Rakhee Kar

Evaluation of eosin-5-maleimide flow cytometric test in diagnosis of hereditary spherocytosis.

A flow cytometry‐based test using eosin‐5‐maleimide (EMA) dye was used for diagnosis of hereditary spherocytosis (HS). The mean fluorescence intensiy (MFI) of EMA tagged erythrocytes is lower in HS than that in other hemolytic and nonhemolytic anemias. We enrolled 114 subjects comprising 20 confirmed HS, 20 suspected HS/hemolytic anemia (HA), 20 normal controls, 20 other hemolytic anemias [13 autoimmune hemolytic anemia, three congenital dyserythropoietic anemia (CDA), one pyruvate kinase deficiency, two microangiopathic hemolytic anemia], 18 microcytic anemia and 16 macrocytic anemia cases. All samples were subjected to flow cytometry as per standard protocol. The mean MFI of normal control subjects was 11 861.5 (SD 883.5) and of confirmed HS was 7949.3 (SD 1304.1). Using this test, of 20 patients suspected to be HS/HA but with no confirmatory diagnosis, eight patients were diagnosed as HS. Using logistic regression analysis, the optimum cut‐off MFI value between HS and normal controls was 10126. The area under the ROC curve was 0.99. The statistical significance of MFI values was obtained by t‐test or Wilcoxon rank sum test as applicable. Compared with normal controls, the MFI values in HS were lower and in megaloblastic anemia were higher which was statistically highly significant (P < 0.01), and the MFI values in CDA were lower which was statistically significant (P < 0.05). False‐positive values were obtained in three cases of AIHA and two cases of CDA. The sensitivity and specificity was 96.4% and 94.2% respectively. The EMA‐based flow cytometry test is a highly sensitive and specific method for the diagnosis of HS.

Spectrum of haemoglobinopathies diagnosed by cation exchange-HPLC & modulating effects of nutritional deficiency anaemias from north India

Background & objectives: The usefulness of cation exchange high performance liquid chromatography (CE-HPLC) as a tool for detection of thalassaemia/haemoglobin variants was evaluated in a prospective study in a tertiary care centre in north India. We also tried to evaluate the effect of concurrent nutritional deficiency on the HPLC pattern in the local ethnic population. Methods: A total of 800 blood samples were analyzed on the Bio-Rad Variant HPLC system by β-thal short program. The retention times, proportion of the haemoglobin (%), and the peak characteristics for all haemoglobin fractions were recorded. Alkaline and acid haemoglobin electrophoresis was performed to document the identities of the haemoglobin variants, wherever necessary. Many cases were subjected to family studies for a definitive diagnosis. Results: Among 800 samples tested, 553 (69.1%) were found to have normal HPLC pattern. Apart from β- thalassaemia, nine additional variants were encountered; HbS (2.8%), HbE (2.5%) and HbD (1.1%) being the most common variants present. Other variants included Hb Q-India, Hb-Lepore, δβ-thalassemia/ HPFH, HbD-Iran, HbJ-Meerut and HbH disease. There was a significant decrease in the level of HbA2 associated with iron deficiency anaemia (IDA) (P=0.004) and increase in megaloblastic anaemia (P<0.001) among subjects with normal HPLC pattern. Interpretation & conclusions: HPLC was found to be a simple, rapid and reliable method for the detection of hemoglobin variants. An accurate diagnosis can be provided in majority of cases by use of retention time, proportion of total haemoglobin, and peak characteristics of HPLC. Haemoglobin electrophoresis and family studies play a valuable role in difficult cases. Concurrent nutritional deficiency also has an effect on HbA2 levels.

Clopidogrel resistance in North Indian patients of coronary artery disease and lack of its association with platelet ADP receptors P2Y1 and P2Y12 gene polymorphisms

Aspirin and Clopidogrel are used in prophylaxis of patients undergoing percutaneous coronary intervention and long-term prevention of cardiovascular and cerebrovascular events. Clopidogrel resistance has been attributed to P2Y1 and P2Y12 adenosine diphosphate (ADP) receptor polymorphisms. This study enrolled 100 patients of coronary artery disease (CAD) who were on the maintenance dose of clopidogrel (75 mg OD) with or without aspirin. In addition, 10 received loading dose (300 mg) prior to percutaneous coronary intervention. Relevant clinical and drug history were elicited. ADP-induced platelet aggregation study and PCR-RFLP for P2Y1 (1622A > G) and P2Y12 (i-T744C) polymorphisms were performed. Two groups of controls were used for defining cut-off for platelet aggregation response. Follow-up data, wherever available was recorded. The most common pattern of aggregation response was disaggregation, either complete (46.4%) or partial (53.6%). A frequency of 13% clopidogrel non-responders and 19% semi-responders was found. All the cases were H1/H1 haplotype for P2Y12 gene polymorphism and 28 (29.2%) patients carried P2Y1 1622A > G (21(21.9%) AG and 7(7.3%) GG) gene polymorphism, the frequency being greater in clopidogrel responders compared to semi/non-responders but difference was not statistically significant. There was no statistically significant difference between responders and semi/non-responders in terms of the history of risk factor for CAD, concurrent atorvastatin use or past history of an acute vascular event. On follow up, the two patients who developed myocardial infarction/acute coronary syndromes (MI/ACS) were clopidogrel semi- and non-responder, respectively. Variability in clopidogrel response with 13% non-responders and 19% semi-responders was seen in this study with adverse outcome (MI/ACS) on follow up seen in two patients. Hence, poor response to clopidogrel may be related to increased likelihood of adverse long-term coronary event that may benefit from additional or alternative anti-platelet therapy. Clopidogrel resistance was not associated with ADP receptor P2Y1 and P2Y12 gene polymorphisms. Hence, it is postulated that clopidogrel resistance in CAD patients is multifactorial and not caused by single-gene polymorphisms.

Clinico-hematological profile of hereditary spherocytosis: experience from a tertiary care center in North India

Abstract Hereditary spherocytosis (HS) is an inherited membranopathy characterized by phenotypic and genotypic heterogeneity. This study describes the clinico-hematological profile of 70 HS patients diagnosed at a tertiary care center in North India over a period of five years. Patients commonly presented with intermittent jaundice (82·9%), pallor (80%) and dark colored urine (11·4%). The common signs were splenomegaly (92·9%), hepatomegaly (50%), cholelithiasis or choledocholithiasis (36·8%) and hemolytic facies (10%). Family history was contributory in 28·6% patients. Blood transfusion (BT) requirement was present in 35·7% patients. Unconjugated and conjugated hyperbilirubinemia was seen in 89·1 and 10·9% patients respectively. At presentation, the hemoglobin ranged from 3–14 g/dl with a mean of 9·37 g/dl (SD2·43). Spherocytes were seen in 88·6% and incubated Osmotic fragility test (OFT) was positive in 88·2% patients. The Eosin-5-maleimide (EMA) flow cytometric test was done in 28 patients. Mean fluorescence intensiy (MFI) for normal subjects was 11861·5 (SD-883·51) and for confirmed HS patients was 7949·3 (SD1304·1). Taking the MFI range of 5341·1–10 557·5 for HS, eight cases of suspected HS/undiagnosed hemolytic anemia with a negative (n=5) or equivocal (n=3) incubated OFT were diagnosed as HS. An increase in HbF level was seen in 10 cases ranging from 2·1 to 17·7% with a mean of 5·66%, three of these had associated β thalassaemia trait. Twelve patients (17%) underwent splenectomy and 91% of them did not require any BT post-splenectomy. Among the patients treated conservatively 49% had persisting pallor and 16·3% had transfusion requirement.

Pseudo Chediak-Higashi anomaly in acute myelomonocytic leukemia

Pseudo Chediak-Higashi anomaly in acute leukemia is a rarely described entity. The significance of this intriguing morphological finding largely remains unknown, although some authors have predicted a poorer outcome in such cases because of a higher susceptibility to fulminant infections. Our case also had a fatal outcome.

Bilirubin Peak can be Mistaken as Hb Bart's or Hb H on High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) has largely replaced electrophoresis for the identification of hemoglobin (Hb) variants. However, one needs to be vigilant to pick up undue interference such as the presence of a bilirubin peak, which can be mistaken for either Hb Barts or Hb H on Hb HPLC. Correlation with the clinical context and biochemical parameters, coupled with repeating the run after washing the sample, can resolve this problem. Herein, we report one such case of an acute on chronic liver failure patient who was found to have heterozygous β-thalassemia (β-thal) on Hb HPLC with an incidental tall unknown peak that was identified as bilirubin.

Anaplastic myeloma: a morphologic diagnostic dilemma

We present a case of a 58-year-old man who was referred to our hospital with complaints of transfusion dependent anemia (20 units in one year) and recurrent fever over a year. There was no history of alteration in urinary or bowel habits. He was previously diagnosed as acute myeloid leukemia and metastatic tumor/anaplastic large cell lymphoma on bone marrow examination on two separate occasions from two different hospitals. He had not received any treatment so far. On examination, he was noticeably pale, but did not reveal any icterus or bleeding manifestations. There was no sternal tenderness, organomegaly or lymphadenopathy. On routine investigations his hemoglobin was 8.8g/dl, total leucocyte count 2200/cmm and platelet count was 47000/ cmm. Peripheral smear showed normocytic normochromic red cells and a normal differential leucocyte count. No rouleaux formation was seen. Erythrocyte sedimentation rate was raised. Bone marrow examination revealed diffuse replacement of marrow elements by poorly differentiated pleomorphic tumor cells arranged in sheets, clusters and scattered singly (Fig. 1) along with numerous bizarre looking tumor giant cells (Figure 1:Inset). Most of the cells were quite anaplastic and primitive in appearance but plasmacytoid differentiation was evident in some areas with many plasmablasts (Fig. 2). A differential diagnosis of Anaplastic myeloma and ? Metastatic tumor deposit was suggested and further investigations were advised on these lines. Radiological investigations did not reveal any occult primary malignancy. Subsequently, on serum electrophoresis, a narrow M-band was detected in β-region (10.2%, 0.4g/dl) with overall reduction of polyclonal γ-globulins. Immunofi xation studies showed ‘M’band to be lambda (λ) light chains. Bone scan revealed diffuse osteopenia. Serum calcium was within normal limits. A fi nal diagnosis of anaplastic plasma cell myeloma was made based on the presence of serum IgGλ paraprotein and proliferation of malignant plasma cells in the marrow. S. Rao · R. Kar · H. P. Pati Department of Hematology, IRCH Building, All India Institute of Medical Sciences, New Delhi 110 029, India

Myelodysplastic syndromes: classification and prognostic scoring systems and their applicability in Indian scenario-experience from a tertiary care center.

Abstract The myelodysplastic syndromes (MDS) are a group of clonal disorders characterized by ineffective haematopoiesis, cytopenias, morphologic dysplasia and leukemic transformation. Difficulties exist in classifying and prognosticating MDS. This study was done to evaluate FAB and WHO classifications and the role of infection especially tuberculosis contributing to secondary myelodysplasia. The clinico-hematological profile of all cases (n=78) of MDS diagnosed over the last one and a half years was analyzed. This included 73 cases of primary MDS and five cases of infection associated myelodysplasia. There were 50 male and 28 female patients. Mean age at presentation was 46·1 years (range: 9 to 82 years). Out of 73 cases, two progressed to AML during the study period. Seventy cases could be classified based on FAB and 62 based on WHO criteria. Five cases of FAB-RAEBt were AML by FAB. One case not classifiable as per FAB could be categorized by WHO and four cases not classifiable as WHO could be categorized by FAB classification. All fulfilled the minimal diagnostic criteria for MDS. The commonest subtype of MDS was RA by FAB (55·7%) and RCMD (21%) and MDS-U (21%) by WHO. Four patients with tuberculosis and one with HIV showed significant myelodysplasia along with reactive changes. The consensus proposal of minimal diagnostic criteria for MDS was most helpful in cases difficult to diagnose and classify. Coexisting infection especially tuberculosis causing secondary myelodysplasia needs to be kept in mind especially in the Indian subcontinent.

Acquired platelet dysfunction in 109 patients from a tertiary care referral hospital.

Background: Acquired platelet function defects (PFDs) remain poorly characterized, underrecognized, and therefore understudied. Patients/Methods: Clinical and laboratory records of 109 patients with acquired PFDs diagnosed over 5 years were analyzed. Screening studies (platelet count, prothrombin time, activated partial thromboplastin time, and thrombin time), template bleeding time, platelet factor 3 (PF-3) availability test, light-transmission aggregometry, and further testing as indicated were performed. Results: 64 patients had mild and 26 had major bleeding. In all, 15 were referred for preoperative testing, whereas 4 had thrombotic events. Causes and associations of PFDs were drug-induced (34), idiopathic (34), hematopoietic neoplasms (15; myeloma 4, Waldenstrom macroglobulinemia 2, chronic myeloid leukemia 4, essential thrombocythaemia 3, and primary myelofibrosis and chronic lymphocytic leukemia 1 each), chronic liver disease (4), postcardiac surgery (2), uremia (2), and thalassemia major (7). Miscellaneous disorders comprised the rest. Conclusions: Acquired PFDs span a wide range of disease settings. Systematic, sequential laboratory testing identifies patterns of dysfunction, excludes inherited disorders, and streamlines management.

Hereditary Spherocytosis with High Fetal Hemoglobin: An Interesting Case

Raised Hb F is occasionally found in stress erythropoiesis associated with hemolytic anemias. In hereditary spherocytosis (HS), elevation of Hb F by 2–5% may be seen but Hb F in the range of 10–20% has not been reported. We present an interesting case of a child, initially presenting with high Hb F, who showed a spontaneous and progressive decline, and was subsequently diagnosed to have HS with raised fetal hemoglobin (Hb F) using an eosin-5-maleimide flow cytometric test.