Neonatal anemia: Revisiting the enigmatic pyknocyte
Authors: Michele L. Nassin, Jo‐Anne Vergilio, Matthew M. Heeney, James L. LaBelle
Journal: American Journal of Hematology (2017)

A 27-day-old, former 31-week, fraternal twin male infant in the neonatal intensive care unit (NICU) was born to a 34-year old gravida 2 para 0 female via cesarean section following spontaneous premature rupture of amniotic membranes. The amniotic fluid was clear and showed no signs of infection or meconium. The mother was blood type O, Rh positive. The patient's Apgar scores at 1 and 5 minutes were 9 and 9 respectively. Physical examination and medical course were unremarkable. Blood cultures were negative and empiric antibiotics were discontinued after 48 hours. On day of life (DOL) 25 the patient's hematocrit (Hct) was 25.5% with an elevated reticulocyte percent (6.4%), unconjugated bilirubin 13.5 mg/dL, and conjugated bilirubin 0.6 mg/dL. Pediatric hematology was consulted on DOL 27 for worsening anemia with increasing reticulocytosis and unconjugated hyperbilirubinemia despite aggressive phototherapy. There was no hematuria, petechiae, ecchymosis, abdominal distention, fever, hematochezia, or cardiorespiratory instability. The patient had not received a red cell transfusion. Parents were nonconsanguineous, Greek Americans without personal or family history of hematologic disorders including cholelithiasis or splenectomy. The patient appeared pale and jaundiced, but in no distress. His exam showed no organomegaly and was overall normal. Laboratory results revealed total bilirubin 9.2 mg/dL, direct bilirubin 0.5 mg/dL, hematocrit 24.8% and reticulocyte count 18.9%. Urinalysis showed 1+ blood without formal microscopic analysis. Newborn screen results demonstrated hemoglobin (Hgb) FA by isoelectric focusing. Full longitudinal laboratory values are depicted in Table 1. Neonatal hyperbilirubinemia can be a common and benign problem in full term neonates.1 Hyperbilirubinemia during the first week of life is more common in premature infants, occurring in up to 80% versus 60% of term infants.2 However, hyperbilirubinemia in the first 24 hours of life is almost always pathologic.3 Bilirubin is the product of heme catabolism in the reticuloendothelial system and is cleared via conjugation in the liver followed by secretion in the bile. Transient physiologic hyperbilirubinemia in neonates is commonly the result of increased red blood cell turnover in combination with transient deficiency in uridine diphosphosphate glucuronosyltransferase (UGT)1A1, the enzyme responsible for hepatic bilirubin conjugation.4 Additionally, neonates have increased enterohepatic circulation compared to children and adults because their gastrointestinal tracts lack bacteria necessary for conversion of bilirubin conjugates into urobilinoids, which are not reabsorbed by the intestine. Therefore, neonates not only have a higher bilirubin burden and decreased ability for conjugation, but they are also more prone to increased enteric reabsorption.5 Physiologic hyperbilirubinemia can be exacerbated with breastfeeding if the neonate is mildly dehydrated until milk production increases. As a result, infants receive fewer calories, a further stimulus for increased enterohepatic bilirubin circulation.4 The most common etiology of acute hyperbilirubinemia in a premature infant is infection. In our case, there were no obvious risk factors, signs, or symptoms of infection in the patient, the dizygotic twin, or the mother. Therefore, other etiologies were considered. Hyperbilirubinemia can be divided into two categories that can aid in determining cause(s): conjugated (direct) or unconjugated (indirect). Conjugated hyperbilirubinemia usually signifies posthepatic obstruction. Conjugated hyperbilirubinemia is not associated with neurotoxicity because conjugated bilirubin cannot cross the blood brain barrier. However, conjugated hyperbilirubinemia can still indicate potentially serious pathologies such as TORCH infections (toxoplasmosis, syphilis, rubella, cytomegalovirus, herpes), biliary atresia, giant cell hepatitis, choledochal cyst, galactosemia, or alpha1-antitrypsin deficiency.2 Hereditary disorders associated with conjugated hyperbilirubinemia, like Dubin-Johnson and Rotor's Syndromes, cause conjugated hyperbilirubinemia secondary to aberrant hepatic bilirubin metabolism.5 While these are important considerations, our patient demonstrated a predominantly unconjugated hyperbilirubinemia. Unconjugated (indirect) hyperbilirubinemia is classically defined when less than 20% of the total bilirubin is conjugated. This occurs when there is an accumulation of unconjugated bilirubin related to increased production (e.g., increased hemolysis) or decreased hepatic conjugation. The differential diagnosis for pathologic unconjugated hyperbilirubinemia in premature neonates is extensive and includes hemolytic anemia, sepsis, hypothyroidism, and pathologic deficiency or defect in UGT1A1 (Gilbert Syndrome and Crigler-Najjar Syndrome).5 The first clinical manifestation of neonatal hyperbilirubinemia is usually jaundice resulting from the accumulation of unconjugated, nonpolar, lipid-soluble bilirubin in the skin. In a subset of infants, acute bilirubin encephalopathy may develop that can progress to kernicterus, a potentially devastating chronic form of cerebral palsy.6 Urgent treatment of hyperbilirubinemia in premature infants should be performed as the neurotoxic threshold of unconjugated bilirubin is significantly lower compared to that of term infants. To minimize neurotoxicity, the American Board of Pediatrics has established a nomogram to guide treatment based on serum unconjugated bilirubin levels, gestational age, hours since birth, and other risk factors.7 The treatment of choice is phototherapy which uses light energy to convert unconjugated bilirubin deposited in the skin to a more water-soluble form that is more easily excreted in the bile and urine.8 Phenobarbital can also be used to help treat hyperbilirubinemia in neonates by enhancing UGT activity.9 However, phenobarbital was not used in our patient because it has been reported to cause respiratory insufficiency in premature infants.9 This patient's elevated bilirubin, improved under phototherapy, decreasing from 6.3 mg/dL to 4.2 mg/dL. Following phototherapy cessation, hyperbilirubinemia returned, requiring re-initiation. This persistent hyperbilirubinemia made physiologic jaundice less likely and necessitated further hematologic evaluation.2 (Table 1). Initial evaluation of hematologic etiologies of unconjugated hyperbilirubinemia, included blood typing, complete blood count, direct antiglobin test (DAT) and antibody screen, reticulocyte count, and review of a peripheral blood smear. Blood typing is helpful to rule out fetal-maternal blood group incompatibility leading to isoimmunization, the most common cause of severe early neonatal hyperbilirubinemia. Immunologic and nonimmunologic forms of hemolytic anemia are distinguished by the DAT. Peripheral blood smear analysis is also critical during evaluation for membranopathies and microangiopathic processes. The patient's blood was type O, Rh positive. DAT and antibody screen tests were negative. The blood smear showed platelets of normal size, number, and granularity. Many erythroblasts were present. RBC morphology revealed marked anisocytosis and polychromasia. Many helmet-shaped erythrocytes, “bite cells,” and RBC fragments were also present. “Bite cells” accounted for approximately 20% of the total RBCs (Figure 1). (A) Peripheral blood smear from the patient containing numerous distorted, irregularly shaped, and spiculated erythrocytes (pyknocytes) comprising approximately 20% of erythrocytes. Also present are a circulating normoblast, myelocyte, characteristic schistocytes (black arrow) and ‘bite cells’ (green arrow). (B) Additional peripheral blood smear containing numerous pyknocytes comprising approximately 45% of the erythrocytes, blister cells (blue arrow), and irregularly contracted/condensed cells (black arrow). [Modified Wright-Giemsa, 100×] [Color figure can be viewed at wileyonlinelibrary.com] The differential diagnosis for hemolytic anemia in the neonatal period is broad and includes both intrinsic and extrinsic pathologies. Evaluation includes comparing blood types of the mother and child and hemoglobin electrophoresis. In our patient, his blood type was matched with his mother and his DAT/antibody screen was negative suggesting a nonimmune mediated pathophysiology. Although the infant's peripheral blood smear was consistent with intravascular hemolysis, serum lactate dehydrogenase and hemoglobinuria were not evaluated. Low haptoglobin and hemopexin levels can be seen in intravascular hemolysis, caused by the binding of free hemoglobin and heme respectively, which is then scavenged by the reticuloendothelial system and hepatocytes. However, in the neonatal period haptoglobin and hemopexin levels can be low at baseline and therefore offer little clinical value.1, 10 Residual free hemoglobin in the plasma undergoes a series of enzymatic reactions within the gut and liver culminating in the production of bilirubin.11 RBCs are phagocytosed by the reticuloendothelial system during extravascular hemolysis, resulting in RBC fragment consumption that leads to anemia and jaundice and a paucity of hemoglobinuria and red cell fragments on the blood smear. Examination of the blood smear can be diagnostic for erythrocyte membrane defects. Hereditary spherocytosis (HS) is the most common membrane defect leading to neonatal hemolytic anemia and is often diagnosed via the presence of spherocytes on the peripheral blood smear. A diagnosis of HS is supported by osmotic fragility (OF) testing and family history, however de novo mutations can arise leading to disease without positive family history. Additionally, OF testing can be inaccurate during the neonatal period because fetal red blood cells are more osmotically resistant than those of adults and up to 33% of neonates with HS do not have significant numbers of spherocytes on their peripheral blood smears.12 Eosin 5-maleimide (EMA) binding testing, although not performed on this patient, could also be done to rule out HS as it has higher sensitivity that OF and genetic testing for HS-associated mutations.13, 14 Other red cell membrane disorders include elliptocytosis, pyropoikilocytosis, and infantile pyknocytosis. A blood smear consistent with intravascular hemolysis can also suggest an intrinsic red cell enzymopathy or unstable hemoglobinopathy. Despite a normal newborn screen, a Heinz body preparation was sent to rule out existence of denaturation of hemoglobin.5 Inherited red cell enzymopathies must also be ruled out given the infant's continued hemolysis. Neonatal red blood cells are more susceptible to oxidative stress than those from older patients. Erythrocyte oxidative stress is regulated by the pentose phosphate pathway. The most common defect in this pathway is glucose-6-phosphate dehydrogenase (G6PD) deficiency.5 In the setting of reticulocytosis, G6PD can be falsely normalized/elevated leading to false negative results. Pyruvate kinase (PK) deficiency is the most common enzymopathy within the glycolytic pathway and its presentation ranges from isolated unconjugated hyperbilirubinemia to profound anemia.15 Significantly rarer causes of congenital hemolytic anemia are glucose phosphate isomerase deficiency and hexokinase deficiency. Both may present with jaundice, reticulocytosis, and splenomegaly, and often require transfusional support.5 A peripheral blood smear revealing schistocytes and thrombocytopenia generally suggests microangiopathic hemolytic anemia. The most common cause of microangiopathic hemolytic anemia is disseminated intravascular coagulation (DIC) where microvascular thromboses consume platelets and coagulation factors and RBC membranes are sheared by fibrin strands.16 Given that this infant remained well and had a normal coagulation profile, DIC was unlikely. Lastly, given the underlying hemolysis, congenital disorders of bilirubin conjugation were unlikely to be causative. G6PD and PK activity testing returned normal. Heinz body preparation returned +1 (negative in the setting of brisk hemolysis). Incubated OF testing was negative. A full red cell enzyme activity panel was sent but results were not readily available. On DOL 29 the patient's hematocrit dropped to 22.6% (Hgb 7.5 g/dL) with a mean corpuscular volume (MCV) of 110fL and reticulocytosis increased to 33% (absolute reticulocyte count 110 × 103/µL). Given the continued decline in hematocrit despite reticulocytosis he was transfused with red blood cells followed by improvement in hyperbilirubinemia and hemoglobin stabilization. The patient was discharged on DOL 33 with a total bilirubin of 5.5 mg/dL and hematocrit of 39.9%. He was prescribed ferrous sulfate and a multivitamin for breast-fed infants. The results of the red cell enzyme activity panel later returned normal. Given the extensive negative enzymatic workup, normal newborn screen, and negative Heinz body preparation, we returned to the initial blood smear. Based upon the negative laboratory evaluation and the presence of pyknocytes, the infant was diagnosed with infantile pyknocytosis (IP). Follow-up at 2 months of age found that the infant's posthospitalization course was unremarkable. His hematocrit was 27.1% (Hgb 9.8 g/dL), MCV 85.5fL, and reticulocyte count 4.5%. Total bilirubin was 1.6 mg/dL with direct bilirubin of 0.2 mg/dL. Repeat G6PD enzyme testing, in the setting of a more normal reticulocyte count, was negative. The peripheral blood smear now revealed normal size and number of platelets, lymphocytes, and red blood cells without presence of red blood cell fragments or pyknocytes (Figure 2). The patient had transient anemia with mild reticulocytosis attributed to the physiologic red cell nadir of infancy. Peripheral blood smear from the patient at two months of life demonstrating normal erythrocyte morphology and no evidence of pyknocytes or hemolysis. [Modified Wright-Giemsa, 100×] [Color figure can be viewed at wileyonlinelibrary.com] Diagnostic algorithm for neonate with hyperbilirubinemia with a focus on hemolytic anemia. The differential diagnosis of hemolytic anemia can be broken into either extrinsic or intrinsic etiologies. Legend: DAT, direct antiglobin test; DIC, disseminated intravascular coagulation; EMA, eosin-5-maleimide; G6PD, glucose-6-phosphate dehydrogenase; INR, international normalized ratio; PK, pyruvate kinase; PTT, partial thromboplastin time; RBCs, red blood cells; UGT1A1, uridine diphosphate glucuronosyltransferase (UGT)1A1 All newborns experience a physiologic hemoglobin level nadir between one and two months of age due to the transition from a relatively hypoxic state in utero to a relatively hyperoxic state with increased tissue oxygenation after birth.5 This leads to a decline in erythropoietin (EPO) concentration that, in combination with the relatively shorter half-life of neonatal erythrocytes, results in a relative anemia. The infant's persistent hyperbilirubinemia was likely due to breast milk jaundice in combination with mild nonimmune mediated hemolysis. Follow-up at 4 months of age revealed a normal CBC and normal bilirubin. The infant continued to grow and develop normally without any subsequent hemolytic episodes. Infantile pyknocytosis was first reported by Tuffy in 1959 in a case series of 11 patients evaluated for hemolytic anemia and was described as, “significant increase in the number of burr cells and associated with an obvious hemolytic anemia.”17 The term “pyknocyte” was included as a red blood cell morphology previously described under a variety of names such as “burr cells,” “irregularly distorted and contracted erythrocytes,” or “poikilocytes characterized by the presence of one or several spiny projections.”18 Although liver and renal diseases predispose to pyknocyte formation, these patients have neither.17 All other evaluations of hemolysis during the work up of these patients are universally negative (Figure 3). This point is critically important as some reports suggest that as many as half of patients diagnosed with G6PD present with pyknocytosis.19 The presence of pyknocytes in the peripheral blood has also been observed in patients who were later diagnosed with hereditary elliptocytosis.20 The actual incidence of IP remains unclear. However, a retrospective review of 149 blood samples diagnosed IP in 9.4% of patients referred for unexplained neonatal hemolytic anemia.21 These patients developed peak bilirubin between 3-4 weeks of life and nearly all symptoms resolved by 4-6 months of age.21 Although periodically reported since Tuffy's initial characterization, the pathophysiology of IP remains unexplained. The brisk hemolysis associated with the disorder is thought to be caused by perinatal extracorpuscular factors. Some believe that alterations in the microenvironment including pH, electrolytes, adenosine triphosphate (ATP) availability, and exposure to viruses and drugs modify the erythrocyte stroma and/or membrane during the neonatal period.22 This theory is supported by a study of Mexican-American infants with persistent or recurrent pyknocytosis following exchange transfusions despite identifying the pyknotic RBCs as donor-derived.23 It has also been shown that normal red blood cells transform into pyknocytes when incubated with plasma from patients with IP.24 Others have theorized that there is a familial predisposition to the development of IP because the process has been observed in multiple siblings. In addition to clustering within families, a majority of mothers with multiple children diagnosed with IP had prior numerous miscarriages.22, 25 Therefore, many continue to suspect that a familial pre-disposition NOT predis-position is responsible for IP and that the transient disease develops following exposure to environmental triggers.25 While the etiology of IP not well understood, its pattern of hemolytic anemia in the perinatal period is uniformly described and it has never been known to occur after the first month of life. Other cases of neonatal anemia, such as hereditary spherocytosis, are associated with elevated free 2,3 diphosphoglycerate (2,3 DPG). Because it does not readily bind fetal hemoglobin. 2,3 DPG elevation can destabilize the red cell membrane and contribute to increased red cell turnover.14 This mechanism could be contributing to the pathophysiology of IP. Given its self-limited nature, supportive care is necessary following a diagnosis of IP. We recommend weekly evaluations with physical examinations and laboratory analysis of hemoglobin, bilirubin, and absolute reticulocyte count. We recommend pRBC transfusion if hemoglobin falls below 7 g/dL or higher if the infant becomes symptomatic. Further, we recommend consideration of EPO if the infant cannot maintain his hemoglobin without transfusional support. EPO has been shown to augment increases in reticulocyte count and hemoglobin, decreasing the need for transfusion in these patients.26