bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet Transfusions in a Medical- Surgical Intensive Care Unit.
Authors: Arnold Donald M. , Molnar Laura A. , Crowther Mark A. , Sigouin Christopher , Carruthers Julie , Heddle Nancy M. , Cook Deborah J.
Published: October 2019

Background: The benefits and harms of platelet transfusions in critically ill patients are unclear.

Objectives: To describe the frequency of, indications for, and effects of platelet transfusions in patients admitted to a medical- surgical intensive care unit (ICU).

Design: Single center cohort study (January 2001 to January 2002).

Methods: We identified all patients who developed thrombocytopenia (platelet count <150 x 109/l) during their ICU admission from a prospective study which enrolled consecutive adults admitted to ICU with an expected length of stay ≥72 hours, and which excluded patients with trauma, orthopedic surgery, cardiac surgery, pregnancy, or receiving palliative care. Retrospectively, using a priori criteria, bleeding severity and the indications for platelet transfusions were assessed; 23.7% of bleeding events and 89.5% of transfusion indications were reviewed in duplicate independently. Initial agreement was calculated using Cohen’s unweighted kappa and all assessments of bleeding severity and transfusion indications were adjudicated by third person.

Results: Of 261 ICU patients, 118 (45.2%) had thrombocytopenia. Initial agreement between primary reviewers for assessments of bleeding severity was good (k= 0.69), and for indications for platelet transfusions was poor (k=0.35); consensus was achieved in all cases. One third of thrombocytopenic patients had major bleeding (37/118, 31.4%), and one fifth had minor bleeding (24/118, 20.3%). Among thrombocytopenic patients, 27/118 (22.9%) received a total of 76 platelet transfusions, 24 (31.6%) of which were administered to treat bleeding, and 52 (68.4%) of which were to prevent bleeding. Of the prophylactic platelet transfusions, 18/52 (34.6%) preceded invasive procedures. The mean ± SD platelet count prior to therapeutic platelet transfusions was 54 ± 40 x109/l; for peri-procedural transfusions, 55 ± 38 x109/l; and for other prophylactic platelet transfusions, 37 ± 21 x109/l. Most transfusions (69/76, 91%) were administered as pools of 5.2 ± 1.2 random donor platelets, and few (7/76, 9.2%) were apheresis platelets. A single platelet transfusion (pool of 5 random donor units or 1 apheresis unit) resulted in a platelet increment of 14 ± 29 x109/l at 6.6 ± 5.9 hours following transfusion. No rise in platelet count was observed following 17 transfusions given to 13 patients.

Conclusions: Platelet transfusions are frequently administered to thrombocytopenic critically ill patients, and, although the indication is not always clear, the most common reason is to prevent bleeding. Nearly half of transfused ICU patients were refractory to one or more platelet transfusion. Further prospective studies are needed on the indications for, and effects of, platelet transfusions in the ICU setting.
Treahlose Loaded, Freeze-Dried Rehydrated Human Platelets Maintain and Regulate Their Intracellular pH.
Authors: Tablin Fern , Tang Minke , Walker Naomi J. , Crowe John H.
Published: October 2019

Maintenance of intracellular pH, a critical cellular function, is required for generation of proton gradients and platelet response to agonist. Previously, we have demonstrated that freeze-dried rehydrated platelets are able to respond to agonists generate a rise in intracellular calcium (Auh et al. 2004 Calcium mobilization in freeze-dried platelets. Cell Preservation Technology in press) and maintain normal membrane and protein secondary structure (Wolkers et al. 2002 Towards a clinical application of freeze-dried platelets. Cell Preservation Technology 1:175–188). As part of our ongoing studies of trehalose loaded freeze-dried rehydrated human platelets we examined their ability to maintain their intracellular pH. Platelet proton regulation is achieved through the Na-H exchanger (NHE1) which is dependent on the concentration of extracellular sodium. Freeze-dried and fresh control human platelets were loaded with the pH ratio dye bis-carboxyfluorescein acetomethyl ester (BCECF-AM), washed over a Sepharose 2B column and examined by fluorescence spectroscopy. Fresh and freeze-dried rehydrated human platelets maintained virtually identical resting intracellular pH, 7.273 +/− 0.015, and 7.270 +/− 0.034 respectively. Both cell types responded to increased extracellular sodium by increasing their pH in a virtually identical manner. The addition of 0.5U/ml thrombin (in the presence of 135 mM NaCl) resulted in an initial acidification and subsequent alkalinzation of both fresh and freeze-dried rehydrated cells. Prior to the addition of thrombin both cell populations had an [pH]i of 6.9, while after thrombin stimulation the pH rose to 7.012 for fresh cells and 7.001 for freeze-dried rehydrated cells. Thrombin stimulation in the absence of extracellular sodium resulted in a significant acidification of both cell populations to a final pH of 6.6 for fresh cells and 6.7 for freeze-dried rehydrated cells. Specific inhibition of the NHE1 transporter by 5-(N-methyl-N-isobutyl) amiloride (MIA) completely abolished the response of all cells to increasing concentrations of sodium. In the parallel control experiment both freeze-dried and fresh cells acidified to pH 6.2 and incubated with 135mM NaCl responded by generating a rise in intracellular pH to 7.1. These results demonstrate that freeze-dried rehydrated platelets are able to maintain normal pH homeostasis and respond to agonist in a specific manner. Studies funded by DARPA.
Plasma Cell Folate Receptor Overexpression Differentiates Multiple Myeloma from Monoclonal Gammopathy of Undetermined Significance and Smoldering Myeloma.
Authors: Greipp Patricia , Cascino Greg , Kimlinger Teresa , Rajkumar Vincent , Prendergast Franklyn G. , Fonseca Rafael , Bergsagel P. Leif , Low Philip S. , Greipp Philip R.
Published: October 2019

Background: Folate receptor (FR) is a recently recognized tumor marker that is being investigated for targeted delivery of drugs, imaging of cancer cells, and tumor immunotherapy. The small size of the molecule, simple conjugation chemistry and lack of immunogenicity make folate an ideal candidate for targeted delivery to tumors bearing the FR (Lu Y, Low PS. Folate-mediated delivery of macromolecular anticancer therapeutic agents. Adv Drug Deliv Rev. 2002 Sep 13;54(5):675–93). FR is overexpressed in 90% of ovarian cancers and in other human tumors including endometrial, colorectal, breast, and neuroendocrine carcinomas, it is negatively or only weakly expressed in most normal tissues including some myeloid cells. In view of the above we wished to learn if overexpression of FR by marrow plasma cells (PC) might differentiate MM from MGUS and SMM.

Methods: We obtained marrow biopsy sections from 20 patients each with MM, MGUS, or SMM, and 11 normals and stained them by immunohistochemistry using a FR-beta antibody. Expression was graded 0 (negative), 1 (equivocal), 2 (positive) or 3 (strong positive) based on immunohistochemical staining of marrow PC. We recorded the percent PC, percent positive PC, and the staining of other cell types including myeloid, erythroid, lymphocytes, macrophages, megakaryocytes and control tissues.

Results: PC of patients with MM positively expressed FR compared to MGUS, SMM and normal PC (p<0.0001) (Figure 1 and 2). Of interest, 6 of 20 SMM patients showed positive expression. Maturing myeloid cells were also positive, acting as internal positive controls. Macrophages were often positive; some endothelial cells were positive; early erythroid precursors were rarely positive; megakaryocytes, maturing erythroid precursors and lymphocytes did not express FR. The chromosomal location of the FOLR gene cluster is 11q13. Gene expression arrays of purified PC failed to show evidence of overexpression of FOLR1, FOLR2, or FOLR3. We are currently performing Western blots to examine levels of protein product of purified PC.

Conclusion: Immunohistochemistal studies show differential expression of FR in MM PC compared to absent or equivocal expression in MGUS and normal PC and variable expression in SMM. The data suggest that FR may be used as a diagnostic tumor marker and has implications for development of folate-based tumor imaging, drug delivery, and immunotherapy.

Figure Figure Figure Figure
Symptomatic and Indolent Waldenström’s Macroglobulinemia Display Different Biological and Cytogenetic but Not Immunophenotypic Characteristics.
Authors: Ocio Enrique M. , Mateo Gema , Vidriales Belen , Lopez-Berges Consuelo , Gutierrez Norma C. , Hernandez Jesus M. , Sanchez-Guijo Fermin M. , Moro Maria J. , Hernandez Jose , Fernandez-Calvo Javier , Garcia-Frade Javier , Orfao Alberto , Miguel J.F. San
Published: October 2019

Waldenström’s Macroglobulinemia (WM) patients are classified in two main subtypes: symptomatic and asymptomatic. In this study we have analyzed the clinical, biological, immunophenotypical and cytogenetic characteristics of 85 WM, including 59 symptomatic WM (SWM) and 26 indolent WM (IWM).

Clinical behaviour of SWM, as expected, was clearly different from that of IWM. Characteristics of the first group included: poor performance status (30%), hyperviscosity syndrome (19%), B symptoms (29%) and neurological symptoms (25%). None of these features were present in IWM.

Regarding biology, most SWM patients (75%) had hemoglobin levels below 12 g/dl while only 8% of IWM showed this feature (p<0.001). Beta2 microglobulin and ESR were significantly higher in SWM than in IWM (3.6 vs 2.3 p=0.008, and 103 vs 62 p<0.001, respectively) and both entities also differed in albumin (3.5 vs 4 p=0.009) and IgM levels (3.4 vs 2.1 p=0.005). Although lymphoid infiltration was higher in SWM, differences did not reach statistical significance (47% vs 34% p=0.06). BM infiltration was predominantly interstitial in IWM (91%) whereas only 44% of SWM had this pattern (p=0.06). Interestingly, SWM patients had higher plasma cell (PC) bone marrow infiltration (2.6% vs 1.5% p=0.006) and a higher proportion of SWM patients showed evidence of mast cells (66% vs 28% p=0.009).

Immunophenotypic characteristics of B cells in SWM and IWM were almost identical, with only slight, but non significant, differences in some markers. The proportion of patients expressing CD27, strong CD24 and bcl-2 were greater in SWM than in IWM (42% vs 28%, 28% vs 17%, and 73% vs 61%, respectively). It should be noted that the frequency of these three markers was even lower in 15 MGUS patients analyzed (18%, 10% and 45% respectively), pointing out to a progressive decrease in expression from SWM to MGUS. Regarding PC, although their number was significantly higher in SWM than in IWM no differences were observed in their immunophenotypic pattern.

Cytogenetic profile was clearly different in SWM as compared to IWM. Thus, 10 out of the 59 SWM (17%) showed chromosomal abnormalities: 8 patients had 6q deletions and 2 patients IgH translocations, while none of the IWM cases displayed 6q deletions. In fact, only 2 out of 26 (8%) IWM showed cytogenetic aberrations: 1 case had IgH translocation and 1 case RB deletion. These results suggest that 6q deletion is associated with a more aggressive course of disease.

In summary, our results show that the different clinical and biological picture of SWM and IWM is associated with a different cytogenetic but not immunophenotypic profile.
Gain of von Willebrand Factor Binding Function Induced by a Mutation within the Leucine-Rich Repeat Region of Platelet Glycoprotein Ibα.
Authors: Peng Yuandong , Shrimpton Corie N. , Lopez Jose A.
Published: October 2019

The first step of platelet adhesion to areas of vessel injury is carried out by the platelet glycoprotein (GP) Ib-IX-V complex, which binds von Willebrand factor affixed to the subendothelial matrix. The GP Ib-IX-V complex comprises four polypeptides, each belonging to the leucine-rich repeat (LRR) family of proteins. Glycoprotein Ibα is the largest of these and contains the VWF binding site within a ~300-amino acid domain at its N-terminus that contains 8 tandem LRRs. In this study, we investigated the role in VWF binding of charged residues in this region of GP Ibα that are conserved across three species: humans, dogs and mice. Each of eleven charged residues was converted to Ala: His37, Glu40, Asp83, His86, Asp106, Glu128, Lys137, Lys152, Glu162, Asp175 and Lys189. All of the GP Ibα mutants expressed normally on the surface of CHO bIX cells, which stably express GP Ibb and GP IX. GP Iba expression was detected by the monoclonal antibody WM23, which binds a region uninvolved by the mutations. In three mutants, His86Ala, Glu128Ala, and Asp175Ala, the mutations appeared to induce large conformational changes, as they markedly altered the binding of the antibodies AN51 and SZ2, with epitopes located N-terminal and C-terminal to the LRRs, respectively. As a result, cells expressing the mutants Glu128Ala and Asp175Ala were unable to adhere to the surface when perfused over immobilized VWF. By contrast, cells expressing mutant His86Ala adhered to the surface and rolled on it much more slowly than did cells expressing wild-type GP Ibα. The His86Ala cells also displayed a left-shifted VWF binding curve in the presence of ristocetin compared to the wild-type cells. We investigated this residue further by converting the His to Glu, a residue of opposite charge. Cells expressing the His86Glu mutant rolled even more slowly over a VWF surface than the His86Ala cells at 20 dynes/cm2, but not at 2.5 dynes/cm2. Previously, mutations of GP Ibα causing gain of VWF-binding function have been confined to the disulfide loop formed by a bond between Cys209 and Cys248, which lies immediately C-terminal to the LRR region of GP Ibα. This is the first report of a gain-of-function phenotype due to a mutation in the LRR region of GP Ibα. Since His86 is more than 20 Å away from the major region of GP Ibα in direct contact with VWF defined by published crystal structures, our data suggests that the leucine-rich repeats regulate the affinity for VWF through long-range allosteric alterations of GP Ibα structure.
Ex Vivo Treatment of Erythroid Cells with Glycolytic Intermediates for Metabolic Correction of Pyruvate Kinase Deficiency.
Authors: Kanno Hitoshi , Aisaki Ken-ichi , Hamada Takako , Okada Shin-ichi , Fujii Hisaichi
Published: October 2019

Glycolysis is an essential metabolic pathway for survival of red blood cells (RBC), and mutations in the RBC-type of pyruvate kinase (R-PK) cause hereditary non-spherocytic hemolytic anemia. We have shown that R-PK deficiency induced apoptosis in erythroid progenitor cells (Blood96:596a, 2000; Am J Hematol74: 68, 2003). In this study, we examined whether ex vivo treatment of erythroid cells with high concentration of glycolytic intermediates ameliorated apoptosis of R-PK deficiency. For this purpose, we used the R-PK deficient Friend erythroleukemic cell, SLC3, which has been established from a mice model of R-PK deficiency (Jpn J Cancer Res90: 171, 1999). SLC3 and a control Friend cell were cultured with 0, 1, 3, and 5mM phosphoenolpyruvate (PEP) or pyruvate (PA). Supplementations of either PEP or PA resulted in about 2-fold accumulation of intracellular PA; however, ATP concentration was not significantly changed. Flow cytometric analysis showed that 24-hours treatment with 5mM PEP significantly reduced annexinV (+)/PI (−) cells. These data suggested that exogenously supplemented glycolytic intermediates increased intracellular PA concentration, resulting in amelioration of erythroid apoptosis. We have shown that a glutathione precursor, N-acetyl cysteine, reduced apoptosis of SLC3. Several studies revealed that PA, which effectively maintained the redox state and also prevented the loss of intracellular antioxidants, antagonized the apoptotic cell death. Taken together, oxidative stress caused by decreased supply of PA might account for erythroid apoptosis due to R-PK deficiency.
Clonality in Granulocytes Detected by an Improved HUMARA Assay: A Good Prognostic Marker in Bone Marrow Failure Patients Exhibiting Chromosomal Abnormalities of Indefinite Significance.
Authors: Ishiyama Ken , Sugimori Chiharu , Yamazaki Hirohito , Takami Akiyoshi , Nakao Shinji
Published: October 2019

Some patients with aplastic anemia (AA) and approximately 40% of patients with refractory anemia (RA) of myelodysplastic syndrome exhibit karyotypic abnormalities in bone marrow dividing cells. Although some of the patients undergo evolution to acute myeloid leukemia (AML), others follow a clinical course similar to AA patients without chromosomal abnormalities. Except for several abnormalities such as −7 and 5q-, the clinical significance of such chromosomal abnormalities in bone marrow failure patients remains unclear. We recently developed a reliable HUMARA assay capable of detecting a clonal population in granulocytes which constitutes 30% or more of total granulocytes (Blood. 2003;102:1211–1216). Studying correlation between chromosomal abnormalities and the presence of clonality may help in understanding the pathogenetic role of chromosomal abnormalities in AA and RA. We thus analyzed 50 acquired AA and 28 RA female patients who were heterozygous for the HUMARA gene. Chromosomal abnormalities such as add(5)(q13), 9q–9q+ and del(7)(q14q22) were found in 8% of AA and 21% of RA patients. Clonality was detected in 38% of AA patients and 39% of RA patients. Incidence of chromosomal abnormalities in patients with clonality (27%) was higher than that in patients without clonality (4%, p<0.01). In two AA patients who respectively exhibited add(5)(q13) in 10% and +8 in 38% dividing cells, clonality was not detected and these abnormal clones became undetectable at the time of subsequent bone marrow examination. Clonality was detected in the other 2 AA patients respectively exhibiting 9q–9q+ in 40% and del(7)(q14q22) in 25% dividing cells, and in all 5 RA patients respectively exhibiting +8 in 10%, del(5)(q13q31), dup(1)(q32q12) in 90%, del(5)(q13), add(11)(q23), inv(9) in 65% and X,-X in 100% of dividing cells. None of the 50 AA patients including 2 patients with clonality and chromosomal abnormalities underwent evolution to AML during 2-year follow up while one of 28 RA patients who exhibited del(5)(q13q31) progressed to AML. The proportion of clonal granulocytes in total granulocytes estimated by the HUMARA assay remained unchanged in most patients with clonality except for the transformed one. These data indicate that the chromosomal abnormality in bone marrow dividing cells is not necessarily associated with presence of clonal granulocyte population in peripheral blood and that detection of clonality in granulcytes in bone marrow failure patients with chromosomal abnormalities of indefinite significance is useful in predicting prognosis of these patients.
Evaluation of Chronic Transfusion (Tx) Practices in Children with Sickle Cell Disease (SCD): A Survey of STOP II Investigators.
Authors: Alvarez Ofelia , Miller Scott , Berman Brian , Brown Clark , Casella James , Coates Thomas , Daeschner Charles , Files Beatrice , Kwiatkowski Janet , Lee Margaret , Wang Winfred , Woods Gerald
Published: October 2019

The follow-up Stroke Prevention Trial (STOP II) attempts to optimize tx therapy for primary stroke prevention in children with SCD who are at increased risk due to an abnormal Doppler ultrasound. A survey of participating investigators (PIs) was performed in order to assess tx practices; 12 PIs out of 26 responded. To begin chronic tx, 42 % of the PIs preferred erythrocytapheresis (ECP), partial or total exchange to bring hemoglobin (Hb) S <30%, 33% used frequent txs over a month, and the rest used either method. For continuation of chronic tx, 5 PIs used only simple tx for their pts, 2 used partial exchange (phlebotomy and simple tx), and 5 used either simple tx or exchange to transfuse their patients (pts). The most common reason to choose a method was intravenous (IV) accessibility. 4 PIs mentioned that they start with simple tx but later might use exchange if unable to keep Hb S <30% or to avoid iron overload. For simple tx, most PIs ordered 10–15 ml/kg leucoreduced, Rh and Kell compatible, S negative packed red blood cells (PRBC). Pts returned for the next tx, depending on prior pre-tx Hb S (goal < 30%), or every 4 weeks unless Hb S was >30%. Most PIs had a post-tx target hematocrit (Hct) 35–36%, but a third of them did not have one. Pre-tx Hct and/or Hb S (but not post-tx values) were used to predict the timing of the next tx by 83% of the group. When performing ECP, the PIs participated in the decision of how much to exchange pts only 33% of the time; generally ECP was planned by a blood bank physician and/or by machine programming. Chelation began either after 12–30 months of tx (median 18 months), or after serum ferritin 1000–2500 ng/ml (median 2000). 67% of the PIs obtained liver biopsies in all or some of the STOP II pts. Indications for liver biopsies were cited as routine for transfused pts (5 PIs) or depending on ferritin values (6 PIs). Deferoxamine 25–50 mg/kg was infused subcutaneously over 8–10 hours 5–7 nights a week in all pts. Eight PIs reported the use of central venous lines (ports) in some pts to facilitate IV access. Barriers cited to effective chronic tx were: pt compliance with chelation (7 reported it as most important), IV access, pt compliance with tx schedule, hypersplenism, and alloimmunization. We conclude that hematologists :(1) Administer leucoreduced Rh and Kell compatible, S negative PRBC to keep pre-tx Hb S levels <30%, (2) use pre-tx Hb S and Hct to predict next tx, usually every 3–4 weeks, (3) monitor and treat iron overload, and (4) report that poor compliance with chelation is a key barrier to an effective tx program. Although liver biopsy to monitor iron stores and partial/total exchange to limit iron overload are accepted interventions, medical practice still varies.
Global ENU Mutagenesis Screen for Genetic Modifiers in Sickle Cell Disease Mice.
Authors: Ryan Thomas M. , Huo Yongliang , McConnell Sean
Published: October 2019

Genetic modifiers of sickle cell disease (SCD) will be identified in an animal model of SCD after mutagenesis with the chemical mutagen N-ethyl-N-nitrosourea (ENU). This phenotype driven approach utilizes a third generation knock-in (KI) mouse model of SCD that reproduces most if not all of the pathology of the disorder. This model was produced by targeted gene replacement of the murine α-globin genes with human α-globin and the murine β globin genes with a human γ- and βs-globin gene cassette that mimics the fetal to adult hemoglobin switch that occurs in man. Sickle embryonic stem (ES) cells were derived from developing blastocysts isolated from female sickle mice that were mated with sickle males. Sickle ES cells were treated with ENU and a mutant library of independent subclones was established and archived. The optimal ENU dosage was empirically determined through a series of pilot experiments that measured the HPRT mutation frequency and the efficiency of producing sickle mice from the mutagenized cells by tetraploid embryo complementation. Animals harboring mutations that affect hematological indices, kidney function, or liver function are identified in the mutagenized sickle mice by comparison to cloned control sickle mice. Microsatellite linkage analyses of mutant offspring outcrossed to congenic SCD mice and direct sequence comparison to the murine genome will allow the positional cloning of modifier genes. Putative modifying factors will be positively confirmed by introducing the exact germline modification discovered during the ENU screen into the unmutagenized ES cells, followed by the direct examination of the phenotype in mice generated from the modified cells by cloning. These studies will define gene(s) responsible for the phenotypic variation in disease severity that is observed in the SCD population. By experimental design, the therapeutic benefit or detriment associated with each modifying gene(s) on the in vivo pathophysiology of sickle cell anemia will be tested directly in our animal model of this disorder.
Renal Involvement in Saudi Patients with Sickle Cell Disease. A Single Institution Experience.
Authors: Abdel-Razeq Hikmat N. , Bajouda Ali , Khalil Manar , Al-Shareef Muneera , Siddiqi Nauman
Published: October 2019

Background: Renal involvement in patients with sickle cell disease has been reported to be as high as 20%. This can take the form of proteinuria, decreased GFR, isothenuria or hematuria. Our own clinical observation of patients attending our sickle cell clinic or admitted to the medical ward at our institution suggests lower rates of renal involvement than previously reported. In this report, we evaluate Saudi patients with sickle cell disease (SS) at our institution for renal involvement.

Patients and Methods: Consecutive afebrile patients attending sickle cell clinic or admitted to inpatient wards were screened. Serum creatinine, urine analysis and 24 hr urine collection for creatinine clearance (GFR) and proteinuria were performed. Data on demographics and co-morbidities (DM, HTN, hepatitis B and C) and medications were obtained. Analysis was performed using SPSS 10.0®.

Results: 62 patients were available for study; results are summarized in tables.

Seven of 48 patients (15%) had proteinuria >300mg/d while two (4.2%) had proteinuria of more than 1.0 gram/24 hrs. Nearly 20% had GFR < 60ml/min. None of the patients had renal tubular acidosis. Serum creatinine was normal in all patients including those with low GFR and proteinuria.

Conclusions: Contrary to our initial believes, significant percentage of Saudi sickle cell patients, like others, have renal involvement with low GFR and proteinuria. Serum Creatinine alone doesn’t indicate this decline in renal function. We recommend annual evaluation for proteinuria and GFR determination in all sickle cell patients, and those at risk should be aggressively managed with nephroprotective agents like angiotensin converting enzyme (ACE) inhibitors.

Age (yrs) M:F BMI Hepatitis C Hydroxyurea NSAID 22.1+/−8.1 23:39 19.3+/−5.1 9 (17.3%) 29 (58%) 33 (66%) Urine Volume (ml/24 hrs) Specific Gravity Serum Creatinine (u mol/L) GFR Proteinuria (mg/24hrs) 1927 +/− 780 1.011 +/− 0.005 40.6 +/− 12.9 113.7 +/− 47.7 256 +/− 371 (20–2390)
A Tool for Predicting Haemoglobin Variants - an Aid to DNA Diagnostics.
Authors: Clark Barnaby E. , Cunningham Juliette C. , Wild Barbara J. , Thein Swee Lay
Published: October 2019

The hemoglobin (Hb) variants represent a well-documented array (>800 in number) of amino acid substitutions, hybrid Hbs and Hbs with extended/shortened amino acid sequences. The majority of these structural alterations have little or no impact on the well being of the individual. However, some can result in severe disease when inherited in a homozygous or compound heterozygous state (e.g. sickle cell disease), while others can give a thalassaemic picture if the globin variant is highly unstable. Kings’s College Hospital (KCH), which is a referral centre for Hb variants, uses automated HPLC (Biorad Variant II) as the first line screening test, followed by further electrophoretic separations (isoelectric focusing (IEF), cellulose acetate membrane (CAM) and acid gel electrophoresis (AGE)) if a variant is present. Definitive diagnosis is then achieved by either mass spectrometry or DNA analysis of the suspected globin gene. The former method is quick and uses small quantities of blood but the equipment is expensive and requires a high degree of expertise, making it unavailable to many institutions. PCR based DNA diagnostics is routine in many service pathology laboratories but it is hampered by the cost of consumables and the time it takes. To minimise these KCH has developed a database that statistically evaluates the phenotypic separations and predicts which Hb variant is present based on past data. A single entry from any one of the tests (HPLC, IEF, CAM, AGE or mass difference) is enough to generate a prediction. Using this prediction only a small region of the putative globin gene will need sequencing or analysis by restriction digest, thereby reducing costs and turnaround times. The database contains the phenotypic separation data of >700 samples collected over six years. Variants were definitively diagnosed using electrospray ionisation mass spectrometry, complemented in some cases by DNA analysis. KCH will continue to enter variant data to increase the data set and improve the tool’s accuracy and aims to make the prediction tool available online so that other laboratories may obtain predictions and add their data. Laboratories will then be able to retrieve predictions based on their own past data or the entire database.
Predictors of Renal Insufficiency in Sickle Cell Disease.
Authors: Keller Margaret A. , Borowski Meggin K. , Devoto Marcella , Li Hui-Hua , Brown Morgan , Surrey Saul , Ballas Samir K.
Published: October 2019

Renal insufficiency is the source of significant morbidity and mortality in adults with sickle cell disease (SCD). A population of adult patients with SCD and end-stage renal disease (ESRD) requiring hemodialysis was examined for factors that predict ESRD. ESRD patients (mean age 36.8 +/− 7.74) were compared with patients without ESRD over 50 years old (mean age 59.3 +/− 8.31). We found that the ESRD population was characterized by more males (14 males, 8 females ESRD vs. 4 males, 15 females non-ESRD), a higher occurrence of the CAR beta-like globin cluster haplotype (44 % ESRD vs. 11 % non-ESRD) and relatively lower fetal hemoglobin levels (3.6 +/− 2.16 ESRD vs. 8.6 +/− 6.35 non-ESRD). In order to further examine potential predictors of renal disease, including genetic modifiers unlinked to the beta-globin cluster, we examined a broader population of patients with elevated creatinine, defined as levels greater than 1.0 mg/dl, who did or did not require hemodialysis. We hypothesize that gender, beta-like globin cluster haplotype, fetal hemoglobin levels and polymorphisms in critical regulators of vasomotor tone are predictors of renal insufficiency in SCD. Genotyping of a single nucleotide polymorphism (SNP) in endothelial nitric oxide synthase (eNOS or NOS3) and an insertion/deletion polymorphism (I/D) in the angiotensin-converting enzyme (ACE) gene will allow us to determine if these polymorphisms are over-represented in this cohort. The eNOS T-786C SNP alters the level of transcription of the eNOS gene and the amount of eNOS activity. We recently showed that this polymorphism was associated with a history of acute chest syndrome in females with SCD (Br. J. Haematol., 2004, 124:240). The ACE I/D polymorphism has been associated with altered plasma ACE levels. Twenty-one adult patients with sickle cell disease with renal insufficiency and 25 age- and gender-matched control patients seen at Thomas Jefferson University are being examined. Genotype results and statistical analysis will be presented. It is hoped that identification of predictors of renal insufficiency in patients with SCD might lead to early identification of at-risk patients, allowing for therapeutic intervention.
Two Novel Mutations in the α-Globin Genes Leading to α-Thalassemia.
Authors: Rund Deborah , Koren Ariel , Zalman Lucia , Ben Barak Ayelet , Oppenheim Ariella , Filon Dvora
Published: October 2019

Although deletional α-thalassemia is the most common form of the disease worldwide, in Israel, point mutations leading to α-thalassemia are relatively common. We routinely sequence the α-globin genes when we fail to find deletions which can account for the hematological phenotype. Our PCR is specific for sequencing either the α1 or α2 globin gene. We here report two novel mutations, one in α1 and the other in α2, which lead to a thalassemia phenotype. Case 1. A 5 yr old Druze girl from northern Israel was referred due to microcytic anemia. Her hematological values were: RBC 5x109/L, Hb 8.8 gr%, MCV 55 fl, MCH 17.6 pg, MCHC 31.7gr/dl, RDW 16.2. HbA 2: 2.2%, HbF: 1%. Iron deficiency was excluded (serum iron: 58 mg/dl, transferrin: 247mg%, and ferritin: 46.2 ng/ml) The patient’s physical examination and mental development were normal, without splenomegaly or jaundice. No therapy was required, and transfusion was unnecessary. The parents had very mild microcytic anemia (mother Hb 11 gr% MCV 75.5, father Hb 13.8, MCV 78.6) with normal Hb electrophoresis. DNA of the parents and propositus was sequenced. The child was found to be homozygous (and the parents, heterozygous) for a deletion of a single cytosine (within a stretch of four) at codons 118/119 of the α2 globin gene, leading to a frameshift at codon 119. Case 2. This family is of mixed ancestry, the mother is from Bukhara. The hematological data is presented in the table below. Sequencing of the α-globin genes of the mother revealed an A to G change at the acceptor site of IVS1 at nt 116 in the α1 gene in compound heterozygosity with the well known 5 nt deletion in IVS1 of α2 (HphI). The same IVS1 nt 116 mutation at this exact nucleotide was previously reported by Harteveld (1996) to be found in the α2 globin gene, causing Hb H inclusion bodies when found in combination with 3.7 kb deletional α-thalassemia. We conclude that rare point mutations can still be identified in the α-globin genes which can be the cause of otherwise unexplained α-thalassemia phenotypes.

Family 2 hematological data and alpha globin genotype

Family member RBC Hb MCV MCH RDW chromosome 1 chromosome 2 del=deletion Mother 5.68 10.7 63.2 18.8 16.4 α2 HphI α1 IVS1-116 Father 6.15 13.4 70.7 21.8 13.9 del 3.7kb del 3.7 kb Son (9 yrs) 5.59 11.1 64.4 19.9 13.7 α2 HphI del 3.7 kb Daughter (7 yrs) 5.44 10.9 64.3 20.0 13.5 del 3.7 kb α1 IVS1-116 Son (2 yrs) 5.55 10.5 61.6 18.9 14.3 del 3.7 kb α1 IVS1-116
Reappraisal of Symptoms and Signs of Uncomplicated Beta Thalassemia Trait.
Authors: Premawardena A. P. , Malewana T. , Arambepola M. , Olivieri N. F. , Weatherall D. J.
Published: October 2019

Although considered a mild disorder, beta thalassemia trait (uncomplicated by any other hematologic abnormality) has been reportedly associated with symptoms of anemia, while in other series hepatosplenomegaly has been reported as associated with this diagnosis. No controlled study has examined the symptoms and signs of individuals with beta thalassemia trait and compared these with age- and sex-matched normal individuals. We administered a questionnaire to 397 parents (67% females) of children with beta thalassemia major attending the National Thalassemia Centre in Kurunagala, Sri Lanka, and to 87 normal volunteers (90% females). All individuals underwent physical examination by the same investigator, as well as testing by automated cell counter (Coulter), and HPLC (Bio Rad). The mean age of the parents (33 years) and controls (29.5 years) was not significantly different. In parents with thalassemia trait, 20% of males and 42% of females complained of reduced exercise tolerance compared to 12.5% of males and 38% of females in the controls (P= NS). The questionnaire revealed that 41% of parents, and 36% of controls, complained of recurrent headache; 18% of parents, and 15% of controls, complained of lethargy (P=NS). A history of malaria infection was reported in 30% of parents with thalassemia trait, and in 24% of controls. Mean quality-of-life score, recorded formally by a linear analog scale, from 0 (poorest) to 10 (greatest) was 7.1 in parents and 7.2 in controls. Splenomegaly was detected in 6% of parents with beta thalassemia trait (mean spleen size 1.5 cm below the costal margin), and 5% of normal controls (mean spleen size 1 cm below the costal margin). These findings suggest that, by contrast to many reports in the older literature, in uncomplicated beta thalassemia trait there appears to be no symptoms or signs greater than those observed in a normal control population and that fatigue, lethargy, exercise intolerance, or splenomegaly in a patient with beta thalassemia trait should not be attributed to the underlying hemoglobinopathy.
Preferential Uptake of Gamma Tocopherol by Mast Cells. Does This Uptake Affect Mast Cell Cytokine Production?.
Authors: Aljitawi Omar S. , Fitzgerald Mathew , Qui Min , Krishnan Koyamangalath , Stone William L. , Krishnaswamy Guha
Published: October 2019

Vitamin E, the main lipid soluble antioxidant, exists in eight different forms, of which α-tocopherol and γ-tocopherol are the two major forms. Previous experiments showed vitamin E uptake by macrophages that contribute to inflammation and immunity. On the other hand, vitamin E has structural similarity to the thiazolidinedione, troglitazone, a peroxisome proliferator-activated receptor (PPAR) agonist. In previous experiments we found that troglitazone (TGZ), a PPAR gamma agonist, had a negative effect on mast cell cytokine production. We therefore wondered whether vitamin E enters human mast cells, and if so, does this modulate mast cell cytokine production? We choose (interleukin) IL-6 for its pro-inflammatory properties and because it’s known to be produced by mast cells in response to stimulants used in the experiment. In this study we try to answer these two questions. Cultured human mast cell line (HMC-1) was first incubated for 24 hrs with pharmacological concentrations of both alpha and gamma forms of vitamin E (10 μM). The mast cells were then activated with phorbol-12-myristate-13-acetate [PMA (50ng/ml)] and ionomycin (5μm) for 24 hours and cell-free supernatants collected. In additional experiments, IL-1ß (10ng/mL) was added to activate mast cells. IL-6 levels in the supernatants were determined in each well utilizing ELISA. Mast cell concentrations of alpha or gamma tocopherol were measured by high pressure liquid chromatography [HPLC] and electrochemical analysis. Mast cells pre-incubated in alpha and gamma forms of vitamin E at 10 μM did not affect mast cell IL-6 production. Mast cells, however, showed uptake of both forms of tocopherols but more pronounced uptake of the gamma form (13181.05 pmole/well of gamma compared to 8742.99 pmole/well of alpha tocopherol). We conclude that mast cells appear to store both alpha and gamma tocopherols but preferentially more gamma tocopherol. Though Vitamin E and PPAR agonists have similar structures, they did not show similar effect on mast cell cytokine production, suggesting they might have different mechanisms of action.
IL-6 Produced by Type I IFN DC Controls IFN-γ Production during the MLR by Blocking the Suppressive Effect of Regulatory T Cells.
Authors: Detournay Olivier , Mazouz Naima , Goldman Michel , Toungouz Michel
Published: October 2019

The dendritic cell family is composed of different subsets able to differentially govern the immune response. Their potent antigen presenting properties make them an attractive candidate for immunization against pathogens or cancer. In that setting, the recently characterized type I IFN DCs present interesting features including a higher expression of molecules involved in antigen presentation and the ability to trigger both the cellular and humoral arms of the immune responses. In view of the pivotal role of regulatory T cells in limiting the effectiveness of effector cells, we analyzed the interactions between these cells and type I IFN DC. DC generated from monocytes in the presence of IFN-β and IL-3 (DCI3) were activated by the maturation agent poly I:C and compared with the classical myeloid DC generated in the presence of GM-CSF and IL-4 (DCG4). Despite the release of lower amounts of IL-12 after maturation, DCI3 were able to induce a higher IFN-γ production by T lymphocytes during the MLR. Analysis at the mRNA level disclosed that DCI3 over transcribed the IL-6 gene leading to the release of high amounts of the protein both after the maturation process and during the MLR itself. Neutralization of IL-6 revealed that this cytokine specifically contributed to the IFN-γ release induced by DCI3. Finally, depletion of CD25+ T cells prior to the MLR identified these cells as a target for IL-6. We conclude from these results that DCI3 are endowed with the unique property of blocking the suppressive effect of regulatory T cells through high IL-6 production during the MLR. This novel mechanism of T cell control is relevant for the use of this DC type in vaccination strategies.
Clinical-Grade Type I IFN-DC Differentiated in the Presence of Either IL-3 or GM-CSF Show Similar Phenotypic and Functional Proprieties.
Authors: Mazouz Naima , Detournay Olivier , Rennesson Joelle , Lambermont Micheline , Marchant Arnaud , Goldman Michel , Toungouz Michel
Published: October 2019

Despite limited clinical efficacy in large trials, dendritic cells (DC)-based immunization has yielded impressive responses in some patients. Key questions remain to be solved in order to optimize this therapeutic vaccine. Among them, the nature of the DC used and its state of maturation are pivotal. Besides myeloid DCs which are the cells essentially used in clinical trials, a new type of DC has been described resulting from the differentiation of monocytes in the presence of type I IFNs. In the present study, we analyzed the features of clinical grade type I IFN DC generated in the presence of either IL-3 (IL-3- DCs) or GM-CSF (GM-CSF-DCs) and compared their capacity to respond to poly I:C, a double-stranded RNA analog that mimics viral infection. The two DC types disclosed a similar immunophenotype characterized by high levels of costimulatory molecules, CCR7, HLA-class-I and class-II molecules. After poly I:C maturation, both DC types displayed a marked upregulation of CD80, CD83 and CD86. In addition, poly I:C stimulated them to secrete IFN-α IL-12 p70, IL-12 p40 and IL-6. Both DC types elicited potent allogeneic reactions. Priming of autologous T cells by IL-3-DCs or GM-CSF-DCs pulsed with an HLA-A2 restricted melan-A derived peptide, lead to expansion of melan-A specific CTL secreting high levels of IFN-γ and displaying a phenotype of memory cells. We conclude that mature clinical grade IL-3-DCs and GM-CSF-DCs share similar phenotype and functional properties including the capacity to prime Ag-specific CTL.
Functional Activity of In Vivo Primed Granulocytes: A Comparative Study.
Authors: Ribeiro Daniel , Laufs Stephanie , Buss Eike B. , Zeller W. Jens , Ho Anthony D. , Fruehauf Stefan
Published: October 2019

Recombinant granulocyte colony-stimulating factor (G-CSF) has been widely used in the treatment of chemotherapy-induced neutropenia as well as in mobilization of peripheral blood stem cells in context with autologous bone marrow transplantation. Recombinant G-CSF expressed in E.coli (Filgrastim) and G-CSF expressed in CHO-cells (Lenograstim), are in clinical use. Here we study the effects of the different G-CSF on functional activity of granulocytes including chemotaxis, oxidative burst and antigen expression.

Granulocytes were obtained from patients with hematological malignancies before and after the administration of one of the three G-CSFs and isolated using a polymorphprep density gradient. Chemotactic properties were assessed using a Boyden chamber assay in combination with an under agarose assay, both using fMLP as chemotactic stimulus. Release of superoxide anions served as measure of the oxidative burst after stimulation with PMA using a chemiluminescence assay. The viability and surface antigen expression were assessed by FACS.

FACS analysis showed a decrease in CD10, CD11b and CD62L of the selectin family contrary to an increased expression of the VLA-5 alpha chain CD49, the LPS-receptor CD14 and the IgG receptor FcγRI (CD64). A stronger effect of lenograstim on CD11b and CD14 could be assessed contrary to filgrastim showing a stronger effect on CD62 and CD64. We observed a decrease of chemotactic activity and non-directed random migration in patients receiving filgrastim, as opposed to the results in patients receiving lenograstim. No obvious differences were found in production of superoxide anions. Whether the superior chemotactic activity and migration of Lenograstim- vs. Filgrastim-primed granulocytes translates into improved antibacterial activity and improved clinical endpoints warrants further study in the clinical setting.
Early Diagnosis of Invasive Aspergillosis in Hematological Patients Using a PCR-ELISA Assay: A Prospective Validation.
Authors: Cornet Muriel , Katsahian Sandrine , Vekhoff Anne , Levy Vincent , Rio Bernard , Marie Jean-Pierre , Bouvet Anne , Florent Martine
Published: October 2019

Purpose: Prospective evaluation of a PCR-ELISA method for the early diagnosis of invasive aspergillosis (IA) in hematological patients.

Patients and Methods: The PCR-ELISA assay was evaluated using 1,494 sera samples from 201 hematological adult patients. Monitoring was performed twice weekly during 256 consecutive high-risk treatment periods.

Results: Underlying diseases were acute leukemia 52.7%, lymphoma 20.9%, multiple myeloma 12.4%. Eighty four patients (41.8%) were stem cell transplant recipients. Fifty-five patients were diagnosed with IA. PCR-ELISA positivity (i) preceded the empirical antifungal therapy in 20/31 patients (median 10.5 days), (ii) preceded or was simultaneous to the radiological diagnosis by chest CT in 21/31 patients (median 8 days), (iii) preceded the mycological/histological diagnosis in 7/12 patients (median 70 days) and, (iv) anticipated or was simultaneous to the galactomannan (GM) antigen positivity in 50% (median 16.5 days) and in 89 % of the patients (median 33 days) using a 0.5 and a 1.5 optical density index (ODI) cut-off for the GM assay, respectively. For at least two positive samples the sensitivity, specificity, positive and negative predictive values of the PCR-ELISA assay, were 63.6%, 82.2%, 57.4% and 85.7%, respectively. When the PCR-ELISA and GM detections were combined, values of the combined test for at least two positive samples with either assay were 80%, 47.3%, 36.4%, 86.2% using the 0.5 GM ODI cutoff, and 67.3%, 82.2%, 58.7% and 87% using the 1.5 GM ODI cutoff.

Conclusion: In addition to serial screening with GM antigenemia and chest CT surveillance, the PCR-ELISA assay may improve the early diagnosis of IA.
Leukemogenic MLL Fusion Proteins Promote MLL Association with HOX Loci Resulting in Persistent Expression.
Authors: Milne Thomas A. , Martin Mary Ellen , Slany Robert K. , Hess Jay L.
Published: October 2019

Rearrangements of the mixed lineage leukemia gene MLL are associated with aggressive lymphoid and myeloid leukemias. The resulting MLL fusion proteins enforce high-level expression of HOX genes including HOX A7 and HOX A9 and the HOX cofactor MEIS1, which is pivotal for leukemogenesis. The mechanism by which this occurs and the relationship to normal MLL function is unknown. To address this we performed a detailed study of where MLL and MLL fusion proteins bind at target genes in hematopoietic cells using quantitative chromatin immunoprecipitation (ChIP). In addition we characterized the histone modifications at the HOX A9 locus that occur with normal down modulation of HOX A9 expression during hematopoietic differentiation and how this is perturbed by induction of conditionally transforming MLL fusion proteins. These studies suggest that a major mechanism of regulating MLL, which is expressed throughout hematopoiesis, is through modulating it’s binding to target promoters. MLL binds directly to the promoters and coding regions of HOX A7, HOX A9, and MEIS1 only in myeloblasts and not in neutrophils, indicating MLL is physically associated with genes only when they are actively transcribed. Expression of A cluster HOX loci and MEIS1 is transiently increased by the addition of IL-3 but remains persistently elevated when MLL-ENL or dimerized MLL fusion proteins are expressed. Expression of either fusion protein is associated with increased binding of wild type MLL accompanied by increases in histone acetylation and histone H3 lysine 4 methylation, marks that are normally almost completely erased during myeloid differentiation. In addition MLL-ENL induces increased lysine 79 methylation. MLL extensively colocalizes with RNA polymerase II, which shows abnormal pausing in the coding regions of HOX genes in Mll null cells. These findings suggest MLL fusion proteins maintain expression of HOX genes critical for leukemogenesis through increased MLL binding resulting in both MLL dependent histone modifications and lysine 79 methylation and ultimately promotion of transcriptional elongation.