), suggesting an infectious or and a left shift. If immature cells are present they are usu- toxic reactive leukocytosis (peripheral blood, Wright-Giemsa stain, ally bands and metamyelocytes. In some instances, the 1000* magnification). Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 455 as is the case with whooping cough caused by Bordetella release toxic intracellular enzymes (granules) and oxygen pertussis infection. Although typically associated with a metabolites. These toxic substances mediate the inflamma- lymphocytosis, viruses can also cause neutrophilia in the tory process by injuring other body cells and propagating earliest stages of infection. the formation of additional chemotactic factors that attract more leukocytes. CASE STUDY (Continued from page 452) Leukemoid Reaction Extreme neutrophilic reactions to severe infections or necrotizing tissue can produce a Laboratory results on Dennis, the trauma patient, 2 leukemoid reaction (Figure 21-1). A leukemoid reaction is days after surgery are as follows: a benign proliferation of leukocytes characterized by (1) a WBC 14.5 * 103/mcL WBC total leukocyte count usually less than 50 * 103/mcL and Differential (2) numerous circulating leukocyte precursors.5 In a neu- trophilic-leukemoid reaction, the number of circulating Hb 12.9 g/dL Segmented 5% neutrophil precursors is increased including bands, meta- neutrophils myelocytes, myelocytes and promyelocytes, and rarely, PLT 180 * 103/mcL Band 50% blasts. neutrophils Leukemoid reactions can produce a blood picture Lymphocytes 40% indistinguishable from that of chronic myeloid leukemia Monocytes 5% (CML; Chapter 24). If a differential diagnosis cannot be determined by routine hematologic parameters, then cyto- Urine, blood, and wound cultures were ordered. genetic studies, molecular analysis, and leukocyte alkaline 1. What results, if any, are abnormal? phosphatase (LAP) stain scores can be helpful (Table 21-2). 2. Contrary to leukemia, a leukemoid reaction is transient, What is the most likely reason for these results? disappearing when the inciting stimulus is removed. A leukemoid reaction can occur in chronic infections (such as tuberculosis) and may present with toxic granules, Döhle Tissue Destruction/Injury, Inflammation, and Metabolic bodies, and vacuoles.5 In addition, leukemoid reactions can Alterations Conditions other than infection that may result accompany carcinoma of the lung, stomach, breast, or liver in a neutrophilia include tissue necrosis, inflammation, cer- and other inflammatory processes.6 tain metabolic conditions, and drug intoxication (Table 21-1). All of these conditions produce neutrophilia by increasing neutrophil egress from the bone marrow into the circulation Checkpoint 21.2 in response to increased neutrophil diapedesis to the tissue. How can CML be distinguished from a leukemoid reaction? Examples of these conditions include rheumatoid arthritis, tis- sue infarctions, burns, neoplasms, trauma, uremia, and gout. Leukoerythroblastic Reaction A leukoerythroblastic Although leukocytes defend the body against foreign reaction (Figure 21-2) is characterized by the presence substances, they also contribute to the inflammatory pro- of nucleated erythrocytes and a neutrophilic left shift in cess. Damaged tissue releases cytokines that act as chemo- the peripheral blood. The total neutrophil count can be taxins, causing neutrophils to leave the vessels and move increased, decreased, or normal. Erythrocytes in this condi- toward the injury site. In gout, for example, deposits of tion often exhibit poikilocytosis with teardrop shapes and uric acid crystals in joints attract neutrophils to the area. anisocytosis. Leukoerythroblastosis is most often associ- In the process of phagocytosis and death, the leukocytes ated with chronic neoplastic myeloproliferative conditions, Table 21.2 Comparison of Laboratory Results in Leukemoid Reactions and Chronic Myeloid Leukemia (CML) Leukemoid Reaction CML Leukocyte count Less than 50 * 103/mcL Usually greater than 50 * 103/mcL Leukocyte differential Shift to the left with bands, metamyelocytes, Shift to the left with immature cells including promyelocytes and myelocytes; neutrophil toxic changes and blasts; increased eosinophils and basophils Erythrocyte count Normal Often decreased Platelets Usually normal Increased or decreased Philadelphia chromosome Absent Usually present BCR/ABL1 gene mutation Absent Present Clinical Related to primary condition Systemic (splenomegaly, enlarged nodes, bone pain) 456 Chapter 21 Checkpoint 21.3 What is the difference between a leukemoid reaction and a leu- koerythroblastic reaction? NEUTROPENIA Neutropenia occurs when the neutrophil count is less than 1.592.0 * 103/mcL (varies depending on ethnicity). Agranulocytosis, a term that refers to a neutrophil count less than 0.5 * 103/mcL is associated with a high prob- ability of infection. Basophils and eosinophils are also com- monly depleted in severe neutropenia. True neutropenia can occur because of (1) decreased bone marrow production, (2) increased cell loss (from immune destruction or increased neutrophil egress to the tissue), or (3) pseudo-neutropenia (increased neutrophilic margination). An artificial neutropenia can result from neutrophil agglutination, disintegration, and laboratory Figure 21.2 A leukoerythroblastic reaction. There are instrument problems. Table 21-3 lists the most common nucleated erythrocytes and a left shift with band neutrophils causes of neutropenia (note that some causes of neutrope- and a myelocyte (peripheral blood, Wright-Giemsa stain, 1000* nia are also seen in neutrophilia). magnification). Decreased Bone Marrow Production Neutropenia can especially myelofibrosis, myelophthisis (replacement of develop as a result of decreased bone marrow production. normal hematopoietic tissue in the bone marrow by fibrosis, In this case, the bone marrow shows myeloid hypopla- leukemia, or metastatic cancer cells), and severe hemolytic sia, and the myeloid-to-erythroid (M:E) ratio is decreased anemias such as Rh hemolytic disease of the fetus and new- (Chapter 38). Defective neutrophil production depletes the born (HDFN; Chapter 19). bone marrow storage pool, decreases neutrophil egress to Stimulated Bone Marrow States Patients whose bone tissues, and reduces both the peripheral blood circulating marrow has been stimulated by hematopoietic growth and marginating pools. Immature cells may enter the blood factors or cytokines such as granulocyte monocyte-colony– in an attempt to alleviate the neutrophil shortage; however, stimulating factor (GM-CSF; CSF2) can produce a rapid cells younger than bands are less efficient in phagocytosis. increase of total white cell counts and release of leukocyte The end result is a lack of an adequate number of neutro- precursors including blasts. Cytokines are used to replenish phils

at inflammatory sites, resulting in increased risk for leukocytes after bone marrow transplant, high-dose chemo- overwhelming infections. therapy, bone marrow failure, or prior to autologous blood Stem Cell Disorders Decreased bone marrow produc- donation or stem cell apheresis7 (Chapter 29). tion can occur when hematopoietic stem cells fail to pro- Corticosteroid therapy produces a neutrophilia that liferate as in aplastic anemia, following radiotherapy or occurs as a result of increased bone marrow output accom- panied by decreased migration of neutrophils to the tis- sues (by inhibition of the ability of neutrophils to adhere Table 21.3 Causes of Leukopenia and/or Neutropenia to vessel walls).8 This inhibition of neutrophil diapedesis can in part explain the increased incidence of bacterial Causes Examples infections in patients on steroid therapy even though the Infections Viral and overwhelming bacterial such as blood neutrophil count is increased. Steroids also decrease sepsis the number and inhibit the function of monocytes/ Physical agents and Radiation, benzene chemicals macrophages.9 Drugs Chemotherapy, certain drugs in the classes Physiologic Leukocytosis Physiologic leukocytosis and of sedatives, anti-inflammatory, antibacterial, antithyroid, and antihistamines neutrophilia are present at birth and for the first few days Hematologic disorders Acute leukemia, megaloblastic and aplastic of life. The leukocytosis can be accompanied by a slight left anemia, splenomegaly shift. Physiologic stressors including exposure to extreme Alloantibodies or Systemic lupus erythematosus temperatures, emotional stimuli, exercise, and labor dur- autoantibodies ing childbirth can cause neutrophilia, generally without a Hereditary or congenital Familial neutropenia, cyclic neutropenia left shift. disorders Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 457 chemotherapy, or with infiltration of hematopoietic tis- SBDS genes.12 Most disorders display neutrophil precursor sue by malignant cells (myelophthisis). Of new leukemia responsiveness to large doses of G-CSF.11 Familial neutrope- cases, 50% present with a total white count that is less than nia is a rare benign anomaly characterized by an absolute 5.0 * 103/mcL and an absolute neutrophil count that is decrease in neutrophils but usually a normal total leuko- less than 1.0 * 103/mcL.10 This leukopenia and neutrope- cyte count, and no infectious complications. It is transmitted nia occurs when normal precursor cells in the bone marrow as an autosomal dominant trait and is usually detected by are replaced by malignant cells, but the malignant cells have chance. Table 21-4 provides a more complete list of congeni- not egressed to the peripheral blood in significant numbers. tal neutropenic disorders.2,12 Megaloblastic Anemia Neutropenia is a characteristic find- Increased Cell Loss Neutropenia can occur as the result of ing in megaloblastic anemia (Chapter 16) and myelodys- increased neutrophil diapedesis. In severe or early infection, plastic syndromes (Chapter 25). In these cases, however, the the bone marrow may not produce cells as rapidly as they marrow is usually hyperplastic. Neutropenia results not from are being utilized, resulting in neutropenia. Various viral, marrow failure but from destruction of abnormal myeloid bacterial, rickettsial, and protozoan infections induce tissue precursors in the bone marrow (ineffective granulopoiesis). damage that increases the demand for and destruction of Chemicals/Drugs A wide variety of drugs and chemicals neutrophils. Marked toxic changes to the granulocytes often are associated with leukopenia and neutropenia if given in accompany neutropenia resulting from severe infections. sufficient dosage (Table 21-3). Chemotherapy and radia- Prognosis in these cases is very poor because the infecting tion treatments for cancer are nonselective and are com- organisms are able to prevail over the body’s immune system. mon causes of not only neutropenia but also pancytopenia. Immune Neutropenia Antibodies directed against neutrophil- The decreased bone marrow production of granulocytes specific antigens can cause a decrease in the number of predisposes patients receiving treatment for malignan- neutrophils. Leukocytes are destroyed in a manner similar cies to frequent and serious infections. Chemotherapeutic to erythrocytes in immune hemolytic anemia (Chapter 17). drugs induce apoptosis in mitotically active cells by a vari- In some cases, drugs precipitate an immunologic response ety of mechanisms including direct DNA damage, altering leading to a sudden disappearance of neutrophils from the folate receptors, or inhibiting enzymes needed for mitosis. circulation. The immunologic mechanism can include direct Although the action of chemotherapeutic drugs reduces cancer cell proliferation, normal hematopoietic precursor cells in the bone marrow also are actively dividing and Table 21.4 Congenital Neutropenic Disorders their proliferation is inhibited as well. Prophylactic use of antibiotics and GM-CSF has reduced mortality in patients Disorder Inheritance receiving chemotherapy, but infections due to neutropenia Disorders of production remain a serious complication.9 Drug-induced neutropenia Cyclic neutropenia AD also can result from allergic (immunologic) reactions to Familial neutropenia AD drugs. Women, older patients, and patients with a history Fanconi pancytopenia AR of allergies are more commonly affected. Reticular dysgenesis AR Congenital Neutropenia Several rare inherited disorders Severe congenital neutrophilia AD, AR cause neutropenia related to decreased bone marrow pro- Wiskott-Aldrich syndrome XLR duction. Periodic or cyclic neutropenia is a curious form of Disorders of RNA synthesis and processing neutropenia that begins in infancy or childhood and occurs Cartilage-hair hypoplasia AR in regular 21- to 30-day cycles. Cyclic neutropenia is inher- Dyskeratosis congenital XLR, AD, AR ited as an autosomal dominant trait and is due to mutations Shwachman-Diamond syndrome AR in the gene for neutrophil elastase (ELANE).10 The severely Disorders of metabolism neutropenic period (less than 0.5 * 103/mcL) lasts for sev- Barth syndrome AR, XLR eral days and is marked by frequent infections. Between Glycogen Storage disease, type 1b AR the neutropenic attacks, the patient is asymptomatic. Severe Pearson’s syndrome Mitochondrial congenital neutropenias include a heterogenous group of Disorders of vesicular transport rare, often fatal disorders marked by extreme neutropenia Chédiak-Higashi syndrome AR (less than 0.2 * 103/mcL). The total leukocyte count is often Cohen syndrome AR within the reference interval. One form of SCN is inherited Griscelli syndrome, type II AR in an autosomal dominant manner and involves mutations in the neutrophil elastase gene. Others, such as Kostmann Hermansky-Pudlak syndrome, type II AR syndrome, display autosomal recessive inheritance, and p14 Deficiency Probable AR involve a number of other genes, including the HAX1 and AD, autosomal dominant; AR, autosomal recessive; XLR, X-linked recessive. 458 Chapter 21 cell lysis or sensitization and subsequent sequestration of False Neutropenia It is important for the laboratory profes- neutrophils in the spleen.2 The two types of immune neu- sional to recognize when neutropenia is a result of labora- tropenia are alloimmune and autoimmune. tory in vitro manipulations of blood. Four in vitro causes of Alloimmune neonatal neutropenia occurs when mater- a low neutrophil count include: nal antibodies are directed against paternal-origin antigens • EDTA-induced neutrophil adherence to erythrocytes on fetal neutrophils and are transferred across the placenta. Affected infants are susceptible to infections for up to 4 • Disintegration of neutrophils over time prior to testing months or until the neutropenia is resolved.2 This immune • Disruption of abnormally fragile leukocytes during process is similar to that found in hemolytic disease of the preparation of the blood for testing fetus and newborn (HDFN) except that the firstborn child • Neutrophil aggregation can be affected. Alloimmune neutropenia can also be the result of a transfusion reaction. Neutrophils can adhere to erythrocytes (rarely) when the Autoimmune neutropenia (AIN) is categorized as blood is drawn in EDTA, causing an erroneously low auto- either primary or secondary. Primary AIN is not associated mated white count. If observed on the stained smears, blood with other diseases and occurs predominantly in young can be recollected by finger stick to make manual dilutions children. This condition of unknown etiology develops as and blood smears without utilizing EDTA (Chapter 37). antibody-coated neutrophils are sequestered and destroyed Neutrophils disintegrate in blood collection tubes faster by the spleen. Patients develop fever and recurrent infec- than other leukocytes. If there is a delay in testing the blood, tions. Spontaneous remission usually occurs after a period the neutrophil count can be erroneously decreased. In some of 13–20 months. Secondary autoimmune neutropenia gen- pathologic conditions, the leukocytes are more fragile than erally occurs in older patients, many of whom have been normal and can rupture with the manipulations of prepar- diagnosed with another autoimmune disorder such as sys- ing blood for testing in the laboratory. Finally, the neutro- temic lupus erythematosus (SLE) or rheumatoid arthritis phil count can be falsely decreased if the neutrophils clump (AR). In secondary AIN, antineutrophil antibodies are not together because of the presence of paraproteins (proteins the only cause of the neutropenia, and the actual target of appearing in large quantity because of other pathological the antibodies is not always known.13 conditions). Infections associated with AIN are not usually life threatening and are treated with routine antibiotic therapy. Checkpoint 21.4 Intravenous doses of immunoglobulin can be used in severe How can the correct white cell count be determined when neu- cases. The total leukocyte count is usually normal or near trophils clump in the presence of EDTA? normal, but the neutrophil count is decreased. G-CSF ther- apy can be indicated for patients with severe infections. Immune neutropenia is difficult to diagnose, but is Qualitative or Morphologic aided by testing for antineutrophil antibodies or neutrophil Abnormalities surface antigens by various methods including agglutina- Automated hematology analyzers do not detect or flag tion tests, immunofluorescence, and enzyme-linked immu- neutrophil morphologic abnormalities. Laboratory profes- nosorbent assay (ELISA). The availability of these complex sionals must microscopically evaluate stained blood smears tests varies widely among laboratories. to identify cytoplasmic and/or nuclear morphologic abnor- Hypersplenism Hypersplenism can result in a selective malities in neutrophils. Cytoplasmic abnormalities are the splenic culling of neutrophils producing mild neutropenia. most common, and most cytoplasmic changes (Döhle bod- The bone marrow in this case exhibits neutrophilic hyper- ies, toxic granulation, and vacuoles) are reactive, transient plasia. Thrombocytopenia and (occasionally) anemia can changes accompanying infectious states. The correct identi- also accompany hypersplenism. fication of alterations such as intracellular microorganisms Pseudo-Neutropenia Pseudo-neutropenia is similar to can lead to the prompt diagnosis and treatment of life- pseudo-neutrophilia in that it is produced by alterations in threatening infections, whereas recognition of Pelger-Huët the circulating and marginated pools. Pseudo-neutropenia or hypersegmented neutrophils can point to the diagnosis results from the transfer of circulating neutrophils to the of specific conditions that can prove elusive without the marginated neutrophil pool with no change in the total morphologic information. peripheral blood neutrophil pool. This temporary shift is NUCLEAR ABNORMALITIES characteristic of some infections with endotoxin production Pelger-Huët Anomaly Pelger-Huët anomaly (Figure 21-3) and of hypersensitivity reactions. Because of the selective is a benign anomaly inherited in an autosomal dominant margination of neutrophils, the total leukocyte count drops fashion and occurs in about 1 in 5000 individuals. The neu- and a relative lymphocytosis develops. trophil nucleus does not segment beyond the two-lobed Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 459 a b Figure 21.3 Pelger-Huët anomaly. (a) Note pince-nez, or eyeglass-shaped, nuclei. These mature cells could easily be confused for bands if not for the highly clumped chromatin. (b) The nucleated neutrophil is the nonsegmented or round nucleus form that can be found (peripheral blood, Wright-Giemsa stain, 1000* magnification). stage and can appear as a single, round nucleus with no likely appear in a blood picture with far more than 10% segmentation. The presence of excessive coarse chroma- (likely more than 50%) of the neutrophils showing three or tin clumping in the nucleus aids in the differentiation of more nuclear lobes and possibly immature myeloid cells.18 bilobed cells from band neutrophils. The bilobed nucleus Hypersegmentation Larger-than-normal neutrophils with has a characteristic dumbbell shape with the two lobes six or more nuclear segments (hypersegmented neutro- connected by a thin strand of chromatin. Cells with this phils) or five or more neutrophils with five lobes are com- appearance are often called pince-nez cells (French name mon and early indicators of megaloblastic anemia. These for the style of eyeglasses without earpieces). Rod-shaped cells are found together with pancytopenia and macro and peanut-shaped nuclei can also be found. Individuals ovalocytes that typically accompany deficiencies of folate heterozygous for Pelger-Huët anomaly have neutrophil or vitamin B12 (Chapter 15). Reported cases of hereditary nuclei that are primarily bilobed, whereas the majority of hypersegmentation of neutrophils, a benign condition, are neutrophils in homozygous individuals have a round or rarely significant but need to be distinguished from multi- oval nucleus with only a few cells having the classic bilobed lobed nuclei that are associated with disease.19 shape.16

The cell is functionally normal, and individu- als with hereditary Pelger-Huët anomaly do not display Pyknotic Nucleus Pyknotic, or apoptotic, nuclei (Figure increased susceptibility to bacterial infections.16 The signifi- 21-4) are found in dying neutrophils in blood or body fluid cance of recognizing this anomaly lies in differentiating the benign hereditary condition from a left shift that can occur during infections. Acquired- or pseudo-Pelger-Huët anomaly can pres- ent in myeloproliferative disorders and myelodysplastic states. The neutrophils in pseudo-Pelger-Huët anomaly (sometimes described as pelgeroid) are frequently hypo- granular because of a lack of secondary granules, and the nuclei acquire a round rather than a dumbbell shape. The chromatin appears with intense clumping, aiding in differ- entiation of these mononuclear cells from myelocytes. It is particularly important that laboratory pro- fessionals distinguish between the Pelger-Huët and pseudo-Pelger-Huët anomalies because the presence of the acquired form can be used to aid in the diagnosis of myelodysplasia and malignancy.17 To distinguish the two, the inherited Pelger-Huët anomaly usually presents with Figure 21.4 The nucleated cell at the top is a dying neutrophil less than 10% of the neutrophils displaying three or more with a pykonotic nucleus. Note the smooth nuclear material that nuclear lobes without the presence of immature myeloid cells is breaking up (peripheral blood, Wright-Giemsa stain, 1000* (meta- and myelocytes). Pseudo-Pelger-Huët neutrophils magnification). 460 Chapter 21 preparations. The nuclear chromatin condenses and the seg- ments disappear, becoming smooth, dark-staining spheres. If the nucleus is round, these apoptotic cells can be confused with nucleated erythrocytes. Checkpoint 21.5 Describe the difference between hypersegmented and hypo- segmented neutrophils and pyknotic nuclei. In what conditions are each seen? CYTOPLASMIC ABNORMALITIES Cytoplasmic inclusions are often found in infectious states and when present, give the health care provider important diagnostic information (Table 21-5). These inclusions are Döhle bodies, toxic granules, vacuoles, and intracellular organisms. Figure 21.5 Arrow points to neutrophil with three bluish inclusions (Döhle bodies) (peripheral blood; Wright-Giemsa stain, Döhle Bodies Döhle bodies are light gray-blue oval 1000* magnification). inclusions in the cytoplasm of neutrophils and eosinophils (Figure 21-5). Found near the periphery of the cell, Döhle bodies are composed of aggregates of rough endoplasmic are discharged to fight bacteria. Toxic granulation is seen reticulum. They can be seen in severe infections, burns, in the same conditions as Döhle bodies. It is important to and cancer and as a result of toxic drugs. Döhle bodies distinguish toxic granules and inclusions from toxic gran- should be looked for whenever toxic granulation or other ules or inclusions can appear as artifacts that can occur reactive morphologic changes are present because they fre- with increased staining time or decreased pH of the buffer quently occur together. Döhle bodies are similar in appear- used in the staining process. True toxic granulation will not ance to the cytoplasmic inclusions found in May-Hegglin appear equally in all neutrophils. anomaly (described later in the “May-Hegglin Anomaly” Cytoplasmic Vacuoles Cytoplasmic vacuoles appear as section). clear, unstained circular areas. Vacuoles probably repre- Toxic Granules Toxic granules are large, deeply stained, sent the end stage of phagocytosis (Figure 21-7). They are blue-black primary granules in the cytoplasm of segmented usually seen in the same conditions as toxic granulation neutrophils and sometimes in bands and metamyelocytes and Döhle bodies. Cytoplasmic vacuoles in neutrophils (Figure 21-6). Primary (nonspecific) granules normally from a fresh specimen correlate highly with the presence lose their basophilia as the cell matures, so even though of septicemia. about one-third of the granules in the mature neutrophil Vacuoles can also appear as an artifact in smears made are primary granules, their presence is not observable by from blood that has been collected and stored in EDTA. light microscopy. In contrast, toxic primary granules retain Vacuoles related to storage are more likely to be smaller their basophilia in the mature neutrophil, perhaps because and more uniformly dispersed than those in toxic states. of a lack of maturation. Additionally, toxic primary gran- Making smears from fresh blood without anticoagulant can ules can become more apparent as the secondary granules eliminate the vacuole artifact. Table 21.5 Cytoplasmic Inclusions Found in Neutrophils in Infectious Conditions Inclusion Morphologic Characteristics Composition Associated Conditions Döhle body Light gray-blue oval near cell periphery Rough endoplasmic reticulum (RNA) Infections, burns, cancer, or inflammatory states Toxic granules Large blue-black granules Primary granules Same as Döhle body Cytoplasmic vacuole Clear, unstained circular area Open spaces from phagocytosis Same as Döhle body Bacteria Small basophilic rods or cocci Phagocytized organisms Bacteremia or sepsis Fungi Round or oval basophilic inclusions Phagocytized fungal organisms Systemic fungal infections often in slightly larger than bacteria immunosuppressed patients Morulae Basophilic, granular; irregularly shaped Clusters of Ehrlichia rickettsial Ehrlichiosis organisms Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 461 Figure 21.6 A segmented neutrophil and a band neutrophil Figure 21.7 Intracellular microorganisms. Note the vacuoles with toxic granulation (peripheral blood, Wright-Giemsa stain, and toxic granulation in the cells (peripheral blood, Wright-Giemsa 1000* magnification). stain, 1000* magnification). agent of human granulocytic ehrlichiosis. Ehrlichia sp. are CASE STUDY (Continued from page 455) tick-borne, small, obligate intracellular, coccobacilli bac- All cultures were negative for bacteria and fungi. teria. They infect leukocytes where they multiply within On further examination of the blood smear, it was phagosomes. The intracellular organisms are pleomorphic, noted that no abnormal cytoplasmic features were appearing as basophilic, condensed, or loose aggregates of observed in the neutrophils. However, the nuclei of organisms, which tend to appear spherical. most of the bands appeared more condensed than Ehrlichiosis is characterized by high fever, leukopenia, normal, and many had two lobes in a dumbbell thrombocytopenia, and elevated liver enzymes.20,21 Most shape. cases of ehrlichiosis occur in April through September when nymphal ticks are most active. The intracellular microcol- 3. Given the leukocyte morphology and cul- onies of Ehrlichia, called morulae, are observed in leuko- tures, what additional condition must now be cytes on stained blood films (Figure 21-8). The presence of considered? morulae in leukocytes can be the first diagnostic finding of 4. Explain the clinical significance of the nuclear Ehrlichia infection. The leukocyte eventually ruptures and anomaly described in Dennis. releases the organisms that then infect other leukocytes. The pathogenesis of ehrlichiosis can be related to direct cellu- 5. Why is the white cell count elevated? lar injury by the bacteria or a cascade of inflammatory or immune events. Intracellular Organisms In most infections, including sep- Confirmation of infection is made through serologic ticemia, the causative agents are not demonstrable in the determination of antibody titers using indirect fluorescent peripheral blood. However, microorganisms seen inside neutrophils should always be considered a significant finding, and the physician should be notified immediately (Figure 21-7). Intracellular Histoplasma, or Candida, is sometimes found on blood smears from patients with HIV or other severe immunosuppression. Organisms found outside cells must be interpreted with care. On stained blood smears, it is cru- cial to distinguish whether the microorganisms came from contaminated equipment or stain or are actually present in the patient’s blood. Organisms must also be distinguished from other cytoplasmic material and precipitated stain. All bacteria and yeasts stain basophilic with Wright’s stain. In the United States, two known species of Ehrlichia Figure 21.8 Morulae in ehrlichiosis. This segmented infect humans. E. chaffeensis infects monocytes and is the neutrophil from a patient with human granulocytic ehrlichiosis causative agent of human monocytic ehrlichiosis (HME), contains two dense, basophilic inclusions called morulae (peripheral whereas E. ewingii infects granulocytes and is the causative blood, Wright-Giemsa stain, 1000* magnification). 462 Chapter 21 antibody (IFA) assays or by identification of DNA sequences by polymerase chain reaction (PCR). Peripheral blood cyto- penia is probably the result of sequestration of infected cells in the spleen, liver, and lymph nodes. The bone marrow is usually hypercellular.22,23 Anaplasma phagocytophilum also infects neutrophils and is the causative agent of human granulocytic anaplasmosis (HGA). The clinical manifestations and laboratory findings are similar to that of Ehrlichia infection, and morulae are observed in the neutrophils.20 INHERITED FUNCTIONAL ABNORMALITIES Functional neutrophil abnormalities are almost always inherited and can be accompanied by morphologic abnor- Figure 21.9 Lymphocyte from Hurler’s disease malities. It is suggested that granulocyte functional abnor- (mucopolysaccharidoses). Note the halo around the granules malities be suspected in patients with recurrent, severe (peripheral blood, Wright-Giemsa stain, 1000* magnification). infections, abscesses, and delayed wound healing and in mutations of the CHS1 gene, which codes for a lysosomal antibiotic resistant sepsis. See Table 21-6 for a summary of trafficking regulatory protein. CHS is characterized by the functional defects and their clinical features. defects in granule morphogenesis, and death usually occurs Alder-Reilly Anomaly Alder-Reilly anomaly is an inherited during the first or second decade of life because of recurrent condition characterized by the presence of large purplish bacterial infections (Figure 21-10). Giant gray-green perox- granules in the cytoplasm of all leukocytes. Similar mor- idase-positive bodies and giant lysosomes are found in the phology is seen in disorders such as Hurler’s syndrome and cytoplasm of leukocytes as well as most granule-containing Hunter’s syndrome, both of which are lysosomal storage cells of other tissues. These bodies are formed by fusion of disorders (discussed later in the “Monocyte/Macrophage primary, nonspecific azurophilic granules. This abnormal Disorders” section; see also Figure 21-9). These disorders are fusion of cytoplasmic membranes prevents the granules characterized by incompletely degraded mucopolysaccha- from being delivered into the phagosomes to participate in rides that accumulate in the lysosomes and appear as large killing of ingested bacteria. Neutropenia and thrombocyto- granules.24 The granules can resemble toxic granulation in penia are frequent complications as the disease progresses. neutrophils and can also appear in lymphocytes. They tend The patients have skin hypopigmentation, silvery hair, and to occur in clusters in the shape of dots or commas and are photophobia from an abnormality of melanosomes. Lymph- surrounded by vacuoles. The inclusions frequently are seen adenopathy and hepatosplenomegaly are characteristic.25 only in cells of the bone marrow, not in the peripheral blood, May-Hegglin Anomaly May-Hegglin anomaly is a rare, but in either case, the cells function normally. inherited, autosomal dominant trait in which granulocytes Chédiak-Higashi Syndrome Chédiak-Higashi syndrome contain inclusions consisting mainly of RNA from rough (CHS) is a rare autosomal recessive disorder associated with endoplasmic reticulum, which are similar in appearance to Table 21.6 Inherited Qualitative Neutrophil Abnormalities Condition Morphologic or Functional Defect Clinical Features Alder-Reilly anomaly Large, purplish cytoplasmic granules in all leukocytes Associated with mucopolysaccharidosis such as Cells function normally Hurler’s syndrome Chédiak-Higashi syndrome Giant fused granules in neutrophils and lymphs Serious, often fatal condition with repeated pyrogenic Cells engulf but do not kill microorganisms infections May-Hegglin anomaly Blue, Döhle-like cytoplasmic inclusions in all Bleeding tendency from associated thrombocytopenia granulocytes Cells function normally Chronic granulomatous disease (CGD) Defective respiratory burst Recurrent infections, especially in childhood Cells engulf but don’t kill microorganisms Myeloperoxidase deficiency Low or absent myeloperoxidase enzyme Usually benign; other bactericidal systems prevent Cell morphology normal most infections Leukocyte adhesion deficiency (LAD) Absence of cell-surface adhesion proteins affecting Serious condition with recurrent infections and high multiple cell functions mortality Cell morphology normal Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 463 from mutations in any of the five genes that encode the sub- units of the NADPH oxidase.25 Patients suffer from recur- rent, often life-threatening, infections with opportunistic pathogens (bacterial and fungal) that result in the forma- tion of granulomas. CGD can be diagnosed in childhood but in some cases, the onset of symptoms may not occur until early adulthood. The affected cells in CGD are morphologically normal but cannot generate antimicrobial oxygen metabolites, such as H2O2. Therefore, the neutrophils can phagocytize micro- organisms but cannot kill them. Ingested microorganisms produce hydrogen peroxide as a by-product of oxidative a b metabolism; those that are catalase-positive are not killed because they destroy the H2O2 of their own metabolism. Figure 21.10 (a) Neutrophil from Chédiak-Higashi syndrome. Catalase-positive organisms continue to grow intracellu- (b) Lymphocyte from the same patient as in a. Note the bluish-gray inclusion bodies (peripheral blood, Wright-Giemsa stain; 1000* larly where they are protected from antibiotics, whereas magnification). catalase-negative organisms kill themselves by generat- ing H2O2 that can be used in the phagocytic cell’s myelo- Döhle bodies (Figure 21-11). The inclusions can be distin- peroxidase-dependent antimicrobial defense mechanisms guished from true Döhle bodies because they are usually (Chapter 7). larger and rounder in shape.26 The Döhle body-like inclu- The peripheral blood

neutrophil count is normal but sions result because of a defect in the myosin heavy-chain increases in the presence of infection. Immunoglobulin lev- type IIA gene (MYHA gene) that leads to the precipitation of els are often increased because of chronic infection.28 Treat- non-muscle myosin heavy chains. The neutrophils function ment involves the use of prophylactic antibiotics and early normally. Variable thrombocytopenia with giant platelets treatment of infections. The nitroblue tetrazolium slide test is characteristic. The only apparent clinical symptom that (NBT) is useful in detecting the abnormal oxygen metabo- patients may exhibit is abnormal bleeding related to the low lism of neutrophils in CGD. Neutrophils are mixed with platelet count. nitroblue tetrazolium and microorganisms. In normal indi- Chronic Granulomatous Disease Chronic granulomatous viduals, the leukocytes phagocytize the microorganisms, disease (CGD) is an inherited disorder (65% X-linked, 35% initiating an increase in oxygen uptake. This process leads autosomal recessive)27 characterized by defects in the respi- to an accumulation of oxygen metabolites that reduce the ratory burst oxidase (NADPH oxidase) system. CGD results NBT to a blue-black compound, which shows in the cell as dark crystals. Neutrophils from individuals with CGD cannot mobilize a respiratory burst, so no dark crystals appear.29 The dihydrorhodamine 123 (DHR123) assay using flow cytometry is replacing the NBT test in some laboratories. The DHR123 assay (also referred to as the neutrophil oxida- tive burst assay), incubates granulocytes with bacteria and the dye DHR123.30 After the neutrophils phagocytize the bacteria, they activate nicotinamide adenine dinucleotide phosphate oxidase and produce reactive oxygen metabo- lites (the respiratory burst). These metabolites oxidize DHR123 to fluorescent rhodamine 123, which is detected by flow cytometry. In healthy adults, the reference interval for granulocytes with phagocytic activity is 80–100%. Myeloperoxidase Deficiency Myeloperoxidase (MPO) deficiency is, for the most part, a benign autosomal reces- sive disorder characterized by an absence of myeloperoxi- dase in neutrophils and monocytes. Although neutrophils Figure 21.11 May-Hegglin anomaly. There is a neutrophil use MPO in the bactericidal process, an increase in bacterial with a Döhle-like structure in the cytoplasm and a large platelet infections is not usually seen in MPO deficiency, even in (arrow; peripheral blood, Wright-Giemsa stain, 1000* magnification). homozygous individuals. The neutrophils are able to utilize 464 Chapter 21 alternative antimicrobial systems to kill the microorganisms thrombasthenia (Chapter 33). LAD III is associated with (although somewhat more slowly; Chapter 7). However, mutations in KINDLIN-3 a protein involved in integrin patients with MPO deficiency and diabetes mellitus can activation,25 experience disseminated fungal infections (usually with Candida albicans).31 Checkpoint 21.6 Certain hematology analyzers can detect the presence Explain how you can determine whether toxic granulation and or absence of MPO in a two-stage cytochemical reaction that vacuoles in the neutrophils are due to the patient’s condition differentiates cells based on stain and size characteristics. or to artifact. Regarding MPO, neutrophils, eosinophils, and monocytes are usually positive and basophils and lymphocytes are negative. The neutrophils in patients with MPO deficiency are depicted as large, unstained cells. On peripheral blood Eosinophil Disorders smears, the neutrophils appear morphologically normal. Disorders involving exclusively eosinophils are rare. An Leukocyte Adhesion Deficiency There are three forms of increase in the circulating number of these cells can indicate leukocyte adhesion deficiency, LAD I, LAD II and LAD III. a potentially serious condition. Because the lower limit of the Features of all three include recurrent soft tissue bacterial reference interval is very low, a decrease is difficult to deter- and fungal infections with persistent leukocytosis and gran- mine and is probably not significant. Eosinopenia can be seen ulocytosis due to increased stimulation of the bone marrow, in acute infections and inflammatory reactions and with the and decreased neutrophil emigration into the tissues. Treat- administration of glucocorticosteroids. Glucocorticosteroids ment includes prophylactic antibiotics and early, aggressive and epinephrine inhibit eosinophil release from the bone treatment of infections. Mortality rate in childhood can be marrow and increase their margination.32 Increased eosin- high for severe cases of Type I and Type III LAD, and bone ophils, hypereosinophilia (HE), can be classified as clonal marrow transplantation is recommended.25 (neoplastic, also called primary) or reactive (non-clonal, also LAD I is a rare, autosomal recessive disorder char- called secondary).33 In either instance, the HE may or may acterized by decreased or absent leukocyte cell-surface not be associated with specific HE-mediated organ damage, adhesion proteins (b2@integrins, also termed CD11/CD18 referred to as hypereosinophilic syndrome (HES).34 In addi- complex; Chapter 7). Neutrophils from patients with LAD tion there are rare cases of familial or hereditary HE. Finally, I have multiple functional defects, including impaired occasionally HE may be found in the absence of evidence of adhesion to endothelial cells, chemotaxis, phagocytosis, the previous three conditions, and is referred to as idiopathic respiratory burst activation, and degranulation. Because of HE, or HE of undetermined/unknown significance.34 defective adhesion proteins, the neutrophils cannot adhere to endothelial cells of the blood vessel walls and exit the Nonclonal (Reactive) circulation. In addition, LAD I neutrophils are not able to recognize the presence of the complement C3bi fragment on Hypereosinophilia microorganisms, so phagocytosis is not stimulated. The fre- Hypereosinophilia refers to an increase in eosinophils more quency and severity of the infections in LAD I depends on than 0.4 * 103/L. Reactive HE appears to be induced by the amount of CD11/CD18 the cells express. Diagnosis can cytokines secreted from T lymphocytes. This type of eosin- be made by flow cytometric analysis of neutrophil CD11b ophilia is polyclonal and the common myeloid progenitor levels using a monoclonal antibody.29 (CMP) cell is normal. Various conditions associated with LAD II also is a rare autosomal recessive disorder the cellular immune response (mediated by T lymphocytes) characterized by failure of endothelial cells to synthesize are characterized by HE including tissue-invasive parasites, the ligand for leukocyte L-selectin (Sialy@Lex). Leukocyte allergic conditions, respiratory tract disorders, gastrointes- function is normal, but the cells fail to adhere to the vessel tinal diseases, and skin and connective tissue disorders and wall endothelium and cannot exit the circulation to enter diseases.34 Parasites are the most common cause of secondary the tissues in host defense responses. LAD II red cells are HE worldwide. When the eosinophil concentration is high Lewis antigen negative, and have the rare Bombay (hh) and immature forms are present, the blood picture can resem- phenotype. These cells are unable to form certain fucose ble that seen in chronic eosinophilic leukemia (Chapter 24). carbohydrate linkages. The mutation in LAD II is in a mem- The conditions associated with HE are listed in Table 21-7. brane fucose transporter, and results in a generalized loss Tissue invasion by parasites produces an eosinophilia of expression of fucosylated molecules on the cell surface.25 more pronounced than parasitic infestation of the gut or LAD III has only been described in a small number of blood. Eosinophils are especially effective in fighting tis- patients. In addition to defects in leukocyte adhesion, the sue larvae of parasites; they readily adhere to larvae coated patients have a bleeding disorder similar to Glanzmann with IgG, IgE, and/or complement. Larvae are too large Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 465 Table 21.7 Conditions Associated with Quantitative Changes of Eosinophils, Basophils, and Mast Cells Eosinopenia Acute infections, inflammatory reactions, administration of glucocorticosteroids Hypereosinophilia (HE) Nonclonal (reactive) Parasitic infection Allergic conditions, especially asthma, dermatitis, and drug reactions Clonal (neoplastic) CEL, NOS; eosinophilia with abnormalities of PDGFRA, PDGFRB, or FGFR1, and eosinophilia that occurs in other MPNs Idiopathic No known cause Familial Unknown Basopenia Inflammatory states following immunologic reactions Basophilia Immediate hypersensitivity reactions Endocrinopathies Infectious diseases Figure 21.12 Hypereosinophilic syndrome. Note that the cells Chronic myeloproliferative disorders, especially are all mature eosinophils (peripheral blood, Wright-Giemsa stain, CML; chronic basophilic leukemia 1000* magnification). Mastocytosis Immediate hypersensitivity reactions Connective tissue disorders Infectious diseases neoplastic disorder with mutations in genes that code for Neoplastic disorders such as lymphoprolifera- platelet derived growth factor receptor (PDGFR) or fibro- tive diseases and hematopoietic stem cell diseases blast growth factor receptor 1 (FGFR1). These disorders are considered myeloproliferative neoplasms and are discussed CEL, NOS, chronic eosinophilic leukemia, not otherwise specified; CML, chronic myeloid leukemia; FGFR1, fibroblast growth factor receptor 1; MPN, myeloproliferative neoplasm; in Chapter 24. PDGFR, platelet-derived growth factor receptors (PDGFRA, PDGFRB) Idiopathic Hypereosinophilia for phagocytosis, so the eosinophil molds itself around the In other cases, the cause for the HE is unknown and clon- larva to destroy it. Intracellular eosinophilic granules fuse ality cannot be proven. These disorders are collectively with the eosinophil membrane and expel their contents into known as idiopathic HE. The identification of genetic muta- the space between the cell and the larva. The granular sub- tions has decreased the number of cases that are considered stances attack the larva wall, partially digesting it.35 idiopathic.34 A moderate increase in eosinophils frequently char- Chronic HE can cause extensive tissue damage as acterizes allergic disorders (asthma, dermatitis, and drug the granules are released from disintegrating eosinophils reactions). Large numbers can also be found in nasal dis- hypereosinophilic syndrome (HES). In many HES cases, charges and sputum of allergic individuals as well as in the large numbers of circulating eosinophils damage the heart. peripheral blood. Charcot-Leyden crystals formed from either eosinophil HE also accompanies a disorder termed pulmonary cytoplasm or granules can be found in exudates and tissues infiltrate with eosinophilia (PIE) syndrome characterized where large numbers of eosinophils migrate and disinte- by asthma, pulmonary infiltrating eosinophils, central ner- grate. Treatment of HES with corticosteroids, hydroxyurea, vous system anomalies, peripheral neuropathy, polyateritis and/or a@interferon is sometimes effective in reducing the nodosa, and local or systemic eosinophilia. This syndrome eosinophil count. HES is most commonly seen in males can be produced by parasitic or bacterial infections, allergic (more than 90%) and is rarely seen in children.37 reactions, or collagen disorders. In some cases, no cause can Idiopathic HES must be differentiated from reactive be found.36 eosinophilias and clonal eosinophilias (such as eosinophilic leukemia). Eosinophilic leukemia usually presents with Clonal (Neoplastic) myeloblasts and eosinophilic myelocytes, whereas idio- Hypereosinophilia pathic HES and reactive eosinophilia present with mature eosinophils. In addition, an abnormal clonal chromosome Primary HE is characterized by a persistent blood eosino- karyotype or molecular mutation suggests eosinophilic leu- philia of more than 1.5 * 103/mcL with tissue infiltration kemia or another clonal variant rather than idiopathic HES (Figure 21-12). It may be a clonal myeloid or lymphoid or reactive eosinophilia (Chapter 24). 466 Chapter 21 Basophil and Mast Cell gene rearrangement suggests the diagnosis of acute baso- philic leukemia, an extremely rare condition. Disorders A decrease in basophils is even more difficult to establish than eosinopenia. Scanning a blood smear with The cells’ cytoplasmic granule-associated preformed media- 100* magnification will reveal a rare basophil in normal tors, including histamine and certain proteases, their lipid individuals. Decreases in basophils are seen in inflamma- mediators (such as prostaglandin D2 and leukotriene C4), tory states and following immunologic reactions. which are generated upon activation of the cells, and their Mastocytosis can be found in a number of disorders cytokines, growth factors, and chemokines contribute to in which the number of mast cells is increased as much as many of the characteristic signs and symptoms of these dis- fourfold in affected tissues (most commonly the skin) and eases. However, mast cells and basophils also contribute to in conjunction with certain neoplastic disorders (Table 21-7). protective host responses associated with IgE production, No clinical disorder that involves a decrease in mast cells especially those directed against parasites. has been identified; however, long-term treatment with glu- Both basophils and mast cells are important in inflamma- cocorticoids can lower the number of mast cells.38 tory and immediate allergic reactions. They both express the receptor for immunoglobulin (Ig) E (FceRI). Their immune Checkpoint 21.7 function can be IgE-dependent (IgE binds to high-affinity Why are the basophil and eosinophil counts important when receptors on these cells) or IgE-independent (triggered by assessing the benign or neoplastic nature of a disorder? complement fragment C5a binding to C5a receptors on these cells). Basophils and mast cells mediate an immune response through granules that contain histamine and some proteases, lipid molecules (i.e., prostaglandin), and cytokines, growth Monocyte/Macrophage factors, and chemokines. Extracellular degranulation can be induced by physical destruction of the cell, chemical sub- Disorders stances (toxins, venoms, proteases), endogenous mechanisms Quantitative disorders are associated with monocytes, (tissue proteases,

cationic proteins), and immune mechanisms. whereas qualitative disorders are associated with both The number of basophils and mast cells increases at monocytes and macrophages. The qualitative disorders are sites of inflammation. Basophils migrate from the blood by inherited lysosomal storage disorders. adhering to the endothelium (mediated by several families of adhesion molecules and receptors).38 Basophilia refers to an increase in basophils greater Quantitative Disorders than 0.2 * 103/mcL and is associated with immediate Monocytosis occurs when the absolute monocyte count hypersensitivity reactions and chronic myeloproliferative is more than 0.8 * 103/mcL (Figure 21-14). It is seen most disorders (Table 21-7, Figure 21-13). An absolute basophilia often in inflammatory conditions and certain malignancies is often helpful in distinguishing CML from a leukemoid (Table 21-8). Monocytosis occurring in the recovery stage reaction or other benign leukocytosis. A basophil count of acute infections and in agranulocytosis is considered a exceeding 80% of the total leukocyte population and the favorable sign. Monocytes also play an important role in the absence of the Philadelphia chromosome and/or BCR/ABL1 cellular response against mycobacterium in tuberculosis. Figure 21.13 Peripheral blood from a patient with CML and Figure 21.14 Peripheral blood from a patient with reactive 30% basophils (peripheral blood, Wright-Giemsa stain, 1000* monocytosis showing two reactive monocytes (peripheral blood, magnification). Wright-Giemsa stain, 1000* magnification). Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 467 In MPS, the accumulating macromolecules are found pri- Table 21.8 Conditions Associated with Quantitative marily in connective tissue. Changes of Monocytes Neoplastic Myelodysplastic/myeloproliferative neoplasms GAUCHER DISEASE Chronic myelomonocytic leukemia Gaucher (pronounced “go-shay”) disease is an inherited Juvenile myelomonocytic leukemia autosomal recessive disorder characterized by a deficiency Chronic myeloid leukemia of b@glucosidase (an enzyme needed to break down the lipid Acute monocytic, myelomonocytic, and myelocytic glucocerebroside). In this disease, the macrophage is unable leukemias to digest the stroma of ingested cells, and the lipid glucocer- Reactive Inflammatory conditions ebroside accumulates. The clinical findings (splenomegaly Collagen diseases Immune disorders and bone pain) of the disease are related to the accumula- Certain infections (e.g., TB, syphilis) tion of this lipid in macrophages mainly in the spleen, liver, Monocytopenia Stem cell disorders such as aplastic anemia and bone marrow. The macrophages (Gaucher cells) are large (209100 mcM[mM]) with small eccentric nuclei, and the cytoplasm appears wrinkled or striated40 (Figure 21-15). Unexplained monocytosis has been reported to be asso- The spleen and liver can become greatly enlarged. Leukope- ciated with as many as 62% of all malignancies. Monocy- nia, thrombocytopenia, and anemia can occur from seques- tosis can be seen in myelodysplastic states, acute myeloid tration by an enlarged spleen. Diagnosis of Gaucher disease leukemias, and chronic myeloid leukemia and in approxi- is determined by the detection of insufficient b@glucosidase mately 25% of Hodgkin lymphomas. In these conditions, enzyme activity in peripheral blood leukocytes.40 the monocyte is probably a part of a reactive process to Cells similar to Gaucher cells can be found in the mar- the neoplasm rather than a part of the clonal neoplasm row of individuals with a rapid granulocyte turnover, itself. Neoplastic proliferation of monocytes occurs in acute especially in chronic myeloid leukemia. The accumulation monocytic leukemia and acute and chronic myelomonocytic of lipid in these disorders does not result from enzyme leukemia (Chapters 24, 26). deficiency but from the inability of the macrophage to Monocytopenia refers to a concentration of monocytes keep up with the flow of fat into the cell from the increased less than 0.2 * 103/mcL and is found in stem cell disorders cell turnover. Differential diagnosis of Gaucher disease such as aplastic anemia. Monocytopenia is difficult to estab- can be confirmed by demonstrating decreased leukocyte lish because of the low normal levels of these cells. b@glucosidase activity, whereas the enzyme level is normal or increased in myeloproliferative disorders. Qualitative Disorders NIEMANN-PICK DISEASE Lysosomal storage disorders include a large group of inher- Niemann-Pick disease includes a group of rare disorders ited disorders. All nucleated cells contain lysosomes used that are related autosomal recessive diseases. Signs of the as a part of the cell’s recycling system. Lysosomes contain disease begin in infancy with poor physical development. various enzymes (including glucosidases, lipases, proteases, The spleen and liver are greatly enlarged. The disease is and nucleases) that are involved in degradative processes. often fatal by 3 years of age. In Niemann-Pick type A and Defects in any of these enzymes can lead to the accumula- tion of either nondegraded substrates or catabolic products that are unable to be transported out of the lysosome. This accumulation can lead to cell dysfunction and pathologi- cal phenotypes. Most of these disorders are inherited in an autosomal recessive pattern.39 The type of storage material that accumulates (glyco- proteins, glycosphingolipids, mucolipids, mucopolysac- charides, etc.) can be used to classify lysosomal storage disorders. Based on this accumulated storage material, there are two main categories: mucopolysaccharidoses (MPS) and lipidoses. Many of these disorders cause detect- able morphology in granulocytes (see the section “Alder- Reilly Anomaly” earlier in this chapter) and monocytes/ macrophages. Disorders based on the presence of abnormal macrophages in hematologic tissue, a common presenting feature, include Gaucher disease, Niemann-Pick disease, Figure 21.15 Gaucher macrophages in bone marrow and sea-blue histiocytosis (see the “Histiocytoses” section). (peripheral blood, Wright-Giemsa stain, 1000* magnification). 468 Chapter 21 type B disease, the defect is a deficiency of sphingomy- (LCH); class II—non-Langerhans histiocytoses (non-LCH), elinase (an enzyme needed to break down lipids), result- which encompasses the histiocytoses of mononuclear ing in excessive sphingomyelin storage. Macrophages with phagocytes; and class III—malignant histiocytoses. a foamy appearance are found in lymphoid tissue and The Langerhans cell is an immature dendritic cell usu- the bone marrow (Figure 21-16). The foam cells are large ally found in the epidermis, oral and vaginal mucosa, and (20–100 mcM) with an eccentric nucleus and globular cyto- lungs. They differ from other tissue cells by characteris- plasmic inclusions. Leukopenia and thrombocytopenia can tic racquet-shaped structural inclusions known as Birbeck occur from increased sequestration by the enlarged spleen, granules. Monocyte/macrophage histiocytes contribute to and blood lymphocytes can contain several vacuoles that reactive (in response to an inflammatory stimulus) and are lipid-filled lysosomes.41 malignant disorders. Reactive macrophage histiocytes can be seen in benign proliferative diseases (xanthoma dissemi- MISCELLANEOUS LYSOSOMAL STORAGE DISORDERS nata, juvenile xanthogranuloma), in nonmalignant hemo- Tay-Sachs disease, Sandhoff disease, and Wolman’s disease phagocytic diseases (fulminant hemophagocytic syndrome, are inherited lipid storage diseases and Hurler’s and Hunter’s histiocytosis with massive lymphadenopathy), and in sev- syndromes are inherited MPS. These disorders are character- eral of the storage disorders (Gaucher, Niemann-Pick, and ized by a deficiency of one or more enzymes that metabolize sea-blue histiocytosis). lipids or mucopolysaccharides. As a result of these enzyme Sea-blue histiocytosis syndrome is a rare inherited deficiencies, abnormal concentrations of these macromole- disorder characterized by splenomegaly and thrombocyto- cules accumulate in the lysosomes of tissue cells. These condi- penia. Sea-blue staining macrophages laden with lipid are tions affect mostly nonhematologic tissue and are often fatal. found in the liver, spleen, and bone marrow (Figure 21-17). There are no specific findings in the peripheral blood. Lipid- laden macrophages can be present in the bone marrow.42 The cell is large (in diameter) with a dense eccentric nucleus and cytoplasm that contains blue or blue-green granules. HISTIOCYTOSES Considerable variation in clinical manifestations is present, Two different types of cells are known as histiocytes: Lang- but in most patients, the course of the disease is benign. Sea- erhans cells/dendritic cells and monocytes/macrophages. blue histiocytes can also be seen in a variety of disorders of Both cell types are antigen processing and antigen present- lipid metabolism and in association with various other dis- ing cells and share a common progenitor. The three classes orders including Niemann-Pick disease, Gaucher disease, of histiocytoses are class I—Langerhans cell histiocytoses some hematopoietic diseases, and in certain infections. Figure 21.16 Macrophage in the bone marrow of a patient with Niemann-Pick disease. Note the foamy cytoplasm with inclusions Figure 21.17 Sea-blue histiocyte from bone marrow (peripheral blood, Wright-Giemsa stain, 1000* magnification). (peripheral blood, Wright-Giemsa stain, 1000* magnification. Summary Leukocytes respond to toxic, infectious, and inflammatory leukocyte types (depending on the cell’s function). Thus, a dif- processes to defend the tissues and limit and/or eliminate the ferential count and the total leukocyte count aid in diagnosis. disease process or toxic challenge. This can involve a change Neutrophilia, an increase in neutrophils, most often in leukocyte concentration, most often increasing one or more occurs as a result of a reaction to a physiologic or pathologic Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 469 process (most commonly, bacterial infection). Tissue injury disease and myeloperoxidase deficiency. Leukocyte adhe- or inflammation can also cause a neutrophilia. Neutropenia, sion deficiency I (LAD I) is identified by the absence of a decrease in neutrophils, is less commonly encountered. It leukocyte cell-surface adhesion proteins that inhibit the can be caused by drugs, immune mechanisms, or decreased adhesion of neutrophils to endothelial cells so the neutro- bone marrow production. Several inherited conditions are phils cannot exit the blood. Other rare leukocyte functional characterized by neutropenia and recurrent infections. Mor- abnormalities include Alder-Reilly, Chédiak-Higashi, and May- phologic abnormalities of neutrophils can be found in infec- Hegglin anomalies. tious states and are important to identify on stained blood Eosinophils increase in infections with parasites, smears. These include Döhle bodies, toxic granulation, and allergic conditions, hypersensitivity reactions, cancer, and cytoplasmic vacuoles. Other morphologic abnormalities chronic inflammatory states. Basophilia is seen in hypersen- include pince-nez cells found in Pelger-Huët anomaly and sitivity reactions, infections, and chronic myeloproliferative morulae found in ehrlichiosis. disorders, especially chronic myeloid leukemia. Monocyto- Functional and morphologic abnormalities of the sis is found in a wide variety of conditions, especially malig- leukocyte characterize a number of inherited conditions. nancies. Macrophage disorders are commonly associated Defects in the generation of oxidizing radicals after phago- with a group of lysosomal storage disorders in which these cytosing bacteria characterize chronic granulomatous cells are unable to completely digest phagocytosed material. Review Questions Level I c. vacuoles 1. Which of the following hematologic values would d. primary granules you expect if the peripheral blood smear revealed 5. Which of the following is a common reason for toxic granulation, Döhle bodies, and vacuoles in neutropenia in hospitalized patients? (Objective 6) neutrophils? (Objectives 1, 2) a. Chronic myeloproliferative disorders such as CML a. WBC: 4.0 * 103/mcL b. Chemotherapy and/or radiation treatment for b. The differential shows 15% bands cancer c. The differential shows 20% eosinophils c. Childbirth d. Hb: 10 g/dL; platelets 20 * 103/mcL d. Lack of exercise 2. The white count in an adult is 2.0 * 103/mcL The 6. Which of the following causes a false neutropenia? differential shows 60% segmented neutrophils. Which (Objective 7) of the following correctly describes these results? a. EDTA-induced agglutination (Objective 6) b. Bone marrow aplasia a. Normal c. Splenomegaly b. Leukocytosis and neutrophilia d. Immune neutropenia c. Leukopenia with normal number of neutrophils 7. Which of the following can be used to distinguish a d. Leukopenia and neutropenia leukemoid reaction from CML? (Objective 5) 3. Which of the following is the most common cause of a. A total WBC count more than 25 * 103/mcL neutrophilia? (Objective 1) b. The presence/absence of immature cells of the a. Bacterial infection myeloid lineage b. Acute leukemia c. Presence/absence of BCR/ABL1 gene mutation c. Chemotherapy d. Presence/absence of autoantibodies d. Aplastic anemia 8. Which of the following is a common cause of 4. Blue-gray oval inclusions composed of RNA near the eosinophilia? (Objective 10) periphery of neutrophils is a description of: a. Parasitic infection (Objective 3) b. Eosinophilic leukemia a. toxic granules c. CML b. Döhle bodies d. Acute hemorrhage 470 Chapter 21 9. What substances build up and are ingested by 3. Which of the following correctly describes the macrophages in qualitative macrophage disorders neutrophil concentration if the patient is an infant? such as Gaucher disease? (Objective 11) (Objective 1) a. Proteins a. Pseudo-neutrophilia b. Carbohydrates b. Leukemoid reaction c. Lipids c. Leukoerythroblastic reaction d. Rough endoplasmic reticulum d. Physiologic neutrophilia 10. Distinct, large, unidentified inclusions are found in 4. Which of the following correctly describes the the cytoplasm of many granulocytes. Select the best differential if the patient is an adult? (Objective 1) course of action. (Objective 9) a. Pseudo-neutrophilia a. Ignore them because they probably are not significant. b. Leukoerythroblastic

reaction b. Report them as intracellular yeast. c. Agranulocytosis c. Suspect rare or unusual conditions such as d. Physiologic neutrophilia Chédiak-Higashi. 5. A leukoerythroblastic reaction can be associated with: d. Report them as toxic granulation. (Objectives 1, 2, 12) Level II a. chronic granulomatous disease b. Chédiak-Higashi syndrome c. severe hemolytic anemia Use the following case study to answer review questions 1–5. d. leukocyte adhesion deficiency A patient’s white count is 30.0 * 103/mcL. The 6. Alder-Reilly anomaly can be differentiated from toxic differential is as follows: granulation by: (Objectives 7, 11) Segmented neutrophils 54% a. presence/absence of other toxic features Band neutrophils 10% (leukocytosis, toxic granulation, Döhle bodies, Metamyelocytes 2% vacuoles) Lymphocytes 26% b. the presence of hypersegmentation in neutrophils Monocytes 5% c. other CBC parameters abnormal (RBC, HCT, or Eosinophils 3% PLT) 6 Nucleated RBCs/100 WBCs d. patient symptoms related to an allergic reaction 7. May-Hegglin can be differentiated from conditions with toxic Döhle bodies by: (Objectives 7, 11) 1. Select the additional information most important to a. presence of thrombocytosis assess whether these results are normal or indicate a b. presence or absence of other toxic features such as disease process. (Objective 1) toxic granulation a. Platelet count c. foam cells in the bone marrow b. RBC count d. patient history of recent trauma c. Patient history 8. Pelger-Huët anomaly can be differentiated from an d. Patient age increase in band neutrophils by: (Objectives 5, 11) 2. Which of the following correctly describes the absolute a. flow cytometry for the presence of CD11b neutrophil count for the patient? (Objectives 1, 12) b. abnormal inclusions found in cells other than a. Normal for both an infant and an adult neutrophils b. Neutrophilia for both an infant and an adult c. other CBC parameters abnormal (RBC, HCT, or c. Normal for an infant and neutrophilia for an adult PLT) d. Normal for an adult and neutrophilia for an infant d. finding all bilobed segmented neutrophils Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 471 9. Chédiak-Higashi can be differentiated from c. The neutrophil count was too high for an adult. intracellular yeasts or morulae by: (Objectives 6, 7, 11) d. The patient suffered from congenital neutropenia. a. presence of giant platelets 11. Leukemia can be differentiated from infection by: b. abnormal inclusions in neutrophils and (Objectives 2, 11) lymphocytes a. lack of glucosidase enzyme activity in monocytes c. other CBC parameters abnormal (RBC, HCT, or PLT) b. a shift to the left in granulocytic cells d. lack of glucosidase enzyme activity c. BCR/ABL1 gene mutation d. flow cytometry for the presence of CD11b 10. An adult patient’s white blood cell count was 10.1 * 103/mcL 12. A sample with toxic vacuoles can be differentiated and the absolute neutrophil count from a sample with prolonged storage by: was 1.3 * 103/mcL. The medical laboratory scientist (Objectives 4, 6, 11) who analyzed the data suspected a false neutropenia and requested a redraw for the patient. Which of the a. presence of other toxic features (leukocytosis, toxic following could have caused the medical laboratory granulation, Döhle bodies, vacuoles) scientist to question the CBC results? (Objective 4) b. abnormal inclusions found in lymphocytes a. 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acknowledge Sue S. Beglinger, MS, who wrote this chapter in previous editions. This chapter retains many of her sections. Objectives—Level I At the end of this unit of study, the student should be able to: 1. Identify the infectious agent and describe 6. Describe clinical symptoms of disorders the clinical symptoms associated with in which a leukocytosis is caused by infectious mononucleosis. lymphocytosis. 2. Describe and recognize the reactive 7. State the complications associated with morphology of lymphocytes found in cytomegalovirus (CMV) infections. infectious mononucleosis. 8. Identify absolute and relative 3. Relate the heterophile antibody test to lymphocytopenia and lymphocytosis, infectious mononucleosis. and list conditions associated with these 4. Given a differential and leukocyte count, cal- abnormal counts. culate an absolute lymphocyte count and dif- 9. Explain the pathophysiology of HIV ferentiate it from a relative lymphocyte count. infections, and describe how it affects 5. Identify reactive cell morphology associated lymphocytes. with viral infections, and compare it to 10. Describe the abnormal hematological normal lymphocyte morphology. findings associated with AIDS. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Assess and resolve/explain conflicting and serologic tests in suspected Epstein-Barr results from peripheral blood morphology virus (EBV) infection. 473 474 Chapter 22 2. Describe the pathophysiology of infectious 8. Assess a patient case using the AIDS case mononucleosis. surveillance criteria of the Centers for 3. Assess and correlate antibody titers found Disease Control and Prevention (CDC) in infectious mononucleosis with respect to and recommend appropriate laboratory the various EBV viral antigens. testing. 4. State the pathophysiology of toxoplasmo- 9. Explain the cytopenia, identify the defect, sis infections, and explain the resulting and recognize the laboratory features lymphocytosis. in congenital qualitative disorders of lymphocytes. 5. Differentiate benign lymphocytic leukemoid reactions from neoplastic lymphoprolifera- 10. Evaluate a case study from a patient with a tive disorders by laboratory results and lymphoproliferative disorder and conclude characteristics of cell morphology. from the medical history and laboratory 6. Define the pathophysiology of CMV results the most likely diagnosis for the infection, and give clinical findings disorder. associated with it. 11. Identify the cytokine associated with 7. In Bordetella pertussis infection, propose hyper IgE syndrome (HIES) and explain the cause of lymphocytosis and recognize its role in the pathophysiology of the laboratory features associated with it. disorder. Chapter Outline Objectives—Level I and Level II 473 Lymphocytosis 475 Key Terms 474 Lymphocytopenia 482 Background Basics 474 Summary 489 Case Study 475 Review Questions 490 Overview 475 References 492 Introduction 475 Key Terms Acquired immune deficiency Hyper IgE syndrome (HIES) Reactive lymphocytosis syndrome (AIDS) Immunosuppressed Severe combined immunodefi- Advanced HIV disease (AHD) Infectious mononucleosis ciency (SCID) syndrome Bordetella pertussis Lymphocytic leukemoid reaction Toxoplasmosis Cytomegalovirus (CMV) Opportunistic organisms Viral load Epstein-Barr virus (EBV) Persistent polyclonal B-cell Heterophile antibody lymphocytosis (PPBL) Background Basics The information in this chapter builds on the concepts Level I learned in previous chapters. To maximize your learning • Describe normal and reactive lymphocyte experience, you should review the following concepts morphology and identify the distinguishing before starting this unit of study: characteristics of T and B lymphocytes. (Chapter 8) Nonmalignant Lymphocyte Disorders 475 • Calculate absolute cell counts and summarize summarize the structure and function of each of the relationship between the hematocrit and the immunoglobulins, and describe the immune hemoglobin. (Chapters 7, 10) response. (Chapter 8) • Define antigen and antibody, describe their roles in • Describe the structure and function of the hemato- infectious and noninfectious diseases, and summa- poietic organs and tissue. (Chapter 3) rize the immune response. (Chapter 8) • Explain the principle and the application of direct Level II antiglobulin tests in diagnosis of immune-mediated • Describe the process of T and B lymphocyte dif- anemia. (Chapter 19) ferentiation and the function of each subtype, lymphocytosis. Some acquired disorders result in a lym- CASE STUDY phocytopenia that can compromise the function of the We refer to this case study throughout the chapter. immune system. In acquired disorders affecting lympho- Heather, a 54-day-old female, was admitted to the cytes, the change in lymphocyte concentration and mor- hospital because of recurrent respiratory distress phology is a reactive process. In contrast, in congenital and failure to gain weight. She was born prema- disorders involving lymphocytes, the primary defect is turely at 35 weeks gestation by urgent Cesarean within the lymphocytic system. section. Her mother was immune to rubella and had negative serologic tests for syphilis. Consider why the lymphocytic system should Lymphocytosis be evaluated in this child and the possible etiology Lymphocytosis, an increase in lymphocytes, can result of repeated respiratory problems. from an absolute or relative increase in lymphocytes. Lymphocytosis can occur with or without a leukocyto- sis. The classification of lymphocytosis varies with age. Overview The lymphocyte concentration in children is normally higher than in adults and varies with the child’s age. The This chapter discusses benign conditions associ- reader is directed to Table B, inside cover, for the reference ated with quantitative and qualitative alterations in interval for such counts for children of various ages and lymphocytes. The first part of the chapter describes adults. Lymphocytosis in young children is defined as an conditions associated with lymphocytosis, includ- absolute lymphocyte count greater than 8.8 * 103/mcL, ing infectious mononucleosis and other acquired while a relative lymphocytosis exists when the differen- disorders c haracterized by l ymphocytosis. The later tial exceeds 65%. Absolute lymphocytosis occurs in adults part of the chapter discusses lymphocytopenia and when the lymphocyte count exceeds 4.8 * 103/mcL, while immune d eficiency states, both acquired and congenital. relative lymphocytosis is present when the lymphocyte Additionally, it discusses laboratory evaluation for the differential exceeds 35% (differs with race). An absolute diagnosis and differentiation of nonmalignant lympho- lymphocytosis can occur without relative lymphocytosis, cytic disorders from neoplastic lymphoproliferative and a relative lymphocytosis can occur without absolute disease. lymphocytosis. Lymphocytosis is usually a self-limiting, reactive pro- cess that occurs in response to an infection or inflamma- Introduction tory condition. Both T and B lymphocytes are commonly affected, but their function remains normal. Occasionally, Evidence of disease, especially infectious disease, can viral infections can cause functional impairment of the lym- be observed by finding abnormal concentrations of lym- phocytes, yielding both a qualitative disorder and quantita- phocytes and/or reactive lymphocytes on a peripheral tive changes. blood smear. This finding helps direct the physician’s Once lymphocytes have been stimulated by an subsequent workup of the patient and aids in the initia- infection or inflammatory condition, they enter vari- tion of appropriate therapy. Most disorders affecting lym- ous states of activation, resulting in a morphologically phocytes are acquired and are characterized by a reactive heterogeneous population of cells on stained blood 476 Chapter 22 smears (Figure 22-1). These activated cells can appear large with irregular shapes, cytoplasmic basophilia, Table 22.1 Conditions Associated with Lymphocytosis granules and vacuoles observed. The nuclear chroma- Benign (Nonmalignant) tin usually becomes more dispersed (Chapter 8). Cells Conditions Neoplastic Conditions with these activation features are commonly referred Viral Infections Acute lymphoblastic leukemia to as reactive or atypical lymphocytes. The preferred • Adenovirus Mature B cell neoplasms Mature T and NK cell term is reactive lymphocytes because these cells are not, • Cytomegalovirus (CMV) neoplasms in fact, “ atypical.” Occasionally, intense proliferation • Coxsackie virus Posttransplant • Epstein-Barr virus (infectious lymphoproliferative disorder of lymphoid elements in the lymph nodes and spleen mononucleosis) occurs, causing lymphadenopathy and splenomegaly, • Human herpesvirus type 6 respectively. • Human herpesvirus type 8 T lymphocytes normally compose about 60–80% of • Human T lymphotrophic virus type 1 peripheral blood lymphocytes. Thus, increases in the • Primary human immunodeficiency concentration of T lymphocytes are more likely to cause virus (HIV) infection changes in the relative lymphocyte count than are increases Nonviral Infections in B lymphocytes. Absolute lymphocytosis is not usu- • Bordetella pertussis ally accompanied by leukocytosis except in infectious • Brucellosis mononucleosis, Bordetella pertussis infection, cytomega- • Rickettsia lovirus infection, and lymphocytic leukemia. A relative • Shigellosis • Toxoplasma gondii lymphocytosis secondary to neutropenia that occurs in a • Tuberculosis variety of viral infections is more commonly found. The Miscellaneous absolute lymphocyte count is calculated as: • Persistent polyclonal B cell lymphocytosis %Lymphocytes (decimal form) * WBC count (* 109/L) • Physiologic stress = Absolute cell count (* 109/L) • Drug reactions It is important to differentiate benign conditions asso- ciated with lymphocytosis from neoplastic or malignant lymphoproliferative disorders (Table 22-1). The presence of heterogeneous reactive lymphocytes, positive sero- CASE STUDY (continued from page 475) logic tests for the presence of specific antibodies against Admission CBC on Heidi was WBC: 7.6 * 109/L; infectious organisms, and absence of anemia and thrombo- Hct: 55%; Plt: 242 * 109/L; and differential: 84% cytopenia favor a benign diagnosis. This chapter includes segs, 2% bands, 4% lymphocytes, 8% monocytes, a discussion of the more common disorders associated and 2% eosinophils. with a reactive lymphocytosis. 1. Does this patient have a leukocytosis, leukope- nia, or neither? Explain. 2. Does this patient have an abnormal lymphocyte count? Explain. Infectious Mononucleosis Infectious mononucleosis is a self-limiting lymphoprolif- erative disease caused by infection with Epstein-Barr virus (EBV). It is one of the most common human viruses and a member of the herpes virus family. EBV infection occurs worldwide, with 90% of people EBV infected at some point in their lives.1 Not considered highly contagious, the disease Figure 22.1 is transmitted through direct contact through body fluids, Forms of stimulated lymphocytes from a case of infectious mononucleosis. There are two immunoblasts (one o’clock primarily saliva. and eight o’clock) and a reactive lymph (three o’clock) (peripheral Infectious mononucleosis was initially described in blood, Wright-Giemsa stain, 1000* magnification). 1920 by Sprunt and Evans as an acute infectious disease Nonmalignant Lymphocyte Disorders 477 accompanied by atypical large peripheral blood lympho- that resemble activated natural killer cells are present early cytes.2 Later, Downey described the morphologic presen- in the disease. Cytotoxic T lymphocytes inhibit the activa- tations of lymphocytes commonly observed in infectious tion and proliferation of EBV-infected B lymphocytes and mononucleosis. The morphology ranged from a small participate in the cell-mediated immune response. The lymphocyte with a lobed nucleus and scant cytoplasm, to majority of the reactive lymphocytes seen in the periph- a large lymphocyte with abundant cytoplasm (“ballerina eral blood are these CD8+ cytotoxic T lymphocytes. Others skirt” cells) that reaches out and abuts adjacent cells, and include, g/d T cells and CD16+ CD56+ natural killer (NK) finally, to a large lymphocyte with finely reticular chromatin cells stimulated in response to EBV-infected B cells. NK cells and abundant cytoplasm.3 These cells represent morpho- are also emerging as important players during infectious logic changes observed in CD8 T lymphocyte activated in mononucleosis.7 response to EBV-infected cells.4 CLINICAL PRESENTATION Infectious mononucleosis is common worldwide and Early symptoms include lethargy, headache, fever, chills, usually affects young adults; the peak age for infection sore throat, nausea, and anoxia. The classic triad presen- is 14–24 years of age. About 90% of adults that have had tation of symptoms are fever, pharyngitis, and lymphade- exposure possess lifelong immunity.5 Infection in children nopathy (Table 22-2).7 Children younger than 10 years of from lower-income groups usually occurs before 4 years of age are often asymptomatic but have reactive lymphocytes age; in more affluent populations, peak infection incidence and elevated C-reactive protein (CRP). Increases in liver occurs during adolescence.6 aminotransferases are generally found in children more A major function of T lymphocytes is to provide cel- than 10 years of age.7 The cervical, axillary, and inguinal lular immunity, thereby limiting the viral growth of EBV- lymph nodes are commonly enlarged. Splenomegaly occurs infected B lymphocytes. Immune-compromised individuals in 50–75% of these patients, and hepatomegaly occurs in are at increased risk of serious infection. EBV-associated B about 25%. Occasionally, jaundice develops. Hematologic cell tumors and lymphoproliferative syndromes can occur complications that can occur during or immediately after in transplant patients and patients with acquired immune the disease include autoimmune hemolytic anemia, throm- deficiency syndrome (AIDS). These patients have severe T bocytopenia, agranulocytosis, and (very rarely) aplas- lymphocyte immunodeficiency. Lacking a T lymphocyte tic anemia. The disease is usually self-limiting, resolving response, male children with the rare X-linked lymphopro- within a few weeks. liferative disorder (XLP) are unable to limit EBV infection of B lymphocytes. As a result,

a fatal polyclonal B lymphocyte LABORATORY EVALUATION proliferation occurs.7 Hematologic findings provide important clues for diagnosis in infectious mononucleosis. Serologic tests can confirm the EBV PATHOPHYSIOLOGY diagnosis. EBV attaches to a receptor on the B lymphocyte mem- Peripheral Blood During active viral infection, an intense brane designated CD21, which is the receptor for the C3d proliferation of lymphocytes occurs within affected complement component.7 The virus infects resting B lym- lymph nodes. The leukocyte count is usually increased phocytes as well as epithelial cells of the oropharynx and (12925 * 103/mcL) primarily because of an absolute lym- cervix. Binding of the virus to the B lymphocyte activates phocytosis. Lymphocytosis begins about 1 week after symp- the cell and induces the expression of the activation marker toms appear, peaks at 2 to 3 weeks, and remains elevated for CD23, which is the receptor for a B lymphocyte growth fac- tor.7 Once internalized, the virus is incorporated into the B lymphocyte genome, instructing the host cell to begin production of EBV proteins. These viral proteins are then Table 22.2 Summary of Typical Clinical Presentation and expressed on the cell membrane. Thus, EBV-infected cells Laboratory Evaluation in Infectious Mononucleosis express markers of activated B-lymphocytes as well as viral Clinical Presentation Laboratory Evaluation markers. The viral genome is maintained in the lymphocyte nucleus and passed on to the cell’s progeny. This results • Lymphadenopathy • Leukocytosis in EBV-immortalized B lymphocytes and possible latent • Fever • Lymphocytosis infection. • Lethargy • Elevated C-reactive protein A complex, multifaceted cellular immune response • Sore throat • More than 20% reactive (atypical) lymphocytes controls acute EBV infection. In the first week of illness, • Splenomegaly • Immunoblasts present a polyclonal increase in immunoglobulins occurs. During the second week, however, the number of immunoglobulin- • Headache • Heterophile antibodies present secreting B lymphocytes decreases because of the action of • Positive antigen tests for EBV CD8+ cytotoxic T lymphocytes. Activated cytotoxic T cells • Elevated aminotransferases (ALT, AST) 478 Chapter 22 2 to 8 weeks. Lymphocytes usually constitute about 50% of Bone Marrow Bone marrow aspirations in EBV infection the leukocyte differential with about 20% reactive lympho- are not indicated, but when performed show hyperplasia of cytes. The platelet count is often mildly decreased; concen- all cellular elements except neutrophils. trations of less than 100 * 103/mcL are rare. Serologic Tests Serologic tests are used to differentiate this Various forms of reactive lymphocytes can be found in disease from similar more serious diseases (e.g., diphtheria, the peripheral blood (Figure 22-2). Typical cells are irregular hepatitis). The blood of patients with infectious mononucle- in shape and have large amounts of spreading cytoplasm osis contains greatly increased concentrations of transient with irregular basophilia. Other reactive cells can have deep heterophile antibodies that agglutinate sheep or horse blue cytoplasm and vacuoles. Immunoblasts are usually erythrocytes but are not specific for EBV8 (a heterophile present early in the disease. Plasmacytoid lymphocytes and antibody reacts with antigens common to multiple species). an occasional plasma cell also can be found. When present, Antibodies specific for EBV can be identified by first absorb- immunoblasts should be distinguished from leukemic lym- ing the nonspecific heterophile antibodies from patient phoblasts to prevent a misdiagnosis. The chromatin pattern serum with guinea pig antigen and testing the absorbed of leukemic lymphoblasts is usually finer than the reticular serum with horse erythrocytes. The infectious mononu- chromatin of immunoblasts. In addition, immunoblasts gen- cleosis IgM antibodies react with horse erythrocytes. Posi- erally have a lower N:C ratio with more abundant, some- tive agglutination of horse erythrocytes by treated serum times vacuolated, cytoplasm. Another important criterion indicates EBV infection. A negative result usually indicates that helps differentiate infectious mononucleosis from leu- infection by some other virus. On occasion, the level of anti- kemia is the morphologic heterogeneity of the lymphocyte body is not yet high enough to be detected; therefore, the population, characteristic of viral infections, whereas leuke- test should be repeated a week later if patient symptoms mia usually has a relatively homogeneous cell population. continue. Other diseases associated with a reactive lymphocyto- Rapid, specific, and sensitive slide agglutination or sis can mimic the blood picture of infectious mononucleo- solid phase immunoassay tests are available to determine sis. These include cytomegalovirus infection, viral hepatitis, the presence of infectious mononucleosis heterophile anti- and toxoplasmosis. bodies. The infected individual also produces antibodies specific for the viral capsid antigen (VCA) of EBV at vari- ous stages of infection, which can be detected earlier than the heterophile antibodies. VCA-IgM rises first, followed by VCA-IgG, which when present, signals the development of immunity.6 Antibodies to EBV nuclear antigens (EBNA) rise during early convalescence and persist together with VCA-IgG (Table 22-3). Thus, the presence of EBNA excludes an acute infection. A patient with all clinical manifestations and peripheral blood findings of infectious mononucleosis occasionally does not have a positive heterophile test (heterophile- negative syndrome). In 10–20% of adult cases and 50% of children younger than 10 years of age, the test is nega- tive in the presence of EBV infection. In other cases, the heterophile-negative syndrome is caused by a non-EBV Figure 22.2 viral infection. The most likely causative agent is cyto- Two reactive forms of lymphocytes from a case of infectious mononucleosis. Note the cytoplasmic vacuoles and megalovirus. Antibody responses might not be detected cytoplasmic basophilia and irregular shapes (peripheral blood, in immunosuppressed individuals (those in whom the Wright-Giemsa stain, 1000* magnification). immune response is suppressed either naturally, artificially, Table 22.3 Antibodies to EBV Found in Infectious Mononucleosis Stage of Infection Heterophile Antibodies VCA-IgM (titer) VCA-IgG (titer) EBNA (titer) Acute (0–3 months) Present 71:160 71:60 Not detected Recent (3–12 months) Present Not detected 71:160 71:10 Past (712 months) Present Not detected 71:40 71:40 VCA, viral capsid antigen; EBNA, EBV nuclear antigen. Nonmalignant Lymphocyte Disorders 479 or pathologically).8 Viral load testing can be important in test is negative. Biopsy of lymph nodes shows a reactive immune-compromised patients who develop fulminant follicular hyperplasia and can play an important role in liver disease when the disease process is not self-limiting.9 diagnosis. Diagnosis of an active infection is confirmed by Other laboratory tests can be abnormal, depending on seroconversion and a rising titer of antibodies to T. gondii. the presence or absence of complications. Hepatitis of some Immunologically compromised hosts have a more severe degree is common and can be a severe complication. An infection.13 The most common hematologic complication is increase in both direct and indirect bilirubin fractions and hemolytic anemia, which can be severe. Molecular tests for an increase in serum liver enzymes are common findings in T. gondii DNA are available. the presence of hepatitis. A rare complication of infectious mononucleosis is hemolytic anemia. The anemia appears Cytomegalovirus to be caused by cold agglutinins directed against the eryth- Cytomegalovirus (CMV) is a worldwide pathogen causing rocyte I-antigen. a spectrum of infection that varies depending on whether THERAPY the host is immunocompetent or immunocompromised. Because the disease is normally self-limited, therapy is sup- CMV is a member of the Herpesviridae family which portive. Bed rest is recommended if fever and myalgia are includes the Epstein-Barr virus, herpes simplex virus, present. Strenuous exercise should be avoided for several varicella-zoster virus, and herpesvirus 6, 7, and 8. These weeks, especially if splenomegaly is present. Antibiotics are viruses have the ability to remain latent within the host, not useful except in the presence of secondary infections. reactivating and shedding when the host’s immune system Antiviral drugs may be helpful for immune-compromised is compromised.14 Serologic tests show that 45 to100% of the individuals.9 U.S. population has prior exposure to CMV. The virus may be shed in body fluids, such as urine, saliva, blood, tears, Checkpoint 22.1 semen, and breast milk. It is also the virus most frequently A patient with lymphocytosis showing reactive lymphocyte transmitted from mother to the developing fetus before or morphology with large, basophilic cells, fine chromatin, and a during birth.15 visible nucleolus has a negative infectious mononucleosis sero- CLINICAL PRESENTATION logic test. What is a possible cause for this altered lymphocyte morphology? In the immunocompetent individual, CMV infection is asymptomatic or produces mild flu-like symptoms. Symptoms, when apparent, develop 9 to 60 days after Toxoplasmosis primary infection. It is characterized by prolonged fever, lymphadenopathy, and splenomegaly. In contrast to infec- Toxoplasmosis is a parasitic disease caused by Toxoplasma tious mononucleosis, pharyngitis is uncommon.16 After gondii (T. gondii). This obligate intracellular parasite can infection, the virus remains alive and is usually dormant multiply in all body cells except erythrocytes, although the within the person's body for life. Recurrent disease rarely preferred target organs are the lymph nodes, brain, heart, occurs unless the person's immune system is suppressed and lungs. Contact with this intracellular protozoan may due to therapeutic drugs or disease. occur through direct ingestion of food or water contami- Infection with CMV can be the result of congenital or nated with cat feces containing oocysts, ingestion of tis- acquired infection. Congenital infection in neonates occurs sue cysts in uncooked meat, transplacental infection of the when the virus from the infected pregnant woman crosses fetus, WBC transfusion, or organ transplantation. Disease the placenta and infects the fetus. The newborn can dem- may occur through acute infection after recent contact with onstrate jaundice, microcephaly, and hepatosplenomegaly. T. gondii cysts or oocysts or through endogenous reactivation.10 However, only about 10% of infected infants exhibit clini- Infections are often asymptomatic, but pose a signifi- cal evidence of the disease. The most common hematologic cant risk to the fetus of a pregnant woman. Transplacental findings in neonates are thrombocytopenia and hemolytic infection can cause abortion, jaundice, hepatosplenomegaly, anemia. chorioretinitis, hydrocephalus, microcephaly, cerebral calci- Acquired infection is spread by close contact, blood fication, and mental retardation.11 Acquired infections can transfusions, and sexual contact. CMV infection in the sometimes cause symptoms resembling infectious mono- immunosuppressed can be life-threatening. It is the most nucleosis. Toxoplasmosis seropositivity is more common in common viral infection complicating tissue transplants and rural than urban children.12 Laboratory evaluations reveal is a significant cause of morbidity and mortality in immu- leukocytosis with a relative lymphocytosis or (more rarely) nocompromised patients.17 However, treatment with anti- an absolute lymphocytosis, and an increase in reactive lym- viral drugs and immunosuppressive therapy dramatically phocytes. Most reactive cells are morphologically similar to decreases the incidence of CMV complications in organ lymphoblasts or lymphoma cells. The heterophile antibody transplantation.18 480 Chapter 22 LABORATORY EVALUATION Most viral infections have the potential to elicit an immune response resulting in lymphocytosis. CMV is the most common cause of heterophile-negative infectious mononu- cleosis. Morphologically, the lymphocytes appear reactive, exhibiting a similar morphology to infectious mononucle- osis.19 The diagnosis is made by serologic testing, cultur- ing the virus, or by detecting the virus's genetic material (CMV DNA). There are two types of CMV antibodies that are pro- duced in response to a CMV infection, IgM and IgG (Chap- ter 8). IgM antibodies are the first to be produced by the body in response to a CMV infection and last 3 to 4 months. CMV IgG is produced early in primary infection and per- sists lifelong. Rising titers of IgG can be used as markers of Figure 22.3 Lymphoproliferation resulting from B. pertussis acute infection. A positive test for CMV IgG indicates that a (whooping cough). Note the numerous small lymphocytes with person was infected with CMV at some time during his or condensed chromatin. This self-limiting peripheral blood picture her life, but does not indicate when a person was infected.20 must be distinguished from that of CLL (peripheral blood, Wright- False-positive reactions have resulted from the presence of Giemsa stain, 1000* magnification). rheumatoid factors. The lymphocytes are small cells with condensed chromatin Laboratory evaluation of the CBC indicates a leukocy- and indistinct nucleoli.21 tosis with an absolute lymphocytosis. Many lymphocytes A pertussis toxin secreted by the bacteria causes an show a reactive morphology, but the heterophile antibody accumulation of lymphocytes in the blood by recruiting test is negative. CMV is thought to infect neutrophils, which lymphocytes into the peripheral circulation and blocking serve as a means of transporting the virus to other body their migration back into lymphoid tissue. The toxin inter- sites.20 Aspartate aminotransferases liver function tests may feres with expression of L-selectin (CD62L) on all leuko- be mildly or moderately

elevated along with evidence of cytes, but lymphocytes are particularly sensitive. Decreased subclinical hemolysis. Diagnosis is confirmed by demon- cellularity of the lymph nodes accompanies rapid periph- strating the virus in the urine or blood using a viral DNA eral lymphocytosis. The ratio of CD4 to CD8 lymphocytes (molecular) assay or by a rise in the cytomegalovirus anti- remains normal.22 Laboratory diagnostic methods include body titer (except in immunocompromised patients). culture, serology, immunophenotyping, and PCR.23 Culture is the gold standard and is specific but not sensitive. The Bordetella Pertussis advantage of culture is that it allows antibiotic sensitivity testing and epidemiological typing. Serologic diagnosis is Pertussis, also known as whooping cough, was one of the based on demonstrating antipertussis toxin antibodies. It is most common childhood diseases in the 20th century and preferred for older children and adults who present after a major cause of childhood mortality. Bordetella pertussis a prolonged coughing illness. PCR for B. pertussis DNA is (B. pertussis), a fastidious, aerobic, Gram-negative cocco- most sensitive in detecting infection when used during the bacillus produces an acute respiratory infection marked first 3 weeks of cough24 and is most useful in acutely ill by severe, spasmodic (whooping) coughing episodes. infants. B. pertussis produces a number of virulence factors, includ- The incidence of whooping cough infections continues ing pertussis toxin, adenylate cyclase toxin, filamentous to rise, suggesting that pertussis immunizations given in hemagglutinin, and hemolysin, which contribute to its childhood do not yield lasting immunity. Studies show that pathologic manifestations.21 Transmission is by droplets 20 to 30% of adults with a cough that lingers 6 to 8 weeks directly from person to person, where the bacteria will are positive for B. pertussis. The increased incidence of infec- colonize the ciliated cells of the respiratory mucosa, and tions also may be due to increased sensitivity of PCR detec- undergo rapid multiplication. tions.24 The infections cause a lingering cough in adults, and Infection with B. pertussis causes a blood picture very passing it on to infants can be fatal.25 similar to that of reactive viral lymphocytosis (Figure 22-3). The leukocyte count typically rises to 15925 * 103/mcL but can reach 50 * 103/mcL. The rise in leukocytes is caused Reactive Lymphocytosis by an absolute lymphocytosis of T, B, and natural killer A reactive lymphocytosis process, previously called infec- (NK) lymphocytes along with increases in neutrophils tious lymphocytosis, is a reactive immune response asso- and monocytes. Granulocytes may show toxic changes. ciated with several common viruses that infect children, Nonmalignant Lymphocyte Disorders 481 usually between the ages of 2 and 10 years (often coxsackie Plasmacytosis and adenovirus). It is characterized by an increase in blood lymphocytes, often up to 30 * 103/mcL and occasionally as Plasma cells are not normally found in the peripheral blood high as 100 * 103/mcL, which might be mistaken for acute and constitute 4% of the cells in the bone marrow. Most leukemia. Eosinophilia may be present. Patients are usually plasma cells are found in the medullary cores of lymph asymptomatic but may have fever, abdominal pain, or diar- nodes, although they can occasionally be found in the rhea. There is no lymph node enlargement or splenomegaly. peripheral blood with intense stimulation of the immune Symptoms only last a few days, but the lymphocytosis may system. This can occur in some viral and bacterial infections persist for several weeks. such as rubeola, infectious mononucleosis, toxoplasmosis, An increased relative lymphocyte count with the syphilis, and lung infections (pneumonia, tuberculosis, presence of reactive or immature-appearing lymphocytes abscesses). Circulating plasma cells can also be found in (lymphocytic leukemoid reaction) characterizes the disorders associated with elevated gamma globulin such lymphocytic reaction. Many of the lymphocytes are large, as multiple myeloma, skin diseases, cirrhosis of the liver, reactive cells with deep blue cytoplasm and fine chroma- collagen disorders, and sarcoidosis. tin and may show cytoplasmic vacuoles (Figure 22-4). Normal plasma cell morphology is included with the Reactive cells are usually nonclonal T lymphocytes and discussion of lymphocyte morphology in Chapter 8. A mor- large granular lymphocytes. In some viral infections, lym- phologic variation of the reactive plasma cell is called the phocytopenia and neutropenia precede lymphocytosis. flame cell, named for its reddish-purple cytoplasm. A glyco- As the infection subsides, plasmacytoid lymphocytes can protein produced in the rough endoplasmic reticulum (RER) be found (Figure 22-5). The heterophile antibodies test is causes the red tinge, and the presence of ribosomes causes negative. the purple tinge. These cells contain more immunoglobulin In some cases, a lymphocytic leukemoid reaction than normal plasma cells. Flame cells have been associated resembles chronic lymphocytic leukemia (CLL; Chapter with IgA plasma cell myeloma but are now recognized in a 29). Bone marrow aspiration, however, shows minimal (if variety of immune pathologies and are occasionally found any) increase in lymphocytes in a lymphocytic leukemoid in normal bone marrow. reaction. In contrast to CLL, lymphadenopathy and spleno- megaly are usually absent. In addition, patients with a lym- Persistent Polyclonal B-Cell phocytic leukemoid reaction are usually young, whereas CLL patients are usually older adults. Lymphocytosis The reactive lymphocytosis can be distinguished from a Persistent polyclonal B-cell lymphocytosis (PPBL) is a lymphoproliferative disorder based on a pleomorphic ver- rare disorder found primarily in the female adult smoker. sus monomorphic presentation, respectively. Most reactive It is characterized by chronic, stable, persistent, polyclonal lymphocytosis shows a spectrum of morphologic changes lymphocytosis with atypical binucleated lymphocytes with including variations in sizes and shapes. abundant cytoplasm in the peripheral blood. There is often Figure 22.4 An immunoblast found in a patient with a Figure 22.5 A plasmacytoid lymphocyte. Note the deep viral infection. This cell must be distinguished from a leukemic basophilic cytoplasm and eccentric nucleus. These cells are associated lymphoblast (peripheral blood, Wright-Giemsa stain, 1000* with infectious states. Note also the neutrophil with toxic granulation magnification). (peripheral blood, Wright-Giemsa stain, 1000* magnification). 482 Chapter 22 evidence of an HLA-DR7 haplotype, partial insertion of to mount an immune response is possible, resulting in chromosome 3, i(3q), and multiple IgH/Bcl-2 rearrange- immunodeficiency. Most individuals with lymphocyto- ments that suggest PPBL could lead to a malignant pro- penia have a reduced absolute number of T cells, particu- cess.26 The disorder is often asymptomatic and found by larly the number of CD4+ T cells. chance on a routine blood analysis. Hematologic evaluation reveals a moderate increase Lymphocyte Sequestration and in the absolute lymphocyte counts (more than 4 * 109/L) without evidence for infection or other conditions that can Destruction increase the lymphocyte count. Specific morphologic fea- Lymphocytopenia results from decreased production or tures predictive of the diagnosis include basophilic vacu- increased destruction of lymphocytes, changes in lympho- olated cytoplasm and monocytoid changes.26 There is an cyte circulation patterns, and other unknown causes (Table expansion of CD27+ immunoglobulin (Ig) M+ IgD+ B@cells. 22-4). Various acquired or inherited diseases, conditions and There is a polyclonal increase in serum IgM but low IgG and factors are associated with lymphocytopenia. Corticosteroid IgA levels. Bone marrow examination reveals lymphocytic therapy causes a sharp drop in circulating lymphocytes infiltrates. within 4 hours. The decrease is caused by sequestration of Symptoms can include fever, fatigue, weight loss, recur- lymphocytes in the bone marrow. Values return to normal rent chest infections, or generalized lymphadenopathy. within 12–24 hours after cessation of therapy. Acute inflam- PPBL may evolve into a variety of malignant lymphop- matory conditions, including viral and bacterial infections, roliferative disorders including non-Hodgkin lymphoma, also can be associated with a transient lymphocytopenia. diffuse large cell lymphoma, splenic marginal zone lym- Malignancies with an associated lymphocytopenia is a phoma, and other clonal solid tumors.26 poor prognostic sign. Systemic lupus erythematosus is fre- Additional plasma cell variants include Mott cells, Rus- quently associated with a lymphocytopenia presumably sell bodies, and Dutcher bodies. These variations are associ- caused by autoantibodies produced against these cells. ated with the malignant disorders plasma cell leukemia and Chemotherapeutic alkylating drugs for malignancy, such as multiple myeloma (Chapter 28). cyclophosphamide, cause the death of T and B-lymphocytes in both interphase and mitosis. Malnutrition is the most common cause of lymphocytopenia. Starvation causes Lymphocytopenia thymic involution and depletion of T lymphocytes. Both congenital and acquired immune deficiency disorders are The lymphocytes are essential for providing immunity and associated with lymphocytopenia.27 protecting the body from infections. Lymphocytopenia is Irradiation causes a prolonged suppression of lym- defined as an abnormally low level of lymphocytes in the phocyte production. CD4+ helper lymphocytes are more blood. The blood value is age dependent and defined as sensitive to radiation than are CD8+ lymphocytes. It an absolute lymphocyte count of less than 1.0 * 103/mcL appears that small daily fractions of radiation are more for adults or less than 3.0 * 103/mcL in children younger damaging to lymphocytes than periodic large doses. With than 5 years of age. In general, lymphocyte counts less periodic radiation, the lymphocytes can renew during than 2 * 103/mcL are abnormal in this population. When periods of nonradiation. Aggressive treatment of hema- the lymphocyte count is decreased, an impaired ability tologic malignancies with chemotherapeutics or ionizing Table 22.4 Conditions Associated with Lymphocytopenia Infectious Diseases Immune Disorders Malignancy Drugs Other Human immunodeficiency virus Systemic lupus Hodgkin’s disease Corticosteroid therapy Acute inflammatory (HIV) erythematosus conditions Severe acute respiratory syn- Ataxia-telangiectasia Diffuse large B cell Radiation and Aplastic anemia drome (SARS) lymphoma chemotherapy Respiratory syncytial virus (RSV) Wiskott-Aldrich syndrome Peripheral T cell lymphoma Steroid therapy Malnutrition West Nile virus encephalitis Severe combined immuno- Stress deficiency disease Measles Sjögren syndrome Acute and chronic renal disease Ehrlichia Salmonella typhi Nonmalignant Lymphocyte Disorders 483 radiation can lead to immunodeficiency by depleting and recombinant viruses. Subtype B predominates in the short-lived, antigen-activated B lymphocytes.27 Americas, Caribbean, and Western Europe.29 Clinical Presentation Patients experience weight loss, Checkpoint 22.2 fever, lymphadenopathy, thrush, chronic rash, and intermit- Why is lymphocytopenia a concern if there is no accompanying tent diarrhea. In the pre-antiretroviral era, patients infected leukopenia? with HIV had a grave prognosis. Current highly active anti- retroviral treatment (HAART) protocols provide effective therapy to reduce viral replication and delay the onset of AIDS for HIV-infected people.29 CASE STUDY (continued from page 476) Not all individuals infected with the HIV virus Heidi weighed 2 pounds 1 ounce at birth. Tests for (HIV-positive individuals) have the clinical condition CMV and toxoplasma infections were negative. known as AIDS. AIDS is defined by the occurrence of At 4 days of age, she was transferred to a special repeated infections with multiple opportunistic organisms facility for feeding and growth m onitoring. She and an increase in malignancies, especially Kaposi’s sar- developed a diaper rash that failed to respond coma, in individuals infected with HIV. to many measures. No thrush was found. Transmission rates between individuals is dictated by At 44 days, she developed pneumonia from the viral load in the HIV-infected source. Transmission of coagulase-negative staphylococci that responded to the virus occurs through sexual intercourse, blood/blood antibiotics. Her WBC count was 12.3 * 103/mcL product transfusion (93%), mother-to-child transmission, with a differential of segs 42%, bands 5%, and needle-sharing. Infants born to HIV-infected mothers lymphocytes 1%, monocytes 28%, eosinophils 23%, are at increased risk for perinatal HIV infection. Estimated and basophils 1%. rates for mother-to-baby transmission in the absence of anti- retroviral therapy is 23%.30 3. What is the absolute lymphocyte count? Surveillance Hiv Case Definition In 1982, the U.S. Centers 4. What possible causes exist for these opportunis- for Disease Control and Prevention (CDC) developed a case tic infections? definition of AIDS for surveillance purposes. It is revised periodically as more data are collected about the disease.31 The 2014 revision of Surveillance Case Definition for HIV infection describes the clinical criteria and “opportunis- Immune Deficiency Disorders tic i llnesses” for presumptive evidence of HIV infection31 (Table 22-5) and standardizes data collection for improved Immune deficiency disorders are characterized by impaired therapeutic treatment. Symptoms of recurrent infections with function of one or more of the components of the immune opportunistic infections warrant suspicion of HIV infection. system, T, B, or NK lymphocytes. In some disorders, the numbers of these cells are also reduced. This results in an inability to mount a normal adaptive immune response. These disorders are characterized clinically by an increase Table 22.5 Opportunistic Illnesses in HIV Infection in infections and neoplasms and can be sub-grouped as Microbial Infections Malignancies Other

acquired or congenital. Recurrent bacterial Cervical cancer AIDS dementia Acquired immunodeficiency syndrome (AIDS) is an infections syndrome infectious disorder characterized by lymphocytopenia. Candidiasis Kaposi’s sarcoma Encephalopathy Because of the frequency with which it is encountered in Coccidioidomycosis Lymphoma Progressive multifocal the clinical hematology laboratory, it is discussed in more leukoencephalopathy detail next and is followed by a discussion of the congenital Cryptococcosis Wasting syndrome immune deficiency disorders. Cryptosporidiosis Cytomegalovirus ACQUIRED IMMUNODEFICIENCY SYNDROME Herpes simplex Acquired Immunodeficiency Syndrome (AIDS) is a Histoplasmosis highly lethal immune deficiency disease first described in 1981.28 The disease is caused by infection with a retro- Isosporiasis virus, human immunodeficiency virus type I (HIV-1) or Mycobacterium type 2 (HIV-2). HIV-1 is comprised of four distinct viruses, Pneumocystis carinii types M, N, O, and P. Group M is the globally predomi- Salmonella nant viral strain and is further divided into nine subtypes Toxoplasmosis 484 Chapter 22 The revised criteria apply to adults and children over by binding to both the CD4 protein that is part of the T-cell 18 months of age and combined the HIV classification sys- receptor (TCR; Chapter 8), and chemokine receptor CCR5.32 tem and AIDS case definition into a single case definition Once in the cell, HIV sheds its viral coat and uses reverse for HIV infection. This revised case definition requires lab- transcriptase to make a DNA copy of the viral RNA. Viral oratory-confirmed evidence of HIV infection. The Council DNA is then integrated into the host cell DNA where the of State and Territorial Epidemiologists (CSTE), in coop- virus replicates. Viral infection of these CD4 lymphocytes eration with pediatricians, also described the criteria for causes cytolysis, rapid selective depletion of this lympho- a negative diagnosis for children …18 months old born to cyte subset, and eventually lymphocytopenia. Monocytes HIV-infected mothers. The child does not meet the criteria and macrophages also have the CD4 and CCR5 proteins and for HIV/AIDS diagnosis if antibody tests or virologic tests are infected but not destroyed by the virus. Rare individuals are negative after 4–6 months of age and the child does not are homozygous for a 32-base pair deletion in the gene for have an AIDS-defining condition.31 Almost all children born CCR5 (CCR5 delta 32), resulting in lack of protein expres- to HIV-infected mothers have anti-HIV IgG antibodies at sion on the cell surface. These individuals are resistant to birth because of placental transmission of maternal antibod- HIV infections.33 ies. However, only 15–30% of the children are infected with Cell-mediated immunity and humoral immunity are HIV. Thus, anti-HIV IgG tests in this population are not reli- abnormal. Cell-mediated immunity declines as CD4+ T able until after 18 months of age. In these cases, tests for lymphocyte-helper function for monocytes, macrophages, the detection of HIV nucleic acid performed after the infant and other T lymphocytes declines. Humoral responses are is at least one month of age are almost always positive if exaggerated with polyclonal B lymphocyte proliferation, the virus is present.30 In addition, the 2014 CDC surveil- increased immunoglobulin production, and hypergam- lance HIV data allow for staging of HIV infections based maglobulinemia. Although the B lymphocytes have a poor on immunophenotyping of CD4 lymphocytes and clinical response to mitogens in vitro, they secrete immunoglobulin conditions (Table 22-6). For surveillance purposes, the stag- spontaneously. This suggests the B lymphocytes are already ing of disease illness only progresses; it is never reclassified activated and unable to react to further in vitro stimulation. as a less severe stage. The CD8+ cytotoxic T lymphocytes attempt to respond to The World Health Organization (WHO) uses similar HIV infection to control viral replication during primary criteria for diagnosing HIV infection as the CDC, but WHO infection and reduce the viral plasma level in chronic infec- splits the CDC stage 2 into two separate stages: stage 2 and tion. Because the immune response to HIV is defective and stage 3. Those patients with decreased lymph counts but does not clear the virus, the immune system remains con- not requiring treatment are classified as stage 2, and those tinually activated with high rates of T cell turnover that patients with decreased CD4+ T lymphs and conditions that eventually leads to T cell exhaustion and depletion.32 require antiretroviral therapy for advanced HIV disease Laboratory Evaluation Multiple hematologic abnormali- (AHD) as stage 3. WHO classifies patients with both clini- ties, including leukopenia, lymphocytopenia, anemia, and cal conditions and the diagnostic lymphopenia as stage 4 thrombocytopenia, are found in HIV infections (Table 22-7). AIDS.31 Leukopenia is usually related to lymphocytopenia, although Pathophysiology The main etiologic agent of AIDS in the neutropenia also can be present. Lymphocytes can include United States is the retrovirus, HIV-1. A related but immuno- reactive forms. Mild to moderate normocytic, normochromic logically distinct virus, HIV-2, is endemic to regions of West anemia is present in the majority of HIV-infected individu- Africa. The virus selectively infects helper T lymphocytes als and worsens as the disease progresses.32 Inflammatory Table 22.6 CDC and WHO Staging of HIV Infection for Adolescents and Adults CD4+ T Lymph CD4+ T Lymph Counta (percentagea of total CDC Staging WHO Staging AIDS-Defining Condition(s) (cells/mcL) lymphocytes) Stage 1 HIV infection Stage 1 HIV infection None CD4+ lymphs Ú 500 CD4+ lymphs Ú 26 Stage 2 HIV infection Stage 2 HIV infection None CD4+ lymphs 2009499 CD4+ lymphs 14925 Stage 3 HIV infection (AHD) Clinical conditions requiring CD4+ lymphs 2009499 CD4+ lymphs 14925 retroviral treatment Stage 3 AIDS Stage 4 AIDS Clinical condition present CD4+ lymphs 6 200 CD4+ lymphs 6 14 a Staging requires either CD4+ T lymph count or percent CD4+ T lymphs of total lymphocytes. Adapted from Appendix, MMWR Recommendations and Reports, 63(RR-03), 1–10, April 2014. Nonmalignant Lymphocyte Disorders 485 Immediate evaluation and treatment for HIV exposure Table 22.7 Laboratory Evaluation in HIV Infections has proven to be very effective in eliminating or significantly • Leukopenia • Thrombocytopenia minimizing the risk of HIV infection and AIDS. Health • Anemia • Decreased CD4 counts care workers with occupational exposure (e.g., needlestick • Macrocytosis (in patients treated • Positive molecular tests for injury) should receive immediate antiretroviral therapy.37 with zidovudine) HIV-1 RNA The CDC recommends initiation of antiretroviral therapy within 36 hours of a needlestick injury from a potential HIV source. Studies show that as time elapses after exposure, cytokines may play a role in suppressing erythropoiesis in viral replications exceed the control of the antiretroviral a manner similar to that found in anemia of chronic disease therapy. After 72 hours, therapy to prevent infection is no (ACD; Chapter 12). Macrocytosis (MCV more than 100fL) longer effective.37 Combination therapy should consist of occurs in up to 70% of patients 2 weeks after receiving zid- two or more antiretroviral drugs and continue for 4 weeks. ovudine.33 Antierythrocyte antibodies can be found in up to Laboratory evaluation for adverse effects should be consid- 20% of patients with hypergammaglobulinemia. These anti- ered after 2 weeks. bodies react like polyagglutinins and cause a positive direct Recent HIV research studies have demonstrated a antiglobulin test (DAT, Coombs’ test; Chapter 19). Immune cross-reacting antibody that develops late in HIV infection thrombocytopenia, indistinguishable from i diopathic of patients with decreased CD4+ T cells and high viral loads, thrombocytopenic purpura (ITP), is common (Chapter 33). which has a neutralizing effect on HIV proteins. The anti- Iron studies are similar to those found in ACD with low body is specifically targeted at the envelope protein, gp120. serum iron and TIBC and increased serum ferritin. This antibody response, although too late to eliminate HIV infection once established, may prove to be effective in find- Disease Monitoring The severity of CD4 lymphocytopenia ing a means for HIV immunization.32,38 and concentration of plasma HIV-1 RNA copies correlate with the severity of disease. The normal CD4:CD8 ratio in periph- eral blood is about 2:1. In AIDS, this ratio reverses progres- Checkpoint 22.3 sively and permanently because of destruction of the CD4+ T Why does infection with HIV result in an increased chance for lymphocytes. The CD4+ T lymphocyte count is performed at opportunistic infections? initial diagnosis and measured periodically to monitor dis- ease progression. AIDS (CDC stage 3 HIV infection) is defined OTHER ACQUIRED IMMUNE DEFICIENCY DISORDERS by a CD4+ T lymphocyte count of less than 200/mcL (mL) or Other acquired immune deficiency disorders are diagnosed a CD4+ T@lymphocyte concentration less than 14% of total based on functional abnormalities of lymphocytes. The lymphocytes.33 In addition, the viral load is monitored by lymphocyte count can be normal but in many cases, it is measuring the number of copies of HIV-1 RNA.34 decreased. Acquired defects of either T or B lymphocytes can result in serious clinical manifestations. Some inflam- Therapy No cure for AIDS currently exists. Treatment with matory states transiently impede the response of T lympho- zidovudine (azodothymidine, AZT) and protease inhibi- cytes to antigen. These include idiopathic granulomatous tors lengthen the time between HIV seropositivity and the disorders and malignancy. Severe infection by one micro- onset of AIDS. Improved antiretroviral treatments have organism sometimes impedes the ability of T lymphocytes postponed the onset of AIDS indefinitely in some patients.34 to react to other infectious organisms. Starvation or severe In 2008, a medical group in Germany reported an protein deficiency also can severely affect the functional apparent cure for an HIV-1 positive patient who had ability of the T lymphocyte. received a bone marrow transplant (BMT) for acute myeloid leukemia.35 The donor was homozygous for the CCR5 delta CONGENITAL IMMUNE DEFICIENCY DISORDERS 32 mutation, and the transplanted hematologic cells were Congenital disorders are usually characterized by a resistant to viral infection by HIV-1. Although widely cov- decrease in lymphocytes and impairment in either cell- ered in both the scientific and popular (lay) literature, this mediated immunity (T lymphocytes), humoral immunity information has not made a significant impact to date on (B lymphocytes), or both (Table 22-8). In contrast to the reac- the treatment strategies for most AIDS patients. BMT is not tive morphologic heterogeneity of lymphocytes associated an option for most AIDS patients, due to both the logistic with viral disorders, lymphocytes in congenital disorders improbability of finding an HLA-compatible/CCR5 delta 32 are usually normal in appearance. The functional impair- homozygous donor and the cost and medical risks involved ment of the immune response is often apparent from birth with the procedure. However, it has sparked interest in the or at a very young age when children present with recurrent possibility of a type of gene therapy in which the CCR5 gene infections. Due to extensive research using molecular tech- is modified in an effort to prevent the HIV virus from being niques, the genetic mutations causing immunodeficiency able to infect hematologic cells.36 are being identified. 486 Chapter 22 Severe Combined Immunodeficiency Syndrome Severe is impaired (Chapter 4). IL-7/IL-7R signaling is required combined immunodeficiency (SCID) syndrome includes for T cell development, and in its absence, T cell lym- a heterogeneous group of rare genetic disorders result- phopoiesis fails. Thus, X-linked SCID is c haracterized by ing in major qualitative immune defects involving both absent T and NK lymphocytes and a hypoplastic thymus.41 humoral and cellular immune functions. These disorders B lymphocytes are normal in number but are nonfunctional include numerous and diverse genetic mutations with dif- due to lack of T-helper function, and immunoglobulin lev- ferent inheritance patterns and varied severity in clinical els are severely depressed. Family history is important in manifestation. Most are inherited as sex-linked or autoso- determining the mode of inheritance of SCID, although a mal-recessive traits, but some autosomal dominant forms negative family history of the disease does not rule out an have been reported. About 75% of individuals with SCID X-linked disease. Up to one-third of the cases present as a are males because the most common form of SCID is an spontaneous mutation.41 X-linked disorder. Females who carry the abnormal X-linked SCID gene Both the T and B lymphoid lineages are functionally have normal immunity. These carriers can be detected deficient. Subgroups of SCID are now defined on the basis by molecular assays for IL-2R mutations in the g@chain of which lymphocytes are absent. If only the T cells are locus using cells other than lymphocytes. The normal absent, it is termed T-B+NK+ SCID; if both T and B cells mature female cell population is a mosaic

with one or are absent, it is T-B-NK+ SCID; and in those patients (usu- the other X chromosome inactivated (X-inactivation [also ally with the most severe lymphopenia) in whom all three called lyonization] is the normal process by which one of lymphocyte subsets are absent, it is T-B-NK- SCID.39 In all the copies of the X chromosome present in the female is cases both the T and B lymphoid lineages are functionally inactivated and transcriptionally silent). In female SCID deficient. Because T cell help is required in generating an carriers, h owever, only lymphocytes carrying normal, antibody response against protein antigens, disorders in non-inactivated X chromosomes are found, rather than the which T lymphocytes are absent result in peripheral blood expected mixture of cells with normal and abnormal X chro- B lymphocytes that are unresponsive to most mitogens, mosome inactivation. Random X chromosome inactivation and immunoglobulin production is decreased. However, occurs in these carriers, but the gene product of the mutant these B lymphocytes respond normally when incubated X chromosome does not support lymphocyte maturation. with normal T lymphocytes in vitro.40 The absolute lym- Thus, lymphocytes with the mutant X chromosome fail to phocyte count is variable but often is decreased to less than develop, and the only lymphocytes found in carriers have 1.0 * 103/mcL. Lymphocyte counts in SCID patients may the active normal X chromosome. be normal in the neonate, especially if they are able to gen- erate B cells and NK cells, but eventually they develop a Checkpoint 22.4 profound lymphopenia. Would you expect female carriers of X-linked SCIDS to be more Lymph node examination reveals a lack of plasma cells, susceptible to infection than the normal population? Why or B lymphocytes, and T lymphocytes. No lymphoid cells are why not? found in the spleen, tonsils, or intestinal tract. The bone marrow also is deficient in plasma cells and lymphocyte Autosomal SCID The autosomal forms of SCID exhibit precursors. severe deficiencies of both T and B lymphocytes. The Frequent recurrent infections, skin rashes, diarrhea, most common form, found in about 30–40% of autosomal- and failure to thrive are characteristic findings in infants recessive SCID (15% of all SCID patients), is due to an with SCID. Death related to overwhelming sepsis usu- adenosine deaminase (ADA) deficiency that results in a ally occurs within the first 2 years of life if untreated. depletion of T, B, and NK lymphocytes, T-B-NK-.41 The Bone marrow transplantation, immunoglobulin therapy, adenosine deaminase gene is located at chromosome and gene therapy are the only options for successful treatment.40 20q13.11 (Table 22-8). Both point mutations and gene dele- tions have been associated with ADA deficiency. Sex-Linked SCID Classic sex-linked (X-linked) SCID is the Another enzyme deficiency, purine nucleoside most common form of inherited severe combined immuno- phosphorylase (PNP), is the cause of ∼2, of SCID cases. deficiency, accounting for about 45% of cases. This form of The gene for PNP is located at chromosome 14q13.1. Both SCID (T-B+NK- SCID) has been mapped to the long arm of these enzymes degrade purines. Without PNP, accumu- of the X chromosome (Xq13.1–13.3) and is associated with lation of toxic DNA metabolites (deoxyadenosine triphos- a loss-of-function mutation in the gene for the g@chain of phate/dATP and deoxyguanosine triphosphate/dGTP) the IL-2 receptor (Table 22-8). Because the g@chain also is an occurs, inhibiting normal T and B cell development. The essential subunit of the receptors for IL-4, IL-7, IL-9, IL-15, two disorders can be differentiated by the presence of NK and IL-21, hematopoietic regulation by all of these cytokines cells and the ADA enzyme in PNP.41 Nonmalignant Lymphocyte Disorders 487 Table 22.8 Laboratory Evaluation in Selected Immunodeficiency Disorders Disorder Immunoglobulins B-Lymphs T-/NK-Lymphs Genetic Loci SCID X-linked T- B+ NK- T IgG, IgE, IgA; N IgM N; abnormal function Absent IL2RG (Xq13.1) Autosomal recessive T- B+ NK+ T IgG, IgE, IgA; N IgM N; abnormal function T-lymphs absent ZAP-70 (2q12); IL7R (5p13) T- B- NK+ T IgG, IgM, IgA, IgE Absent T-lymphs absent RAG-1 and RAG-2 (11p13); PNP (14q13.1) T- B+ NK- T IgG, IgE, IgA; N IgM N; abnormal function Absent JAK3 (19p13.1) T- B- NK- T IgG, IgM, IgA, IgE Absent Absent ADA (20q13.11, ADA deficiency); AK2 (1p34, reticular dysgenesis) MHC class II deficiency T IgG, IgE, IgA; N IgM N N or T T-lymphs CIITA (16p13); RFXANK (19p12); T+/- B+ NK+ RFX5 (1q21); RFXAP (13q14) Other Immunodeficiency Syndromes X-linked X-linked agammaglobulinemia T IgG, IgM, IgA Mature cells absent N BTK mutations (Xq21.3–q22) (T+ B- NK+) Wiskott-Aldrich syndrome T IgM; N/T IgA; N; abnormal function N or T T-lymphs WAS mutations (Xp11.22–11.3) (T+ B- NK+) c IgE; N/c IgG Autosomal diGeorge syndrome N or c IgG; IgM; IgA N number; delayed T-lymphs TBX1 (del 22q11.2) (T- B+ NK+); AD maturation Absent Ataxia-telangiectasia; AR N/c IgM; N/c IgG; IgE; IgA N; abnormal function T T-lymphs ATM (11q22–23) Hyper IgE syndrome (HIES); N IgM, IgG, IgA; c IgE N T T-lymphs DOCK8 (9p24.3); STAT3 AR and AD (17q21.31) T , decreased; c , increased; N, normal; ADA, deaminase; AK2, adenylate kinase 2; AD, autosomal dominant; AR, autosomal recessive; ATM, ataxia telangiectasia mutated; BTK, Bruton’s tyrosine kinase; CIITA, Class II MHC transactivator; DOCK8, dedicator of cytokinesis 8; IL2RG, interleukin 2 receptor, gamma chain; IL7R, interleukin 7 receptor 7 alpha chain; JAK3, Janus kinase 3; PNP, purine nucleoside phosphorylase; RAG, recombination activating gene; RfX5, Regulatory factor X-5; RfXAP, RfX-associated protein; RfXANK, RfX-associated ankyrin containing protein; SCID, severe combined immunodeficiency syndrome; STAT3, signal transducer activator of transcription 3; TBX1, T-box-containing transcription factor; WAS, Wiskott-Aldrich syndrome; ZAP70, z@chain associated protein kinase-70. Other defects include a deficiency of MHC class II gene expression, interleukin-2 receptor a@chain (IL@2Ra) defi- 7. Are the lymphocytes more likely to be morpho- ciency, mutations in the RAG-1 and RAG-2 genes (which logically heterogeneous or homogeneous? Why? catalyze VDJ recombination), and defective assembly of the 8. What confirmatory test is indicated? T cell receptor–CD3 complex (Chapter 8).40 Wiskott-Aldrich Syndrome Wiskott-Aldrich syndrome CASE STUDY (continued from page 483) (WAS) is a sex-linked recessive disease characterized by the All of Heidi’s immunoglobulin levels were triad of eczema, thrombocytopenia, and immunodeficiency, decreased. T and B lymphocyte counts were severely resulting in recurrent infections. The first signs of WAS are decreased. The peripheral blood smear showed usually petechiae and bruising, resulting from a low plate- anisocytosis, poikilocytosis (schistocytes), polychro- let count. Spontaneous nosebleeds and bloody diarrhea are matophilia, and two nucleated RBCs/100 WBCs. common. Eczema develops within the first month of life Her thymus was not detectable on chest films. while recurrent bacterial infections develop by 3 months.42 5. Is this child more likely to have a congenital or About two-thirds of affected children have a family history acquired immune deficiency? of the disease; in one-third there is an apparent spontane- ous mutation. Untreated, most children die before 10 years 6. If she has a congenital immune deficiency, is it of age as a result of infection or bleeding. Those who sur- more likely that she has X-linked or autosomal vive longer can develop neoplasms of the histiocytic, lym- SCIDS? phocytic, or myelocytic lineages. EBV infections in WAS patients can lead to lymphoma.43 488 Chapter 22 Laboratory evaluation plays an important role in the or maternal X chromosomes and are asymptomatic. This diagnosis of WAS (Table 22-9). There is a progressive decrease nonrandom inactivation pattern is found in the carrier’s in thymic-dependent immunity and depletion of paracorti- T and B lymphocytes, granulocytes, monocytes, and mega- cal areas in the lymph nodes, leading to abnormal lympho- karyocytes, indicating that all hematopoietic cells in WAS cyte function. The absolute numbers of CD4+ and CD8+ are affected by the presence of the WAS gene. lymphocytes and their ratio is normal to variable. Typically, Therapy includes treatment for bleeding and infection. CD8+ lymphocytes are decreased, but numbers vary over Splenectomy usually results in correction of the platelet time for individuals. Circulating B-lymphocyte numbers are count and normalized platelet volume and significantly normal, but antibody production is abnormal.43 Serum IgM reduces the risk of bleeding complications. Bone marrow levels are decreased, but IgE and IgA levels are increased. transplant before 5 years of age has an 85% cure rate.43 IgG concentrations are usually normal (Table 22-8). One of the most consistent findings is the low or absent Checkpoint 22.5 level of circulating antibodies to the blood group antigens.43 What laboratory findings suggest WAS in a child, and how is the These children are unable to produce antibodies to polysaccha- diagnosis confirmed? ride antigens, a T lymphocyte-independent phenomenon. This suggests that there is an intrinsic B lymphocyte abnormality. DiGeorge Syndrome DiGeorge syndrome is a congenital A low platelet count with abnormal bleeding in the neo- immunodeficiency marked by the absence or hypoplasia of natal period is one of the first clinical signs of WAS. Patients the thymus, hypoparathyroidism, heart defects, and dys- have a severe thrombocytopenia (less than 70 * 103/mcL) morphic facies. Hypocalcemia is typical, and the presenting with small-size platelets.43 Platelets are intrinsically abnor- symptom can be seizure resulting from hypocalcemia. Usu- mal with decreased expression of surface glycoproteins ally a decrease in peripheral blood T lymphocytes occurs IIb, IIIa, and IV and the defective expression of CD62P and as well as a decrease in the cellularity of the T lymphocyte CD63. The prothrombin time and activated partial throm- regions of peripheral lymphoid tissue. The low lymphocyte boplastin time are normal, indicating the coagulation factor count is related to a decreased number of CD4+ lympho- proteins are adequate (Chapters 31 and 32). Megakaryocytes cytes (Table 22-8). T lymphocyte function varies. Children in the bone marrow are normal or increased in number and with a hypoplastic thymus may be able to produce enough morphologically normal. lymphocytes with normal function to maintain immuno- Genetic mutations involve the Wiskott-Aldrich competence. B lymphocytes are normal in number and syndrome (WAS) gene on the short arm of the X chromo- function, and immunoglobulin levels are normal. Infants some between Xp11.22 and Xp11.3. The WAS gene codes exhibit increased susceptibility to viral, fungal, and bacterial for the WAS protein (WASp), which is mainly expressed infections that are frequently overwhelming. Death occurs in hematopoietic cells. Females are carriers; affected males in the first year unless thymic grafts are performed. do not pass the deficiency to their male children. PCR tech- Cytogenetic studies on these children show a chromo- niques detect 98% of affected males and are the primary some 22q11.2 deletion; this deletion is thought to affect an diagnostic tests when WAS is suspected.43 Molecular analy- estimated 1 in 4,000 people, and is considered autosomal sis using restriction fragment length polymorphisms reveals dominant because a deletion in one copy of chromosome 22 that female carriers have selective inactivation of the WAS in each cell is sufficient to cause the condition.44 This defect X chromosome rather than random inactivation of paternal also is found in a parent of a child with DiGeorge syndrome in 25% of the cases. Table 22.9 Sex-Linked Agammaglobulinemia Sex-linked (X-linked) Laboratory Features in Wiskott-Aldrich Syndrome (WAS) agammaglobulinemia (Bruton’s disease) is inherited as a sex-linked disease characterized by frequent respiratory Feature Description and skin infections with extracellular, catalase-negative, Platelets Decreased concentration, small size, pyogenic bacteria. Molecular analysis has revealed that abnormal platelet function the genetic defect is on the long arm of the X chromo- Lymphocytes Decreased or normal concentration, some, (Xq21.3–22; Table 22-8).39 More than 90% of patients T lymphocytes variable, B lymphocytes usually normal have a loss-of-function mutation of a tyrosine kinase gene, Immunoglobulin IgM decreased, IgE and IgA increased, IgG Bruton’s tyrosine kinase (BTK). The genetic mutation results normal/increased in a block in B lymphocyte maturation at the pre-B lympho- Antibodies to blood group Absent or decreased cyte stage. The variable and constant regions of the IgM antigens immunoglobulin chain fail to connect. PCR Detects WAS gene mutation Peripheral blood lymphocyte counts are normal as are T Ig, immunoglobulin; PCR, polymerase chain reaction. lymphocytes; there is, however, a decrease in B lymphocytes Nonmalignant Lymphocyte Disorders 489 and an absence of plasma cells in lymph nodes. The serum skin abscesses, recurrent lung infections, and eosinophilia. concentrations of IgG, IgM, and IgA are decreased or absent. The disorder has both autosomal dominant and recessive Cell-mediated immune function is normal. Monthly injec- forms. These HIESs are associated with several mutations tions of gamma globulin

are effective in preventing severe (Table 22-8). There are two forms of HIES: a dominant form infections. caused by mutations in STAT3 and a recessive form, for Female carriers of this disease have normal immunity. which a genetic cause is unclear. All of their B lymphocytes carry the paternal, normal X Autosomal-dominant HIES, the most common disease chromosome, suggesting that the normal X chromosome in this group, results from STAT3 gene mutations and has confers a survival advantage. a variety of connective tissue and skeletal abnormalities.46 JAK-STAT (Chapter 4) mutations alter cytoplasmic signal- Ataxia-Telangiectasia Ataxia-telangiectasia (AT) is inher- ing pathways, which cause deficiencies in multiple cell lin- ited as an autosomal-recessive disease that results from eages. CD4+ lymphocytes, B lymphocytes, myeloid cells, mutations in the ataxia-telangiectasia mutated (ATM) gene keratinocytes, osteoclasts, and monocytes are all decreased. at chromosome 11q22–23. The ATM protein is involved Autosomal-dominant HIES additionally results in connec- in signaling pathways involved in cellular responses to tive tissue, skeletal, vascular, and dental abnormalities. It is DNA damage. The disease is characterized by progressive the most frequent cause of immune deficiency with HIES.46 neurologic disease, immune dysfunction, and predisposi- STAT3 is necessary for normal IL-17 cytokine signaling and tion to malignancy. Affected individuals are ataxic and in regulation. Deficiencies in IL-17 due to STAT3 mutations childhood or adolescence develop telangiectasias. Ataxia lead to decreased differentiation of T helper 17 (Th17) lym- refers to uncoordinated movements, such as when walking. phocytes and result in a primary immunodeficiency. Both A telangiectasia is a vascular lesion formed by a dilation of a T and NK lymphocytes are decreased. IL-17 also controls group of blood vessels that appears as a red line or r adiating eosinophilotropic chemokines that drive eosinophil pro- limbs (spider). Telangiectasias appear as tiny, red, spider- duction. Patients have an eosinophilia, numerous tissue like veins. Chronic respiratory infection and lymphoid abnormalities including facial, dental, skeletal, and soft malignancy are the most common causes of death. tissue abnormalities, short limbs, and osteoporosis and are Patients with AT have a defect in cell-mediated immu- susceptible to repeated infections. Recurrent infections are nity with hypoplasia or dysplasia of the thymus gland and characterized by increased levels of serum IgM but deficien- depletion of T lymphocyte areas in the lymph nodes. B lym- cies in the other immunoglobulins, especially IgG. phocyte function also is abnormal with impaired isotype Autosomal-recessive HIES is caused by homozygous switching (Chapter 8). Lymphocytopenia with a reversed null mutations in TYK2 (tyrosine kinase 2) or DOCK8 CD4:CD8 ratio exists along with deficiencies in IgA, IgG, (dedicator of cytokinesis 8) genes.47 This mutation impairs and IgE. IgM levels are increased. Cytogenetic analysis T lymphocyte function, which results in a lack of specific reveals excessive chromosome breakage and rearrange- antibody formation, recurrent viral infections, candidiasis, ments in cultured cells and clonal abnormalities of chro- mosome 7 or 14.45 dermatitis, and the increased serum IgE. Both X-linked and autosomal recessive mutations have Hyper IgE Syndrome The hyper IgE syndromes (HIES) been identified that affect T lymphocyte activation because (originally named Job's syndrome) are rare, primary of abnormalities in cytokine signaling. Patients have quali- immunodeficiency diseases that involve both humoral tative defects in both T and B lymphocytes with recur- and cellular immunity deficiencies. They are characterized rent infections and extreme susceptibility to opportunistic by elevated serum IgE, eczema, recurrent staphylococcal infections.46 Summary Lymphocytes mount an immune response in inflammatory response to eliminate foreign antigens, the cell itself also or infectious states. In these states, the lymphocyte morphol- can serve as the site of infection for some viruses that use ogy often includes various reactive forms, immunoblasts, lymphocyte membrane receptors to attach to and invade and possibly plasmacytoid cells. Lymphocytosis is defined the cell. as an absolute lymphocyte count exceeding 4 * 103/mcL, Infectious mononucleosis is a common self-limiting whereas lymphocytopenia is defined as a total lymphocyte lymphoproliferative disorder caused by infection with EBV. count less than 1.0 * 103/mcL. Quantitative changes (either Laboratory diagnosis of this disorder includes serologic increased or decreased) in the total lymphocyte concentra- testing for heterophile antibodies and identification of reac- tion occur. Although the lymphocyte induces an immune tive lymphocytes on Romanowsky-stained blood smears. 490 Chapter 22 Lymphocytopenia, on the other hand, typically reflects currently is no cure, but extensive research is investigating depletion of T cells, the most abundant lymphocyte sub- a neutralizing immune response that may lead to vaccine type in the blood. The most common cause of such T cell development. depletion is a viral infection, such as infection with the Congenital qualitative disorders of lymphocytes human immunodeficiency virus, although other causes include a wide variety of immunodeficiency disorders. exist. AIDS is a disease caused by infection of the CD4+ T Either the T or B lymphocyte or both can be affected. These lymphocyte with the retrovirus HIV-1. The virus suppresses are usually very serious disorders in which most affected the immune response by replicating within and destroying individuals succumb to the disease in childhood. As molec- CD4+ lymphocytes. CD4+ lymphocyte levels and viral loads ular testing improves detection, more genetic mutations will are used to monitor disease progression in HIV-infected elucidate the origins of these primary immunodeficiencies patients. Antiretroviral treatments in combination with pro- so that treatment options can be explored. Bone marrow/ tease inhibitors slow the progression of the disease. There stem cell transplant is the only treatment in many cases. Review Questions Level I 5. A 29-year-old female patient was seen for abdomi- nal fullness, fever, and lethargy. Her total leuko- 1. Advanced HIV disease (AHD) patients have numer- cyte count was 11.8 * 103/mcL. The differential ous infections because the HIV virus has infected: showed 32% neutrophils, 65% lymphocytes, and (Objective 9) 3% monocytes. A few of her lymphocytes were a. neutrophils small round lymphocytes with a high N:C ratio and b. CD8+ lymphocytes overall size similar to the red cells, but many of her lymphocytes appeared large with basophilic cyto- c. CD4+ lymphocytes plasm and increased N:C ratio. This patient should d. B-lymphocytes be considered for: (Objective 5) 2. According to the CDC definition, a patient is consid- a. a urinary tract infection ered to have progressed from AHD to AIDS when the b. hepatitis following is detected: (Objective 10) c. bacterial meningitis a. a leukopenia d. Streptococcus pneumonia b. a CD4:CD8 ratio of 2:1 6. The morphology of lymphocytes found in infectious c. the absolute CD4+ count of less than 200/mcL mononucleosis is described as: (Objective 2) d. the absolute CD4+ count of less than 500/mcL a. segmented nucleus with pinkish-tan cytoplasm 3. Which of the following infectious agents is associated b. horseshoe-shaped nucleus with blue/gray with a newborn found to have jaundice and micro- cytoplasm cephaly with an enlarged spleen and decreased plate- let and red cell counts? (Objective 7) c. irregular shaped nucleus with spreading, deep blue cytoplasm a. Cytomegalovirus d. small cells with condensed chromatin and deep b. Epstein-Barr virus blue cytoplasm c. Toxoplasmosis gondii 7. A 2-year-old child has a total leukocyte count of d. Bordetella pertussis 10 * 103/mcL and 60% lymphocytes. Which of the 4. A teenager is seen at the clinic for a sore throat and following best describes the child’s blood count? enlarged cervical lymph nodes. He has reactive, atypi- (Objectives 4, 8) cal lymphocytes along with an elevated CRP and a. Absolute lymphocytosis elevated aminotransferases. He should also be tested b. Relative lymphocytosis for: (Objectives 1, 3) c. Normal lymphocyte count for the age given a. pertussis antitoxin antibodies d. Absolute lymphocytopenia b. HIV antibodies c. Toxoplasmosis antibodies d. heterophile antibodies Nonmalignant Lymphocyte Disorders 491 c. decreased reactive B lymphocytes with decreased Use this case study for questions 8–10. cytotoxic T lymphocytes A 19-year-old female college student went to student d. increased reactive B lymphocytes with increased health complaining of lethargy and a sore throat for cytotoxic T lymphocytes the past two weeks. Physical exam shows pharyn- 3. Epstein-Barr virus infects lymphocytes by attaching to gitis, lymphadenopathy, and splenomegaly with a which receptor? (Objective 2) total leukocyte count of 11 * 103/mcL and 70% lym- phocytes (50% of lymphs are reactive). a. CD4 b. CD8 c. CD21 d. TCR 8. She probably has: (Objective 1) a. HIV b. hepatitis Use this case study to answer questions 4 and 5. c. X-linked SCIDS A young male sees his physician for an ongoing d. infectious mononucleosis cough. He is found to have a total leukocyte count of 9. Her absolute lymphocyte count is: (Objective 4) 23 * 103/mcL with a differential of 22% neutrophils, a. 10 * 103/mcL 72% lymphocytes, 5% monocytes, and 1% eosino- b. 11 * 103/mL phils. The majority of the lymphocytes appear small with dense chromatin. c. 5.5 * 103/MCL d. 7.7 * 103/MCL 10. The best description of this patient’s leukocyte count is a(n): (Objectives 4, 8) 4. Which of the following describes his blood count? (Objective 7) a. relative lymphocytopenia a. Absolute neutrophilia b. relative neutrophilia b. Relative neutrophilia c. absolute lymphocytosis c. Absolute lymphocytosis d. absolute neutrophilia d. Relative eosinophilia Level II 5. The most likely cause of illness is: (Objectives 5, 7, 10) 1. A 17-year-old female patient is seen at the clinic because she has a lingering cough. She had a total leukocytosis a. Epstein-Barr virus of 23 * 103/mcL with 79% normal- looking lympho- b. Bordetella pertussis cytes. She should be screened for: (Objective 6) c. Cytomegalovirus a. pertussis antitoxin antibodies d. hepatitis b. VCA IgM 6. After taking care of a pair of cats, a 25-year-old male c. EBNA saw his physician for fever of unknown origin. He d. CMV antibodies had a total leukocyte count of 16 * 103/mcL with 78% lymphocytes, many showing reactivity, baso- 2. A patient with lethargy, pharyngitis, and lymphade- philia, and increased cytoplasm. His monospot test nopathy is found to have many lymphocytes that are was negative. What other testing should be consid- large with spreading, irregular cytoplasm and some ered? (Objective 4) with deep basophilia. A study of the lymphyocyte subsets would be expected to show: (Objective 2) a. Screening for Toxoplasmosis gondii a. decreased reactive B lymphocytes with increased b. Screening for HIV cytotoxic T lymphocytes c. Detection of pertussis antitoxin antibodies b. increased reactive B lymphocytes with decreased d. Detection of hepatitis B antigen cytotoxic T lymphocytes 492 Chapter 22 c. AHD Use this case study for questions 7–9. d. AIDS A 39-year-old male went to the clinic with com- 8. Which lymphocytes are periodically counted to moni- plaints of nagging cough, weight loss, diarrhea, and tor the disease? (Objective 8) low-grade temperature. Results of physical exami- nation showed lymphadenopathy, congested lungs, a. Infected B lymphocytes and increased heart rate. Slight splenomegaly and b. CD4+T lymphocytes hepatomegaly were noted. A CBC and flow cytome- c. CD8+T lymphocytes try studies were ordered. Histologic examination of d. Natural killer lymphocytes sputum with Gomori’s methenamine silver nitrate stain revealed Pneumocystis carinii. 9. Which laboratory test will be used to follow this patient’s disease? (Objective 8) Laboratory data Differential WBC 2.8 * 103/mcL Segmented 68% a. HIV-1 viral load count neutrophils b. Throat swab RBC 3.86 * 106/mcL Lymphocytes 21% c. Serologic test for heterophile antibody count d. PCR for genetic mutations Hb 13.6 g/dL Monocytes 10% 10. Immune deficiency associated with HIES is attributed (136 g/L) to the abnormal signaling or loss of cytokine: Hct 0.41 L/L Eosinophils 1% (Objective 11) Platelet 104 * 103/mcL Positive for HIV-1 a. IL-2 count antibodies b. IL-7 c. IL-15 d. IL-17 7. What clinical condition does this patient have? (Objectives 8, 10) a. Congenital immune deficiency b. Infectious mononucleosis References 1. Zhang, X., Dawson, C. W., He, Z., & Huang, P. (2012). Immune 7. Balfour, H. H. Jr., Dunmire, S. K., & Hogquist, K. A. 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D., Jing, H., & Zhang, Q. (2011). Dock8 deficiency. Infectious Disease Journal, 32(Suppl. 2), i-KK4. Annals of the New York Academy of Science, 1246, 26–33. This page intentionally left blank Section Five Neoplastic Hematologic Disorders 495 Chapter 23 Introduction to Hematopoietic Neoplasms Gideon H. Labiner, MS Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define and differentiate the terms neoplasm 5. Compare and contrast the laboratory and malignant and identify hematopoi- findings of the acute and chronic leukemias etic disorders that can be included in each and myeloid and lymphoid leukemias. category. 6. Define and differentiate proto-oncogenes 2. Compare and contrast the general and oncogenes and summarize their characteristics of the myelodysplastic relationship to neoplastic processes. syndromes (MDSs), myeloproliferative 7. Correlate patient age to the overall incidence neoplasms (MPNs), and acute and chronic of the hematopoietic neoplasms. leukemias. 8. Explain the usefulness of immunological 3. Describe the World Health Organization (WHO) techniques, chromosome analysis, and classification system used for MDSs, MPNs, molecular genetic analysis, in the diagnosis the leukemias, and lymphoid neoplasms. and prognosis of hematopoietic neoplasms. 4. List the various laboratory methods used 9. State the prognosis and survival rates of the to classify the hematopoietic neoplasms. hematopoietic neoplasms. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Explain how proto-oncogenes are activated and 2. Describe the effects of radiation on the inci- the role that oncogenes and tumor suppressor dence of leukemia. genes and their protein products play in the 3. Differentiate between the acute and etiology of hematopoietic neoplasms. chronic myeloid and lymphoid leukemias 496 Introduction to Hematopoietic Neoplasms 497 based on their clinical and hematologic leukemia (AML, ALL), and mature lymphoid findings. neoplasms, and justify a patient diagnosis 4. Reconcile the use of chemotherapy for based on these features. treatment of leukemia. 8. Select laboratory procedures appropriate 5. Compare and contrast treatment options for confirming cell lineage and diagnosis for the hematopoietic neoplasms including in hematopoietic neoplasms. possible complications. 9. Define epigenetics and explain its role 6. Name the leukemogenic factors of leukemia in cancer. and propose how each contributes to the 10. Define cancer stem cell and explain its development of leukemia. similarities to the hematopoietic stem 7. Compare and contrast laboratory features cell (HSC). of MDS, MPN, acute myeloid and lymphoid Chapter Outline Objectives—Level I and Level II 496 Laboratory Evaluation 506 Key Terms 497 Hematopoietic Neoplasm Classification 506 Background Basics 498 Laboratory Procedures for Diagnosing and Case Study 498 Classifying Neoplasms 507 Overview 498 Prognosis and Treatment of Neoplastic Disorders 509 Introduction 498 Summary 512 Pathophysiology 499 Review Questions 513 Epidemiology 505 References 515 Clinical Presentation 505 Key Terms Acute lymphocytic leukemia (ALL) Cytogenetic remission Mature neoplasm Acute myeloid leukemia (AML) Epigenetics Minimum residual disease (MRD) Auer rod French-American-British (FAB) Molecular remission Benign Hematologic remission Myelodysplastic syndrome (MDS) Cancer-initiating cell Induction therapy Myeloproliferative neoplasm Cancer stem cell Leukemia (MPN) Chronic lymphocytic leukemia Leukemic stem cell (LSC) Neoplasm (CLL) Leukemogenesis Oncogenes Chronic myelogenous leukemia Lymphoma Precursor neoplasm (CML) Maintenance chemotherapy Proto-oncogene Consolidation therapy Malignant Tumor suppressor gene 498 Chapter 23 Background Basics The information in this chapter serves as a general introduc- Level II tion to the hematopoietic neoplasms (Chapters 24–28). To • Describe the actions of cytokines, cytokine recep- maximize your learning experience, you should review the tors, signaling pathways, and transcription factors. following concepts before starting this unit of study: (Chapters 2, 4) • Summarize the cell cycle and identify factors that Level I affect it. (Chapter 2) • Summarize the origin and differentiation of the • Describe the normal structure and function of the hematopoietic cells. (Chapters 2, 4) bone marrow, spleen, and lymph nodes. (Chapter 3) • Describe the morphologic characteristics of the hematopoietic cells. (Chapters 5, 7, 8) syndromes. The acute myeloid neoplasms are described in CASE STUDY Chapter 26, the acute lymphoid neoplasms are described We refer to this case study throughout the chapter. in Chapter 27, and Chapter 28 includes a discussion of the Agnes, a 72-year-old female, saw her physician mature lymphoid neoplasms. The chapters on flow cytom- for a persistent cough and fatigue. She had always etry (Chapter 40), chromosome analysis (Chapter 41), and been in good health and played golf regularly. molecular analysis (Chapter 42), are referenced frequently Upon examination, she was noted to be pale and and provide additional information on laboratory proce- had slight splenomegaly. A CBC revealed the WBC dures used in diagnosis, prognosis, and treatment. count was

83.9 * 109/L. Consider possible explanations for this test result and which reflex tests should be performed. Introduction Neoplasm (tumor) means “new growth.” Neoplasms arise as a consequence of dysregulated proliferation of a single Overview transformed cell. Genetic mutations in the transformed cell reduce or eliminate the cell’s dependence on external cyto- This chapter is the first in a section that discusses the hema- kines (growth factors) to regulate proliferation. topoietic neoplasms. It provides a general introduction to Neoplasms are either benign or malignant. Benign neoplastic hematologic disorders that are discussed in neoplasms are formed from highly organized, differenti- detail in Chapters 24–28. It describes oncogenesis includ- ated cells and do not spread or invade surrounding tis- ing how oncogenes are activated and their association with sue. Malignancy means “deadly” or “having the potential hematopoietic disease. This is followed by a description of for producing death.” A malignant neoplasm is a clone how neoplasms are classified and characterized according of abnormal, anaplastic, proliferating cells, which often to cell lineage, degree of cell differentiation, morphology, have the potential to metastasize (spread). Only malignant cytochemistry, immunophenotype, and genetic abnormali- tumors are correctly referred to as cancer. Although cancer ties. The etiology and pathophysiology of the leukemias are is actually a malignancy of epithelial tissue, common use examined using general clinical and laboratory findings. of the term includes all malignant neoplasms. A benign Finally, prognosis and treatment modalities for the disor- neoplasm can be premalignant and progress with further ders are considered. genetic mutations to a malignant neoplasm (Figure 23-1). A comprehensive table that includes the diagnosis, Malignant neoplasms of the bone marrow are collec- immunophenotype, chromosome translocation, and geno- tively known as leukemia. These neoplasms are grouped typic finding is included in Appendix B. This table should according to cell lineage as lymphoid, myeloid, and histio- be referred to when reading the chapters in this section. cytic/dendritic cell.1 The myeloid and lymphoid neoplasms This introductory chapter should be read before progress- are further subgrouped as precursor neoplasms (acute) and ing to the other chapters in this section. The other chapters mature neoplasms (usually chronic). A comparison of clini- are organized according to the World Health Organization cal and laboratory findings associated with these precursor (WHO) classification of hematopoietic and lymphopoi- and mature subgroups is listed in Table 23-1. etic neoplasms. Chapter 24 includes the myeloprolifera- Precursor neoplasms are characterized by genetic tive neoplasms. Chapter 25 includes the myelodysplastic mutations that promote proliferation and survival and/or Introduction to Hematopoietic Neoplasms 499 MPN Reactive leukocytosis MDS Acute Leukemoid reaction CLL Leukemia Plasma cell neoplasms Figure 23.1 The spectrum of hematopoietic proliferation ranges from benign to malignant outcomes. Benign myeloid and lymphoid proliferation is usually a reactive process (reactive leukocytosis; leukemoid reaction). Mature myeloid and lymphoid neoplasms include myeloproliferative neoplasms (MPNs), and myelodysplastic syndromes (MDSs), chronic lymphocytic leukemia (CLL), and plasma cell neoplasms. These neoplasms are derived from a mutated precursor cell that divides incessantly but has Figure 23.2 Acute myeloid leukemia. Note the large number some capacity to mature. Acute leukemia is a malignant precursor of myeloblasts with no mature granulocytes present (Wright stain, neoplasm characterized by unregulated cell proliferation and a 1000* magnification, peripheral blood). block in maturation. Mature neoplasms can progress with additional genetic mutations and terminate in acute leukemia. Abnormal proliferation of lymphoid cells sometimes occurs within the lymphatic tissue or lymph nodes. These that block differentiation into mature hematopoietic cells. solid tumors are referred to as lymphoma. If the lymphoma Thus, in acute leukemia (AL), a precursor neoplasm, there affects the bone marrow and lymphoma cells are found in is a gap in the normal maturation pyramid of cells and the peripheral circulation, the leukemic phase of lymphoma presents with many blasts (Figure 23-2) and some mature is present. forms of the affected lineage, but a decrease in intermedi- Failure of normal hematopoiesis is the most serious ate maturational stages. The mature cells seen in the bone consequence of malignant neoplasms. As the neoplastic marrow and peripheral blood arise from proliferation of cell population increases, the concentration of normal cells the residual normal hematopoietic stem cells (HSCs) in decreases, resulting in the inevitable cytopenias of normal the bone marrow. The excess of blasts primarily reflects blood cells (Figure 23-4). If the neoplasm is not treated, the proliferation of the abnormal malignant clone that fails to patient usually succumbs to infections secondary to granu- undergo maturation. locytopenia or bleeding secondary to thrombocytopenia. Mature neoplasms are also characterized by an increase in proliferation and survival of neoplastic cells, but in con- trast to precursor neoplasms, maturation of progenitor cells is nearly normal. The result is leukocytosis with the pre- Pathophysiology dominant leukemic cells being amitotic, mature or partially Cancer is a disease of gene mutations. Hematopoietic mature cells, with normal function. Thus, the bone marrow neoplasms are believed to occur as the result of a somatic and peripheral blood usually show an increase in concen- mutation(s) of a single hematopoietic stem or progenitor tration of cells at all maturation stages (Figure 23-3). The cell.2 Evidence for the clonal evolution of neoplastic cells mature myeloid neoplasms include subgroups in which the comes from cytogenetic studies. More than 50% of individu- cells exhibit dysplastic features (myelodysplasia). als with leukemia show an acquired abnormal karyotype in Table 23.1 Comparison of Precursor and Mature Hematopoietic Neoplasms Precursor (acute neoplasm) Mature (chronic neoplasm) Age All ages Adults Clinical onset Sudden Insidious Course of disease (untreated) Weeks–months Months–years Predominant cell Blasts, some mature forms Mature forms Anemia Mild–severe Mild Thrombocytopenia Mild–severe Mild WBC Variable Increased 500 Chapter 23 normal HSCs (Chapter 3). While more restricted progeni- tor cells, such as committed lymphoid progenitor (CLP) or committed myeloid progenitor (CMP) cells, do not normally have self-renewal capacity, they may acquire mutation(s) that reactivate the self-renewal program(s) and that allow them to become cancer-initiating cells.2 Many of the signal- ing pathways that have been shown to regulate normal stem cell development also play a role in cancer cell proliferation.2,3 The cancer-initiating cell gives rise to the cancer stem cell that divides incessantly to generate a tumor of “identi- cal” sibling cells or clones and sustains malignant growth. Most cancers are not truly clonal but consist of hetero- geneous cell populations. Only small subsets of cells are capable of extensive proliferation (cancer stem cells) within Figure 23.3 Chronic myelogenous leukemia. Note the a tumor. Identification of the hematopoietic form of a can- large number of granulocytic cells in various stages of maturation cer stem cell, or leukemic stem cell (LSC), for each type of including blasts, metamyelocyte, bands, and segmented neutrophils leukemia has become a major focus of research because the (Wright stain, 1000* magnification, peripheral blood). LSC is responsible for propagating the leukemia. hematopoietic cells whereas other somatic cells are normal. Using cytogenetic markers, normal and malignant cells can Checkpoint 23.1 be demonstrated to populate the marrow simultaneously. In A patient has 50% monoblasts in the bone marrow. Which untreated leukemias and during relapse, the leukemic cells precursor cell could be the cancer-initiating cell? dominate, whereas during remission, usually only normal cells can be detected. The mutations leading to malignant transformation of the cancer stem cell or LSC often are associated with a chro- Cancer Stem Cells mosome alteration that is observed as an abnormal karyotype Hematopoietic neoplasms arise when a normal hematopoi- when studying cells in mitosis (Chapter 41). When chromo- etic precursor cell acquires a cancer-initiating mutation. The some studies are normal, aberrations in DNA at the molecu- cell in which this genetic mutation(s) occurs is termed the lar level may be found (Chapter 42). In either case, genomic cell of origin or cancer-initiating cell (Figure 23-5). The can- changes in the cancer cells lead to a survival and/or prolifera- cer-initiating cell can be a hematopoietic stem cell (HSC) or tion advantage over normal cells and to the neoplastic expan- more differentiated progenitor cells.2,3 A hallmark of hemato- sion of the affected cancer stem cell and its progeny. In acute logic malignancies is the capacity for unlimited self-renewal leukemia, this unregulated proliferation is accompanied by of the cancer-initiating cell, which is also a characteristic of an arrest in maturation at the blast cell stage (Figure 23-5). Checkpoint 23.2 Neoplastic cells in peripheral blood A 62-year-old male presents with an elevated leukocyte count, mild anemia, and a slightly decreased platelet count. His phy- sician suspects leukemia. Explain why the erythrocytes and platelets are affected. Molecular Basis of Cancer The cancer cell genotype is generally maintained (stably inherited) during cell division. This implies that the tumor Normal cells in peripheral blood cell DNA determines the disease phenotype. Time ONCOGENES Figure 23.4 When inoculated into animals, certain viruses are capable of Clonal expansion of neoplastic cells in the bone marrow over a period of time leads to a decrease in the causing tumors. These tumor viruses carry discrete genetic concentration of normal cells in both the bone marrow and elements, oncogenes, which are responsible for inducing peripheral blood. malignant cell transformation. The proteins encoded by Bone marrow stem cell pool Introduction to Hematopoietic Neoplasms 501 Development of a Development of a normal hematopoietic neoplasm hematopoietic cell HSCs Leukemic Progenitors mutation(s) Blasts AL Figure 23.5 A hematopoietic neoplasm (left) is derived from a single cancer-initiating cell. The leukemic mutation(s) (black arrows) that transform a normal hematopoietic precursor cell to a cancer-initiating cell can occur at the hematopoietic stem cell (HSC) or more committed progenitor. If the cancer-initiating cell is a committed progenitor, then the mutations must include the capacity for self-renewal (blue arrows). If the mutation also can result in a block to terminal differentiation—the resulting malignancy will be an acute leukemia (AL). Residual “normal” HSCs and committed progenitors in the marrow will still be capable of producing mature cells (right). the oncogenes play important roles in the cell cycle, such • Growth factor receptors When activated to an onco- as initiation of DNA replication and transcriptional control gene, the mutated receptors are capable of trigger- of genes. Importantly, many viral oncogenes have normal ing growth-promoting signals, even in the absence of counterparts in the human genome, called proto-oncogenes. ligand (cytokine) binding. Thus, the human genome carries genes with the potential • Signal transducers The normal function of these pro- to dramatically alter cell growth and to cause malignancy teins (the largest class of proto-oncogenes) is to pass when altered or activated to an oncogene.4 receptor signals to downstream targets. Many of these One of the defining features of cancer cells is their abil- proto-oncogenes encode protein-tyrosine kinases found ity to proliferate under conditions in which normal cells on the inner surface of the membrane. Often the onco- do not.5 The proteins encoded by proto-oncogenes func- genic form of these genes produces signaling molecules tion in the signaling pathways by which cells receive and that exist in a constantly activated state in the absence execute growth instructions. The mutations that convert of growth factor/receptor interaction and signaling. proto-oncogenes to oncogenes (referred to as oncogene acti- • Transcription factors These proteins bind DNA and vation) are often either structural mutations resulting in the function to control the expression of cellular genes continuous (constitutive) activity of a protein without an required for proliferation. incoming signal or mutations in gene regulation that lead to the production of a protein at the wrong place or time. The Thus, proto-oncogenes are genes that regulate the ini- result in either case is a persistent internal growth signal tiation of DNA replication, cell division, the commitment to that is uncoupled from environmental controls. It is pos- cellular differentiation, and/or apoptosis (Chapter 2). Their sible that any gene playing a key role in cellular growth activation to an oncogene disrupts the growth-control appa- can become an oncogene if mutated in an appropriate way. ratus of the cell. Proto-oncogene activation occurs by one of In general, the proto-oncogenes that have been identi- three genetic mechanisms: mutation, gene rearrangement, fied serve one of the following functions in normal growth or gene amplification. The result is either (1) a qualitative control4 (Chapters 2, 4): change in function of the gene’s protein product, resulting in enhanced activity, (2) a protein that is no longer subject to • Growth factors These molecules provide the signals to the control of regulatory factors, or (3)

a quantitative change grow and when activated to an “oncogene” result in an (increased production) of an otherwise normal protein. autocrine growth stimulation. 502 Chapter 23 TUMOR SUPPRESSOR GENES in most cells. Acquired mutations of RB (i.e., nonfamilial) Cancer is widely accepted to be a multi-hit phenomenon, are found in about 25% of sporadic cancers and have been resulting from several independent genetic alterations observed in various hematopoietic neoplasms. occurring sequentially within a single cell. Specific tumor Inactivation of the p53 gene, also a tumor suppressor suppressor genes function to inhibit cell growth in normal gene, is seen in more than half of all human cancers, mak- cells. Thus, in addition to oncogene activation that results ing it the most common genetic defect detectable in human in growth-promoting activity, tumor cells often have inac- tumors.8,9 Interestingly, a damaged p53 gene can be inherited tivating mutations of growth-suppressing genes that can (like familial retinoblastoma) resulting in Li-Fraumeni syn- also contribute to tumor development. Mutations in tumor drome and an inherited susceptibility to a variety of cancers suppressor genes behave differently from oncogene muta- including hematopoietic neoplasms.10,11 In affected individ- tions (Table 23-2). Oncogene mutations tend to be activating uals, 50% develop cancer by age 30 and 90% by age 70. The mutations, which functionally are dominant to wild-type function of p53 in cell cycle regulation (Chapter 2) is to block (nonmutated) gene products; they produce proliferation cell cycle progression in the event of damaged DNA or to signals even when a single copy of the oncogene is pres- trigger apoptosis if the damaged DNA cannot be repaired. ent. Tumor suppressor mutations, on the other hand, are The p53 protein is a major component of the body’s anti- recessive, loss-of-function mutations. Mutation in one gene tumor army, serving as a “molecular policeman” monitor- copy usually has no effect as long as a sufficient amount of ing the integrity of the genome. Loss of function of the p53 normally functioning wild-type protein remains. However, gene facilitates tumor formation by allowing damaged cells mutations in both copies of a tumor suppressor gene lead to to proceed through the cell cycle and continue to replicate. complete loss of its normal, growth-inhibitory effect. EPIGENETICS Understanding the function of tumor suppressor genes In addition to the mutations observed in various oncogenes has been greatly aided by studies of rare cancers that run and tumor suppressor genes, epigenetic alterations may also in families in which affected family members appear to be observed in a malignancy.12,13 Epigenetics is defined as inherit susceptibility to and develop certain kinds of tumors heritable changes in gene expression not caused by changes at rates much higher than the normal population. The first in DNA sequence, but rather to the ability of a gene to be of these familial cancers to be explained at the molecular expressed. Epigenetic changes are stable from one cell gen- level was the inherited susceptibility to retinoblastoma (a eration to the next after each mitotic event. These changes tumor of the eye) in certain families.6,7 Although retinoblas- play an important role in normal development and differ- toma can occur sporadically, about one-third of the cases entiation and are associated with “silencing” genes and occur in related siblings, suggesting an inherited suscep- chromatin condensation into heterochromatin (Chapter 2). tibility to the disease. The development of retinoblastoma Major epigenetic changes include DNA methylation of CpG requires two mutations that inactivate both of the RB loci dinucleotides, histone acetylation/deacetylation reactions, on each of the chromosomes 13. In the familial form of the and microRNAs. disease, the affected children inherit one mutant RB allele and one normal allele. Retinoblastoma (or another malig- DNA METHYLATION nancy) develops when acquired mutations eliminate the The methylation pattern of a gene determines its expression. function of the remaining normal (wild-type) allele. Thus, In a malignancy, there can be regions of the genome that are the RB gene acts as a tumor suppressor gene that normally inappropriately demethylated whereas other regions may functions to arrest excessive growth of cells. As is typically be aberrantly methylated. The list of genes that acquire true of tumor suppressor genes, one RB copy is sufficient hypermethylation of CpGs in their promoter regions and to keep growth in check, but loss of both copies eliminates contribute to tumorigenesis is growing.14 This hypermeth- the tumor suppressor function, and a tumor develops. The ylation is associated with transcriptional silencing of these protein product of the RB gene (Rb protein) is not specific genes and is the explanation for one of the most common to retinal tissue but serves as a universal cell cycle brake causes of loss of function of key tumor suppressor genes. Table 23.2 Properties of Oncogenes and Tumor Suppressor Genes Property Oncogenes Tumor Suppressor Gene Nature of mutation Dominant (one mutated allele displays the phenotype) Recessive (both alleles must be mutated to display the phenotype) Gain of function Loss of function Inherited mutant allele Never observed Rare—basis for inherited predisposition in cancers Somatic mutations in cancers Yes Yes Introduction to Hematopoietic Neoplasms 503 HISTONE DEACETYLATION Amplification Modifications to histone proteins are also seen in malignan- Translocation Retroviral insertion cies. Histones that are acetylated allow for chromatin to be loosely compacted and therefore allows for gene expres- + sion. Hypo-acetylated histones bind tightly to the phos- G1 phate backbone of DNA and help maintain chromatin in Cyclin D1 p16 Rb an inactive state, thus silencing gene expression. Various Cdk4 types of malignant cells use enzymes called histone deacety- S – – lases (HDACs) to hypo-acetylate key histone proteins. This + results in the silencing of tumor suppressor genes and, Mutation Mutation Deletion Amplification Deletion likewise, cell proliferation rather than differentiation. One Methylation Mutation Tumor viruses approach to treating cancer patients involves demethylat- ing agents or HDAC inhibitors to reverse epigenetic changes Figure 23.6 Alterations of the G1 checkpoint that can lead associated with certain types of cancer.14 to malignancy. Loss-of-function alterations in cell cycle negative regulators (i.e., the tumor suppressor gene products p16 or Rb) can MICRORNAs contribute to uncontrolled proliferation. Similarly, gain-of-function MicroRNAs (miRNAs) are a class of non-protein coding, mutations of positive regulators of proliferation can contribute to single-stranded RNA molecules (about 22 nucleotides in uncontrolled proliferation (i.e., the proto-oncogene gene products length) that help to control cell differentiation through post- Cyclin D, Cdk4). = inhibition of the pathway; + = an alteration that transcriptional degradation of their target mRNAs (Chap- increases activity of the indicated proteins; - = an alteration that decreases activity of the indicated proteins. ter 2). Aberrant expression of miRNAs leads to malignant transformation in a variety of cancers including hematologic malignancies by two general mechanisms: (1) increased in cyclin-dependent kinases can also lead to a loss of cell miRNA expression can lead to the degradation of tumor cycle regulation. For instance, mutations that lead to overex- suppressor gene transcripts, and (2) decreased miRNA expres- pression of Cdk4 (a kinase regulated by cyclin D) have been sion can lead to the over-expression of proto- oncogenes. reported in a number of human tumors and contributes MiRNAs are increasingly being used as biomarkers of diag- to the excessive growth characteristics of those diseases. nosis, prognosis, and prediction of response to therapy.15 Therefore, the p16-cyclin D-Rb pathway, which controls the G1 checkpoint in cell cycle regulation, is believed to play a pivotal role in tumorigenesis (Figure 23-6). Some investiga- Checkpoint 23.3 tors have proposed that a mutation involving at least one Mutations in proto-oncogenes predisposing to malignancy are member of this checkpoint must occur in order for a malig- nant phenotype to be established.16 said to be dominant mutations, whereas mutations in tumor suppressor genes are said to behave as recessive mutations, requiring loss of both alleles. Explain this difference in behavior of the gene products. Checkpoint 23.4 A cell contains a mutation that blocks expression of p16. What is the effect (if any) on the daughter cells produced? CELL CYCLE CHECKPOINTS AND CANCER A common feature of many cancer cells is the loss of regu- lation of cell cycle checkpoints (Chapter 2), either by over- APOPTOSIS AND CANCER expression of positive regulators (for example, cyclins and The accumulation of an excess number of cells, a characteris- Cdks) or the loss of function of negative regulators (the Cdk tic of malignancies, can result from increased cell proliferation inhibitors, p53, or Rb).15 Cyclin D, cyclin E, and cyclin A are (see previous discussion of cell cycle checkpoints and can- overexpressed in a variety of human cancers and function cer) and/or to decreased cell death (apoptosis).5 Thus, muta- as oncogenes in their mutated configuration. Often, spe- tions of genes important in regulating apoptosis have also cific chromosomal translocations activate the expression been identified as oncogenes and tumor suppressor genes. of a cyclin gene by placing it under the influence of other These include loss-of-function mutations that initiate apop- transcriptional control elements. For example, the t(11;14) tosis such as p53, Bax, and other pro-apoptotic Bcl-2 family translocation seen in some B-lymphocyte malignancies members as well as overexpression of antiapoptotic proteins places the cyclin D gene under regulatory control of the such as Bcl-2 and other Bcl-2 family members that function immunoglobulin heavy chain locus, resulting in activation to inhibit apoptosis17 (Chapter 2). Bcl-2 is overexpressed of cyclin D expression at inappropriate times during a cell’s in most cases of B-cell follicular lymphoma, many cases life. Thus, the result of the t(11;14) translocation is an onco- of B-cell chronic lymphocytic leukemia (CLL), and some genic form of cyclin D (Chapter 41). Furthermore, mutations cases of acute myeloid l eukemia (AML)18 (Chapters 26–28). # 504 Chapter 23 Mutations in Bax (resulting in loss of pro-apoptotic func- Radiation, some chemicals, and drugs can cause chro- tion) have been reported in about 20% of leukemic cell lines. mosome mutations. Ionizing radiation has long been rec- The result is production of cells with an extended life span, ognized as capable of inducing leukemia, which is evident increased proliferation capacity, and diminished cell death. from observations of human exposure to radiation from nuclear reactions, therapeutic radiation, and occupational Leukemogenesis exposure to radiation. An increase in leukemia has been observed after treatment with alkylating agents and other From studies on laboratory animals, several factors have chemotherapeutic drugs used in treatment of many kinds been suggested as playing roles in leukemogenesis: (1) of malignancy. The only chemical that is specifically impli- genetic susceptibility, (2) somatic mutation, (3) viral infec- cated in causing leukemia other than those used as medica- tion, and (4) immunologic dysfunction (Table 23-3). tions is benzene. GENETIC SUSCEPTIBILITY The outcome of chromosome breaks and translocations Strong evidence suggests that hereditary factors and abnormal can lead to the activation of oncogenes and ultimately result genetic material have important leukemogenic effects. A num- in the aberrant expression of the protein product (Appen- ber of individuals who have congenital abnormalities associ- dix B). Furthermore, breaks and translocations within gene ated with karyotypic abnormalities have a markedly increased sequences can produce hybrid (fusion) genes and a new pro- risk of developing acute leukemia. Each of these genetic events tein product. For example, the balanced translocation between has the potential to activate proto-oncogenes or eliminate the chromosomes 9 and 22 in chronic myelogenous leukemia function of a tumor suppressor gene. The best known of the (CML) results in the formation of a BCR-ABL fusion gene that genetic abnormalities associated with leukemia is Down syn- encodes an abnormal tyrosine kinase (TK) protein. The TK is drome in which the extra chromosome 21 may participate in constitutively activated to transduce signals of cell survival, translocations.19,20 Various other congenital disorders also are proliferation, and resistance to apoptosis. Consequently, the associated with an increased risk for leukemia.21,22 cell containing the translocation is able to persist under condi- tions that would not normally support its survival. SOMATIC MUTATION A somatic cell mutation is an acquired change in the genetic VIRAL INFECTION material of cells other than those involved in reproduction. Retroviruses have been shown to cause leukemia in labo- Mutations in the chromosome near proto-oncogenes likely ratory animals, and a few malignancies can be traced to a play a role in neoplasm development. More than 50% of viral infection of cells in humans.23 Retroviruses contain a patients with leukemia have acquired abnormal karyotypes reverse transcriptase that

allows them to produce a DNA and cytogenetic studies have revealed specific, consistent copy of the viral RNA core. The DNA can then be copied mutations in certain subgroups of hematopoietic neoplasms.23 to produce more viral cores or can be incorporated into the host cell’s nuclear DNA. The strongest support for the exis- tence of a leukemogenic virus in humans comes from the Table 23.3 Factors that May Play a Role in isolation of several human retroviruses known as human Leukemogenesis T-cell leukemia/lymphoma virus (HTLV-I, II, V) and human Factor Example immunodeficiency virus (HIV-1) from cell lines of patients with mature T-cell malignancies.24 Exactly how viruses Genetic susceptibility Down syndrome induce leukemia is unclear, but the incorporation of the Fanconi anemia viral genome into host DNA is suspected to lead to activa- Kleinfelter syndrome tion of proto-oncogenes. Bloom syndrome Wiskott-Aldrich syndrome IMMUNOLOGIC DYSFUNCTION Diamond-Blackfan syndrome An increased incidence of lymphocytic leukemia has been observed in both congenital and acquired immunologic Xeroderma pigmentosum disorders. These disorders include the hereditary immu- Li-Fraumeni syndrome nologic diseases Wiskott-Aldrich syndrome, Bruton type Somatic mutation Radiation X-linked agammaglobulinemia, and ataxia telangiectasia. Chemicals An association between long-term treatment of patients Drugs with immunosuppressive drugs (e.g., renal transplant) Viral infection Retroviruses—HTLV-I, II, V, HIV-1 and leukemia also has been observed. Possibly, a break- Immunologic disorders Wiskott-Aldrich syndrome down in the cell-mediated immunologic self-surveillance Bruton type X-linked agammaglobulinemia system and/or deficient production of antibodies against Ataxia telangiectasia foreign antigens leads to the emergence and survival of Immunosuppressive therapy neoplastic cells. Introduction to Hematopoietic Neoplasms 505 MISCELLANEOUS FACTORS Although acute leukemia occurs at all ages, peak incidence Certain hematologic diseases appear to pose a leukemo- occurs in the first decade, particularly from the ages of 2 to genic risk. Leukemia development in some patients appears 5 followed by a decreasing incidence in the second and third to be related to the treatment used for the primary disease, decade. Thereafter, the incidence begins to increase, rising but in others, no such relationship can be found. The high- steeply after age 50. The cellular type of leukemia occur- est incidence of acute leukemia is found in individuals with ring at these peak periods differs significantly. Most child- other neoplastic bone marrow disorders, such as myelo- hood acute leukemias are of the lymphoid type, whereas proliferative neoplasms (MPNs) and myelodysplastic those occurring in adults are typically myeloid. Chronic syndrome (MDS),25,26,27 prompting some hematologists to leukemias are rare in children. Chronic myelogenous leu- use the words preleukemia or premalignant for these disor- kemia occurs most often in young to middle-aged adults, ders. Additional genetic mutations (or epigenetic altera- and chronic lymphocytic leukemia is diagnosed primarily tions) occur as the preleukemic disease progresses to the in older adults. MPN and MDS occur most often in middle- malignancy, leukemia. Other hematopoietic diseases with age to older adults. an increased incidence of leukemia include paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia, and multiple myeloma. Interestingly, all of these hematologic disorders are considered stem cell disorders in which the Clinical Presentation primary hematologic defect lies in the myeloid or lymphoid Failure of the normal triad of hematopoiesis is the most progenitor cells or in the pluripotential stem cells. serious consequence of hematopoietic neoplasms and the No single factor is responsible for the genetic altera- most frequent symptoms are related to erythrocytopenia, tions that result in hematopoietic neoplasms; rather, a vari- thrombocytopenia, and/or neutropenia. The major clini- ety of etiologic factors produce the malignancy.28,29,30 The cal problems are anemia, infection, and bleeding episodes. cause varies from patient to patient, and some individuals Weight loss and bone pain that results from marrow expan- are more susceptible than others to malignancies. sion are also common complaints. Physical examination can show hepatosplenomegaly and, occasionally, lymph- adenopathy. Organomegaly is more common in mature Checkpoint 23.5 cell leukemias (chronic leukemias) than in the precursor Does a 3-year-old child with Down syndrome have an increased (acute) forms. risk of developing leukemia? Why or why not? Although the disease originates in the bone marrow, neoplastic cells can infiltrate any tissue of the body, especially the spleen, liver, lymph nodes, central nervous Epidemiology system, and skin. The lesions produced vary from rashes to tumors. Skin infiltration is most commonly found in The Leukemia and Lymphoma Society predicted that in acute myeloid leukemia (AML), particularly those with 2016, about 171,000 new cases of leukemia, lymphoma, and a monocytic component. Central nervous system (CNS) myeloma would have been reported, accounting for 10% of involvement is common in acute lymphoblastic leukemia new cases of cancer in the United States.31 About 31% more (ALL) of childhood.32 Chloromas, which are green tumor males are living with leukemia than females. masses of immature leukocytes, are associated with AML Approximately 50% of all leukemias are diagnosed and CML and are usually found in bone but can be found as acute. Although some difference in the incidence of the throughout the body. The green color, which fades to a dirty acute leukemias exists between countries and regions of yellow after exposure to air, is responsible for the descrip- countries, the differences are not great. tive name given to this unique clinical finding. Presumably, Of particular interest are the incidence and morpho- the green color results from the myeloperoxidase content of logic variation of leukemia among age groups (Table 23-4). the malignant cells. Table 23.4 Age Groups Typically Found in Acute (Precursor) and Chronic (Mature) Leukemias Neoplasm Age Acute lymphocytic leukemia (ALL) Children 2–5 years old Chronic lymphocyte leukemia (CLL) Adults more than 50 years old Acute myeloid leukemia (AML) Adults Chronic myeloid leukemia (CML) and other myelopro-liferative neoplasms Adults 506 Chapter 23 Laboratory Evaluation Maturation abnormalities are commonly present in all three cell lines. Megaloblastoid erythropoiesis can be Cell counts and morphology are variable in hematopoietic prominent but is unresponsive to vitamin B12 or folic acid neoplasms (Table 23-5). A normocytic (occasionally macro- treatment. cytic) normochromic anemia is often present at diagnosis. Because of the intense increase in cell turnover, other If not present initially, anemia invariably develops during laboratory tests reflecting cell destruction could be abnor- progression of the disease. mal. An increase in uric acid, which is a normal product of The platelet count varies. Thrombocytopenia is usu- nucleic acid metabolism, is a consistent finding in all types ally present at diagnosis in precursor neoplasms (acute of leukemia. The rate of excretion can increase to 50 times leukemia). Thrombocytosis is a common initial finding in normal. Serum lactic dehydrogenase (LD) levels appear to some of the mature neoplasms but can decrease with dis- correlate closely with the concentration of leukemic cells. ease progression. Platelet morphology and function may Isoenzyme studies reveal that the LD is derived from imma- be abnormal. Large hypogranular platelet forms are com- ture leukocyte precursors. Muramidase (lysozyme) is a mon, and circulating micromegakaryocytes occasionally lysosomal enzyme present in monocytes and granulocytes. are present. The serum and urine muramidase concentration in leuke- The leukocyte count can be normal, increased, or mia is highly variable and is related to the cellular type. decreased. More than 50% of patients with AML do not The highest concentrations are found in neoplasms with a have a significant leukocytosis at diagnosis. However, if monocytic component. left untreated, leukocytosis eventually develops. On the other hand, in the mature myeloproliferative and lym- phoproliferative neoplasms, leukocytosis at diagnosis is a prominent finding. Normal or decreased leukocyte Checkpoint 23.6 counts are typical in myelodysplastic syndrome (MDS). Why is finding Auer rods an important factor in the diagnosis Regardless of the leukocyte count, an increase in imma- of leukemia? ture precursors is found in most cases. Blasts are especially prominent in the precursor leukemias. Unique pink-stain- ing granular inclusions called Auer rods can be found in the blast cells and promyelocytes of some acute myeloid Hematopoietic Neoplasm leukemias. These Auer rods are believed to be formed from fused primary granules. When AML is suspected, finding Classification Auer rods can help to establish the diagnosis because they Classifications of hematopoietic neoplasms are considered are not found in ALL. to be important for three reasons: The bone marrow is hypercellular but occasionally is 1. They provide clinicians and researchers a way to study, normocellular or hypocellular. Reticulin is increased, often select, and compare various therapeutic regimens. worsening with disease progression. Blasts are usually increased. A minimum of 20% blasts is recommended for a 2. They provide a system for diagnosis using clearly defined diagnosis of AML. The cutoff of less than 20% blasts is used clinical features and laboratory findings. to differentiate the mature myeloid neoplasms (MPN and 3. They permit meaningful associations of genetic abnor- MDS) from precusor myeloid neoplasms. malities with pathogenesis of neoplastic disease. Table 23.5 Characteristic Hematologic Findings in Hematopoietic Neoplasms Neoplasm Leukocytes Platelets Bone Marrow Other Acute leukemia Normal or increased; blasts Decreased Hypercellular, more than 20% Auer rods in AML present blasts MDS Normal or decreased; blasts can Variable Hypercellular, occasionally hypo- Dysplastic be present cellular, less than 20% blasts MPN (includes chronic Increased; shift to left but predomi- Usually increased Hypercellular, occasionally hypo- myelogenous leukemia (CML) nance of mature forms cellular, less than 20% blasts MDS/MPN Increased blasts and other imma- Usually Hypercellular, less than 20% blasts Dysplastic ture forms decreased CLL Increased mature lymphocytes; Variable Hypercellular, less than 20% blasts Smudge cells; less than neutropenia 10% prolymphocytes MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; CLL, chronic lymphocytic leukemia Introduction to Hematopoietic Neoplasms 507 Before 2001, hematopoietic neoplasm classification was primarily based on cell lineage as determined by the CASE STUDY (continued from page 498) morphology and cytochemistry of the neoplastic cells. The CBC results on Agnes were: The system was known as the FAB (French-American- WBC 83.9 * 103/mcL Differential British) classification system.33 Later, immunopheno- RBC 3.15 * 106/mcL Segs 12% typing was added to the schema. More recently, genetic Hb 9.5 g/dL (95 g/L) Lymphs 88% features, prior therapy, and a history of myelodysplasia Hct 29% (0.29 L/L) are recognized to have a significant impact on the clini- Platelets 130 * 103 cal behavior of the hematopoietic neoplasms. Thus, in /mcL 2001, the Society for Hematopathology and the Euro- 1. Given Agnes’s laboratory results, would this pean Association of Hematopathologists developed the most likely be considered an acute or chronic World Health Organization (WHO) classification to bet- leukemia? Explain. ter define the hematopoietic neoplasms; this classifica- 2. What group of leukemia (cell lineage) is sug- tion was updated in 2008 and again in 2016.34,35,36,37 This gested by Agnes’s blood cell differential results? classification uses morphologic, immunophenotype, and molecular analyses to determine cell lineage and degree 3. What would you expect the blast count in the of maturation (Table 23-6). These analyses also help to bone marrow to be? determine whether cell proliferation is effective or ineffec- tive and to detect whether cells are cytologically normal or dysplastic. Clinical features such as prior therapy, age, and history of MDS are correlated with genetic findings, morphology, and immunophenotype to define subgroups Laboratory Procedures for of myeloid and lymphoid neoplasms.35,36,37,38 Cytogenetic and molecular genetic studies are necessary to categorize Diagnosing and Classifying those neoplasms that are genetically defined, to establish a baseline, to assess genetic evolution and progression, Neoplasms and to monitor response of the disease to therapy. In some Initial evaluation of patients with a hematopoietic neoplasm cases, molecular analysis can detect gene mutations when should include collection of peripheral blood and bone mar- the cytogenetic profile is normal. These studies that define row specimens prior to therapy. Bone marrow should include cell lineage, degree of differentiation, and dysplasia and aspirate as well as biopsy specimens. Morphology and blast detect genetic abnormalities should be performed at ini- count are performed on Romanowsky-stained smears. In tial evaluation.36 addition to obtaining a blast count, these cells must be differ- The classification according to lineage of the neoplastic entiated. Differentiation of leukemic blasts allows classifica- cells includes three groups: myeloid, lymphoid, and his- tion of acute leukemia into cell lineage (lymphoid or myeloid) tiocytic/dendritic (Table 23-6). Mast cells are derived from and various subtypes (Table 23-7). In the acute leukemias, hematopoietic progenitor cells and possess myeloid cell identification of the cell lineage of leukemic blasts is often characteristics. Thus, mast cell disease can also be consid- difficult when performed by morphology alone unless Auer ered a myeloid disorder.

rods are present. However, the distinction of cell lineage is Table 23.6 WHO Classification of Hematopoietic, Lymphopoietic, and Histiocytic/Dendritic Neoplasms Classification Diseases Included Hematopoietic Myeloproliferative neoplasms (MPNs) Myeloid and lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB, FGFR1, or PCM1-JAK2 Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) Myelodysplastic syndromes (MDSs) Acute myeloid leukemia (AML) and related neoplasms Acute leukemia of ambiguous lineage Lymphopoietic Precursor lymphoid neoplasms (T and B lymphoblastic leukemia/lymphoma) Mature B-cell neoplasms (including chronic lymphocytic leukemia, CLL) Mature T-cell and NK-cell neoplasms Hodgkin lymphoma Posttransplant lymphoproliferative disorders (PTLD) Histocytic and Dendritic Histiocytic and dendritic cell neoplasms Subgroups not included. See Appendix C for classification that includes subgroups. 508 Chapter 23 Table 23.7 Comparison of Acute Lymphoblastic Leukemia (ALL) and Acute Myeloid Leukemia (AML)a ALL AML Age Common in children Common in adults Hematologic presentation Anemia, neutropenia, thrombocytopenia, lympho- Anemia, neutropenia, thrombocytopenia, myeloblasts, blasts, prolymphocytes promyelocytes Prominent cell morphology Small-to-medium lymphoblasts, fine chromatin with Medium-to-large myeloblasts with distinct nucleoli, fine nuclear scanty to abundant cytoplasm, indistinct nucleoli chromatin, and abundant basophilic cytoplasm, possible Auer rods Cytochemistry PAS positive, peroxidase negative, SBB negative, TdT PAS negative, peroxidase positive, SBB positive, TdT negative or positive, chloroacetate esterase (specific) negative positive, chloroacetate esterase positive Immunophenotype B Cell: CD19, CD20, cytoplasmic CD79a and CD22, CD34, HLA-DR, CD117, CD13, CD33, CD15 CD10, CD19 (can also have CD13 and CD33); T cell: CD1a, CD2, CD3, CD4, CD5, CD7, CD8 PAS = periodic acid Schiff; SBB = Sudan black B; TdT = terminal deoxynucleotidyl transferase a Genetic abnormalities are also used to differentiate ALL and AML. important for selecting the appropriate therapy. Morphology, that of megaloblastic cells. As a result, although the nucleus immunologic marker analysis (immunophenotype), and/or appears very immature, the cytoplasm of leukemic blasts may genetic studies can differentiate blasts. Cytogenetic analysis contain constituents normally present only in more mature on a bone marrow specimen should be performed, and addi- cells or can lack one or more constituents expressed by their tional molecular genetic studies should be guided by clinical, normal counterparts. In addition, abnormal accumulation laboratory, and morphologic information. and distribution of cellular metabolites is possible. Distinction of the mature neoplasms is not as difficult as in acute leukemias because the maturation of the neo- Immunologic Analysis plastic cell in mature neoplasms is not blocked, and the cell matures into recognizable blood cells. Immunophenotyping Immunologic analysis is based on identifying specific is not usually necessary in classification of MDS and MPN, membrane antigens (surface markers) characteristically but genetic studies are helpful in subgrouping. All data are found on a particular cell lineage. Immunologic techniques correlated into a report that gives the WHO diagnosis.35,36 with monoclonal antibodies are widely used to identify cell membrane antigens on a variety of cells (Chapter 40). The Cytochemical Analysis development of a large number of monoclonal antibodies that react with surface antigens on normal and neoplastic In hematology, cytochemistry refers to in vitro staining of cells cells and the technical advances in flow cytometry have that allows microscopic examination of the cells’ chemical greatly enhanced the ability to define leukemic cell lineage, composition. Cell morphology is not significantly altered in the the stage of cell development, and clonality. By utilizing a staining process. Most cellular cytochemical markers represent panel of monoclonal antibodies, a more complete picture of organelle-associated enzymes and other proteins. The cells are the cells’ lineage can be determined. incubated with substrates that react with specific cellular con- Immunophenotyping plays a central role in diagnosing stituents. If the specific constituent is present in the cell, the hematopoietic and lymphoid neoplasms. It includes identify- constituent’s reaction with the substrate is confirmed by the ing the lineage of blasts in the blast phase of CML, subtyping formation of a colored product. The stained cells are examined the myeloid leukemias, and identifying the lineage of those and evaluated on smears with a light microscope, although leukemias that lack specific morphologic characteristics such electron microscopy occasionally is necessary to identify very as minimally differentiated AML from ALL and subtyping weak reactions at the subcellular level. The results of these the lymphoid neoplasms as T or B cell. Immunologic patterns cellular reactions in normal and disease states are well estab- can help plan molecular analysis because some immunophe- lished. Cytochemistry is particularly helpful in differentiating notypes are associated with particular genetic mutations. The the lymphoid or myeloid lineage of the blasts in precursor identification of cell antigens can also help direct treatment leukemias when morphologic identification on Romanowsky- because some antigens are targeted for specific therapy (e.g., stained smears is difficult (Table 23-7). Cytochemistry is also rituximab, an anti-CD20 drug). If marrow cell suspensions helpful in subgrouping AML and related precursor neoplasms. are not available, immunophenotyping can be done using Staining procedures are described in Chapter 37. immunohistochemical analysis on bone marrow biopsy It is important to remember that the blast cells seen in specimens. See Chapter 40 for a more thorough discussion leukemias are neoplastic and can therefore differ from normal of monoclonal antibodies and their use in the identification blasts in both morphology and metabolic activity. Leukemic of neoplastic hematopoietic disorders. Appendix B includes cells often display nuclear/cytoplasmic asynchrony similar to the antigenic patterns in hematologic neoplasms. Introduction to Hematopoietic Neoplasms 509 Genetic Analysis Molecular analysis also is helpful in providing clues to the pathogenesis of hematopoietic neoplasms. For Genetic analysis includes cytogenetics to determine the cell example, the specific t(15;17) (q22;q12) mutation found in karyotype and molecular methods to identify specific gene acute promyelocytic leukemia produces an abnormal form mutations—both of which can be key to differentiate and of the nuclear hormone receptor, retinoic acid receptor@a define some neoplasms. In some cases, the karyotype will (RAR@a). The abnormal receptor is involved in the matu- appear normal but mutations can be found using molecu- ration blockade seen in the neoplastic promyelocytic cells. lar techniques such as polymerase chain reaction (PCR), When patients who possess this mutation are treated with DNA sequencing, and fluorescent in situ hybridization retinoic acid derivatives, the cells are induced to differenti- (FISH) (Chapter 42). ate into mature granulocytes. CYTOGENETICS Advances in cytogenetics have enabled cytogeneticists to Checkpoint 23.7 identify characteristic nonrandom abnormal karyotypes in A patient has 35% blasts in the bone marrow. They do not the majority of the acute leukemias and in some MPNs and show any specific morphologic characteristics that will allow MDSs.39 Some specific chromosome changes are consistently them to be classified according to cell lineage. What are the associated with a particular neoplastic subgroup and thus next steps that the clinical laboratory scientist should take with are helpful in diagnosis. For example, the t(15;17)(q22;q12) is this specimen? diagnostic of acute promyelocytic leukemia, and the Philadel- phia chromosome characterized by t(9;22)(q34;q11.2) confirms a clinical diagnosis of CML. In the lymphoid leukemias, non- Prognosis and Treatment random chromosome changes are often associated with either the B or T lymphocyte lineage and provide important prog- of Neoplastic Disorders nostic as well as diagnostic information. Chromosomal rear- rangements and their accompanying molecular abnormalities Studies of the hematopoietic neoplasms that have eluci- can identify distinct clinical groups with a predictable clinical dated the pathogenesis of these disorders have led to new course and response to specific therapy. In addition to helping treatment modalities that significantly changed the prog- physicians evaluate their patients, cytogenetic studies provide nosis for patients. new insights into the pathogenesis of neoplastic diseases. When cytogenetic abnormalities are present before therapy, their presence or absence can be used to identify Prognosis remission, relapse, and minimal residual disease after ther- Before the 1960s, a patient diagnosed with acute leukemia apy. If the cytogenetic abnormality identified before therapy could expect to die within a few months. With new treat- is still present after therapy, it is evidence that neoplastic ment modalities, remission rates for both ALL and AML cells remain in the bone marrow. In some cases, additional have improved dramatically. Remission was defined origi- chromosome aberrations can be identified during the course nally as a period of time in which there were no clinical or of the disease or after a period of remission. The finding of hematologic signs of the disease. More recently with the var- additional chromosome aberrations is not usually a signal ious diagnostic and monitoring modalities available, several of disease progression. Thus, physicians may order multiple levels of remission can be defined. For treatment purposes, cytogenetic analyses (Chapter 41). complete remission (response) is defined as the total absence of disease according to the test used (e.g., hematologic ver- MOLECULAR ANALYSIS sus cytogenetic versus molecular). Hematologic remission Molecular genetic analysis, the process of using DNA tech- refers to the absence of neoplastic cells in the peripheral nology to identify genetic defects at the molecular level, blood and bone marrow and the return to normal levels of is being used increasingly as a diagnostic tool in studying hematologic parameters. Cytogenetic remission refers to the neoplasms. In some cases, the chromosome karyotype is absence of recognized cytogenetic abnormalities associated normal, but a genetic mutation can be identified. About 5% with a given neoplastic disease. Molecular remission refers of patients with CML do not show the typical Philadelphia to the absence of detectable molecular abnormalities using chromosome on karyotyping, but the BCR/ABL1 mutation PCR or related molecular technologies. can be identified by molecular techniques (Chapters 24 Sensitive molecular testing such as PCR can detect the and 42). When present, this helps establish or confirm the presence of less than 1 in 106 tumor cells (Chapter 42), whereas diagnosis. The JAK2 mutation, JAK2(V617F), plays a piv- the sensitivity of detection of malignant cells using cytoge- otal role in the pathogenesis of many BCR/ABL1–negative netics is much lower. Thus, a complete molecular response MPNs. Thus, diagnostic algorithms for MPN now consider is highly desirable and the most promising evidence that the the mutational status of JAK2. neoplasm has been eliminated. A combination of negative 510 Chapter 23 “traditional” tests (peripheral blood and bone marrow blast also kills many normal cells. Complications of traditional count and cytogenetics) and positive molecular tests (PCR/ therapy include bleeding because of decreased platelet FISH) is sometimes referred to as a state of minimum resid- counts, infections from suppression of granulocytes, and ual disease (MRD). The designation “partial response” is anemia because of erythrocyte suppression in the marrow. used when the relevant laboratory values have a significant Supplemental support with recombinant growth factors can decrease without achieving a total absence of disease. A “major sometimes be used to mitigate the cytopenias. response” can include either a complete or partial response. Most drugs used to treat leukemia are included in three Survival in acute leukemias varies with age and group— groups: antimetabolites, alkylating agents, and antibiotics. ALL or AML. Approximately 90% of children treated for ALL The antimetabolites are purine or pyrimidine antagonists, can be expected to enter a prolonged remission with an indef- which inhibit the synthesis of DNA. These drugs kill cells inite period of survival.39 The prognosis for ALL in adults is in cycle, affecting any rapidly dividing cell. In addition to not as good as that for children. Only 10–15% of patients typi- leukemic cells, the antimetabolites also kill cells lining the cally achieve long-term survival. Patients who receive bone gut, germinal epithelium of the hair follicles, and normal marrow or stem cell transplants, especially younger patients, hematopoietic cells. This leads to complications of gastro- have a better prognosis. Patients who had a previous MDS intestinal disturbances, loss of hair, and life-threatening or chronic MPN respond poorly to standard chemotherapy. cytopenias. The alkylating agents (chemical compounds Survival in the chronic neoplasms is longer. For CML containing alkyl groups) are not specific for cells in cycle patients receiving imatinib as initial therapy, overall survival but kill both resting and proliferating cells. These drugs at 60 months is 89%.40 After onset of blast crisis, survival is gen- attach to DNA molecules, interfering with DNA synthesis. erally only 1–2 months. Survival in CLL depends on its sever- As a class, they are mutagenic and carcinogenic; they frag- ity at diagnosis and ranges from 30 to more than 120 months. ment and clump chromosomes, inactivate DNA viruses, Patients with other MDS and MPN diseases usually survive and inhibit mitosis but not protein function. The side without treatment for a year or more and even

longer for some effects of these compounds include myelosuppression, subtypes. Prognosis for the lymphomas depends on the cell stomatitis, and nausea and vomiting. Antibiotics bind to type and can range from 6 months to 10 years or longer. both DNA and RNA molecules, interfering with cell repli- cation. Toxic effects of this therapy are similar to those of alkylating agents. Since the 1970s, various drug combinations have been CASE STUDY (continued from page 507) found to be more effective than single drug administration. 4. Would you expect Agnes to survive more than The drugs commonly used, and their modes of action are 3 years or succumb fairly quickly after treatment? included in Table 23-8. Therapy for most leukemias is divided into several phases. The induction therapy phase is designed to induce the disease into complete remission (i.e., eradicating the Treatment leukemic blast population). Once a complete remission has been achieved, it is often followed by a continuation Therapeutic success rates differ by disease and the patient’s of treatment, referred to as maintenance chemotherapy or condition at diagnosis. Often a complete hematologic or consolidation therapy. The purpose of maintenance ther- cytogenetic remission is achieved initially, only to be fol- apy is to eradicate any remaining leukemic cells. lowed by a return of the disease (relapse) after a period of The treatment regimen for AML and ALL is similar, time. Most currently used treatment regimens target the although the combination of antileukemic agents differs. In actively proliferating cancer cells and might not, in fact, be both diagnoses, the purpose of chemotherapy is the same: effective against the leukemic stem cells (LSCs) (also referred to eradicate the leukemic blasts. Central nervous system to as the cancer stem cells). Thus, the relapse seen in some (CNS) involvement is a common feature of ALL but not patients following a complete remission is likely because of of AML. Therefore, CNS prophylactic treatment (cranial the reemergence of the disease from a quiescent LSC, which irradiation and/or intrathecal chemotherapy) is part of the was not eliminated by the treatment regimen utilized. therapy regimen for ALL. CHEMOTHERAPY Permanent remission in CLL is rare. Treatment is Chemotherapy remains the treatment of choice for many conservative and usually reserved for patients with more leukemias. The goal of this type of therapy is to eradicate all aggressive forms of the disease. Treatment for MPN and malignant cells within the bone marrow, allowing repopu- MDS also is primarily supportive and designed to improve lation by residual normal hematopoietic precursors. The the quality of the patient’s life. Drugs designed to reverse problem with this type of therapy is that the drugs used epigenetic alterations in MDS recently have been intro- in treatment are not specific for leukemic cells. Treatment duced (see “Epigenetic Therapy”). Introduction to Hematopoietic Neoplasms 511 Table 23.8 Chemotherapeutic Agents Usually Used in Acute Leukemia (AL) Treatment Drug Class Action Doxorubicin Anthracycline antibiotic Inhibits DNA and RNA synthesis Daunorubicin Anthracycline antibiotic Inhibits DNA and RNA synthesis Idarubicin Anthracycline antibiotic Inhibits DNA and RNA synthesis 5-Azacytidine Pyrimidine antimetabolite Inhibits DNA and RNA synthesis 6-Thioguanine Purine antimetabolite Inhibits purine synthesis Methotrexate Folic acid antimetabolite Inhibits pyrimidine synthesis 6-Mercaptopurine Purine antimetabolite Inhibits pyrimidine synthesis Cytosine arabinoside Pyrimidine antimetabolite Inhibits DNA synthesis Prednisone Synthetic glucocorticoid Lyses lymphoblasts Vincristine Plant alkaloid Inhibits RNA synthesis and assembly of mitotic spindles Asparaginase Escherichia coli enzyme Depletes endogenous asparagines Cyclophosphamide Synthetic alkylating agent Cross-links DNA strands MOLECULAR-TARGETED THERAPY removing some of the patient’s marrow while the patient is As the genetic mysteries of hematologic neoplasms are in complete remission. The marrow specimen is then treated being resolved, novel therapies that target genetic muta- to remove any residual leukemic cells and cryopreserved. tions are used to silence the gene’s expression or that of Chemotherapy and/or radiotherapy are administered to the mutated protein or to reactivate silenced genes. The the patient to remove all traces of leukemia, and the treated therapies appear to be better tolerated than the traditional marrow is given back to the patient. chemotherapy regimens. Two targeted therapies are in cur- Autologous bone marrow transplantation has been rent use as first-line therapy: imatinib for CML and all-trans applied to patients in remission and to those in early relapse. retinoic acid (ATRA) for acute promyelocytic leukemia.41 Overall survival appears to be better in those transplanted Although hematopoietic stem cell transplantation had been during the first complete remission. Bone marrow transplan- recommended as first-line therapy for CML because it was tation appears to be successful in many cases; the number of the only treatment with the potential for cure, recent stud- patients who are undergoing this type of therapy is increasing. ies reveal that survival is superior in patients receiving drug treatment (interferon and/or imatinib).42 Rituximab, STEM CELL TRANSPLANTS cytolytic monoclonal anti-CD20 antibody, is a treatment Peripheral blood as well as bone marrow stem cells can directed against cells that have the CD20 protein (primarily be used to re-establish hematopoiesis in the marrow after B cells). These therapies are discussed in Chapters 24 and 26. intensive chemotherapy or radiotherapy in a process called stem cell transplantation. In this procedure, apheresis is used EPIGENETIC THERAPY to collect stem cells from the peripheral circulation, usu- With the recognition of the contribution of epigenetic altera- ally after they have been mobilized (induced to exit the tions to the neoplastic process, a number of drugs have been marrow) by cytokines such as G-CSF. These stem cells can developed and are in various stages of clinical trials. Both come from either the patient (autologous) or from a suit- demethylating agents (e.g., azacitidine) and histone deacet- able donor (allogeneic). People who receive allogeneic stem ylase inhibitors (HDAC-I) (e.g., valproic acid, phenylbutyr- cells are given drugs to prevent rejection. Production of ate) are being evaluated with promising early results.43,44,45 new blood cells usually becomes established in 10–21 days BONE MARROW TRANSPLANT following infusion of the stem cells. Stem cell transplanta- Bone marrow transplants have provided hope as a possible tion is still a fairly new and complex treatment for leuke- cure for hematopoietic disorders; the highest rate of success mia. A more thorough discussion of hematopoietic stem in transplant patients has occurred with those younger than cell transplantation can be found in Chapter 29. 40 years of age in a first remission with a closely matched donor. In this procedure, drugs and irradiation are used to induce remission and eradicate any evidence of leukemic CASE STUDY (continued from page 510) cells. Bone marrow from a suitable donor is then transplanted into the patient to supply a source of normal stem cells. 5. Is Agnes a suitable candidate for a bone marrow Autologous transplants have been used when a com- transplant? Why or why not? patible donor cannot be found. This procedure involves 512 Chapter 23 HEMATOPOIETIC GROWTH FACTORS In excessive amounts, the uric acid precipitates in renal Recombinant hematopoietic growth factors are used in sup- tubules, leading to renal failure (uric acid nephropathy). portive care of acute leukemia patients. Erythropoietin is Lysed cells also can release procoagulants into the vas- used in the treatment of chemotherapy-related anemia.46 cular system, leading to disseminated intravascular coagu- Granulocyte colony-stimulating factor (G-CSF) and gran- lation. In this case, the resulting decrease in platelets and ulocyte-macrophage colony-stimulating factor (GM-CSF) coagulation factors can lead to hemorrhage. This compli- aid in decreasing the incidence of severe neutropenia and cation is especially prevalent in acute promyelocytic leu- infection in patients receiving myelosuppressive chemo- kemia. The granules of the promyelocytes contain potent therapy.44 Interleukin-11 promotes the maturation of mega- activators of the coagulation factors (Chapter 33). karyocytes by stimulating stem cells and megakaryocyte Since chemotherapeutics destroy normal as well as leu- progenitor cells.47 Research in the area of identifying addi- kemic cells. The cytopenias that develop during aggressive tional hematopoietic growth-stimulating factors continues chemotherapy can lead to death from infection, bleeding, in the hope of accelerating hematopoietic recovery in che- or complications of anemia. To prevent these life-threaten- motherapy patients. ing episodes, the patient may need supportive treatment including transfusions with blood components and/or COMPLICATIONS OF TREATMENT Treatment for leukemia actually can aggravate the patient’s cytokines as well as antimicrobial therapy. clinical situation. Although uric acid levels are commonly elevated in leukemia because of the increase in cell turnover, the concentration of uric acid can drastically increase dur- CASE STUDY (continued from page 511) ing effective therapy because of the release of nucleic acids by lysed cells. Uric acid is a normal end product of nucleic 6. What types of treatment are available for Agnes? acid degradation and is excreted mainly by the kidney. Summary A neoplasm is an unregulated production of cells that can in the regulatory mechanisms of cell proliferation and dif- be either benign or malignant. The WHO classification of ferentiation. Epigenetic alterations also play an important neoplastic disorders of the bone marrow hematopoietic role in silencing tumor suppressor genes in neoplastic cells. cells groups the disorders according to lineage of the neo- Differentiation and classification of acute leukemias plastic cells: myeloid, lymphoid, and histiocytic/dendritic. depend on accurately identifying the blast cell popula- The myeloid and lymphoid neoplasms are subgrouped tion. Because the lineage of blast cells is sometimes diffi- as precursor and mature neoplasms. Morphology, genetic cult to differentiate using only morphologic characteristics, abnormalities, immunophenotype, and clinical features immunologic phenotyping using monoclonal antibodies define these major groups. The mature myeloid subgroups are employed routinely to help identify blast phenotypes include myeloproliferative neoplasms, myeloproliferative/ and stage of cell differentiation. Chromosome and molecu- myelodysplastic neoplasms, and myelodysplastic syn- lar genetic analyses are helpful because specific mutations dromes. The precursor myeloid group includes the acute often are associated with specific types of leukemias. myeloid leukemias. The lymphoid group includes precur- Hematologic findings of hematopoietic neoplasms sor B-cell, mature B-cell, precursor T-cell, and mature T-cell include anemia, thrombocytopenia (in acute leukemia and neoplasms. The mature lymphoid group includes CLL. The MDS), and often leukocytosis. A leukocytic shift to the left is precursor lymphoid group includes ALL. consistently found with a combination of blasts and mature Oncogenes are mutated forms of proto-oncogenes and cells in acute leukemia. In the chronic leukemias and other are known to contribute to tumorigenesis. Many proto- chronic neoplastic stem cell disorders, cells appear more on oncogene protein products are involved in regulating cell a continuum from immature to mature. Morphologic abnor- growth and include hematopoietic growth factors as well malities of neoplastic cells are not unusual. Auer rods can as their cellular receptors, signaling proteins, and tran- be found in blasts of AML. scription factors. Proto-oncogenes can be mutated to onco- Historically, hematopoietic neoplasms have been genes by mutagens, viruses, or chromosome breaks and treated using a combination of cytotoxic drugs (che- translocations. Oncogenes can cause production of abnor- motherapy). The goal is to induce remission by eradi- mal growth factors, abnormal amounts of growth factors, cating the leukemic cells. Hematopoietic stem cell abnormal growth factor receptors, or other abnormalities transplants can be used to restore the marrow after Introduction to Hematopoietic Neoplasms 513 intense chemotherapy or radiotherapy. New approaches factors is used in some cases to stimulate leukemic cells include drugs to reverse epigenetic modifications char- to proliferate, making them more susceptible to cytotoxic acteristic of neoplastic cells and other drugs targeted drugs. This therapy also has been used to decrease the at the exact molecular abnormality associated with the neutropenic, anemic, and thrombocytopenic period after neoplastic cell. Treatment with hematopoietic growth chemotherapy or radiotherapy. Review Questions Level I c. ALL 1. Auer rods are inclusions found in: (Objective 5) d. CLL a. myeloblasts 7. Acute lymphoblastic leukemia occurs with greatest b. lymphoblasts frequency in which age group? (Objective 7) c. erythrocytes a. 2–5 years d. prolymphocytes b. 10–15 years c. 20–30 years 2. Chromosome changes in hematologic neoplasms are: (Objective 8) d. More than 50 years a. used to aid in diagnosis 8. Chronic leukemias primarily affect: (Objectives 2, 7) b. present in AL but not MPN or MDS a. adults, progress slowly, and have mature cells c. associated with a poor outcome in circulation d. not usually present b. children, progress rapidly, and have mature cells in peripheral circulation 3. Genes that can cause tumors if activated are: c. young adults, progress slowly, and have immature (Objective 6) cells in peripheral circulation a. cancer genes d. all ages,

progress rapidly, and have immature cells b. proto-oncogenes in peripheral circulation c. preleukemia genes 9. The WHO system classifies a 19-year-old patient’s d. tumor suppressor genes bone marrow as a precursor B-cell leukemia. Which of the following best describes this leukemia? 4. A common characteristic of acute lymphoblastic (Objectives 2, 3, 5) leukemia is: (Objective 2) a. CLL a. BCR/ABL1 gene mutation b. CML b. bone pain c. AML c. many blast cells with Auer rods d. ALL d. leukocytopenia 10. Immunophenotyping of blast cells is important to: 5. Which of the following does the WHO c lassification (Objective 8) use to subgroup the ALL into T- and B-cell neoplasms? (Objective 3) a. identify the leukemia’s etiology a. Clinical presentation b. help determine cell lineage b. Immunophenotype c. determine whether cytogenetic analysis is necessary c. Cytogenetics d. replace the need for molecular analysis d. Morphology 11. The minimum percentage of blast cells required for a 6. A leukemia that shows a profusion of granulocytes at diagnosis of acute myeloid leukemia using the WHO all stages of development from blasts to segmented classification is: (Objective 3) neutrophils is: (Objectives 2, 5) a. 20% a. CML b. 30% b. AML 514 Chapter 23 c. 40% 4. A patient with a hypercellular, dysplastic bone mar- d. 50% row; anemia; and neutropenia in peripheral blood most likely has which of the following neoplasms? 12. Proteins encoded by proto-oncogenes serve to: (Objective 7) (Objective 6) a. MPN a. demethylate oncogenes b. AML b. inactivate tumor suppressor genes c. MDS c. cause unregulated cell proliferation d. ALL d. provide signaling pathways for normal cell growth control 5. A 52-year-old female was admitted to the hospital for minor elective surgery. Her pre-operative CBC was: 13. Which of these genes cause unregulated cell growth? WBC 49.4 * 103/mcL WBC Differential (Objective 6) RBC 4.50 * 106/mcL 3% segs a. Oncogenes Hb 12.7 g/dL 97% lymphs b. Antioncogenes Hct 38% PLT 213 * 103/mcL c. Proto-oncogenes d. Tumor suppressor genes What is the best explanation for the cause of this patient’s leukocytosis and lymphocytosis? Level II (Objectives 3, 7) 1. If the cell of origin for a neoplastic tumor undergoes a. CLL genetic mutations that gives the cell the ability to b. AML self-renew and blocks terminal differentiation, the c. ALL resulting malignancy will be: (Objective 11) d. CML a. acute leukemia b. chronic lymphocytic leukemia c. chronic myelogenous leukemia Use the following information to answer questions 6–8: d. myeloproliferative neoplasm A 43-year-old male had been working with the 2. Which of the following factors has not been proposed Peace Corps in Mexico for the past 10 years. His as playing a role in causing leukemia? (Objective 6) primary responsibilities were taking radiographs a. Benzene and doing laboratory work at the various clin- ics. He had been complaining of weakness and b. Radiation fatigue for about a month and had had several c. Living at high altitudes severe nosebleeds. His CBC upon admission to d. Chromosome translocations the hospital was: 3. A 3-year-old child with Down syndrome pres- WBC 25.6 * 103/mcL Differential ents with pallor, fatigue, lymphadenopathy, and RBC 3.11 * 106/mcL 75% blasts with Auer rods hepatosplenomegaly. The initial CBC results were: Hb 8.9 g/dL 20% lymphs (Objectives 3, 7) Hct 26.7% 3% monos WBC 18.7 * 103/mcL WBC Differential PLT 13 * 103/mcL 2% segs RBC 2.34 * 106/mcL 27% segs Hb 5.8 g/dL 10% lymphs Hct 17.4% 63% blasts PLT 130 * 103/mcL 6. Which leukemia is this patient most likely to have? These findings are suggestive of: (Objectives 3, 7) a. MDS a. ALL b. CLL b. AML c. AML c. CLL d. ALL d. CML Introduction to Hematopoietic Neoplasms 515 7. What would be the most likely causative agent of the 9. Heritable changes in gene expression not from leukemia? (Objective 2) changes in DNA are known as: (Objective 10) a. 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Chapter 24 Myeloproliferative Neoplasms Tim R. Randolph, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Identify the major lineages involved with 5. Describe and recognize the peripheral blood the various myeloproliferative neoplasms findings in CML patients and in those with (MPNs): chronic myeloid leukemia blast crisis. (CML), polycythemia vera (PV), essential 6. List and recognize laboratory findings thrombocythemia (ET), and primary typically associated with PMF. myelofibrosis (PMF). 7. Define criteria that indicate a 2. Recognize abnormal complete blood transformation of an MPN into a blast crisis. count (CBC) results that suggest an MPN. 8. Compare the lab findings in primary PV to 3. Explain the diagnostic chromosome those in secondary polycythemia. abnormality associated with CML and 9. Describe and recognize the characteristic its significance in acute lymphoblastic peripheral blood picture found in ET. leukemia (ALL). 10. Describe and recognize the peripheral 4. Identify the peak incidence of CML blood findings in patients with clonal according to age and sex distribution. hypereosinophila and mastocytosis. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Differentiate the subgroups of 2. Contrast laboratory findings in MPNs and myeloproliferative neoplasms (MPNs) from myelodysplastic syndromes (MDSs) as well other reactive and neoplastic diseases based as acute leukemia (AL). on laboratory findings in the peripheral 3. Describe molecular gene mutations in blood, bone marrow, and other diagnostic MPNs and hypothesize how these changes laboratory tests. in the pluripotential stem cell lead to MPNs 517 518 Chapter 24 and blast crisis (Philadelphia c hromosome, 8. Use laboratory results and clinical findings BCR/ABL1 rearrangement p53, p16, JAK2 to differentiate essential thrombocythemia (V617F), MPL (W515), CALR, FIP1L1/ from nonmalignant conditions that result in PDGFRA, ETV6-PDGFRB, and FGFR1 thrombocytosis. mutations, and expression of growth factor 9. Use the criteria suggested for diagnosing receptors). CML, chronic eosinophilic leukemia, not 4. Differentiate CML from a benign leukemoid otherwise specified (CEL-NOS), and chronic reaction using laboratory tests. neutrophilic leukemia (CNL) to distinguish 5. Assess the role of platelet-derived growth these disorders. factor (PDGF) in the fibrosis associated with 10. Differentiate unclassifiable myeloprolifera- primary myelofibrosis (PMF). tive neoplasms from other subgroups of 6. Assess laboratory results including the MPNs based on laboratory features. evaluation of peripheral blood and bone 11. Differentiate eosinophilic disorders using marrow smears using the diagnostic criteria laboratory results and genetic mutations. associated with PMF. 12. Describe systemic mastocytosis by giving 7. Compare clinical and laboratory findings, the criteria for its diagnosis. and interpret laboratory findings in 13. Using peripheral blood, bone marrow find- relative polycythemia and absolute ings, and patient medical history, determine polycythemia. the classification of MPNs. Chapter Outline Objectives—Level I and Level II 517 Chronic Myeloid Leukemia (CML) 522 Key Terms 518 Chronic Neutrophilic Leukemia (CNL) 530 Background Basics 519 Essential Thrombocythemia (ET) 531 Case Study 519 Polycythemia Vera (PV) 535 Overview 519 Primary Myelofibrosis (PMF) 540 Introduction 519 Myeloproliferative Neoplasm, PART I: Overview of Myeloproliferative Unclassifiable (MPN, U) 545 Neoplasms (MPNs) 520 Clonal Hypereosinophilia 545 Classification 520 Mast Cell Disease (Mastocytosis) 548 Pathophysiology 521 Summary 549 General Features 521 Review Questions 550 PART II: Subgroups of MPNs 522 References 552 Key Terms Blast crisis Janus kinase 2 (JAK2) gene Chronic eosinophilic leukemia, not otherwise Leukemic hiatus specified (CEL-NOS) Panmyelosis Chronic myeloid leukemia (CML) Plethora Chronic neutrophilic leukemia (CNL) Polycythemia vera (PV) Clonal hypereosinophilia Primary myelofibrosis (PMF) Essential thrombocythemia (ET) Spent phase Hypereosinophilic syndrome (HES) Systemic mastocytosis Myeloproliferative Neoplasms 519 Background Basics The information in this chapter builds on concepts Level II learned in previous chapters. To maximize your learn- • Describe the influence of growth factors on hemato- ing experience, you should review these related concepts poietic cell proliferation. (Chapter 4) before starting this unit of study: • Explain the evaluation of red cell mass based on changes in fluid volume. (Chapter 11) Level I • Explain the hemoglobin–oxygen dissociation curve. • Outline the cell cycle; describe stem cell differen- (Chapter 6) tiation and maturation for the various myeloid lin- eages. (Chapters 2, 3) • Explain how proto-oncogenes are activated and their role in the etiology of hematopoietic neoplasms. • Describe and recognize morphology for the various (Chapters 2, 23) stages of myeloid maturation. (Chapter 7) • Describe how cell marker panels can be used to dif- • Calculate red cell indices. (Chapters 10, 36) ferentiate hematopoietic neoplasms. (Chapters 23, 40) • Use appropriate morphologic terms to describe size • Discuss the value of cytogenetic studies in suspected and chromia of red cells in anemic states. (Chapters hematopoietic neoplasms. (Chapters 23, 41) 10, 11) • Explain how the use of molecular tests can assist in • Outline and explain the classification of hematopoi- diagnosing hematopoietic neoplasms. (Chapters 23, 42) etic neoplasms. (Chapter 23) • Summarize the relationship of oncogenes to the neo- plastic process (Chapter 23) laboratory evaluation, and therapy are included for each CASE STUDY subgroup. These are followed by an explanation of how to We refer to this case throughout the chapter. differentiate MPNs from diseases with similar laboratory Roger, a 52-year-old man with hyperuricemia, findings. This is a lengthy chapter, so it is divided into two went to the clinic for a follow-up evaluation for sections to help the reader organize the study of this group splenomegaly. His palpable spleen, noted 18 of important disorders. months earlier, had been gradually enlarging. He originally denied fatigue, fever, and discomfort. He was examined, and a CBC was ordered. The Introduction results revealed leukocytosis, thrombocytosis, and Hematopoiesis is a highly regulated process whereby a anemia. normal steady-state production of hematopoietic cells in Consider how reflex laboratory testing can the bone marrow and destruction of senescent cells in the assist in diagnosing this patient. tissues maintain a constant peripheral blood cell concentra- tion (Chapters 2, 3). Hematopoietic neoplasms result from acquired mutations in hematopoietic stem cells, allowing them to escape the regulatory controls for proliferation, Overview natural cell death (apoptosis), and/or differentiation in the bone marrow (Chapters 2, 23). This chapter presents the group of neoplastic but not truly In the former French-American-British (FAB) classification malignant disorders called myeloproliferative neoplasms system, neoplastic disorders of hematopoietic cells typically (MPNs). MPNs must be distinguished from other neoplas- were grouped into three main categories: m yeloproliferative tic and benign hematologic disorders for the physician disorders (MPDs), myelodysplastic states or syndromes to select appropriate therapy for the patient. The chapter (MDSs), and acute leukemias (ALs), including both myeloid begins with the classification, pathophysiology, and general and lymphoid subtypes. This classification system used the characteristics of MPNs, which provide the groundwork for blast count, lineage commitment, cell morphology, level of a more detailed explanation and description of each sub- differentiation of the neoplastic cells, cytochemistry, and group. More specific pathophysiology, clinical presentation, immunophenotyping to classify the diseases. 520 Chapter 24 The World Health Organization (WHO) developed a Hematologic classification is based on the most affected cell newer classification system for hematopoietic neoplasms line (Table 24-1). In the WHO classification,2 the spectrum that integrates cytogenetics, DNA analysis, and clinical of myeloproliferative neoplasms includes: features with cytochemistry and immunophenotyping. In • Chronic myeloid leukemia (CML), Philadelphia (Ph) 2016, the WHO published an update to this classification chromosome positive with BCR-ABL1 fusion gene pres- system. The WHO classification of myeloid disorders ent, t(9;22) (q34;q12) includes the same three categories as the original FAB system and the previous WHO system, AML, MPN, and • Chronic neutrophilic leukemia (CNL), BCR-ABL1- MDS, but adds additional categories to include: myeloid/ negative lymphoid neoplasms with eosinophilia and rearrange- • Essential thrombocythemia (ET) ment of PDGFRA, PDGFRB, or FGFR1; the myelodys- • Polycythemia vera (PV) plastic/myeloproliferative neoplasms (MDS/MPN); • Primary myelofibrosis (PMF, formerly known as myelo- blastic plasmacytoid dendritic cell neoplasm; and acute fibrosis with myeloid metaplasia [MMM] and chronic idio- leukemias of ambiguous lineage. In addition, each group pathic myelofibrosis [CIMF]) has s ubcategories based on clinical history, genetic and laboratory f indings. The WHO proposed MDS/MPN cat- • PMF, prefibrotic/early stage egory includes neoplasms that are proliferative like MPNs • PMF, overt fibrotic stage but have dysplastic features like MDS. MDS/MPNs are • Myeloproliferative neoplasm, unclassifiable (MPN, U) discussed in Chapter 25 with MDS. • Myeloid and lymphoid neoplasms associated with In the WHO classification, the percentage of blasts, eosinophilia and PDGFRA, PDGFRB, or FGFR1 degree of cell maturation, and dysplasia are critical assess- mutations ments initially used to classify the hematopoietic neo- • Chronic eosinophilic leukemia, not otherwise specified plasms. A blast count of more than 20% is necessary for (CEL-NOS) an AL diagnosis, whereas the nonacute leukemia disorders are diagnosed with less than 20% blasts. MPNs and MDS • Mastocytosis are characterized by an autonomous, neoplastic clonal Important criteria in the WHO classification system of proliferation of hematopoietic precursors. Increased num- the MPNs are (1) Ph chromosome must be present or if not bers of erythrocytes, leukocytes, and/or platelets in both the identified by routine cytogenetic analysis, the BCR/ABL1 bone marrow and peripheral blood generally distinguish fusion gene must be detected for a diagnosis of CML and (2) MPNs from MDS. MDS, on the other hand, is most com- the minimum bone marrow blast count to distinguish MPNs monly characterized by a hyperproliferative bone marrow, from AL is 20% (MPNs have less than 20% blasts). Because dysplastic maturation, and increased apoptosis that result identification of Ph is required for a diagnosis of CML, Ph- in peripheral blood cytopenias. Both MPNs and MDS have negative cases with myelodysplastic and myeloproliferative the potential of evolving into acute leukemia. features are included in the WHO MDS/MPN group and are called atypical CML (aCML). The classification of these PART I disorders is not always clear because of overlapping clinical OVERVIEW OF MYELOPROLIFERATIVE NEOPLASMS (MPNs) Table 24.1 Classification of Myeloproliferative Neoplasms (MPNs) by Predominance of Cell Types Classification Involved Cell Line Myeloproliferative Neoplasm Myeloid Chronic myeloid leukemia (CML) and chronic The term myeloproliferative syndrome, coined by William neutrophilic leukemia (CNL), and sometimes primary myelofibrosis (PMF) Dameshek in 1951, describes a group of disorders that Megakaryocytic Essential thrombocythemia (ET) result from an unchecked, autonomous clonal proliferation of cellular elements in the bone marrow.1 Under the WHO Erythroid Polycythemia vera (PV) classification system, myeloproliferative neoplasms are gen- Fibroblasta Primary myelofibrosis (PMF) erally characterized by panhypercellularity (panmyelosis) Eosinophil Chronic eosinophilic leukemia, not otherwise specified (CEL-NOS) of the bone marrow accompanied by erythrocytosis, granu- Mast cell Mastocytosis locytosis, and/or thrombocytosis in the peripheral blood. Variable Myeloproliferative neoplasm, unclassifiable Although trilineage cell involvement (erythrocytic, granu- (MPN, U) locytic, thrombocytic) is characteristic of MPNs, one cell a The fibroblast in PMF is not a part of the neoplastic process but is increased because of a line is usually more prominently affected than the others. reactive process. Myeloproliferative Neoplasms 521 Table 24.2 Differential Features of Myeloproliferative Neoplasms Parameter CML CNL ET PV PMF CEL-NOS Peripheral blood Hematocrit N or c N or c N or T c c c T T Leukocyte c c c c N or c N or c c or c c c Platelets c or T N or c c c c c N,c , T N Immature c c c Slight c (610%) Rare Absent or c c c N or c granulocytes LAP T c N or c N or c N,c , T - Philadelphia Present Absent Absent Absent Absent Absent chromosome Spleen size N or c c N or c c c c c N Bone marrow

fibrosis Absent or c Absent Absent or c Absent or c c c c Absent or c N, normal; T , decreased; c , c c , c c c , slight, moderate, marked increase, respectively; LAP, leukocyte alkaline phosphatase; CML, chronic myeloid leukemia; CNL, chronic neutrophilic leukemia; ET, essential thrombocythemia; PV, polycythemia vera; PMF, primary myelofibrosis; CEL-NOS, chronic eosinophilic leukemia, not otherwise specified. and laboratory features between subgroups at different times during the disease course (Table 24-2). General Features Myeloproliferative neoplasms usually occur in middle-aged and older adults (peak frequency in the fifth to seventh Pathophysiology decades of life). They are rare in children. The onset of the disease is gradual, evolving over months or even years. The The primary defect in MPNs appears to be in the pluripoten- clinical, laboratory, and morphologic findings frequently tial hematopoietic stem cell (HSC)3 (Figure 24-1). A clone of overlap among the specific MPN disorders. Clinical find- abnormal hematopoietic stem cells and their progeny pref- ings can include hemorrhage, thrombosis, infection, pallor, erentially expand until normal hematopoietic cell growth and weakness. Anemia or polycythemia, leukoerythroblas- is inhibited and the majority of functioning bone marrow tosis, leukocytosis, thrombocytosis with bizarre platelet is derived from the abnormal clone. Excessive prolifera- morphology, and bone marrow fibrosis are common labo- tion of hematopoietic cells occurs through various mecha- ratory findings that can occur in almost any of the MPN nisms that require multiple genetic mutations3 (Chapter 23). subtypes. Commitment, differentiation, and maturation of the abnormal clone are generally preserved, leading to increased numbers of mature cells in the peripheral blood, usually with one lin- eage (erythroid, myeloid, or megakaryocytic) predominating. Erythrocyte Finding uniform biochemical, cytogenetic, or molecu- lar genetic abnormalities in hematopoietic cells from the HSC Platelets bone marrow or peripheral blood of patients with MPNs suggests that these cells were derived from a single mutant MPN stem cell (i.e., clonal origin) (Chapter 23). The abnormali- Granulocytes ties are not present in other somatic cells, indicating that the mutations are acquired rather than inherited. The most consistent chromosome abnormality, the Ph chromosome, is found in all hematopoietic blood cells in patients with CML. The Janus kinase 2 (JAK2) gene, which codes for Monocyte Macrophage a tyrosine kinase involved in cell signaling, is mutated in almost all cases of PV and some cases of ET and PMF. These Figure 24.1 This schematic of hematopoietic cell mutations are discussed in more detail later in the chap- development shows that a mutation in the pluripotential ter. The application of molecular biology in the study of hematopoietic stem cell, HSC (CD34+ ), results in clonal proliferation hematopoietic neoplasms has been useful in identifying the of the progeny of that cell, all containing the mutation. Thus, all cell lines will be affected. The HSC is thought to be the original specific genetic abnormality at the DNA, mRNA, and pro- neoplastic cell in CML and other MPNs. Arrows indicate intermediate tein levels (Chapter 42). These studies support the clonality steps to mature cells that are not shown. MPN, myeloproliferative hypothesis of MPNs. neoplasm. 522 Chapter 24 When present, anemia is caused by ineffective eryth- Hepatosplenomegaly is a common clinical finding. Extra- ropoiesis, marrow fibrosis, and/or a shortened red cell medullary hematopoiesis can also occur in benign diseases survival because of splenic sequestration resulting in such as chronic hemolytic anemias, but the hematopoiesis splenomegaly. While decreased bone marrow iron can be in these disorders is confined to the erythrocyte lineage. In observed, it does not reflect true iron deficiency.4 For this contrast, all cell lineages are present in the extramedullary reason, serum ferritin and serum iron are more reliable esti- masses that accompany an MPN. Marrow fibrosis likely mates of iron deficiency in the presence of MPNs than is the causes distortion of marrow sinusoids, which permit HSCs evaluation of bone marrow iron. to escape into the sinusoids and gain entry to the peripheral Thrombocytosis may be present, and if it is classi- blood.7 The HSCs then lodge in extramedullary sites, such fied as the primary disorder, it is believed to result from as the spleen, to proliferate and differentiate. an autonomous, unregulated proliferation of megakaryo- MPNs carry a significant risk of terminating in acute cytes. The level of interleukin-6 (IL-6) and thrombopoietin leukemia. This transition may result from chromosome (TPO), cytokines that promote megakaryopoiesis, is normal instability in the original mutant stem cell8 or as the result in MPNs, whereas the level is usually increased in second- of leukemogenic chemotherapy and radiotherapy for the ary or reactive thrombocytosis.5 Platelet membrane pro- original myeloproliferative neoplasm (Chapter 23). teins (glycoprotein IIb/IIIa, von Willebrand factor [VWF], fibrinogen, fibronectin, and vitronectin) are significantly Checkpoint 24.1 decreased in many patients with MPNs.5 Genetic changes In essential thrombocythemia, all hematopoietic lines have altering growth factor receptors, including the thrombopoi- increased cell proliferation. Which lineage has the greatest etin receptor (myeloproliferative leukemia virus oncogene increase? [MPL]), and mutations in the Janus kinase (JAK) signal- ing pathways result in increased megakaryopoiesis and enhanced activation and aggregation of platelets.6 PART II The bone marrow is usually hypercellular at onset SUBGROUPS OF MPNs of the MPN but often becomes fibrotic during the course of the disease. Fibroblasts are a part of the bone marrow stroma, which provides a suitable microenvironment for developing hematopoietic cells (Chapters 3, 38). Fibroblasts Chronic Myeloid Leukemia produce reticular fibers that form a three-dimensional sup- porting network for vascular sinuses and hematopoietic (CML) elements. Fibrosis refers to an increase in fibroblasts and Chronic myeloid leukemia (CML), also known as chronic reticular fibers. Fibrosis in MPNs is thought to be a reac- granulocytic leukemia (CGL), chronic myeloid leukemia, and chronic tive process that is secondary to the increased production myelocytic leukemia, is the best-defined MPN. It is characterized of cytokines from the abnormal hematopoietic cells (primar- by a neoplastic growth of primarily myeloid cells in the bone ily megakaryocytes). As evidence of their benign prolifera- marrow with an extreme elevation of these cells in the periph- tion, fibroblasts exhibit normal karyotypes. Although often eral blood. Erythrocytic and megakaryocytic lineages can also considered the hallmark of PMF, fibrosis can be seen in the expand and extramedullary granulocytic proliferation in the other MPNs as well. spleen and liver reflects progression of the disease. Reactive fibrosis can result from the intramedullary The natural course of the disease occurs in three phases: release of cytokines from platelets, megakaryocytes, and chronic, accelerated, and blast crisis. The initial chronic phase malignant cells that are mitogenic for fibroblasts. Human responds well to therapy. Use of traditional chemotherapy platelet-derived growth factor (PDGF) stimulates growth can usually restore and maintain normal health for months and cell division of fibroblasts as well as other cells. The or years. CML can eventually t ransform into an accelerated platelet concentration of PDGF in patients with MPNs is phase and then into blast phase or acute leukemia. The acute significantly decreased, likely because of excessive release. leukemic phase can be either acute myeloid leukemia (AML) The serum PDGF level is significantly higher in patients or lymphoblastic leukemia (LL, also called acute LL [ALL]). with PMF and ET than in other MPNs or in normal con- After progression to the blast crisis phase, the prognosis is trols. Not surprisingly, PMF and ET are the two MPNs with poor with a survival of less than 6 months using traditional the most significant degree of fibrosis.5 Increased fibroblast chemotherapy. H owever, 70–90% of patients who are treated proliferation and function leads to an increase in collagen, in the chronic phase with tyrosine kinase inhibitors (TKIs; laminin, and fibronectin in the medullary cavity. e.g., imatinib mesylate) experience a 5-year progression- When marrow fibrosis supervenes over the course of free survival. It is anticipated that long-term survival will the disease, the major sites of hematopoiesis become the improve with the use of second- and third-generation TKIs extramedullary tissues, particularly the liver and spleen. used as rescue therapy and as first-line treatment. Myeloproliferative Neoplasms 523 Etiology and Pathophysiology Philadelphia chromosome translocation The Ph chromosome (Ph) is an acquired chromosomal Chrs 9 Chrs 22 translocation that results in a fusion gene called BCR/ ABL1. This fusion gene is present in all neoplastic hemato- poietic cells in CML except T lymphocytes and sometimes q11.2 BCR B lymphocytes and is necessary for the diagnosis of CML (Figure 24-2) (Chapters 41, 42). The Ph chromosome was the first chromosome abnormality found consistently asso- Chrs 22q2 ciated with a malignant disease. It is not found in other BCR/ABL1 (Ph+chromosome) somatic cells or fibroblasts. In some cases, the Ph chromosome can be detected months before the diagnosis of CML. Once the chromo- some is identified, it will rise and fall as a reflection of tumor burden and can be used as a measure of disease pro- gression, remission, and relapse. Because all hematopoietic q34 ABL1 cells are involved in the neoplastic process (evidenced by the presence of Ph in those cells), the original neoplastic Chrs 9q1 cell (cell of origin) is most likely the HSC (Figure 24-1). ABL1/BCR Molecular biology has dramatically increased under- Normal CML standing of the role of the Ph in CML. The Ph results from a balanced reciprocal translocation between the Figure 24.3 The Philadelphia translocation in CML. long arms of chromosomes 9 and 22, t(9;22)(q34;q11.2) Arrows indicate the chromosome breakpoints at 9q34 (ABL1 (Figure 24-3). On chromosome 9, the breakpoint is spread gene) and 22q11.2 (BCR gene) in the genes directly involved in across a 90 kb region within the first exon of ABL1, trans- the translocation. The translocation results in a lengthened 9q+ locating the 3′ end of exon 1 and exons 2–11 (in which chromosome and a shortened 22q- chromosome (Ph chromosome). the tyrosine kinase domains reside) to chromosome 22. The breakpoint in chromosome 22 occurs within the BCR exon 13 or 14 of BCR. The resulting Ph has a 5′ BCR head gene, a large 70-kb gene with 25 exons. The breakpoint (exons 1-13 or 1-14) from chromosome 22 and a 3′ ABL1 usually occurs in an area known as the major breakpoint tail (exons 2–11) from chromosome 9 (Figure 24-4). This cluster region (M-BCR). When the breakpoint occurs in hybrid gene is then transcribed into an 8.5-kb fusion the M-BCR, two different fusion genes are formed (BCR/ ABL1 and ABL1/BCR). Both fusion genes join the translo- cated portion of the ABL1 gene (exons 2–11) with either ABL1 gene (Chromosome 9) 1b 1a a2 a11 5' 3' BCR gene (Chromosome 22) e1 e1e2 e6 e13 e14 e19 5' 3' m-BCR M-BCR micro-BCR e1a2 BCR ABL1 p190 e13a2 BCR ABL1 p210 e14a2 BCR ABL1 Figure 24.4 Exon maps of the normal BCR and ABL1 genes, and the BCR/ABL1 fusion gene formed by the Ph chromosome translocation found in CML and de novo acute leukemias. Breakpoints in the ABL1 gene occur upstream of exon 2 (a2, downward arrow). Breakpoints in the major breakpoint cluster region (M-BCR) of the BCR gene occur in exon 13 (e13) and exon 14 (e14) in CML; breakpoints can Figure 24.2 Karyotype from a patient with CML showing also occur in the minor breakpoint cluster region (m-BCR, 3′ of exon1 the Ph chromosome translocation, t(9:22)(q34;q11.2). The Ph [e1]) in acute leukemias. Following BCR/ABL1 fusion, the resulting chromosome is chromosome 22. protein of 210 (p210; e13a2 or e14a2) or 190 (p190; e1a2) kD is formed. 524 Chapter 24 mRNA, in contrast to the normal ABL1, which is tran- scribed to a 6- or 7-kb mRNA.9 Table 24.4 Clinical Uses of Molecular Analysis for the BCR/ABL1 Gene Rearrangement10 Translation of the 8.5-kb fusion mRNA creates a new, abnormal fusion protein, with a molecular mass of 210 • Differential diagnosis of CML kD (p210, normal ABL1 is a 145-kD protein; F igure 24-4). • Diagnosis of CML when Ph is absent This fusion protein produces constitutive tyrosine kinase • Differentiation of CML in blast crisis from de novo ALL when Ph is present activity, increasing autophosphorylation within the cell • Confirmation of a diagnosis of CML when the patient presents in the that activates intracellular signal transduction pathways, blast crisis phase of CML and results in abnormal gene expression (Chapters 2, • Monitoring of CML patients on tyrosine kinase inhibitor (TKI) therapy to determine response, resistance, or relapse 23). Tyrosine kinase enzymes regulate metabolic path- •

Detection of minimal residual disease in post-bone marrow transplant ways by transferring phosphate groups from phosphate patients donors (ATP and GTP) to target proteins at tyrosine residues, activating the protein. Some serve as recep- tors for growth factors (RTK [receptor tyrosine kinase]). Table 24.5 Genetic Rearrangements and Related The oncogenic role of p210 is found in association with Proteins Found in Philadelphia Chromosome Positive CML, increased granulocyte colony–stimulating factor (G-CSF) ALL, and Philadelphia Chromosome Negative MPN, or ALL and PDGF and suppression of apoptosis in hematopoi- etic cells.9 Over a period of several years, the t(9;22) cell Clinical Condition Involvement of Ph/BCR Size of TK line replaces the normal marrow cells, and the clinical presentation of CML is observed. The reciprocal fusion Normal Ph- , BCR- 145 kD gene, ABL1/BCR1, is transcribed in approximately two- CML Ph+ , BCR+ 210 kD thirds of CML patients, although its significance is not Ph- , BCR+ 210 kD clear. Characteristics of the ABL1 and BCR proto-onco- Other MPNs Ph- , BCR- 145 kD genes, as well as the BCR/ABL1 fusion oncogene, are ALL Ph+ , BCR+ 210 kD (CML blast described in Table 24-3. crisis) Detection of the BCR gene rearrangement has sev- Ph+ , BCR- (minor BCR+ ) 190 kD (? de novo ALL) eral clinical uses in the diagnosis and prognosis of CML Ph- , BCR- 145 kD (? de novo ALL) (Table 24-4).9,10 About 5–10% of patients with the CML Ph+ , BCR- (micro BCR+ ) 230 kD (variant CML) phenotype lack Ph. In these cases, the translocation is not TK, tyrosine kinase; CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; detected at the karyotypic level by cytogenetic studies but Ph+ , Philadelphia chromosome positive; Ph- , Philadelphia chromosome negative; BCR+ , rearrangement within the M-BCR region; BCR- , no rearrangement within the M-BCR is detected at the molecular level by reverse transcriptase region. polymerase chain reaction (RT-PCR) or fluorescent in situ hybridization (FISH), verifying that the molecular BCR/ ABL1 fusion has occurred (Chapters 41, 42). The malig- DISEASE PROGRESSION AND ADDITIONAL CHROMO- nant cells in these cases also express the 8.5-kb chimeric SOME/MOLECULAR MUTATIONS mRNA and the p210 protein (Table 24-5). Historically, or in untreated cases, progression of CML Cases of phenotypic CML that are both Ph chromo- is marked by an accelerated phase followed by an acute some negative and BCR/ABL1-negative are usually CNL, phase (blast crisis). In about 80% of patients, this progres- aCML, or possibly cases of chronic myelomonocytic leu- sion is preceded or accompanied by the development of kemia (CMML) if absolute monocytosis and leukocytosis additional chromosomal abnormalities (Table 24-6).9 Thus, are present. The last two disorders are now considered repeated chromosome analysis in patients with CML can MDS/MPN but all have high proliferative rates.11 be helpful in predicting disease progression. Table 24.3 Characteristics of the Proto-Oncogenes and Oncogenes Involved in the BCR/ABL1 Gene Rearrangements in CML Gene Size No. Exons Breakpoint Transcript(s) Protein Location Activity ABL1 More than 230 kb 11 5′ of exon II 6 kb; 7 kb p145 Nucleus Tyrosine kinase (normal) (100–200 kb) BCR More than 100 kb 25 BCR (5.8 kb) 4.5 kb; 6.7 kb p160 Cytoplasm Serine/Threonine kinase BCR/ABL1 Varies Varies Fusion hybrid 8.5 kb p210 Plasma Tyrosine kinase fusion membrane (increased) Kb, kilobases. Myeloproliferative Neoplasms 525 and older. It is most prevalent in the seventh, eighth, and Table 24.6 Additional Chromosomal Changes in Blast ninth decades of life and is almost equally distributed Crisis of CML between sexes. CML can occur in young adults more so Rate of Occurrence Chromosomal Change than other MPNs. Although rare, CML can occur in child- Frequent Duplication of Ph chromosome hood. The incidence of CML is highest in countries that Trisomy 8 are more economically advanced. The question arises, Isochromosome 17 however, as to whether the higher incidences reflect the Loss of Y chromosome ability to detect and diagnose the disease because of Rare Translocation (15;17) increased availability of advanced medicine and diag- Translocation (3;21)(q26;q22) nostic tools in these countries versus a higher mutagen exposure. Translocation (3;3)/inversion (3) The disease has an insidious onset with the most com- Very rare Deletion of chromosome 5(-5) or the long arm (5q- ) mon symptoms being increased weakness, loss of stamina, Deletion of chromosome 7(-7) or the long unexplained fever, night sweats, weight loss, and feelings arm (7q- ) of fullness in the abdomen (hepatosplenomegaly). Gastro- intestinal tract bleeding or retinal hemorrhages are occa- sionally the first signs of the disease. Physical examination At the molecular level, mutations in the p53 gene, a reveals pallor, tenderness over the lower sternum, spleno- tumor suppressor gene, are found in at least 25% of patients megaly, and occasionally hepatomegaly. Lymphadenopa- in blast crisis, especially in those with myeloid blast crisis,9 thy is not typical but when present, it suggests an onset while mutations/alterations of p53 in the chronic state of of the acute phase of the disease. Petechiae and ecchymo- CML are rare. Other tumor suppressor genes such as p16 ses reflect the presence of quantitative and/or qualitative and RB and oncogenes such as BCL-2 and AML1 can also platelet abnormalities. Some individuals are asymptom- acquire mutations that contribute to the development of atic, and CML is found incidentally during examination blast crisis9 (Chapter 23). for other medical problems or during a routine physical PHILADELPHIA (PH) CHROMOSOME IN ACUTE examination. LEUKEMIAS Any organ eventually can be infiltrated with myeloid About 2–5% of childhood ALL, 25% of adult ALL, and elements, but extramedullary masses in areas other than some cases of AML carry the ph chromosome at diagnosis the spleen and liver are uncommon findings in the chronic (Chapters 25, 26). Ph-positive AML cases may actually be phase. On fresh incision, extramedullary masses appear CML in blast crisis that were not diagnosed in the chronic green, presumably because of the presence of the myeloid (CML) stage. In about 50% of the Ph-positive ALLs, the enzyme myeloperoxidase. These greenish tumors have BCR/ABL1 protein, p210, is present and these cases prob- been called chloromas. The green color fades to a dirty yel- ably represent the blast crisis phase of CML. In the remain- low when the tissue is exposed to air. ing 50% of Ph-positive all, the breakpoints on chromosome Without intervention, symptoms worsen over the 22 fall 5′ to the M-BCR within the first intron of the BCR next 3–5 years, and increased debilitation heralds the gene (minor breakpoint cluster region, M-BRC). These onset of the blast phase. With the onset of blast crisis, leukemias express a distinct translation product from the response to therapy is poor and survival is less than 6 mRNA hybrid termed the p190 kD protein9 (Figure 24-4). months using traditional chemotherapy. Treatment with These Ph-positive ALLs may actually be de novo acute leu- tyrosine kinase inhibitors has dramatically improved kemia cases (Table 24-5). Like p210, the p190 protein also patient outcomes. shows an increased tyrosine kinase activity, but its role in the development of the ALL phenotype is unclear. Laboratory Evaluation PERIPHERAL BLOOD Checkpoint 24.2 The most striking abnormality in the peripheral blood A patient has the CML phenotype, but the genetic karyotype does not show the Ph chromosome. If this is truly a CML, what is the extreme leukocytosis. The white count is usually should molecular analysis show? greater than 100 * 103/mcL, with a median of about 170 * 103/mcL. Patients diagnosed early may have a Clinical Presentation leukocyte count of 25-75 * 103/mcL. Thrombocytosis, which can exceed 1000 * 103/mcL, and variation in plate- CML is the most common MPN, accounting for 15–20% let shape are found in more than half of the patients. If of all leukemia cases. It can occur at any age, but the inci- thrombocytosis is a new observation, blast crisis is prob- dence increases dramatically among those 55 years of age ably imminent; during blast crisis, thrombocytopenia is 526 Chapter 24 a common finding. Platelet function also is frequently The marrow differential count of leukocyte precursors often abnormal. Megakaryocyte fragments and micromega- is within the reference interval. The typical leukemic hiatus karyocytes may be found (Figure 24-5). (lack of developing cells between immature cells [blasts] and At the time of diagnosis, a mild to moderate normo- mature cells [segmented neutrophils]) that is characteristic cytic, normochromic anemia is typical with a hemoglobin of AL is not present in CML. Auer rods may be found in the concentration in the range of 9–13 g/dL. The severity of myeloblasts during blast crisis, but this is an unusual find- anemia is proportional to the increase in leukocytes. Eryth- ing. Erythropoiesis is normoblastic because the increased rocyte morphology is generally normal, but nucleated tyrosine kinase activity does not directly affect erythropoi- erythrocytes can be found. Reticulocytes are normal or esis, but normoblasts can be decreased. Megakaryocytes slightly increased. are usually increased with frequent immature and atypi- Blood smears exhibit a shift to the left with all stages cal forms. In contrast to the large megakaryocytes found of granulocyte maturation present (Figure 24-6). The pre- in other subgroups of MPNs, CML typically reveals small dominant cells are the segmented neutrophils and myelo- megakaryocytes. cytes. Promyelocytes and blasts do not usually exceed 20% Gaucher-like cells (histiocytes exhibiting a wrinkled of the leukocytes in the peripheral blood. Eosinophils and tissue paper appearance of the cytoplasm) have been basophils are often increased in both relative and abso- observed in the bone marrow (Chapter 21). However, these lute terms. Increasing blast or basophil numbers herald Gaucher-like cells do not occur because of the lack of the blast crisis. Monocytes are moderately increased. Signs of b@glucocerebrosidase enzyme as in Gaucher’s d isease myeloid dysplasia including pseudo–Pelger-Huët anomaly but because of the overload of cerebrosides caused by (hyposegmentation of the neutrophil nucleus [Chapter 21]) increased cell turnover. The histiocytes in CML have nor- and decreased leukocyte alkaline phosphatase (LAP) are mal to increased amounts of b@glucocerebrosidase, but the frequent (Chapters 23, 37). Low or absent LAP is charac- cells cannot process the excess cerebrosides fast enough to teristic but not specific for CML. Monocytosis, myeloid dys- prevent them from accumulating. The marrow can become plasia, and micromegakaryocytes are overlapping features fibrotic late in the course of the disease. If the patient does found in both CML and chronic myelomonocytic leukemia not see a physician until the fibrosis is prominent, an inap- (CMML), but the presence of the Ph will differentiate the propriate diagnosis of PMF could be considered. At this two disorders. point, chromosomal (Ph) or molecular (BCR/ABL1) analysis can establish the correct diagnosis. BONE MARROW Other nonspecific findings related to the increased The bone marrow is 90–100% cellular with a striking increase proliferation of cells can be present. Total serum in the myeloid-to-erythroid ratio (10:1 to 50:1) reflective of cobalamin and the unsaturated cobalamin binding capac- the myelopoiesis. The active red marrow can extend into ity are increased. Serum haptocorrins are often elevated the long bones. Cortical thinning and erosion of the tra- (Chapter 15). These findings are probably related to the beculae can be present. The hematopoietic marrow cells are increased number of granulocytes that are thought to syn- primarily immature granulocytes with less than 20% blasts thesize these proteins. Uric acid and lactate dehydrogenase by WHO classification, an important characteristic that (LD) are elevated, secondarily to increased cell turnover. distinguishes CML from all forms of acute leukemia (AL).2 Muramidase is normal or only slightly increased. Figure 24.5 Arrow points to a micromegakaryocyte in the Figure 24.6 CML with leukocytosis and a shift to the left peripheral blood of a patient with CML (peripheral blood, Wright- (peripheral blood, Wright-Giemsa stain, 1000* magnification). Giemsa stain, 1000* magnification). Myeloproliferative Neoplasms 527 Checkpoint 24.3 Describe the peripheral blood differential of a CML patient. CASE STUDY (continued from page 519) Physical examination revealed a slightly enlarged liver and palpable spleen. Roger had hyperurice- mia. Blood counts showed: Hb 11.6 g/dL Hct 35% RBC 3.6 * 106/mcL WBC 26.2 * 103/mcL Figure 24.7 Peripheral blood film from a patient with CML in Platelets 853 * 103 accelerated phase. There is an increase in blasts. Arrows point to /mcL blasts (Wright-Giemsa stain, 1000* magnification). The blood cell differential showed marked aniso- cytosis, poikilocytosis with many teardrops, and at any time after the initial diagnosis, and often precedes numerous nucleated red blood

cells. Immature the final stage of blast transformation (blast crisis).2 Clinical myeloid cells were found along with basophilia features reflect an increase in debilitation including pyrexia and large platelets. (fever), night sweats, weight loss, increased weakness, mal- 1. What are Roger’s MCV and MCHC? aise, bone pain, and lymphadenopathy. About 30% of those patients in the accelerated phase die before developing 2. How would you classify his anemia blast crisis. morphologically? Although blast crisis typically develops after a short, 3. Based on Roger’s history and current laboratory accelerated phase, about one-third of cases abruptly data, what other tests should be performed? develop a blast transformation. After onset of blast crisis, survival is about 1–2 months using classical chemotherapy and is dramatically improved for most patients receiving Terminal Phase TKI therapy. The clinical features in blast crisis are similar to those of acute leukemia (Chapters 26, 27). If untreated, the typical CML course progresses to the The hematologic criteria for identifying blast crisis is accelerated stage approximately 3–5 years after diagnosis made by finding moderate to marked diffuse bone mar- (Figure 24-7). Transition to the accelerated phase is h eralded row fibrosis and greater than or equal to 20% blasts2 in the by one or more of the changes listed in Table 24-7, can occur peripheral blood or bone marrow of a patient previously Table 24.7 Accelerated Phase of CML Characterized by the presence of one or more of the following: Peripheral Blood/Bone Marrow Findings Molecular Findings Therapy-Specific Findings Other Persistent or increasing WBC count (more than Additional clonal chromosomal Hematologic resistance to the Persistent or increasing 10 * 103/mcL), unresponsive to therapy abnormalities in Ph+ cells at diagnosis first TKI splenomegaly (second Ph, trisomy 8, isochromosome (unresponsive to therapy) 17q, trisomy 19), complex karyotype, or abnormalities of 3q26.2 Ten to 19% myeloblasts in peripheral blood or Any new clonal chromosomal Any hematological, cytogenetic, bone marrow abnormality in Ph+ cells that occurs or molecular indications of during therapy resistance to two sequential TKIs Basophils in peripheral blood Occurrence of two or more mutations in BCR/ABL1 during TKI therapy Persistent thrombocytopenia (less than 100 * 103/mcL), unresponsive to therapy Persistent thrombocytosis (greater than 1000 * 103/mcL), unresponsive to therapy 528 Chapter 24 Table 24.8 Blast Crisis Phase of CML Characterized by the presence of one or more of the following: • Greater than or equal to 20% blasts in the bone marrow or nucleated cells in the peripheral blood • Extramedullary blast proliferation • Presence of large clusters of blasts in the bone marrow biopsy specimen diagnosed as having CML (Table 24-8 and Figure 24-8). Any type of blast involvement is possible including myeloid, lymphoid, erythroid, or megakaryocytic cells. Because blast morphology alone is often not sufficient to identify the type of blast involved, cytochemical, enzymatic, ultra- structural, and immunophenotyping and molecular studies Figure 24.8 Peripheral blood from a patient with CML in blast are necessary (Chapters 23, 37, 40). crisis. Note the cluster of blasts with vacuoles (Wright-Giemsa stain, About 65–75% of blast crises are myeloblastic, and 25–35% 1000* magnification). of blast crises are lymphoblastic. The lymphoblasts in blast cri- sis are immunologically typed as common acute lymphocytic leukemia antigen positive (CALLA+/CD10+ ) (Chapters 8, quinone 2 (NQ02), and c-raf. Imatinib b inding inhibits 27, 40) and demonstrate an elevated terminal deoxynucleoti- kinase activity and prevents cell signal transduction.14,15,16 dyl transferase (TdT) suggesting that these cells belong to the This range of binding explains the potential use of imatinib B lymphocyte lineage (Chapter 8). Erythroblastic and mega- in other conditions but raises questions about clinical effi- karyoblastic crises are uncommon. cacy and toxicity.14 Imatinib is generally well tolerated with mild to moder- Therapy ate side effects, primarily involving the gastrointestinal tract and skin. However, it is teratogenic in laboratory animals. The purpose of CML therapy is to reduce the leukocyte mass, Imatinib is also effective in delaying disease progression in restore bone marrow function, reduce splenomegaly, and patients in blast crisis.12,13 abolish symptoms. Leukapheresis is sometimes used initially Patients initially responsive to imatinib therapy can to reduce the leukocyte mass when excessive numbers of cells develop resistance (secondary resistance or loss of response), result in a significant increase in blood viscosity. Supportive whereas other patients might not respond to initial therapy measures during therapeutic regimens include transfusion to (primary resistance; Table 24-9).13 The majority of patients in treat severe anemia and antibiotics to treat infections. the advanced stages of CML show a measurable response to Before the discovery of molecularly targeted therapy imatinib although fewer of them reach remission compared for malignant diseases, the use of interferon@a, plus cyta- with those treated in the chronic phase. rabine was considered standard therapy for patients with In the majority of cases, loss of response (second- CML who were not candidates for an allogeneic HSC trans- ary resistance; Table 24-9) is related to development plant. Interferon@a, a glycoprotein, has a myelosuppressive of additional mutations in the kinase domain of BCR/ effect directly inhibiting myeloid progenitor cells. It induces a remission in 55–75% of CML patients and, in some cases, eliminates the Ph clone. A cytogenetic response is more Table 24.9 Resistance to Imatinib in Patients with CML favorable in patients who are treated early in the chronic Resistance Definition phase of the disease.9 Primary resistance • Complete hematologic response is not IMATINIB achieved by 3–6 months Currently, the tyrosine kinase inhibitor (TKI) imatinib • Cytogenetic response is not achieved by 6 months (more than 95% Ph+ metaphases mesylate (Gleevec®), is considered the first treatment option persist) except in pregnant patients.12,13 It is a molecular-targeted • Partial cytogenetic response is not achieved therapy. Imatinib competitively binds to the ATP binding by 12 months (more than 35% Ph+ metaphases persist) site of the tyrosine kinase of ABL1 (BCR/ABL1), stem cell fac- Secondary resistance • Loss of a complete hematologic response tor receptor (c-Kit), platelet-derived growth factor receptor • Loss of a partial or complete cytogenetic (PDGFR), lymphocyte-specific kinase (Lck), vascular endo- response thelial growth factor receptor (VEGFR), colony s timulating • Increasing levels of BCR/ABL1 as assessed factor receptor-1 (CSFR-1), NAD(P)H dehydrogenase, by real-time PCR Myeloproliferative Neoplasms 529 ABL1.9 A particular ABL1 protein mutant, T315I, in which Ponatinib is considered a third-generation TKI and was threonine is mutated to isoleucine at the gatekeeper posi- also FDA approved in 2012 as a rescue therapy for patients tion of the kinase domain does not respond to any of the who are resistant to imatinib, dasatinib, and nilotinib. TKIs currently used in CML therapy and represents about Most notable is that Ponatinib is effective against the T315I 15% of i matinib resistant patients.14 Mutation screening is mutation. It can inhibit a wide range of tyrosine kinases to suggested for those who do not have an adequate initial include PDGFR, c-Kit, FGFR, VEGFR, and Flt-3.14 response or who experience a loss of response to imatinib Baseline assessment for CML patients prior to start- because the position of the m utation within the kinase ing imatinib therapy should include bone marrow mor- domain can be clinically r elevant. Some mutations have phologic analysis, cytogenetics, and RT-PCR to determine inferior survival (P-loop of BCR/ABL1 protein kinase muta- baseline level of BCR/ABL1 transcripts. Measuring BCR/ tions) compared with mutations at other sites.15 ABL1 mRNA levels in plasma has been found to correspond Several therapeutic approaches for patients who with patient tumor burden and is suggested as a monitor- experience imatinib resistance exist. Dose escalation is ing standard for patients.16 The RT-PCR assay of blood often the first course of action. Remission can be recovered should be performed at least every 3 months after therapy by imatinib dose escalation when the loss of remission is begins, or a single assay at 6 months can be performed if the caused by the development of a second Ph in the HSC or patient is responding.17 Bone marrow cytogenetics should BCR/ABL1 gene duplication. Occasionally, relapse because be performed every 6 months until a major c ytogenetic of certain mutations in the ATP binding site can be over- response (MCyR; less than 35% Ph+ ), is achieved. A com- come through imatinib dose escalation.16 plete cytogenetic response (0% Ph+ ) and BCR/ABL1- When dose escalation fails, switching to an alterna- negative results (complete molecular response) in treated tive tyrosine kinase inhibitor is the treatment of choice. patients predict a favorable outcome. Incomplete response Dasatinib (BMS354825) is a tyrosine kinase inhibitor that, (primary resistance) indicates the presence of minimal like imatinib, attaches to the ATP binding site of the ABL1 residual disease, and increasing BCR/ABL1 results indicate portion of the BCR/ABL1 protein. It is effective against all the potential for disease relapse (secondary resistance). imatinib resistant mutants currently identified except the HSC TRANSPLANTATION T315I mutation. Allogenic HSC transplantation can be an option for patients Nilotinib (AMN107) is a structural derivative of ima- who meet the bone marrow transplant criteria. Limitations tinib that binds to and stabilizes the ABL protein ATP bind- are based on patient age (less than 65 years) and availability ing site with 30-times greater potency than imatinib. Like of a compatible donor.9 High-dose chemoradiotherapy or dasatinib, nilotinib is effective against all imatinib resistant imatinib therapy is followed by HSC transplantation from mutants currently identified except the T315I mutation. syngeneic or allogenenic donors. Stem cell transplantation is In September 2012, the U.S. Food and Drug Administra- most successful when administered during the first chronic tion approved bosutinib for the treatment of chronic, accel- phase of CML. Of patients receiving HLA-matched sibling erated, or blast phase Philadelphia chromosome positive stem cells, 86% have survival rates that exceed 3 years.9 (Ph+ ) chronic myeloid leukemia (CML) in adult patients with resistance or intolerance to prior therapy. Like dasat- inib and nilotinib, it is effective against most imatinib-resis- Differential Diagnosis tant mutants except the T315I mutation. Preliminary results Many infectious, inflammatory, or malignant disorders and suggest that second-generation TKIs could be superior first- severe hemorrhage or hemolysis can cause a leukemoid line therapies over imatinib for new CML patients.16 reaction (Chapter 21) that resembles CML (Table 24-10). Table 24.10 Comparison of Peripheral Blood Features of CML and Leukemoid Reactions Laboratory Parameter CML Leukemoid Reaction Leukocytes Moderate to marked leukocytosis, blasts and promyelocytes in Mild to moderate leukocytosis, toxic granulation, peripheral blood (deep left shift), toxic changes usually absent, Döhle bodies and vacuoles present, blasts absent, eosinophilia and basophilia, neutrophils with single-lobed nuclei promyelocytes rare, no absolute basophilia or and hypogranular forms can be present eosinophilia Platelets Often increased with abnormal morphological forms present, occa- Usually normal sional micromegakaryocytes Erythrocytes Anemia usually present, variable anisocytosis, poikilocytosis, NRBC Anemia rarely, NRBC not typical present LAP Low Increased Chromosome karyotype Ph or BCR/ABL1 translocation present Normal 530 Chapter 24 At times, the clinical findings of a leukemoid reaction per- mit an accurate diagnosis, but in some cases, differential Table 24.11 Diagnostic Criteria for Chronic Neutrophilic Leukemia2 diagnosis requires further investigation. In a leukemoid reaction, leukocytosis is generally accompanied by a pre- Peripheral blood leukocytosis greater than or equal to 25 * 103/mcL dominance of segmented neutrophils and bands on the • Bands and segmented neutrophils more than 80% white blood count (WBC) blood smear (myelocytes, metamyelocytes, promyelo- • Immature granulocytes less than 10% WBC cytes, and blasts are few in number compared with CML). • Myeloblasts less than 1% WBC Toxic granulation, cytoplasmic vacuoles, and Döhle bod- • No dysgranulopoiesis ies in granulocytes often accompany benign toxic leukocy- Hypercellular BM tosis in leukemoid reactions and are not common in CML. • Increased percent of neutrophilic granulocytes In contrast to CML, monocytes, eosinophils, and baso- • Less than 5% myeloblasts phils are generally not elevated in a leukemoid reaction. • Normal neutrophil maturation pattern Other diagnostic tests (elevated LAP score and • Normal or left shift of megakaryocytes absence of Ph) are helpful in differentiating a leukemoid Absence of Ph chromosome or BCR/ABL1 mutation Absence of mutations of PDGFRA, PDGFRB, or FGFR1 reaction from CML. Splenomegaly is uncommon in a leu- No evidence of other MPNs kemoid reaction. Although a bone marrow examination No evidence of an MDS or MDS/MPN disorder; monocytes less than is rarely necessary to make a differential diagnosis, the 1 * 103/mcL; no dysplastic changes in granulocytes or other myeloid cells marrow in a leukemoid reaction can be

hypercellular, but Presence of CSF3R T618I or other activating CSF3R mutation in contrast to CML, the maturation of granulocytic cells If CSF3R T618I is absent, persistent neutrophilia for at least 3 months, splenomegaly, no identifiable cause of reactive neutrophilia including is orderly. the absence of a plasma cell neoplasm OR demonstration of clonality CML occasionally resembles PMF. Distinguishing of myeloid cells by cytogenetic or molecular studies. features of PMF include markedly abnormal erythrocyte morphology with nucleated erythrocytes and immature leukocytes (leukoerythroblastic reaction), an increased Etiology and Pathophysiology LAP score, bone marrow fibrosis, and the absence of the Ph. The cause of CNL has not been identified. About 20% of CNL is another MPN that may resemble CML but does cases are associated with an underlying neoplasm, par- not have the BCR/ABL1 mutation. It must be distinguished ticularly multiple myeloma. However, clonality of the from CML because in the absence of the BCR/ABL1 muta- neutrophils when neutrophilia is associated with multiple tion, imatinib is not an effective treatment. Because of the myeloma has not been demonstrated. Thus, neutrophil pro- similarity of CNL to CML, it is discussed next. liferation may be related to abnormal cytokine production in this setting. The CNL cell of origin is probably the HSC. Normal apoptotic signaling is likely disrupted. Checkpoint 24.4 Cytogenetics are normal in most patients, but up to 25% What clinical, peripheral blood, and genetic features differentiate have mutations including 20 q- , 21+ , 11q- , and +8, +9. Ph CML from an infectious process? and BCR/ABL1 fusion gene are absent. If plasma cell prolifera- tion is present, clonality of the neutrophils should be established by cytogenetic or molecular studies for a diagnosis of CNL. Chronic Neutrophilic Clinical Presentation Leukemia (CNL) CNL is a rare MPN, with approximately 150 reported cases. The median age at diagnosis is 65 years. The male-to-female Chronic neutrophilic leukemia (CNL) is an MPN ratio is about 1:1. Most patients are asymptomatic at the time characterized by a sustained increase in neutrophils in of diagnosis, but fatigue, weight loss, easy bruising, bone the peripheral blood with a slight shift to the left and pain, and night sweats can occur. Hepatosplenomegaly is no Ph or BCR/ABL1 mutation. Monocytosis and baso- usually present. philia are absent, which helps to distinguish CNL from myelodysplasia and CML, respectively. The bone mar- row is hypercellular because of increased neutrophilic Laboratory Evaluation granulocyte proliferation, but dysplasia is absent. All The most notable feature is neutrophilia (more than causes of a reactive neutrophilia and other MPNs must 25 * 103/mcL) (Figure 24-9). Mature segmented forms be ruled out for a diagnosis of CNL, so it is a diagnosis of and bands predominate, and more immature cells account exclusion. The diagnostic criteria for CNL18 are defined for less than 10% of the leukocytes. Neutrophils can appear in Table 24-11. toxic but not dysplastic. Both the RBC count and platelet Myeloproliferative Neoplasms 531 Essential Thrombocythemia (ET) Essential thrombocythemia (ET) is a myeloprolifera- tive neoplasm affecting primarily the megakaryocytic lineage. Sustained proliferation of megakaryocytes in the marrow and extreme thrombocytosis in the peripheral blood with thrombocytopathy (a qualitative disorder of platelets) occurs. Previously, considerable contro- versy existed concerning the inclusion of ET as a spe- cific entity in the myeloproliferative disorders because thrombocytosis is often a component of CML, PMF, and PV. However, ET is now firmly established as a hemato- Figure 24.9 logic neoplasm with d istinct clinical manifestations and Peripheral blood from a patient with CNL. Note complications.19 the increased numbers of mature neutrophils (Wright-Giemsa stain, 1000* magnification). Synonyms of ET include primary thrombocythemia, hemorrhagic thrombocythemia, primary thrombocytosis, and idiopathic thrombocytosis. morphology are normal. Platelets are usually present in normal concentration, but thrombocytopenia can develop Etiology and Pathophysiology as the disease progresses and the spleen enlarges. Bone mar- row is hypercellular with an M:E ratio that can reach 20:1 or ET is a neoplastic disorder of the HSCs usually resulting higher. Granulocytic hyperplasia is present, but dysplasia, in clonal hematopoiesis affecting all three lineages, but Auer rods in blasts, and increased blasts are absent. in some cases, involving only the megakaryocytes.20 The Excessive erythroid and megakaroycytic p roliferation may clonal population of cells appears hypersensitive to some be present. The LAP score is usually increased. A muta- cytokines, including IL-3 and IL-6, but the clones are not tion in the colony stimulating factor 3 receptor (CSF3R) is hypersensitive to GM-CSF. Sensitivity to the inhibitory strongly associated with CNL. The Ph chromosome and effects of TGF@b is decreased, minimizing inhibition of BCR/ABL1 fusion gene are absent. thrombopoiesis. Thus, a combination of increased sensi- tivity to some cytokines that promote platelet production coupled with a decreased sensitivity to negative regulators Therapy could account for the increased megakaryocyte prolifera- Hydroxyurea is a first-line therapy for CNL. Response lasts tion characteristic of ET. for about 12 months. Second-line therapy is interferon@a. Expression of MPL, the TPO receptor, and its mRNA Allogeneic stem cell transplantation is a potentially curative are generally decreased in ET, serum levels of thrombopoi- treatment for those patients who are eligible. etin are normal or slightly elevated in most patients, yet Median survival is 2.5 years. CNL also has an accel- proliferation of progenitor cells ensues.20 erated phase that is marked by progressive neutrophilia unresponsive to treatment, anemia, thrombocytopenia, and JAK2(V617F) splenomegaly.9 Blasts and other immature cells can be pres- The normal JAK2 protein is a cytoplasmic protein kinase ent in the peripheral blood. closely associated with cytokine receptors and thus is dis- tributed almost exclusively near the cell membrane. When Differential Diagnosis a receptor binds cytokines, the JAK2 protein is transphos- phorylated and activated. In turn, JAK2 phosphorylates CNL must be differentiated from CML and physiologic signal transducers and activators of transcription (STAT) causes of neutrophilia such as infection or inflammation. proteins. The JAK–receptor complex also activates other Differential diagnosis from other myeloid neoplasms signaling pathways. Several inhibitory control mecha- requires an absence of circulating blasts, absolute monocy- nisms constrain the normal JAK2/STAT activation path- tosis, eosinophilia, and basophilia. way. The JAK2 protein has two homologous domains: JH1, which has functional (kinase domain) activity, and JH2, Checkpoint 24.5 which lacks kinase activity (pseudo-kinase domain). The What is the most important feature that separates all other JH2 domain normally interacts with the JH1 domain to forms of MPNs from CML? inhibit kinase activity and to modulate or regulate recep- tor signaling.20 532 Chapter 24 In 2005, a gain-of-function mutation in the JAK2 Clinical Presentation tyrosine kinase gene, JAK2(V617F), was identified in patients with PV and later in ET and PMF.20 JAK2(V617F) is Although a relatively rare disorder, the incidence of ET found on chromosome band 9p24 and encodes a mutated peaks primarily from 50–60 years of age and secondarily tyrosine kinase activator of cell signal transduction. The from 20–30 years of age. The older group of patients has single nucleotide somatic mutation in exon 14 (substitution no gender predilection, but the younger age group pre- of thymine for guanine) results in the substitution of the dominantly involves women. The overall incidence is about 1.592.4/100,000 people annually.20 amino acid valine for phenylalanine at position 617. The amino acid substitution occurs in the pseudo-kinase JH2 The presenting symptoms of patients with ET are vari- domain of the JAK2 protein. This reduces the JH2 inhibitory able. Extreme thrombocytosis is frequently detected. Many function and results in constitutive activation. Furthermore, of these patients are asymptomatic, and their diagnosis is the mutation allows for downstream phosphorylation of made incidentally.20 Symptomatic patients most commonly STAT molecules independent of cytokine interaction with present with thrombosis (primarily involving the microvas- the receptor (i.e., autonomous signaling; Chapter 3). This culature) that can result in pulmonary embolisms or minor JAK2(V617F) gain-of-function mutation gives the cells a bleeding. Neurologic complications are common (e.g., proliferation advantage. headache, paresthesias of the extremities) and are associ- Presence of the JAK2(V617F) mutation appears to ated with platelet-mediated ischemia and thrombosis. Cir- increase the risk of thrombosis proportional to the degree culatory insufficiency involving the microvasculature of the of leukocytosis with no correlation to platelet count.20 The toes and fingers is frequent and associated with pain and connection between leukocytosis and thrombosis may occasionally gangrene. Hemorrhagic episodes can occur, be the result of tissue factor expression by neutrophils, primarily involving the gastrointestinal tract, skin, urinary platelet P-selectin expression, and aggregate formation tract, and oral mucosal membranes. These problems appear between platelets and leukocytes that lead to interac- to be more frequent in patients older than 59 years of age tion with the endothelium in areas of inflammation and with thrombosis occurring more frequently than bleeding at the lower platelet concentrations.20,29 injury.21,22 A single JAK2(V617F) mutation is more commonly About half of the patients have a palpable spleen, but encountered in ET, whereas two JAK2(V617F) mutations splenomegaly is usually slight. Occasionally, splenic atro- are associated with PV.23,24,25,26 This dosage effect is asso- phy resulting from repeated splenic thrombosis and silent ciated with disease transformation from ET to PV and infarctions is seen. When this occurs, it is associated with with disease progression in patients with PV. Therefore, typical morphologic alterations on the peripheral blood JAK2(V617F) burden is implicated as having a dominant smear as discussed in the next section. role in the pathophysiology of Ph negative MPNs.27 The mutation also results in increased responsiveness to eryth- Laboratory Evaluation ropoietin (EPO) and IL-3. JAK2(V617F) is found in about PERIPHERAL BLOOD 50% of patients with PMF and most patients (greater than The most striking finding in the peripheral blood is extreme 95%) with polycythemia vera.20,29,28 and consistent thrombocytosis (Figure 24-10). Platelet counts Mutations in cytokine receptors for EPO, TPO, are greater than 450 * 103/mcL and often range from 1000 and G-CSF may activate JAK2 in those cases that are to 5000 * 103/mcL. The peripheral blood smear can show JAK2(V617F) negative. giant bizarre platelets, and platelets can appear in aggre- gates. Megakaryocytes and megakaryocyte fragments may MPL MUTATIONS be present. However, in many cases, platelet morphology Two mutations in the thrombopoietin receptor MPL appears normal. Abnormalities in platelet aggregation and (MPLW515L and MPLW515K) occur in approximately 4% of adhesion suggest defects in platelet function (Chapter 36). patients with JAK2(V617F)-negative ET and 10% with PMF. Anemia, if present, is generally proportional to the This mutation results in cytokine-independent growth and constitutive downstream signaling pathways.20 severity of bleeding and is usually normocytic; however, long-standing or recurrent hemorrhagic episodes can lead CALRETICULIN (CALR) MUTATIONS to iron deficiency and a microcytic, hypochromic anemia. Calreticulin (CALR) mutations also affect the JAK-STAT In about one-third of the patients, slight erythrocytosis is pathway. At least four mutations have been described in present and can cause confusion with polycythemia vera. CALR; the mutations are associated with a higher platelet Aggregated platelets can lead to an erroneous increase in count and lower WBC and hemoglobin levels compared the erythrocyte count on automated cell counters. Therefore, to ET patients with a JAK2 mutation.22 Thrombosis occurs hemoglobin determinations are better to assess the patient’s at half the rate of ET patients with a JAK2 mutation and anemic status. The reticulocyte count can be increased if patients do not progress to PV.22,31 bleeding is present, in which case mild polychromatophilia Myeloproliferative Neoplasms 533 Table 24.12 Platelet Abnormalities Found in Essential Thrombocythemia • Decreased or mutated MPL receptors for thrombopoietin (TPO) • Shortened platelet survival • Increased plasma b@thromboglobulin (b@TG) • Increased urinary thromboxane B2 (TXB2) • Acquired Von Willebrand disease • Defective epinephrine, collagen, ADP-induced platelet aggregation • Decreased ATP secretion • Acquired storage pool deficiency because of abnormal in vivo platelet activation common findings including defective platelet aggregation Figure 24.10 Essential thrombocythemia. Platelets are with epinephrine, ADP, and collagen. A loss of platelet markedly increased, and a giant form (arrow) is present (peripheral a@adrenergic receptors associated with reduced epineph- blood, Wright-Giemsa stain, 1000* magnification). rine-induced aggregation is characteristic of an MPN and is useful in differentiating ET from secondary thrombocytosis. Spontaneous in vitro platelet aggregation, or hyperaggrega- may be noted. Peripheral blood abnormalities second- bility, is a common finding. In vivo platelet aggregation is ary to autosplenectomy can occur if the spleen has been likely if increased plasma b@thromboglobulin and platelet infarcted (Chapter 3). These abnormalities include Howell- factor 4 (released from platelet a@granules) levels are found. Jolly bodies, nucleated erythrocytes, and poikilocytosis. Other platelet

abnormalities that have been described in A leukocytosis ranging from 22 to 40 * 103/mcL is association with ET are included in Table 24-12.32 almost always present. Occasional metamyelocytes and A form of acquired von Willebrand disease (VWD) has myelocytes can be found with ET. Mild eosinophilia and been described in association with excessively high platelet basophilia also are observed. The LAP score may be nor- counts and ET. The increase in number of circulating plate- mal or increased; it is rarely low. Nucleated erythrocytes are lets is associated with adsorption of larger VWF multim- found in 25% of patients. ers and their removal from the circulation. The laboratory ET patients must not meet any criteria for features are characteristic of Type 2 VWD with a decrease BCR/ABL1 + CML, PV, PMF, MDS, or other myeloid or absence of large VWF multimers and reduced levels of neoplasms and must demonstrate JAK2(V617F), MPL, or ristocetin cofactor activity (Chapters 34, 36). CALR mutations. Minor criteria include presence of a clonal MOLECULAR GENETICS marker or absence of evidence of reactive thrombocytosis.2 A low incidence of clonal chromosomal cytogenetic abnor- BONE MARROW malities (about 5%) is found in ET. However, mutations in The bone marrow exhibits marked hyperplasia with a JAK2, MPL, and CALR genes function to drive the disor- striking increase in megakaryocytes often with clustering der and are found in both sporatic and familial cases.22 The of megakaryocytes along the sinusoidal borders. The back- JAK2(V617F) and MPL(W515L) mutations are the most com- ground of stained slides shows many platelets. The mega- mon.22,32 Most triple-negative patients express other somatic karyocytes are large with abundant mature cytoplasm and mutations in JAK2 and MPL.32,33 frequently increased nuclear lobulation. Mitotic forms are OTHER LABORATORY EVALUATION increased. Erythroid and myeloid hyperplasia also are evi- Other laboratory tests may be abnormal. Serum cobala- dent. Stains for iron reveal normal or decreased stores in min and the unsaturated cobalamin binding capacity are the context of normal serum ferritin levels. In about 25% of increased. An increase in cell turnover can cause serum uric cases, reticulin is increased, but significant fibrosis is gener- ally not seen.20 acid, lactate dehydrogenase (LD), and acid phosphatase to be elevated. Serum potassium can be elevated as a result TESTS OF HEMOSTASIS of in vitro release of potassium from platelets (pseudohy- Laboratory tests alone are unreliable in predicting bleeding perkalemia). The spurious nature of this hyperkalemia can or thrombotic complications in ET. The prothrombin time be verified by performing a simultaneous potassium assay (PT) and activated partial thromboplastin time (APTT) are on plasma, which should be normal. Arterial blood gases usually normal, but evidence of low-grade disseminated may reveal a pseudohypoxia if the sample is not tested intravascular coagulation (DIC) may be present. Platelet promptly because of the in vitro consumption of oxygen by aggregation studies are frequently abnormal with the most the increased numbers of platelets. 534 Chapter 24 Prognosis and Therapy Table 24.13 Conditions Associated with Thrombocytosis Median survival for patients with ET is 19.8 years.22 Essential thrombocythemia (ET) Transformation into primary myelofibrosis or acute leu- Polycythemia vera (PV) kemia occurs at a rate of 10% and 5%, respectively, and a Chronic myeloid leukemia (CML) Primary myelofibrosis (PMF) JAK2 mutation increases the risk of transformation into Secondary thrombocytosis polycythemia vera.20 The prognosis appears to be better in • Chronic inflammatory disorders younger patients. The most common causes of death are • Acute hemorrhage thrombosis and bleeding. • Hemolytic anemia Controversy exists as to which patients with ET require • Hodgkin’s disease therapy. It is generally agreed that patients with a history • Metastatic carcinoma of thrombosis or cardiovascular risk factors require therapy • Lymphoma • Postsplenectomy to reduce the platelet count. Plateletpheresis can quickly • Postoperative reduce the platelet count below 1000 * 103/mcL for con- • Iron deficiency trol of vascular accidents. Anticoagulants and drugs to inhibit platelet function are used to control thrombosis, and aspirin therapy is recommended for all ET patients unless contraindicated.20 is normal as are leukocytes and erythrocytes, and spleno- CHEMOTHERAPY megaly is absent. Cytoreductive therapy frequently includes use of Differentiating ET from PV can be difficult. However, hydroxyurea and anagrelide. Hydroxyurea is an effec- marked erythrocytosis with clinical findings suggestive tive form of cytoreductive therapy in patients to lower of hypervolemia is more typical of PV. The Polycythemia both the leukocyte and platelet counts and lower the risk Vera Study Group (PVSG) proposed a set of diagnostic of thrombosis. Although anagrelide (inhibitor of mega- criteria for ET, which were adopted as diagnostic criteria karyocyte differentiation) can be used to reduce only the in the WHO classification (Table 24-14). The first criterion, platelet count, its side effects are not well tolerated.20 The a platelet count greater than 600 * 103/mcL later reduced leukemogenic potential of these therapeutic agents is of by the WHO group to 450 * 103/mcL excludes many concern. The benefit of specific therapy in asymptom- cases of secondary thrombocytosis. The second criterion, atic patients has not been established. Therapeutic trials increased megakaryocytes in the bone marrow, is necessary with b@interferon show improvement in both hematologic to confirm the diagnosis of ET and rule out other causes of clonal thrombocythemia.36 parameters and clinical symptoms on nearly all patients.32 Withdrawal of interferon, however, leads to recurrence of thrombocytosis. MOLECULAR TARGETS Table 24.14 WHO Diagnostic Criteria for Essential Thrombocythemia (ET)2Use of JAK inhibitors is being investigated primarily in PMF but is beginning to be studied in patients with advanced Major Criteria* ET and PV. These include ICNB018424 (ruxolitinib) and • Platelet count greater than 450 * 103/mcL CEP-701 (lestaurtinib).30,31 Patient response to lestaurtinib • Bone marrow biopsy shows megakaryocytic hyperplasia (enlarged, mature megakaryocytes). No significant increase or left shift in neu- is not as promising as that for ruxolitinib,35 a JAK i nhibitor trophil granulopoiesis or erythropoiesis and very rarely minor (grade 1) that binds the ATP-binding site of JAK (wild-type and increase in reticulin fibers. JAK2[V617F]).9,14 • Not meeting WHO criteria for BCR/ABL1 + CML, PV, PMF, MDS, and other myeloid neoplasms • Presence of JAK2, CALR, or MPL mutations Differential Diagnosis Minor Criteria Although the other MPNs have certain diagnostic markers, • Presence of a clonal marker or absence of evidence for reactive thrombocytosis ET is largely a diagnosis of exclusion. Essential thrombo- cytosis must be differentiated from a secondary, reactive * Diagnosis of ET requires meeting all four major criteria or the first three major criteria and one minor criteria. thrombocytosis (Table 24-13) associated with many acute and chronic infections, inflammatory diseases, carcino- mas, and Hodgkin’s disease. The platelet count in ET often Checkpoint 24.6 exceeds 1000 * 103/mcL and is persistent over a period Is a patient who has a platelet count of 846 * 103/mcL, spleno- of months or years. Secondary or reactive thrombocyto- megaly, and abnormal platelet function tests of hyperaggrega- sis rarely reaches 1000 * 103/mcL and is transitory. In tion likely to have reactive or essential thrombocytosis? Explain. addition, platelet function in secondary thrombocytosis Myeloproliferative Neoplasms 535 Polycythemia Vera (PV) Table 24.15 Classification of Polycythemia The term polycythemia literally means an increase in the cel- Classification Associated Conditions lular elements of the blood. However, it is most commonly Polycythemia vera (primary) Normal or decreased erythropoietin used to describe an increase in erythrocytes exclusive of leu- levels; autonomous cell proliferation kocytes and platelets. Polycythemia vera (PV) is a myelo- Secondary polycythemia High altitude proliferative neoplasm characterized by an unregulated (erythropoietin driven) Chronic obstructive pulmonary disease proliferation of primarily the erythroid elements in the bone Obesity (Pickwickian syndrome) marrow and an increase in erythrocyte concentration in the Inappropriate erythropoietin production peripheral blood. In addition to autonomous proliferation Tumors (e.g., hepatoma, uterine of erythroid cells, leukocytes and platelets can also prolifer- fibroma, renal carcinoma) ate uncontrolled, resulting in a pancytosis (an increase in all Renal ischemia hematopoietic cells in the blood). PV has several synonyms, Familial erythrocytosis including polycythemia rubra vera, primary polycythemia, ery- Hemoglobins with high oxygen affinity thremia, and Osler’s disease. Congenital decrease in erythrocyte 2,3-BPG Relative polycythemia Gaisböck’s syndrome (stress poly- Classification cythemia, spurious polycythemia, pseudopolycythemia) Polycythemia is a general term used to describe erythrocyto- Dehydration sis resulting in an increase in both hemoglobin concentra- tion and red cell mass (RCM) or hematocrit (Chapter 10). When evaluating a patient for polycythemia, it is impor- stimulus of erythrocytosis—hence, the name secondary tant to determine whether these parameters are elevated polycythemia—and is associated with elevated plasma because of an absolute increase in total erythrocyte mass EPO levels. PV results from a primary, unregulated, or (absolute erythrocytosis) or from a decrease in plasma vol- dysregulated increase in erythrocyte production. Rela- ume (relative erythrocytosis) (Figure 24-11). Although an tive polycythemia is characterized by a normal or even absolute erythrocytosis suggests a PV diagnosis, polycy- decreased RCM and occurs as a result of a decreased themia that is secondary to tissue hypoxia, cardiac or pul- plasma volume. It is generally a mild polycythemia result- monary disease, and abnormal hemoglobins should also ing from dehydration, hemoconcentration, or a condition be considered. known as Gaisböck’s syndrome. In an attempt to clarify the pathogenesis of the disor- der, polycythemia is classified into three different groups: Etiology and Pathophysiology PV, secondary polycythemia, and relative polycythe- mia (Table 24-15). Both PV and secondary polycythemia The panhyperplasia often associated with PV suggests a result from an absolute increase in the total body RCM. clonal stem cell defect, and cytogenetic studies have con- Secondary polycythemia can be distinguished from PV firmed its clonal nature.37 Evidence of clonality persists in by a distinct, although not always apparent, physiologic cells even during complete remission. Although all lineages in the peripheral blood can be increased in PV, an increase in erythropoiesis is the out- standing feature. Possible mechanisms for this increase are suggested in Table 24-16.38 In vitro studies using cell culture systems show that PV bone marrow cells can form erythroid colonies without the addition of erythropoietin, suggesting 60% Hct that increased proliferation results from an unregulated neoplastic proliferation of stem cells.37 Other patients’ bone 45% Hct 55% Hct Table 24.16 Possible Mechanisms for Increased Normal Absolute Relative Erythropoiesis in Polycythemia Vera polycythemia polycythemia • Erythropoietin-independent proliferation of neoplastic progenitor cells Figure 24.11 The hematocrit can be increased because of • Hypersensitivity of erythroid progenitor cells to erythropoietin an absolute increase in erythrocyte mass, a condition known as • Hypersensitivity of erythroid progenitor cells to growth factors other than erythropoietin absolute polycythemia (center) or a decrease in plasma volume, a condition known as relative polycythemia (right). • Inhibition of apoptosis in progenitor cells 536 Chapter 24 marrow cells show increased sensitivity to EPO, insulin-like These include MPL,48 the lymphocyte-specific adapter growth factor 1, and IL-3, forming in vitro colonies at sig- protein LNK,49 and an anti-apoptotic protein member of nificantly reduced cytokine concentrations and likely giving the Bcl-2 family called Bcl - xL.43 PV progenitor cells a growth advantage.39,40 The erythroid MPL mutations promote thrombopoietin independent maturation is morphologically normal and the erythrocytes activation of the MPL protein and the resulting throm- function normally and have a normal lifespan. bocytosis associated with ET and PV. Furthermore, the protein LNK functions to downregulate JAK/STAT signal- JAK2 ing following normal erythropoietin and thrombopoietin Several mutations, most affecting the Janus kinase–STAT sig- stimulation to return cell production to the steady state. nal transduction pathway, have been identified in patients Loss of LNK function results in continued erythroid and with PV. As is the case in ET, three mutations in particular thrombocytic production.49 Finally, increased expression drive the disease (JAK2, MPL, and CALR) and can occur of Bcl - xL leads to inhibition of apoptosis in progenitor in both sporatic and familial forms.22 Some patients pres- cells43 (Chapter 2). The defect in programmed cell death ent with none of these three mutations and are called triple creates an accumulation of altered, hypersensitive stem negative. The majority of triple-negative patients possess a and progenitor cells. Thrombopoietin receptor hyperre- somatic mutation in JAK2 and/or MPL.37 The JAK2(V617F) sponsiveness and resistance to apoptosis are also found in mutation is seen in almost all patients (more than 95%) with the megakaryocytic lineage, resulting in the common find- PV.40 In addition, about 5% of PV cases carry other muta- ing of thrombocytosis associated with PV.6,43 tions in exon 12 of JAK2

that disrupt normal JAK2 function. Since the JAK2 (V617F) is not found in all PV patients, it is EPIGENETICS hypothesized that JAK2(V617F) is not the sole transform- Epigenetic changes contribute to the malignant transfor- ing mutation, and many suspect it is not even the initiat- mation of PV. In addition to the TET2 mutation described ing event.41,42 This theory is supported by evidence that in earlier, somatic mutations in the gene for the isocitrate some patients with JAK2 mutated myeloproliferative neo- dehydrogenase (IDH) 1 enzyme that functions in the citric plasms that evolved into AML, the resulting myeloblasts acid cycle manifest epigenetic modifications. IDH1 nor- do not bear the JAK2 mutation.43 Therefore, one or more mally functions to convert isocitrate to a@ketoglutarate with mutations are likely to precede the JAK2(V617F) mutation the associated production of NADPH. Mutations have also and are necessary to predispose patients to developing the been identified in IDH2 in patients with MPNs. Epigenetic JAK2(V617F) mutation and the resulting MPN. analysis of IDH1/2 mutations in AML patients has demon- Two mutations that may precede the JAK2(V617F) strated hypermethylation. Mutations in these two enzymes mutation include TET2 (ten-eleven translocation) and a occur in PV, ET, and PMF at a rate of 1–5% and are much germline single nucleotide polymorphism (SNP) in the more frequent (26%) in the later stages of an MPN and after JAK2 locus (rs10974944).44 The TET2 protein functions as transformation to acute leukemia.33 It has been speculated a catalyst in the hydroxylation of 5-methylcytosine (5-mC), that mutational analysis of IDH1 and IDH2 might be useful converting 5-mC to 5-hydroxylmethylcytosine (5-hmC).45,46 as a marker for disease progression in patients with MPNs. It is thought that 5-hmC is an intermediate base in the demethylation of DNA and therefore serves as an epigen- GENES AND PRESENTATION OF PHENOTYPE etic regulator that participates in driving myeloid prolif- Because the JAK2(V617F) mutation is found in nearly all eration (Chapter 23). Mutations that delete or lead to loss patients with PV and in about half of the patients with ET of function in TET2 are thought to occur in the HSC and and PMF, it is unclear as to how one mutation can result contribute to myeloid rather than lymphoid neoplasms.47 in three different conditions. It is possible that a dosage Clonal analysis of immature progenitor cells in patients effect of the JAK2 mutation may play an important role in with JAK2 and TET2 mutations suggest that TET2 precedes determining disease phenotype since most patients with ET JAK2 with the exception of one report of familial MPN for are heterozygous for JAK2, whereas PV patients tend to be which the opposite is suspected.48 homozygous. Given the suspicion of a pre-JAK2 mutation in A germline polymorphism in the JAK2 gene (rs10974944) the MPNs and the discovery of mutated MPL, the following is found between exons 12 and 13. This polymorphism mutational sequence has been proposed50: increases the likelihood of developing the JAK2(V617F) • A pre-JAK2 mutation produces a hyperproliferative mutation and the resulting MPN by three- to four-fold.44 clone with increased susceptibility to additional muta- tions. Either the MPL or JAK2 mutation occurs, trigger- OTHER MUTATIONS ing the ET phenotype. The JAK2 mutation may produce In the absence of the JAK2(V617F) mutation, other mutations preferential triggering of the MPL receptor over the have been identified that disrupt the normal function of EPO receptor because MPL receptors have a higher the JAK/STAT pathway resulting in myeloid proliferation. density on the HSC surface. Myeloproliferative Neoplasms 537 • When a second JAK2 mutation occurs, sufficient JAK2 survival exceeds 10–20 years.37 Factors that adversely affect proteins exist to trigger the EPO receptors converting survival are age, abnormal karyotype, leukocytosis, and a the disease phenotype to PV. history of thrombosis.20 • Additional mutations evolve the phenotype to that of PMF, but thrombocytosis remains because of chemo- Laboratory Evaluation kines released in response to the fibrosis. PERIPHERAL BLOOD Clinical Presentation The most striking peripheral blood finding in PV is an abso- lute erythrocytosis in the range of 6910 * 106/mcL, with a Polycythemia vera has an estimated prevalence of hemoglobin concentration more than 18.5 g/dL in males 44957/100,000 people.20 Familial cases of ET have been and 16.5 g/dL in females. The hematocrit in females is usu- identified.20,31 It occurs most often between the ages of ally greater than 48% and in males is greater than 52%. The 40–60 years with a peak incidence in the sixth decade of total RCM is increased, greater than 25% of mean normal; life. The disease is rare in children. It occurs more frequently the plasma volume can be normal, elevated, or decreased. in males than females and is more common in whites than Early in the disease, the erythrocytes are normocytic, nor- blacks, particularly in those of Jewish descent.40 PV has been mochromic; however, after repeated therapeutic phlebot- reported to occur in several members of the same family, omy, iron-deficient erythropoiesis can result in microcytic suggesting a familial predisposition may exist. hypochromic cells. Patients with PV occasionally present The disease onset is usually gradual with a history of with iron deficiency secondary to occult blood loss resulting mild symptoms for several years. In some cases, PV is found from abnormal platelet function. This can create a confusing in asymptomatic individuals. When symptoms are present, peripheral blood picture because the concentration of eryth- they are typically related to the increased erythrocyte mass rocytes is normal to increased with significant microcytosis, and the associated cardiovascular disease because of the simulating a thalassemia (Chapter 14). Nucleated erythro- hyperviscosity of the blood. Headache, weakness, pruritus, cytes can be found. On the blood smear, the erythrocytes weight loss, and fatigue are the most common symptoms. typically appear crowded even at the feathered edge. The Pruritus is attributed to hyperhistaminemia that can be reticulocyte count is normal or slightly elevated. spontaneous or induced by hot showers or baths. Itching is Leukocytosis in the range of 129 20 * 103/mcL occurs generalized with absence of a rash. in about two-thirds of the cases because of an increase in About one-third of the patients experience throm- granulocyte production. Early in the disease, there can be botic or hemorrhagic episodes; however, the CALR muta- a relative granulocytosis and a relative lymphopenia with tion imparts a reduced thrombotic risk especially in ET a normal total leukocyte count. A shift to the left can be patients.20,37 Myocardial infarctions, retinal vein thrombo- found with the presence of myelocytes and metamyelo- sis, thrombophlebitis, and cerebral ischemia can occur at cytes, but finding promyelocytes, blasts, or excessive num- any stage of the disease and occasionally may be the first bers of immature myeloid cells is unusual. Relative and indication of the disease. absolute basophilia is common. The LAP score is usually When the hematocrit exceeds 60%, the blood viscos- higher than 100. ity increases steeply, decreasing blood flow and increasing Megakaryocytic hyperplasia in the bone marrow peripheral vascular resistance. These interactions produce accompanied by an increase in platelet production is a hypertension in about 50% of the patients with PV. Plethora consistent finding in PV. In some patients, the megakaryo- (a florid complexion resulting from an excessive amount of cytes proliferate without overexpression of the receptor blood), especially on the face but also on the hands, feet, and for thrombopoietin (those patients with a mutation in the ears, is a common finding on physical examination. MPL gene) and have decreased apoptosis as do the ery- After 2–10 years, bone marrow failure may develop, throid progenitor cells.40,43 The platelet count is greater accompanied by clinical findings that include an increase in than 400 * 103/mcL in 20% of PV patients and occasion- splenomegaly, anemia, and bleeding. Laboratory results are ally exceeds 1000 * 103/mcL. Giant platelets can be found likely to reveal a decreased platelet count and decreasing on the blood smear. Qualitative platelet abnormalities are hematocrit. Together, these findings are known as the spent reflected by abnormal aggregation to one or more aggregat- phase and often herald the transition to AML. Postpolycy- ing agents—epinephrine, collagen, adenosine diphosphate themic myelofibrosis develops in about 30% of PV cases (ADP), or thrombin (Chapters 33, 36). Lack of aggregation and splenomegaly is a characteristic finding at the spent with epinephrine is the most common abnormality. The PT phase. Acute leukemia develops as an abrupt transition in and APTT are usually normal (Chapters 34, 36). Abnormal 5–10% of patients. Leukemia appears to develop at a higher multimeric forms of von Willebrand factor (VWF) are found rate in patients treated with myelosuppressive drugs than in about half of PV patients and may lead to a diagnosis of in those treated with phlebotomy alone. Overall median acquired VWD40 (Chapter 34). 538 Chapter 24 Advanced disease is accompanied by striking morpho- resulting from tissue hypoxia, EPO levels are elevated, and logic changes in erythrocytes (Figure 24-12). The peripheral arterial oxygen saturation levels are decreased. When sec- blood picture may resemble that of myelofibrosis with a leu- ondary polycythemia occurs from an inappropriate increase koerythroblastic anemia, poikilocytosis with dacryocytes, in EPO, oxygen saturation levels are usually normal. and thrombocytopenia. In cases that advance to acute leuke- Other laboratory tests also can be abnormal. Serum mia, the blood picture exhibits anemia with marked eryth- uric acid is greater than 7 mg/dL in two-thirds of the rocyte abnormalities, thrombocytopenia, and blast cells. patients and can cause symptoms of gout. The increase probably reflects an increase in the turnover of nucleic BONE MARROW acids from the increased number of blood cells. Serum Most patients with PV have a moderate to marked increase cobalamin-binding capacity in most of the untreated PV in bone marrow cellularity. The hypercellularity is greater patients is increased, primarily because of the increase in than is seen in secondary polycythemia, and hematopoietic haptocorrins derived from granulocytes. Serum cobalamin marrow can extend into the long bones. Granulopoiesis as also is increased but not in proportion to the unsaturated well as erythropoiesis is often increased; consequently, the binding capacity. M:E ratio is usually normal. Although the relative number of myeloblasts is not increased, one of the most significant MOLECULAR GENETICS findings is an increase in megakaryocytes. Eosinophils Because of the high frequency of the JAK2(V617F) muta- are also often increased. Sometimes bone marrow biop- tion in PV, peripheral blood screening for JAK2(V617F) in sies reveal a slight to marked increase in fibrotic material the initial evaluation of patients suspected of having PV or reticulin, but it is generally directly proportional to the has been recommended. Cytogenetic abnormalities includ- degree of cellularity (e.g., more cellular marrows demon- ing chromosomal aneuploidy and partial deletions may strating more reticulin). Iron stores are usually absent, pre- be found. The most consistent abnormality is a trisomy 8 sumably because of a diversion of iron from storage sites to or 9, an abnormally long chromosome 1, and partial dele- the large numbers of developing erythroblasts. In the post- tions of chromosome 13 and 20. The frequency of multiple polycythemic stage, the bone marrow reveals reticulin and karyotypic abnormalities increases after years of treatment collagen fibrosis. Cellularity varies but is often hypocellular and occurs in more than 80% of patients who develop acute with prominent clusters of megakaryocytes. Erythropoiesis leukemia. Thus, progression from a normal to an abnormal and granulopoiesis decrease. A shift to the left can be pres- karyotype is an adverse prognostic indicator. ent, but blasts are usually less than 10% and dysplasia is unusual. Prognosis and Therapy OTHER LABORATORY EVALUATION There is no known cure for PV, but treatment usually pro- EPO levels are normal or low in PV, and arterial oxygen longs survival. Without treatment, 50% of the patients saturation levels are normal. In secondary polycythemia survive about 18 months. With only phlebotomy as the palliative treatment, survival extends to about 14 years. Thrombosis is the most frequent complication, and often patients are given antiplatelet therapy.37 PHLEBOTOMY Two types of therapy, phlebotomy and myelosuppressive therapy, have been used historically. Therapeutic phle- botomy is performed to keep the hematocrit below 45% in men and 42% in women and to intentionally reduce iron supplies. Lack of iron is expected to slow the production of erythrocytes, reducing erythrocytosis and the accompany- ing symptoms. CHEMOTHERAPY Myelosuppressive or cytoreductive therapy utilizing hydroxyurea, alkylators, interferon, radioactive phosphorus (32P), and anagrelide have been used to

reduce the amount of proliferating hematopoietic cells and for patients at risk Figure 24.12 Peripheral blood from a patient with PV for vascular events.36,37 Hydroxyurea inhibits ribonucleo- showing thrombocytosis and large platelets. A reactive lymphocyte and segmented neutrophil are in the center. Poikilocytosis with target tide reductase and carries less risk of secondary leukemia cells, pencil cells, and acanthocytes is present (Wright-Giemsa stain, than does busulphan, an alkylating agent. Younger patients 600* original magnification). who are intolerant or resistant to hydroxyurea can be treated Myeloproliferative Neoplasms 539 with a@interferon; busulfan is used for older patients.37 blood oxygen saturation can also occur in severe obstructive Low-dose pegylated interferon a@2a has been shown to lung disease and in obesity. The hematocrit is generally not normalize blood counts in patients with PV and ET, and higher than 57% in these cases. on sustained therapy, a 30–40% chance that patients could Inappropriate Increase in Erythropoietin A nonphysio- develop a significant molecular response and a significant logic increase in EPO (inappropriate) has been described decrease in the JAK2(V617F) allele burden. Pegylated inter- in association with certain tumors that appear to secrete feron a@2a can retard the progression of ET and PV toward EPO or an EPO-like substance. About 50% of patients who PMF.51 Patients receiving 32P therapy have a mean survival experience this have renal tumors. Other tumors that have of 12 years, yet these patients show a progressive incidence been associated with erythrocytosis include those of the of malignant complications. liver, cerebellum, uterus, adrenals, ovaries, lung, and MOLECULAR TARGETS thymus. In almost all cases, EPO levels return to normal Research to find a molecularly targeted therapy specific and the erythrocytosis disappears after resection of the for the abnormal JAK2 kinase similar to the use of ima- tumor. Renal cysts are also associated with polycythe- tinib in CML is ongoing. The JAK2 inhibitor, ruxolitinib, mia, possibly because of localized pressure and hypoxia showed promising results in clinical trials. In PV patients, to the juxtaglomerular apparatus, resulting in increased 97% achieved phlebotomy independence and many EPO secretion. In some patients with hypertension, renal showed complete remission.52 CEP-701 (lestauritinib, a artery disease, and renal transplants, renal ischemia can JAK inhibitor) therapy also revealed a decreased need occur, resulting in erythrocytosis secondary to increased for phlebotomy and a reduction in JAK2(V617F) allele EPO production. burden.53 Familial Polycythemia Inherited hemoglobin variants with increased oxygen affinity cause tissue hypoxia and are Differential Diagnosis associated with a secondary erythrocytosis. Because of the It is essential that PV be differentiated from the more benign increased oxygen affinity, less oxygen is released to the tis- causes of secondary erythrocytosis and relative polycythe- sues, stimulating erythropoietin production. Inherited defi- mia so that effective therapy can be initiated. ciency of 2,3-BPG also results in decreased oxygen release to tissues. These inherited conditions are usually found in SECONDARY POLYCYTHEMIA young children and in other family members as well. Secondary polycythemia can be classified into the follow- ing groups: Neonatal Polycythemia In neonates, hematocrits of 48% are common (Table A in Appendix). The etiology is attributed 1. Polycythemia caused by an increase in EPO as a normal to placental transfusion that occurs as a result of late cord physiologic response to tissue hypoxia clamping (7–10 seconds after delivery) and/or increased 2. Polycythemia caused by an inappropriate, nonphysi- erythropoiesis stimulated by intrauterine hypoxia.40 ologic increase in erythropoietin production 3. Familial polycythemia associated with high oxygen- Checkpoint 24.7 affinity hemoglobin variants Renal tumors can produce an inappropriate amount of EPO, 4. Neonatal polycythemia associated with intrauterine resulting in what type of polycythemia? hypoxia or late cord clamping Tissue Hypoxia A decreased arterial oxygen saturation Relative Polycythemia and subsequent tissue hypoxia are the most common cause Relative polycythemia is a mild polycythemia resulting from of secondary polycythemia. The polycythemia disappears dehydration, hemoconcentration, or a condition known as when the underlying cause is identified and effectively Gaisböck’s syndrome, which has several synonyms, includ- treated. Residents of high-altitude areas demonstrate a ing spurious polycythemia, pseudopolycythemia, and stress significant increase in hemoglobin and hematocrit that is erythrocytosis. Patients with these conditions have a rela- progressively elevated at high altitudes. The decrease in tive polycythemia and hypertension with nephropathy or barometric pressure at high altitudes decreases the inspired relative polycythemia associated with emotional stress.40 oxygen tension. As a result, less oxygen enters the eryth- RCM is essentially normal. High hematocrit and hemo- rocytes in the pulmonary alveoli, and the arterial blood globin concentrations appear to result from a combination oxygen saturation decreases (Chapters 6, 11). Chronic of high-normal erythrocyte concentrations with a low- hyperventilation partially compensates for the reduced pO2 normal plasma volume. The most common symptoms in the lungs. Compensation at the cellular level involves an are light-headedness, headaches, and dizziness. Plethora increase in 2,3-BPG, facilitating the transfer of oxygen to the is common but splenomegaly is rare. These patients tissues. Tissue hypoxia secondary to a decrease in arterial have a high incidence of thromboembolic complications 540 Chapter 24 Table 24.17 Polycythemia Vera Diagnostic Criteria as Table 24.18 Differential Features of Polycythemia Defined by WHO2 Feature PV Secondary Relative Major criteria 1. Hb greater than 16.5 g/dL in men or 16.0 g/dL in women OR Spleen size c N N Hct greater than 49% in men and 48% in RCM Greater than or c N women OR equal to 36 mL/kg Increased RBC mass greater than 25% (males) mean normal Greater than or 2. Bone marrow showing hypercellularity for age equal to 32 mL/kg with trilineage growth (panmyelosis) i ncluding (females) prominent erythroid, granulocytic, and Leukocyte count c N N megakaryocytic proliferation with pleomorphic, mature megakaryocytes (difference is size) Platelet count c N N 3. Presence of JAK2(V617F) or JAK2 exon Serum cobalamin c N N 12 mutation Arterial O2 saturation N T N Minor criterion 1. Subnormal serum erythropoietin level Bone marrow Panhyperplasia, Erythroid N reticulin deposits hyperplasia LAP N to c N N and cardiovascular disease. Although the hemoglobin, Iron stores T N N hematocrit, and e rythrocyte counts are increased, leuko- cytes and platelets are normal. Bone marrow cellularity is EPO N, T N, c N normal with no increase in megakaryocytes or reticulin. Chromosome Abnormal, greater N N Bone marrow iron stores are absent in 50% of the patients, studies than 90% JAK2 (V617F)+ but serum iron studies are normal. Chromosome karyo- types are almost always normal. N, normal; c , increased; T , decreased; LAP, leukocyte alkaline phosphatase; EPO, erythropoietin. LABORATORY DIFFERENTIATION OF POLYCYTHEMIA The WHO-defined diagnostic criteria for PV include ini- tial determination of total RCM or hemoglobin to establish Genetic Studies Studies should include molecular analysis the presence of an absolute polycythemia37 (Table 24-17). for the JAK2(V617F) mutation found in exon 14. In addi- However, in some cases with hemoglobin levels below the tion, 10 different mutations have been identified in exon 12 value used for a diagnosis of PV and thrombocytosis, the of JAK2 that are associated with erythrocytosis. Karyotype RCM may be higher than required, thus revealing an occult screening for trisomies of chromosome 8 and 9 as well as erythrocytosis.54 Without the RCM, these cases would have deletions of 13 or 20 may also be performed.40 been diagnosed as essential thrombocythemia. This sug- gests that RCM is an important test to perform when PV Bone Marrow Changes A bone marrow assessment is suspected. Determination of serum EPO levels is impor- can be helpful in patients in whom EPO is not low and tant because it helps to distinguish between primary and JAK2(V617F) is not detected, but an elevated RCM and secondary polycythemia. If EPO levels are low, screening clinical symptoms suggest PV. Histologic changes for PV for the JAK2(V617F) mutation and bone marrow histology include hypercellularity with increased erythroid precur- should be performed.40 An elevated EPO indicates second- sors, increased granulocytic and megakaryocytic cells with ary polycythemia. megakaryocyte clusters, and reticulin fibrosis.40 In any classification scheme, recognizing the possibility of two coexisting disease states is imperative. For instance, Checkpoint 24.8 a patient can have both PV and a secondary polycythemia Which of these conditions—iron deficiency, smoking, emphy- as occurs in chronic obstructive pulmonary disease. Refer sema, pregnancy, dehydration—are associated with an abso- to Table 24-18 for differentiating features of polycythemia lute increase in RCM? vera from secondary and relative polycythemia. Erythropoietin Measurement EPO is critical in differ- entiating PV from secondary polycythemias. With the JAK2(V617F) mutation, erythropoiesis occurs without the Primary Myelofibrosis (PMF) need for EPO stimulation. Serum EPO levels are usually Primary myelofibrosis (PMF) is a clonal hematopoietic very low or not detectable in PV. Secondary causes of poly- stem cell disorder with splenomegaly, leukoerythroblastosis, cythemia are related to elevated EPO levels either because extramedullary hematopoiesis (myeloid metaplasia), and of hypoxia or an inappropriate release of EPO from the kid- progressive bone marrow fibrosis.55,56 It is the least common neys (tumors and renal carcinomas). but most aggressive of the myeloproliferative neoplasms. Myeloproliferative Neoplasms 541 The proliferation of hematopoietic cells in the early stages Most PMF patients have a somatic mutation in one of of the disease is neoplastic and presents either as a de novo three driver mutations that affect the JAK-STAT pathway condition or as an evolutionary consequence of PV or ET. (JAK2, MPL, CALR).51 A small subset of PMF patients do not The fibroblast (collagen-producing cell) is an important express any of the three driver mutations, termed triple nega- component of normal bone marrow in which the fibro- tive, and are associated with a worse prognosis. Mutations blasts provide a support structure for hematopoietic cells. in ten-eleven-translocation-2 (TET2), additional sex comb Fibroblast proliferation in PMF is reactive and secondary to like 1 (ASXL1), DNA methyltransferase 3A (DNMT3A), and the underlying disorder. Fibrotic tissue eventually disrupts enhancer of Zeste homolog 2 (EZH2) have also been identified the normal architecture and replaces hematopoietic tissue in patients with PMF, but these mutations affect epigenetic reg- in the bone marrow. Excessive marrow fibrosis inhibits ulation rather than the JAK-STAT pathway.33 These mutations normal hematopoiesis, producing marrow hypoplasia and constitutively activate cell signaling and DNA replication. stimulates myeloid metaplasia. Myeloid metaplasia usually CYTOKINE INVOLVEMENT occurs in both the spleen and the liver. These organs can Megakaryocytes play an important role in the pathogenic become massive in size as the result of islands of proliferat- development of the abnormal PMF marrow. In areas of ing erythroid, myeloid, and megakaryocytic elements. The megakaryocyte necrosis, fibroblast proliferation and colla- extramedullary hematopoiesis is similar to that occurring gen deposition often are prominent. This stromal reaction during embryonic hematopoiesis. is a cytokine-mediated process.57 PDGF, epidermal growth PMF has been known by many synonyms. Most of the factor (EGF), and transforming growth factor beta (TGF@b) different names were attempts to describe the typical blood, are contained in the a@granules of megakaryocytes and bone marrow, and spleen abnormalities. Some of the terms platelets, and all stimulate the growth and proliferation of that have been used include agnogenic myeloid metaplasia, fibroblasts. Reduced platelet concentrations of PDGF and myelofibrosis with myeloid metaplasia, chronic idiopathic myelo- increased levels of serum PDGF are characteristic of PMF. fibrosis, aleukemic myelosis, myelosclerosis, splenomegalic myelo- This condition is thought to represent the abnormal release phthisis, and leukoerythroblastic anemia. or leakage of the growth factor from the platelet. PDGF does not stimulate synthesis of collagen, laminin, or fibro- Etiology and Pathophysiology nectin, but TGF@b stimulates increased expression of genes for fibronectin and collagen while it decreases synthesis of PMF is associated with a profound hyperplasia of mor- collagenase-like enzymes. Thus, the net effect is the accu- phologically abnormal megakaryocytes (dysplastic and mulation of bone marrow stromal elements.57 necrotic). In most cases, only megakaryocytes and granulo- cytes are involved, but all three lineages, including erythro- GENE MUTATIONS cytes, can be involved in the disease process. PMF is often About 50% of patients can have a somatic mutation in preceded by a hypercellular phase of variable duration. The either the JAK2 or MPL genes in a hematopoietic stem cell. disease evolves from this prefibrotic stage with minimal JAK2(V617F) allele homozygosity is identified more often reticulin fibrosis to a fibrotic stage with marked reticulin in PV and PMF than in ET. The JAK2(V617F) allele burden or collagen fibrosis. Thus, at diagnosis, the bone marrow has been associated with disease progression.58,59,60,61 On the can exhibit varying degrees of fibrosis. The fibrosis is

not other hand, PMF patients negative for JAK2(V617F) and with considered part of the primary abnormal clonal prolifera- lower JAK2(V617F) burden experience shorter survival. This tion but is a secondary reactive event occurring in response may indicate that there is another clone as yet not identified to the progeny of the clonal hematopoietic cells and, in fact, that overrides the V617F-negative clone and results in a more fibroblasts do not contain the chromosome abnormalities aggressive disease phenotype.59 The proto-oncogene CBL is found in the hematopoietic cells. mutated primarily in juvenile myelomomocytic leukemia Understanding of this disease has increased consid- (JMML) or chronic myelomonocytic leukemia (CMML) and erably with a better understanding of normal bone mar- has been identified in 6% of patients with PMF.62 Chromosome row structure and the changes that occur in myelofibrotic aberrations, when present, are restricted to cells derived from marrow associated with megakaryocyte growth factors the mutated HSCs. Some of these cells are highly sensitive to that mediate fibrogenesis.57 The bone marrow extracellular or independent from regulation by their respective stimula- matrix or microenvironment (stroma) supports hematopoi- tory factors. The disorder has been reported to terminate in etic cell proliferation (Chapter 3). Myelofibrotic stroma is ALL as well as AML in some patients. characterized by an increase in total collagen, fibroblasts, vitronectin (a cytoadhesion molecule), fibronectin (a cytoad- Checkpoint 24.9 hesion molecule normally limited to megakaryocytes and What growth factors are primarily responsible for stimulating walls of blood vessels), and laminin (a glycoprotein that fibrogenesis in the bone marrow? supports adhesion and growth of cells). 542 Chapter 24 Clinical Presentation and the effectiveness of extramedullary hematopoiesis. The anemia becomes more severe with the progression PMF generally affects individuals older than 50 years of the disease and is aggravated in some patients by the of age and seems to occur equally between sexes. It combination of splenomegaly, which causes sequestration rarely occurs in childhood. Its onset is gradual, and of erythrocytes, and expanded plasma volume (dilutional the disease is chronic. Early in the disease process, the anemia). patient might not have symptoms, making the time of The presence of abnormal erythrocyte morphol- onset difficult to determine. If symptoms are present, ogy is an important feature of PMF. The most typical they are usually related to anemia or pressure from an poikilocyte is the dacryocyte, although elliptocytes and enlarged spleen. Bleeding occasionally is a present- ovalocytes are also present (Figure 24-13). A few nucle- ing symptom. Patients complain of weakness, weight ated erythrocytes are usually found and sometimes can loss, loss of appetite, night sweats, pruritus, pain in be numerous. B asophilic stippling is a common finding. the extremities, bone pain, discomfort in the upper left Reticulocytosis is typical, ranging from 2 to 15%. The quadrant, and fever. The major physical findings are majority of patients have an absolute reticulocyte more splenomegaly (in 90%), h epatomegaly (in 50%), pallor, than 60 * 103/mcL. and petechiae.63,64 Myeloid metaplasia is found in the The leukocyte count is usually elevated but can be spleen and frequently in the liver and can be found in normal or, less often, decreased upon initial presentation. the kidney, adrenal glands, p eritoneal and extraperi- The count generally ranges from 15 to 30 * 103/mcL. toneal s urfaces, skin, lymph nodes, and spinal cord. A leukocyte count above 60 * 103/mcL prior to splenec- Osteosclerosis is a frequent finding and, when found in tomy is rare. As the disease progresses, the leukocyte association with splenomegaly, suggests a diagnosis of concentration does not decrease as quickly as the erythro- myelofibrosis. cytes and platelets. An orderly progression of immature An atypical acute form of the disease has been described granulocytes is characteristically found. Blasts generally with a rapid and progressive course of a few months to 1 compose less than 5% of circulating leukocytes. Other year. Anemia develops rapidly, and the leukocyte count is common findings include basophilia, eosinophilia, and decreased. The bone marrow in these cases exhibits a pro- pseudo–Pelger-Huët anomaly. The LAP is elevated or liferation of reticular and collagen fibers. normal but occasionally is decreased. Low LAP scores Patients with systemic lupus erythrematosus (SLE) correlate with leukopenia. When elevated, the LAP score can present with myelofibrosis that is morphologically helps to differentiate this disease from CML. The Ph chro- indistinguishable from the myelofibrosis of PMF. These mosome is not present. patients also have various peripheral blood cytopenias Platelets can be decreased, normal, or increased. similar to those found in PMF but not splenomegaly. The Higher counts are associated with early disease stages; myelofibrosis in SLE has been referred to as autoimmune thrombocytopenia is usually found in the later stages. myelofibrosis. Patients with myelofibrosis and an absence of splenomegaly should have an antinuclear antibody (ANA) test to rule out SLE.65 Laboratory Evaluation PERIPHERAL BLOOD The typical peripheral blood findings for PMF reflect both qualitative and quantitative cellular abnormali- ties. At diagnosis, there may be anemia, leukocytosis with a left shift, and thrombocytosis. Pancytopenia and leukoerythroblastosis with striking anisocytosis and poikilocytosis is typical at later stages of the disease (Figure 24-13). The anemia is usually normocytic, normo- chromic, but hypochromia may be found if the individual has a history of hemorrhage or hemolysis. Between 35 and 54% of patients with PMF have a hemoglobin level of less than 10 g/dL.56 Folic acid deficiency can develop as a result of increased utilization by the neoplastic clone and Figure 24.13 Peripheral blood from a patient with PMF. Leukoerythroblastic picture with an erythroblast and a myeloblast is associated with a macrocytic anemia. Anemia uncom- below it. The arrow points to a large platelet. Other abnormal plicated by iron deficiency or folic acid deficiency cor- platelets are present. Note the poikilocytosis with dacryocytes relates directly with the extent of bone marrow fibrosis (Wright-Giemsa stain, 1000* magnification). Myeloproliferative Neoplasms 543 Thrombocytopenia is often attributed to excessive splenic pooling. The platelets can appear dysplastic: typically CASE STUDY (continued from page 527) giant, bizarre, and frequently hypogranular. Circulating During the office visit, Roger stated his symptoms of megakaryocyte fragments, mononuclear micromega- fatigue, weakness, dyspnea, bone pain, and abdomi- karyocytes, and naked megakaryocyte nuclei can exist. nal discomfort. A bone marrow biopsy was ordered. The micromegakaryocytes can present an identification The aspiration was unsuccessful, but the marrow problem because they frequently resemble lympho- biopsy showed moderate to marked hyperplasia, cytes. However, important differentiating features of clusters of platelets, abnormal megakaryocyte mor- micromegakaryocytes are the presence of demarcation phology, and fibrotic marrow spaces. membranes with bull’s-eye granules in the cytoplasm and 4. What diagnoses do these results suggest? cytoplasmic blebbing. Qualitative platelet abnormalities including abnormal aggregation and adhesiveness, and 5. Give a reason for the unsuccessful, dry-tap bone defective platelet procoagulant activity on exposure to marrow aspiration. collagen are consistent findings. 6. What characteristic peripheral blood morpholo- Among patients with PMF, 15% have major hemolytic gies correlate with the bone marrow picture and episodes during the course of their disease.56 Hemosiderin- physical exam? uria and decreased haptoglobin are found in about 10% of patients, suggesting intravascular hemolysis. The cause of hemolysis can be hypersplenism, PNH-like defective eryth- rocytes, and antierythrocyte antibodies. A bleeding diathesis ranging from petechiae and ecchy- moses to life-threatening hemorrhage can be found in some patients, likely resulting from a combination of throm- bocytopenia and/or abnormal platelet function. Hemo- static abnormalities suggestive of chronic DIC, including decreased platelet count, decreased concentration of factors V and VIII, and increased fibrin degradation products, can be present.56 Other laboratory tests for PMF are frequently abnor- mal. Serum uric acid and LD are elevated in most patients. Serum cobalamin can be slightly increased but is usually normal. Checkpoint 24.10 What erythrocyte morphologic feature is a hallmark for Figure 24.14 Bone marrow stained with reticulin stain shows myelofibrosis? increased collagen (black-staining fibers) in a patient with PMF (BM biopsy, reticulin stain, 100* magnification). BONE MARROW The bone marrow is difficult to penetrate and frequently MOLECULAR GENETICS yields a dry tap. If aspiration is successful, smears may No specific cytogenetic abnormality is diagnostic for PMF. show no abnormalities; biopsy specimens are needed However, cytogenetic analysis is important to differentiate to reveal the extent of fibrosis. In most PMF cases, myelofibrosis from other myeloproliferative disorders, par- the marrow is hypercellular with varying degrees of ticularly CML, which also can have some degree of fibrosis. diffuse fibrosis and focal aggregates of megakaryocytes The Ph chromosome is not present in PMF. Trisomy or dele- (Figure 24-14). tion of group C chromosomes (chromosomes 6–12) is also Three bone marrow histologic patterns have associated with myelofibrosis. Complete or partial loss of been described: (1) panhyperplasia with absence of chromosomes 5, 7, and 20 is associated with PMF patients myelofibrosis but a slight increase in connective tissue treated with chemotherapy. reticulin, (2) myeloid atrophy with fibrosis, prominent Most patients with PMF have mutations in one of the collagen and reticulin fibers, and cellularity less than three driver genes, (JAK2, MPL, and CALR).51 JAK2(V617F) 30% and (3) myelofibrosis and myelosclerosis with bony is the most common of the three driver mutations fol- trabeculae occupying 30% of the biopsy and extensive lowed by CALR, then MPL. In addition to the driver muta- fibrosis. tions, most PMF patients bear mutations in genes affecting 544 Chapter 24 Table 24.19 WHO Diagnostic Criteria for Overt PMF2 Major criteria* 1. Megakaryocytic proliferation and atypia, accompanied by either reticulin and/or collagen fibrosis grades 2 or 3 2. Not meeting the WHO criteria for BCR/ABL1 + CML, PV, ET, MDS, or other myeloid neoplasms 3. Presence of JAK2, CALR, or MPL mutation or in the absence of these mutations, presence of another clonal marker, or absence of minor reactive BM myelofibrosis Minor criteria 1. Presence of at least one of the following, confirmed in two consecutive determinations a. Anemia not attributed to a comorbid condition b. Leukocytosis greater than 11 * 103/mcL c. Palpable splenomegaly d. LDH increased to above upper limit of institution’s reference range e. Leukoerythroblastosis * Diagnosis of prePMF requires meeting all three major criteria and at least one minor criteria. epigenetic modifications like TET2, ASXL1, DNMT3A, and Differential Diagnosis EZH2.51 The diagnostic algorithm for PMF was revised by WHO in 2016 (Table 24-19). Differentiating PMF from other conditions associated with fibrosis is essential to ensure appropriate therapeutic Prognosis and Therapy regimens (Table 24-20). Splenomegaly, anemia, and a leu- koerythroblastic blood picture are significant findings in The average survival time after diagnosis is 4–5 years. The both myelofibrosis and CML. In myelofibrosis, the leuko- main causes of death are infection, hemorrhage, thrombosis, cyte count is generally less than 50 * 103/mcL, whereas in and cardiac failure. About 10–15% of patients progress to an CML, the count is expected to be higher. In myelofibrosis, acute myeloid leukemia and some to acute lymphoblastic the granulocyte left shift is less pronounced and poikilo- leukemia. cytosis is striking. The bone marrow in myelofibrosis is Therapy is selected to achieve two particular outcomes: fibrous with large numbers of megakaryocytes. In CML, the improve cytopenia and reduce massive splenomegaly. Cor- bone marrow can also exhibit some fibrosis, but the most ticosteroids, androgens, and recombinant erythropoietin abnormal finding is the myeloid hyperplasia. The serum are used to stimulate erythropoiesis to reduce anemia, but cobalamin level is not as elevated in myelofibrosis as it is in patients may still require periodic transfusions.56 When ane- CML. The LAP score in myelofibrosis is variable, but when mia cannot be controlled, splenectomy can be considered. elevated, it is strong evidence against CML. The most reli- Irradiation has been suggested to decrease spleen size able test to differentiate CML and PMF is cytogenetic analy- in an attempt to relieve symptoms or to decrease excessive sis for the Ph chromosome. erythrocyte destruction. Hydroxyurea has been used, but it Differentiating PMF from polycythemia vera, espe- can cause severe pancytopenia. Thalidomide and lenalido- cially in the later stages, is more difficult. The later stages mide also have been used to treat splenomegaly as well as of PV can be accompanied by increased marrow fibrosis chemotherapeutic agents such as 2-chlorodeoxyadenosine and actual transformation to PMF can occur. When throm- (2-CdA or Leustatin) when the splenomegaly is advanced.66 bocytosis is the principal initial hematologic finding, PMF Neuropathy has been reported with thalidomide, and can be confused with ET. A bone marrow biopsy aids in myelosuppression

can occur with lenalidomide. A similar the differentiation, revealing fibrosis in PMF. agent, pomalidomide, has produced anemia and symptom relief without neuropathy and myelosuppression.67 Alloge- neic stem cell transplantation is the only curative therapy for PMF, but the advanced age of many patients is a factor Table 24.20 Conditions Associated with Marrow Fibrosis in the high mortality rate. Neoplasms Other JAK inhibitors are being investigated with varying Primary myelofibrosis Miliary tuberculosis success. Ruxolitinib, a JAK1/2 inhibitor that when admin- Chronic myeloid leukemia Fungal infection istered to patients with advanced PMF at maximum toler- Polycythemia vera Granulomas ated doses daily, resulted in clinical improvement for some Essential thrombocythemia Marrow damage by radiation or patients.68 However, ruxolitinib did not impact JAK2(V617F) Megakaryocytic leukemia chemicals allele burden. CYT387, another JAK1/2 inhibitor, is associ- Metastatic carcinoma ated with splenic reduction and some degree of symptom Hairy cell leukemia Lymphoma relief.36 Clinical trials with drugs that are JAK2/FLT3 inhibi- Hodgkin’s disease tors have variable clinical effects.69 Acute megakaryocytic leukemia Myeloproliferative Neoplasms 545 the action of eosinophilic cytokines, GM-CSF, IL-3, and CASE STUDY (continued from page 543) IL-5. IL-5 is relatively lineage specific for eosinophils Over the next several months, Roger experienced (Chapters 4, 7). The cells are released into the peripheral increasing splenomegaly and abdominal discom- blood and rapidly migrate to tissues where they per- fort. Cytogenetic studies revealed a trisomy 8. form their p hysiologic function. Diagnosis of disorders associated with eosinophilia may be urgent because of 7. What is the most likely explanation for the potential damage to organs infiltrated with eosinophils increased splenomegaly? and subsequent release of cytokines, enzymes, and other 8. What are possible outcomes of this disorder? proteins. The term hypereosinophilic syndrome (HES) describes a group of disorders that demonstrate an abso- lute eosinophil count of greater than 1.5 * 103/mcL that persists for more than 4 weeks.70 The eosinophilia can result Myeloproliferative from a clonal (neoplastic) disorder or a nonclonal (benign or reactive) disorder or can be of unknown origin (idiopathic- Neoplasm, Unclassifiable hypereosinophilic syndrome, I-HES) (Table 24-21). Reactive, nonclonal (benign) hypereosinophilic dis- (MPN, U) orders result from increased production of eosinophil cytokines secondary to or associated with another diag- Myeloproliferative neoplasm, unclassified (MPN, U) is the nosis (Chapters 7, 21). Diseases typically associated with diagnosis for cases that have the characteristic clinical, lab- benign eosinophilia must be ruled out before considering oratory, and morphologic features of an MPN but do not meet the specific criteria for one of the other MPN catego- ries or have features that overlap two or more categories. The Ph chromosome and BCR/ABL1 fusion gene are absent, Table 24.21 Conditions of Hypereosinophilia and the cell of origin is most likely the HSC. Conditions Examples Most cases of MPN, U are either very early stages of Neoplastic Myeloproliferative neoplasms PV, PMF, or ET or are at a late stage of advanced MPN in conditions (clonal) • Chronic eosinophilic leukemia, not otherwise which extensive myelofibrosis, osteosclerosis, or transfor- specified (CEL-NOS) mation to an aggressive stage obscures the true disorder. • Chronic myeloid leukemia (CML) Follow-up at intervals can permit an accurate diagnosis. • Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) A specific diagnosis should be made as soon as possible • Chronic myelomonocytic leukemia (CMML) because of the therapeutic implications for each of the neo- • Myelodysplastic syndromes plastic disorders. • Systemic mastocytosis (SM) The incidence of MPN, U is unknown but can be • Myeloid and lymphoid neoplasms associated as high as 20% of MPNs. Clinical features are similar to with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1 those found in other MPNs, including splenomegaly and • Myeloid and lymphoid neoplasms associated hepatomegaly. with PDGFRA rearrangement Leukocytes and platelets can be increased. Hemo- • Myeloid neoplasms associated with PDGFRB globin is variable. The bone marrow is hypercellular and rearrangement • Myeloid and lymphoid neoplasms associated often shows megakaryocytic hyperplasia and variable with FGFR1 abnormalities granulocytic and erythroid proliferation. In advanced Acute leukemias (AML or ALL) stages, the bone marrow is fibrotic or osteomyeloscle- Benign, reactive Parasitic infection rotic. If there are 10–19% blasts in the peripheral blood, conditions the disease is diagnosed as an accelerated stage. No spe- (nonclonal) cific cytogenetic or molecular abnormalities are identified Asthma with this disorder. Allergies Skin diseases Loeffler’s syndrome Clonal Hypereosinophilia Vasculitis Drug hypersensitivity Eosinophils are derived from the colony-forming units Idiopathic- generating granulocytes, erythroblasts, macrophages, hypereosinophilic and megakaryocytes (CFU-GEMM and CMP) through syndrome 546 Chapter 24 factor receptor 1 (FGFR1). These gene mutations result in Table 24.22 Suggested Information and Testing to Exclude constitutive activation of intrinsic tyrosine kinase activ- Diseases Associated with Reactive (secondary) Eosinophilia ity (Chapter 4). All three disorders can present as a chronic Clinical Patient history myeloproliferative disorder. Less frequently, they can Physical examination present as AML or precursor T lymphoblastic leukemia/ Laboratory tests Complete blood count and differential lymphoma. Recognition of these disorders is important Bone marrow aspiration and biopsy because the mutations result in abnormal tyrosine kinases Routine chemistries that may respond to treatment with imatinib or other TKIs. Serum IgE Eosinophilia is variable but present in most cases. Cobalamin MYELOID AND LYMPHOID NEOPLASMS WITH PDGFRA HIV serology REARRANGEMENT Serology testing and stool analysis for parasites A gain of function tyrosine kinase fusion gene was discov- Other tests Pulmonary function tests ered when some HES patients who were unresponsive to Chest and abdominal CT scan corticosteroid therapy were found to respond to imatinib therapy. The gene fusion was found to occur between the FIP1L1 gene and the PDGFRA gene. The mutation results a neoplastic diagnosis (Table 24-21). See Table 24-22 from a small interstitial deletion in chromosome 4, del(4) for tests to exclude diseases associated with reactive (q12q12), resulting in the FIP1L1/PDGFRA (F/P) gene70 and (secondary) eosinophilia.71 It is especially important an abnormal, constitutively activated tyrosine kinase pro- to perform serological testing to rule out infection with tein. The cell of origin is the HSC. The detection of a fusion Strongyloides sp. because patients with this infection who gene in a particular lineage does not always correlate with are given corticosteroids (common treatment for I-HES) the morphological presentation. can experience dissemination of the disease, which can be fatal.72 Once it is found that the hypereosinophilic condi- Clinical Presentation Most of the cases involving the F/P tion is not clonal, testing to identify reactive conditions mutation are found in males (17:1 male-to-female ratio), and such as allergies, parasitic infections, and asthma, must age at onset is 25–55 years.70 There is evidence of eosinophil- be performed. In some cases, no underlying cause for the related tissue damage and tissue fibrosis. Splenomegaly is eosinophilia can be found and clonality cannot be proven. present. This disorder is known as I-HES (Table 24-21). Many dis- Laboratory Evaluation The initial laboratory finding is orders that were previously considered as I-HES are now usually hypereosinophilia but may be AML or T lympho- included in subgroups with specific genetic abnormalities. blastic leukemia/lymphoma. Eosinophils are mature with Clonal hypereosinophilia is considered a progenitor few myelocytes or promyelocytes. Eosinophil granulation cell disorder (primary) that includes a new myeloid/lym- may be sparse with vacuoles present. Hyper- or hyposeg- phoid neoplasm group added by the WHO in 2008 termed mentation of the nucleus can be seen. A few patients have myeloid and lymphoid neoplasms with eosinophilia and an increase in blasts, but the peripheral blood and/or bone abnormalities of platelet-derived growth factor receptor a marrow contain less than 20% blasts. Anemia and throm- (PDGFRA), PDGFR b (PDGFRB), or fibroblast growth fac- bocytopenia may be present. Increased serum tryptase tor receptor 1 (FGFR1)70 and chronic eosinophilic leukemia, and cobalamin are typical. The bone marrow is hypercel- not otherwise specified (CEL-NOS), a subgroup of MPNs lular with increased eosinophil precursors. Charcot-Ley- (Table 24-21). The eosinophil may also be a part of the neo- den crystals may be seen. Many cases show an increase in plastic clone of cells found in other neoplasms such as MPNs, spindle-shaped atypical mast cells with a CD25+ immuno- MDS, AML, and ALL. Cytogenetics or molecular analysis phenotype. These findings also are present in a significant can help clarify the origin of eosinophils in these neoplasms. number of patients who have systemic mastocytosis with increased atypical (spindle-shaped) mast cells in the bone Myeloid and Lymphoid Neoplasms marrow and a mutation in the stem cell factor receptor c-Kit. Associated with Eosinophilia and The F/P gene can be demonstrated by RT-PCR or FISH tech- niques (Chapters 41, 42). PDGFRA, PDGFRB, or FGFR1 Mutations Therapy Although most patients who harbor the F/P mutation respond to imatinib therapy, variations within These are a group of neoplastic eosinophil disorders caused the F/P mutation have recently been identified as resistant by mutations in genes that encode the a@ or b@chains of the to imatinib. The second generation nilotinib and the third protein tyrosine kinases platelet-derived growth factor generation ponatinib TKIs have shown promising results in receptors (PDGFRA, PDGFRB) or in the fibroblast growth imatinib-resistant patients.73 Myeloproliferative Neoplasms 547 MYELOID NEOPLASMS WITH PDGFRB that incorporate part of the FGFR1 gene. All fusion genes REARRANGEMENT encode a tyrosine kinase that has constitutive activity. Other The myeloid neoplasms with PDGFRB rearrangement are cytogenetic abnormalities can occur. The postulated cell of characterized by a t(5;12) (q33;p13) rearrangement (most origin is the HSC. commonly) to form the ETV6-PDGFRB fusion gene. Other Clinical Presentation There is a male predominance with translocations with a 5q31–33 breakpoint can occur and lead the myeloid and lymphoid neoplasms in the FGFR1 abnor- to other fusion genes, but they are not included in this sub- malities subgroup (1.5:1), and a wide age range at onset group and are not likely to respond to imatinib therapy. The (3–84 years) is observed. postulated cell of origin is a multipotential HSC that is able to differentiate to neutrophils, monocytes, eosinophils, and Laboratory Evaluation This neoplasm presents as a probably mast cells. hypereosinophilia, or if transformed to an acute neo- plasm, as an AML or precursor T or B lymphoblastic Clinical Presentation Myeloid neoplasms with PDGFRB leukemia/lymphoma or as a mixed phenotype acute rearrangement can present as CMML with eosinophilia, leukemia. In patients who are diagnosed in acute trans- aCML, or as MPN with eosinophilia. Acute (blast) transfor- formation, the myeloblasts, lymphoblasts, and eosino- mation can occur. There is a 2:1 male predominance and a phils belong to the neoplastic clone. The chronic phase wide variability in age (8–72 years) at onset. usually has eosinophilia, neutrophilia, and occasionally Laboratory Evaluation The leukocytes are increased with monocytosis. a variable increase in eosinophils, neutrophils, monocytes, Therapy Prognosis is poor. Tyrosine kinase inhibitors are and precursor cells. Anemia and thrombocytopenia may be not currently effective although second- and third-line TKIs present. The bone marrow is hypercellular with an increase are showing promising results.73 Bone marrow transplant in mast cells that can be spindle shaped. Reticulin can be can be indicated in the chronic phase. increased. The peripheral blood and bone marrow have less than 20% blasts. Molecular analysis using primers for all known breakpoints to confirm ETV6-PDGFRB is recom- Chronic Eosinophilic Leukemia, Not mended. Cytogenetic analysis usually reveals the t(5;12). Otherwise Specified (CEL-NOS) Therapy Before imatinib therapy, survival with myeloid CEL-NOS is a clonal myeloproliferative neoplasm that neoplasms with PDGFRB rearrangement was less than 2 presents with eosinophilia not classified as another years. Patients usually respond to imatinib treatment.73 neoplastic condition and does not have the PDGFRA, Survival is expected to improve with early diagnosis and PDGFRB, or FGFR1 mutations. In addition, reactive treatment before organ damage occurs. eosinophilia, diseases associated with abnormal release MYELOID AND LYMPHOID NEOPLASMS WITH FGFR1 of cytokines (IL-2, IL-3, IL-5, or GM-CSF), or presence of ABNORMALITIES aberrant T cells must be ruled out. The diagnostic crite- The myeloid and lymphoid neoplasms with FGFR1 abnor- ria for CEL-NOS70 are listed in Table 24-23. The cell of malities subgroup of eosinophilic neoplasms are charac- origin for this leukemia is probably the HSC. In some terized by an 8p11 breakpoint, which, depending on the cases, cytogenetic abnormalities such as trisomy 8 can chromosome partner, results in a variety of fusion genes be identified.70 Table 24.23 Diagnostic Criteria for Chronic Eosinophilic Leukemia, Not Otherwise Specified (CEL-NOS)—Persistent Eosinophilia Greater Than or Equal to 1.5 * 103/mcL in Blood • Less than 20% blasts in blood and bone marrow; no inv(16)(p13.1q22) or t(16;16)(p13.1:q22) or other diagnostic feature of

AML • Exclude eosinophilic disorders that bear the PDGFRA, PDGFRB, or FGFR1 mutations • No BCR/ABL1 fusion gene; absence of other MPNs, MDS/MPN • Exclude all secondary causes of eosinophilia • Exclude neoplastic disorders with secondary eosinophilia • Exclude neoplastic disorders when eosinophils are a part of the neoplastic clone (e.g., AML) • Exclude presence of an aberrant phenotypic T-cell population • With all of the preceding conditions and evidence of clonality or greater than 2% blasts in the peripheral blood and greater than 5% blasts in the bone marrow, diagnosis is CEL If a patient has persistent eosinophilia but does not meet these criteria, the diagnosis can be reactive eosinophilia, idiopathic hypereosinophilia, or idiopathic hypereosinophilic syndrome. Gotlib, J. (2012). World Health Organization-defined eosinophilic disorders: 2012 update on diagnosis, risk stratification and management. American Journal of Hematology, 87(9), 903–914; Vardiman, J. W., Thiele, J., Arber, D. A., et al. (2009). The 2008 revision of the World Health Organization (WHO) classification of the myeloid neoplasms and acute leukemia: Rationale and important changes. Blood, 114, 937–951. Bain, B. J., Gilliland, D. G., Vardiman, J. W., et al. (2008). Chronic eosinophilic leukemia, not otherwise specified. In S. H. Swerdlow, E. Campo, N. L. Harris, et al. (Eds.), WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (4th ed.). Lyon, France: IARC Press. 548 Chapter 24 CLINICAL PRESENTATION Idiopathic Hypereosinophilic CEL-NOS is most often diagnosed in middle-aged men (male-to-female ratio is 9:1). Presenting symptoms include Syndrome (I-HES) fever and significant weight loss. Clinical features include I-HES is a diagnosis of exclusion. If the patient has central nervous system (CNS) irregularities, hepatospleno- persistent eosinophilia (eosinophil count greater than megaly, congestive heart failure, pulmonary fibrosis, and or equal to 1.5 * 103/mcL for at least 6 months) and occasionally lymphadenopathy. Release of excessive eosin- does not meet the criteria for CEL (Table 24-23) or a ophilic granules in the blood causes fibrosis of the endo- familial eosinophilia (autosomal dominant disorder), thelial cells resulting in peripheral vasculitis, gangrene of the d iagnosis may be I-HES. I-HES usually has organ- digits, and organ damage, particularly of the heart and associated i nvolvement and no evidence of clonality lungs.70 or T-cell abnormality (T cells secrete eosinophil cyto- kines). Pathogenesis is unknown. As more molecular LABORATORY EVALUATION genetic mutations have been found and more diagnos- CEL-NOS is characterized by a peripheral blood tic techniques have become available, this diagnosis eosinophilia of 1.5 * 103/mcL or more. There is evidence has been made less frequently. First-line therapy for of clonality and/or more than 2% blasts in the peripheral most patients with I-HES is the use of c orticosteroids.74 blood or 5–19% blasts in the bone marrow. The leukocyte Patients with I-HES should be m onitored regularly count is usually greater than 30 * 103/mcL with 30–70% because follow-up evidence may show that the condi- eosinophils. Anemia and thrombocytopenia can be present. tion is leukemic. The LAP score is normal, but the serum cobalamin, uric acid, and muramidase are frequently elevated. The bone marrow shows a left shift with many eosinophilic myelo- cytes. Marrow fibrosis is a common finding, and Charcot- Mast Cell Disease Leyden crystals are often seen. (Mastocytosis) THERAPY When therapy is ineffective, the prognosis for patients with Mast cell disorders are heterogeneous and are c haracterized CEL-NOS is poor. Few live beyond 1 year after diagnosis. by the abnormal proliferation of mast cells in one or more The major cause of death is congestive heart failure from organ systems. Mast cells share a c ommon progenitor cell tissue injury. with myelomonocytic cells. Based on the 2016 revised WHO diagnostic criteria for MPNs, m astocytosis is no longer DIFFERENTIAL DIAGNOSIS considered an MPN; however, the WHO classification is Ruling out conditions associated with reactive eosinophilia included in Table 24-24. is important. CEL-NOS also should be differentiated from The two major groups of mast cell disorders are clonal eosinophilia found in other hematologic disorders cutaneous and systemic. Cutaneous disease is based (Table 24-21). If there is no evidence of a reactive eosino- on the presence of histological skin lesions and typical philia, clonality, or increase in blasts, the diagnosis should clinical signs but no systemic involvement. It is typically be I-HES. found in children. Table 24.24 Criteria for Diagnosis of Systemic Mastocytosis (SM) and Mast Cell Leukemia (MCL) SM requires the major criteria and one minor criteria or three minor criteria. Major criteria • Bone marrow or other organs that show multifocal, dense infiltrates of tryptase positive mast cells (more than 15 cells in aggregates) Minor criteria • Biopsy of bone marrow or other organs shows spindle-shaped or atypical morphology in more than 25% of mast cells or more than 25% of all mast cells in the bone marrow are immature or atypical • Presence of c-Kit point mutation (codon 816) in mast cells from bone marrow or other organ • Mast cells in peripheral blood, bone marrow, or other tissue coexpress CD2 and/or CD25 in addition to normal mast cell markers • In absence of associated clonal myeloid disorder, total serum tryptase is greater than 20 ng/mL Mast cell leukemia criteria • Criteria for SM fulfilled • Diffuse infiltration by atypical immature mast cells shown in bone marrow biopsy Myeloproliferative Neoplasms 549 Systemic disease or systemic mastocytosis (SM) cells in bone marrow, and multiorgan failure (Table 24-24). involves multifocal histological lesions in the bone marrow The aleukemic mast cell leukemia variant has less than and other organs. There may be anemia and an increase or 10% mast cells in the peripheral blood. The bone marrow decrease in leukocytes and platelets, and eosinophilia can mast cells can be immature and blast-like. Mast cells with be marked. bilobed or polylobed nuclei can be found. Signs of myelo- Genetic studies reveal that SM is a clonal disorder proliferation and dysplasia can be p resent, but the criteria characterized by a somatic mutation of the c-Kit proto- for another hematologic disorder are not. The mast cells are oncogene. Stem cell factor receptor is encoded for by c-Kit tryptase positive and express c-Kit. Serum tryptase levels (tyrosine kinase receptor) and is a major regulator of mast are elevated. Pancytopenia characterizes later stages of the cell development, and hematopoietic stem cells. When disease when bone marrow failure occurs. mutated, the receptor is constitutively activated, giving Most patients are adults with symptoms related to the cell a proliferative advantage.75 proteins or mediators released from mast cells, which One subgroup of systemic mast cell disease is mast include hypotension, flushing, and diarrhea. Weight loss, cell leukemia (MCL), an aggressive disease with a short bone pain, and organomegaly occur later in the disease. No survival. The criteria for its diagnosis are the fulfillment standard therapy or long-term cure exists. Antimediator of the criteria for systemic mastocytosis, circulating mast drugs such as aspirin and antihistamines are used to relieve cells in the peripheral blood (greater than or equal to 10%) symptoms. Experience with chemotherapy and bone mar- mast cells comprising greater than 20% of the nucleated row transplant is lacking.75 Summary The myeloproliferative neoplasms (MPNs) are character- in gene expression and abnormal control of cell growth. ized as a group of clonal stem cell disorders differentiated Abnormal karyotypes in hematopoietic cells can be found by neoplastic production of one or more of the hemato- in any of the subgroups. The best-characterized abnormal- poietic lineages in bone marrow and peripheral blood. ity is the Philadelphia (Ph) chromosome found in all indi- The WHO classification of MPN includes the subgroups viduals with CML. The Ph chromosome is the result of a chronic myeloid leukemia (CML); chronic neutrophilic translocation of genetic material between chromosomes leukemia (CNL); essential thrombocythemia (ET); poly- 9 and 22 [t(9;22) (q34;q11.2)]. Results of molecular testing cythemia vera (PV); primary myelofibrosis (PMF); myelo- of CML patients, whether positive or negative for the Ph proliferative neoplasm, unclassifiable (MPN, U); myeloid chromosome, reveals a BCR/ABL1 fusion gene encoding an and lymphoid neoplasms associated with eosinophilia abnormal tyrosine kinase protein, p210. The function of the and PDGFRA, PDGFRB, or FGFR1 mutations; chronic abnormal tyrosine kinase results in the pathogenesis of CML. eosinophilic leukemia, not otherwise specified (CEL- JAK2(V617F), MPL(W515), and CALR mutations are com- NOS). The 2016 revised WHO criteria excluded mastocy- mon in other MPNs. Each driver mutation modulates the tosis from the MPNs but it is still discussed in this section. JAK-STAT pathway affecting cellular signal transduction. Although all hematopoietic cell lineages can be The survival of patients with MPNs varies according involved in the unregulated proliferation in MPNs, one lin- to the subgroup and complications of thrombosis. Patients eage is usually involved more than the others. The myeloid with PV and ET appear to survive longer than patients cells are primarily affected in CML, CNL, and eosinophil with CML or PMF. Any of the subgroups can evolve into disorders; the erythrocytes are affected in PV; the plate- acute leukemia with or without specific therapy. Currently, lets/megakaryocytes are affected in ET; and mast cells are stem cell transplantation is the only cure, but few patients elevated in mastocytosis. The most characteristic finding of qualify for the procedure. However, molecular-targeted PMF is a nonmalignant proliferation of fibroblasts in the therapy and antiplatelet drugs give favorable outcomes and bone marrow. Splenomegaly, bone marrow fibrosis, and improve prognosis. Imatinib mesylate revolutionized CML megakaryocytic hyperplasia are findings common to all therapy and a cadre of second- and third-generation TKIs subgroups. are being tested for potential use as first-line therapy, rescue The underlying pathophysiology appears to be chro- therapy, or combination therapy. Several JAK2 inhibitors mosomal rearrangements that occur in the regions of proto- are being tested for effectiveness against the JAK2(V617F)- oncogenes leading to qualitative or quantitative alterations positive MPNs. 550 Chapter 24 Review Questions Level I 8. A 50-year-old man was admitted to the e mergency room for chest pain and a blood count was ordered. 1. The most prominent cell line in CML is the: (Objective 1) The results showed erythrocyte count 6.5 * 106/mcL; a. erythroid hematocrit 0.60 L/L (60%); leukocyte count b. myeloid 15 * 103/mcL; and platelet count 500 * 103/mcL. These results indicate: (Objective 2) c. megakaryocyte d. fibroblast a. the need for further investigation of a possible diagnosis of MPN 2. The most prominent cell line found in polycythemia b. normal findings for an adult male vera (PV) is: (Objective 1) c. the patient has experienced a thrombotic episode a. erythroid d. a malfunction of the cell-counting instrument b. myeloid 9. A patient previously diagnosed with CML now has a c. megakaryocyte platelet count of 540 * 103/mcL and a leukocyte count d. fibroblast of 350 * 103/mcL with a peripheral blood differential showing 15% segmented neutrophils, 23% bands, 2% 3. The Philadelphia chromosome (Ph) results from a metamyelocytes, 35% blasts, 6% lymphocytes, 4% translocation of chromosomes: (Objective 3) monocytes, 6% eosinophils, and 8% basophils. These a. 8 and 14 results are most consistent with: (Objective 5) b. 9 and 22 a. CML c. 12 and 17 b. ET d. 15 and 17 c. CML in blast crisis d. PMF 4. The peak age for CML is: (Objective 4) 10. A patient presenting in the ER with a platelet count of a. <5 years greater than 1000 * 103/mcL and a leukocyte count of b. 15–30 years 25 * 103/mcL with a normochromic, normocytic ane- c. 40–59 years mia should be evaluated for: (Objective 9) d. 60 years or more a. Ph chromosome 5. The Philadelphia chromosome (Ph) can be found in b. essential thrombocythemia patients with: (Objective 3) c. PMF d. primary polycythemia a. CML b. ALL Level II c. ET d. PV Use this case study to answer questions 1–5. A 45-year-old Caucasian female was admitted to the 6. Which of the following represent a characteristic hospital from the emergency room. She had experi- peripheral blood finding in patients with PMF? enced pain in the upper quadrant and bloating for the (Objective 6) past several weeks. She had multiple bruises on her a. Elliptocytes legs and arms. She also stated that her gums bled easily b. Dacryocytes when she brushed her teeth. She had been unusually c. Target cells tired and lost about 10 lb in the last 2 months. Results of physical examination showed a massive spleen. The d. Schistocytes following laboratory results

were noted on admission. 7. PV can be distinguished from secondary polycythe- Hb 7.4 g/dL WBC Differential mia by measuring: (Objective 8) Erythrocyt 2.9 * 106/mcL 31% segmented a. hematocrit ount neutrophils b. plasma volume Hct 22% 26% bands c. hemoglobin concentration RDW 18.0 8% metamyelocytes d. erythropoietin Myeloproliferative Neoplasms 551 c. Leukopenia Leukocyte 520 * 103/mcL 11% myelocytes d. Myelodysplastic count 5. What is the best description of the bone marrow? Platelet 960 * 103/mcL 4% promyelocytes (Objective 1) count a. Decreased M:E ratio and increased cellularity 2% blasts b. Increased M:E ratio and decreased cellularity 4% lymphocytes c. Increased M:E ratio and increased cellularity 3% monocytes d. Decreased M:E ratio and decreased cellularity 5% eosinophils 6% basophils 6. Extensive bone marrow fibrosis, leukoerythroblastic peripheral blood, and the presence of anisocytosis 4 nucleated with dacryocytes are most characteristic of which erythrocytes/100 MPN? (Objective 6) leukocytes a. CML Occasional micro- megakaryocytes b. PV c. ET Anisocytosis and poikilocytosis were moderate. d. PMF A bone marrow aspiration was performed. The marrow was 90% cellular with a myeloid-to-ery- 7. A 68-year-old man was seen in the clinic for lethargy, throid ratio of 10:1. The majority of the cells were dyspnea, and light-headedness. Results of his blood neutrophilic precursors. Eosinophils and basophils counts were erythrocyte count 5.0 * 106/mcL; hema- were increased. Myeloblasts accounted for 10% of tocrit 55%; leukocyte count 60 * 103/mcL; platelet the nucleated cells. Megakaryocytes were increased. count 70 * 103/mcL. His differential showed a shift to the left in myeloid elements with 40% eosinophils. The bone marrow revealed 10% blasts. He does not 1. What findings suggest that this patient has a defect in have the PDGFRA, PDGFRB, or FGFR1 mutations or the pluripotential stem cell rather than a benign pro- the Ph chromosome. Secondary causes of eosinophilia liferation of hematopoietic cells? (Objective 4) have been ruled out. He most likely has: (Objective 10) a. The presence of a leukoerythroblastic blood picture a. PV b. The involvement of several cell lineages in the prolifer- b. CML in blast crisis ative process including neutrophilic cells and platelets c. essential thrombocythemia c. The shift to the left in the neutrophilic cell line d. chronic eosinophilic leukemia d. An increase in the RDW 8. A molecular test should be performed on the patient 2. Molecular analysis (RT-PCR) revealed the presence of in question 7 for which mutation? (Objective 3) a BCR/ABL1 fusion product. Based on this informa- tion, what myeloproliferative disorder is present? a. JAK2(V617F) (Objectives 1, 3) b. BCR/ABL1 a. CML c. FIP1L1/PDGFRA b. PV d. MPL(W515) c. ET 9. Which of the following does not cause secondary d. PMF polycythemia? (Objective 7) 3. What cytochemical stain is used to help differentiate a a. Chronic obstructive pulmonary disease leukemoid reaction from CML? (Objectives 1, 4) b. Smoking a. Myeloperoxidase c. Emphysema b. New methylene blue d. Dehydration c. Leukocyte alkaline phosphatase d. Perl’s Prussian blue 10. Which of the following gene mutations are uniquely associated with clonal hypereosinophilia? (Objective 11) 4. Which of the following terms most accurately describes this patient’s peripheral blood picture? a. JAK2(V617F) (Objectives 10, 13) b. F1P1L1/PDGFRA a. Leukemoid reaction c. MPL(W515) b. Leukoerythroblastic d. BCR/ABL1 552 Chapter 24 References 1. Dameshek, W. (1951). Some speculations on the 17. Guilhot, F., Roy, L., & Tomowiak, C. (2012). Current treatment myeloproliferative syndromes. Blood, 6(4), 372–375. strategies in chronic myeloid leukemia. Current Opinion in 2. Arber, D. A., Orazi, A., Hasserjian, R., Thiele, J., Borowitz, M. Hematology, 19(2), 102–109. doi: 10.1097/MOH.0b013e32834ff610 J., Le Beau, M. M., . . . Vardiman, J. W. (2016). 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A., Pardanani, A. D., Metcalfe, D. D., . . . Valent, P. (2017). Mastocytosis. In: S. H. Cortes-Franco, J., Thomas, D. A., . . . Tefferi, A. (2010). Safety Swerdlow, E. Campo, N. L. Harris, E. S. Jaffe, S. A. Pileri, & H. and efficacy of INC018424, a JAK1 and JAK2 inhibitor, in Stein, eds. World Health Organization Classification of Tumours (4th myelofibrosis. New England Journal of Medicine, 363(12), ed., pp. 61–70). Lyon, France: International Agency for Research 1117–1127. doi: 10.1056/NEJMoa1002028 on Cancer. Chapter 25 Myelodysplastic Syndromes Sara A. Taylor, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define myelodysplastic syndrome (MDS) and 4. Define the WHO category of myelodysplastic/ list general characteristics of these diseases. myeloproliferative neoplasms (MDS/MPNs), 2. List the six subgroups of MDS recognized and list its general characteristics. by the 2016 World Health Organization 5. List the five subgroups of MDS/MPNs and (WHO) Classification System and identify identify key morphological and clinical key morphological and clinical criteria that criteria as well as laboratory findings that distinguish each group. distinguish each group. 3. Describe laboratory findings and recognize changes in morphology that are characteristic of this group of disorders. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Describe the pathophysiology of MDS. 5. Assess the results of cytogenetic and 2. Distinguish among agranular blasts, molecular tests and correlate them with a granular blasts, and promyelocytes. diagnosis of MDS. 3. Summarize the treatment and prognosis of 6. List and briefly describe the MDS variants MDS. not listed in the six WHO subgroups. 4. Explain the relationship between MDS and 7. Differentiate MDS from MPN, acute acute leukemia. leukemia, and other hematologic 555 556 Chapter 25 abnormalities using peripheral blood, bone 9. Select laboratory tests that are helpful in marrow, and cytogenetic characteristics. diagnosing and differentiating MDS and 8. Differentiate MDS/MPNs from MDS, MDS/MPNs. MPN, acute leukemia, and other 10. Evaluate a patient’s laboratory and c linical hematologic abnormalities using peripheral findings, and propose a diagnostic MDS blood, bone marrow, and cytogenetic or MDS/MPNs subgroup based on those characteristics. findings. Chapter Outline Objectives—Level I and Level II 555 Classification 567 Key Terms 556 Description of MDS Subgroups 568 Background Basics 556 Variables of MDS Subgroups 571 Case Study 557 Differential Diagnosis 571 Overview 557 Prognosis 572 Introduction 557 Therapy 573 Pathophysiology 557 Myelodysplastic/Myeloproliferative Neoplasms Incidence 560 (MDS/MPNs) 574 Clinical Presentation 560 Summary 576 Laboratory Evaluation 560 Review Questions 577 Blast and Precursor Cell Classification 565 References 580 Key Terms Atypical chronic myeloid leukemia Juvenile myelomonocytic leukemia Myelodysplastic syndrome with (aCML, BCR/ABL1-) (JMML) ring sideroblasts (MDS-RS) CHIP Micromegakaryocyte Myelodysplastic syndrome Chronic myelomonocytic leukemia Myelodysplastic syndromes (MDS) with multilineage dysplasia (CMML) Myelodysplastic/ (MDS-MLD) Dysplasia myeloproliferative neoplasms Myelodysplastic syndrome Dyspoiesis (MDS/MPNs) with single lineage dysplasia Endomitosis Myelodysplastic syndrome with (MDS-SLD) excess blasts (MDS-EB) Ring sideroblasts ICUS Background Basics The information in this chapter builds on the concepts • Describe and recognize the cell morphology described learned in previous chapters. To maximize your learning by these terms: ring sideroblasts (Chapter 12), experience, you should review the following concepts Pelger-Huët anomaly (Chapter 21), and megaloblas- before starting this unit of study: toid erythropoiesis (Chapter 15). • Explain what ineffective hematopoiesis means. Level I (Chapters 11, 24) • Create a schematic depicting derivation of different • Summarize the history and basis of the classification types of blood cells from the pluripotent hematopoi- system to classify malignant leukocyte disorders. etic stem cell. (Chapter 2) (Chapter 23) Myelodysplastic Syndromes 557 • Describe normal leukocyte development, differ- • Explain how oncogenes and hematopoietic growth entiation, and concentrations in the peripheral factors affect cellular maturation and prolifera- blood. (Chapter 8) tion. (Chapters 3, 23) • Explain the role of epigenetics in cellular develop- Level II ment. (Chapter 2) • Describe the use of immunological features that dis- • Explain the use of cytogenetics and flow cytometry in tinguish the different types of blasts. (Chapters 23, 40) the diagnosis of malignant disorders. (Chapters 40, 41) and, in some cases, transformation into a condition indis- CASE STUDY tinguishable from acute leukemia. MDS occurs most com- We refer to this case study throughout the chapter. monly in the elderly, although it is diagnosed occasionally in children.1,2 Hancock, a 65-year-old white male, was seen in tri- age with complaints of fatigue, malaise, anorexia, Before the 1980s, much confusion and disagreement and hemoptysis of recent onset. A complete blood existed in the literature concerning the criteria for defining, count (CBC) was ordered and revealed anemia and subgrouping, and naming MDS. Because of the predisposi- a shift to the left in granulocytes. Hematopoietic tion of MDS to terminate in leukemia, the term preleukemia cells showed dysplastic features. commonly was used to describe these disorders. However, Consider diagnostic probabilities and reflex the evolution of MDS to acute leukemia is not obligatory, testing that could provide differential diagnostic and, in fact, many patients die of intercurrent disease or information. complications of the cytopenia before evolving to leukemia. Most hematologists currently consider the terms dysmyelo- poietic syndrome and myelodysplastic syndrome to be more acceptable than preleukemia or other synonyms in describing these hematologic disorders. In this book, the term myelo- Overview dysplastic syndrome is used. This chapter describes the neoplastic hematopoietic dis- orders known as the myelodysplastic syndromes (MDS). It Pathophysiology begins with a description of the pathophysiology, inci- dence, and general clinical and laboratory features, includ- Myeolodysplastic syndromes arise from proliferation of ing peripheral blood and bone marrow findings.

Genetic stem cells that have acquired a defect (somatic mutation) findings common to MDS are discussed. This is followed that prevents them from differentiating, maturing, and pro- by a discussion of the classification of the disorders into liferating normally. The stem cell abnormality most often morphologic subgroups according to the 2016 classification involves the myeloid progenitor cell; the lymphocytic cell of the World Health Organization. Each subgroup is then lines are rarely affected. Many of the afflicted stem cells do described with specific features that allow it to be classi- differentiate into mature cells but exhibit flawed and ineffec- fied. Variants are also described. Prognosis and therapy are tive progression so that their numbers decrease in the periph- discussed and the chapter concludes with a discussion of eral blood (cytopenia), a phenomenon known as ineffective myelodysplastic/myeloproliferative overlap syndromes. hematopoiesis. In addition to decreased numbers, the myelo- dysplastic cells develop abnormal morphology and exhibit compromised function. Gradually, the stem cells lose their Introduction ability to differentiate, and in some cases, they transform into cells that are consistent with acute leukemia.2,3 The myelodysplastic syndromes (MDS) are considered pri- Somatic mutations in hematopoietic cells that lead to mary, neoplastic, pluripotential stem cell disorders. They are clonal expansion of the abnormal cells can be detected in characterized by one or more peripheral blood cytopenias people with normal blood counts, without dysplasia, and with prominent maturation abnormalities (dyspoiesis or with no apparent disease. These mutations are commonly dysplasia) in the bone marrow. The peripheral blood cyto- acquired during the aging process. The presence of these penias result from ineffective hematopoiesis and increased mutations confers an increased risk of a hematologic malig- apoptosis as evidenced by an accompanying bone mar- nancy diagnosis. This condition is called clonal hematopoiesis row hyperplasia. These relatively common entities evolve of indeterminate potential (CHIP), analogous to monoclonal progressively, leading to the aggravation of the cytopenias gammopathy of undetermined significance4 (Chapter 28). 558 Chapter 25 On the other hand, there are patients with unexplainable, ABNORMAL KARYOTYPES persistent cytopenia, but no abnormal marrow morphology Nearly every chromosome has been shown to exhibit and no known MDS-associated somatic mutation or abnor- genomic aberration in MDS. Moreover, abnormal cytogenetic mal karyotype. This is known as idiopathic cytopenia of unde- patterns in MDS are heterogeneous; chromosome abnor- termined significance (ICUS). These patients are monitored; malities can present either as the sole aberration in MDS or some will subsequently be diagnosed with MDS or AML.4 can occur as part of a complex karyotype. The most com- Myelodysplastic syndromes are described as either monly encountered abnormal karyotypes in MDS are the primary (de novo origin) or secondary (developing after -5/del(5q), -7/del(7q), +8, del(20q), and -Y, suggesting treatment for another malignancy). The cause of either is that genes located in these regions contribute to the patho- uncertain, but myelodysplastic syndromes are acquired physiology of this syndrome.7,8 Table 25-1 describes the most neoplasms. When the MDS is the primary type, environ- frequently mutated genes in MDS. Many mutations and mental insults such as exposure to genotoxic chemicals karyotypic abnormalities implicated in the development of (benzene), radiation, and viral infection are all etiologic MDS are epigenetic regulators, splicing factors, signaling pro- agents that could contribute to MDS development. teins, and tumor suppressors.9,10 MDS patients often display If MDS develops after chemotherapy with alkylat- more than one functional type of mutation, and only 10% of ing agents or topoisomerase II inhibitors either in conjunc- MDS patients lack any of the commonly aberrant genes. tion with radiation treatment or alone, the MDS is known The cytogenetic abnormalities associated with MDS can as the secondary type.1,3 Regardless of whether the MDS be MDS-defining in a cytopenic patient even in the absence of develops after environmental insult or previous therapy, a diagnostic morphologic dysplasia. However, the abnormality multistep pathophysiology is believed to be necessary for must be demonstrated by conventional karyotyping and not MDS to develop. MDS appears to develop as a result of FISH or gene sequencing. On the other hand, the presence complex interactions between compromised hematopoietic of +8, -Y, or del(20q) is not considered as MDS-defining if stem cells and the microenvironment of the bone marrow.5,6 there are no diagnostic morphologic findings associated with Although nearly 50% of MDS patients present with normal MDS.11 The only cytogenetic or molecular genetic abnormal- chromosomes, genetic instability of the myeloid stem cell ity that defines a specific MDS subtype is del(5q). almost certainly contributes to the development of MDS. −5 and del(5q) -5 and del(5q) are among the most fre- That MDS is primarily a disease present in elderly patients quently noted genetic abnormalities in MDS, found in lends credence to the implication of genomic instability as 10–40% of myelodysplastic syndromes.7,8,9 The WHO clas- a contributing factor toward disease progression. sification of myeloid neoplasms includes an MDS subtype named for this cytogenetic abnormality when it exists as Checkpoint 25.1 the sole aberration: MDS with isolated del(5q). Based on How does the pathophysiology of primary MDS differ from sec- data that shows no adverse effect if there is one additional ondary MDS? chromosomal abnormality in addition to the del(5q), MDS with del(5q) may be diagnosed if there is one additional cytogenetic abnormality except if that abnormality is mono- Cytogenetics, Epigenetics, and Single somy 7 or del(7q).11 Gene Mutations Three commonly deleted regions (CDRs) are found on the long arm of chromosome 5, located among bands The abnormal MDS clone is characterized by altered func- 5q31–33.2,9 The loss of many of the genes in these CDRs tion of genes that result from chromosomal abnormalities, seems to have a role in the pathophysiology of hematopoi- gene silencing (epigenetic changes), or single gene muta- etic malignancies; their mechanisms of action include the tions. “Unbalanced” genetic abnormalities (which result control of cell signaling, transcription, cell cycle, antioxi- in a larger or smaller amount of DNA in the cell, such as dant levels, apoptosis, cell differentiation/proliferation, and trisomies or whole or partial chromosomal deletions) are tumor suppression. Whole or partial losses of chromosome characteristically seen in the MDS and are thought to be 5 are mostly somatically acquired; often they are associated responsible for the ineffective hematopoiesis and cytope- with earlier treatment with radiotherapy, alkylating agents, nias.2 These chromosomal abnormalities are in contrast to or DNA topoisomerase II antagonists.11 the balanced abnormalities that maintain the normal quan- tity of DNA (e.g., reciprocal translocations) seen primarily −7 and del(7q) Karyotypic abnormalities on chromosome in myeloproliferative neoplasms (MPNs) and acute myeloid 7 appear with up to 50% incidence in MDS patients.7,8,9 The leukemia (AML) (Chapters 24 and 26, respectively). More- -7 karyotype presents with severe refractory cytopenias over, many chromosomal alterations involve gene deletions, and increased incidence of severe infections.6 Three distinct suggesting that tumor suppressor genes or DNA repair CDRs have been identified on chromosome 7, one at 7q22, genes are altered (Chapter 23). and the other two at 7q312–33 and 7q35–36.8 It is very likely Myelodysplastic Syndromes 559 that the long arm of chromosome 7 is well populated with deletions of chromosome 5, chromosome 7, or both are fre- tumor suppressor genes.8,9 quently components of a complex karyotype.9 +8 Trisomy 8 occurs with about 5% frequency in MDS. EPIGENETIC CHANGES Individuals with MDS who present with trisomy 8 in their Epigenetic changes to the genome refer to changes in genetic progenitor cells robustly express apoptosis genes as well as information that is not encoded in the DNA sequence itself, compromised immune response genes, which could under- but influenced by molecules “around” the nucleotides lie their MDS phenotype.9 of DNA (Chapter 23). Epigenetic modifications to DNA del(20q) A deletion of the long arm of chromosome 20 is changes the way that the genes are expressed. In the past seen in about 5% of MDS cases. Genes located in this area several years, studies of epigenetic changes to DNA have of the genome that are crucial to normal hematopoietic pro- concentrated mostly on DNA methylation and histone gression include ASXL1 (Table 25-1).7,8,12 modification. DNA methylation is the addition of methyl groups to cytosine-rich regions (CpG regions) commonly −Y MDS patients can present with a loss of the Y chromo- found within gene-promoter regions. CpG hypermethyl- some, but the frequency is variable, and the significance of ation silences the transcript; the contribution of hypermeth- the missing chromosome remains uncertain. -Y is found in ylation of tumor suppressor gene promoters in MDS has many malignant diseases but also in healthy, aging men.13 been established for many years. Complex Karyotype Approximately 20% of patients Histone acetylation adds acetyl groups to DNA, relax- exhibiting primary MDS and nearly 90% of MDS patients ing it and making it more available to transcriptional diagnosed with treatment-related MDS exhibit a complex machinery, which allows genes to be expressed. Histone karyotype. One of the significant differences between pri- methylation can be either repressive or activating, depend- mary and secondary MDS is the degree of genomic abnor- ing on the gene involved. mality between the two. Primary MDS is associated with More recently, the concept of epigenetic changes to single chromosomal abnormalities, whereas complex chro- DNA has been expanded to include demethylation, ubiq- mosomal abnormalities are often seen in secondary MDS.9 uitinylation, microRNAs, and long non-coding RNAs.14,15,16 Complex karyotypes generally involve the presence of more The putative mechanisms of epigenetic influence on the than three chromosomal abnormalities. Whole or partial development of MDS include increased self-renewal and Table 25.1 Significant Mutated Genes in MDS Gene Location Frequency Significance ASXL1 20q11.1 ∼11% Epigenetic control (histone modification) BCOR Xp11.4 ∼5% Transcription repressor CBL 11q23 2% Negative regulator of signal transduction; frequent in MPN/MDS DNMT3A 2p23 ∼8% Epigenetic control (CpG methylation) ETV6 12p13 Less than 5% Transcription factor EZH2 7q36 ∼5% Tumor suppressor signaling and prevention of apoptosis IDH1/2 2q33/15q26 Less than 5 % Cell metabolism; epigenetic control (DNA methylation) K-RAS 12p12.1 Less than 1% Activating mutations stimulate MAPK signaling JAK2 9p24 ∼5% Kinase signaling NPM1 5(q35) ∼5% Putative proto-oncogene; product inactivates p53 N-RAS 1p13.2 Less than 5 % Activating mutations stimulate MAPK signaling PRPF8 17p13.3 Less than 5 % Splicing factor RUNX1 21q22.12 ~ 10 % Transcription factor important for hematopoietic differentiation SETBP1 18q21.1 2–5% Proto-oncogene; mutations associated with proliferation SF3B1 2q33 ∼30% Splicing factor SRSF2 17q25 Less than 15% Splicing factor TET2 4q24 ∼20% DNA methylation; tumor suppressor TP53 17p13.1 Less than 10% Transcription factor; tumor suppressor U2AF1 21q22 ~ 7 % Splicing factor ASXL1, additional sex combs like 1; BCOR, BCL6 corepressor; CBL, Casitas B-lineage lymphoma; ETV6, ets variant 6; EZH2, enhancer of zeste 2 polycomb repressive complex 2 s ubunit; IDH1/2, isocitrate dehydrogenase 1/2; JAK2, janus kinase 2; NPM1, nuclephosmin 1; N-RAS, neuroblastoma rat sarcoma; K-RAS, Kirsten rat sarcoma; PRPF8, pre-mRNA processing factor 8; RUNX1, runt-related transcription factor 1; SF3B1, splicing factor 3b; SRSF2, serine/arginine-rich splicing factor 2; TET2, tet methylcytosine dioxygenase 2; TP53, tumor protein p53; U2AF1, U2 small nuclear RNA auxiliary factor 1. 560 Chapter 25 inhibited differentiation of the hematopoietic stem cell (HSC). Multiple genes, including those for cell cycle regu- Incidence lators, tumor suppressors, DNA repair, and apoptosis, have MDS occurs primarily in individuals older than 50 years of been identified as targets for methylation. Silencing any of age and has a slight male predominance, except in the form these critical genes could contribute to the development of with isolated 5q deletion, which predominates in women. MDS; it is believed that epigenetic changes constitute a sig- The incidence rate of MDS in the United States is 4.4 per nificant factor in the progression of MDS into AML.14,15,16 100,000, but the incidence increases with age. The median The influence of epigenetic changes in the development age at diagnosis is 65–70 years.2 The frequency of MDS diag- of MDS will certainly be more fully elucidated in the future. noses seems to be rising. This rise could be the result of an Treatment with drugs that modify epigenetic transforma- increase in the awareness of MDS on the part of physicians tion, such as hypomethylating agents, are examined in the and clinical laboratory professionals, in the application of section on MDS treatment. diagnostic procedures in elderly individuals, or in the num- ber of elderly in the population. ONCOGENES AND TUMOR SUPPRESSOR GENES Control of cellular differentiation, maturation, and prolifer- ation occurs through properly functioning proto-oncogenes and tumor suppressor genes

(Chapter 23) and when these Clinical Presentation genes are mutated, neoplasia can result.3 Although muta- The most frequent presenting symptoms, fatigue and tions in the proto-oncogenes JAK2 (Chapter 24), RAS, and weakness, are related to an anemia that is nonresponsive RUNX1 and the tumor suppressor gene TP53 are most com- to treatment. Less commonly, hemorrhagic symptoms and monly observed, the overall incidence of single gene muta- infection precede diagnosis. These symptoms are related to tions is low7,11,16,17 (Table 25-1). thrombocytopenia and neutropenia. Many individuals can N- and K-RAS mutations have been observed in 10–15% be asymptomatic, and their disease is discovered on rou- of patients with MDS. The most common mutation is a tine laboratory screening.18 Infection is a common complica- single base change in codon 12 of the N-RAS (neuroblas- tion in patients diagnosed with MDS, particularly for those toma-ras) family. RAS proteins normally function in growth patients diagnosed with an aggressive MDS subgroup or factor-mediated signaling, and mutation is associated with who present with neutropenia (less than 1 * 109/L). Infec- a higher risk of AML transformation and a worse progno- tion and bleeding are the most common causes of death.18 sis. RUNX1 is a regulatory transcription factor for hema- Splenomegaly and/or hepatomegaly are uncommon. topoietic differentiation, and point mutations have been observed in 20% of MDS patients. Mutations in the tumor suppressor gene TP53 (Chapter 23) appear in approximately 20% of MDS patients Laboratory Evaluation and are most commonly associated with secondary MDS. MDS presents with cytopenia(s) and a range of abnormal Normal p53 protein functions to maintain genetic stability morphologic features (dysplasia) that can be demonstrated in cells and TP53 mutations in MDS patients are linked to a on stained peripheral blood and bone marrow smears. poor prognosis.7,11,16,17 The quality of the peripheral blood smear and bone mar- row specimen is very important as poor quality smears can Proliferation Abnormalities result in misinterpretation of dysplasia. Smears should be made from blood that has been exposed to anticoagulants In vitro studies suggest that excessive premature intramed- for less than 2 hours. Criteria for classification of MDS into ullary cell death of hematologic precursors via apoptosis subtypes based on morphology and cell counts are pre- contributes to ineffective hematopoiesis and peripheral sented in a later section of this chapter. Included here are blood cytopenias seen in early MDS. The various gene the general hematologic features used to initially define the alterations of the MDS clone result in an intrinsic increase presence of an MDS (Table 25-2). in susceptibility of the clone to apoptosis.2,5 Increased levels of tumor necrosis factor (TNF)@a in MDS patients has been linked to increased apoptosis of the MDS clone and normal Peripheral Blood hematopoietic cells.2,5 Progression to leukemia is associated The peripheral blood characteristics of MDS are cytope- with a reduction in apoptosis, thereby allowing the expan- nias and dysplasia. Cytopenias are defined as hemoglo- sion of the neoplastic clone. bin less than 10 g/dL, absolute neutrophil count less than 1.8 * 109/L, and platelets less than 100 * 109/L. Anemia Checkpoint 25.2 is the most consistent finding, occurring in more than 80% How do epigenetics contribute to the pathophysiology of MDS? of the cases. Bicytopenia occurs in 30% of the cases and pan- cytopenia in 15%.19 Less commonly, an isolated neutropenia Myelodysplastic Syndromes 561 Table 25.2 Hematologic Abnormalities in Myelodysplastic Syndromes Erythroid Series Myeloid Series Thrombocyte Series Peripheral blood Anemia Neutropenia Thrombocytopenia or thrombocytosis Anisocytosis, dimorphism Hypogranulation, abnormal granulation Giant forms Poikilocytosis Shift to the left Hypogranulation Macrocytes, oval macrocytes Nuclear abnormalities Micromegakaryocytes Basophilic stippling Pseudo-Pelger-Huët Functional abnormalities Nucleated RBCs Ring nuclei Howell-Jolly bodies Monocytosis Sideroblasts Reticulocytopenia Bone marrow Megaloblastoid erythropoiesis Abnormal granules in promyelocytes Micromegakaryocytes Nuclear fragmentation and budding Increase in granular and agranular blasts Megakaryocytes with multiple separated Karyorrhexis Absence of secondary granules nuclei Multiple nuclei Nuclear abnormalities Large mononuclear megakaryocytes Defective hemoglobinization Decreased myeloperoxidase Hypogranulation or large abnormal gran- ules in megakaryocytes Vacuolization Auer rods in blasts Ring sideroblasts or thrombocytopenia is found. Dysplastic features of one or a common initial finding in the refractory anemia with more cell lines are typical. Because of the critical importance ring sideroblasts (RARS) subgroup. Reticulocytes show of recognizing dysplasia in the diagnosis of MDS, smears an absolute decrease in number but can appear normal should be made from samples that have been anticoagu- if only the uncorrected or relative number (percent) is lated for 2 hours or less. Morphologic changes from pro- reported. longed exposure to anticoagulant can easily be confused In addition to anemia, qualitative abnormalities indica- with dysplastic changes in cells. Functional abnormalities tive of dyserythropoiesis are present. These include aniso- of hematologic cells are also common. Studies show that cytosis, poikilocytosis, basophilic stippling, Howell-Jolly the higher the degree and number of cytopenias, the worse bodies, and nucleated erythrocytes. Often hemoglobin F is the prognosis.19 increased (5–6%) and distributed in a heterogeneous pattern. Acquired hemoglobin H has also been found in MDS.19 Other ERYTHROCYTES erythrocyte changes include altered A, B, and I antigens, The degree of anemia is variable, but the hemoglobin is enzyme changes, and an acquired erythrocyte membrane less than 10 g/dL. The erythrocytes are usually macrocytic change similar but not identical to that found in paroxysmal (Figure 25-1) and less often normocytic. Oval macrocytes nocturnal hemoglobinuria (PNH)19 (Chapter 17). similar to those in megaloblastic anemia are frequently present, but patients have normal vitamin B12 and folate LEUKOCYTES levels. A dimorphic anemia with both oval macrocytes Neutropenia is the second most common cytopenia or normocytes and microcytic hypochromic cells is also observed in MDS and is present in approximately 50% of patients at the time of diagnosis.20 Neutropenia can be accompanied by a shift to the left with the finding of meta- myelocytes and myelocytes on the peripheral blood smear. Blasts and promyelocytes can also be present. Morphologic abnormalities in granulocytes indicative of dysgranulopoiesis are considered a hallmark finding in MDS. Dysgranulopoiesis is characterized by agranular or hypogranular neutrophils, persistent basophilia of the cyto- plasm, abnormal appearing granules, hyposegmentation (pseudo-Pelger-Huët) (Figure 25-2), or hypersegmentation of the nucleus and donut- or ring-shaped nuclei. Care should be taken to distinguish neutrophils with the pseudo-Pelger- Huët anomaly and hypogranulation from lymphocytes and Figure 25.1 A peripheral blood film of a patient with MDS. to distinguish neutrophilic-band forms with hypogranula- Note macrocytic cells and anisocytosis (Peripheral blood, Wright- tion from monocytes. Neutrophils can also demonstrate Giemsa stain, 1000* magnification). enzyme defects, such as decreased myeloperoxidase (MPO) 562 Chapter 25 Figure 25.4 Arrow points to a large agranular platelet in a Figure 25.2 Peripheral blood film from a patient with MDS peripheral blood film from a patient with MDS (Wright-Giemsa stain, showing neutrophils with the pseudo-Pelger-Huët nucleus. One 1000* magnification). cell’s nucleus is eye glass shaped (pince-nez) and the other is single lobed. The nuclear chromatin is condensed, and the cells contain MICROMEGAKARYOCYTES granules, making identification possible. In many cases, these types of neutrophils are agranular, making differentiation of them Sometimes circulating micromegakaryocytes (small abnor- from lymphocytes difficult (Peripheral blood, Wright-Giemsa stain, mal megakaryocytes), also called dwarf megakaryocytes, 1000* magnification). can be found in MDS and MPNs. Micromegakaryocytes can be difficult to identify and are frequently overlooked and decreased leukocyte alkaline phosphatase (LAP). In unless cytoplasmic tags or blebs are present (Figure 25-5). some cases, neutrophils exhibit severe functional impair- Micromegakaryocytes are believed to represent abnor- ment, including defective bactericidal, phagocytic, or che- mal megakaryocytes that have reduced ability to undergo motactic properties. Absolute monocytosis is a common endomitosis (megakaryocytes undergo a unique matura- finding even in leukopenic conditions. tion process whereby the DNA content duplicates without cell division, resulting in a polyploid nucleus (endomitosis; PLATELETS Chapter 31). Most micromegakaryocytes have a single-lobed Qualitative and quantitative platelet abnormalities are nucleus and are the size of a lymphocyte. Morphologi- often present. The platelet count can be normal, increased, cally, they can be confused with lymphocytes but can be or decreased. Approximately 25–50% of patients have distinguished by cytoplasmic tags of one or more platelets mild to moderate thrombocytopenia when diagnosed.21 attached to a nucleus. Some can have pale blue, foamy, or Giant platelets, hypogranular platelets, and platelets with vacuolated cytoplasm that resembles a nongranular plate- large fused granules can be seen in the peripheral blood let. The nuclear structure is variable, but many cells have (Figures 25-3 and 25-4). Functional platelet abnormalities include abnormal adhesion and aggregation. As a result, platelet function tests can be abnormal. Figure 25.5 Arrow points to a micromegakaryocyte in a peripheral blood film from a patient with MDS. Note the dense chromatin structure and the irregular rim of cytoplasm with Figure 25.3 Arrows point to large platelets in a peripheral cytoplasmic tags. An agranular platelet in the field matches the blood film from a patient with MDS (Wright-Giemsa stain, 1000* appearance of the micromegakaryocyte cytoplasm (Wright-Giemsa magnification). stain, 1000* magnification). Myelodysplastic Syndromes 563 very densely clumped chromatin and stain dark blue-black complex and variable, but one of their functions is to secrete with Wright-Giemsa stain. Others can have a finer, looser growth factors and cytokines that are necessary for hema- chromatin. These cells also are found in the bone marrow. topoietic cell development and survival. It is presumed that atypical synergy of the hematopoietic cells and the ancil- Checkpoint 25.3 lary bone marrow cells contribute to the ineffective hema- How does the typical peripheral blood picture in MDS differ from topoiesis observed in MDS. An unfavorable bone marrow that in aplastic anemia (Chapter 16)? microenvironment may be hypoxic, inflamed, lacking in appropriate cytokines and/or growth factors, and gener- ally contrary to normal hematopoietic development.5,6 Collection of bone marrow samples are necessary for CASE STUDY (continued from page 557) morphological examination since identification of the char- The results of the CBC on Hancock were: acteristic dyshematopoietic element of MDS is important in RBC 1.60 * 1012/L WBC Differential establishing a diagnosis. Moreover, bone marrow aspirate provides material for molecular diagnostic testing, which Hb 5.8 g/dL (58 44% segmented may be essential for diagnosis. In most cases, the bone g/L) neutrophils marrow is hypercellular; the hypercellular bone marrow in Hct 0.17 L/L (17%) 7% band conjunction with peripheral blood pancytopenia is a nota- neutrophils ble feature of myelodysplastic syndromes. Asynchrony in WBC 10.5 * 109/L 6% lymphocytes nuclear and cytoplasmic development, known as dyshema- Platelets 39 * 109/L 28% eosinophils topoiesis, is evident in all the hematopoietic cell lines.2,21 In MDS, the number of myeloblasts is less than 20%. Reticulocyte 0.8% 1% metamyelocytes count 1% myelocytes DYSERYTHROPOIESIS The most common bone marrow finding in MDS is nuclear- 9% promyelocytes cytoplasmic asynchrony similar to that seen in megaloblas- 4% blasts tic anemia (Chapter 15). However, the chromatin is usually The neutrophilic cells show marked hypo- hypercondensed. This nuclear chromatin pattern is often segmentation and hypogranulation. Red blood described as megaloblastoid (Figure 25-6). The abnormal cell (RBC) morphology includes anisocytosis and erythrocytic maturation is not responsive to vitamin B12 or poikilocytosis, teardrop cells, ovalocytes, and folic acid therapy. Giant, multinucleated erythroid precur- schistocytes. sors can be found (Figure 25-7). Other nuclear abnormalities include fragmentation, abnormal shape, budding, karryo- 1. In what cell lines is cytopenia present? hexis, and irregular staining properties. The cytoplasm of 2. What abnormalities are present in the differential? erythroid precursors can show defective hemoglobiniza- tion, vacuoles, and basophilic stippling. The presence 3. What evidence of dyspoiesis is seen in the leuko- of ring sideroblasts, reflecting the abnormal erythrocyte cyte morphology? metabolism, is a common finding. The International Work- 4. Calculate the mean cell volume (MCV). What ing Group on Morphology of MDS (IWGM-MDS) defines peripheral blood findings are helpful to rule out ring sideroblasts as erythroblasts in which at least five megaloblastic anemia? 5. What features of the differential resemble chronic myeloid leukemia (CML)? What helps to distin- guish this case from CML? Bone Marrow The bone marrow microenvironment contains pluripotent hematopoietic stem cells, capable of differentiating into all the peripheral blood cells. There are many other types of cells in the bone marrow that are ancillary to the devel- opment of hematopoietic progenitor cells; these include Figure 25.6 Megaloblastoid erythroblasts in the bone marrow osteoblasts, vascular endothelial cells, mesenchymal stem of a patient with MDS. Note the condensed chromatin and Howell- cells, and macrophages. The roles of these ancillary cells are Jolly bodies (Wright-Giemsa stain, 1000* magnification).

564 Chapter 25 Figure 25.8 Bone marrow from a patient with MDS. Note the metamyelocytes (arrows) and bands with a lack of secondary granules. The bands have light bluish-pink cytoplasm, and the Figure 25.7 Arrow points to a multinucleated metamyelocytes have a darker bluish cytoplasm (Wright-Giemsa polychromatophilic erythroblast in the bone marrow of a patient stain, 1000* magnification). with MDS. It is also megaloblastoid (Wright-Giemsa stain, 1000* magnification). DYSMEGAKARYOPOIESIS Megakaryocytes can be decreased, normal, or increased. mitochondrial iron deposits encircle at least one-third or The presence of micromegakaryocytes, large mononuclear more of the circumference of the nucleus.22 megakaryocytes, and megakaryocytes with other abnormal nuclear configurations (Figure 25-9) reflect abnormalities in DYSGRANULOPOIESIS maturation. Megakaryocyte nuclei can be hyperlobulated Granulopoiesis is usually normal to increased in patients or hypolobulated and can show multiple widely separated with MDS unless the overall marrow is hypocellular. Abnor- nuclei. The lack of granules or presence of giant abnormal mal granulocyte maturation (dysgranulopoiesis), however, granules is also characteristic. Although dysmegakaryo- is almost always present (Figure 25-8). One of the major poietic morphology is potentially useful for diagnosis and findings of dysgranulopoiesis in the bone marrow is abnor- prognostication, identification of dysplasia is subject to mal staining of the primary granules in promyelocytes and individual interpretation.23 myelocytes. Sometimes the granules are larger than normal, and at other times are absent. Secondary granules can be absent in myelocytes and other more mature neutrophils, Molecular Diagnostics giving rise to the hypogranular peripheral blood neutro- Morphology contributes greatly to the diagnosis of MDS, phils. Irregular cytoplasmic basophilia with a dense rim of but genomic instability figures importantly in the develop- peripheral basophilia is also characteristic. Nuclear abnor- ment of myelodysplastic syndromes. Although it is becom- malities similar to those found in the peripheral blood ing increasingly essential to obtain information about a granulocytes can be present in bone marrow granulocytes. patient’s genetic profile for prognosis of MDS and overall a b Figure 25.9 (a) Abnormal megakaryocyte (arrow) from a patient with MDS. (b) Arrow points to a micromegakaryocyte in the bone marrow of a patient with MDS. Note the megaloblastoid features of the surrounding cells (Both are bone marrow; Wright-Giemsa stain, 1000* magnification). Myelodysplastic Syndromes 565 survival, at the present time, gene mutations are insufficient for a diagnosis of MDS and cannot be used as presumptive 6. Which of the hematopoietic cell lines exhibit evidence for a diagnosis. Information about chromosome dyshematopoiesis in the bone marrow? aberrations provides useful information to clinicians for diagnosing, prognosticating, tracking, and treating MDS. 7. How would you classify the bone marrow To date, it hasn’t been possible to assign specific genetic cellularity? abnormalities to the development of MDS, but certain aber- 8. What does the M:E ratio indicate? rations repeatedly have been identified in MDS patients. In broad terms, mutations associated with MDS affect 9. Identify at least two features of the bone marrow DNA methylation, DNA repair, chromatin/histone modi- that are compatible with a diagnosis of MDS. fication, transcription regulation, tumor suppression, and 10. What chemistry tests would be helpful to rule signal transduction.11,14–17 It is interesting to note that the out megaloblastic anemia? genetic profiles of patients with primary versus secondary MDS differ; those with primary MDS tend to have single chromosomal abnormalities, whereas patients with second- ary MDS are more likely to exhibit complex chromosomal abnormalities.9 DNA amplification, hybridization tech- Blast and Precursor Cell niques, and gene sequencing are molecular techniques that are employed to help identify genetic irregularities. Cyto- Classification genetic studies (karyotyping; Chapter 41) are also useful in For patients with MDS, the blast count appears to be the the diagnosis of MDS. most important prognostic indicator of survival and pro- gression to acute leukemia. The maximum number of blasts Additional Laboratory Evaluation compatible with a diagnosis of MDS is 19%, whereas the Serum iron and ferritin levels are normal or increased, minimum criterion for a diagnosis of acute leukemia is at and the total iron-binding capacity (TIBC) is normal or least 20% blasts. Correctly identifying blasts and type and decreased, distinguishing MDS from iron-deficiency ane- degree of dysplasia is important in MDS because these char- mia (IDA). Cobalamin (vitamin B acteristics are the basis of classifying MDS subtypes and dif- 12) and folic acid levels are normal to increased, a feature that helps to differentiate ferentiating them from AML with myelodysplasia-related MDS with megaloblastoid features from megaloblastic ane- changes (AML-MRC; Chapter 26).24 It is essential that suffi- mias with the typical megaloblastic features. Lactic dehy- cient cells be evaluated in order to accurately identify blasts drogenase (LD) and uric acid levels can be increased as a and precursor cells in suspected cases of MDS.25 The blast result of ineffective hematopoiesis. count in myeloid neoplasms is expressed as a percentage of all nucleated cells in the bone marrow, including eryth- roblasts. A total of 500 nucleated cells should be counted Checkpoint 25.4 in the bone marrow and 200 nucleated cells counted in the Why is serum cobalamin serum folate level or bone marrow iron peripheral blood. If cytopenia in the peripheral blood is stain important in diagnosing MDS? severe, a buffy coat should be used to perform the count. In 2005 and 2006, IWGM-MDS developed new diagnostic cri- teria for MDS and redefined MDS myeloblast morphology. CASE STUDY (continued from page 563) In addition to redefining myeloblast morphology, it pro- posed criteria necessary to define normal and dysplastic A bone marrow was performed on Hancock. The promyelocytes and ring sideroblasts. The morphological marrow showed a cellularity of about 75%. There categories of early cells in MDS established by the IWGM- was myeloid hyperplasia with 9% blasts, 26% MDS are granular myeloblasts, agranular myeloblasts, promyelocytes, 18% myelocytes, 6% metamyelo- normal promyelocytes, dysplastic promyelocytes, and ring cytes, 4% bands, and 37% eosinophils. The ratio of sideroblasts22 (Figure 25-10). myeloid-to-erythroid precursors (M:E) was 12:1. The myelocytes were hypogranular, and some had two nuclei. The erythroid precursors showed Myeloblasts megaloblastoid changes. Megakaryocytes were Myeloblasts are defined as either granular or agranu- adequate in number but showed abnormal forms lar. Granular blasts possess azurophilic granules and can with nuclear separation and single nucleated exhibit Auer rods, although this is an unusual finding. Gran- forms. ular blasts include the former type II and type III blasts, but today there is no differentiation of blasts based on the 566 Chapter 25 Dysplastic Agranular blast Granular blast Promyelocyte promyelocyte • High • Same morphologic • Central or eccentric • Irregular cytoplasmic nuclear/cytoplasmic characteristics as nucleus with nucleoli basophilia ratio agranular blast • Clearly visible Golgi • Poorly developed • Nucleoli and fine • Cytoplasmic granules zone Golgi zone nuclear chromatin • Azurophilic granules • Hyper- or • Basophilic cytoplasm • Basophilic cytoplasm hypogranularity or irregular distribution (clumps) of granules Figure 25.10 Blasts and promyelocytes as defined by the IWGM-MDS. number of granules observed in the cytoplasm. Agranular which can complicate the morphologic differentiation of blasts correspond to the earlier type I classification of MDS granular blasts and promyelocytes. Dysplastic promy- blasts. Whether granular or agranular, the blasts of MDS elocytes differ from normal promyelocytes in that their should exhibit visible nucleoli, fine nuclear chromatin, scant chromatin can vary from fine and lacy to coarse, their azu- basophilic cytoplasm, and, very importantly, should lack a rophilic granules can be of variable quantity and distribu- Golgi zone22,23 (Figure 25-11). tion, their cytoplasm can stain irregularly, and their Golgi zone may be only faintly visible.22,23 Promyelocytes Differentiating between granular blasts and promyelocytes Ring Sideroblasts is essential. Promyelocytes present with most of the features The IWGM-MDS has defined three types of sideroblasts, of granular blasts, including fine azurophilic cytoplasmic types 1, 2, and 3. granules. The IWGM-MDS has determined that the distin- Type 1 sideroblasts are defined as possessing fewer guishing feature between the two cells is the presence of a than five siderotic cytoplasmic granules. distinct nonstaining, perinuclear Golgi zone seen in pro- Type 2 sideroblasts have five or more siderotic granules, myelocytes. Promyelocytes of MDS can exhibit dysplasia, but they lack perinuclear distribution. Type 3 sideroblasts a b Figure 25.11 (a) Agranular myeloblast. (b) Granular myeloblasts (arrows) (Both: peripheral blood, Wright-Giemsa stain, 1000* magnification). Myelodysplastic Syndromes 567 exhibit five or more siderotic granules arranged around the nucleus of the erythroblast so that they encircle at least one- Classification third of the nuclear perimeter. A system to describe, define, and name diseases is impor- The IWGM-MDS recommends that a minimum of tant so that they can be diagnosed, treated, and studied. In 100 nucleated erythrocytes at all stages of maturation be 1982, the French-American-British (FAB) group proposed a included in the count. Note in the section “Classification” classification scheme for the myelodysplastic syndromes. that the WHO still defines the required number of ring sid- This classification defined five subgroups based on morpho- eroblasts as 15%.22,23 logical characteristics, such as the blast count and degree of dyspoiesis in the peripheral blood and bone marrow.2,24 Although the FAB classification system was the bench- Checkpoint 25.5 mark for diagnosing MDS for the past two decades, it did not Why is it important to correctly identify the number of blasts when evaluating the peripheral blood or bone marrow smear of incorporate the newer diagnostic technologies such as cytoge- a patient suspected of having MDS? netics and immunophenotyping.2 In 2001, the WHO published a new classification system developed by a group of American and European pathologists, hematologists, and oncologists. Immunological Identification This new system incorporated morphology, immunopheno- of Blasts type, and genetics with clinical and prognostic features into a classification of all neoplastic diseases of the hematopoietic In 2008, a new classification system devised by the WHO and lymphoid tissues (Chapter 23). This resulted in signifi- included immunophenotype abnormalities in the bone cant changes in the classification of MDS. The WHO system marrow cells of suspected MDS as an additional diagnostic of classification is updated and revised periodically. Major and prognostic tool (this classification system was updated changes in the 2008 revision eliminated previous categories in 2016). Immunophenotyping with flow cytometry detects of MDS and added new groups; the current subclasses of MDS the expression of antigen abnormalities in all cell lines. It are listed in Table 25-4. The most recent 2016 update mostly is most useful when marrow morphology and cytogenet- changes the terminology used to identify the various sub- ics are inconclusive, and to distinguish MDS cases with groups included in MDS. Terms such as “refractory anemia” a hypoplastic bone marrow from other bone marrow fail- and “refractory cytopenia” have been replaced with the term ure disorders, such as aplastic anemia.24,25 Aberrant anti- “myelodysplastic syndrome” amended with the modifiers: genicity observed in MDS includes lymphoid antigens single/multilineage dysplasia, ring sideroblasts (and single/ on myeloid cells, over- or under-expression of expected multilineage dysplasia), excess blasts, unclassifiable, and antigens, the presence of immature antigens on mature del(5q).11 A provisional category, refractory cytopenia of child- cells, and expression of mature antigens on immature cells hood (RCC), was added in the 2008 revision and remains in (Table 25-3).24,25 place in the 2016 update. The 2016 WHO Classification System Table 25.3 Characteristic Flow Cytometric Analysis of Myelodysplastic Syndromes Abnormal Expression Decreased Expression CD5, CD7, CD13, CD33, CD56 Progenitors, neutrophils, monocytes CD10 Neutrophils CD36, CD71 Erythrocyte precursors Side Scatter Neutrophils, monocytes CD14/CD36, CD16, CD36 Monocytes CD45 Progenitors, neutrophils, monocytes CD19 Neutrophils, Monocytes CD11b, CD14, HLA-DR Monocytes Increased Expression Loss of Synchronized Expression CD11b, CD15 Progenitors CD11b/CD13, CD11b/CD16, CD13/ Neutrophils CD36 Neutrophils CD16, CD15/CD10 Erythrocyte precursors CD117 Neutrophils, monocytes CD71/CD235 HLA-DR Neutrophils Side Scatter Progenitors CD11b/HLA-DR, CD34 Neutrophils, monocytes Decreased Populations Increased Populations CD19+/CD10+ B@cells CD34+/CD19@ Progenitors CD19+/CD34+ B@cells CD34+/CD38@/dim Progenitors CD117+ Progenitors CD117+ Erythrocyte progenitors CD34+/HLA@DR Progenitors 568 Chapter 25 Table 25.4 2016 World Health Organization Classification Criteria for Myelodysplastic Syndromes Classification Peripheral Blood Bone Marrow MDS with single lineage Unicytopenia or bicytopenia Unilineage dysplasia dysplasia (MDS-SLD) Rare or no blasts (less than 1%) 10% or more of cells in affected lineage are dysplastic No Auer rods Less than 5% blasts, less than 15% ring sideroblast No Auer rods MDS with ring sideroblasts (MDS-RS) • MDS-RS and single lineage dys- Anemia Erythroid dysplasia plasia (MDS-RS-SLD) Rare or no blasts (less than 1%) Less than 5% other blasts No Auer rods 15% or more ring sideroblasts • MDS-RS and multilineage Anemia No Auer rods dysplasia (MDS-RS-MLD) Rare or no

blasts (less than 1%) Dysplasia in 10% or more of cells of two or more lineages No Auer rods less than 5% other blasts 15% or more ring sideroblasts No Auer rods MDS with multilineage Uni-, bi-, or pancytopenia Dysplasia in 10% or more of cells of two or more lineages dysplasia (MDS-MLD) Rare or no blasts (less than 1%) Less than 5% blasts in marrow No Auer rods Variable percentage of ring sideroblasts Less than 1 * 109/L monocytes No Auer rods Refractory anemia with excess blasts (MDS-EB) • MDS-EB-1 Uni-, bi-, or pancytopenias Unilineage or multilineage dysplasia Less than 5% blasts 5–9% blasts • MDS-EB-2 No Auer rods No Auer rods Uni-, bi-, or pancytopenia Unilineage or multilineage dysplasia 5–19% blasts 10–19% blasts { Auer rods { Auer rods MDS with isolated del(5q) Anemia and/or neutropenia Normal to increased megakaryocytes with hypolobulated nuclei Rare or no blasts Unilineage or multilineage dysplasia less than 5% blasts No Auer rods Isolated del(5q) cytogenetic abnormality No Auer rods MDS, unclassifiable (MDS-U) Uni-, bi-, or pancytopenia Unequivocal dysplasia in less than 10% of cells lines less than 5% blasts Uni-, bi-, pancytopenia No ring sideroblasts Rare or no blasts (less than 1%) Cytogenetic abnormalities characteristic of MDS Childhood MDS Less than 2% blasts; dysplasia in Less than 5% blasts; hypocellular Clusters of 20 or more erythroid precursors • Refractory cytopenia of childhood neutrophils More than 10% cells with uni-dysplasia, bi-, or multilineage dysplasia criteria for each subgroup of MDS are shown in Table 25-4. • MDS-RS and single lineage dysplasia (MDS-RS-SLD) Studies to evaluate the clinical usefulness of the WHO clas- • MDS-RS and multilineage dysplasia (MDS-RS-MLD) sification conclude that it clearly identifies more homogenous 3. MDS with multilineage dysplasia (MDS-MLD) subgroups and enables clinicians to select the best treatments and better predict prognosis and clinical responses.11 At this 4. MDS excess blasts (MDS-EB) time, most clinicians have adopted the 2016 WHO Classifica- • MDS-EB-1 tion System, and it is followed in this chapter. • MDS-EB-2 5. MDS with isolated del(5q) 6. MDS, unclassifiable Description of MDS 7. Childhood MDS Subgroups • Refractory cytopenia of childhood (provisional) The 2016 WHO Classification System identifies the follow- MDS with Single Lineage Dysplasia ing myeloplastic syndromes: (MDS-SLD) 1. MDS with single lineage dysplasia (MDS-SLD) The category MDS with single lineage dysplasia is for 2. MDS with ring sideroblasts (MDS-RS) cases with isolated cytopenia or bicytopenia accompanied Myelodysplastic Syndromes 569 by unilineage dysplasia. It includes refractory anemia MDS with Multilineage Dysplasia (RA) and less common cases of refractory neutropenia and refractory thrombocytopenia. These cases together make up (MDS-MLD) approximately 10–20% of all types of MDS; refractory neu- MDS with multilineage dysplasia (MDS-MLD) is char- tropenia and refractory thrombocytopenia represent only acterized by dysplastic features in at least 10% of the cells 1–2%. In general, these cases have an extended course with in two or more cell lineages, less than 5% blasts in the bone the rate of progression to AML of less than 5% at 5 years. marrow, and less than 1% blasts in the peripheral blood. Most patients present with a normocytic, normochomic, or This is one of the most common subgroups, representing sometimes macrocytic anemia. Variation in size and shape 30–40% of all MDS cases. Studies have shown that individ- of the red blood cells is common. Less than 1% blasts are uals with MDS-MLD have a worse prognosis than those present in the peripheral blood and less than 5% blasts with dysplasia in only one lineage; thus, a new subgroup are in the bone marrow. The bone marrow is hypercellular was added in 2001.25 Patients usually display cytopenias in because of increased erythropoiesis or sometimes normo- two or more lineages and have less than 1 * 109/L mono- cellular. Dyserythropoiesis is present without prominent cytes present in the peripheral blood. Ring sideroblasts can ring sideroblasts. In MDS with single lineage dysplasia, exceed 15% of the nucleated red blood cells in the marrow. dysplasia is seen in less than 1–2% in the granulocytic and Chromosomal abnormalities can be present in up to 50% of thrombocytic cells. cases in this category, some being complex. The frequency Chromosomal analysis is frequently helpful in diagnos- of evolution to AML is approximately 10% within 2 years ing MDS with single lineage dysplasia because of the mini- of onset. mal morphologic changes in some cases. Excluding other reactive causes of cytopenias and associated dysplasia is also important. Patients with pancytopenia and morpho- MDS with Excess Blasts (MDS-EB) logic dysplasia of only one cell line should be classified as In MDS with excess blasts (MDS-EB), cytopenia occurs MDS, unclassified rather than this category. in at least two lineages and conspicuous qualitative abnor- malities are in all three lineages. The anemia is normocytic or slightly macrocytic with reticulocytopenia. Evidence MDS with Ring Sideroblasts of dysgranulopoiesis is prominent. Monocytosis without (MDS-RS) leukocytosis can be present, but the absolute monocyte count does not exceed 1 * 109/L, and serum and urinary The 2016 revision of the WHO classification of MDS changes lysozyme levels are normal. Platelet abnormalities include the name of refractory anemia with ring sideroblasts to giant forms, abnormal granularity, and functional aberra- MDS with ring sideroblasts (MDS-RS) and divides this tions. Sometimes circulating micromegakaryocytes can be subgroup further, into MDS-RS with single lineage dyspla- found. sia and MDS-RS with multilineage dysplasia.22 Mutations to The bone marrow is hypercellular but less often is a spliceosome gene SB3B1 is often seen in MDS, it appears normocellular with varying degrees of granulocytic and to be an early mutation event in the development of MDS. erythrocytic hyperplasia. All three lineages show signs of This mutation correlates well with the appearance of ring dyshematopoiesis. Blast numbers below 20% define MDS; sideroblasts. The presence or absence of a mutated SF3B1 blasts in excess of this number changes the neoplasm to gene and the number of ring sideroblasts both contribute to AML. Abnormal promyelocytes can be present. These the inclusion of an MDS syndrome into the MDS-RS class; abnormal cells have blast-like nuclei with nucleoli and no if there are between 5 and 15% ring sideroblasts among the chromatin condensation, and the cytoplasm contains large erythroid precursors, the SF3B1 mutation must be present bizarre granules. Ring sideroblasts may be increased, but for inclusion in MDS-RS. If there are at least 15% ring sid- the elevated blast count differentiates MDS-EB from MDS- eroblasts, the presence of mutated SF3B1 becomes irrelevant RS. In some cases, differentiating MDS-EB from acute leuke- since the presence of at least 15% ring sideroblasts qualifies mia is difficult. Serial examinations are sometimes necessary the syndrome as MDS-RS.2,22 The anemia is usually macro- to make an accurate diagnosis. cytic and is less often normocytic. Sometimes evidence of a There are two subgroups of MDS-EB based on the blast dual population of normochromic and hypochromic cells count, emphasizing the importance of determining blast exists. The peripheral blood shows reticulocytopenia and quantity for purposes of therapy.26 In MDS-EB-1 there are often leukopenia. less than 5% blasts in the peripheral blood and 5–9% blasts The bone marrow is hypercellular with megaloblastoid in the bone marrow. Auer rods are not present in the blood dyserythropoiesis if the subclassification is determined to be or bone marrow. In MDS-EB-2 there are 5–19% blasts in the a single lineage dysplasia. If dysgranulopoiesis and/or dys- peripheral blood and 10–19% blasts in the bone marrow. megakaryopoiesis are also present, the classification becomes MDS-EB-2 may have Auer rods present. Generally, the num- MDS with ring sideroblasts and multilineage dysplasia. ber of bone marrow blasts serves as a prognostic indicator, 570 Chapter 25 with patients having 5–9% bone marrow blasts experienc- third type of MDS-U, one that is based on defining cytoge- ing a longer median survival time than those patients with netic abnormality. In this unclassifiable MDS type, there are 10–19% blasts.27 Up to 50% of patients with the increased rare or no blasts in the peripheral blood, dysplastic changes blast count progress on to AML, and even patients with to single or multiple cell lineages, and Auer rods are not the lower blast count have a 25% likelihood of progress- observed. Up to 15% ringed sideroblasts may be present, ing to acute leukemia. Current WHO classification of MDS but other blasts in the bone marrow are less than 5%. To be is based on the percentage of blasts in total bone marrow placed in this category the cells must exhibit an MDS defin- nucleated cells, but there is a movement toward calculating ing abnormality.26 blast percentage from nonerythroid nucleated cells only.17,27 Recent studies indicate that enumeration of blasts from Refractory Cytopenia of Childhood nonerythroid nucleated cells rather than from total nucle- ated cells in the bone marrow allows a superior prognostic Childhood MDS is rare and accounts for less than 5% of assessment of MDS.27 cases of hematopoietic neoplasms in children. The mani- festation of MDS in adults and in children has major dif- MDS with Isolated del(5q) ferences, and pediatric hematologists deemed previous classification systems not useful.1,2 MDS patients with an isolated deletion of the long arm of The 2008 WHO classification of MDS introduced chromosome 5 (del[5q] or 5q- ) and no other chromosomal a provisional entity, refractory cytopenia of childhood abnormalities appear to have a unique disease course char- (RCC), specifically for childhood cases of MDS with less acterized by a favorable prognosis and low risk of transfor- than 2% blasts in the peripheral blood and less than 5% mation into AML. Since there is no adverse effect associated in the marrow and persistent cytopenias associated with with this subgroup having one additional chromosomal dysplasia in any or all cell lines (this is retained in the 2016 abnormality, MDS with isolated del(5q) can be diagnosed WHO classification). Other childhood cases of MDS with if there is an additional cytogenetic abnormality except if 2–19% blasts in the peripheral blood and 5–19% blasts that abnormality is monosomy 7 or del(7q). Women have a in the bone marrow should be categorized the same as marked predominance of cases (70%), and the mean age at for adults. Dysplasia in children with MDS is less pro- presentation is 66 years. The main features are macrocytic nounced, and the more aggressive subtype (MDS-EB) pre- anemia, moderate leukopenia, normal to increased plate- dominates; progression to acute leukemia is faster than let count, hypolobulated megakaryocytes, and less than in adults. Age of 2 years or less and a hemoglobin F level 1% blasts in the peripheral blood and less than 5% in the of 10% or higher are associated with a poor prognosis.30 bone marrow. The bone marrow is usually hypercellular or Cytogenetic abnormalities are seen in approximately 70% normocellular with normal to increased megakaryocytes, of cases, and monosomy 7 is the most common cytoge- some with hyperlobulated nuclei.28 The JAK2 V617F muta- netic change. Unlike adults, abnormalities of chromosome tion has recently been found in a small subset of patients 7 do not seem to be associated with a poor prognosis in with isolated del(5q). These patients have slightly higher children. One-third of children with MDS have genetic WBC and platelet counts but no separate classification has predisposition syndromes, such as Down syndrome.30 been established to date.29 These cases have been moved to a new category, myeloid leukemia in Down syndrome, and are now excluded from MDS, Unclassifiable (MDS-U) the MDS.31 MDS-U has variable presentation, and cases assigned to this category do not fit into any of the defined WHO subgroups. Checkpoint 25.6 There are several types of unclassifiable myelodysplastic When a diagnosis of MDS is considered for a patient, why must syndromes, and they present with variable findings. MDS-U other causes of reactive cytopenias be ruled out? What type of with 1% peripheral blood blasts also presents with uni-, bi-, testing helps determine the diagnosis of MDS? or pancytopenia and dysplastic changes in one or more cell lines. Auer rods are not seen in either the peripheral blood or in bone marrow cells. There are no ringed sideroblasts and the percentage of other blasts in the bone marrow is CASE STUDY (continued from page 565) less than 5%. MDS-U with single lineage dysplasia has rare Review Hancock’s peripheral blood and bone mar- or no blasts in the peripheral blood, but pancytopenia, and row features previously identified.

dysplastic changes in a single cell line. Auer rods are not seen in either the peripheral blood or in bone marrow cells. 11. What is the most likely MDS subgroup, and on There are no ringed sideroblasts and the percentage of other what criteria is the answer based? blasts in the bone marrow are less than 5%. Finally, there is a Myelodysplastic Syndromes 571 Variables of MDS typically have pancytopenia, excess blasts, hypocellular bone marrow with fibrosis, trilineage dysplasia, small Subgroups megakaryocytes with hypolobulated nuclei, and no hep- atomegaly or prominent splenomegaly.34 The increased A number of patients have blood and/or marrow find- fibrosis is thought to be produced by the liberation of cyto- ings that cause diagnosis and/or classification problems. kines such as transforming growth factor@b (TGF@b) and Some of these occur often enough to consider them as platelet-derived growth factor (PDGF) from dysplastic variables within MDS subgroups, but the 2008 WHO megakaryocytes.33, 34 classification does not formally recognize them. The recommendation is that these cases be classified accord- ing to one of the subgroups and note the unusual mor- Therapy-Related Myelodysplasia phologic features, such as “MDS-EB-2 with hypoplastic In 2008, the WHO Classification of Hematopoietic Neo- marrow.”3,18 plasms and the International Agency for Research on Cancer placed neoplasms that develop after a patient has received Hypoplastic MDS chemotherapy or radiation therapy for malignancies into a special category, therapy-related myeloid neoplasm (t-MN). Although most MDS cases are associated with hypercellular The category includes therapy-related myelodysplastic syn- or normocellular bone marrows, about 5–10% have hypocel- dromes (t-MDS) and therapy-related acute myeloid leuke- lular marrows. The hypocellular marrow does not appear mia (t-AML).35 to affect survival rate, nor does it influence progression to Development of MDS or acute leukemia (AL) appears AML.29 In these cases, bone marrow biopsy is necessary to to be related to the therapy’s duration, amount, and repeti- exclude a diagnosis of aplastic anemia or hypoplastic AML. tion as well as the patient’s age. The highest incidence of This distinction is important because the diagnosis influ- t-MDS occurs 3–8 years after treatment, and long-term use ences treatment and prognosis. of DNA alkylating agents appears to present the highest Hypoplastic MDS should be considered when the risk.2,7 On a molecular level there is a high frequency of bone marrow cellularity is less than 30% or less than 20% TP53 gene mutations, suggesting that aberrant DNA repair in patients older than 60 years of age. The criteria for MDS and dysregulated cell apoptosis underlie development of must be met in hypoplastic cases as well as in the hyper- myeloid neoplasms.36 Mutational analysis in patients who cellular or normocellular cases. Dysplasia can be difficult have developed t-MDS reveal aberrant gene expression to identify, and dyserythropoiesis has been described in related to mitochondrial function, protein synthesis, DNA aplastic anemia. Dysmegakaryopoiesis and dysgranulo- repair, and regulation of hematopoiesis. poiesis, however, are most characteristic of MDS and can be helpful findings. In addition, atypical localization of immature precursors (ALIP), indicating abnormal bone marrow architecture, is typical of MDS. If present, chromo- Differential Diagnosis somal abnormalities help distinguish MDS from aplastic anemia as does immunophenotyping by flow cytometry MDS must be differentiated from other causes of cytope- for CD34 and the presence of megakaryocytes.28 The dis- nia and dysplasia that mimic MDS.4,37 Possible reactive tinction of MDS from AML can be made based on the etiologies of dysplasia should be considered, especially blast count. A count greater than 19% indicates AML. when the dysplasia is limited to a single cell line. Dys- Although the pathophysiology of hypoplastic MDS is plastic cells in excess of 10% (the MDS threshold) may unknown, secretion of inhibitory cytokines by autoreactive occur in normal individuals and in non-neoplastic causes or clonal-involved T cells is believed to suppress normal of cytopenia. A smaller percentage of dysplastic nor- hematopoiesis.32 moblasts and megakaryocytes may be seen in the bone marrow of all age groups37 and disorders with erythroid MDS with Fibrosis hyperplasia can be associated with significant erythroid dysplasia. The differential diagnosis of MDS from other Mild to moderate fibrosis has been described in up to causes of dysplasia is particularly important in, but not 50% of patients with MDS; marked fibrosis can be seen limited to, deficiencies of vitamin B12, folate, and copper, in 10–15% of cases.27,33 The incidence of fibrosis appears abusive alcohol consumption, chemotherapeutic drugs, to be even higher in therapy-related MDS. If fibrosis is and certain infections. present, other diagnoses including primary myelofibrosis Nutritional deficiency of vitamin B12 and folate are (Chapter 24), chronic myelogenous leukemia (Chapter 24), associated with cytopenias and dysplasia of all three cell and acute megakaryoblastic leukemia (Chapter 26) should lineages; dysplastic changes include macro-ovalocytes, be considered and excluded. MDS patients with fibrosis giant metamyelocytes and bands, nuclear cytoplasmic 572 Chapter 25 asynchrony, and hypersegmented neutrophils. Vitamin The WHO Classification-Based Prognostic Scoring B12 and serum methylmalonic acid (MMA) should be mea- System (WPSS) was published in 2011 (Table 25-5).This sured to evaluate vitamin B12 if the classic morphologic prognostic scoring system utilizes the WHO category of abnormalities of hypersgmented neutrophils and macro- MDS classification, karyotype, and transfusion require- ovalocytes are found. ments and assigns patients to five risk groups of survival Copper deficiency may masquerade as MDS with ane- and probability of leukemic progression.1,44 mia, neutropenia, and thrombocytopenia in the peripheral In 2012 a revised International Prognostic Scoring blood.38,39 The bone marrow reveals vacuoles in erythroid System (IPSS-R) was published that integrated additional and myeloid precursors, iron containing plasma cells, and clinical features to improve prognostic assessment1,44 decrease in granulocyte precursors and ring sideroblasts; (Table 25-6). The IPSS-R incorporated additional new these changes are often found in copper-deficient individu- cytogenetics and clinical features including the degree of als who have increased zinc supplementation. cytopenia of three lineages (erythroid, platelets, and neu- Alcohol abuse can cause direct suppression of the bone trophils). The karyotypes were split into five cytogenetic marrow, resulting in cytopenias. Associated nutritional prognostic subgroups, rather than the three subgroups deficiencies of folate and copper can lead to morphologic used in the original IPSS, and the marrow blast percent- changes, as described above. Vacuolated erythroblasts in age values were adjusted. It was further refined by cat- the bone marrow is a transient feature of acute alcohol use. egorizing patients into five rather than four prognostic Many medications including chemotherapeutic agents, categories. The IPSS-R is based on a much larger combined antibiotics, and some immunosuppressive agents can cause database of patients provided by the International Working hematopoietic dysplasia. G-CSF can cause an increase in Group for Prognosis in MDS (IWG-PM) and is expected to peripheral blood blasts as well as hypergranular and hypol- provide an improved prognostic categorization model.1,44 obular neutrophils. Infection with Parvovirus B19 can cause decreased ery- throid precursors and giant pronormoblasts.40 HIV infection CASE STUDY (continued from page 570) is associated with trilineage dysplastic changes and abnor- malities of bone marrow cellularity.41 Hepatitis C infection Hancock’s karyotope showed multiple complex may cause dyserythropoiesis.42 abnormalities. In some diagnostically ambiguous cases the patient is 12. Using the IPSS-R, what is the prognosis for diagnosed with CHIP or ICUS and followed until further Hancock? information on their condition is available.4 Prognosis Table 25.5 WHO Classification-Based Prognostic Scoring System (WPSS) of Myelodydysplastic Syndromes The median survival for all types of MDS is less than 2 years; however, some patients can survive many years Points WHO Category Karyotypea Transfusion requirementbwith continuous transfusion therapy. The mortality rate varies from 58–72%. Leukemic transformation ranges in 0 MDS-SLD, MDS-RS- Good None incidence from 10–40%. The likelihood of transformation to SLD, MDS-5q AML increases with the presence of severe cytopenias, more 1 MDS-MLD, Intermediate Regular MDS-RS-MLD overt dysplastic features of cells, and complex chromosome 2 MDS-EB-1 Poor – abnormalities.43,44 3 MDS-EB-2 – – The International Prognostic Scoring System (IPSS) Use summed points to determine risk category was developed in 1997 to assist physicians in predicting WPSS Risk Group Score prognosis and selection of optimal therapy for individuals diagnosed with MDS. This system was developed using Very low 0 data from seven large studies that had previously gen- Low 1 erated prognostic systems and was accepted worldwide. Intermediate 2 The prognostic score was based on the percentage of blasts High 3–4 in the bone marrow, cytogenetic abnormalities, and num- Very high 5–6 ber of cytopenias, and patients were divided into four risk a Karyotypes: Good = normal, -Y, del(5q- ) only, del(20q- ) only. Poor = complex (3 or groups with distinct risks of death and leukemic trans- more abnormalities), or chromosome 7 abnormalities. Intermediate = all others. b Transfusion dependency is defined as at least one red blood cell transfusion every 8 formation. Low scores indicate prolonged survival.1,17,43,44 weeks over a period of 4 months. Myelodysplastic Syndromes 573 Table 25.6 International Prognostic Scoring System-Revised (IPSS-R) for the Myelodysplastic Syndromes Prognostic Variable 0 0.5 1 1.5 2 3 4 Marrow blasts % …2 72965 5–10 710 Karyotypea Very Good Good Intermediate Poor Very Poor Hemoglobin (g/dL) Ú10 8–10 68 Platelets (*109/L) Ú100 5096100 650 Absolute neutrophil Ú0.8 60.8 count (*109/L) IPSS Risk Group Median Survival (years); Score no treatment Very low 8.8 …1.5 Low 5.3 71.593 Intermediate 3.0 7394.5 High 1.6 74.596 Very high 0.8 76 a Cytogenetic prognostic subgroups include: Very Good [-Y, del(11q)]; Good [normal, del(5q), del(12p), del(20q), double including del(5q)]; Intermediate [del(7q), +8, +19, i(17q), any other single or double independent clones]; Poor [-7, inv(3)/t(3q)/del(3q), double including -7/del(7q), complex: three abnormalities]; Very Poor (complex: more than three abnormalities). Therapy myelocytic cell lines may contribute to the development of MDS.2 Myelodysplastic syndromes are so heterogeneous that The U.S. Food and Drug Administration (FDA) has therapy must be based on the patient’s risk stratifica- approved three drugs for the treatment of MDS: azacitidine, tion, transfusion needs, the number of blasts seen, and decitabine, and lenalidomide. Azacitidine and its deoxy more recently, the patient’s cytogenetic profile. Patients derivative decitabine are pyrimidine nucleoside analogues should be stratified into lower-risk patients and higher- of cytidine. As such, they incorporate into DNA to act as risk patients. Lower-risk patients include those whose risk false substrates and in this way reversibly inhibit DNA score is determined to be Low or Intermediate-1 (IPSS); methyltransferase to block DNA methylation. The result- Very Low, Low, or Intermediate (IPSS-R); or Very Low, ing hypomethylation can activate tumor suppressor genes Low, Intermediate (WPSS). Higher-risk patients are those silenced by hypermethylation, producing an antitumor whose risk score is Intermediate-2 or High (IPSS); Inter- effect. Although the mechanism of action of lenalidomide mediate, High, or Very High (IPSS-R); or High, Very High remains incompletely understood, it appears to have multi- (WPSS).2 ple mechanisms of action including direct antitumor effect, The therapeutic strategy for patients evaluated as inhibition of the microenvironment that supports tumor lower risk is to improve their relevant cytopenias. Lineage cells, and an immunosuppressive function. Interestingly in appropriate growth factors may be effective in counter- MDS with del(5q), lenalidomide inhibits the malignant cell ing the cytopenias of MDS. Antibiotics are necessary for line, but in MDS without 5q deletions, lenalidomide exerts infections that are likely to follow neutropenia but are not its effect by promoting erythropoiesis.44,45 recommended as a prophylactic treatment. Since transfu- The treatment options for patients with higher-risk sions are often indicated to relieve anemia in MDS, iron MDS could include the same treatment options for lower overload is a concern. The National Comprehensive Can- risk patients but traditionally have also utilized classi- cer Network (NCCN) recommends the use of iron chela- cal combination therapy with anthrocycline and cytara- tion therapy in patients with ferritin levels 2500 mg/L or bine. The most important prognostic factor governing more.2,25,43 the response of patients to such AML-like therapy is the Immunosuppressive therapy such as that with cyclo- patient’s karyotype. Patients with -7, del (7q), or com- sporine or antithymocyte globulin (ATG) is controver- plex karyotype do not respond favorably to this regimen.45 sial but may be considered in some lower-risk patients. Allogeneic stem cell transplantation is usually not recom- ATG consists of pooled antibodies against human mended for patients with lower-risk MDS but remains a T-l ymphocytes. This treatment can be effective because viable treatment option for children and higher risk patients abnormal autoimmune activity of activated T-cells against and is potentially curative.2,25,44 574 Chapter 25 Myelodysplastic/ Clinical symptoms can

result from cytopenia(s) when present, dysplastic cells that do not function properly, and Myeloproliferative leukemic infiltration of various organs. Splenomegaly and hepatomegaly are commonly found, but the clinical pre- Neoplasms (MDS/MPNS) sentation is highly variable. The incidence varies widely, depending on the specific disease, as do the prognosis and The category of myelodysplastic/myeloproliferative neo- tendency for clonal evolution and disease progression. The plasms (MDS/MPNs) includes clonal hematopoietic neo- disorders included in MDS/MPN are: plasms that, at the time of initial presentation, have some clinical, laboratory, or morphologic findings of both an MDS • Chronic myelomonocytic leukemia (CMML) and an MPN.24,46 Typically, patients with MDS/MPN have • Atypical chronic myeloid leukemia, BCR/ABL1- a hypercellular bone marrow associated with proliferation • Juvenile myelomonocytic leukemia (JMML) in one or more of the myeloid lineages. Frequently, the pro- liferation is effective and results in increased numbers of • MDS/MPN with ring sideroblasts and thrombocytosis circulating cells. However, these cells commonly are mor- (MDS/MPN-RS-T) phologically and functionally dysplastic. While one or more • MDS/MPN, unclassifiable (Table 25-7) lineages can show hyperproliferation, one or more other lineages can exhibit ineffective proliferation and resulting Chronic Myelomonocytic Leukemia cytopenia(s). The percentage of blasts in the bone marrow and peripheral blood is always less than 20% At present, (CMML) there are no identified genetic defects specific for any of the Chronic myelomonocytic leukemia (CMML) is a clonal entities included in this category. However, recurring chro- hematopoietic neoplasm associated with a persistent mosomal and molecular abnormalities have been described monocytosis (greater than 1 * 109/L) in the peripheral (Table 25-7). blood (Figure 25-12). Although the finding of absolute Table 25.7 The Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPNs) Classification Peripheral Blood Bone Marrow Genetics Immunophenotype Chronic myelomonocytic Monocytosis 610% blasts (including BCR/ABL1- CD33, CD13+ , leukemia (CMML-1) More than 1 * 109/L; less promonocytes); dysplasia CD14, CD68, than 5% blasts (including of one or more myeloid CD64 variable; lysozyme+ promonocytes) lineages CMML-2 5–19% blasts (including 10–19% blasts (including (same as CMML-1) (same as CMML-1) promonocytes) promonocytes or when Auer rods present, regardless of blast/promonocyte count) Atypical chronic Leukocytosis (less than Hypercellular; less than 20% BCR/ABL1- CD33, CD13+ , MPO+ myeloid leukemia 10% monocytes); less than blasts; dysgranulopoiesis; No evidence of PDGFA, (aCML, BCR/ABL1-) 20% blasts; more than 10% without dyserythropoiesis PDGFRB, or FGFR1 promyelocytes, myelocytes, or megakaryopoiesis rearrangement metamyelocytes No PCM1-JAK2 No/minimal basophilia Juvenile myelomonocytic Monocytosis Hypercellular; less than 20% BCR/ABL1- CD33, CD13+ , leukemia (JMML) More than 1 * 109/L; less blasts; dysgranulopoiesis; Somatic mutation in CD14, CD68, than 20% blasts occasionally dyserythropoiesis PTPN11, KRAS, NRAS, CD64 variable; lysozyme+ or NF1 mutation, germ line CBL mutation, loss of heterozygosity of CBL, monosomy 7 Myelodysplasic/ Anemia with or without More than 15% ring SF3B1 mutation; myeloproliferative multilineage dysplasia; less siderblasts BCR@ABL1-; neoplasm with ring than 1% blasts; persistent Less than 5% blasts no rearrangement of sideroblasts and thrombocytosis PDGFRA, PDGFRB, thrombocytosis FGFR1; no PCM1-JAK2, (MDS/MPN-RS-T) no 3;3(q21;q26), inv(3)) (q21;q26), del(5q) Myelodysplastic/ Anemia, leukocytosis and/ Hypercellular; proliferation in BCR/ABL1- Nondiagnostic myeloproliferative or thrombocytosis; less than any or all myeloid lineages neoplasm, unclassifiable 20% blasts (MDS/MPN, U) Myelodysplastic Syndromes 575 characterized by primary involvement of the neutrophil series with leukocytosis involving dysplastic immature and mature neutrophils. Multilineage dysplasia is common. The annual incidence of aCML, BCR/ABL1- is about 3 in 100,000. Most patients are older with the median age at diagnosis in the seventh to eighth decade. Median survival time is less than 20 months, and progression to acute leuke- mia is seen in 25–40% of cases. The cell of origin is believed to be the common myeloid progenitor cell (CMP). Reported cytogenetic abnormalities are similar to CMML. The BCR/ABL1 fusion gene is absent, and the specific genes SETBP1 and ETNK1 are mutated in about 30% of the cases.47 For a diagnosis of aCML, BCR/ABL1-, the peripheral blood WBC must be in excess of 13 * 109/L with increased Figure 25.12 dysplastic neutrophils and their precursors. Promyelocytes, Increased monocytes in the peripheral blood myelocytes, and metamyelocytes comprise more than 10% from a patient with CMML. (Wright stain, 1000* magnification) and blasts constitute less than 20% of the peripheral blood leukocytes. Absolute monocytosis and basophilia are usu- monocytosis is required for diagnosis, other hematologic ally absent, but their presence doesn’t preclude a diagnosis findings are remarkably variable. Leukocytosis, leukopenia, of aCML. Dysplasia and ineffective hematopoiesis in other neutrophilia, and neutropenia are all possibilities in CMML. cell lines frequently result in anemia and thrombocytopenia. Dyshematopoiesis can range from minimal expression in a aCML, BCR/ABL1- typically presents with a hypercel- single lineage to marked dysplasia in all the lineages. lular bone marrow. Dysplastic granulocytes account for most The cell of origin is believed to be the HSC. Clonal cyto- of the hypercellularity, dysplasia of the erythrocytic and genetic abnormalities are found in 20–40% of patients, but megakaryocytic lineages may or may not be present. Immu- none are specific. The most frequently reported abnormali- nophenotype results are typical for the neutrophil lineage. ties include SRSF2, TET2, and ASXL1. Somewhat less fre- quently seen gene mutations include SETBP1, NRAS/KRAS, RUNX1, CBL, and EZH2 (Table 25-1). Checkpoint 25.7 The WHO recommendations of diagnostic criteria for How do the laboratory findings of aCML, BCR/ABL1- differ from CMML are as follows: peripheral blood monocytosis greater those observed in CML (Chapter 24)? than 1 * 109/L, less than 20% blasts (myeloblasts, monoblasts, and promonocytes) in the peripheral blood and bone marrow, dysplasia in one or more myeloid lineages, absence of the BCR/ Juvenile Myelomonocytic Leukemia ABL1 fusion gene, and no rearrangement of PDGFRA, PDG- Juvenile myelomonocytic leukemia (JMML) is an aggres- FRB, or FGFR1 genes.23,46 The WHO recommends classifying sive childhood myeloproliferative disorder characterized CMML into two subgroups based on blasts or blast equiva- by proliferation and immaturity of the granulocytic and lents (promyelocytes) seen in the bone marrow (Table 25-7), monocytic lineages in the peripheral blood and bone mar- but recently stratification of CMML into three blast-based row. Dysmyelopoiesis and dyserythropoiesis but rarely groups has been suggested for better prognostication. The dysmegakaryopoiesis are evident in the bone marrow. Con- three proposed groups are CMML-0 when there are less than comitantly, as a result of the myelomonocytic expansion in 2% blasts in the PB and less than 5% BM blasts; CMML-1 the bone marrow, JMML patients often present with anemia when PB/BM blasts are 2–4%/5–9%; and CMML-2 for cases and thrombocytopenia. with blasts of 5–19% in the PB and 10–19% in the BM.46 The cell of origin is believed to be the HSC. Gain of function mutations of N-RAS and K-RAS and disruption Atypical Chronic Myeloid Leukemia of tumor suppressor genes are essential initiating events in (aCML, BCR/ABL1−) the development of JMML. Moreover, somatic mutations in PTPN11 (that encodes a protein tyrosine phosphatase) have The 2008 revision of the WHO classification of myeloid been found in 35% of JMML cases and have been recom- neoplasms renamed atypical chronic myeloid leukemia mended for inclusion in the diagnostic criteria.48 Monosomy (aCML) to aCML BCR/ABL1- to emphasize that the disor- 7 can also be included for diagnostic purposes. der is not merely an atypical CML. Atypical chronic myeloid The annual incidence of JMML is 0.13/100,000 children leukemia (aCML, BCR/ABL1-) is a variant of MDS/MPN (age 0–14 years of age). It accounts for less than 2–3% of all 576 Chapter 25 leukemia in children. The age at diagnosis is variable (from in this subtype even if the SF3B1 mutation is detected, 1 month to early adolescence), but 75% of cases are diag- whereas a myelodysplastic syndrome may be classified as nosed in children less than 3 years old. A significant associa- MDS-RS with less than 15% ring sideroblasts as long as the tion of JMML with neurofibromatosis type 1 (NF-1) exists, SF3B1 mutation is present. MDS/MPN-RS-T often presents with about 10% of cases of JMML occurring in children with with the JAK2V617F mutation as well. The JAK2 mutation is this diagnosis. The overall prognosis is poor, although sur- seen in several myeloproliferative diseases, and putatively vival times can vary based on the type of therapy chosen. underlies the thrombocytosis seen in these neoplasms.32 Without effective treatment, most children die from organ failure as the result of leukemic infiltration. About 10–20% Myelodysplastic/Myeloproliferative progress to acute leukemia. The following criteria have been established for the Neoplasm, Unclassifiable (MDS/ diagnosis of JMML: absolute monocytosis (more than MPN, U) 1 * 109/L), less than 20% blasts in the peripheral blood This subcategory, MDS/MPN, U, is used for cases that have and bone marrow, absence of the BCR/ABL1 fusion gene clinical, laboratory, and morphologic features that support a in addition to at least two of the following: myeloid pre- diagnosis of both MDS and MPN but do not meet the crite- cursors in the peripheral blood, leukocytosis of more than ria of the other entities included in the MDS/MPN category. 10 * 109/L, increased fetal hemoglobin for the patient’s No cytogenetic or molecular genetic findings are specific for demographic, or hypersensitivity of the hematopoietic pro- this group. The presence of the BCR/ABL1 fusion gene must genitor to GM-CSF. always be excluded. The MDS/MPN, U disorders are characterized by the MDS/MPN with Ring Sideroblasts proliferation of one or more of the myeloid lineages that and Thrombocytosis (MDS/ are ineffective and/or dysplastic. Proliferation of the other MPN-RS-T) myeloid lineages is generally effective with or without dysplasia. Anemia (with or without macrocytosis) usually In earlier classifications, this category was known as refrac- occurs, occasionally with a dimorphic population of RBCs. tory anemia with ring sideroblasts and thrombocytotosis The bone marrow is hypercellular and can show prolifera- (RARS-T).23 MDS/MPN-RS-T is very similar to MDS-RS tion in any or all of the myeloid lineages. Dysplastic features with respect to the ring sideroblasts present in the erythroid are present in at least one lineage. Typically, the patient has precursors of the bone marrow. In MDS-RS, the presence of a less than 20% blasts in the blood and bone marrow and mutated SF3B1 gene is well correlated with the appearance prominent myeloproliferative features associated with of ring sideroblasts and this is true for MDS/MPN-RS-T as either thrombocytosis or leukocytosis. The bone marrow well. With MDS/MPN with ring sideroblasts and thrombocy- and peripheral blood are always involved; extramedullary tosis, at least 15% ring sideroblasts are required for inclusion tissues (liver, spleen) are sometimes also involved. Summary The myelodysplastic syndromes are pluripotential hema- bone marrow is abnormal with megaloblastoid features com- topoietic stem cell disorders characterized by one or more monly present. Neutrophils can show hyposegmentation of peripheral blood cytopenias and prominent cellular matura- the nucleus and hypogranulation. Megakaryocytes also show tion abnormalities. The bone marrow is usually normocellular megaloblastoid features. Platelets can be large and agranular. or hypercellular, indicating a high degree of ineffective hema- The WHO group has classified the MDSs into six sub- topoiesis. The most commonly encountered abnormal karyo- groups depending on the blast count, degree of dyspoie- types in MDS are the -5/del(5q), -7/del (7q), +8, del(20q), sis and cytopenias, and presence of abnormal cytogenetic -Y, and complex karyotype. Mutated genes of significance findings. These include MDS-SLD, MDS-RS (subclasses involve RNA splicing machinery, signaling pathways, tran- MDS-RS-SLD and MDS-RS-MLD), MDS-MLD, MDS-EB scription regulation, DNA repair, and epigenetic alterations (subclasses MDS-EB-1 and MDS-EB-2), MDS with isolated to the genome. Some of the most frequently mutated genes del(5q), and MDS, unclassifiable. Subgroups with higher include TET2, IDH1/2, ASXL1, EZH2, ETV6, FLT3, JAK2, blast counts and involvement of multiple lineages in dys- NPM1, N-RAS, K-RAS, RUNX1, TP53, and SETBP1. poiesis and complex cytogenetic abnormalities are more Although anemia is the most common cytopenia, neu- aggressive disorders. MDS frequently terminates in acute tropenia and thrombocytopenia also occur. Erythrocytes are leukemia; treatment is variable because of the heteroge- macrocytic or less frequently normocytic. Erythropoiesis in the neous presentation of the disease. Treatment options are Myelodysplastic Syndromes 577 made based on multiple clinical parameters including the myelodysplastic syndrome simultaneously. This category patient’s cytogenetic profile. includes CMML, aCML BCR/ABL1-, JMML, MDS/MPN- The myelodysplastic/myeloproliferative neoplasm RS-T, and MDS/MPN, U. An important diagnostic consid- category (MDS/MPN) includes disorders that have eration is absence of the BCR/ABL1 fusion gene in these features of both a myeloproliferative neoplasm and a patients. Review Questions Level I c. hypercellular 1. Which subgroup of MDS has refractory cytopenia d. fibrotic with unilineage dysplasia? (Objective 2) 7. The most common dyserythropoietic

finding in the a. MDS-MLD bone marrow in MDS is: (Objective 3) b. MDS-EB-1 a. megaloblastoid development c. MDS associated with isolated del(5q) b. impaired hemoglobinization d. MDS-SLD c. pseudo-Pelger-Huet cells d. agranular cytoplasm 2. Dysplasia in 10% or more of cells of two or more lineages, less than 5% other blasts, 15% or more ring 8. Which of the following help define myelodysplastic sideroblasts, and no Auer rods in the bone marrow syndrome? (Objective 1) suggests a diagnosis of: (Objective 5) 1. Characterized by one or more peripheral blood a. MDS-RS-MLD cytopenias b. MDS-RS 2. Maturation abnormalities (dyspoiesis) in the bone c. MDS-MLD marrow blood cells d. MDS-EB 3. Microcytic, hypochromic RBCs in the peripheral blood 3. The WHO classification system of MDS is based on: a. 1 and 2 only (Objective 2) b. 2 and 3 only a. morphology c. 1 and 3 only b. immunophenotype d. 1, 2, and 3 c. genetics 9. Which test(s) is(are) most helpful to differentiate a sus- d. all of the above pected MDS from megaloblastic anemia? (Objective 3) 4. The type of anemia usually seen in MDS is: a. MCV and MCHC (Objective 1) b. Serum ferritin a. macrocytic, normochromic c. Serum folate and cobalamin b. normocytic, normochromic d. bone marrow cellularity c. microcytic, hypochromic 10. Which MDS subgroup occasionally may have Auer d. normocytic, hypochromic rods? (Objective 2) 5. The most common “cytopenia(s)” seen in MDS is/are: a. MDS-MLD (Objectives 3 and 5) b. MDS with isolated del(5q)- syndrome a. leukopenia c. MDS-EB-1 b. thrombocytopenia d. MDS-EB-2 c. anemia 11. A dimorphic red blood cell population is most d. a combination of two of the above characteristic of which MDS subgroup? (Objective 2) 6. The typical bone marrow cellularity in MDS is: a. MDS-SLD (Objective 3) b. MDS-RS a. hypocellular c. MDS-EB b. normocellular d. CMML 578 Chapter 25 12. A patient presents with the following symptoms and a. 1 and 3 laboratory data: Weak, e asily fatigued, night sweats, and b. 2 and 4 dyspnea on exertion. The spleen was slightly enlarged. c. 1, 2, and 3 WBC = 6.0 * 103/mcL, RBC = 2.80 * 106/mcL, Hb = 85g/L, MCV = 103 fL, Plt = 220 * 103/mcL. d. 1, 2, 3, and 4 The peripheral blood film showed macro-ovalocytes 17. Dysgranulopoiesis may be seen on a peripheral blood and hypersegmented neutrophils. Less than 5% blasts film of an MDS patient as: (Objective 3) and a few ringed sideroblasts were seen in the bone marrow. Serum folate and cobalamin levels were 1. agranular or hypogranular neutrophils normal. What is the most likely diagnosis? 2. hypersegmented neutrophils (Objective 2) 3. pseudo-Pelger-Huet cells a. MDS, subgroup MDS-SLD 4. lingering cytoplasmic basophilia in mature b. MDS, subgroup MDS-RS neutrophils c. Megaloblastic anemia a. 1 and 3 d. Sideroblastic anemia b. 2 and 4 c. 1, 2, and 3 13. Which MDS subgroup is found predominantly in women? (Objective 2) d. 1, 2, 3, and 4 a. MDS with isolated del (5q) 18. Evidence of dysmegakaryocytopoiesis may be seen on a peripheral blood film as: (Objective 3) b. MDS-SLD c. MDS-MLD 1. Micromegakaryocytes d. MDS-RS 2. Increased platelets 3. Hypogranular platelets 14. Which of the following terms are currently acceptable a. 1 and 3 terminologies for MDS? (Objective 1) b. 2 1. Preleukemia c. 1, 2, and 3 2. Myelodysplastic syndrome d. 1 and 2 3. Smouldering leukemia 4. Dysmyelopoietic syndrome Level II a. 1 and 3 1. A cell resembling a blast that contains primary b. 2 and 4 granules and lacks a Golgi zone would be classified c. 1, 2, and 3 as a(n): (Objective 2) d. 1, 2, 3, and 4 a. granular blast b. agranular blast 15. Which of the following, when present in the bone marrow, indicate a typical MDS? (Objective 1) c. promyelocyte d. dysplastic promyelocyte 1. Hypercellularity 2. Abnormal maturation (dyspoiesis) 2. The contrast between a hypercellular bone marrow 3. Less than 20% blasts and a cytopenic peripheral blood film seen in MDS is attributed to: (Objective 1) 4. Increased amounts of fibrotic tissue a. 1 and 3 a. premature destruction of abnormal cells in the bone marrow (ineffective hematopoiesis) b. 2 and 4 b. production of blood cells outside the bone marrow c. 1, 2, and 3 (extramedullary hematopoiesis) d. 1, 2, 3, and 4 c. splenic sequestration 16. Which of the following are peripheral blood signs of d. immune destruction of cells in the peripheral an MDS? (Objective 1) blood 1. Macro-ovalocytes 3. Which of the following would be most helpful to dif- 2. Pseudo-Pelger-Huet cells ferentiate CMML from CML? (Objective 8) 3. Giant platelets with decreased granulation a. Karyotype 4. Cytopenia, alone or in combination b. Presence of nucleated RBCs Myelodysplastic Syndromes 579 c. Elevated leukocyte count 8. The most common chromosomal abnormality found d. Percentage of bone marrow blasts in the MDS is: (Objective 5) 4. The most effective treatment for MDS is currently a. 5q- considered to be: (Objective 3) b. 7q- a. hematopoietic growth factors c. trisomy 8 (+8) b. chemotherapy d. t(9;22) c. bone marrow transplant 9. 5q- syndrome is frequently associated with which d. immunotherapy morphological abnormality? (Objective 5) a. pseudo-Pelger-Huet cells b. hyperlobulated megakaryocytes Use the following information to answer questions 5–7, c. ring sideroblasts A patient presents with the following laboratory data: d. megaloblastoid erythropoiesis RBC 2.30 * 106/mcL Case Study Hb 78 g/L Hct 24% Use this information to answer Questions 10–12. MCV 104 fL RDW 20 A patient’s primary presenting symptoms were fatigue and shortness of breath. WBC 8.5 * 103/mcL Plt 140 * 103/mcL RBC decreased Hb 8.5 g/dL The differential was normal except for 2% metamyelocytes. MCV 104 fL Oval macrocytes, a few abnormal NRBC, and a few siderocytes WBC were seen. The bone marrow contained 3% blasts and exhibited 3.4 * 103/mcL hypercellularity with megaloblastoid development in erythroid Plt 180 * 103/mcL cells. Previous treatment with cobalamin and folate was not effective. The peripheral blood film showed macrocytes, atypical platelets, 5. What is the most probable MDS subgroup? (Objective hypogranulation of neutrophils, and pseudo-Pelger-Huet cells. 10) The differential revealed a few myelocytes, metamyelocytes, and 2% blasts. The bone marrow was hypercellular with erythroid a. MDS-EB-1 and granulocytic hyperplasia. Dysplasia was evident in all b. MDS-RS three cell lines. There were 8% myeloblasts present and 20% c. MDS-SLD ring sideroblasts. d. MDS-EB-2 6. What other hematologic disorder does this peripheral 10. Ringed sideroblasts are defined as nucleated red blood picture resemble? (Objective 7) blood cells in which iron deposits encircle at least how much of the nucleus? (Objectives 1 [Level I] a. Iron-deficiency anemia and 5) b. Aplastic anemia a. One-third c. Megaloblastic anemia b. One-half d. Anemia of chronic disease c. One-fourth 7. In reference to Question 6, what laboratory test(s) d. The whole nucleus would be helpful to distinguish the two disorders? (Objective 9) 11. Which MDS subgroup is most likely? (Objective 8) a. Lactate dehydrogenase a. MDS-RS b. Uric acid b. MDS-EB-2 c. Serum folate and cobalamin c. MDS-EB-1 d. Serum ferritin d. MDS-MLD 580 Chapter 25 12. What morphological finding distinguishes MDS-EB-1 17. According to the IPSS, which factors have a major from MDS-EB-2? (Objective 8) impact on disease prognosis? (Objective 3) a. MDS-EB-2 presents with monocytosis 1. Degree of cytogenetic abnormalities b. MDS-EB-2 presents with an increased platelet 2. Number of blasts in the bone marrow count 3. Number of cytopenias in the peripheral blood c. MDS-EB-2 has a greater number of blasts in the 4. Number of blasts in the peripheral blood bone marrow a. 2 and 4 d. MDS-EB-2 presents with erythroid hyperplasia in b. 1, 2, 3, and 4 bone marrow c. 1, 2, and 3 13. Complex karyotype abnormalities are present in what d. 1 and 3 percentage of therapy-related MDS? (Objective 5) 18. Atypical CML can best be distinguished from CML by a. 50% which of the following? (Objective 8) b. 90% a. Dysplastic cells in the bone marrow c. 20% b. Leukocytosis d. 10% c. Absence of BCR/ABL1 fusion gene 14. Presence of micromegakaryocytes may be distin- d. Abnormal karyotype guished from lymphocytes by: (Objective 9) 19. MDS is usually diagnosed in elderly patients. This is a. cytogenetics evidence that which of the following is a contributing b. presence of cytoplasmic tags on the nucleus factor to disease development? (Objective 1) c. Wright’s stain a. Genetic instability of myeloid stem cell d. epigenetics b. Epigenetic changes 15. The origin of the MDS is considered to be: (Objective 1) c. Changes in bone marrow microenvironment a. enzyme deficiency d. Inherited chromosomal disorders b. abnormal stem cell 20. Which of the following would distinguish an c. abnormal DNA synthesis MDS/MPN from MDS? (Objective 8) d. congenital genetic defect a. Less than 20% blasts in the bone marrow 16. Which would help to distinguish MDS with a b. Hypercellular bone marrow hypoplastic marrow from aplastic anemia? c. Leukocytosis (Objective 7) d. Presence of dysplastic cells 1. Chromosomal abnormalities 21. A 70-year-old patient is suspected to have an MDS. 2. Pancytopenia His peripheral blood shows dysplasia, cytopenias in 3. Dysgranulocytosis and dysmegakaryocytosis two cell lines, and an increased number of monocytes. 4. Absolute decrease in reticulocytes This would best be classified as: (Objectives 6 and 8) a. 2 and 4 a. JMML b. 1, 2, and 3 b. MDS-MLD c. 1 and 3 c. aCML d. 1, 2, 3, and 4 d. CMML References 1. National Comprehensive Cancer network. NCCN clinical 4. Steensma, D. P., Bejar, R., Jaiswal, S., Lindsley, R. C., Sekeres, M. 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P., & Bennett, J. M. (2006). The myelodysplastic 37. Steensma, D.P. (2012). Dysplasia has a differential diagnosis: syndromes: Diagnosis and treatment. Mayo Clinic Proceedings, Distinguishing genuine myelodysplastic syndroms (MDS) from 81(1), 104–130. mimics, imitators, copycats and impostors. Current Hematologic 21. Bejar, R., & Steensma, D. P. (2010). Myelodysplastic syndromes. Malignancy Reports, 7, 310–320. In Kaushansky, K., Lichtman, M., Prchal, J., Levi, M., Press, O., 38. Halfdanarson, T. R., Kumar, N., Li, C-Y., & Phyliky, R. L. (2008). Burns, L., Caligiuri, M., eds. Williams Hematology 9th ed. Haematological manifestations of copper deficiency: A (pp. 1341–1272). New York: McGraw-Hill. retrospective review. European Journal of Haematology, 80, 22. Mufti, G. J., Bennett, J. M., Goasguen, J., Bain, B. J., Baumann, I., 523–531. Brunning, R., . . . Jinnai, I. (2008). Diagnosis and classification 39. Gabreyes, A. A., Abbasi, H. N., Forbes, K. P., McQuaker, G., of myelodysplastic syndrome: International working group Duncan, A., & Morrison, I. (2013). Hypocupremia associated on morphology of myelodysplastic syndrome (IWGM-MDS) cytopenia and myelopathy: A national retrospective review. consensus proposals for the definition and enumeration of European Journal of Haematology, 90, 1–9. myeloblasts and ring sideroblasts. Haematologica, 93(11), 40. Young, N. S., & Brown, K. E., (2004). Parvovirus B19. NEJM, 350, 1712–1717. 586–597. 23. Giagounidis, A., & Haase, D. (2013). Morphology, cytogenetics 41. Bain, B. J. (1997). The haematological features of HIV infection. and classification of MDS. Best Practice & Research Clinical British Journal of Haematology, 99, 1–8. Haematology, 26(4), 337–353. 42. Klco, J. M., Geng, B., Brunt, E. M., Hassan, A., Nguyen, R-D., 24. Ossenkoppele, G. J., van de Loosdrecht, A. A., & Schuurhuis. G. J. Kreisel, F. H., & Frater, J. L. (2010). Bone marrow biopsy in (2011) Review of the relevance of aberrant antigen expression patients with hepatitis C virus infection: Spectrum of findings 582 Chapter 25 and diagnostic utility. American Journal of Hematology, 85, 46. Foucar, K. (2009). Myelodysplastic/myeloproliferative n eoplasms. 106–110. American Journal of Clinical Pathology, 32(2), 281–289. 43. Jonas, B. A., & Greenberg, P. L. (2015). MDS prognostic scoring 47. Sochacki, A. L., Fischer, M. A., & Savona, M. R. (2016). systems-Past, present, and future. Best Practice & Research Clinical Therapeutic approaches in myelofibrosis and myelodysplastic/ Haematology 28(1), 3–13. myeloproliferative overlap syndromes. Oncological Targets and 44. Meers, S. (2015). The myelodysplastic syndromes: The era of Therapy, 9, 2273. understanding. European Journal of Haematology, 94(5), 379–390. 48. Niemeyer, C. M., & Kratz, C. P. (2008). Paediatric myelodysplastic 45. Bhatia, R., & Deeg, H. J. (2011). Treatment-related myelodysplastic syndromes and juvenile myelomonocytic leukaemia: Molecular syndrome-Molecular characteristics and therapy. Current Opinion classification and treatment options. British Journal of H aematology, in Hematology, 18(2), 77. 140(6), 610–624. Chapter 26 Acute Myeloid Leukemias Kyle Riding, PhD Objectives—Level I By the end of this unit of study, the student should be able to: 1. Define acute leukemia (AL) and acute myeloid leukocytes, thrombocytes, and blasts) seen leukemia (AML). in AML. 2. State the differences between acute myeloid 5. Describe the typical bone marrow picture leukemia (AML) and acute lymphoblastic (cellularity, M:E ratio, blasts) seen in AML. leukemia (ALL). 6. Give the typical cytochemistry and immu- 3. List and define the common variants of AML nophenotype results that help differentiate as defined by the World Health Organiza- AML from other ALs. tion (WHO) classification. 7. Describe Auer rods and their significance. 4. Describe and recognize the typical peripheral blood picture (erythrocytes, Objectives—Level II At the end of this unit of study, the student should be able to: 1. Compare and contrast the various presenta- 4. Correlate Wright stain morphology of the tions of AML. AML subgroups with cytochemical stains, 2. Predict the most likely leukemia subtype flow cytometry and genetic testing. based on patient history, physical assess- 5. Evaluate peripheral blood results in relation ment, and laboratory findings. to oncological therapy. 3. Correlate cellular presentation of AML with 6. Evaluate patient data from the medical his- prognosis and common complications. tory and the laboratory results to determine 583 584 Chapter 26 whether a disorder can be classified and, 7. Explain the criteria used for the 2016 WHO if not, specify the additional testing to be classification of AML. performed. Chapter Outline Objectives—Level I and Level II 583 Laboratory Evaluation 585 Key Terms 584 Classification 587 Background Basics 584 Therapy 601 Case Study 584 Summary 602 Overview 585 Review Questions 602 Introduction 585 References 604 Etiology and Pathophysiology 585 Key Terms Acute leukemia (AL) B-lymphoid cell antigens Recurrent genetic abnormalities Acute myeloid leukemia (AML) Dysplasia T-lymphoid cell antigens Auer rods Leukemias of ambiguous lineage WHO classification Background Basics The information in this chapter builds on concepts learned Level II in previous chapters. To maximize your learning experi- • Summarize the role of oncogenes and growth fac- ence, you should review and have an understanding of the tors in cell proliferation, differentiation, and matura- following concepts before starting this unit of study: tion. (Chapters 2, 4, and 23) • Describe the role of molecular analysis in diagnosing Level I and treating acute leukemia. (Chapters 23, 42) • Describe the origin and differentiation of hematopoi- • Describe the use of immunophenotyping in acute etic cells. (Chapter 4) leukemia. (Chapters 23, 40) • Summarize the maturation, differentiation, and • Describe the role of cytogenetics in diagnosis, function of leukocytes. (Chapter 7) treatment, and prognosis of acute leukemia. • Outline the classification and general laboratory (Chapters 23, 41) findings of acute leukemias. (Chapter 23) • List and describe the criteria used to d ifferentiate acute leukemias from other hematologic neoplasms. (Chapter 23) CASE STUDY a chemistry panel. He was sent to the local emer- We refer to this case study throughout the chapter. gency room when his CBC revealed a WBC count of 120 * 106/mcL and potential blasts being noted Jonathan, a 12-year-old Caucasian male, had been by the physician office analyzer. He was admitted in excellent health until he was seen at his primary to the hospital for further evaluation. care physician for a sore throat, lack of energy, and Consider what additional laboratory testing a purple rash. The physician noted splenomegaly could assist in diagnosing Jonathan. and ordered a CBC with differential along with Acute Myeloid Leukemias 585 Overview older FAB system to fall out of favor with healthcare practi- tioners now using the WHO classification in its place. This chapter describes the acute myeloid (also referred to as myelogenous) leukemias (AMLs). It begins with the general laboratory findings in AML followed by a specific descrip- tion of each subgroup in the World Health Organization Etiology And (WHO) classification. Clinical and laboratory findings are Pathophysiology described with an emphasis on defining characteristics. The chapter concludes with the current types of therapy used AML is a disease characterized by two fundamental cellular to treat AML. features: the ability to proliferate continuously and aberrant or arrested development1 (Figure 26-1). Excessive prolifera- tion can be the result of mutations affecting growth factors, growth factor receptors, signaling pathway components, and Introduction transcription factors that regulate genes involved in cell sur- All acute leukemias (ALs) are stem cell disorders character- vival and proliferation (Chapter 23). More than half of the ized by malignant neoplastic proliferation and accumula- cases of AML display cytogenetic abnormalities. Most are tion of immature and nonfunctional hematopoietic cells in balanced, reciprocal chromosomal translocations with many the bone marrow. The disease arises due to a succession of of the translocation break points located at the loci for genes somatic mutations in a hematopoietic stem or progenitor encoding transcription factors and other signaling mole- cell. The neoplastic cells show increased proliferation and/ cules. The most common consequence of the translocation is or decreased programmed cell death (apoptosis). The net the generation of a fusion gene that codes for a novel fusion effect is expansion of the leukemic clone and a decrease in protein. The fusion proteins that are formed usually alter normal cells.1 the normal function of one or both of the rearranged genes Acute leukemia is a clonal expansion of a single trans- and modify the normal programs of cell proliferation, dif- formed cell; therefore, all ALs begin long before any clinical ferentiation, and survival. In addition, other types of genetic signs and symptoms appear. As the leukemic cells expand abnormalities (e.g., epigenetic alterations) likely interact in number, the classic triad of anemia, infection, and bleed- with the cytogenetic mutations (called the multistep origin of ing seen in acute leukemia occur as a result of “normal” malignancy), resulting in the full leukemic transformation.1 hematopoietic cell cytopenias. Death often occurs from either infection or hemorrhage in weeks to months unless therapeutic intervention occurs.2 Laboratory Evaluation The two major categories of acute leukemias are clas- sified according to the origin of the cell with the primary Peripheral Blood defect: acute myeloid leukemia (AML) and acute lympho- The peripheral blood picture is variable in AML. Although blastic leukemia (ALL). If the defect primarily affects the it is traditional to describe leukemias as having elevated maturation and differentiation of the common myeloid pro- leukocyte counts, 50% of the cases have decreased counts or genitor (CMP) cell, the leukemia is classified as AML. If the counts within the reference interval at the time of diagnosis. defect primarily affects the common lymphoid progenitor The leukocyte count ranges from less than 1 * 103/mcL to (CLP) cell, the leukemia is classified as ALL (Chapter 27). greater than 100 * 103/mcL. Regardless of the leukocyte The acute leukemias are classified into subtypes. The concentration, the presence of blasts on the blood smear most reliable parameters for defining and classifying neo- suggests the AL diagnosis. The current WHO definition of plastic cells in AML into subtypes are cell markers (cell sur- AL requires 20% or more blasts in the peripheral blood or face or internal antigens) defined by immunologic probes bone marrow.5 Upon preliminary diagnosis, cases must be and genetic abnormalities identified with cytogenetic and classified based on a combination of immunologic, cyto-

molecular studies (Chapters 40–42). In the late 1970s, the genetic and molecular genetic methods and cytochemical first internationally accepted classification for the acute leu- tests (Chapter 23). Typically, the myeloblast seen in AML kemias, the French-American-British (FAB) classification, is approximately 20 mcM in diameter with variably promi- was based on a combination of neoplastic cell morphol- nent nucleoli in a nucleus composed primarily of dispersed ogy and cytochemical cellular reactions.3 This classification chromatin (euchromatin or transcriptionally active DNA). remained essentially unchanged until 1999 when the WHO A Golgi apparatus is present but is not easily visualized. and the International Society of Hematology proposed a Although blasts normally do not have granules visible by new classification.4 The incorporation of cytogenetic and bright-field microscopy, neoplastic blasts do not always molecular information as well as myelodysplasia and clini- develop normally and can have granules in the RNA-rich cal findings into the characterization of AL has caused the cytoplasm (Chapter 25). 586 Chapter 26 AML AML CML MDS AML AML NP Granulocyte GMP MP Monocyte HSC CMP MkP Platelets MkEP EP Erythrocyte Figure 26.1 AML may originate from any of the cells that fall within the paths of the downward arrows. Importantly, the AML cell of origin acquires the capacity for self-renewal and maturation arrest. AML, acute myeloid leukemia; CML, chronic myeloid leukemia; MDS, myelodysplastic syndrome; CMP, common myeloid progenitor cell; NP, neutrophil progenitor; MP, monocyte progenitor; MkP, megakaryocyte progenitor; EP, erythroid progenitor; MkEP, megakaryocyte erythrocyte progenitor; GMP, granulocyte monocyte progenitor; CMP, common myeloid progenitor; HSC, hematopoietic stem cell. Erythrocytes are typically decreased, and a hemoglobin differentiated monocytic or myelocytic cells (promyelo- value less than 10 g/dL is common. Erythrocytes can be cytes). They are not found in lymphoid blasts. slightly macrocytic because of their inability to successfully Other abnormal findings on the blood smear can compete with neoplastic cells for folate or cobalamin and/ include monocytosis and neutropenia. Monocytosis fre- or early release of marrow reticulocytes. The red cell distri- quently precedes overt leukemia. Neutrophils can dem- bution width (RDW) is often elevated. Erythrocyte inclu- onstrate signs of dysplasia including hyposegmentation, sions, such as Howell-Jolly bodies, Pappenheimer bodies, hypogranulation, and small nuclei with hypercondensed and basophilic stippling, reflective of erythrocyte matura- chromatin. Signs of myelodysplasia are especially common tion defects, may be present. Nucleated erythrocytes may in promyelocytic leukemia.6 Eosinophils and basophils can also be present. be mildly to markedly increased. When present, basophilia Platelets are typically decreased. Hypogranular plate- can help to differentiate leukemia from a leukemoid reac- lets and occasional enlarged forms (giant platelets) may be tion. Absolute basophilia is not present in a leukemoid present. As the disease progresses, more immature platelet reaction. A blast phase of chronic myeloid leukemia (CML) forms, such as megakaryocytic fragments, may be seen. The should be excluded by evaluating BCR/ABL1. platelet count might not correlate with the potential compli- cation of bleeding because qualitative platelet defects may also be present. Bone Marrow If the physician suspects AL but no blast cells are When a diagnosis of AML, MDS, or MPN is suspected, detected on the peripheral blood smear or if the leukocyte the first step is to evaluate the bone marrow. Bone mar- count is low (less than 2 * 103/mcL) a buffy-coat smear row (BM) testing should include both aspirate and biopsy may be prepared and often reveals the presence of blast specimens. The quality of marrow specimen is critical for cells when they are present in very low concentrations. all subsequent analyses. Typically, the BM presentation is Finding blasts with azurophilic granules is helpful in iden- hypercellular with decreased fat content (relative to age- tifying the myeloid nature of the leukemia. The presence of related normals), a predominance of blasts, and sometimes Auer rods (fused or coalesced primary granules) in blasts an increase in fibrosis. excludes a diagnosis of ALL. Auer rods are primarily found According to the WHO criteria for AL, blasts must in myeloblasts and on rare occasions in monoblasts or more compose 20% or more of the nonerythroid nucleated cells Acute Myeloid Leukemias 587 to distinguish AL from myelodysplastic syndromes. Fre- quently, the blast count is close to 100%. Auer rods are Classification present in BM blasts in about half of AML cases. Normal The 2016 revision to the WHO classification is the result- erythropoiesis, megakaryocytopoiesis, and granulopoiesis ing work of a worldwide clinical advisory committee that are decreased or absent in the marrow aspirate. Additional updated the 2008 classification of the hematopoietic neo- assessment of the subtypes of AML evaluates only the plasms. As in the past, the 2016 WHO classification expands myeloid cells, not lymphocytes, plasma cells, mast cells, the parameters used to classify the neoplastic disorders.7 The macrophages, or nucleated erythrocytes. parameters include microscopic morphology, cytochemistry Cells may be clumped together, occasionally forming flow cytometry, cytogenetics, molecular genetic abnormali- sheets of infiltrate that disturb the usual marrow architec- ties, and clinical findings. The WHO major AML subgroups, ture. A larger infiltrate that disrupts marrow architecture each with variants or subtypes, are listed in Table 26-1. may make it difficult to obtain appropriately spiculated bone marrow aspirates. In addition to light microscopic Identification of Cell Lineage morphologic evaluation, BM samples should be sent for Because blast cells are immature cells, they are often difficult flow cytometry, cytogenetics, and molecular genetics. AML to identify by morphology alone using light microscopy. is the diagnosis if blasts possess a t(8;21, inv(16), or t(15;17), Cytochemistry, molecular testing, and immunophenotyping regardless of the blast percentage, when certain clinical give additional information that can help define cell lineage parameters are met.6 Table 26.1 2016 WHO Classification of Acute Myeloid Checkpoint 26.1 Leukemias (AML) What results would you expect to find on the CBC and differen- tial in a suspected case of AL? 1. AML with recurrent genetic abnormalities • AML with t(8;21)(q22;q22.1), RUNX1-RUNX1T1 • AML with inv(16)(p13.1;q22) or t(16;16)(p13.1;q22); CBFB-MYH11 Other Laboratory Evaluation • AML with t(9;11)(p21.3;q23.3); MLLT3-KMT2A • APL (promyelocytic) with PML-RARA Other laboratory findings can reflect the increased prolifera- • AML with t(6;9) (p23;q34.1); DEK-NUP214 tion and turnover of cells. Hyperuricemia and increased • AML with inv(3)(q21.3;q26.2) or t(3;3)(q21.3;q26.2); GATA2, lactate dehydrogenase (LD) are common findings resulting MECOM • AML (megakaryoblastic) with t(1;22)(p13.3;q13.3); RBM15-MKL1 from the increased cell turnover. When present, hypercalce- • AML with mutated NPM1 mia is thought to be caused by increased bone resorption • AML with biallelic mutation of CEBPA associated with leukemic proliferation in the bone marrow. • AML with BCR-ABL1 (new provisional entry) Increased serum and urine muramidase are typical findings • AML with mutated RUNX1 (new provisional entry) in leukemias with a monocytic component. 2. AML with myelodysplasia-related changes AML arising from a previous myelodysplastic or myelodysplastic/ myeloproliferative neoplasm CASE STUDY (continued from page 584) 3. Therapy-related myeloid neoplasms Includes (t-AML), (t-MDS), and (t-MDS/MPN) in patients who receive Admission laboratory data on Jonathan are as cytotoxic therapies (alkylating agents, ionizing radiation, topoisomer- follows: ase, and others) 4. AML not otherwise specified RBC 3.2 * 106/mcL WBC Differential • AML minimally differentiated Hb 9.7 g/dL Blasts 75% • AML without maturation Hct 30 % Neutrophils 6% • AML with maturation PLT 31 * 103 / mcL Promyelocytes 2% • Acute myelomonocytic leukemia • Acute monoblastic and monocytic leukemia WBC 120 * 103/mcL Myelocytes 8% • Pure erythroid leukemia Metamyelocytes 9% • Acute megakaryoblastic leukemia Note: Auer Rods seen in some blasts • Acute basophilic leukemia Erythrocyte morphology: Rare schistocytes and dacryocytes seen. • Acute panmyelosis with myelofibrosis 1. What clues do you have that this patient could 5. Myeloid sarcoma have an acute leukemia? Tumor mass consisting of myeloid blasts with or without maturation occurring in an extramedullary site 2. Based on the presenting data, what additional 6. Myeloid proliferations related to Down syndrome testing might be of value? • Transient abnormal myelopoiesis • Myeloid leukemia associated with Down syndrome 588 Chapter 26 (Chapters 40, 41, 42). When both cytochemistry and immu- a limited, representative panel of monoclonal antibodies. nophenotyping are used, most AL cases can be classified as Common cell markers used in identification include CD2, being of lymphoid or myeloid origin. Rarely, a population CD3, CD4, CD5, CD7, CD10, CD13, CD14, CD15, CD16, of malignant blasts is cytochemically negative by conven- CD19, CD22, CD33, MPO, HLA-DR, CD34, CD45, CD56, tional methods and nonreactive with both lymphoid and CD64, and CD117.9 myeloid monoclonal antibodies. Such leukemias are classi- The first panel of monoclonal antibodies should differ- fied as undifferentiated.5 entiate AML from ALL and T-cell ALL (T-ALL) from B-cell The acute leukemias of ambiguous lineage encompass ALL (B-ALL). Use of a panel of antibodies such as those leukemias that show no clear evidence of differentiation to listed in Table 26-2 can usually discriminate AML from ALL. a single lineage. This group includes leukemias without Individual facilities have their own preferred panels of anti- lineage-specific antigens (acute undifferentiated leukemia) bodies. Panels should include typing for the myeloid anti- and leukemias that express antigens of more than one lin- gens, the B-lymphoid cell antigens (CD19, CD20, CD22, eage, making it impossible to determine any one lineage and CD79a), and the T-lymphoid cell antigens (CD2, CD3, (mixed phenotype acute leukemias, MPAL). These leuke- CD5, and CD7). Several aberrant antigens are sometimes mias are discussed in Chapter 27. found on neoplastic cells (e.g., the CD7 [T-lymphoid] anti- gen can be found on neoplastic myeloid cells in AML). CYTOCHEMISTRY The CD34 marker is also present on the least differentiated The common cytochemical stains include the myeloperoxi- myeloid cells and early lymphoid cells. The monoclonal dase (MPO), Sudan black B (SBB), naphthol AS-D chloroac- antibodies that react with most cases of AML include CD13, etate esterase (specific esterase), and a@naphthyl esterases CD15, CD33, CD64, and CD117.8 (nonspecific esterase) (Chapter 34). Granulocytic cells stain The CMP cell is capable of differentiation into granulo- positive with MPO and with SBB; lymphoblasts are nega- cytes, erythrocytes, monocytes, and megakaryocytes. Thus, tive. Thus, these stains help differentiate the acute myeloid if the neoplastic clone has “early” myeloid antigens, a sec- leukemias from the acute lymphoblastic leukemias. The ond panel of monoclonal antibodies should include anti- esterase stain helps differentiate precursor granulocytic bodies to subtype the AML into granulocytic, monocytic, cells from precursor monocytic cells. Granulocytic cells erythrocytic, and megakaryocytic lineages (Table 26-3). stain positive with naphthol AS-D chloroacetate esterase, and cells of monocytic lineage stain positive with nonspe- cific esterase. Most institutions use immunophenotyping and cytogenetics as first-line tests to define cell lineage. CASE STUDY (continued from page 588) Flow cytometry or immunohistochemistry can be used 4. If the cells from Jonathan’s BM were immu- to demonstrate terminal deoxynucleotidyl transferase (TdT) nophenotyped, which of these—CD13, CD33, in individual cells. Although originally thought to be a lym- CD20, CD2, CD7, CD10, CD19—would you phoid specific marker, TdT is found on more immature expect to be positive? hematopoietic cells, sometimes including those of myeloid lineage. Therefore, TdT cannot be the sole determinant of lymphoid lineage. CYTOGENETIC ANALYSES Two-thirds of patients with AML have detectable cytoge- netic abnormalities that include aneuploidies (variation in CASE STUDY (continued from page 587) total chromosome number) and translocations.1 Commonly 3. Based on the peripheral blood examination, what observed aneuploidies include trisomy 8, monosomy 7, cytochemical stain results would you expect to monosomy 21, trisomy 21, and loss of an X or Y chromo- find on Jonathan’s neoplastic cells? some. Translocations result in fusion genes that are either Table 26.2 Differentiation of ALL from AML Using IMMUNOPHENOTYPING Immunophenotyping with Selected Monoclonal Antibodies Immunophenotyping by flow cytometry has become a nec- Cell Marker essary component of AL classification, particularly when the presence of more than one neoplastic cell population Leukemia CD13, CD19, CD20, CD2, CD3, HLA-DR CD10 Type CD33 CD22, CD79a CD5, CD7 is suspected.8 Immunophenotyping by flow cytometry follows a specific sequence of testing with monoclonal AML + + - - - antibodies. The use of extensive panels is costly and time B-lymphocyte + - + + - consuming. In most cases, lineage can be determined using T-lymphocyte - - - { + Acute Myeloid Leukemias 589 Table 26.3 Morphologic, Immunophenotypic, and Cytochemical Results Used to Classify AML Subcategories Cell Markers with Monoclonal Antibodies CD71 CD41, CD42, Other Markers That AML Subgroup Morphology Cytochemistry HLA-DR CD117 CD34 CD13 CD33 CD11b CD14 Glycophorin A CD61 May Be Present AML not otherwise

specified AML minimal Minimal evidence of Myeloperoxidase - + + + + + + + + - - - - CD7, CD38 TdTa differentiated myeloid differentiation (less than 3% of blasts) No maturation seen Sudan black B- Agranular cytoplasm (less than 3% of blasts) Specific esterase - AML without Evidence of myeloid dif- Myeloperoxidase + + + + + + + + + +/- + - - CD7, Lysozyme maturation ferentiation, no matura- (greater than 3% of blasts) tion seen, Auer rods or Sudan black B+ some granulation (greater than 3% of blasts) Specific esterase + AML with All stages of neutrophil Myeloperoxidase + +/- + + +/- + + + + + - - - - CD4, CD15, CD19, maturation maturation Sudan black B+ Lysozyme Pseudo–Pelger-Huët Specific esterase + Hypogranulation Esoinophilic precursors possible Acute myelomono- Neutrophil and mono- Myeloperoxidase + - + - + + + + + + +/- - - CD4, CD11c CD36, cytic leukemia cytic precursors present Nonspecific esterase +/- CD64, CD68, (AMML) Vacuolization Specific esterase + Lysozyme Acute monoblastic/ Monoblast/monocyte Myeloperoxidase - + + - + + + + + + - +/- CD11c, CD15 monocytic leukemia dominance Nonspecific esterase +/- CD65,CD4 (AMoL) Hemophagocytosis Specific esterase + CD64, Lysozyme present Nuclear lobulation Pure erythroid 80% or more immature Myeloperoxidase + +/- + - + + + + - - + - - leukemia erythroid precursors; Sudan black B+ 30% or more are pro- Nonspecific esterase +/- erythroblasts; less than PAS + (erythroblasts) 20% myeloblasts pres- ent amongall cells in the bone marrow Acute megakaryo- Cytopenia with or with- Myeloperoxidase - + + + - + - - - +/- - blastic leukemia out thrombocytopenia Sudan black B- (AMkL) Basophilic agranular Nonspecific esterase +/- blasts with pseudopods Specific esterase - Micro-megakaryocytes PAS+ Platelet peroxidase + Acute basophilic Basophilic precursors Metachromatic positivity + + + - + - - - + + CD123, CD203, leukemia (ABL) Vacuolization with toluidine blue CD9 Acute panmyelosis Pancytopenia Myeloperoxidase + + + + + + - - +/- +/- Lysozyme with myelofibrosis Dysplastic changes in neutrophils and platelets (Continued) 590 Chapter 26 Table 26.3 Continued Cell Markers with Monoclonal Antibodies CD71 CD41, CD42, Other Markers That AML Subgroup Morphology Cytochemistry HLA-DR CD117 CD34 CD13 CD33 CD11b CD14 Glycophorin A CD61 May Be Present Therapy-related myeloid neoplasms (t-MDS, t-AML, t-MDS/MPN) Most have multilineage Myeloperoxidase + No con- CD13, CD33, CD34, dysplasia Sudan black B+ sistent CD7, Basophilia is often Increased iron in ring pheno- CD56 present formation type Pancytopenia or isolated PAS+ cytopenia Specific esterase + Associated with Nonspecific esterease + unbalanced loss of genetic material often involving chromosomes 5 and or 7 AML with myelodysplasia-related changes Following MDS or Dysplasia required in Myeloperoxidase + No con- CD13, CD33, CD34, MDS/MPNs 50% or more of two cell Sudan black B+ sistent CD7, CD56, Multidrug With a cytogenetic lines Increased iron in ring pheno- resistance glycoprotein abnormality charac- Hypogranular neutro- formation type receptor (MDR-1) teristic of MDS phils, pseudo-Pelger- PAS+ With multilineage Huët nuclei dysplasia without Dyserythropoiesis with NPM1 or biallelic megaloblastoid changes, CEBPA mutations ringed sideroblasts, nuclear fragments or vacuoles Dysmegakaryopoiesis with micromegakaryo- cytes, hypolobulated nuclei, and other dysplastic signs a TdT is usually used to identify early lymphoid precursors but can also be found in 10–20% of AML Specific esterase = Napthol AS-d chloroacetate esterase; nonspecific esterase = Alpha - naphthyl esterase Acute Myeloid Leukemias 591 beneficial for proliferation and survival or disrupt differ- sensitivity than cytogenetics for diagnosing and following entiation and maturation. Additional genetic abnormalities the progression of the disease. The AMLs in this category can develop in subclones as the disease progresses. generally have a high rate of complete remission and a more Characteristic, nonrandom cytogenetic abnormalities favorable prognosis. Table 26-4 outlines the morphologies are observed in the WHO classifications of AML with recur- frequently encountered for each recurrent abnormality. rent genetic abnormalities and frequently in therapy- AML with T(8;21)(Q22;Q22.1); RUNX1-RUNX1T1 This sub- related myeloid neoplasms. These nonrandom chromosome category is found in 5–10% of AML cases (previously clas- abnormalities are discussed in subsequent sections. If the sified as AML with maturation). It occurs predominantly in expected abnormal karyotype is not found, fluorescence in children and adults younger than 60 years of age.10 Myelo- situ hybridization (FISH) or molecular analysis is some- blasts are usually large with abundant basophilic cytoplasm times helpful. containing numerous azurophilic granules, perinuclear clearing, and Auer rods. Some blasts may show very large granules (pseudo-Chédiak-Higashi granules). Promyelo- Checkpoint 26.2 cytes, myelocytes, and mature neutrophils with variable Explain why molecular analysis is not performed on all sus- dysplasia are usually present in the bone marrow. Dyspla- pected cases of acute leukemia. sia in other cell lines is uncommon. Eosinophilia can be present. Cytochemical and immunophenotypic results are typical for myeloblasts (Appendix B). The leukemic “cell of WHO Classification of AML origin” is believed to be the hematopoietic stem cell (HSC; Figure 26-1). AML WITH RECURRENT GENETIC ABNORMALITIES The t(8;21)(q22;q22) translocation repositions the 5′ The AML with recurrent genetic abnormalities classi- region of the RUNX1 gene (chromosome 21, previously fication is used when chromosomal or molecular-level termed AML1) with the 3′ region of the RUNX1T1 gene abnormalities, are found. In most cases, the chromosomal (chromosome 8; also called ETO). The result is a fusion rearrangements create a fusion gene, encoding a novel protein called RUNX1-RUNX1T1 (also known as RUNX1- fusion (chimeric) protein while the molecular-level muta- ETO). RUNX1 is a member of a family of genes called tions involve genes that impair cellular differentiation and core@binding factor@a (CBFa). CBFa proteins such as RUNX1 apoptosis. Molecular techniques (RT-PCR) have a higher normally form a heterodimeric complex with CBFb proteins Table 26.4 Cytogenetic and Morphologic Results Used to Classify AML with Recurrent Cytogenetic Abnormalities AML with recurrent genetic abnormalities Morphology AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 Maturation in the neutrophil lineage Auer rods AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22) or t(16;16) Acute myelomonocytic leukemia (AMML Eo) (p13.1;q22); CBFB-MYH11 Maturation in both the neutrophil and monocytic lineage, abnormal eosinophils Eosinophils 5% or more Abnormal eosinophilic granules present in promyelocyte or myelocyte stage AML with t(9;11)(p21.3;q23.3); MLLT3-KMT2A Monoblasts and promonocytes predominate, can see vacuoles and granules Acute promyelocytic leukemia with PML-RARA Hypergranular variant Promyelocytes dominate Multiple Auer rods in one cell Microgranular variant Bi-lobed nuclei No visible granularity AML with t(6;9)(p23;q34.1); DEK-NUP214 With or without monocytic features and often associated with basophilia and multilineage dysplasia AML with inv(3)(q21.3;q26.2) or t(3;3) (q21.3;q26.2); GATA2, Associated with elevated platelets and atypical megakaryocytes (mono or MECOM bilobed) and multilineage dysplasia AML with t(1;22)(p13.3;q13.3), RBM15-MLK1 Megakaryocytic lineage with maturation AML with mutated NPM1 Myelomonocytic or monocytic features presents in older people with a normal karyotype AML with biallelic mutated CEBPA Myeloid leukemia with or without maturation AML with mutated RUNX1 (provisional entity) Can show myelomonocytic or monoblastic features AML with BCR-ABL1(provisional entity) 592 Chapter 26 in order to bind to DNA as functional transcription factors. detectable by conventional cytogenetics, so FISH or molecu- The RUNX1-RUNX1T1 fusion protein acts as a transcrip- lar studies must be performed.7 tional repressor by blocking the normal DNA binding func- tion of RUNX1, resulting in its inability to regulate cellular differentiation. Furthermore, the RUNX1-RUNX1T1 fusion Checkpoint 26.3 What does recurrent genetic abnormality mean in the context of leads to the activation of other genes involved in cellular AML, and what is the general outcome of these abnormalities? proliferation. The outcome is blocked differentiation and increased proliferation of the leukemic cells.11 AML with INV(16)(P13.1;Q22) OR T(16;16)(P13.1;Q22); CBFB- AML with PML-RARA Acute promyelocytic leukemia MYH11 This subcategory is identified in 7% of patients (APL) with PML-RARA is an AML in which abnormal pro- with AML12 and generally presents with monocytic and myelocytes predominate. Both hypergranular (“typical” granulocytic maturation and the presence of abnormal APL) and hypogranular or microgranular presentations eosinophils in the bone marrow. The most striking abnor- can be seen. APL can occur at any age, but most patients mality of the eosinophils is the presence of immature (baso- are adults in middle life. APL constitutes 5–8% of AML.7 philic) granules that would normally predominate at the The presenting signs for both hypergranular and hypo- promyelocyte and myelocyte stages (Chapter 7). The immu- granular APL often include acute disseminated intravas- nophenotypic and cytochemical results reflect the presence cular coagulation (DIC; Chapter 34).15 The most common of both neutrophilic and monocytic lineages (Appendix B). clinical finding at initial diagnosis is bleeding. The release of The leukemic cell of origin is believed to be an HSC. procoagulant material from promyelocytic granules likely The inv(16)(p13.1;q22) and t(16;16)(p13.1;q22) both initiates DIC, a serious complicating factor of the disease. result in the fusion of the core-binding factor gene CBFB Cytotoxic therapy for APL can potentiate or aggravate this (16q22) to the smooth muscle myosin heavy chain gene complication. Not only do the lysed promyelocytes release SMMHC (MYH11) at 16p13.1. Because of difficulties in large amounts of procoagulant material but also tissue correctly identifying these mutations with traditional cyto- factor–containing particles are released from dying cells genetics, FISH or RT-PCR may be necessary to document during cytotoxic therapy. Evidence of secondary fibrinoly- the mutation. The CBFb - MYH11 fusion protein binds to sis as a component of the DIC syndrome is also present. RUNX1 and represses its transcription factor function.13 Heparin therapy can be administered with initiation of che- motherapy to prevent or modulate the DIC. Other abnor- AML with T(9;11)(P21.3;Q23.3); MLLT3-KMT2A These malities of coagulation can be present. leukemias are usually associated with monocytic features (monoblasts and promonocytes). They can occur at any Hypergranular APL. Most patients with the hypergranular age but are more common in children (up to 25% of child- subtype are leukopenic or exhibit only slightly increased hood AML cases and 2–5% of all AML cases).14 Identical leukocyte counts. Most cells in the BM are abnormal pro- cytogenetic abnormalities can also be found in therapy- myelocytes with heavy azurophilic granulation. The gran- related AML (see the section “Therapy-Related Myeloid ules can be so densely packed that they obscure the nucleus Neoplasms”). Patients can have extramedullary myeloid (Figure 26-2a). Some cells filled with fine dustlike granules (monocytic) sarcomas (extramedullary leukemia) and tissue also can be present. Cells with multiple Auer rods, some- infiltration (gingiva, skin) and may present with dissemi- times occurring in bundles, are characteristic with cyto- nated intravascular coagulation. Monoblasts and promono- plasm that is frequently clear and pale blue, but cells can cytes can have scattered azurophilic granules and vacuoles contain only a few azurophilic granules or lakes of clear and give phenotypic results typical for the monocytic lin- pink material16 (Figure 26-3). In some cases, the typical eage (Table 26-4). The leukemic cell of origin is believed to hypergranularity of promyelocytes is less evident in the be the HSC. peripheral blood than in the bone marrow.17 The nucleus The KMT2A (formerly MLL) gene (11q23) is involved in varies in shape but is often folded or indented or sometimes a number of leukemia-associated translocations with differ- bilobed. Often a large number of promyelocytes appear to ent partner chromosomes. The KMT2A protein is a DNA- be disrupted on the blood smear with free azurophilic gran- binding protein that interacts with other nuclear proteins ules and Auer rods intermingled with intact cells. Anemia and permits the association of transcription factors that help and thrombocytopenia are typical findings. regulate transcription. The most common translocations Microgranular APL Variant. In contrast to typical APL, the involving 11q23 seen in childhood AML are t(9;11)(p21;q23), leukocyte count in microgranular APL is usually markedly t(11;19) (q23;p13.1), and t(11;19)(q23;p13.3). More than 80 increased.16 The predominant cell in the peripheral blood different translocations involving KMT2A are described is a promyelocyte with a bilobular, reniform, or multilobed with more than 50 different translocation partner genes. Up nucleus (resembling that of a monocyte) and cytoplasm with to one-third of KMT2A translocations in AML are not an apparent paucity of granules on Wright-stained smears Acute Myeloid Leukemias 593 a b Figure 26.2 (a) Peripheral blood film from a patient with acute promyelocytic leukemia (APL), hypergranular variant. These promyelocytes have an irregularly shaped nucleus and numerous azurophilic granules. (b) Peripheral blood film from a patient with APL, microgranular variant. Note the bilobed nuclei and absent or fine granules in these abnormal promyelocytes. (Wright-Giemsa stain, 1000* magnification) (Figure 26-2b). The apparent absence of granules is because A

form of APL in which some promyelocytes contain of their submicroscopic size, but they are readily visible metachromatic granules (when stained with toluidine blue) with cytochemistry (myeloperoxidase, MPO positive). has been described.18 Distinctive features include folded The nuclear chromatin is fine with nucleoli often visible. nuclei, hypergranular cytoplasm, and coarse metachromatic A small abnormal promyelocyte with a bilobed nucleus, granules. deeply basophilic cytoplasm, and sometimes cytoplasmic Diagnostic Gene Rearrangement. The PML-RARA fusion projections is present as a minor population of cells in is consistently associated with APL. The break point most cases and occasionally is the predominant cell. When involves the retinoic acid receptor@a (RARa) gene on chro- cytoplasmic projections are present, the cells can resemble mosome 17 and the promyelocytic leukemia (PML) gene megakaryoblasts. Cells with multiple Auer rods are scarce on chromosome 15 and is limited to the neoplastic cells. or absent, but single Auer rods can be found. In contrast Some APL patients have a cytogenetically normal karyo- to the hypogranular appearance of peripheral blood pro- type, but molecular analysis has shown submicroscopic myelocytes, the BM promyelocytes may be more typical insertion of RARa into PML, leading to the expression of the cells found in the hypergranular form of APL. See of the PML/RARa transcript. These findings suggest Appendix B for cytochemical and immunophenotypic that the t(15;17) translocation/fusion protein is involved results for APL. in the pathogenesis of the disease. However, other vari- ant translocations have been reported in APL, all involv- ing the RARa gene translocated with different target genes, including promyelocytic zinc finger (PLZF) on chromosome 11(t[11;17][9q23;q21]), nuclear mitotic apparatus (NuMA) on chromosome 15 (t[11;17][q13;q21]), nucleophosmin (NPM1) on chromosome 5 (t[5;17][q23;q12]), and signal transducer and activator of transcription 5b (STAT5b) on chromosome 17 (t[17;17]).19 The complex routes by which the PML-RARA fusion protein can be found led the 2016 WHO classification to be more inclusive and not focus on the t(15;17) muta- tion in the diagnosis and instead rely on the presence of the fusion protein.7 The typical t(15;17) gene rearrangement results in a fusion PML/RARa gene and a reciprocal RARa/PML gene. The PML/RARa mRNA has been identified in all APL Figure 26.3 A cell from promyelocytic leukemia. Notice patients and the reverse translocation RARa/PML mRNA the bundle of Auer rods (arrow). (Wright-Giemsa stain, 1000* is found in about 75% of APL patients.20 Molecular analysis magnification) of the PML/RARa gene is important in monitoring therapy 594 Chapter 26 and relapse because the gene disappears with successful destroys the dividing stem cells that bear the t(15;17) trans- treatment but returns as an early marker of relapse. Also, location and induces a durable remission.1 some patients have molecular evidence of the fusion protein About 25% of patients given RA therapy become acutely product (fusion gene) in the absence of a detectable cytoge- ill associated with the death and lysis of the large number netic abnormality. of cells. The mortality rate in these individuals can be high. The RARa protein is a nuclear hormone receptor that The illness is similar to capillary leak syndrome with fever, binds to specific DNA sequences (RA-responsive elements) respiratory disease, renal impairment, and hemorrhage.23 and controls transcription of specific target genes under the control of retinoid hormones. RARa forms a complex with a second protein, retinoid-X receptor (RXRa). In the Checkpoint 26.4 absence of RA, the RARa/RXRa complex represses tran- Why is it important to perform molecular studies on patients scription by recruiting corepressor proteins and inducing with AML with t(15;17)? histone deacetylation.20 Physiologic concentrations of RA normally induce a conformational change in the retinoic acid receptors, causing release of the corepressor molecules AML with T(6;9)(P23;Q34.1); DEK-NUP214 This AML pres- and recruitment of coactivator molecules, resulting in gene transcription and granulocytic differentiation.21 ents with or without monocytic features often associated with basophilia and multilineage dysplasia. It is detected in PML is a growth suppressor nuclear protein normally 0.7–1.8% of AML and occurs in both children and adults.7 found in complex macromolecular structures containing AML with t(6;9)(p23;q34) usually presents with anemia and numerous other nuclear proteins. The PML/RARa fusion thrombocytopenia, and often with pancytopenia. In adults, protein binds to the RA-responsive elements of the target the white blood cell count is generally lower than other genes. Like the native protein, the fusion protein recruits AML categories.24 Morphologically, they most commonly corepressor molecules and in the absence of RA inhibits present with AML with maturation or acute myelomono- transcription of RA-responsive genes. Although the fusion cytic leukemia. Auer rods are present in one-third of cases. protein can bind RA, physiologic concentrations of RA are Marrow and peripheral blood basophilia is seen in 44–62% not sufficient to induce the release of the corepressor pro- of cases. Most cases show evidence of granulocytic and ery- teins from PML/RARa and repression of gene transcrip- throid dysplasia. tion is maintained.21 Thus, the cells do not differentiate into The t(6;9)(p23;q34) results in a fusion of the DEK gene granulocytes. on chromosome 6 with the NUP214 (formerly known as Pharmacological concentrations of RA (all-transretinoic CAN) gene on chromosome 9. This results in a nucleoporin acid [ATRA]) have proven to be an effective treatment for fusion protein that acts as an aberrant transcription factor inducing complete hematologic remission in APL (but the and alters nuclear transport by binding to soluble transport remission is generally short lived). ATRA induces the neo- factors.25 The t(6;9) is the sole abnormality in the majority of plastic promyelocytes to differentiate to mature granulo- cytes, thus overcoming the maturation arrest.22 cases, but some patients have the t(6;9) in association with It is thought a complex karyotype. that a high concentration of RA, as is given in induction therapy for APL, overcomes the interference with receptor AML with INV(3)(Q21.3;Q26.2) OR T(3;3)(Q21.3;Q26.2); activation (it promotes release of the corepressor molecules GATA2, MECOM This AML can present de novo or arise and assembly of the coactivator molecules on the target from a prior MDS. It is often associated with normal or genes, allowing gene transcription and cellular differentia- elevated peripheral blood platelet count and has increased tion). Cells previously unable to mature and initiate apop- atypical megakaryocytes with mono- or bi-lobated nuclei tosis are then able to do so, causing a transitory situation and associated multilineage dysplasia. AML with inv(3) known as hyperleukocytosis. accounts for 1–2% of all AML.26 It occurs most commonly Unfortunately, the stem cells bearing the t(15;17) trans- in adults with no sex predilection. Patients present with location are unaffected by ATRA and continue to proliferate, anemia and a normal or elevated platelet count. Some pres- resulting in relapse of the disease. The current approach ent with hepatosplenomegaly. A subset of cases may have to therapy is to induce hematologic remission with ATRA less than 20% blasts at the time of diagnosis with features and then administer traditional chemotherapy (to try to of chronic myelomonocytic leukemia. Marrow eosinophils, eradicate the leukemic stem cells). When the promyelocytes basophils, and mast cells may be increased. The BM shows are first induced to differentiate and are no longer pres- small hypo-lobated megakaryocytes. ent in large numbers, the subsequent chemotherapy does A variety of abnormalities in the long arm of chromo- not result in the massive death of promyelocytes (and the some 3 occur in myeloid malignancies; inv(3)(q21;q26.2) release of procoagulants), thus avoiding or minimizing the and t(3;3)(q21;q26.2) are the most common. The t(3;3) life-threatening effects of DIC. Instead, the chemotherapy (q21;q26.2) involves the repositioning of a GATA2 enhancer Acute Myeloid Leukemias 595 that simultaneously causes activation of the EVI1 gene or show extramedullary involvement with the most frequently the MECOM complex locus (MDS1-EVI1 genes) and leads affected sites being the gingiva, lymph nodes, and skin. to leukemogenesis, and ihematopoietic cell transforma- NPM1 mutation is found in approximately 35% of AML tion.27–29 Secondary karyotypic abnormalities are common patients.34 NPM1 mutations can also be seen in AML with with inv(3)(q21;q26.2) and t(3;3)(q21;q26.2) with monosomy and without maturation and in acute erythroid leukemia. 7 most common, occurring in approximately 50% of cases, The diagnosis of AML with mutated NPM1 is made followed by 5q deletions and complex karyotypes. based on the identification of the mutation by molecular studies and is usually associated with a normal karyotype; AML with T(1;22)(P13.3;Q13.32); RBM15-MKL1 This AML 5–15% show chromosomal abnormalities including trisomy generally shows maturation in the megakaryocytic lineage. 8 and del(9q). It is uncommon, representing less than 1% of all cases. It occurs de novo and is restricted to infants and young AML with Biallelic Mutations of CEBPA Acute myeloid children with most cases occurring in the first 6 months leukemia with mutated CEBPA is usually seen in AML of life. The clinical presentation is marked organomegaly, with or without maturation. Some cases can show myelo- especially hepatosplenomegaly. Patients present with ane- monocytic or monoblastic features. The CEBPA protein is mia and thrombocytopenia with a moderately elevated normally expressed at low levels in HSCs, increases in con- white blood cell count. The blasts are similar to those of centration as cells mature to the CMP stage, and decreases acute megakaryoblastic leukemia. Small and large mega- again as cells mature to terminally differentiated neutro- karyoblasts may be present along with morphologically phils or monocytes. Loss of CEBPA gene expression leads undifferentiated blasts resembling lymphoblasts. The to maturation arrest at the CMP.35 megakaryoblasts are usually medium to large in size with CEBPA mutations usually present de novo, and are seen an indented nucleus, fine reticular chromatin, and one to in 5–15% of AML.36 There is no age or sex difference. Patients three nucleoli. The cytoplasm is basophilic and often agran- present with a higher hemoglobin level and lower platelet ular and may show distinct blebs or pseudopod formation. counts, lower LD levels, and higher peripheral blood blast Micromegakaryocytes are common. Dysplasia in the ery- count when compared with CEBPA non-mutated AML. throid and granulocytic cell lines is usually not present. AML with a normal karyotype and biallelic CEBPA muta- The t(1;22)(p13;q13) translocation results in the fusion tion is associated with a favorable prognosis while those of the RNA-binding motif protein-15 (RBM15) gene (1p13) with only a single CEBPA mutation do not have the same with the megakaryoblastic leukemia-1 (MKL1) gene (22q13) outcome.7 This favorable outcome is only associated with and ultimately, expression of the RBM15–MKL1 fusion pro- biallelic mutations, thus the 2016 WHO classification distin- tein. In most cases, this is the sole abnormality. guishes between single and double mutation disease. The function of RBM15 is not well defined. It appears to act as a transcriptional repressor that negatively regu- Provisional Subclasses and Miscellaneous Mutations lates megakaryocyte development. When RBM15 is lost or As in prior editions, the 2016 WHO classification uses the nonfunctional, megakaryocytes are increased in number in provisional entity designation to introduce new subclasses the BM and spleen.30 MKL1 encodes a transcriptional coacti- to the recurrent genetic abnormalities subtypes. These enti- vator of genes involved in megakaryocyte differentiation ties are included based on new data that highlights the role and maturation.31 The precise role of the fusion protein these mutations play in disease progression and response in AML is unclear; however, it may modulate chromatin to therapy. The FLT3 mutation is also discussed below due organization (epigenetic control) in regions important for to its importance in prognosis. megakaryocytic development.32 AML With BCR-ABL1 BCR-ABL1 mutations are found in a AML with Mutated NPM1 This form of AML usually number of neoplastic hematopathologies including an esti- involves mutations in exon 12 of the NPM1 gene. The NPM mated 1% of de novo AML. As is the case in other disease protein is a chaperone of proteins that shuttles between the processes, this mutation is more prevalent among males in nucleus and cytoplasm. NPM1 gene mutations appear to the sixth decade of life.37 Without appropriate clinical his- cause loss-of-function (Chapter 23) of the NPM protein to tory, differentiating Philadelphia chromosome positive de the extent that NPM remains in the cytoplasm and can no novo AML from blastic transformations of CML may be dif- longer translocate to the nucleus.33 ficult, but its presence in AML is important as it indicates This AML frequently has myelomonocytic or mono- that tyrosine kinase therapy may be beneficial.7 cytic features and presents de novo in older adults with Examination of peripheral blood often shows variation a normal karyotype. Usually there is no

history of a prior in the white blood count, but median counts are approxi- MDS or MPN. Patients often have anemia and thrombocy- mately 19.0 * 103/mcL. Typically the cells present are topenia and a higher white blood cell count than other AML immature myeloid cells, although occasional cases involve types. There is a slight female predominance. Patients can a predominance of monocytic or megakaryocytic elements. 596 Chapter 26 Statistically significant differences in the presence of In these cases, AML occurs as a late complication of cyto- splenomegaly and absolute basophilia have been noted toxic chemotherapy and/or radiation therapy administered between CML and AML patients with this mutation. for a prior malignancy. This group of neoplasms is best con- However a more definitive approach for separating de sidered together as a unique clinical syndrome. novo AML from blast transformation in CML is being Therapy-related neoplasms are thought to be the sought for those patients with limited clinical information consequence of mutations induced by cytotoxic therapy. present at diagnosis.7,37 Cytotoxic agents commonly implicated include alkylating agents, topoisomerase II inhibitors, and ionizing radia- AML with Mutated RUNX1 This new provisional subtype tion therapy. Two subsets are recognized. The most com- is defined by RUNX1 gene mutation and is found pre- mon occurs 5–10 years after receiving the alkylating drug dominantly in patients who develop AML with minimal or radiation. The patient often presents with t-MDS, and a differentiation. Megakaryoblastic subtypes of AML may minority will present with t-MDS/MPN or t-AML. Patients also demonstrate the mutation. Myelodysplastic-related commonly have an unbalanced loss of genetic material alterations in hematopoietic lineages are often noted, even involving chromosome 5 and/or 7. In the second subset, the in cases of de novo AML. The RUNX1 gene is located on latency period is shorter, about 1–5 years, and follows treat- the long arm of chromosome 21 and plays an important ment with topoisomerase II inhibitors. Most of the patients role in the development of hematopoietic stem cells during embryonic and fetal development.38 in this category present with acute leukemia. Approximately 70% of patients have unbalanced chro- While the gene mutation itself is not known to be leu- mosomal losses, mainly with chromosome 5 and/or 7, and kemogenic, it is often found with other genetic mutations or are often associated with additional abnormalities including aneuploidy states.39 It is included in the 2016 WHO classi- del(13q), del(20q), del(11q), del(3p), -17, -18, -21, +8. This fication because its presence is associated with unfavorable outcomes and resistance to therapy.7,39 type of AML generally has a poor prognosis.1 AML WITH MYELODYSPLASIA-RELATED CHANGES FLT3 and AML Accurate assessment of prognosis is central This category is an acute leukemia with morphological to the management of AML. The FLT3 gene mutations are features of a myelodysplastic neoplasm, a prior history of associated with a worse prognosis. The FLT3 gene is located myelodysplastic neoplasm, a myelodysplastic/myelopro- at 13q12 and encodes a tyrosine kinase receptor involved liferative neoplasm or an MDS-related cytogenetic abnor- in hematopoietic stem cell differentiation and proliferation. mality. To classify as AML with myelodysplasia-related The FLT3 protein is expressed on progenitor cells and blasts changes by morphology, the dysplasia must be present in most cases of AML. FLT3 mutations may occur with any in 50% of cells in at least two cell lineages. Dyserythro- AML type and in MDS and can occur with or without addi- poiesis is characterized by megaloblastosis, karyorrhexis, tional cytogenetic abnormalities.40 Mutations lead to con- nuclear irregularity, multinucleation, ring sideroblasts, or stitutively activated FLT3 that transduces signals for cell cytoplasmic vacuoles. Dysgranulopoiesis is characterized growth and inhibition of apoptosis. FLT3 gene mutations by neutrophils with hypogranular cytoplasm, hyposeg- occur in approximately 30% of adult and 12% of pediatric patients with de novo AML.40 mentation (pseudo–Pelger-Huët anomaly), or abnormal segmentation. Dysmegakaryopoiesis is characterized by The two primary types of FLT3 mutations are internal micromegakaryocytes, megakaryocytes with non-lobated tandem duplication (FLT3-ITD) seen in 30–40% of cases, and nuclei, or multiple nuclei. The chromosomal abnormalities mutations affecting the second tyrosine kinase domain found in this category are similar to those found in MDS (TKD).5 FLT3-ITD mutations are associated with an adverse and often involve complex karyotypes with -7/del(7q) and outcome, but the significance of FLT3-TKD mutations -5/del(5q) being the most common (Chapter 25). Trisomy remains controversial.7 Although FLT3 mutations are not 8 and del(20q) are common in MDS but are not considered included as a defining criterion for WHO classification, they are important for prognosis and should be evaluated.5 to be disease-specific abnormalities and would not be suf- ficient by themselves to be considered a case of AML with myelodysplasia-related changes. The 2016 WHO classifica- Checkpoint 26.5 tion has determined that multilineage dysplasia with either Would the FLT3, NPM1, and CEBPA genes be classified as NPM1 or bialleic mutations of CEBPA cannot be classified tumor suppressor or proto-oncogenes? under AML with myelodysplastic-related changes.7 AMLS NOT OTHERWISE SPECIFIED THERAPY-RELATED MYELOID NEOPLASMS This AML category includes cases of AML that do not fulfill This category includes therapy-related acute myeloid leuke- criteria for any of the other described groups.5 The subcat- mia (t-AML), myelodysplastic syndrome (t-MDS), and myelo- egories in this group are primarily differentiated on mor- dysplastic/ myeloproliferative neoplasms (t-MDS/MPNs). phology and cytochemical features. The defining criterion Acute Myeloid Leukemias 597 is 20% or more blasts (the blast percentage includes promy- elocytes in APL and promonocytes in AML with monocytic Table 26.5 Cytochemical Reactions for Auer Rods differentiation). For pure erythroid leukemias, the blast SBB + percentage is based on the percentage of abnormal erythro- Myeloperoxidase (MPO) + blasts. Immunophenotypic and cytochemical results for this Napthol AS-D chloroacetate esterase { category of AML are included in Table 26-3. PAS { AML WITH MINIMAL DIFFERENTIATION AML with Romanowsky + or - (occasionally seen only with MPO or SBB) minimal differentiation is a rare leukemia (less than 5% of all AML cases) and is characterized by lack of evidence of myeloid differentiation by morphology (absence of gran- The defining feature is its lack of cellular maturation; less ules) or cytochemistry (63, of the blasts are positive for than 10% of all granulocytic cells show evidence of matura- SBB or MPO).41 This subtype is generally associated with a tion beyond the myeloblast stage (Figure 26-4). poor prognosis. The blasts, however, demonstrate myeloid The predominant cell in the peripheral blood is usually differentiation by flow cytometry for the myeloid lineage a poorly differentiated myeloblast with fine lacy chromatin (CD13 and CD33). Most cases are also CD34, CD38, and and nucleoli. Myeloblasts are usually 90% or more of non- HLA@DR+ . No unique chromosomal abnormalities are erythroid cells in the BM. with more than 3% of blasts posi- associated with this subtype. The most common previously tive for MPO and/or SBB. The blasts may have azurophilic reported abnormalities are complex karyotypes and unbal- granules, Auer rods, and vacuoles. If no evidence of gran- anced abnormalities such as -5/del(5q), -7/del(7q), +8, ules or Auer rods is present, the blasts can resemble lym- and del(11q), but the presence of these abnormalities now phoblasts and must be differentiated by immunophenotype. places them in a different category (Table 26-1).10 Because Cytochemical staining reactions for Auer rods are similar to the blasts in the minimally differentiated AML group have reactions for myeloblasts (Table 26-5). There is no demon- no morphologic differentiating features, immunophenotyp- strated association between AML without maturation and ing should be used to exclude the lymphoid lineage. The specific chromosomal abnormalities. cell of origin is thought to be the HSC.42 AML WITHOUT MATURATION Characterized by a high percentage of BM blasts without significant evidence of maturation to more mature neutrophils, this variant AML Checkpoint 26.6 without maturation can occur at any age but most com- What hematologic features help distinguish AML minimally differentiated from AML without maturation? monly in adulthood. It accounts for 5–10% of the cases of AML. Patients usually present with BM failure; however, there can be a leukocytosis with markedly increased blasts. AML WITH MATURATION AML with maturation is characterized by 20% or more blasts with evidence of mat- uration to more mature neutrophils (greater than 10% of cells at differentiated stages of maturation: promyelocytes, myelocytes, metamyelocytes). Monocytes and their pre- cursors constitute less than 20% of the marrow cells. This type of AML occurs in all age groups (20% of patients are younger than 25 years of age, and 45% are older than 60 years of age) and accounts for about 10% of AML cases.40 Patients often present with anemia, thrombocytopenia, and neutropenia. Blasts with and without azurophilic granulation can be present, and blasts frequently contain Auer rods. Vari- able dysplasia, including myeloid hypogranulation, nuclear hypo- and hypersegmentation, and occasionally binucle- ated myeloid cells, can be seen (Figure 26-5). The BM is hypercellular, and myeloblasts make up 20–89% of the Figure 26.4 Peripheral blood film from a patient with AML. nonerythroid nucleated cells. Eosinophils and sometimes The large mononuclear cells are myeloblasts. The cell at the left (arrow) is a myeloblast with an Auer rod. Note the high nuclear- basophils can be increased. No demonstrated association to-cytoplasmic ratio, the fine lacy chromatin, and the prominent between AML with maturation and specific recurrent chro- nucleoli. (Wright-Giemsa stain, 1000* magnification) mosomal abnormalities exists. 598 Chapter 26 Figure 26.5 Figure 26.6 Peripheral blood film from a case of acute Peripheral blood film from a patient with AML myelomonocytic leukemia (AMML). Monoblasts and promonocytes with maturation. Myeloblasts are at the bottom and hypogranulated are present. (Wright-Giemsa stain, 1000* magnification) segmented neutrophils are at the top. (Wright-Giemsa stain; 1000* magnification) Additional laboratory testing is required when (1) the Checkpoint 26.7 BM findings are as previously described, but the peripheral A patient with AML has a peripheral blood differential that blood monocyte count is less than 5 * 103/mcL or (2) the includes 91% myeloblasts, 4% promyelocytes, 3% granulo- peripheral blood monocyte count is 5 * 103/mcL or more, cytes, and 2% monocytes, and 30% of the blasts are positive but the BM has less than 20% monocytic cells. In these cases, with MPO. Which category of AML is the most likely diagnosis? Explain. ancillary laboratory tests such as lysozyme levels or cyto- chemical methods can be utilized to confirm the presence of a significant monocytic component and establish a diag- Acute Myelomonocytic Leukemia (AMML) Characterized nosis of AMML (Table 26-6). by proliferation of both myelocytic and monocytic precur- sors, the BM in acute myelomonocytic leukemia (AMML) is hypercellular with 20% or more blasts; neutrophils and their precursors as well as monocytes and their precursors each Table 26.6 Nonimmunophenotypic Criteria for Diagnosis compose 20% or more of marrow cells. AMML accounts for of AMML 5–10% of AML cases. Some patients have a history of chronic 1. Bone marrow Blasts 20% or more of nonerythroid cells myelomonocytic leukemia. Infiltrations of leukemic cells in Monocytic cells 20% or more of nonerythroid cells extramedullary sites are more frequent than in the pure Granulocytic cells 20% or more of nonerythroid cells granulocytic variants. Serum and urinary levels of murami- and peripheral blood 5 * 103/mcL or more monocytic cells dase (lysozyme) are usually elevated because of the mono- or if peripheral blood cytic proliferation. Less than 5 * 103/mcL monocytic cells The peripheral blood leukocyte count in AMML is Requires ancillary Serum or urinary lysozyme 3* normal tests usually increased. Monocytic cells (monoblasts, promono- or naphthol AS-D chloroacetate esterase and cytes, monocytes) are increased to 5 * 103/mcL or more a@naphthyl acetate esterase (+ ) in blasts (Figure 26-6). Anemia and thrombocytopenia are present or naphthol AS-D acetate esterase with and in almost all cases. The myeloblasts appear similar to blasts without NaFl reveal more than 20% monocytic cells in bone marrow in AML with differentiation. Monoblasts are usually large with abundant bluish-gray cytoplasm and can show pseu- 2. Peripheral blood 5 * 103/mcL or more monocytic cells dopods. Scattered fine azurophilic granules and vacuoles and bone marrow Blasts 20% or more of nonerythroid cells may be present. The nucleus is round or convoluted with Monocytic cells less than 20% of nonerythroid cells delicate chromatin and one or more prominent nucleoli. Requires ancillary Serum or urinary lysozyme 3* normal tests The monoblasts and myeloblasts demonstrate the expected cytochemical and immunophenotypic results for their or naphthol AS-D chloroacetate esterase and a@naphthyl acetate esterase (+ ) in blasts respective lineages

(Table 26-5). The BM can reveal eryth- or naphthol AS-D acetate esterase with and rophagocytosis by monocytes. The cell of origin is thought without NaFl reveal more than 20% monocytic to be the hematopoietic stem cell.42 cells in bone marrow Acute Myeloid Leukemias 599 Acute Monoblastic Leukemia and Acute Monocytic Leuke- aspect of this disease is the degree of extramedullary leuke- mia (AMOL) These subtypes are myeloid leukemias in mic proliferation (gingival and cutaneous infiltration, cen- which 80% or more of the leukemic cells are monocytic tral nervous system involvement). As with AMML, serum (including monoblasts, promonocytes, monocytes). The and urine muramidase are moderately elevated. Monocytic neutrophilic component, if present, is less than 20% of the cells account for 80% or more of cells in the bone marrow. cells. The majority of the monocytic cells (usually 80% or Monocytes in the peripheral blood are increased, and mono- more) found in acute monoblastic leukemia are monoblasts blasts are often present. The monoblasts are large (up to 40 (Figure 26-7). The majority of the monocytic cells in acute mcM) with abundant, variably basophilic cytoplasm. Pseu- monocytic leukemia are promonocytes (Figure 26-8). Acute dopods with translucent cytoplasm are common, and fine monoblastic leukemia accounts for 5–8% of AML cases, and azurophilic granules can be present. The nucleus is round acute monocytic leukemia accounts for 3–6% of them.42 This or oval with delicate chromatin and one or more prominent disease is usually seen in children or young adults. nucleoli, but Auer rods are usually not found. Dyshemato- The most common clinical findings are weakness, poiesis is not conspicuous. Promonocytes have a more irreg- bleeding, and a diffuse erythematous skin rash. One notable ular and convoluted nucleus, and nucleoli can be present. a b Figure 26.7 (a) A smear from a bone marrow aspirate of a patient with acute -monoblastic leukemia. The cells are predominately monoblasts and promonocytes. (b) Monoblasts in peripheral blood from a case of acute monoblastic leukemia. (Wright-Giemsa stain, 1000* magnification) a b Figure 26.8 (a) A smear from a bone marrow aspirate of a patient with acute monocytic leukemia. There is a predominance of promonocytes and monoblasts (Wright-Giemsa stain, 1000* magnification). (b) Monocytic cells in peripheral blood, including promonocytes and monoblasts, from acute monocytic leukemia (Wright-Giemsa stain; 1000* magnification). 600 Chapter 26 The cytoplasm is less basophilic than that of the monoblast with a ground glass appearance. Fine azurophilic granules Checkpoint 26.8 are often present. What are the WHO2016 criteria for a diagnosis of pure erythroid Abnormalities of the long arm of chromosome 11 with leukemia? translocations or deletions are often found in monocytic leu- kemias. The t(8;16) abnormality is also found and is asso- Acute Megakaryoblastic Leukemia (AMKL) The mega- ciated with hemophagocytosis. The expression of c-FOS karyoblastic subgroup of AML is an acute leukemia in proto-oncogene on chromosome 14 appears to be enhanced which 50% or more of the leukemic blasts are of the mega- in acute leukemias with monocytic lineage involvement.1 karyocytic lineage. It occurs in both adults and children and This gene has been linked to normal monocyte–macrophage constitutes less than 5% of all AML cases.42 This category differentiation. The cell of origin for this AML subtype is excludes AML with myelodysplasia-related changes, AML believed to be an HSC.7 with t(1;22)(p13;q13), inv(3)(q21;q26), t(3;3)(q21;q26.2), and Down syndrome-related cases. Pure Erythroid Leukemia (PEL) The only change to the Patients usually present with cytopenias; although category of AML not otherwise specified in the 2016 WHO most have thrombocytopenia, some can have thrombocy- classification involves AL involving the erythrocytic lin- tosis. Dysplastic features in the neutrophils and platelets eage. Classification of erthrocytic leukemias has always may be present. There is no significant organomegaly. The been challenging due to overlapping features with myelo- BM megakaryoblasts are usually medium to large (12–18 dysplastic syndromes and other subtypes of AML. The 2016 mcM) with basophilic, often agranular, cytoplasm. The cells classification has simplified the criteria for this subtype, frequently show distinct pseudopod formation. Small blasts now known as pure erythroid leukemia.7 resembling lymphoblasts can also be present. On careful Bone marrow analysis shows more than 80% of bone examination of the peripheral blood smear, circulating marrow cells are erythroid precursors with 30% or more micromegakaryocytes and undifferentiated blasts can be being proerythroblasts. Cases with fewer erythroid pre- found. However, the finding of cytoplasmic blebs suggests cursors or an increase in myeloblasts may be classified as that these cells are megakaryocytic. Megakaryocytic frag- AML or myelodysplastic syndrome, based on other criteria ments also can be present. according to the 2016 WHO classification system. The BM BM aspiration often results in a dry tap because of erythroblasts are distinctly abnormal with bizarre morpho- extensive marrow fibrosis associated with an expanded logical features. Giant multilobular or multinucleated forms megakaryocyte lineage. In these cases, marrow biopsy are common. Other features include nuclear budding and may be required and usually reveals increased fibro- fragmentation, cytoplasmic vacuoles, Howell-Jolly bodies, blasts and/or increased reticulin as well as more than 20% ringed sideroblasts, and megaloblastoid changes. Erythro- blasts. It has been suggested that megakaryocytes secrete phagocytosis of the abnormal erythroblasts is a common a number of mitogenic factors that stimulate fibroblast finding. Dysmegakaryopoiesis is common with mononu- proliferation. clear forms or micromegakaryoblasts present. Neutrophils Blasts can be identified as megakaryocytic by immu- can exhibit hypogranularity and pseudo–Pelger-Huët nophenotyping flow cytometry for CD41, CD61, and anomaly. The leukocyte alkaline phosphatase score is nor- CD42b, and cytochemistry. The blasts are highly variable, mal or increased. Leukocytes and platelets are usually ranging from small round cells with scant cytoplasm and decreased. dense heavy chromatin to cells with moderately abundant The most dominant changes in the peripheral blood cytoplasm with or without granules and nuclei with lacy are anemia with striking poikilocytosis and anisocytosis. chromatin and prominent nucleoli. Some blasts can have There are a large number of nucleated erythrocytes that are cytoplasmic blebs. Megakaryocytes with shedding platelets dysplastic with megaloblastoid nuclei and/or bi- or multi- occasionally are present. Dysplasia of all cell lines is a com- nucleated cells in the more immature stages. The cytoplasm mon finding. frequently contains vacuoles. No unique chromosomal abnormality is associated Normoblasts are typically periodic acid-Schiff (PAS) with acute megakaryoblastic leukemia in adults. negative; however, in erythroleukemia, erythroblasts can demonstrate coarse positivity in either a diffuse or granu- Acute Basophilic Leukemia (ABL) The primary differen- lar pattern. PAS-positive erythroblasts are occasionally also tiation of this acute leukemia is along the basophil lineage. found in MDS, other subgroups of AML, iron-deficiency Some cases develop as a blast transformation phase of CML. anemia, thalassemia, severe hemolytic anemia, and some- This is a very rare form of AML with few reported cases times in megaloblastic anemia. Erythroblasts usually react (less than 1% of all AML cases). The most characteristic positively with antibodies to glycophorin A (CD71) or feature by cytochemistry is metachromatic positivity with hemoglobin A. toluidine blue.7 The blasts can show diffuse staining with Acute Myeloid Leukemias 601 acid phosphatase and occasionally PAS positivity. They are in reticulin fibrosis resulting in a dry tap. The blasts are usually negative for typical cytochemical stains for myeloid positive for the same markers as in TAM except that 50% cells but express myeloid markers (e.g., CD13, CD33). No of cases are negative for CD34, and 30% of cases are nega- consistent chromosomal abnormality has been identified tive for CD56. In addition to trisomy 21, somatic mutations with this subtype of AML. The cell of origin is believed to of the gene encoding the transcription factor GATA1 are be a myeloid precursor committed to the basophil lineage. considered pathognomonic of TAM or MDS/AML of The differential diagnosis includes a blast phase of a myelo- Down syndrome. proliferative neoplasm and other AML with basophilia such as AML with t(6;9)(p23;q34). ACUTE PANMYELOSIS WITH MYELOFIBROSIS CASE STUDY (continued from page 588) This disorder is associated with an acute panmyeloid pro- liferation and fibrosis of the bone marrow. A rare form of A BM aspirate and biopsy were performed on Jon- AML, it occurs primarily in adults. The major differential athan to aid in diagnosis. The BM biopsy revealed diagnosis is with acute megakaryoblastic leukemia (with a hypercellular marrow and an M:E ratio of 9.1:1. fibrosis) or other types of AML with associated marrow The predominant cells were blasts with Auer rods fibrosis and/or with chronic idiopathic myelofibrosis. frequently present. This diagnosis is indicated when the proliferative process 5. Based on the morphology, what is the most likely involves all major myeloid lineages (granulocytes, erythroid AML classification? cells, and megakaryocytes [i.e., panmyelosis]). Dysplastic, 6. What are at least two recurrent genetic mutations small megakaryocytes are characteristic. The number of seen in this type of presentation? blasts varies but is usually between 20 and 25%. Diagnosis requires BM biopsy and immunohistology. MYELOID SARCOMA This designation refers to a disease process in which a tumor mass of myeloblasts or immature myeloid cells occurs in an Therapy extramedullary site or in bone. It can occur concurrently Traditional chemotherapy for AML is designed to reduce with acute or chronic myeloid leukemias or other myelo- tumor load as rapidly as possible. Newer treatment modal- proliferative or myelodysplastic disorders. The immuno- ities include molecularly targeted therapies (e.g., ATRA), phenotype and cytochemistry reflect the specific myeloid epigenetic-targeted therapies (demethylating agents, his- cell(s) involved in the malignant process (i.e., myeloblasts, tone deacetylase inhibitors), and autologous or allogeneic promyelocytes, occasionally monocytes). BM transplants and infusions of donor lymphocyte cells with total body irradiation to increase leukemic cell destruc- MYELOID PROLIFERATIONS RELATED TO DOWN tion. Current research is focusing on novel therapies includ- SYNDROME ing the use of monoclonal antibodies and gene therapy and Individuals with Down syndrome have been found to the destruction of the cellular matrix that supports the neo- have an increased risk of leukemia, estimated at 10- to 100- plastic tissue. fold. They also are at risk for developing transient abnor- Evaluation of peripheral blood counts is essential to mal myelopoiesis (TAM), which is a unique disorder of support patients during chemotherapy. Development of at- Down syndrome. The newborn with TAM presents with risk stages such as pancytopenia, severe granulocytopenia, clinical and morphologic findings indistinguishable from and/or thrombocytopenia must be monitored so that early AML. The blasts are of the megakaryocytic lineage. At pre- intervention can occur (growth factors to stimulate hema- sentation, thrombocytopenia is the most common feature. topoietic cell recovery and/or transfusions). There may be a marked leukocytosis with an increase in BM transplantation (Chapter 29), whether allogeneic or blasts. Hepatosplenomegaly may be present. In the major- autologous, remains the only therapeutic choice that cur- ity of cases, spontaneous remission occurs within the first rently provides the potential for a prolonged (10+ years) three months of life. In most cases, the blasts are positive disease-free survival for most patients with AML. for CD34, CD56, CD117, CD13, CD33, CD7, CD4, CD41, CD42, and CD61. In addition to trisomy 21, acquired GATA1 mutations are present in blast cells of TAM. Chil- dren with Down syndrome have a 50-fold increase in inci- Checkpoint 26.9 dence of acute leukemia during the first 5 years of life. Predict the peripheral blood picture of a patient on antifolate About 1–2% of children with Down syndrome will develop chemotherapy. AML during this time. The BM core may show an increase 602 Chapter 26 Summary The ALs compose a heterogeneous group of neoplastic ALL (MPO- ). Subgrouping the AMLs requires additional stem cell disorders characterized by unregulated prolif- cytochemical stains, immunophenotyping, and cytoge- eration and blocked maturation. The two major groups of netic analysis. The AML cases known as AML not other- AL are AML and ALL, which the 2016 WHO Classifica- wise specified are primarily classified by morphology and tion System further categorizes into subtypes based on cytochemistry. morphologic criteria, cytochemical stains, immunologic The onset of AML is usually abrupt; without treatment, analysis, and cytogenetic and molecular abnormalities. it progresses rapidly. Symptoms are related to anemia, The 2016 WHO classification system recognizes AML thrombocytopenia, and/or neutropenia. Splenomegaly, subtypes with recurrent genetic abnormalities, with mul- hepatomegaly, and lymphadenopathy are common find- tilineage dysplasia, and following cytotoxic therapy. It ings. Hematologic findings include a macrocytic or nor- replaces the FAB classification system that was based on mocytic, normochromic anemia, thrombocytopenia, and a morphology and cytochemistry and updates the prior decreased, normal,

or increased leukocyte count. Blasts are WHO classifications based on updated knowledge related almost always found in the peripheral blood. A BM exami- to AML diagnosis and treatment. MPO and/or SBB cyto- nation is always indicated if leukemia is suspected. The BM chemical staining help differentiate AML (MPO+ ) from reveals 20% or more blasts. Review Questions Level I 4. An Auer rod is best described as (Objective 7): 1. A differential report notes the presence of more than a. threads of chromosomal DNA found within the 20% blasts. This number supports the diagnosis of: cytoplasm (Objective 1) b. inappropriately fused primary granules a. CML c. excessive accumulations of ribosomal RNA b. AML d. Golgi apparatus that have not been enzymatically c. CLL degraded d. MDS 5. The leukemia that belongs to the WHO classifica- tion of AML with recurrent genetic abnormalities is: 2. The presence of blasts with no evidence of myeloid (Objective 3) differentiation by morphology and less than 3% posi- tive for SBB or MPO is commonly seen in: a. APL with PML-RARA (Objective 2) b. megakaryoblastic leukemia a. AML—minimally differentiated c. pure erythroid leukemia b. AML—without maturation d. AML minimally differentiated c. acute basophilic leukemia 6. Which of the following subcategories of AML give a d. myeloid sarcoma positive result with a@naphthyl esterases? (Objective 6) 3. Which of the following best describes the bone marrow of a patient with AML? (Objective 5) a. AML without maturation a. M:E ratio of 1:4, normocellular, and 15% b. Acute monocytic leukemia lymphoblasts c. Pure erythroid leukemia b. M:E ratio of 10:1, hypercellular, and 35% d. APL with PML-RARA myeloblasts c. M:E ratio of 1:5, hypocellular, and 11% myeloblasts d. M:E ratio of 4:1, hyperceullar, and 40% lymphoblasts Acute Myeloid Leukemias 603 7. A patient who had been treated previously for myelo- 2. The WHO classification of AML is based on: dysplasia developed AML. What subgroup is most (Objectives 2, 7) appropriate for a diagnosis in this case? (Objective 3) a. genetic abnormalities a. AML with recurrent genetic abnormalities b. morphology and cytochemistry of blasts b. AML with MDS-related changes c. immunophenotyping of blasts c. t-AML d. all of the above d. AML with differentiation 3. Which of the following leukemias is associated with 8. The minimum number of blasts in the peripheral cells containing an abundance of Auer rods occurring blood or bone marrow needed for a diagnosis of AML in bundles? (Objective 2) is: (Objectives 1, 4) a. APL a. 30% b. AML with multilineage dysplasia b. 10% c. AML t(8;21) c. 50% d. AML therapy related d. 20% 4. Which of these leukemic blasts demonstrate t(1;22) 9. Pure erythroid leukemia must demonstrate which (p13;q13) positivity? (Objective 4) of the following features within the bone marrow? (Objectives, 3, 5) a. Erythroblasts a. More than 20% myeloblasts b. Myeloblasts b. More than 30% orthrochromic erythroblasts c. Megakaryoblasts c. Less than 20% megakaryocytes d. Monoblasts d. Less than 80% erythroid precursors 5. A bone marrow from a 20-year-old male revealed 10. The presence of de novo AML with BCR-ABL1 as a 40% agranular blasts. Cytochemical stains were provisional entity was added to the WHO classifica- negative for MPO, SBB, and specific and nonspecific tion of AML due to: (Objective 3) esterases. What immunophenotype panel should be used to help identify the blast cell lineage? a. the presence of the mutation definitively differenti- (Objective 6) ates de novo AML from CML a. CD13, CD33, CD34, CD10, CD19, CD2, CD3, CD5 b. therapy with tyrosine kinase inhibitors may improve outcomes b. CD11a, CD14, CD10, CD2, CD3, CD71, CD61 c. data suggests that BCR-ABL1 is more appropri- c. CD13, CD33, CD34, CD11b, CD71, CD41 ately linked to AML rather than CML d. CD41, CD61, CD13, CD15, CD38, CD117 d. the prognosis for this mutation is better using standard therapies 6. A patient has a leukocyte count of 75 * 103/mcL. A large number of blast cells are present in the Level II peripheral blood smear. Cytochemical stain reveals positive staining with SBB. Immunophenotyping is 1. In attempting to subtype a case of acute l eukemia, CD13+ , CD33+ , CD19- , CD22- , CD3- , CD5- . a laboratory professional noted that the blasts were Karyotyping reveals t(8;21)(q22;q22). Based on the negative with myeloperoxidase, SBB, specific and results, these cells would be considered: nonspecific esterase but positive when stained (Objective 4) with PAS and platelet peroxidase. These blasts should show positivity with which of the following a. Myeloblasts monoclonal antibody(ies)? (Objectives 2, 4) b. Stem cells a. CD41, CD42, CD61 c. Monoblasts b. CD24 d. Promyelocytes c. CD7 d. Glycophorin A 604 Chapter 26 7. A patient with leukemia is receiving chemotherapy 9. The presence of CD34, CD13, and CD14 is typically including a drug that is an antagonist to folic acid associated with a positive: (Objective 4) metabolism. Which of the following types of erythro- a. PAS stain cytes will be produced? (Objective 5) b. TdT a. Macrocytes c. CD71 b. Microcytes d. myeloperoxidase c. Spherocytes d. Codocytes 10. All transretinoic acid therapy is used in leukemia patients with this genetic abnormality: 8. Hypergranular promyelocytic leukemia is character- (Objectives 3, 5) ized by which cytogenetic abnormality? (Objective 2,4) a. t(9;22) a. t(15;17) b. t(8;21) b. 11q23 abnormality c. trisomy 8 c. t(8;21) d. t(15;17) d. inv(16)(p13;q22) References 1. Lichtman, M. A., & Liesveld, J. L. (2015). Acute myelogenous leu- translocation: Clinical consequences and biological implications. kemia. In: O. W. Press, ed. 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N., Sundermann, J., 8461). Blood, 100(13), 4325–4336. doi: 10.1182/blood-2002-03-0772 Alpermann, T., . . . & Haferlach, T. (2011). RUNX1 mutations are 27. Groschel, S., Sanders, M. A., Hoogenboezem, R., de Wit, E., Britta, frequent in de novo AML with noncomplex karyotype and confer B. A., Erpelinck, C., . . . & Rombouts, E. J. (2014, June). A single an unfavorable prognosis. Blood, 117(8), 2348–2357. doi: 10.1182/ oncogenic enhancer rearrangement causes concomitant EVI1 and blood-2009-11-255976 GATA2 deregulation in leukemia. Cell, 157, 369–381. 39. Gaidzik, V. I., Bullinger, L., Schlenk, R. F., Zimmermann, A. S., 28. Yamazaki, H., Suzuki, M., Otsuki, A., Shimizu, R., Bresnick, E. H., Röck, J., Paschka, P., . . . & Späth, D. (2011). RUNX1 mutations in Engel, J. D., & Yamamoto, M. (2014). A remote GATA2 hemato- acute myeloid leukemia: results from a comprehensive genetic poietic enhancer drives leukemogenesis in inv (3)(q21; q26) by and clinical analysis from the AML study group. Journal of Clini- activating EVI1 expression. Cancer Cell, 25(4), 415–427. cal Oncology, 29(10), 1364–1372. 29. Lahortiga, I., Vázquez, I., Agirre, X., Larrayoz, M. J., Vizmanos, J. L., 40. Kadia, T. M., Ravandi, F., Cortes, J., & Kantarjian, H. (2015). Gozzetti, A., . . . & Odero, M. D. (2004). Molecular heterogeneity in Toward individualized therapy in acute myeloid leukemia. JAMA AML/MDS patients with 3q21q26 rearrangements. Genes, Chro- Oncology, 1(6), 820–828. doi: 10.1001/jamaoncol.2015.0617 mosomes and Cancer, 40(3), 179–189. doi: 10.1002/gcc.20033 41. Patel, K. P., Khokhar, F. A., Muzzafar, T., You, M. J., Bueso-Ramos, 30. Raffel, G. D., Mercher, T., Shigematsu, H., Williams, I. R., Cullen, C. E., Ravandi, F., . . . & Medeiros, L. J. (2013). TdT expression in D. E., Akashi, K., . . . & Gilliland, D. G. (2007). Ott1 (Rbm15) has acute myeloid leukemia with minimal differentiation is associ- pleiotropic roles in hematopoietic development. Proceedings of the ated with distinctive clinicopathological features and better over- National Academy of Sciences, 104(14), 6001–6006. all survival following stem cell transplantation. Modern Pathology, 31. Cheng, E. C., Luo, Q., Bruscia, E. M., Renda, M. J., Troy, J. A., 26(2), 195–203. doi: 10.1038/modpathol.2012.142 Massaro, S. A., . . . & Sun, Y. (2009). Role for MKL1 in megakaryo- 42. Jaffe, E. S., Harris, N.L., & Vardiman. J.W., eds. (2001). World cytic maturation. Blood, 113(12), 2826–2834. Health Organization Classification of Tumours: Pathology and genet- 32. Lee, J., & Skalnik, D. G. (2012). Rbm15-Mkl1 interacts with the ics of tumours of haematopoietic and lymphoid tissues. Lyon, France: Setd1b histone H3-Lys4 methyltransferase via a SPOC domain IARC Press. Chapter 27 Precursor Lymphoid Neoplasms Kyle Riding, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define acute lymphoblastic leukemia (ALL) 4. Give the typical results of flow cytometric and lymphoblastic lymphoma (LBL), and analysis and genetic findings in ALL. differentiate them from acute myeloid 5. Summarize the clinical signs and symptoms leukemia (AML). and the most frequent age groups associated 2. List and define the variants of ALL/LBL with ALL. according to the WHO classification. 6. Define the rare acute leukemias that are not 3. Describe and recognize the typical p eripheral included in the AML and ALL groups. blood picture (erythrocytes, leukocytes, blasts, thrombocytes) seen in ALL. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Compare and contrast the various 4. Correlate Wright stained blast morphology presentations of ALL/LBL. in the ALL subgroups with flow cytometry 2. Predict the most likely WHO or and genetic testing results. immunophenotypic subgroup based on 5. Evaluate peripheral blood results in relation patient history, physical assessment, and to response to oncological therapy (e.g., laboratory findings. complete or partial remission, relapse). 3. Correlate cellular presentation with 6. Identify acute leukemia (AL) from a prognosis and common complications in peripheral blood smear and recommend ALL/LBL. laboratory tests that may be useful in 606 Precursor Lymphoid Neoplasms 607 differentiation of acute leukemia of 8. Correlate clinical and laboratory findings ambiguous lineage, AML, ALL, and the with prognosis in ALL/LBL. subgroups of ALL. 9. Contrast the clinical and laboratory findings 7. Define the phases and purposes of of ALL to LBL. chemotherapy for ALL. Chapter Outline Objectives—Level I and Level II 606 Laboratory Evaluation 609 Key Terms 607 Identification of Cell Lineage 611 Background Basics 607 WHO Classification 612 Case Study 608 Therapy 617 Overview 608 Summary 618 Introduction 608 Review Questions 618 Etiology and Pathophysiology 608 References 620 Clinical Presentation 608 Key Terms Acute undifferentiated leukemia Biphenotypic acute leukemia Minimal residual disease (MRD) (AUL) Consolidation therapy T cell acute lymphoblastic B cell acute lymphoblastic Induction therapy leukemia leukemia Maintenance chemotherapy Bilineage acute leukemia Background Basics The information in this chapter will build upon concepts Level II learned in previous chapters. To maximize your learning • Summarize the role of oncogenes and growth factors experience, you should review and have an understand- in cell proliferation, differentiation, and maturation. ing of these concepts before starting this unit of study: (Chapters 4, 23) • Diagram the maturation pathway for T and B lym- Level I phocytes. (Chapter 8) • Summarize the origin and differentiation of hemato- • Describe the role of molecular analysis in diagnosis poietic cells. (Chapter 4) and treatment of acute leukemia. (Chapter 42) • Describe the maturation, differentiation, and func- • Describe the use of immunophenotyping in acute tion of the lymphocytes. (Chapter 8) leukemia. (Chapters 23, 40) • Outline the classification and general laboratory find- • Describe the role of cytogenetics and diagnosis and ings of the acute leukemias. (Chapter 23) treatment of acute leukemia. (Chapters 23, 41) • Summarize the typical laboratory findings that define acute myeloid leukemia. (Chapter 26) 608 Chapter 27 two entities are not always distinct. Tissue involvement CASE STUDY can occur in ALL, and peripheral blood and bone marrow We refer to this case study throughout the chapter. involvement can occur in LBL. It is thought that ALL and Dan, a 4-year-old white male, was seen by his LBL are different clinical expressions of the same disease physician for symptoms of easy fatigue and bruis- and therefore are grouped together in the WHO classifica- ing. His mother stated that until one month ago, tion. They arise from a malignant change in the hematopoi- he was a “typical kid.” Since then she has noticed etic stem cell, common lymphoid progenitor (CLP), or more increased lassitude, a regression to more babylike differentiated progenitors of the T or B cell lineage. behavior, and loss of appetite. For the past two days, his temperature has been 100°F. Upon physi- cal examination, the child presented as pale, quiet, Etiology and and of appropriate size for his age. Most systems were unremarkable with the exception of several Pathophysiology small firm lymph nodes felt in the cervical and Similar to AML (Chapter 26), the ALLs are hematologic dis- auxiliary regions. His CBC revealed a WBC count orders characterized by malignant neoplastic proliferation of 40.2 * 109/L with 90% blasts. and accumulation of immature and dysfunctional hema- Consider the laboratory testing that could help topoietic cells in the bone marrow. Multiple somatically in diagnosing Dan’s illness. acquired mutations within a stem cell prior to its differen- tiation into a lymphoid progenitor or within a lymphoid precursor cell gives rise to a clone of malignant lympho- cytes. Thus, ALL/LBL can be either T or B cell phenotypes. Overview How the cells acquire these mutations is unknown, but a combination of leukemogenic factors could be responsible This chapter discusses the acute lymphoblastic leukemias (Chapter 23). The types of genetic alterations include chro- and lymphomas. A summary of clinical signs and symp- mosomal rearrangements and abnormalities of cell ploidy as toms is juxtaposed with the hematologic findings. The 2016 well as point mutations in oncogenes or tumor suppressor World Health Organization (WHO) Classification System genes that alter cellular functions (Chapter 23). These geneti- is used. The emphasis is on the laboratory techniques and cally abnormal cells proliferate in an unregulated manner results used to diagnose and classify the neoplasms. The with altered responses to growth and antigrowth signals rare acute leukemias that are not grouped into either acute and exhibit maturation arrest at any one of several early myeloid leukemia (AML) or acute lymphoblastic leukemia stages of differentiation. They divide and synthesize DNA (ALL) are termed acute leukemias of ambiguous lineage and are more slowly than normal hematopoietic cells, but they have also reviewed. ALL may also be referred to as lymphoblastic enhanced self-renewal and increased resistance to apoptotic leukemia (LL) since the term acute is considered by some to signals.1 Thus, the leukemic clone expands and the neoplas- be redundant with the term lymphoblastic. tic cells accumulate. Impairment of normal hematopoiesis as the leukemic clone expands is the primary cause of concern. Introduction Lymphoid malignancies include a wide spectrum of syn- Clinical Presentation dromes from disorders that primarily involve the bone Although ALL has been diagnosed in all age groups, the inci- marrow and peripheral blood (leukemias) to those that ini- dence of the disease demonstrates two clear frequency clus- tially present as solid tissue masses (lymphomas) primarily ters, one between the ages of 2 and 5 years and another in the involving lymphoid organs (lymph nodes, tonsils, spleen, sixth decade. In elderly people, the signs and s ymptoms— thymus, and the lymphoid tissue of the gastrointestinal complaints of fatigue, infections, and b ruising—are typically tract). These lymphoid neoplasms may be composed primar- more acute than in children. The onset of the disease in chil- ily of mature or precursor lymphoid cells, a distinction that dren can be insidious or abrupt with nonspecific signs and serves as a means for classification. The mature lymphoid symptoms. leukemias and lymphomas are discussed in Chapter 28. This The clinical picture of anemia, increased infections, and chapter includes a description of the precursor or immature thrombocytopenia results from bone marrow failure because lymphoid leukemias (ALL) and lymphomas (lymphoblastic of the replacement of normal marrow elements by leukemic lymphoma [LBL]). Although ALL is primarily a neoplasm lymphoblasts. Pallor, fatigue, and shortness of breath reflect of the bone marrow and peripheral blood and LBL is a neo- the anemia, which tends to become symptomatic below plasm that presents as a solid mass in lymphoid tissue, the 10 g/dL of hemoglobin. Fever can result from an infection Precursor Lymphoid Neoplasms 609 or remain classified as a fever of unknown origin, perhaps because of the release of endogenous pyrogens and other Table 27.1 Initial Laboratory Findings Characteristic of ALL inflammatory compounds. Both petechiae and ecchymoses are present in more than 50% of the patients. Bone pain, especially Peripheral blood • Leukocyte count usually increased but can be normal or decreased tenderness of the long bones, presents in about 80% of chil- • Neutropenia dren with ALL, and is often excused as “growing pains” but • Lymphoblasts present is actually from the expansion of marrow space by leukemic • Normocytic, normochromic anemia cells. Weight loss with or without anorexia is common because • Thrombocytopenia of the negative nitrogen balance of metabolically abnormal Bone marrow • Hypercellular cells. Leukemia infiltrates are responsible for splenomegaly, • Increased lymphoblasts, usually more than hepatomegaly, and lymphadenopathy when present. 25%, not less than 20% B cell ALL is characterized by inappropriate secretion of monoclonal immunoglobulins that can increase blood most commonly in patients with T cell ALL. The absolute viscosity and impair blood flow through the microcircula- neutrophil count is decreased in 20–40% of patients, even tion (hyperviscosity syndrome). These immunoglobulins when there is leukocytosis, and correlates strongly with also can impair granulocyte and platelet function, induce the incidence of infection.1 Although no established mini- pathologic rouleaux, interact with coagulation proteins, and mum percentage of blasts defines ALL (as there is for AML), interfere with coagulation. Production of autoantibodies can WHO recommends that the diagnosis of ALL not be made lead to autoimmune hemolytic anemia, autoimmune throm- if there are less than 20% blasts within the bone marrow.3 bocytopenia, and autoimmune neutropenia. Extramedul- Many treatment protocols use the criterion of a minimum of lary involvement is common, affecting the central nervous 25% blasts within the bone marrow for a diagnosis of ALL. system, lymph nodes, spleen, liver, and testis. T and B lymphoblasts are not distinguishable by mor- The neoplasm can present with primary involvement

of phology. Circulating lymphoblasts are found in the periph- nodal and extranodal sites with minimal or no involvement eral blood in 90% of cases.1 They vary from small cells with of bone marrow and peripheral blood. The disease is then scant to moderate light basophilic or gray-blue cytoplasm, referred to as LBL. Sites of involvement in B cell LBL include a high N:C ratio, slightly condensed chromatin, and indis- skin, soft tissue, central nervous system, and lymph nodes. tinct nucleoli to larger cells with a moderate amount of In T cell LBL, mediastinal (thymic) involvement is com- cytoplasm, finely dispersed chromatin, and distinct nucle- mon. When the patient presents with a neoplastic lymphoid oli1 (Figures 27-1 and 27-2). The nucleus can be irregularly mass and lymphoblasts in the bone marrow, the distinction shaped. between lymphoma and leukemia is arbitrary. However, The larger blasts may be intermixed with the smaller when peripheral blood and bone marrow are extensively blasts (Figure 27-2). The cytoplasm can contain vacuoles involved, the disease is known as lymphoblastic leukemia. and amphophilic granules (that stain a pinkish to purple Without leukemia, LBL is usually asymptomatic. color) that are probably lysosomal in origin.4 These granules T cell ALL and LBL tend to have more aggressive clini- can make it difficult to distinguish these lymphoblasts from cal behavior than B cell malignancies. T cell malignancies myeloblasts. often involve extranodal and extramedullary sites.1 Lymph- Unless accompanied by bleeding, a normochromic adenopathy and hepatospenomegaly are usually present. normocytic anemia is common. Anisocytosis, poikilocyto- sis, and nucleated RBCs are usually not present.5 There is Checkpoint 27.1 typically thrombocytopenia (48952 * 109/L). Compare the typical age groups in which AML and ALL are found. CASE STUDY (continued from page 608) The physician ordered a CBC on Dan. The results Laboratory Evaluation were as follows: Evaluation of the peripheral blood and bone marrow is criti- WBC 40.2 * 109/L WBC Differential cal for making a diagnosis of ALL (Table 27-1). RBC 3.45 * 1012/L Neutrophils 8% Peripheral Blood Hb 9.7 g/dL Monocytes 1% Hct 32% Eosinophils 1% The white blood cell count can be increased, decreased, or MCV 92.7 fL Blasts 90% within the reference interval.2,3 Hyperleukocytosis (greater than 100.0 * 109/L) occurs in 11–23% of all patients and 610 Chapter 27 a b Figure 27.1 (a) Lymphoblasts in peripheral blood from acute lymphoblastic leukemia. Notice the nuclear cleavage, condensed chromatin, and high N:C ratio. (b) Lymphoblasts in the bone marrow from the patient with ALL in (a). Note cells with a high N:C ratio, fine chromatin, and nucleoli (both Wright-Giemsa stain, 1000* magnification). The bone marrow in most patients reveals a marrow densely MCH 28.1 pg populated with lymphoblasts (greater than 65%) and aspi- ration may not be possible. In this case, biopsy is indicated. MCHC 30.3 The morphology of the cells in the marrow mirrors that RDW 17.3 seen in the peripheral blood. Auer rods are not present in PLT 63 * 109/L lymphoblasts; if Auer rods are seen, the blasts are distin- On a scan of the peripheral blood smear, rare guished as myeloblasts. nucleated erythrocytes were seen, and the platelets appeared decreased in number. Tissue Involvement 1. Based on these data, what would be the initial Lymphoma is the term used to describe lymphoid neoplasms interpretation of Dan’s presentation? when there is mass lesion of lymphoid (Chapter 3) or other tissue with no or little involvement of the bone marrow and peripheral blood. In B-LBL, lymph nodes and other Bone Marrow involved tissue show diffuse or paracortical patterns of infiltration by malignant lymphoid cells. There are usually The bone marrow is preferred for diagnosis because there numerous mitotic figures. There may be a focal “starry sky” are no circulating blasts at diagnosis in up to 10% of patients pattern due to macrophage phagocytosis of apoptotic tumor with ALL. Marrow cells also are better for genetic studies. cells. In T-LBL, lymph nodes can show total architectural a b Figure 27.2 (a) Lymphoblasts in peripheral blood from acute lymphoblastic leukemia. Note the fine chromatin and nucleoli (Wright- Giemsa stain, 1000* magnification). (b) Lymphoblasts in the bone marrow from patient in (a). Note the blasts with vacuoles (Wright-Giemsa stain, 500* magnification). Precursor Lymphoid Neoplasms 611 effacement that involves the capsule. Sometimes the morphologic examination of Romanowsky-stained smears involvement in the paracortical area is only partial, spar- alone. When morphologic examination cannot differenti- ing the germinal center. A multinodular pattern and “starry ate lymphoblasts from other blasts, cytochemistry may be sky” effect can be present. helpful (Chapter 37). In lymphoblasts, the myeloperoxi- dase (MPO) and Sudan black B (SBB) stains are negative, Other Laboratory Evaluation which are usually sufficient to differentiate lymphoblasts from myeloblasts. The periodic acid-Schiff (PAS) reaction As with other leukemias, other laboratory findings are con- usually demonstrates a coarse granular positivity in lym- sistent with increased cellular metabolism and, in general, phoblasts, especially those with a T cell immunophenotype. the extent of abnormality of the various laboratory param- The PAS reactivity may be present in AML, but the granular eters is proportional to the tumor burden. Hyperurice- pattern is superimposed on a diffusely positive background, mia and an increased lactate dehydrogenase are common whereas there is no background positivity in lymphoblasts. expressions of cell turnover. Hypercalcemia may occur, which is thought to be from bone resorption associated with leukemic infiltration in the bone marrow. The typical con- Terminal Deoxynucleotidyl sequence of leukemic infiltration of the kidney is impaired Transferase (TdT) renal function with increased serum creatinine, phospho- Certain intracellular enzymes are helpful in identifying rus, blood urea nitrogen, and uric acid. Because the central cellular subtypes. The most important of these is terminal nervous system (CNS) is a frequent site for extramedullary deoxynucleotidyl transferase (TdT), a DNA polymerase metastasis of ALL, cerebrospinal fluid analysis may be used found in cell nuclei. Its presence may be determined by throughout treatment to investigate the possibility of leu- direct enzyme assay, by indirect immunofluorescence, or kemic infiltration. with monoclonal antibodies. This enzyme is not present in normal mature lymphocytes but can be found in 65% of the Checkpoint 27.2 total thymic population of lymphocytes with the TdT posi- Explain the difference between ALL and LBL. tive cells localized in the cortex.6 It can also be found in very early B cells and occasionally very early myeloblasts and therefore, about 1–3% of normal bone marrow cells are TdT positive. Its value in ALL is to differentiate early precursor Identification of Cell lymphoblasts from more mature cells. Lineage Immunophenotyping The first step in classifying acute leukemia (AL) is to differ- entiate ALL from AML, usually by identifying the lineage The immunologic phenotype of neoplastic cells is helpful of the blasts. Although cell morphology on Romanowsky- in determining lineage of the neoplastic cells, differentiat- stained smears and cytochemistry can help distinguish ing a benign from a neoplastic process, classification and ALL from AML, classification of ALL/LBL into subgroups prognosis of the disease, and detection of minimal residual relies on immunologic, cytogenetic, and molecular genetic disease (MRD) after treatment. The appearance of specific methods. cell markers (antigenic determinants) is developmentally Immunophenotyping is used to determine the cells’ regulated in normal lymphoid cells (Chapter 8). Some anti- lineage (myeloid or lymphoid), subtype (T or B cell), and genic determinants appear at a very early stage of devel- maturation stage. Cytogenetics and molecular analysis opment and disappear with age, while others appear on can provide evidence of clonality, reveal distinct genetic mature cells. Studies of surface and intracellular markers abnormalities associated with subgroups of ALL/LBL, reveal that lymphoblasts in ALL share markers with nor- mal lymphoid counterparts in various stages of maturation7 and provide important prognostic information. Identifica- tion of these cellular characteristics can reveal the biologic (Chapter 8). However, immunophenotyping also has dem- subtypes, which is important for optimizing treatment onstrated that while some leukemic cells have phenotypes outcomes. of normal cells, others can show asynchronous gene expres- sion, resulting in an inappropriate combination of antigens Morphology and Cytochemistry or lineage plasticity8 (Figure 27-3). Because no one marker is specific for any neoplasm, a panel of antibodies must be Morphology is important since some neoplasms have used for accurate diagnosis. characteristic or diagnostic cell features. However, differ- Immunophenotyping is important in differentiat- entiation of ALL from AML (AML minimally differenti- ing ALL from minimally differentiated AML and in iden- ated or AML without maturation) may not be possible by tifying subtypes of the neoplastic lymphoblast (T or B). 612 Chapter 27 Progenitor Pro-B ALL Intermediate Pre-B ALL HCL Multiple cell pre-B ALL CLL myeloma PLL (plasma cell) TdT TdT TdT (mature) CD34 cCD22 CD19 CD19 CD19 CD38 CD19 CD34 CD10 CD20 CD20 cIg CD19 CD201/2 sCD22 CD22 cCD22 cIg sIg Figure 27.3 B lymphocyte maturation pathway with leukemic and other lymphoproliferative counterparts. ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; PLL, prolymphocytic leukemia; HCL, hairy cell leukemia; clg, cytoplasmic immunoglobulin; slg, surface immunoglobulin. Immunophenotyping also is valuable in recognizing leuke- signaling pathways that govern cell proliferation, such as mias of mixed lineage. After therapy, immunophenotyping RAS (Chapter 2).10,11 is used in combination with molecular studies (e.g., poly- merase chain reaction [PCR]/fluorescence in situ hybridiza- CASE STUDY (continued from page 609) tion [FISH]; Chapter 42) to determine MRD. 1. What tests should be used as the initial follow Checkpoint 27.3 up in this case? Why is it necessary to immunophenotype the lympho- blasts in ALL if they have been identified as lymphoblasts morphologically? Who Classification Cytogenetic Analysis The WHO Classification defines two subgroups of pre- Chromosomal abnormalities are present in 75% of ALL. cursor ALL/LBL: Precursor B cell and precursor T cell This information has diagnostic and prognostic implica- neoplasms (leukemia/lymphoma; Table 27-2). These tions. Some specific abnormalities are recurrent and used precursor neoplasms with bone marrow and peripheral to subgroup the B cell ALL/LBL. blood involvement are acute lymphoblastic leukemias, whereas precursor T cell and precursor B cell neoplasms Molecular Analysis presenting as solid tumors are lymphoblastic lymphomas (Chapter 28). Molecular analysis can determine the presence of rear- B cell ALL/LBLs that are characterized by recurrent rangements of immunoglobin heavy (IGH) chain genes genetic abnormalities are subgrouped as ALL with recurrent and/or light chain genes and T cell receptor (TCR) genes cytogenetic abnormalities (see Appendix B for these subgroups (Chapter 8). Rearrangement of IGH chain genes occurs and cellular characteristics). The remainder are subgrouped before cellular expression of immunoglobulin (Ig) and is as ALL not otherwise specified (NOS). Although Burkitt an early genetic marker of B cell ontogeny.9 Rearrangement lymphoma was classified as ALL in the FAB classification of the TCR polypeptide genes is an early sign of T cell lin- (L3), it is suggested that the B cell ALL designation not be eage. Molecular analysis of these gene rearrangements can used to designate Burkitt leukemia/lymphoma.1 Burkitt be used to establish clonality of the T or B cell populations lymphoma is included in the WHO classification of mature as well as cell lineage. Clonal gene rearrangement helps to B cell neoplasms and is discussed in Chapter 28. There are differentiate neoplastic lymphoid cells from normal lym- no recurrent cytogenetic changes observed that are specific phoid cells. However, IGH gene rearrangement is seen in to LBL.9 The subgrouping of T cell ALL has been a topic of some instances of T cell ALL, and TCR gene rearrangement considerable debate but recently had one subset created in occurs in some cases of B cell ALL.9 Thus, identification of the 2016 WHO update classification (Appendix B).12 additional markers of B and T cell lineage commitment is required for diagnosis. Point mutations are less frequently B Lymphoblastic Leukemia/ observed but may include genes of major cell-cycle reg- ulator proteins such as p53, various cyclin-dependent Lymphoma kinase inhibitors (CDKi), including p16, p15, and p14, Precursor B cell leukemia (B cell ALL) is a neoplasm of lym- and activating mutations of proto-oncogenes involved in phoblasts committed to the B cell lineage, which involves Precursor Lymphoid Neoplasms 613 Table 27.2 Classification of Lymphoid Leukemia and Lymphoma by the World Health Organization Subgroup Subgroup Subgroups B cell neoplasms Precursor B cell neoplasms Lymphoblastic leukemia B cell ALL /LBL with recurrent genetic abnormalities Lymphoblastic lymphoma (see Appendix B) B cell ALL /LBL not otherwise specified Mature B cell neoplasms (See Chapter 28) (See Chapter 28) T

cell neoplasms Precursor T cell neoplasms Lymphoblastic leukemia Lymphoblastic lymphoma Mature T cell neoplasms (See Chapter 28) (See Chapter 28) the bone marrow and peripheral blood. Occasionally, the IMMUNOPHENOTYPING disease can present with primary involvement of the lymph Immunophenotyping can identify markers on neoplastic nodes or extranodal sites, in which case it is called B lym- B cells that correlate to normal stages of maturation within phoblastic lymphoma (B-LBL). Although arbitrary and with the B cell lineage (pro-B, intermediate pre-B, and pre-B; exceptions, if a patient has a mass lesion and 25% or fewer Table 27-3). However, for therapeutic purposes, it is neces- lymphoblasts in the bone marrow, the term lymphoma is sary to distinguish only between precursor B cell, mature preferred. B cell, and T cell immunophenotypes.13 It is recommended B cell ALL accounts for 80–85% of the cases of ALL that panels for cell identification include at least one highly in children and about 70% of the cases of ALL in adults. sensitive marker, CD19, and a highly specific marker, cyto- In general, children between the ages of 2 and 10 years plasmic CD79a and CD22, for B cell lineage.12 of age diagnosed with precursor B cell ALL have a good The earliest stage of differentiation is the pro-B cell prognosis (long-term event-free survival more than 80%). and is characterized by an immunophenoptype pattern Adult event-free survival is lower, 30–50%, depending of TdT+ , HLA@DR+ , and almost always CD19+ and on the disease subtype and the patient’s age at diag- CD79a+ . The intermediate pre-B cell (also known as the nosis (there is an inverse relationship between age and common ALL cell) expresses CD10 (the common ALL anti- prognosis). gen, or CALLA).14,15 The most mature precursor B cell Table 27.3 Summary of Laboratory Features Helpful in Classification of ALL Common ALL Characteristic Pro-B Pre-B Precursor T (intermediate pre-B) Gene rearrangement Immunoglobulin (Ig) +/- + + -a T cell receptor (TCR) - - - +b Immunologic features Cytoplasmic Ig - + + - Surface Ig - - +/- - Immunophenotype CD34 + +/- - + CD19 + + + - CD22 +1c2 +1c2 +1s2 - CD10 - + +/- - CD20 - +/- + - CD2, CD3, CD5, CD7 - - - + Cytochemistry TdT + + + + PAS - - - + a About 20% of T ALL exhibits rearrangement of Ig gene b A small number of B ALL exhibits rearrangement of TCR gene + , positive; - , negative; (c), cytoplasmic; (s), surface. 614 Chapter 27 differentiation stage is the pre-B cell, which can be CD10 The rearrangements are more heterogeneous than originally negative but positive for CD20, surface CD22 (sCD22), thought which has caused a shift in the nomenclature used and cytoplasmic immunoglobulin (clg). However, surface by the 2016 WHO classification system. The presence of a immunoglobulin is absent. Malignancies involving more rearrangement in this gene is known to carry prognostic mature stages of B cell maturation are discussed in the clas- implications depending on the age of the patient and may sification of mature lymphoid neoplasms (Chapter 28). warrant hematopoietic stem cell transplantation in younger The majority of ALL/LBL presentations in both chil- patients to improve prognosis.20 dren and adults arise from precursor or immature B cells The t(9;22)(q34.1;q11.2)/BCR-ABL1 translocation is that typically have the same immunophenotype as normal more common in adults (10–15%) than children with B cell immature B cells (CD10, CD19, CD34, CD22, and TdT). This ALL.21 In most childhood cases, the BCR/ABL1 translocation makes the distinction between normal and neoplastic cells results in a p190 kD fusion protein, whereas in adult cases, challenging, especially when assessing minimal residual about one-half of the translocations produce the p210 kD disease unless the abnormal cells have an additional or protein present in CML. The remainder produce the p190 distinct marker.16 Up to 50% of B cell ALL cells coexpress protein (Chapter 24). a myeloid associated antigen—CD13, CD15, or C33—that The t(5;14)(q31.1;q32.3) results in a translocation can help in the differentiation between normal and neoplas- between the IL3 gene and the IGH2 gene, which results in a tic. Chapter 40 and Appendix B describe immunopheno- variable eosinophilia that is reactive, not clonal. Blasts are types associated with specific leukemias. CD19+ and CD10+ . The diagnosis can be made on immun- ophenotypic and genetic information regardless of the blast CYTOGENETICS AND PATHOGENESIS count in the bone marrow. Cytogenetic abnormalities associated with B cell ALL include translocations, hypodiploidy, and hyperdiploidy.10 Hyperdiploid B cell ALL is common in children and is seen in 20–26% of B ALL cases.23 Almost all cases The most significant translocations that have distinctive of hyperdiploid B cell ALL have mutations in the recep- clinical or phenotypic properties and important prognostic information are:17 tor tyrosine kinase FLT-3, resulting in constitutive activa- tion of the receptor.24 Hypodiploid clones may also have • t(12;21)(p13.2;q22.1)/ETV6-RUNX1 structural abnormalities but none are specific for diag- • t(v;11q23.3); KMT2A rearrangement nosis. Cytogenetic abnormalities considered to be poor • t(9;22)(q34;q11)/BCR-ABL1 prognostic factors are t(4;11)(q21;q23)/MLL-AFF1 (9% of cases), t(1;19)(q23.3;p13.3)/PBX1-E2A (5% of cases), t(9;22) • t(5;14)(q31;q32.3)/IL3-IGH2 (q34;q11.2)/BCR-ABL1 (4% of cases), and hypodiploidy (5% • t(1;19)(q23.3;p13.3)/E2A-PBX1(TCF3-PBX1) of cases).1,25 • Translocations involving tyrosine kinases or cytokine B ALL with intrachromosomal amplification of chromo- receptors (BCR-ABL1-like mutations) some 21 (iAMP21) and BCR-ABL1-like mutations are the • Intrachromosomal amplification of chromosome 21 newest provisional entities within the 2016 WHO classifi- (iAMP21) cation. iAMP21 involves chromosome 21 containing five or more copies of the RUNX1 gene being present that requires The most common translocation (present in about 25% aggressive therapy and is found predominantly in children. of cases) in childhood B cell ALL is t(12;21)(p13;q22.3), pro- Similarly, the BCR-ABL1-like mutations are found mostly in ducing the ETV6(TEL)-RUNX1(AML1) fusion gene.18,19 Both children and again carry a poor prognosis but may respond the ETV6 and RUNX1 genes are translocated in other types well to tyrosine kinase inhibitors.12 of leukemia—including t(5;12), t(8;21), and t(3;21)—that are found in various subtypes of AML. The ETV6-RUNX1 PROGNOSIS fusion protein is believed to initiate and drive leukemo- Precursor B cell ALL generally has a good prognosis. In genesis in cells carrying the translocation by disrupting the pediatric population, the complete remission rate the normal processes of cell differentiation, apoptosis, cell approaches 95%; in adults, it is 60–85%.26 Long-term, event- adhesion, and response to DNA damage.17 Expression of free survival is lower for both patient groups (∼80% and the ETV6-RUNX1 fusion protein in B cell ALL is associ- 30–50%, respectively). Generally, hyperdiploidy (more ated with an excellent prognosis with event-free survival than 50 chromosomes) is a better prognostic finding than approaching 90%. hypodiploidy (less than 45 chromosomes) with event-free Infants who present with ALL commonly carry the survival rates nearly 90%. Patients with hyperdiploid chro- t(v;11q23.3) translocation that rearranges the KMT2A gene mosome counts less than 50 have a worse prognosis.7,8 (formerly MLL gene). The protein encoded by the KMT2A Positive predictive factors in children include diagnosis gene is involved in chromatin remodeling and epigenetic at ages 1–9, hyperdiploid chromosomes or t(12;21), and a transcriptional control of cell-cycle regulatory proteins.18 low or normal white blood cell (WBC) count at diagnosis.1 Precursor Lymphoid Neoplasms 615 Adverse factors include very young age (less than 1 year), high WBC counts at diagnosis, and other cytogenetic Checkpoint 27.4 abnormalities listed in the section “Cytogenetics and A patient has 50% blasts in his bone marrow. Immunophe- Pathogenesis” (Table 27-4).27 notyping is CD19 positive, but CD20, CD2, CD10, and CD7 negative. What additional testing can be helpful to distinguish the immunologic subgroup of this leukemia? T Lymphoblastic Leukemia/ Lymphoma CYTOGENETICS AND PATHOGENESIS Accounting for approximately 15% of the cases of childhood T cell lineage can be determined by detecting rearrange- ALL and approximately 25% of the cases of adult ALL, pre- ment of the TCR genes using molecular methods. There are cursor T cell leukemia (T cell ALL) involves lymphoblasts four TCR genes that are capable of rearranging and encode committed to T cell lineage in the bone marrow and periph- the a@, b@, g@, and d@chains of the TCR (Chapter 8). Detection eral blood. If the primary site of involvement is a lymph of a monoclonal TCR gene rearrangement suggests a neo- node or extranodal site, the disease is termed T lymphoblastic plasm of the T cell lineage, but about 20% of T cell ALL cases lymphoma (T-LBL). exhibit simultaneous rearrangement of one of the immuno- Precursor T cell ALL usually presents with a high WBC globulin genes. In addition, a few cases of B cell ALL have count and often a mediastinal mass (because of leukemic shown TCR gene rearrangement. infiltration of the thymus) or other tissue masses. The lym- About one-third of the cases of T cell ALL have translo- phoblasts are similar to those seen in B cell ALL, although cations involving the a and d T cell receptor loci (14q11.2), they are more likely to be variable in size, and cytoplasmic the b locus (7q35), or the g locus (7p14–15). The translo- vacuoles can be present. The cytochemistry is also similar cations involve a variety of partner genes, including the to that seen in B cell ALL, but acid phosphatase can show transcription factors MYC (8q24.1), TAL1 (1p32), RBTN1 focal intense positivity in T cell ALL. (11p15), RBTN2 (11p13), HOX11 (10q24), and HOX11L2 (5q35).31 The result is usually a dysregulation of the part- IMMUNOPHENOTYPING ner gene, resulting in growth enhancement. In addition, The lymphoblasts in T cell ALL are usually TdT+ , CD7+ , molecular mutations of the NOTCH1 gene are found in and CD3+ (Table 27-5). However, only CD3 is considered more than 50% of cases of T cell ALL.32 NOTCH1 is a trans- lineage specific. There is variable expression of CD1, CD2, membrane receptor that is involved in the regulation of CD4, CD5, CD8, and CD10.28 Lymphoblasts frequently normal early T cell development. MYC, the downstream coexpress CD4 and CD8, indicative of the cortical stage of target of NOTCH1, is involved in the growth of neoplastic thymocyte differentiation (“double-positive cells”).25 At the cells. However, the NOTCH1 mutation is rare in ETP ALL; medullary stage of differentiation, the cells are either CD4+ mutations more frequently associated with myeloid malig- or CD8+ . CD79a, generally considered to be a B lineage nancies (e.g., FLT3, IDH1, and GATA2) are seen in this form marker, has been observed in some cases. One or more of of T cell ALL.12,29 the myeloid-associated markers (CD13, CD15, CD33) can occasionally be seen. As in B cell ALL, T cell ALL also can PROGNOSIS be stratified into differentiation stages, with cytoplasmic Although acute precursor T cell leukemia was considered a CD3, CD2, and CD7 appearing in the earliest stage followed high-risk disease with a poorer prognosis than B cell ALL, by CD5, CD1a, and subsequently, the appearance of mem- current therapeutic protocols are improving the prognosis brane CD3.25 for this disease. T cell ALL in children increases the risk for The 2016 WHO classification differentiates early T induction failure and relapse. In adults, the prognosis of precursor (ETP) ALL as a unique group. The characteristic T cell ALL may be better than of B cell ALL. This may be immunophenotype of ETP ALL is expression of CD7 while related to a lower incidence of adverse cytogenetic muta- lacking expression of CD1a and CD8 and expression of at tions in T cell ALL. least one myeloid/stem cell marker such as CD34, CD117, or CD65.12 The prognosis for these patients is exceptionally poor.29,22 CASE STUDY (continued from page 612) 3. If the flow cytometry pattern showed a positive Table 27.4 Poor Prognostic Indicators in ALL CD10, what would be the classification of this acute leukemia? Clinical findings Laboratory findings • Infants (less than 1 year old) • High blast counts 4. In this situation, would the therapeutic outcome • Patients beyond puberty • Presence of BCR-ABL1 or be considered as favorable or bleak? Why? • CNS or mediastinal involvement BCR-ABL1-like mutations 616 Chapter 27 Table 27.5 Cellular Markers Useful in Diagnosis and Classification of T Cell ALL T Cell CD19, Developmental CD20, Ig genes TCR genes Stage HLA-DR TdT CD34 CD2 CDla CD3 CD7 CD8 CD4 CD10 CD22 rearrangement rearrangement Pro-T +/- +/- +/- - - + (c) + - - +/- - - +/- Pre-T +/- +/- +/- +

- + (c) + - - +/- - - +/- Cortical T +/- +/- - + + + (c) + + + +/- - - +/- Medullary T +/- +/- - + - + (c) + +a or - +a or - +/- - - +/- Early-T +/- +/- +/- +/- - +/- + - +/- - - - +/- a Either CD4 or CD8+ . (c), cytoplasmic; - , absent; + , present; +/- , may be present; Ig, immunoglobulin; TCR, T cell receptor. morphologic and/or immunophenotypic characteristics of Checkpoint 27.5 both myeloid and lymphoid cells (or of both T and B lym- A 3-year-old patient has 45% lymphoblasts in the bone marrow. phocytes). Terminology regarding these disorders has been If the cells tested positive for CD19, CD10, and CD34, what is confusing. If two distinct populations of blasts are identifi- the most likely immunologic subgroup? Why should cytogenet- able, each expressing markers of a distinct lineage (i.e., one ics be performed on this patient? population myeloid and one population lymphoid or one population T lymphoid and one population B lymphoid), Acute Leukemias of Ambiguous the disease has been called bilineage acute leukemia. If the blasts coexpress myeloid and T or B lineage-specific Lineage markers or concurrent T and B lineage markers, the disease This WHO group of leukemias include malignant neo- has been called biphenotypic acute leukemia.29 Also rec- plasms in which the morphology, cytochemistry, undefined ognized is a leukemia with myeloid/natural killer (NK) cell genetic alterations, and immunophenotype lack sufficient acute leukemia with markers of both NK cells (CD56) and information to classify them within a given lineage or in myeloid lineage (CD13/CD33/MPO).29 WHO classification which the blasts have morphologic and/or immunopheno- attempts to clarify the terminology confusion and refers to typic characteristics of more than one lineage. these biphenotypic and bilineage leukemias by the gen- eral term mixed phenotype acute leukemia (MPAL). The terms ACUTE UNDIFFERENTIATED LEUKEMIA B/myeloid leukemia and T/myeloid leukemia are used to des- Although determination of the myeloid or lymphoid origin of ignate leukemias containing the two lineages specified blasts is important for treatment decisions, doing so is some- regardless of whether one or two populations of blasts are times not possible with current methods. The acute undiffer- seen. Specific mutations in the BCR-ABL1 gene and rear- entiated leukemia (AUL) category includes acute leukemias rangement of the KMT2A gene are distinct subtypes of in which the morphology, cytochemistry, and immunophe- MPAL according to the WHO classification mainly due to notype of the proliferating blasts lack sufficient information prognosis (i.e., poor prognosis of KMT2A rearrangement) to classify them as either myeloid or lymphoid origin. In this and therapeutic (i.e., TKI therapy for BCR-ABL1 muta- case, the leukemia is classified as acute undifferentiated leu- tions) reasons.12 However, they carry the same ambiguity kemia.28,33 Electron microscopic studies can sometimes detect in regard to cellular lineage of the MPAL leukemias not oth- ultrastructural evidence of primary granules and/or peroxi- erwise specified. dase, a finding that would indicate acute myeloid leukemia. The MPAL designation is reserved for cases in which Immunophenotyping should be done with a comprehensive lineage assignment is ambiguous. It should not be used panel of monoclonal antibodies to exclude neoplastic cells when an ALL aberrantly expresses one or two myeloid of unusual lineages such as basophils and NK-cell precur- antigenic markers or an AML aberrantly expresses one or sors. Absence of primary granules and/or MPO with elec- two lymphoid antigenic markers. These are considered tron microscopy with other negative or ambiguous findings myeloid antigen–positive ALL and lymphoid antigen–positive indicates a diagnosis of AUL. It is predominantly diagnosed AML, respectively. Because many markers are only lineage in adults, and only about one-third of patients respond to the associated, not lineage specific, coexpression of only one or chemotherapy regimens for AML and ALL. two cross-lineage antigens is insufficient for a diagnosis of ACUTE LEUKEMIAS WITH LINEAGE HETEROGENEITY biphenotypic leukemia. The category acute leukemias with lineage heterogene- Lack of lineage specificity (lineage infidelity) could be ity includes those leukemias in which the blasts have the result of genetic misprogramming, or the leukemic clone Precursor Lymphoid Neoplasms 617 could represent a bipotential cell that has retained both lym- express inappropriate combinations of antigens, making phoid and myeloid markers during development. Although diagnosis challenging. Treatment protocols and prognosis mixed lineage leukemias are uncommon, their identifica- are proving to be more effective and accurate when the leu- tion is important to determine their actual occurrence, to kemic cell lineage is accurately classified immunologically. identify appropriate therapy, and to correlate karyotypic Therapy response and detection of residual leukemic cells abnormalities.34,35 A large percentage of the acute leukemias (MRD) is possible using immunophenotyping and genetic of ambiguous lineage has associated cytogenetic abnormali- testing. ties. The cell of origin is thought to be the multipotential Chemotherapy for ALL is divided into several phases. hematopoietic stem cell. The induction therapy phase is designed to reduce the dis- Current methodology using two or more immuno- ease to complete remission (i.e., eradicating the leukemic logic markers in a double-labeling technique distinguishes blast population). This is followed by a CNS prophylactic whether the lymphoid and myeloid antigens are on the phase. CNS leukemia is the most common form of relapse in same cell or separate cells.28 This can be done with flow young children who have not undergone specific treatment cytometry or immunohistochemistry on tissue sections. to the brain and spinal column early in remission. The two Another procedure is to stain smears with MPO to identify potential modes of treatment in the CNS prophylactic phase myeloid cells coupled with flow cytometry to detect the T or are cranium irradiation and/or intrathecal chemotherapy. B lymphoid population. Cranial irradiation is seldom a component of most current treatment protocols because of the risk of neurocognitive deficits and endocrinopathy and of inducing a second can- Checkpoint 27.6 cer.1 The third phase is maintenance chemotherapy, also A patient with acute leukemia has two morphologically different types of blasts. One population is positive for CD7 and CD2. called cytoreductive therapy or remission consolidation therapy.1The other is positive for CD33 and CD13. What is the most The need for this type of therapy is controver- appropriate classification of this leukemia? sial. Some studies have shown a slight increase in survival with its use, whereas others reveal no improvement. Before the institution of CNS prophylactic treatment, there was a Natural Killer Cell Lymphoblastic high incidence of relapse. The maintenance therapy was designed to prevent this relapse and prolong remission. The Leukemia/Lymphoma purpose of maintenance therapy is to eradicate any remain- Natural killer (NK) cell lymphoblastic leukemia is a neo- ing leukemic cells. Drug treatment usually continues for 2–3 plasm that is difficult to define because (1) no specific mark- years. The relapse rate after cessation of all therapy is about ers for human NK-cell progenitors have been identified and 25% in the pediatric patient population. (2) NK-cell progenitors can express markers that are seen Allogeneic hematopoietic stem cell transplantation in T cell ALL (CD7, CD2, CD5, and cytoplasmic CD3). The (HSCT) remains controversial. Currently, most clinicians CD56 marker was previously thought to identify NK leu- consider it to be of benefit to some high-risk adult patients kemia but many of these cases are now known to be plas- and has shown promise in infants with KMTA2 gene rear- macytoid dendritic cell leukemia. More specific markers for rangements.1,20 Patients who relapse while on therapy or precursor NK cells are CD94 and CD161, but they are not after only a short remission are often considered candidates usually tested. Until more specific panels for NK cells are for HSCT. The use of umbilical cord blood as a source of developed, the 2016 WHO recommends that NK-cell lym- hematopoietic stem cells is being considered more fre- phoblastic leukemia remain a provisional entity but distinct quently, especially in the pediatric patient population and separate from other lymphoblastic or ambiguous lin- because it does not require the same degree of histocompat- eage leukemia subtypes.12,36 ibility as do transplants using peripheral blood or marrow stem cells. Relapse is defined as the reappearance of leukemic cells Therapy anywhere in the body, although the bone marrow is the most common site. Leukemic relapse occasionally occurs New treatment protocols have raised the complete at extramedullary sites. Most relapses occur during treat- remission rate for B cell ALL to more than 95% in chil- ment or within the first two years after completion. Rarely, dren and 60–85% in adults. The prognosis of T cell ALL/ relapses have been observed up to ten years after the induc- LBL is not as favorable as B cell ALL/LBL. This may be tion of remission. Relapse indicates a poor outcome for most in part because of the presence of high-risk clinical fea- patients, especially if it occurs during therapy or after only tures (Table 27-4). A significant number of acute leukemias a brief initial remission. 618 Chapter 27 Summary Acute leukemias represent a heterogeneous group of pre- Regardless of subtype, the onset of ALL is usually cursor hematopoietic neoplasms characterized by unregu- abrupt and without treatment, progresses. Symptoms are lated proliferation, arrested maturation, and/or ineffective related to anemia, thrombocytopenia, and/or neutropenia. apoptosis. The World Health Organization classification Splenomegaly, hepatomegaly, and lymphadenopathy are categorizes two major groups: precursor (acute) myeloid common findings. Hematologic findings of ALL include a and precursor (acute) lymphoid. The precursor lymphoid normocytic, normochromic anemia, and thrombocytope- group includes lymphoblastic lymphomas (LBL) and acute nia and a decreased, normal, or increased leukocyte count. lymphoblastic leukemia (ALL). LBL presents with a mass of Blasts are almost always found in the peripheral blood. malignant lymphoid cells in nodal or extranodal sites with Although the WHO classification does not require a mini- little or no bone marrow and peripheral blood involvement. mum number of blasts for a diagnosis of ALL/LBL as it ALL presents with bone marrow and peripheral blood does for AML, it is recommended that a diagnosis of ALL involvement. The two groups are considered to be differ- not be made for blast counts less than 20%. ent clinical presentations of the same disease. Acute leukemia with lineage heterogeneity includes ALL/LBL is subdivided into precursor B cell and pre- two situations in which ALs have characteristics of T and cursor T cell based on the lineage of the neoplastic blast B cells or myeloid and lymphoid cells. Mixed phenotype population. Further subgrouping is based on morphology, acute leukemia (MPAL) describes ALs with blasts that pos- genetic or karyotypic mutational status, and immunophe- sess markers of multiple lineages or two different popu- notype of blasts. Various cytogenetic and molecular changes lations of blasts. While natural killer cell lymphoblastic are described in ALL, including t(12;21)(p13;q22)/ETV6- leukemia is considered a unique group of lymphoblastic RUNX1 most commonly and t(9;22)(q34;q11.2)/BCR-ABL1 leukemia, difficulties in creating specific panels that detect that is associated with a poor prognosis. their presence is an ongoing diagnostic challenge. Review Questions Level I c. acute lymphoblastic leukemia 1. Acute lymphoblastic leukemia is characterized by the d. acute leukemia of ambiguous lineage presence of: (Objective 1) 4. The presence of CD19 and CD22(c) and absence of a. less than 20% small resting lymphoblasts in the CD10 (CALLA) on neoplastic lymphoblasts is most bone marrow often a sign of: (Objective 4) b. hypercellular bone marrow with increased a. biphenotypic leukemia lymphoblasts b. pro-B ALL c. 30% or more lymphoblasts with erythroid c. pre-B ALL dysplasia in the bone marrow d. T cell ALL d. more than 30% myeloblasts in the peripheral blood 5. The anemia most often seen in patients with ALL is: 2. Acute lymphoblastic leukemia is most often seen in (Objective 3) patients: (Objective 5) a. microcytic hypochromic a. older than 60 years b. normocytic normochromic b. between the ages of 35 and 60 years c. macrocytic normochromic c. between the ages of 10 and 35 years d. macrocytic hypochromic d. younger than 5 years 6. Leukemia of ambiguous lineage is most often described as the presence of: (Objectives 4, 6) 3. ALL characterized by the presence of small basophilic lymphoblasts with a round nucleus, finely g ranular a. large, basophilic blasts with slight to moderate chromatin, and inconspicuous nucleoli can be granulation classified as: (Objective 3) b. blasts demonstrating both CD10 and C33 a. ALL—Burkitt’s type c.

CD3 and CD4 markers b. acute biphenotypic leukemia d. no demonstrable markers using immunophenotyping Precursor Lymphoid Neoplasms 619 7. The oversecretion of monoclonal immunoglobulins is a. precursor B ALL most often seen in: (Objectives 3, 5) b. AUL a. B cell ALL c. T cell ALL b. T cell ALL d. bilineage ALL c. B cell LBL 4. An adult patient with splenomegaly has an increase in d. T cell LBL mononuclear cells in the peripheral blood. The bone 8. CD2, CD3, and CD4 are present on the cells found in marrow was filled with a heterogeneous collection a patient with a previously undiagnosed leukemia. of blasts with no granulation. Flow c ytometry shows These markers are characteristic of: (Objective 4) a positive CD20 and CD10 and negative surface immunoglobulin. Which of the following conditions a. pre-B cell ALL is most likely? (Objective 4) b. pro-B cell ALL a. Intermediate pre-B ALL (CALLA) c. T cell ALL b. Pro-B ALL d. ALL—Burkitt’s type c. Pre-T cell ALL 9. Acute biphenotypic leukemia is characterized by the d. Cortical T cell ALL presence of: (Objective 6) 5. Which of the following represents the most common a. ultrastructual evidence of primary granules chromosomal translocation found in ALL? b. CD19, CD20, and CD23 (Objective 4) c. CD2, CD4, CD8, and presence of myeloperoxidase a. t(1;19)(q23;p13.3)/PBX1-E2A d. negative immunophenotyping b. t(12;21)(p13;q22)/ETV6-RUNX1 10. Which of the following laboratory results are most c. t(4;11)(q21;q23)/MLL-AFF1 commonly found in ALL? (Objective 3) d. t(9;22)(q34;q11)/BCR-ABL1 a. Eosinophilia and basophilia 6. The CD10 antigen found in ALL is also known as the: b. Neutropenia and thrombocytopenia (Objective 4) c. Neutrophilia and thrombocytopenia a. B cell ALL antigen d. Lymphocytosis and thrombocytosis b. T cell ALL antigen Level II c. CALLA antigen 1. Cells that are positive for t(9;22) are characteristic of d. NK-cell ALL antigen which of the following? (Objective 4) 7. The most likely patient presentation for ALL in an a. T cell ALL adult includes: (Objective 2) b. Reactive lymphocytosis a. male aged 18–30 with mild anemia and no c. Acute undifferentiated leukemia thrombocytopenia d. B cell ALL b. male older than 60 years with significant anemia and thrombocytopenia 2. Monoclonal rearrangement of the TCR genes is associated with blasts that have the following c. female 10–25 years old with significant bone pain immunophenotype: (Objective 2) and hemolytic anemia d. female 30–50 years old with hemorrhage and bulky a. CD2+ , CD4+ , CD19+ nodes b. CD19+ , CD20+ , CD10+ c. CD3+ , CD13- , CD34+ 8. The objective of induction therapy includes: (Objective 7) d. CD2+ CD3+ , CD4+ a. reduction in the tumor burden 3. A peripheral blood smear is noted to have 80% blasts. b. elimination of tumor cells in the central nervous There are two different types of blast cells present: system one that is small, with a round nucleus, indistinct nucleoli, and scanty cytoplasm while the second is a c. replacement with peripheral blood stem cells from large blast with basophilic cytoplasm. One possible another person interpretation for this is: (Objectives 1, 6) d. prevention of relapse 620 Chapter 27 9. Poor prognostic findings for patients with ALL 10. A large mediastinal mass is found in a 60-year-old include: (Objective 8) male. The bone marrow has 15% lymphoblasts. This is most characteristic of: (Objective 9) a. young age at time of diagnosis b. presence of chromosomal mutations such as a. precursor T cell LBL BCR/ABL1 b. ALL—Burkitt’s type c. hyperploidy c. precursor T cell ALL d. presence of TEL-AML1 gene d. precursor B cell ALL References 1. Larson, R. A. (2015). Acute lymphoblastic leukemia. In K. 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Scott, A. A., Head, D. R., Kopecky, K. J., Appelbaum, F. R., Theil, Independent prognostic value of BCR-ABL1-like signature and K. S., Grever, M. R., . . . Licht, J. D. (1994). HLA-DR-, CD33, CD56, IKZF1 deletion, but not high CRLF2 expression, in children with CD16- myeloid/natural killer cell acute leukemia: A previously B-cell precursor ALL. Blood, 122(15), 2622–2629. doi: 10.1182/ unrecognized form of acute leukemia potentially misdiagnosed blood-2012-10-462358 as French-American-British acute myeloid leukemia-M3. Blood, 84(1), 244–255. 28. Lewis, R. E., Cruse, J. M., Sanders, C. M., Webb, R. N., Tillman, B. F., Beason, K. L., . . . Koehler, J. (2006). The immunophenotype 35. Vela, J. A., Monteserin, M. C., Delgado, I., Benito, L., & Oña, F. of pre-TALL/LBL revisited. Experimental and Molecular Pathology, (2000). Aberrant Immunophenotypes Detected by Flow Cytom- 81(2), 162–165. doi: 10.1016/j.yexmp.2006.06.006 etry in Acute Lymphoblastic Leukemia. Leukemia and Lymphoma, 29. Coustan-Smith, E., Mullighan, C. G., Onciu, M., Behm, F. G., 36(3–4), 275–284. doi: 10.3109/10428190009148848 Raimondi, S. C., Pei, D., . . . Campana, D. (2009). Early T-cell 36. Borowitz, M. J., Bene, M. C., Harris, N. L., Porwit, A., Matutes, E., & precursor leukaemia: A subtype of very high-risk acute Arber, D. A. (2017). Acute leukemias of ambiguous lineage. In S. lymphoblastic leukaemia. The Lancet Oncology, 10(2), 147–156. Swerdlow, E. Campo, N. Harris, E. Jaffe, S. Pileri, H. Stein, & H., doi: 10.1016/s1470-2045(08)70314-0 Thiele, J. (Eds.), WHO Classification of Tumours of Haematopoietic and 30. Inukai, T., Kiyokawa, N., Campana, D., Coustan-Smith, E., Lymphoid Tissues (4th ed., pp. 179–187). Lyon, France: International Kikuchi, A., Kobayashi, M., . . . Ohara, A. (2011). Clinical Agency for Research on Cancer. Chapter 28 Mature Lymphoid Neoplasms Katalin Kelemen, MD, PhD Fiona E. Craig, MD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Describe the clinical presentation of patients 7. List and describe the

chronic leukemic with mature lymphoid neoplasms. lymphoproliferative disorders. 2. Describe how the diagnosis of a lymphoid 8. Recognize and differentiate abnormal and neoplasm is made. normal lymphocytes on a stained peripheral 3. Differentiate among chronic lymphocytic blood smear and associate their presence leukemia (CLL), lymphoma, and multiple with a clinical diagnosis. myeloma based on peripheral blood 9. Describe and apply a multidisciplinary findings and ancillary studies. approach to the classification and staging 4. Describe the histology of a normal lymph of lymphoid neoplasms. node. 10. Compare the laboratory and clinical 5. Summarize the causes of lymphadenopathy. findings of multiple myeloma and lymphoplasmacytic lymphoma. 6. Contrast the morphology of Hodgkin and non-Hodgkin lymphoma. 11. Define monoclonal gammopathy. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Describe the use of immunophenotyping 3. Compare and contrast the laboratory and genotyping in detecting clonality and features characteristic of the following list the characteristic results of the more non-Hodgkin lymphomas: common mature lymphoid neoplasms. a. Small lymphocytic lymphoma 2. Contrast the features of low-grade and b. Follicular lymphoma high-grade lymphoma. c. Mantle cell lymphoma 622 Mature Lymphoid Neoplasms 623 d. MALT lymphoma 6. Compare and contrast the laboratory e. Lymphoplasmacytic lymphoma features characteristic of the following f. Diffuse large B cell lymphoma plasma cell disorders: g. Burkitt lymphoma a. Plasmacytoma h. Anaplastic large cell lymphoma b. Symptomatic plasma cell myeloma i. Peripheral T cell lymphoma c. Monoclonal gammopathy of undetermined 4. Compare and contrast the laboratory significance features characteristic of Hodgkin 7. Recognize and identify the peripheral blood lymphoma (HL) subtypes. abnormalities associated with CLL, B-PLL, 5. Compare and contrast the clinical HCL, T-LGL, Sézary syndrome, plasma cell and laboratory features characteristic myeloma, and non-Hodgkin lymphoma. of the following chronic leukemic 8. Describe the etiology and pathophysiology lymphoproliferative disorders: of lymphoid neoplasms. a. Chronic lymphocytic leukemia (CLL) 9. Differentiate reactive from malignant b. B-prolymphocytic leukemia (B-PLL) proliferations of lymphoid cells using c. Hairy cell leukemia (HCL) clinical and laboratory data. d. T-prolymphocytic leukemia (T-PLL) e. Large granular lymphocytic leukemia (T-LGL) f. Sézary syndrome Chapter Outline Objectives—Level I and Level II 622 Diagnosis and WHO Classification 625 Key Terms 623 Mature B Cell Neoplasms 626 Background Basics 624 Mature T and NK Cell Neoplasms 636 Case Study 624 Hodgkin Lymphoma (HL) 640 Overview 624 Summary 642 Introduction 624 Review Questions 642 Etiology and Pathophysiology 624 References 644 Key Terms Anaplastic large cell lymphoma Leukemia Richter’s transformation (ALCL) Lymphoepithelial lesion Rouleaux BCL-2 gene Lymphoma Sézary cell Bence-Jones proteinuria Monoclonal gammopathy of unde- Smudge cell Butt cell termined significance (MGUS) Stage Dutcher body Plasma cell neoplasm Starry-sky appearance Flame cell Plasmacytoma Tingible body macrophages Hairy cell Popcorn cell (L&H cell) Waldenström macroglobulinemia Hallmark cell Prolymphocyte Lacunar cell Reed-Sternberg (R-S) cell 624 Chapter 28 Background Basics The information in this chapter builds on concepts • Outline the classification and summarize the general learned in previous chapters. To maximize your learn- characteristics of acute leukemia. (Chapter 23) ing experience, you should review these concepts before starting this unit of study: Level II • Explain the pathophysiology of neoplasia. Level I (Chapters 2, 23) • Describe the structure and function of lymph • Identify the etiology of acute leukemia. (Chapter 23) nodes. (Chapter 3) to the lymphoid lineage (Chapters 23, 27). Although, in gen- CASE STUDY eral, these broad categories are useful, distinction between We refer to this case study throughout the chapter. leukemia and lymphoma is not always clear-cut. Leukemia Julia, a 56-year-old female, presented with a cells can infiltrate tissue, and lymphoma can involve the 4-month history of generalized painless lymphade- bone marrow and peripheral blood. nopathy and fatigue. A CBC revealed leukocytosis resulting from lymphocytosis. Consider laboratory features that can help dif- Etiology and ferentiate a reactive lymphocytosis from a neoplas- tic lymphocytosis. Pathophysiology The genesis of lymphoid neoplasms is thought to be a mul- tistep process involving acquired genetic, inherited genetic, and environmental factors. The lymphoma cells are thought Overview to derive from a single precursor cell (they are clonal). Lymphoid neoplasms can be divided into mature and pre- cursor neoplasms, based on the stage of maturation of the Acquired Genetic Factors neoplastic cells. The precursor lymphoid neoplasms are dis- As described for the acute leukemias, acquired alterations cussed in Chapter 27. This chapter focuses on the mature of proto-oncogenes and tumor suppressor genes have been lymphoid neoplasms, and discusses their etiology and associated with the development of mature lymphoid pathophysiology, how a diagnosis is established, the clas- neoplasms. Additional targets for genetic damage are the sification scheme, and unique features of selected subtypes. genes involved in programmed cell death (apoptosis [e.g., BCL-2]; Chapter 2). The BCL-2 gene on chromosome 18 is involved in the pathophysiology of follicular lymphoma. Introduction Translocation of the BCL-2 gene to the region of the immu- noglobulin heavy chain gene, t(14;18), causes overexpres- Mature lymphoid neoplasms represent a heterogeneous sion of the BCL-2 gene. The resulting increase in BCL-2 group of disorders that can be divided into two broad cat- protein leads to an inhibition of apoptosis. Decreased cell egories based on the distribution of disease: lymphoma death results in an accumulation of lymphocytes within and leukemia. Lymphomas are malignant neoplasms that the lymph node. Therefore, low-grade follicular lymphoma present as tumor masses primarily involving the lymphoid appears to arise from cell persistence rather than uncon- organs, including lymph nodes, tonsils, spleen, thymus, trolled cell proliferation. and lymphoid tissue of other organs. Although most lym- phomas are composed of mature lymphoid cells, blastic malignancies do occur (lymphoblastic lymphoma). Leukemia Inherited Genetic Factors is a malignant neoplasm that primarily involves the bone Some inherited immunodeficiency syndromes are associ- marrow and peripheral blood. The chronic leukemic lym- ated with an increased incidence of malignant lymphoma, phoid neoplasms are composed of mature lymphocytes and including ataxia telangiectasia, Wiskott-Aldrich syndrome, usually have an insidious onset and an indolent course. In severe combined immunodeficiency (SCID), X-linked lym- contrast, lymphoblastic leukemia (also referred to as acute phoproliferative disorder, and autoimmune lymphoprolif- lymphoblastic leukemia [ALL]) is composed of blasts belonging erative syndrome (ALPS). Mature Lymphoid Neoplasms 625 Environmental Factors cell type. Less frequently, malignant lymphoid cells can be recognized under the microscope because of an abnormal Viral and bacterial infections may play a role in lym- or bizarre cell appearance. Mature lymphoid neoplasms phoma genesis. Epstein-Barr virus (EBV) is associated have widely different clinical symptoms and aggressive- with the development of several forms of lymphoid neo- ness, making the implementation of a generic grading sys- plasm including African Burkitt lymphoma, some cases of tem difficult. endemic Burkitt lymphoma, Hodgkin lymphoma (HL), and lymphoma associated with inherited or acquired immuno- deficiency. EBV infection is acquired orally and is often Laboratory Evaluation manifest clinically as infectious mononucleosis. The virus Clonal rearrangement of immunoglobulin or T cell recep- infects B lymphocytes where it remains latent under the tor genes or the presence of an abnormal translocation can immune system’s control. The EBV-infected cells can pro- be used to identify neoplastic lymphocytes (Chapter 42). liferate if the host becomes immunocompromised and/or The presence of a specific translocation can also assist in the B lymphocytes acquire additional genetic abnormalities determining the lymphoma subtype, as in the BCL-2 gene such as the C-MYC translocation. rearrangement in follicular lymphoma (Chapters 41 and 42). Another infectious agent associated with the devel- Flow cytometric studies can be used to demonstrate clonal- opment of non-Hodgkin lymphoma (NHL) is Helicobacter ity by demonstrating expression of only one of the immu- pylori. Patients with H. pylori–induced inflammation of noglobulin light chains (k or l) on B cells. The presence of the stomach have a high incidence of gastric lymphoma an abnormal phenotype such as CD5- T lymphocytes can of mucosa-associated lymphoid tissue (MALT) lymphoma support the diagnosis of a T cell lymphoma (Chapter 40). type. Chronic Helicobacter infection leads to antigen-driven Immunophenotyping of the lymphoma cells assists in sub- T lymphocyte stimulation and subsequent B lymphocyte classification, determining prognosis, and detecting resid- activation. The B lymphocytes initially are polyclonal and ual disease following treatment. depend entirely on T lymphocyte stimulation. With time, the B cell population can proliferate autonomously. If the Prognosis B lymphocyte proliferation still depends on T lymphocyte stimulation, the lymphoma can regress following removal The 2008 World Health Organization (WHO) scheme uses of the antigenic stimulus with antimicrobial therapy. Lym- morphologic, phenotypic, and genotypic features to clas- phoma that is proliferating independent of antigenic stim- sify mature lymphoid neoplasms into distinct disease ulation requires more drastic therapy including excision entities that have a predictable prognosis and response to and/or chemotherapy. therapy (Table 28-1). The latest revision of the WHO clas- sification published in 2016 incorporates new molecular advances derived from whole genome sequencing while Checkpoint 28.1 retaining the principles of the 2008 classification.1 Some of How does the BCL-2 gene rearrangement promote lymphoma these disease entities have a long indolent course and are genesis? often referred to as being low-grade (e.g., small lympho- cytic lymphoma). In contrast, some subtypes are clinically aggressive and, without treatment, kill the patient rapidly. Diagnosis and Who These are referred to as aggressive or high grade (e.g., Burkitt lymphoma). Classification The diagnosis and classification of mature lymphoid neo- Therapy plasms involve integration of clinical information with Current therapeutic regimens are actually more effective at the morphologic appearance and the results of ancillary treating high-grade than low-grade lymphoid neoplasms, studies, such as flow cytometry, chromosome analysis, and many patients treated for high-grade lymphoma are and molecular analysis. For most cases of leukemia, mor- cured of their disease. Often patients with low-grade lym- phologic evaluation is performed on peripheral blood and phoid neoplasms are treated for symptomatic relief rather bone marrow (aspirate and biopsy) samples. A diagnosis than the intent to cure. In general, histologic sections from of lymphoma requires a tissue biopsy or a fine-needle aspi- lower-grade lymphoma more often demonstrate a nodular rate of a mass. In general, normal or reactive proliferations growth pattern, smaller cells, lower mitotic activity, and of lymphocytes contain a mixture of cells varying in size, an absence of apoptosis. Higher-grade lymphoma usually shape, and staining characteristics. The cells present in has a diffuse growth pattern, is often composed of larger lymphoid neoplasms are usually more homogeneous than cells, and displays more numerous mitoses and apoptotic reactive lymphocytes because of the expansion of a single bodies. 626 Chapter 28 Table 28.1 WHO 2016 Classification of Selected Mature Table 28.2 Ann Arbor Staging System for Malignant Lymphoid Neoplasms Lymphoma Mature B Cell Chronic lymphocytic leukemia/small lymphocytic I Single lymph node region or single extralymphatic site 1IE2 Neoplasms lymphoma II Two or more lymph node regions on same side of diaphragm or B cell prolymphocytic leukemia with involvement of limited contiguous extralymphatic site 1IIE2 Hairy cell leukemia III Lymph node regions on both sides of diaphragm, which can Follicular lymphoma include spleen 1IIIS2 and/or limited contiguous extralymphatic Mantle cell lymphoma site 1IIIE2 Extranodal marginal zone lymphoma of mucosa- IV Multiple or disseminated foci of involvement of one or more associated lymphoid tissue (MALT lymphoma) extralymphatic organs or tissues with or without lymphatic Splenic marginal zone lymphoma involvement Nodal marginal zone lymphoma Lymphoplasmacytic lymphoma Diffuse large B cell lymphoma (DLBCL), not other- wise specified Mature B Cell Neoplasms Primary mediastinal (thymic) B cell lymphoma Chronic Lymphocytic Leukemia/ Burkitt lymphoma Plasma cell myeloma Small Lymphocytic Lymphoma Monoclonal gammopathy of undetermined Chronic lymphocytic leukemia (CLL) and small lymphocytic significance lymphoma (SLL) represent different clinical manifestations of Monoclonal immunoglobulin deposition diseases one disease entity (CLL/SLL). Patients with CLL present with Mature T and NK T cell prolymphocytic leukemia cell Neoplasms peripheral blood lymphocytosis but often develop lymph T cell large granular lymphocytic leukemia Sézary syndrome node involvement. In contrast, patients with SLL present Adult T cell leukemia/lymphoma with lymphadenopathy but often develop peripheral blood Extranodal NK/T cell lymphoma, nasal type and bone marrow disease. At first diagnosis of CLL/SLL, Enteropathy-associated T cell lymphoma patients are often asymptomatic. As the disease progresses, Hepatosplenic T cell lymphoma anemia, thrombocytopenia, and neutropenia may develop Subcutaneous panniculitis-like T cell lymphoma due to replacement of the bone marrow hematopoietic cells Angioimmunoblastic T cell lymphoma by neoplastic lymphocytes, hypersplenism, and immune- Anaplastic large cell lymphoma (ALCL), ALK positive mediated cell destruction, among other causes (Chapter 19). Anaplastic large cell lymphoma

(ALCL), ALK negative Peripheral T cell lymphoma, not otherwise specified PERIPHERAL BLOOD Hodgkin Nodular lymphocyte-predominant Hodgkin A diagnosis of CLL requires a sustained absolute lymphocyto- lymphoma lymphoma sis more than 5 * 103/mcL with characteristic morphologic Classical Hodgkin lymphoma: nodular sclerosis, and phenotypic findings. The lymphocytes are small and mixed cellularity, lymphocyte rich, and lymphocyte depleted appear mature with scant cytoplasm. The nuclei are usually round, and the chromatin is regularly clumped. Nucleoli are inconspicuous (Figure 28-1a). A few large p rolymphocytes Staging are usually present but represent 610, of all lymphocytes (Figure 28-1a). Prolymphocytes have abundant pale-staining The prognosis of a patient with a lymphoid neoplasm is cytoplasm and a large central prominent nucleolus. Smudge not only related to the pathologic findings but also clinical cells represent CLL cells that burst open during smear prepa- parameters, such as the extent and distribution of disease ration (Figure 28-1a). Though common in CLL, smudge cells (stage). Patients with widespread lymphoma usually have are nonspecific and they should not be used to diagnose CLL. a worse prognosis. Determining the stage of disease usu- The number of smudge cells can be reduced by mixing a drop ally involves radiologic studies, peripheral blood exami- of albumin with a drop of blood prior to making the smear. nation, and bone marrow aspiration and biopsy. The Ann Arbor scheme is often used to stage malignant lymphoma IMMUNOPHENOTYPING (Table 28-2). Bone marrow involvement indicates dissemi- Although a provisional diagnosis usually can be made fol- nated disease, Stage IV. lowing smear examination, immunophenotyping is often used to establish a definitive diagnosis. CLL is characterized by aberrant expression of the T lymphocyte antigen CD5. Checkpoint 28.2 The CLL cells usually have weak surface expression of CD20 How does staging differ from grading in characterizing the lym- and monoclonal immunoglobulin. Expression of CD23 and phoid neoplasms? CD200, and lack of FMC-7 positivity distinguishes CLL from leukemic mantle cell lymphoma (MCL). A recently Mature Lymphoid Neoplasms 627 a b Figure 28.1 (a) Chronic lymphocytic leukemia. Small round lymphocytes with clumped chromatin, a larger prolymphocyte with a prominent nucleolus, and numerous smudge cells (arrows) (peripheral blood, Wright stain, 1000* magnification). (b) Small lymphocytic lymphoma. The periphery of the image displays many small lymphocytes with round nuclei and clumped chromatin. The center of the image contains a vague nodule containing pale-staining larger cells (proliferation center; lymph node biopsy, H&E stain, 50* magnification). recognized useful marker is the Lymphoid enhancer bind- status of the immunoglobulin (Ig) heavy chain gene variable ing factor 1 (LEF-1), which is expressed in CLL/SLL but not region by a molecular sequencing method1VH2. CLL with a in mantle cell lymphoma, marginal zone lymphoma and more aggressive behavior is associated with higher levels follicular lymphoma.1 of expression of CD38 and ZAP-70 as determined by flow Flow cytometric studies occasionally detect a small cytometry and an unmutated Ig VH gene. population of cells with a phenotype characteristic of CLL but representing less than the required absolute count of CYTOGENETICS AND PROGNOSIS 5 * 103/mcL. If there is no clinical evidence of an overt The use of routine cytogenetic studies is limited in CLL/SLL lymphoid neoplasm, the diagnosis of monoclonal B lympho- because the leukemic cells do not proliferate well in culture. cytosis (MBL) is rendered. MBL precedes virtually all cases Fluorescence in situ hybridization (FISH), a more suitable of CLL. Based on recent recommendations of the revised cytogenetic technique, is able to detect chromosomal abnor- WHO classification, “low-count” MBL (defined as less than malities in approximately half of all CLL/SLL patients. The 0.5 * 103/mcL) does not require a clinical follow-up other most common chromosomal abnormality, del 13q14-23.1, is than routine medical care, while a “high-count” MBL (more associated with a relatively good prognosis. The following than 0.5 * 103/mcL) requires a yearly follow-up.1 chromosomal abnormalities, listed in order of decreasing frequency, are associated with a worse prognosis: trisomy LYMPH NODE BIOPSY 12, deletion 11q22.3-23, deletion 6q21-23, and deletions at A lymph node biopsy performed in CLL/SLL reveals an 17p13.1 (p53 aberrations), 14q abnormalities and complex essentially diffuse infiltrate of small mature lymphocytes. chromosomal abnormalities. The small lymphocytes have dense, regularly clumped chromatin and lack nucleoli (Figure 28-1b). At low power, TRANSFORMATION vaguely nodular structures, the so-called proliferation cen- Less than 10% of patients with CLL/SLL develop trans- ters (Figure 28-1b) containing larger cells with pale-staining formation to a B cell lymphoid neoplasm with a worse cytoplasm and prominent eosinophilic nucleoli can be seen.1 prognosis. Over time, the number of prolymphocytes can increase, often in association with acquired genetic abnor- MOLECULAR PROGNOSTICS malities, although progression to prolymphocytic leukemia The majority of patients with CLL/SLL experience a slowly is unusual. Richter’s transformation is the transition to an progressing indolent course and require treatment only for aggressive large B cell lymphoma, which occurs in approxi- symptom relief. It has been recently recognized that CLL/SLL mately 2–8% of patients with CLL/SLL.2 is a rather heterogeneous disorder and a subset of patients requires earlier treatment due to a more aggressive course THERAPY and a shorter overall survival. Prognostic markers are labo- The Rai and Binet prognostic staging systems, which are ratory markers that correlate with a certain clinical behavior based on physical examination and blood counts, can be and are often evaluated at diagnosis in an attempt to predict used to assess the need for therapy and the specific type outcome. Commonly used prognostic markers include the of therapy.3 Typically, patients with early stage CLL do not expression of proteins CD38 and ZAP-70 and the mutational receive therapy but are monitored for disease progression. 628 Chapter 28 Standard therapy for those who are symptomatic or have advanced disease is chemotherapy with fludarabine, cyclo- phosphamide, and rituximab (anti-CD20). New small- molecule inhibitors (e.g., ibrutinib) that work inside the cell to block activity of enzymes responsible for cell signaling may change the standard treatment of B cell malignancies. B Cell Prolymphocytic Leukemia B cell prolymphocytic leukemia (B-PLL) is an aggressive, rare, leukemic disorder that often does not respond to treat- ment. B-PLL can arise de novo, or as a progression of CLL or another low-grade lymphoma, such as splenic marginal zone lymphoma. Patients with B-PLL usually have promi- nent splenomegaly but minimal lymphadenopathy. PERIPHERAL BLOOD The CBC in patients with B-PLL reveals marked absolute Figure 28.2 Prolymphocytic leukemia. Numerous large lymphocytosis, often more than 300 * 103/mcL, anemia, lymphoid cells with prominent nucleoli (prolymphocytes) (peripheral blood, Wright stain, 1000* magnification). and thrombocytopenia. The neoplastic cells have a charac- teristic appearance of “prolymphocytes”: large cells with moderate amounts of pale basophilic cytoplasm, moder- ately condensed chromatin, and a single prominent nucleo- Hairy Cell Leukemia lus (Figure 28-2). Prolymphocytes represent less than 10% Hairy cell leukemia (HCL; Figure 28-3) is an uncommon of the lymphocytes seen in CLL and more than 55% of the B cell neoplasm presenting in middle age. Males have a lymphocytes present in PLL. Cases with 11–55% prolym- significantly higher incidence (male-to-female ratio = 7:1). phocytes have an unpredictable course. At presentation, patients usually have massive spleno- megaly but lack lymphadenopathy. Extensive bone mar- IMMUNOPHENOTYPING row involvement often exists; therefore, patients with HCL The phenotype of B-PLL differs from that of CLL in demon- usually present with pancytopenia. strating more variable expression of CD5, stronger intensity expression of surface immunoglobulin and CD20, positivity PERIPHERAL BLOOD with FMC-7, and absence of CD23. B-PLL is not associated The white blood cell (WBC) count is low because of both with a characteristic chromosome abnormality, but the short neutropenia and monocytopenia; patients thus have an arm of chromosome 17 is often deleted, del(17p), resulting increased susceptibility to infection, especially with myco- in inactivation of the p53 gene (a tumor suppressor gene). bacterial organisms. Although neoplastic cells (hairy cells) B-PLL does not respond well to chemotherapy and has a are usually present on a peripheral smear, they are usually poor prognosis. too few to elevate the WBC count. The hairy cells have a a b Figure 28.3 Hairy cell leukemia. (a) Two abnormal lymphocytes with abundant pale-staining cytoplasm with hair-like projections and relatively finely distributed chromatin (peripheral blood, Wright stain, 1000* magnification). (b) Replacement of hematopoietic precursors by abnormal small lymphocytes with abundant clear cytoplasm (“fried-egg” appearance) (bone marrow, H&E stain, 1000* magnification). Mature Lymphoid Neoplasms 629 characteristic abnormal appearance with abundant pale- Follicular Lymphoma staining cytoplasm, circumferential cytoplasmic projections (“hairs”), usually oval or reniform nuclei, and relatively Follicular lymphoma (FL; grade 1; Figure 28-4) is one of fine chromatin (Figure 28-3a). Flow cytometric immuno- the most common types of mature B cell lymphoid neo- phenotyping is used to establish a diagnosis of HCL. HCL plasm within the United States, second only to diffuse cells are mature B lymphocytes that are positive for CD19, large B cell lymphoma. Patients usually present with gen- CD20 (strong intensity), CD22, CD25, CD103, and CD11c. eralized painless lymphadenopathy, most have advanced There is usually strong-intensity monoclonal surface stage disease (Figure 28-4a) with bone marrow involve- immunoglobulin present. Molecular diagnostics reveal the ment (Figure 28-4a), and a few have peripheral blood BRAF V600E mutation in the vast majority of cases (nearly involvement (Figure 28-4b). Involvement of other extra- 100%) and is generally recognized as the causal genetic nodal sites is unusual. event of HCL.4 LYMPH NODE BIOPSY BONE MARROW Lymph node biopsy of FL reveals an infiltrate of lymphoid Bone marrow involvement is often diffuse, and the neoplas- cells forming poorly circumscribed nodules that resemble tic cells are usually surrounded by fibrosis, preventing their follicular germinal centers (Figure 28-4c). In addition, the aspiration from the bone marrow (resulting in a “dry tap”). lymphoma cells grow in a diffuse pattern in some areas. Bone marrow biopsy sections reveal a monotonous infil- Neoplastic follicles differ from reactive follicles in that trate of abnormal lymphocytes with small nuclei and abun- neoplastic follicles lack evidence of lymphocyte apoptosis. dant pale-staining cytoplasm (“fried egg” appearance)5 This is manifest on histologic sections as a lack of macro- (Figure 28-3b). phages engulfing fragments of dead cells (tingible body macrophages), which are usually found in areas of exten- TISSUE INVOLVEMENT sive apoptosis (reactive germinal centers and high-grade Splenectomy specimens reveal marked expansion of the red lymphoma). The neoplastic infiltrate always contains a pulp because of an infiltrate of abnormal cells with the fried mixed population of small cleaved (centrocytes) and large egg appearance. Lakes of erythrocytes are often formed cells (centroblasts) but is often more homogeneous than a between the tumor cells (pseudosinuses). Immunohisto- normal germinal center (Figure 28-4d). The proportion of chemical stains for the B cell antigen CD20 can be used to large cells varies and is used to separate FL into three grades highlight the infiltrate. Other features characteristic of HCL (1, 2, or 3). include Annexin A1 and CD123 staining and BRAF V600E In situ follicular neoplasia (ISFN) is a term used for non- mutation.4,5 HCL is an indolent disease. Long-lasting com- neoplastic follicles colonized by follicular lymphoma cells. plete remissions are often obtained with the chemotherapy ISFN exhibits a low-rate progression, but it may be associ- agent 2-chlorodeoxyadenosine (2-CDA/cladribine), while ated with lymphoma elsewhere and therefore, it requires more recently the use of BRAF inhibitors and immunother- additional clinical assessment.1 FL cells in the peripheral apy (anti-CD20 monoclonal antibody) are used for long- blood and bone marrow usually have very irregular nuclear lasting clinical responses. outlines and a deep indentation (cleft) of the nuclear mem- brane (“buttock” or “butt” cells; Figure 28-4b). Checkpoint 28.3 IMMUNOPHENOTYPING AND CYTOGENETICS Chronic lymphoid malignancies compose a heterogeneous Conventional histologic evaluation is usually insufficient group. What characteristics allow these malignancies to be to establish a diagnosis of FL. Immunophenotyping can grouped together? be used to confirm the presence of lymphoma (monoclo- nal lymphocytes) and confirm the subtype of lymphoma 1CD10+2. Most FL cases arise because of a chromosome CASE STUDY (continued from page 624) translocation t(14;18) involving the BCL-2 gene that leads Julia’s CBC revealed a WBC count of 20 * 103/mcL to overexpression of BCL-2 protein in lymphocytes. with 60% lymphocytes. Examination of the periph- BCL-2 protein inhibits individual cell death (apoptosis), eral blood revealed mature lymphocytes with allowing follicle center cells to accumulate and produce scant cytoplasm, clumped chromatin, and irregular lymphadenopathy. nuclear outlines. PROGNOSIS AND TREATMENT 1. What is the differential diagnosis? Most patients with FL have an indolent

disease with a median survival of 7–9 years. Patients with grade 3 FL 2. What studies could be performed to establish the are sometimes cured. However, the lower-grade FLs diagnosis? (grades 1 and 2) are incurable with current therapies and are therefore treated for relief of symptoms. Duodenal 630 Chapter 28 a b c d Figure 28.4 Follicular lymphoma, grade 1. (a) Bone marrow involvement by low grade follicular lymphoma displaying a characteristic paratrabecular growth pattern (bone marrow biopsy, H&E stain, 20* magnification). (b) Circulating lymphoma cells with irregular nuclear outlines (peripheral blood, Wright stain, 1000* magnification). (c) Loss of the normal lymph node architecture. The abnormal infiltrate forms numerous poorly defined nodules (follicles) (lymph node biopsy, H&E stain, 20* magnification). (d) Neoplastic follicle composed of a relatively homogeneous population of small lymphoid cells containing angulated, twisted nuclei (lymph node biopsy, H&E stain, 200* magnification). follicular lymphoma and pediatric-type follicular lym- CASE STUDY (continued from page 629) phoma are two recently recognized subtypes that are Julia’s cervical lymph node biopsy reveals efface- often localized and may not require systemic treatment ment of the normal architecture by a lymphoid other than local excision.1 Low-grade FL can progress to infiltrate with a nodular growth pattern. The a diffuse large cell lymphoma with a median survival of nodules contain a relatively homogeneous popu- less than 1 year. lation of small lymphocytes and a few admixed large cells. The infiltrating lymphoid cells have Mantle Cell Lymphoma (MCL) irregular nuclear outlines. Tingible body macro- phages are lacking, and very few mitotic figures Mantle cell lymphoma usually presents with disseminated are present. disease involving multiple lymph node groups, bone mar- row, peripheral blood, spleen, liver, and gastrointestinal 3. What is the cause of the lymphadenopathy? Is tract. Gastrointestinal tract involvement can present as this process low grade or high grade? multiple polyps involving the small bowel (lymphomatous polyposis; Figure 28-5a). Mature Lymphoid Neoplasms 631 a b c d Figure 28.5 Mantle cell lymphoma. (a), (b) Abnormal lymphoid infiltrate in a lymph node with a uniform infiltrate of small lymphoid cells with slightly irregular nuclear outlines (lymph node biopsy, H&E stain, (a) 200* , (b) 500* magnification). (c) Cyclin D1 nuclear overexpression (lymph node biopsy, immunohistochemistry stain, 500* magnification). (d) SOX11 nuclear overexpression (lymph node biopsy, immunohistochemical stain, 500* magnification). PERIPHERAL BLOOD IMMUNOPHENOTYPING AND CYTOGENETICS MCL is a B cell lymphoma composed of small- to medium- Establishing an MCL diagnosis requires the use of ancillary sized lymphoid cells with irregular nuclear contours studies. The following immunophenotype is characteris- (Figure 28-5). MCL is associated with a t(11;14)(q13;q32) tic of MCL: CD19+ , CD5+ , CD23- , FMC-7+ , and sIg+ IgH/CCND1 translocation resulting in overexpression of (strong intensity), and almost all are positive for cyclin D1 Cyclin D1, a regulatory protein participating in progression and SOX11 by immunohistochemistry6 (Figure 28-5d). The of cells from the G1 to S phase of the cell cycle (Chapter 2). IgH/CCND1 translocation can be demonstrated by FISH An indolent clinical variant is the leukemic, non-nodal studies in the vast majority of cases of MCL. MCL, which involves peripheral blood, bone marrow, PROGNOSIS and spleen without lymph node involvement. Blastoid MCL is a relatively aggressive lymphoma with a poor and pleomorphic MCL (composed of blast-like or large response to current therapies. However, recent findings cells, respectively) represent more aggressive lymphomas regarding the pathophysiology of the disease may help and usually carry additional molecular and cytogenetic in the development of new therapeutic approaches.1 Cur- abnormalities. rently, the overall median survival is approximately 3–4 LYMPH NODE BIOPSY years. Therefore, distinction of MCL from the other small On histologic sections, MCL can demonstrate either a dif- lymphoid B cell neoplasms (follicular lymphoma, SLL, and fuse or vaguely nodular growth pattern (Figure 28-5b). The MALT lymphoma [see “Extranodal Marginal Zone Lym- mantle zone pattern is characterized by a neoplastic infil- phoma of Mucosa-Associated Lymphoid Tissue” below]) trate surrounding reactive germinal centers (Figure 28-5c). is important. 632 Chapter 28 Extranodal Marginal Zone immunoglobulin on lymphocytes) or paraffin section Lymphoma of Mucosa-Associated immunohistochemistry (cytoplasmic immunoglobulin in cells demonstrating plasma cell differentiation). In contrast Lymphoid Tissue to FL and small lymphocytic lymphoma, MALT lymphoma Patients with extranodal marginal zone lymphoma of is usually negative for CD10 and CD5. mucosa-associated lymphoid tissue (MALT) usually pres- TREATMENT ent with localized extranodal disease (e.g., involving the MALT lymphoma is an indolent disease, though it has the stomach, salivary gland, lacrimal gland, thyroid, lung). A potential to transform to higher-grade, large B cell lym- preceding chronic inflammatory disorder such as chronic phoma in a subset of patients. Although, patients with gastritis from Helicobacter pylori infection or autoimmune H. pylori–associated gastric MALT lymphoma are often disease (Sjögren’s syndrome or Hashimoto’s thyroiditis) cured with antimicrobial therapy, the polymerase chain often occurs. reaction (PCR) assay for a monoclonal B cell population can LYMPH NODE BIOPSY remain positive after complete remission. MALT lymphoma A biopsy of MALT lymphoma (Figure 28-6a) reveals an infil- that is unresponsive to antimicrobial therapy or that occurs trate of small to intermediate size lymphocytes intimately at other sites is often treated with local excision or radiation associated with epithelial cells (e.g., gastric mucosa, salivary therapy. gland ducts). Epithelial structures infiltrated by neoplastic lymphocytes are referred to as lymphoepithelial lesions Lymphoplasmacytic Lymphoma (Figure 28-6a). MALT lymphoma is composed predomi- Lymphoplasmacytic lymphoma (LPL) is a neoplasm that nantly of small lymphocytes with round or slightly cleaved primarily involves the bone marrow and sometimes the nuclei that often have abundant pale-staining cytoplasm spleen and lymph nodes and is composed of a mixture of and are referred to as monocytoid B cells because of their neoplastic small lymphocytes and plasma cells. LPL must resemblance to monocytes (Figure 28-6b). MALT lymphoma be distinguished from small B cell lymphoid neoplasms, can contain admixed neoplastic plasma cells. In addition other types of lymphoma that can have plasmacytic dif- to the neoplastic cells, the infiltrate often contains benign ferentiation, such as MALT lymphoma, and neoplasms germinal centers. Infiltration of benign germinal centers by composed entirely of plasma cells (plasma cell neoplasms). neoplastic cells is referred to as follicular colonization. Unlike those with plasma cell neoplasm multiple myeloma, IMMUNOPHENOTYPING patients with LPL lack lytic bone lesions. The neoplas- The differential diagnosis includes other lymphomas tic plasma cells in LPL often secrete monotypic immuno- composed of small lymphocytes, including SLL, MCL, globulin, leading to a paraprotein, often of IgM type. High and FL. Ancillary studies can assist in obtaining the cor- plasma levels of the large pentamer IgM result in hypervis- rect diagnosis. Restriction of immunoglobulin light chains cosity that can lead to poor circulation in small blood ves- can be demonstrated by immunophenotyping (surface sels, visual impairment, headache, dizziness, and deafness. a b Figure 28.6 Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). (a) Parotid salivary gland involved by MALT lymphoma. Two lymphoepithelial lesions are present demonstrating infiltration of ducts by neoplastic lymphoid cells (parotid gland biopsy, H&E stain, 50* magnification). (b) Lymphoid infiltrate of MALT lymphoma. Many of the neoplastic cells have abundant pale- staining cytoplasm (i.e., a “monocytoid” appearance) (parotid gland biopsy, H&E stain, 100* magnification). Mature Lymphoid Neoplasms 633 Waldenström macroglobulinemia is the combination of transformation from a lower-grade lymphoma (e.g., follicu- LPL with bone marrow involvement and an IgM monoclo- lar lymphoma or small lymphocytic lymphoma), whereas nal paraprotein. others arise de novo. Though several histologic variants are recognized, morphologic and immunophenotypic features LYMPH NODE BIOPSY have limited value in predicting prognosis in DLCL. Histologic sections usually demonstrate a diffuse infiltrate composed of lymphocytes, plasma cells, and lymphoid cells GENOMIC PROFILING AND IMMUNOHISTOCHEMISTRY that demonstrate some evidence of differentiation toward Gene expression profiling has divided DLBCL into germi- plasma cells (plasmacytoid lymphocytes). Immunohisto- nal center B cell–like (GCB) and activated B cell–like (ABC) chemistry can be performed to demonstrate the presence subgroups. These subgroups differ in their chromosomal of cytoplasmic monoclonal immunoglobulin light chain (k alterations, molecular pathways and clinical outcome, with or l) and IgM heavy chain. the ABC type DLBCL associated with a worse outcome then the GCB type. Since genomic profiling is difficult to MOLECULAR EVALUATION perform, a simple immunohistochemical approach using While no specific chromosomal abnormalities are recog- antibodies to CD10, BCL6, and IRF4/MUM1 (so-called nized in LPL, about 90% of LPL or Waldenstrom macroglob- Haas algorithm) provide a useful surrogate to differentiate ulinemia have MYD88 L265P mutations. This mutation is between ABC and GCB.8 also found in a significant proportion of IgM but not IgG or IgA monoclonal gammopathy of undetermined significance MOLECULAR PROGNOSTICS (MGUS), and in certain diffuse large B cell lymphomas, but MYC gene alterations play an important role in the prog- not in plasma cell myeloma.7 nosis of DLBCL. The MYC gene is rearranged in 5-15% of DLBCL and is frequently associated with BCL2 or, less fre- TREATMENT quently, with BCL6 (so-called “double-hit” or “triple-hit” Treatment of symptomatic patients includes rituximab, lymphomas).9 These lymphomas have been recently recog- which can be used in combination with chemotherapy. nized as a separate entity designated as “High-grade B cell Autologous bone marrow transplant is a recent treatment lymphoma with rearrangement of MYC and BCL2 and/or option. Plasmapheresis to treat hyperviscosity is used to BCL6.”1 Additional important prognostic markers are the reduce paraprotein levels. The median survival for patients expression of MYC and BCL2 proteins by the lymphoma, with LPL is typically 5–10 years. which are commonly expressed without the presence of a translocation. “Double expressor” lymphomas (MYC Diffuse Large B Cell Lymphoma and BCL2 positive by immunohistochemistry) are also Diffuse large B cell lymphoma (DLBCL) is a heteroge- associated with a worse outcome, though not as aggres- neous group of tumors composed of large B lymphoid sive as the “double-hit” or “triple-hit” DLBCL.10 Finally, cells (Figure 28-7). Some DLBCLs develop as a result of the importance of Epstein-Barr virus in the pathogenesis and prognosis is acknowledged by recent introduction of the category “EBV-positive DLBCL.”11 With multi-agent chemotherapy and anti-CD20 monoclonal antibody ther- apy (rituximab), the long-term remission rate of DLBCL is 50–60%. CASE STUDY (continued from page 630) The previous biopsy had established a diagnosis of low-grade NHL, follicular type. Julia received multi-agent chemotherapy for symptomatic relief. Two years following the diagnosis, she returned with rapidly expanding lymph nodes in her neck. Repeat biopsy revealed effacement of the lymph node architecture by a diffuse infiltrate of large B cells. Figure 28.7 Diffuse large B cell lymphoma. Abnormal infiltrate 5. What is the diagnosis? composed of large lymphoid cells with pale-staining, vesicular 6. What is the relationship of this disease to the pre- chromatin, irregular nuclear outlines, and basophilic nucleoli. Several vious diagnosis? mitotic figures are present (lymph node biopsy, H&E stain, 200* magnification). 634 Chapter 28 Burkitt Lymphoma lymphoblastic leukemia/lymphoma and DLBCL. Most cases of Burkitt lymphoma have an isolated chromosome Burkitt lymphoma is a high-grade NHL with a high inci- translocation leading to rearrangement of the MYC and IGH dence in Africa (endemic subtype). It represents approxi- genes, typically t(8;14) or less frequently t(2;8) or t(8;22). mately one-third of all pediatric lymphomas occurring MYC gene rearrangement occurs in only 5% of DLBCL. This outside Africa (sporadic Burkitt lymphoma). Many adult distinction is important because the therapeutic regimen cases occur in immunocompromised individuals such as used for Burkitt lymphoma usually differs from that used those infected with the HIV virus. Burkitt lymphoma often for DLBCL. With the appropriate aggressive multiagent involves extranodal sites. Endemic Burkitt lymphoma has chemotherapy, Burkitt lymphoma is potentially curable. a predilection for involvement of the facial bones and jaw; sporadic Burkitt lymphoma often presents with disease involving the intestine, ovaries, or kidney. EBV is thought Plasma Cell Neoplasms to play a role in the pathophysiology of Burkitt lymphoma. The plasma cell neoplasms are considered to be a group of DNA of EBV is present in most cases of endemic Burkitt lym- diseases composed of immunoglobulin-secreting cells in the phoma and approximately one-third of the HIV-associated absence of neoplastic B lymphocytes. It is important to dis- tumors. EBV is found less frequently in the sporadic form. tinguish this group of neoplasms from the mature lymphoid neoplasms that can demonstrate plasmacytic differentiation LYMPH NODE BIOPSY (e.g., LPL). A biopsy of Burkitt lymphoma usually reveals a diffuse infil- Serum or urine protein electrophoresis (SPEP or UPEP) trate of neoplastic

cells demonstrating a “starry sky” appear- reveal increased protein with a narrow range of electropho- ance (Figure 28-8). The “sky” represents the blue nuclei of the retic mobility (M spike) (Figure 28-9a, b). The M spike can neoplastic lymphocytes; the “stars” are formed by scattered be further characterized by immunofixation electrophoresis pale-staining tingible body macrophages. The infiltrating (IFE) to confirm the presence of a monoclonal protein and lymphoid cells are intermediate in size with nuclei approxi- determine the immunoglobulin class (Figure 28-9c). The mately the same size as those of the tingible body macro- monoclonal protein usually contains one immunoglobulin phages. Multiple small nucleoli are usually present, and light chain (k or l) and one heavy chain with the follow- mitotic figures and apoptotic bodies are frequent. The latter ing incidence: IgG 7 IgA 7 IgM 7 IgD 7 IgE. Normal two features are characteristic of high-grade lymphoma. immunoglobulin production is usually decreased, leading IMMUNOPHENOTYPING AND MOLECULAR to functional hypogammaglobulinemia. Plasma cell neo- EVALUATION plasms can produce an excess of immunoglobulin light Burkitt lymphoma exhibits a germinal center B cell-type chains, light chains only, heavy chains only, or no immu- immunophenotype: CD19+ , sIg+ , CD10+ , CD5- . The noglobulin (nonsecretory). Patients with light chain only differential diagnosis for Burkitt lymphoma includes B disease can have a normal SPEP because the protein passes into the urine. The presence of free immunoglobulin light chains in the urine is referred to as Bence-Jones proteinuria. The plasma cell neoplasms can be divided into disease entities based on the distribution and extent of disease (soli- tary versus multiple lesions and bone versus extraosseous) and the characteristics of the immunosecretory protein produced (class of heavy chain and the presence of amy- loid production, immunoglobulin heavy, or light chain; Table 28-3). PLASMA CELL MYELOMA Plasma cell myeloma is a bone marrow based, multifocal plasma cell neoplasm associated with an M protein in serum and/or urine. Several clinical variants range from asymp- tomatic to an aggressive disease that is identified on the presentation, radiologic findings, and laboratory informa- Figure 28.8 Burkitt lymphoma. Intermediate size lymphocytes tion. Symptomatic plasma cell myeloma is characterized by an with multiple nucleoli and scant cytoplasm. Numerous mitotic figures M protein in the urine or serum, clonal plasma cells in the are present. The presence of apoptotic bodies indicates individual bone marrow whose symptoms are hypercalcemia, renal cell necrosis. There is a “starry-sky” appearance because of pale- staining tingible body macrophages scattered in an infiltrate that insufficiency, anemia, lytic bone lesions (CRAB), and evi- appears basophilic because of staining of the tumor cell nuclei dence of related organ or tissue impairment. Patients often (lymph node biopsy, H&E stain, 100* magnification). present with bone pain and/or pathologic fractures because Mature Lymphoid Neoplasms 635 9/10/99 ABN 1392 Monoclonal spike 1681 Albumin 1685 Monoclonal serum protein Normal control 723 a1 a2 b g a b Free k light chains IgG k SPEP IgG IgA IgM K l c Figure 28.9 Monoclonal gammopathy. (a) Serum protein electrophoresis. Sample 1681 displays a band in the early gamma region. (b) Densitometry scan reveals a “spike” in the gamma region. A similar spike was found in the urine. (c) Immunofixation electrophoresis performed on a urine sample reveals two monoclonal bands composed of IgG kappa and free kappa light chains (Bence-Jones proteins). of tumor infiltration. Asymptomatic (smoldering) myeloma is replacing the normal hematopoietic cells (Figure 28-10a, b). characterized by M protein in serum at a level more than Neoplastic plasma cells vary from normal to abnormal forms 30 g/L and/or 10% or more of clonal plasma cells in the bone with more finely distributed chromatin or nucleoli. Intra- marrow but no organ or tissue impairment or symptoms. nuclear inclusions composed of immunoglobulin (Dutcher bodies; Figure 28-11a) or red-tinged cytoplasm (flame cells; Cytogenetics Cytogenetic evaluation by FISH distin- Figure 28-11b). Intracytoplasmic inclusions composed of guishes two genetic subtypes based on chromosome immunoglobulin can occur as single inclusions (Russell content: hyperdiploid and nonhyperdiploid.12 The hyper- body) or multiple inclusions (Mott cell; F igure 28-11c), they diploid group includes trisomies of chromosomes 3, 5, 7, 9, can also be seen in reactive plasma cells. Neoplastic plasma 11, 15, 19, and 21. The nonhyperdiploid group is character- cells rarely circulate in the blood, but the peripheral smear ized by reciprocal translocations, deletions, and complex is often abnormal because of stacking of the erythrocytes karyotypes. The most frequent translocation involves the (rouleaux formation; Figure 28-11d; Chapter 10). immunoglobulin heavy chain variable region on chromo- some 14q32 and involves cyclin D1. MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE (MGUS) Peripheral Blood and Bone Marrow Examination of the Monoclonal gammopathy of undetermined significance bone marrow reveals an abnormal infiltrate of plasma cells (MGUS) is used to describe a low level of serum monoclonal g b a2 a1 Albumin 636 Chapter 28 for symptomatic plasma cell myeloma. In contrast, plasmacy- Table 28.3 Key Features of Selected Plasma Cell toma of extraosseous locations, such as the upper respiratory Neoplasms tract, have a good prognosis with only rare recurrence after Neoplasm Features excision and a low incidence of dissemination. Sometimes, Symptomatic plasma M spike in serum or urine distinguishing extraosseous plasmacytoma from MALT lym- cell myeloma Clonal plasma cells in bone marrow phoma with plasmacytic differentiation is difficult. Related organ or tissue damage (CRAB) Patients under 65 years of age can be treated with high- Asymptomatic plasma M spike in serum or urine (more than 30 g/L) dose chemotherapy followed by autologous or allogeneic cell myeloma Clonal plasma cells in bone marrow greater stem cell transplant. Those over the age of 65 can be treated than 10% with chemotherapy (melphalan) and prednisone. The prog- No related organ or tissue damage nosis of symptomatic plasma cell myeloma is poor with a Monoclonal gammop- M spike in serum or urine (less than 30 g/L) athy of undetermined median survival of 3–4 years. Patients with asymptomatic Clonal plasma cells in bone marrow 610, significance (MGUS) plasma cell myeloma can have stable disease for many years No related organ or tissue damage but often progress to symptomatic myeloma. Plasmacytoma Solitary collection of clonal plasma cells Can have M spike No clonal plasma cells elsewhere in bone marrow No related organ or tissue damage Mature T and NK Cell CRAB, hypercalcemia renal insufficiency, anemia, bone lesions. Neoplasms T cell and NK cell lymphomas are less common than B cell protein without evidence of an overt neoplasm. Therefore, lymphomas, as they comprise only 10–15% of NHLs in MGUS is a diagnosis that requires exclusion of other plasma the United States and Western Europe. Despite their low cell and lymphoid malignancies. The prevalence of a mono- prevalence, they represent a highly heterogeneous group clonal serum spike increases with age and is present in 3% of diseases. T cell lymphoma and systemic anaplastic large of asymptomatic patients over 70 years of age. There is no cell lymphoma arise in peripheral lymphoid organs (mainly treatment for MGUS. Although most patients have stable lymph nodes) and resemble CD4+ effector T cells. Other, disease and die of other causes, MGUS can progress to an less common T cell lymphomas (hepatosplenic T cell lym- overt plasma cell neoplasm. phoma, subcutaneous panniculitis-like T cell lymphoma PLASMACYTOMA and enteropathy-associated T cell lymphoma) are related A plasmacytoma is a localized, tumorous collection of clonal to the innate immune system and have a tendency to arise plasma cells. The prognosis of a plasmacytoma is related to its in mucosal or cutaneous sites, more commonly express location. Many patients with plasmacytoma of bone develop gamma/delta T cell receptors, and cytotoxic granule- additional plasmacytomas and ultimately meet the criteria associated proteins. Cytogenetic translocations, specific a b Figure 28.10 Symptomatic plasma cell myeloma. (a) Skull x-ray demonstrating multiple lytic bone lesions giving a “moth-eaten” appearance. (b) Replacement of bone marrow hematopoietic precursors by an infiltrate of plasma cells (bone marrow aspirate, Wright stain, 1000* magnification). Mature Lymphoid Neoplasms 637 a b c d Figure 28.11 Symptomatic plasma cell myeloma. (a) Plasma cell with intranuclear inclusion indicated with arrow (Dutcher body) (H&E stain, 1000* magnification). (b) Many plasma cells with a red tinge to the cytoplasm imparting a flame-cell appearance (bone marrow, Wright stain, 1000* magnification). (c) Plasma cell with multiple cytoplasmic inclusions (Mott cell) (bone marrow, Wright stain, 1000* magnification). (d) Stacking of peripheral blood erythrocytes resulting from the presence of increased immunoglobulin (Rouleaux) (peripheral blood, Wright stain; 1000* magnification). cytogenetic abnormalities, and disease-defining mutations classified as peripheral T cell lymphoma, not otherwise specified are uncommon in T and NK cell lymphomas. The WHO (PTCL NOS). The morphologic appearance of PTCL, NOS classification divides the mature T and NK cell neoplasms is heterogeneous with the neoplastic cells often exhibiting based on their primary clinical presentation into nodal, abundant clear or pale-staining cytoplasm and irregular extranodal and leukemic categories. Association with nuclear outlines. Histiocytes, plasma cells, and eosinophils Epstein-Barr virus, immunophenotype (CD4+ versus are admixed with the neoplastic cells. Immunophenotyping CD8+ ) and the presence of cytotoxic or NK-associated demonstrates expression of pan–T cell antigens. The pres- markers also play a role in the classification. ence of an abnormal immunophenotype (CD5- or CD7- ) or clonal rearrangement of the T cell receptor can assist in Nodal T and NK Cell Lymphomas the distinction of T cell lymphoma from a reactive process. Nodal cases usually show a CD4+/CD8- phenotype. Cyto- PERIPHERAL T CELL LYMPHOMA, NOT OTHERWISE genetics usually reveals a complex karyotype. PTCL, NOS SPECIFIED (NOS) usually presents with peripheral lymph node involvement A large category of peripheral T cell lymphoma (30% in though bone marrow, spleen, and liver can be also involved. Western countries) cannot be placed into one of the other In general, peripheral T cell lymphoma is an aggressive dis- subtypes in the current classification. This category is ease with a poor response to therapy and frequent relapses. 638 Chapter 28 Combination chemotherapy and stem cell transplantation systemic multiagent chemotherapy, but the rate of relapse is offer cure in a subset of patients. high. Brentuximab-vedotin is a recently emerged anti-CD30 antibody-drug conjugate that has shown great success in ANGIOIMMUNOBLASTIC T CELL LYMPHOMA (AITL) the treatment of ALCL and other CD30+ lymphomas.13 Angioimmunoblastic T cell lymphoma (AITL) presents with Systemic ALCL, ALK negative is a lymphoma morphologi- generalized lymphadenopathy, hepatosplenomegaly, ane- cally and immunophenotyically similar to ALCL, ALK+ , but mia and hypergammaglobulinemia. Lymph node biopsy lacks ALK translocations and ALK protein expression. The shows effacement of the paracortex by a polymorphic patients are typically older and clinically have a worse prog- inflammatory infiltrate, frequent large immunoblasts and nosis than ALCL, though still better prognosis than PTCL, a marked vascular proliferation. Residual B cell follicles are NOS. Other subtypes of ALK negative ALCL include primary often present and irregular proliferation of follicular den- cutaneous ALCL and a recently introduced category of ALCL dritic cells is typical. It has been recently recognized that the associated with breast implant.1 Both of these entities resemble neoplastic cells in AITL may be derived from intrafollicular systemic ALCL morphologically and immunophenotypically, T cells; they express a number of T follicular cell markers, but they are usually localized to their respective extranodal including CD10, CXCL13, and CD270 (PD-1). Virtually all location and show an indolent course. cases of AITL contain increased numbers of EBV-infected B cells. A clonal T cell population is usually present and in a subset of cases, a B cell clone also may be detected. Extranodal T and NK Cell ANAPLASTIC LARGE CELL LYMPHOMA (ALCL) Lymphomas One of the best-characterized subtypes of T/NK cell lym- HEPATOSPLENIC T CELL LYMPHOMA (HSTCL) phoma is anaplastic large cell lymphoma (ALCL). ALCL is HSTCL a rare extranodal lymphoma that arises from the composed of large bizarre anaplastic cells with horseshoe or cytotoxic g/d or a/b T cells of the splenic red pulp. Most wreath-like appearance (“hallmark” cells; Figure 28-12a). cases are young adult males who present with massive ALCL is positive for the CD30 antigen, may express pan-T hepatosplenomegaly, anemia, thrombocytopenia but no cell antigens such as CD2 and CD3 and frequently, cytotoxic lymphadenopathy. A subset of cases arise after solid organ granule proteins. Although ALCL is derived from T cells, transplantation or in the setting of treatment of inflamma- frequent loss of T cell antigen expression can result in a tory bowel disease. This lymphoma

infiltrates cords and null phenotype (lack of expression of either T or B cell anti- sinuses of the splenic red pulp, hepatic sinusoids and bone gens). Most ALCL cases involving lymph nodes contain the marrow sinuses. The characteristic immunophenotype is translocation t(2;5) that joins the nucleophosmin (NPM1) CD2+ , CD3+ , CD4- , CD5- , CD7+ and CD8+ . Most cases and anaplastic large-cell kinase (ALK) genes. This leads to exhibit g/d T cell receptors (TCR), though a minor subset is abnormal expression of ALK protein that can be detected a/b TCR positive. Karyotypic studies often show isochro- by immunohistochemistry (Figure 28-12b). Approximately mosome 7q. The disease is aggressive, and most patients 70% of patients with systemic ALCL go into remission with die within two years. a b Figure 28.12 Anaplastic large cell lymphoma. (a) Abnormal infiltrate of large cells with eosinophilic cytoplasm, including cells with multilobated nuclei (lymph node biopsy, H&E stain, 100* magnification). (b) ALK expression (lymph node biopsy, immunohistochemical stain, 200* magnification). Mature Lymphoid Neoplasms 639 EXTRANODAL NK/T CELL LYMPHOMA, NASAL TYPE T CELL LARGE GRANULAR LYMPHOCYTIC (T-LGL) Nasal NK/T cell lymphomas are rare in the United States and LEUKEMIA they are most frequent in East Asia and among the Native T-LGL leukemia often presents with a modest lymphocytosis American populations of Mexico and Central and South composed of cells with abundant pale-staining cytoplasm, America. This lymphoma is aggressive with an angiocentric azurophilic cytoplasmic granules, and nuclei with mature and angiodestructive growth producing areas of geographic clumped chromatin. Large granular lymphocytes are normal necrosis. Most of these lymphomas are EBV-associated and components of the peripheral blood and represent NK-like represent either true NK cell lymphomas or cytotoxic T cell T cells (CD3+ ) or NK cells (CD3- ) (Chapter 8). T-LGL leu- lymphomas. Expression of the NK cell associated antigen kemia cells show the following phenotype: CD2+ , CD3+ , CD56 is usually present. Non-nasal presentation can occur CD4- , CD5- , CD7+ , CD8+ , CD16+ , CD56+/- , and in skin, soft tissue, gastrointestinal tract and testis. CD57+/- . Loss of pan T cell antigens and demonstration of clonal T cell receptor rearrangement supports the diagnosis of SUBCUTANEOUS PANNICULITIS-LIKE T CELL a lymphoma as opposed to a reactive proliferation. In addition LYMPHOMA to lymphocytosis, anemia, neutropenia, and thrombocytope- This rare subtype of T cell lymphoma presents as multiple nia are common features. Although splenomegaly is com- erythematous subcutaneous nodules on the legs, trunk or mon, lymphadenopathy and hepatomegaly are uncommon. both. This lymphoma tends to remain localized to the sub- Approximately 25% of patients with T-LGL leukemia have cutaneous tissue, but may be complicated by a severe and symptomatic rheumatoid arthritis. The triad of rheumatoid often fatal hemophagocytic syndrome. Histologically the arthritis, splenomegaly, and neutropenia defines Felty’s syn- lymphoma exhibits a growth pattern similar to the inflam- drome. Patients with T-LGL leukemia usually have an indo- matory condition panniculitis. Lymphoma cells are usually lent course with more than 80% overall survival after 10 years. CD8 positive, a/b TCR positive and express cytotoxic gran- ule proteins. ADULT T CELL LEUKEMIA/LYMPHOMA (ATLL) ATLL is a T cell neoplasm caused by Human T cell leukemia ENTEROPATHY-ASSOCIATED T CELL virus-1 (HTLV-1) and it is more prevalent where the ret- LYMPHOMA (EATL) rovirus is endemic, in Southwestern Japan, Central Africa, EATL is a rare aggressive lymphoma that arises from the and the Caribbean basin. The disease involves peripheral intraepithelial lymphocytes of the small intestine as a blood, bone marrow, lymph nodes, and skin. Circulating long-term complication of celiac disease. Patients present lymphoma cells have hyperlobated, cloverleaf-like nuclei. with abdominal pain and weight loss. Acute obstruction The neoplastic cells are CD4+ and express CD25 and FoxP3, or perforation is less common. Histologically the tumors a phenotype characteristic of regulatory T cells. ATLL exhib- vary from proliferation of abnormal intraepithelial lym- its clonal T cell gene rearrangements and clonal integration phocytes to single or multiple tumor masses along the of the HTLV-1 genome. small intestine. The tumor cells are pleomorphic and show brisk mitotic activity. The phenotype is often CD2+ , CD3+ , SÉZARY SYNDROME CD4- , CD5- , CD7+ , CD8 and a/b TCR positive. Many Sézary syndrome, the leukemic presentation of cutane- cases demonstrate a clonal T cell gene rearrangement. ous T cell lymphoma, is defined by the combination of erythroderma (red skin), generalized lymphadenopathy, Leukemic T and NK Cell and neoplastic T cells (Sézary cells) in the skin, lymph Lymphomas nodes, and peripheral blood. Sézary cells, the neoplastic T lymphocytes, have irregular, convoluted (cerebriform) T CELL PROLYMPHOCYTIC LEUKEMIA (T-PLL) nuclear outlines (Figure 28-13). Abnormal cells can be T-PLL is a rare leukemic disorder of adults that presents counted in a blood smear leukocyte differential to deter- with marked lymphocytosis and splenomegaly. Lymph- mine a Sézary count; an absolute count of greater than adenopathy, hepatomegaly, and skin infiltration may be 1000 cells/mm3 (1 * 103/mcL) is required for diagnosis. present. The lymphoma cells are small or medium in size Sézary cells are mature helper T cells (CD3+ , CD4+ ) but and can have convoluted nuclear outlines and a prominent usually lack expression of CD7. The presence of a CD4:CD8 nucleolus. T-PLL has a mature phenotype with expression ratio of greater than 10, aberrant antigen expression by flow of CD3, CD2, CD5, and CD7. Most cases are CD4+ , though cytometry, and a clonal T cell receptor gene rearrangement some cases express both CD4 and CD8. The most frequent support the diagnosis. Sézary syndrome is closely related to cytogenetic abnormalities include inv(14)q11q32, del(11q), the primary cutaneous T cell lymphoma mycosis fungoides i(8q), and trisomy 8q. T-PLL is an aggressive disorder with (MF). MF is usually characterized by cutaneous patches and a median survival time of only 7.5 months. A subset of cases plaques, rather than diffuse erythroderma, and has less fre- shows a good clinical response to anti-CD52 monoclonal quent blood involvement. Treatment is usually palliative to antibody therapy. relieve symptoms and improve the quality of life. Clinical 640 Chapter 28 Table 28.4 Classification of Hodgkin Lymphoma Subtype Sclerosis Lymphocytes Tumor Cells Variants NLPHL - + + + + + L&H CHL NS Present + + + + Lacunar MC - + + + + - LR - + + + + + - LD -/+ + + + + + - NS, nodular sclerosis classical HL; MC, mixed cellularity classical HL; LD, lymphocyte depletion classical HL; LR, lymphocyte-rich classic HL; LP, lymphocyte-predominant HL; L&H, malignant cells characteristic of LP HL; + , few; + + + + , many. The reactive small lymphocytes that surround the neoplas- tic cells of NLPHL often include an unusual subset of CD57 positive T cells. Figure 28.13 Sézary syndrome. Abnormal large lymphocytes with relatively finely distributed chromatin and numerous nuclear folds (Sézary cell) (peripheral blood, H&E stain, 1000* Classical Hodgkin Lymphoma (CHL) magnification). The tumor cells in classical Hodgkin lymphoma (CHL) have large prominent eosinophilic nucleoli and coarse nuclear trials with ultraviolet B radiation and high-dose chemo- membranes. Reed-Sternberg (R-S) cells have two or more therapy and radiation with stem cell transplant are ongoing. nuclear lobes containing inclusion-like nucleoli and an area of perinucleolar clearing imparting an owl’s-eye appear- ance (Figure 28-15a). The reactive cells accompanying the Hodgkin Lymphoma (HL) neoplastic cells of classical HL include a heterogeneous Hodgkin lymphoma is a lymph node based neoplasm com- mixture of small lymphocytes, histiocytes, eosinophils, and posed of a small number of large mononucleated or mul- plasma cells. The tumor cells of classical HL represent acti- tinucleated neoplastic cells residing in a heterogeneous vated post-germinal center B lymphocytes. They express non-neoplastic cell infiltrate. HL is classified into two main CD30 and PAX5 and express CD15 in 75–80% of cases. They diseases, namely, nodular lymphocyte predominant HL may variably express other B cell markers CD20 or CD79a, (NLPHL) and Classical HL (CHL). Separation of these two but strong uniform positivity for these markers is not char- broad categories of lymphoma is important because they acteristic and should suggest an alternative diagnosis. CHL are treated with different combinations of chemotherapeutic cells carry mutated IgV genes, indicating that they represent agents. CHL is further classified into nodular sclerosis (NS), B cell lineage-derived cells, though they do not express the mixed cellularity (MC), lymphocyte-rich (LR), and lympho- transcription factors OCT2 and BOB1 together. They can cyte depletion (LD) subtypes (Table 28-4) based on the cel- also be positive for EBV. lular background and the extent of fibrosis. Nodular Lymphocyte-Predominant Hodgkin Lymphoma (NLPHL) Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is composed of nodular or nodular and diffuse prolifera- tion of large neoplastic cells known as popcorn cells (L&H cells) residing in a meshwork of follicular dendritic cells and many small B cells. The popcorn cells characteris- tically have a large multilobated nucleus with delicate nuclear membranes, finely granular chromatin, and small indistinct nucleoli (Figure 28-14). Unlike the neoplastic cells of classical HL, those of NLPHL have a B cell phe- Figure 28.14 Nodular lymphocyte predominant Hodgkin lymphoma. Large L&H (“popcorn”) cell with multilobated nucleus, notype—CD45+ , CD19+ , CD20+ , CD79a+ , and PAX5+— delicate nuclear membranes, and small basophilic nucleoli. The and they express the B cell transcription factors OCT2 and background contains many histiocytes with pale-staining eosinophilic BOB1. By definition, they lack staining for CD30 and CD15. cytoplasm and small lymphocytes (H&E stain, 200* magnification). Mature Lymphoid Neoplasms 641 A diagnosis of mixed cellularity HL is made after exclud- Checkpoint 28.4 ing the other classic subtypes: NS, LR, and LD. However, Name and describe the cell that is characteristic of Hodgkin the histologic subtypes of classical HL do not have inde- lymphoma. pendent prognostic significance. The stage of HL is one of the most important prognostic markers. The 5-year survival for patients with Stage I or IIA Hodgkin lym- The nodular sclerosis subtype of CHL is character- phoma currently is approximately 90% and for Stage ized by C-shaped bands of birefringent fibrous tissue IV is 60–70%. Treatment options depend on the stage of (Figure 28-15b), nodular aggregates of cells, and tumor disease and can include chemotherapy with or without cell variants with cytoplasmic clearing and delicate, radiation.1 multilobated nuclei (lacunar cells; Figure 28-15c). The lymphocyte-rich form of CHL has a background of reactive cells including many small lymphocytes, but in contrast to Checkpoint 28.5 NLPHL, the neoplastic cells have the phenotype of classi- What clinical finding differentiates multiple myeloma from other cal HL. Lymphocyte-depleted HL is a rare subtype that has plasma cell neoplasms? many tumor cells and only rare reactive lymphocytes. a b c Figure 28.15 Classical Hodgkin lymphoma, nodular sclerosis. (a) Reed-Sternberg cell with two nuclear lobes and prominent, eosinophilic nucleoli giving an owl’s eye appearance. The background contains many small lymphocytes and a few pale eosinophilic histiocytes (lymph node biopsy, H&E stain, 200* magnification). (b) Bands of fibrous tissue isolate nodules containing an abnormal cellular infiltrate (nodular sclerosing pattern) (lymph node biopsy, H&E stain, 20* magnification). (c) Lacunar cells with abundant clear-staining cytoplasm, delicate nuclear membranes, and small basophilic nucleoli (lymph node biopsy, H&E stain, 100* magnification). 642 Chapter 28 Summary The mature lymphoid neoplasms represent a diverse group usually requires a combination of conventional morphol- of neoplasms that vary in clinical presentation, morpho- ogy, immunophenotyping, molecular analysis, and cyto- logic appearance, immunophenotype, and genotype. The genetics. HL differs from other lymphomas clinically and WHO classifies these neoplasms into mature B cell and histologically; it is composed of large tumor cells that do mature T/NK cell neoplasms. As in the precursor B cell not resemble a normal cell counterpart and are accom- and T cell neoplasms, the leukemic entities are character- panied by many reactive cells. HL has several histologic ized by peripheral blood and bone marrow involvement subtypes that differ in phenotype, appearance of the large whereas the lymphomas have tumorous masses involving neoplastic cells, and nature of the reactive component. the lymph nodes and other lymphoid tissue. The malignant Although the classification of lymphoid malignancies is cells often resemble one or more stages of normal lympho- complicated, it is important to separate distinct disease cyte development and, therefore, must be distinguished entities that have a predictable outcome and response to from reactive proliferations of these cells. The diagnosis specific therapy. Review Questions Level I 4. A patient with absolute lymphocytosis of 4.0

* 103/mcL was found to have a monotypic B cell 1. Which of the following is the most likely distribution population by flow cytometry. The i munophenotype of disease in a patient with leukemia? was consistent with CLL/SLL. This indicates: (Objective 3) (Objective 9) a. Lytic bone lesion a. CLL/SLL b. Tumorous mass involving lymph nodes b. monoclonal B lymphocytosis (MBL) c. Tumorous mass involving the tonsil c. Richter’s transformation d. Widespread involvement of the bone marrow d. prolymphocytic transformation of CLL 2. A 60-year-old male undergoing a routine CBC 5. You are asked to perform a peripheral blood smear was found to have lymphocytosis. The peripheral review on a 56-year-old male with a history of lytic smear revealed a uniform population of small bone lesions, hypercalcemia, and a recent diagnosis mature l ymphocytes. Which of the following is of plasmacytoma. Which of the following is the most helpful in differentiating a benign from a malignant likely peripheral blood finding? (Objective 10) lymphocytosis? (Objectives 8, 9) a. Plasmacytosis a. Cytochemistry b. Lymphocytosis b. Immunophenotyping c. Agglutination c. Morphology d. Rouleaux d. Absolute lymphocyte count 6. A bone marrow biopsy was performed on a patient 3. Diagnosis of a lymphoid neoplasm is supported by with bone pain. The bone marrow differential which of the following findings? (Objective 2) revealed 40% plasma cells. Serum electrophoresis a. Clonal immunoglobulin gene rearrangement showed an increased protein with an M spike. What b. Population of lymphocytes expressing only one would you expect to find on the peripheral blood immunoglobulin light chain smear in this patient? (Objectives 3, 10) c. Population of lymphocytes expressing an a bnormal a. Rouleaux phenotype b. Spherocytes d. All of the above c. Plasma cells d. More than 20% blasts Mature Lymphoid Neoplasms 643 7. The WHO classification of lymphoid neoplasms rec- 3. A lymph node biopsy is performed on a patient with ommends which of the following studies to identify lymphadenopathy. Which of the following findings neoplastic cells and type of lymphoid malignancy? is(are) characteristic of a reactive proliferation rather (Objective 9) than malignant lymphoma? (Objective 9) a. Genotype a. A mixed population of cells varying in size, shape, b. Morphology and color c. Phenotype b. Clonality of cells d. All of the above c. Presence of large, bizarre cells d. Many cells with mitotic activity 8. The Reed-Sternberg cell is found in what type of lym- phoid malignancy? (Objective 6) 4. A CBC performed on a 65-year-old female reveals a. Chronic lymphocytic leukemia lymphocytosis. A bone marrow reveals replacement of the hematopoietic precursors by an infiltrate b. Multiple myeloma of mature lymphocytes. Which of the following c. HL is the most likely course of the disease? d. NHL (Objective 5) 9. A peripheral smear performed on a 35-year-old male a. Cure following therapy with lymphocytosis reveals numerous smudge cells. b. Rapid progression Which of the following does this finding indicate? c. Slow progression (Objectives 3, 8) d. Spontaneous remission a. Chronic lymphocytic leukemia b. Infectious mononucleosis 5. Which of the following phenotypes is characteristic of chronic lymphocytic leukemia? (Objective 1) c. Lymphoblastic leukemia d. None because the finding is not diagnostic a. CD19+ , CD5+ , CD23+ b. CD19+ , CD5- , CD23+ 10. Peripheral smear examination of a patient with c. CD19+ , CD5+ , CD23- chronic lymphocytic leukemia reveals 8% prolympho- cytes. This finding is consistent with which of the fol- d. CD19+ , CD5- , CD23- lowing diagnoses? (Objective 8) 6. A CBC performed on an 80-year-old female reveals a. Chronic lymphocytic leukemia an absolute lymphocyte count of 8 * 103/mcL. b. Prolymphocytic leukemia Examination of the peripheral blood smear reveals a uniform population of small lymphocytes. Which of c. Prolymphocytoid transformation of CLL the following procedures is most likely to establish a d. Richter’s transformation diagnosis? (Objectives 1, 5) Level II a. Cytogenetics 1. Which of the following is(are) involved in the patho- b. Flow cytometry immunophenotyping physiology of low-grade follicular lymphoma? c. Immunoglobulin gene rearrangement (Objective 8) d. Lymph node biopsy a. Genes involved in apoptosis 7. A patient presents with pancytopenia and massive b. Inherited immunodeficiency splenomegaly but no lymphadenopathy. Unusual c. Proto-oncogenes cells noted on the blood smear had abundant d. Tumor suppressor genes pale-staining cytoplasm and cytoplasmic projections. The nuclei were oval with a fine chromatin pattern. 2. Which type of lymphoma can antimicrobial therapy Which of the following test results would be helpful cure? (Objective 8) to establish the diagnosis? (Objective 5) a. Burkitt lymphoma a. Clonal T cell gene rearrangement b. Hodgkin lymphoma b. t(11;14) IGH/CCND1 by FISH c. MALT lymphoma c. BRAF V600E mutation d. HIV-associated non-Hodgkin lymphoma d. POSITIVE myeloperoxidase cytochemical staining 644 Chapter 28 8. A CBC performed on a 60-year-old male with a his- b. The patient has a new lymphoid neoplasm: chronic tory of rheumatoid arthritis revealed neutropenia lymphocytic leukemia. and an absolute lymphocytosis. Examination of the c. The lymphoma has transformed. peripheral smear revealed many lymphocytes with d. The patient has developed a therapy-related abundant pale-staining cytoplasm and cytoplasmic neoplasm. granules. Which of the following is the most likely phenotype? (Objectives 1, 5) 10. A lymph node biopsy revealed an infiltrate of small a. CD3+ , CD2+ , CD57+ lymphoid cells with a vaguely nodular growth pat- tern. Flow cytometry revealed a monoclonal popula- b. CD19+ , CD5+ , CD57- tion of B cells expressing CD5. Immunohistochemistry c. CD2+ , CD3- , CD56+ revealed nuclear staining for cyclin-D1 protein. Which d. CD19+ , CD10+ , CD34+ of the following translocations is associated with these neoplastic cells? 9. A patient previously diagnosed with SLL is found to (Objective 3) have peripheral blood lymphocytosis. The lympho- cytes are small and have round nuclei with clumped a. t(11;14) chromatin. Which of the following is the most appro- b. t(15;17) priate interpretation? (Objective 3) c. t(1;14) a. This is a different presentation (CLL) of the same d. t(8;14) follicular lymphoma disease process. References 1. Swerdlow, S. H., Campo, E., Pileri S. A., Harris, N. L., Stein, H., Waldenstrom’s macroglobulinemia. New England Journal of Siebert, R., . . . , Zelenetz, A.D. (2016). The 2016 revision of the Medicine, 367, 826–833. World Health Organization classification of lymphoid neoplasms. 8. Hans, C. P., Weisenburger, D. D., Greiner, T. C., Gascoyne, R. D., Blood, 127, 2375–2390. Delabie, J., Ott, G., . . . Chan, W.C. (2004). Confirmation of the 2. Tandon, B., Peterson, L., Gao, J., Nelson, B., Ma, S., Rosen, S., & molecular classification of diffuse large B-cell lymphoma by Chen, Y.H. (2011). Nuclear overexpression of lymphoid-enhancer- immunohistochemistry using a tissue microarray. Blood, 103, binding factor1 identifies chronic lymphocytic leukemia/small 275–282. lymphocytic lymphoma in small B-cell lymphomas. Modern 9. Swerdlow, S. H. (2014). Diagnosis of “double-hit” diffuse large Pathology, 24, 1433–1443. B-cell lymphoma and B-cell lymphoma, unclassifiable, with 3. Hallek, M. (2013). Chronic lymphocytic leukemia: 2013 update features intermediate between DLBCL and Burkitt lymphoma: on diagnosis, risk stratification and treatment. American Journal of When and how, FISH versus IHC. Hematology American Society of Hematology, 88, 803–816. Hematology Educational Program, 2014, 90–99. 4. Falini, B., Martelli, M. P., & Tiacci, E. (2016). BRAF-V600E 10. Kabube, K., & Campo, E. (2015). MYC alterations in diffuse large mutation in hairy cell leukemia: from bench to bedside. Blood. B-cell lymphomas. Seminars of Hematology, 52, 97–106. doi: 10.1182/blood-2016-07-418434 11. Said, J. (2015). The expanding spectrum of EBV+ lymphomas. 5. Blombery, P. A., Wong, S. Q., & Hewitt, C. A. (2012). Detection of Blood, 126, 827–828. BRAF mutations in patients with hairy cell leukemia and related 12. Chesi, M., & Bergsagel, P. L. (2013). Molecular pathogenesis lymphoproliferative disorders. Haematologica, 97, 780–783. of multiple myeloma: Basic and clinical updates. International 6. Mozos, A., Royo, C., Harmann, E., De Jong, D., Batro, C., Valera, Journal of Hematology, 97, 313–323. A., . . . Chuang, S. (2009). SOX11 expression is highly specific 13. Younes, A., Bartlett, N. L., Leonard, J. P., Kennedy, D. A., for mantle cell lymphoma and identifies the cyclin D1-negative Lynch, C. M., Sievers, E. L., & Forero-Torres, A. (2010). subtype. Haematologica, 94, 1555–1562. Brentuximab vedotin (SGN-35) for relapsed CD30-positive 7. Treon, S. P., Xu, L., Yang, G., Zhou, Y., Liu, X., Cao, Y., . . . lymphomas. New England Journal of Medicine, 363, 1812–1821. Tripsas, C. (2012). MYD88 L265P somatic mutation in Chapter 29 Hematopoietic Stem Cell Transplantation Aamir Ehsan, MD Andrea Yunes, MD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Describe the sources of hematopoietic stem 5. Identify the infections that are serious cells and characteristics of each source. complications during the peritransplant 2. Identify the diseases that can be treated with period. different sources of hematopoietic stem cells. 6. Describe the significance and effects of graft- 3. List characteristics of stem cells. versus-host disease (GVHD), and compare them to those of the graft-versus-leukemia 4. Describe the significance of ABO and HLA (GVL) process. antigens for stem cell transplant. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Summarize the collection and processing of 5. Formulate the sequence of events for hematopoietic stem cells, and assess the suc- a patient who will receive a stem cell cess of these procedures. transplant. 2. Explain the role of the clinical laboratory 6. Select laboratory tests used to determine professional in stem cell transplantation. engraftment and assess engraftment given 3. Select and outline methods to enumerate the results obtained by these tests. hematopoietic stem cells. 7. Differentiate between the types of stem cell 4. Explain the complications of stem cell transplantation (SCT), and select the most transplant, and assess the patient’s risk of appropriate type for the patient’s clinical developing them. condition. 645 646 Chapter 29 Chapter Outline Objectives—Level I and Level II 645 Collection Target for Stem Cells 653 Key Terms 646 Hematopoietic Engraftment 654 Background Basics 646 Role of the Clinical Laboratory Professional in Stem Case Study 647 Cell Transplantation 654 Overview 647 Graft-versus-Host Disease and Graft-versus-Leukemia Introduction 647 Effect 655 Origin and Differentiation of Hematopoietic Stem Complications Associated with Stem Cell Cells 647 Transplantation 656 Sources of Hematopoietic Stem Cells and Types of Increased Availability and Success of Stem Cell Stem Cell Transplants 648 Transplantation 657 Collection and Processing of Hematopoietic Stem Summary 658 Cells 650 Review Questions 658 Quantitation of Hematopoietic Stem Cells 652 References 660 Key Terms Allogeneic Cryopreserved Polymorphic Apheresis Engraftment Progenitor cells Autologous Graft-versus-host disease (GVHD) Purging Chimerism Graft-versus-leukemia (GVL) Syngeneic Clonogenic Passenger lymphocyte syndrome Conditioning regimen (PLS) Background Basics This chapter builds on concepts learned in previous Level II chapters. To maximize your learning experience, you • Describe the role of cytokines and bone marrow should review the following material before starting this microenvironment in maturation and differentiation unit of study: of hematopoietic stem cells; explain the role of onco- genes in cancer development. (Chapters 2, 4, 23) Level I • Explain the use of molecular genetic technology and • Describe the origin and differentiation of hematopoi- cytogenetics in diagnosis and prognosis of neoplastic etic cells. (Chapters 2, 3, 4) hematopoietic disorders. (Chapters 41, 42) • Outline the classification and explain the etiology • Correlate subgroups of neoplastic hematologic and pathophysiology of neoplastic hematologic disorders with laboratory findings and prognosis. disorders. (Chapter 23) (Chapter 23) • Explain the role of chemotherapy and radiotherapy in treatment of neoplastic hematologic disorders. (Chapters 23–28) Hematopoietic Stem Cell Transplantation 647 CASE STUDY Introduction We refer to this case study throughout the chapter. Hematopoietic stem cell transplantation (SCT) is a recog- Brandon, a 35-year-old male (weight 80 kg), was nized therapeutic modality for leukemias, lymphomas, recently diagnosed with acute myeloid leukemia solid organ tumors, and a variety of metabolic and immu- (AML) and received induction chemotherapy. Day nologic disorders (Table 29-1). The concept of SCT came 21 bone marrow revealed no evidence of residual from experiments done over six decades ago when it was leukemia. Two weeks later, circulating blasts observed that mice given intravenous marrow infusions could overcome lethal doses of radiation.1,2 were seen in the peripheral blood. Brandon was Later, in the evaluated for a hematopoietic stem cell transplant 1960s, the first successful bone marrow transplant was per- (SCT). formed on a leukemia patient using marrow donated by the Consider the laboratory’s role in evaluating the leukemic patient’s brother. transplant, collecting