Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 DOI 10.1007/s12288-012-0199-y

ABSTRACTS

53rd National Conference of Indian Society of Hematology & Blood Transfusion (ISHBT) 2012, 9–11 November 2012, Puri, India

Ó Indian Society of Haematology & Transfusion Medicine 2012

Acute Leukemias

Abstract P 002

Abstract P 001

Studies with the Different Extracts of Ruellia tuberosa Leaves Against AML Patients’ Cells and Normal Human Lymphocytes

Anti-Cancer Study of a Popular NSAID, Lornoxicam on Human Leukemic Cell Line and Human Hepatocellular Carcinoma Cell Line

Sayantan Dey1, Subhadeep Roy1, Moumita Ray1, Nilanjana Deb1, Aparna Gomes1, Chinmay Chowdhury2, Prithvish Banerjee3, Santanu Bose3, Shila Elizabeth Besra1

Subhadeep Roy1, Sayantan Dey1, Moumita Ray1, Nilanjana Deb1, Aparna Gomes1, Shila Elizabeth Besra1 1

Drug Development/Diagnostic & Biotechnology Division, Indian Institute of Chemical Biology (CSIR), 4 Raja S.C. Mullick Road, Kolkata-700032, West Bengal, India Objective: The aim of the present study was intended on cancer because they are the most responsible disease for causing significant morbidity, mortality, and incurring healthcare costs worldwide. Recent data have expanded the concept that many cancers arise from sites of infection, chronic irritation and inflammation. These observations imply that anti-inflammatory agents should have a potential in both the prevention and treatment of cancer. These drugs can also be used as adjuvant to the currently available chemotherapy and radiotherapy. Lornoxicam is used for the treatment of various types of pain, especially resulting from inflammatory diseases of the joints, osteoarthritis, surgery, sciatica, and other inflammations but on cancer no work done so far. We have studied the anti-proliferative, cytotoxic and apoptotic activity of Lornoxicam on human leukemic cell line and human hepatocellular liver carcinoma cell line. Method: We studied cell viability by Trypan blue exclusion, cytotoxicity study by MTT assay, morphological study by fluorescence microscopy and DNA fragmentation was studied by gel electrophoresis on U937 cell line and HepG-2 cell line. Result: Lornoxicam significantly inhibited the cell viability and cytotoxicity (MTT) in a time and concentration dependent manner. The fluorescence showed characteristic features of membrane blabbing, chromatin condensation the sign of early and late apoptotic changes in the cells after treatment with Lornoxicam. Gel electrophoresis study showed fragmented DNA in the form of ladder. Conclusion: The present study reveals that the Lornoxicam possesses potent anti-leukemic activity. Studies are in progress to identify the mechanism of anti-leukemic activity of Lornoxicam.

1 Drug Development/Diagnostic & Biotechnology Division, Indian Institute of Chemical Biology (CSIR), 4 Raja S.C. Mullick Road, Kolkata-700032, India; 2Department of Chemistry, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India; 3 Employee State Insurance Hospital Sealdah, 301/3, A.P.C. Road, Kolkata-700009

Objective: In the present scenario, the demand for herbal products is growing exponentially throughout the world. This enthusiasm seems to be a result of people all over the world looking to various alternative systems of medicine, especially herbal drugs which are claimed to be safe, equally effective in comparison to allopathic drugs and which provide some answer to some of the chronic diseases like cancer. Ruellia tuberose leaf has a wide range of biological activities which includes anti-diabetic, anti-inflammatory, antipyretic, analgesic etc. Therefore, we have studied the anti-leukemic activity on AML patient’s cell and compared with normal healthy human leukocytes with methanol and methanol–water extracts of Ruellia tuberosa leaves. Methods: Collection, identification and extraction of Ruellia tuberosa leaves and designated as RTLE. AML PBMNCs was isolated by Histopaque (Sigma), cell viability by Trypan blue exclusion and cytotoxicity study by MTT assay in both the cells. Morphological study was determined by Fluorescence microscopy and gel electrophoresis of fragmented DNA was observed. Results: The cell viability and MTT assay showed the methanol and methanol–water extracts of RTLE significantly inhibit the PBMNCs of AML patients in a time and concentration dependant manner but no toxicity towards normal lymphocytes after 24 h treatment. The Fluorescence microscopic images show early and late apoptogenic changes in the leukemic cells than control, treated with IC50 doses. DNA bands confirmed apoptosis with both the treatment of RTLEs. Conclusion: The present study reveals that both the extracts of Ruellia tuberosa leaf have potent antileukemic activity.

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Abstract P 003 Apoptotogenic Activity of Sulfonoquinovosyldiacylglyceride (SQDG): A Constituent of Azadirachta indica (Leaves) Against PBMNCs of AML and CML Patients Shila Elizabeth Besra1, Moumita Ray1, Subhadeep Roy1, Sayantan Dey1, Aparna Gomes1, Sukdeb Banerjee2, Nirup Bikas Mondol2, Prithvish Banerjee3, Santanu Bose3 1

Drug Development/Diagnostic & Biotechnology Division, Indian Institute of Chemical Biology (CSIR), 4 Raja S.C. Mullick Road, Kolkata-700032, West Bengal, India; 2Chemistry Division, Indian Institute of Chemical Biology (CSIR), 4 Raja S.C. Mullick Road, Kolkata-700032, West Bengal, India; 3Employee State Insurance Hospital Sealdah, 301/3, A.P.C. Road, Kolkata-700009 Objective: Cancer, a dreadful disease characterized by uncontrolled growth and spread of abnormal cells without apoptosis, is a leading cause of death worldwide. Elimination of cancer cells through apoptosis is the key target of cancer therapy. A sulfonoglycolipid identified as a sulfonoquinovosyldiacylglyceride (SQDG) isolated from leaves of Azadirachta indica showed significant anti- leukemic activity in U937 and K562 human leukemic cell lines. Therefore, we studied the apoptogenic activity of SQDG against PBMNC of ALL and AML patients. Methods: In vitro cell proliferation assay were done by MTT assay, morphological determination of the cells undergoing apoptosis by Fluorescent dye staining, DNA fragmentation by gel electrophoresis, quantization of apoptosis by flow cytometric analysis and PBMNCs isolated by lymphocyte separating fluid Histopaque (Sigma). Results: After treatment, SQDG significantly inhibited the metabolically active cell growth and cytotoxicity of both the AML and CML patient’s cells. The effect of SQDG on normal human peripheral blood mononuclear was studied by cell viability and cytotoxicity and was found to be lower than that on AML and CML cells, indicating its specificity towards cancer cells. The morphological study showed the characteristic features of apoptotic changes in the treated cells than control cells after 24 h. The induction of apoptosis was confirmed by using Annexin-FITC/PI staining by flow cytometric analysis and fragmented DNA was found in the form of ladder after treatment with SQDG. Conclusion: Study reveals that, sulfonoquinovosyldiacylglyceride (SQDG) may be used as novel chemotherapeutic agent in future for better treatment of cancer without systemic toxicity.

Abstract P 004 Treatment Related Complications in Patients with Acute Lymphoblastic Leukemia Rajesh Kashyap, Mukul Agarwal, Pradeep Kumar, Garima Agarwal

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 protocol (BFM 90 of BFM 95 protocol). Result: One hundred and one patients had 147 events of non-hematological complications. Gastrointestinal toxicity was the most frequent complication and occurred predominantly as hepatitis. Hyperglycemia was seen in 25 cases (24.7 %) and was most frequent in patients above the age of 20 years. Neurological complication was predominantly seen in patients below the age of 20 years (76.2 %) and seizures was the most common presentation. Osteonecrosis involving the hip joints was seen two adult young males. Conclusion: Non-hematological complications are quite frequent during the late phase of treatment of ALL patients. The Gastrointestinal tract, nervous system and endocrine system most frequently affected. Many of these events are missed because of low levels of clinical suspicion. This study highlights the incidence of these complications as they are associated with high morbidity and mortality.

Abstract P 005: Poster Presentation Acute Erythroid Leukaemia: A Clinico-Hematological Review of 5 Case Series Nidhi Rai, M Deepak Nayak1, Chethan Manohar, Sushma V Belurkar Department of Pathology, Kasturba Medical College, Manipal; Department of Pathology, Melaka Manipal Medical College, Manipal

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Introduction: Acute erythroid-leukaemia is a rare form of acute myeloid leukaemia (AML), characterized by abnormal proliferation of erythroid precursors (proerythroblasts and basophilic erythroblasts). It comprises \5 % of AML cases. Objective: To review the clinico-hematological features of erythroleukaemia cases with review of literature. Materials and Methods: We report 5 such cases seen in our institution over a period of 1 year. 4 out of these 5 cases presented with pallor, easy fatigability and hepatosplenomegaly, while one case reported with non-classical symptoms. Peripheral smear examination and complete haemogram revealed pancytopenia with circulating blasts ([20 %) in all five cases. Bone marrow study yielded [50 % proerythroblasts and basophilic erythroblasts and dyspoiesis in other lineages. Results: Final diagnosis of Erythroleukaemia (erythroid/ myeloid) was made in four cases and one case with non-classical presentation was typed morphologically as Pure Erythroleukaemia. Conclusion: Erythroleukaemia is an uncommon hematopoietic neoplasm. Among these, pure erythroleukaemia is seldom reported in literature and is known to have poor response to standard chemotherapy. In the present case series, clinical presentation did not differ from other types of acute myeloid leukaemia cases. These five cases presented in the most unobtrusive manner. Thus awareness of its hematological and morphological features is necessary to avoid a diagnostic dilemma since treatment protocols and prognosis are varied.

Department of Hematology, SGPGIMS, Lucknow, UP Aim: To study the different non-haematological complications occurring during the late phase (2 weeks of induction therapy to remission) treatment of ALL patients. Materials and Methods: Patients diagnosed with ALL and being treated at department of haematology, SGPGIMS were subject of the study. The diagnosis of ALL was made based on complete hemogram, bone marrow examination with cytochemistry and immunophenotyping by flow cytometry. The clinical records of the patients were analysed for occurrence of non-haematological complications occurring 2 weeks after the start of therapy. The patients were treated as per the BFM

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Abstract P 006 Clinical, Hematological, Cytogenetic & Molecular Profile of Acute Myeloblastic Leukemia J Latha Fathima, S Sitalakshmi, Parimala Puttaiah, Poornima D Rao, A Vanamala, AM Shanthala Devi, Karuna Ramesh Kumar Department of Clinical Pathology, St. John’s Medical College Hospital, Bangalore;

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Introduction: Acute myeloid leukemia is a disease resulting from the clonal expansion of myeloid blasts in the peripheral blood, bone marrow or in other tissue. It is a clinically, morphologically, genetically and prognostically heterogeneous disease. In India the incidence of AML is 2.8 to 3.5 cases per 100,000 population per year. The course and prognosis of the disease in the western countries and India differs. Objectives: 1. To study the clinical and hematological features of AML. 2. To study the cytogenetic and molecular abnormalities of AML. 3. To compare the course and prognosis of the disease with western countries. Methods: This retrospective study was done from the year 2010 to July 2012 for a period of 30 months in the department of Clinical Pathology, St.John’s Medical College Hospital, Bangalore. The clinical details were retrieved from the patient records in the medical records department. The laboratory parameters were obtained from the department of Clinical Pathology. Result: The total number of cases were 131. The age ranged from 2 to 84 years. The peak incidence was in fourth decade. The most common presentation was fever. Morphologically AML-M2 was the common subtype followed by acute promyelocytic leukemia. Immunophenotypically, CD 13 was the most commonly expressed (62 %) myeloid marker. CD 7 was aberrantly expressed in 28 %. PML-RARA translocation t(15:17) was the common cytogenetic abnormality. One case of therapy related AML was also encountered. Conclusion: The common subtype is AML M2 followed by APML. The commonest cytogenetic abnormality noted is PML-RARA translocation. Keywords Acute myeloid leukemia, Acute promyelocytic leukemia, Cytogenetics

Abstract P 007 Extensive Extramedullary Involvement in a Child with Acute Myeloid Leukemia Anand Prakash1, Athira Ramakrishnan2, Balasubramaniam2, Suravi Mohanty3, Anuradha Ananthamurthy3

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Abstract P 008: Poster Presentation CD133 and MLL in the Same Cup G Smeeta1, Anita Chopra1, C Jagan1, Sameer Bakhshi2, Sunu Lazar Cyriac2, Rajive Kumar1 1 Laboratory Oncology Unit, 2Department of Medical Oncology, Dr. B.R.A Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, Ansari Nagar, New Delhi

Introduction: CD133 positivity has been described in, but is rarely ever emphasized as a facet of mixed lineage leukemia (MLL)+ pro-B acute lymphoblastic leukemia (ALL). Nuclear cupping is a feature that hardly ever finds mention outside acute myeloid leukemia (AML). We present two cases where these coexisted. Case Summary: The first patient, a 5-year old girl, diagnosed B-lineage ALL 4 years back and treated on UK-MRC-ALL-2003 protocol till 2010, and symptom-free for 2 years, presented to our institution in June 2012 with fever and lymphadenopathy. Peripheral blood smear showed 80 % blasts; with over 80 % having prominent cup-like nuclear invagination. The second patient was a 10-month old male, presented with fever and splenomegaly. Peripheral blood smear showed 90 % blasts, 20 % exhibiting cup-like morphology. In both patients, blasts were positive for CD34, CD45, CD19, HLA DR, CD133, CD38, CD15, cCD79a, CD117dim and negative for CD10, cCD22, CD20, CD13, CD33, CD56, CD2, CD4, CD64, CD14, cCD3 and cMPO. NG2 was positive in 10 and 100 % blasts in first and second case, respectively. Both the patients were diagnosed as pro-B ALL. MLL gene rearrangement was identified in both by fluorescent in situ hybridization using a dual color, break apart rearrangement probe. Conclusion: We believe ours is the first case showing florid nuclear cupping in association with CD133 positivity and MLL rearrangement in a setting of pro-B phenotype. We conclude that nuclear cupping, even when present in nearly every blast cell, may not necessarily mean AML and may be a pointer toward CD133 positive MLL translocated pro-B ALL. CD133 should be evaluated as part of a B-lineage ALL flow cytometric panel with a view to define its role as a reliable indicator of MLL rearrangement.

St Johns Medical College Hospital, Department of Pediatrics, Bangalore, India; 2St Johns Medical College Hospital, Department of Otorhinolaryngology, Bangalore, India; 3St Johns Medical College Hospital, Department of Pathology, Bangalore, India

Keywords Cupping, CD133, MLLL

Purpose: Extramedullary involvement can occasionally be the presenting feature of Acute Myeloid Leukemia in children. We report a child who presented with features of extensive paranasal, orbital, lymph node and dural involvement of chloromas with facial nerve paralysis. Method: Case Report: A 12 year old girl presented with fever, loss of hearing, proptosis and headache of 2 months duration. Clinical examination revealed mild left orbital proptosis with painless cervical lymphadenopathy. She had no hepatosplenomegaly. Cranial imaging showed dural enhancement in the left frontoparietal region with extensive maxillary, ethmoid and sphenoid sinus obliteration. There were minimal deposits in both retro-orbital areas. Peripheral blood film and bone marrow aspirate revealed myeloblasts of 10 %. A biopsy of the paranasal mass showed features of a granulocytic sarcoma. Immunohistochemistry was strongly positive for Myeloperoxidase and CD117 and was negative for CD20 and CD3. The Ki 67 index was about 50 %. CSF was negative for malignant cells. Conventional Cytogenetics was attempted but unsuccessful. Results: The patient has completed chemotherapy (induction with cytarabine and daunorubicin and consolidation with three cycles of high dose cytarabine). She had a complete resolution of her proptosis, facial palsy and the paranasal sinus mass. She is currently well and in remission. Conclusion: Extensive extramedullary manifestations of AML can present with paranasal sinus extension and cranial nerve palsy.

Acute Promyelocytic Leukemia—from Morphology to Molecular Diagnosis

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Abstract P 009: Oral Presentation

Tathagata Chatterjee1, Srishti Gupta2, Ajay Sharma2 1 Armed Forces Medical College, Pune-40; 2Army Hospital (R&R), Delhi Cantt-10

Introduction: Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) with typical clinicohematological features. Cytogenetically, it is predominantly characterized by balanced reciprocal translocation between chromosome 15 and 17 which results in fusion between promyelocytic leukemia (PML) gene and Retinoic acid receptor a (RARa) gene. There are 3 possible isoforms caused by these translocations. The breakpoint in chromosome 17 is consistently found in intron 2, but varies in chromosome 15. The 3 breakpoints on the PML gene can occur at intron 3 (L-long form), intron 6 (S-short form), and exon 6 (V form). The study on molecular characterization was undertaken due to lack of sufficient data in Indian patients. Aims and Objectives: (1) To study the clinic-hematological and morphological profile in APL patients. (2) To study molecular characterization of BCR subtypes in Indian APL patients. Materials and Methods: A prospective study of

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194 fifteen APL patients presenting to Army Hospital (R&R) were taken for study between November 2010 to March 2012. The clinical features, hematological parameters and morphology were analyzed. Peripheral blood and bone marrow aspirate were stained with Leishman-Giemsa, myeloperoxidase (MPO) and chloroacetate esterase (CAE) and non specific esterase (NSE) using Merck’s Diagnostic reagents. Flowcytometric evaluation was done on bone marrow aspirate/peripheral blood using Beckman Coulter FC 500, 4 color flow cytometer using standard lyse wash technique. For molecular studies, peripheral blood sample was collected and Real Time PCR was performed using Fusion QuantÒ kits for bcr-1, bcr-2 and bcr-3 and Rotor GeneTM 3000 software. Results: Patients presented with fever, loss of appetite, bleeding manifestations (petechial, conjunctiva hemorrhage, gum bleed, vaginal bleed and bleeding per rectum) and pallor. Median age was 42 years, TLC—4,500/mm3, Hb—7.5 g/dl and platelet—35,000/mm3. Three patients were micro granular variants and rest were hyper granular variants. All cases stained for MPO and CAE and 33.33 % for NSE. Flowcytometric analysis revealed classic high SSC with dim CD45 scatter with hyper granular variants and low SSC with dim CD45 with micro granular variants. Molecular analysis revealed BCR1 in 6/15 (40 %), BCR2 in 3/15 (20 %) and BCR 3 in 6/15 patients (40 %). Conclusions: In our study no correlation was found between age, sex, TLC, hemoglobin and platelet counts with different BCR isoforms. There was no significant increase in particular isoform in Indian population unlike published data. Morphological, cytochemical, flow cytometry and molecular diagnosis were achieved within 48 h of admission.

Abstract P 010 Trisomy Chromosome 6 as a Sole Cytogenetic Abnormality in Acute Myeloid Leukemia

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Paediatrics, 4Bioststistics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi Introduction: Early T cell precursor acute lymphoblastic leukemia (ETP-ALL), a newly identified subtype of T-ALL, is characterized by a strikingly distinct immature immunophenotype: CD5-, CD1a, CD8and expression of C1 myeloid or stem cell markers (CD117, CD34, HLA-DR, CD13, CD33, CD11b, and/or CD65) on C25 % of lymphoblasts. It has been reported to be associated with poor prognosis. A very few studies, none from India, have evaluated the clinical and prognostic significance of ETP-ALL cases. Objective: To determine the incidence and clinical significance of ETP-ALL. Methods: Fifty five cases of T-ALL were retrieved from the records of Laboratory Oncology, AIIMS. The clinical and immunophenotypic characteristics of all cases were noted. The immunophenotypic markers used were: CD3, CD45, CD5, CD8, CD1a, CD13, CD33, CD117, HLA-DR, CD34, CD65 and CD11b. The cases were subclassified based on expression of CD5: CD5+ and CD5-. These were further subclassified into CD8+/CD1a+ and CD8-/CD1a- subgroups. In all these groups, presence of myeloid and/or stem cell markers were also noted. The remission rate, relapse rate and overall survival of these groups were compared. Results: CD5 was positive in 48 and negative in 7 cases. In the CD5+ group, 31 cases were CD8+/CD1a+ (myeloid/stem cell markers: positive 19 and negative 12) and 17 were CD8-/CD1a- (myeloid/stem cell markers: positive 16 and negative 1). All CD5- cases (n = 7) were CD8-/ CD1a- and myeloid/stem cell markers positive. All CD5- cases (12.7 %) fulfilled the criteria for ETP-ALL. ETP-ALL cases had a poor remission rate as compared to non- ETP-ALL cases (40 vs. 86.7 %; p = 0.042). Death rate was also higher in ETP-ALL group (28.6 vs. 6.25 %; p = 0.02). Conclusion: ETP-ALL is a distinct, high risk subtype of T-ALL, that must be identified by correctly designed immunophenotyping panels, so that therapy appropriate to the disease and distinct from the non-ETP- type T-ALL can be instituted. Recognition of the disease provides a fresh and meaningful perspective on the nebulous concept of lineage infidelity of acute leukemias.

Monika Gupta, Nita Radhakrishnan, Manoranjan Mahapatra, Renu Saxena Department of Haematology, All India Institute of Medical Sciences, New Delhi, India Abstract: Identification of cytogenetic abnormalities plays an important role in the diagnosis and prognosis of leukemias. Isolated trisomy 6 is a rare abnormality, the prognostic significance of which is not well established. We report one case of acute myeloid leukemia (AML M5 variant) with trisomy 6 as the sole cytogenetic abnormality. Previously, trisomy 6 has been reported in aplastic anaemia, myelodysplastic syndrome and AML and they are usually associated with hypocellular marrow. However our patient had a very short history and a hypercellular marrow infiltrated with blasts. We report this case due to the rarity of the condition. More studies are required to ascertain the role of trisomy 6 in the development of leukemia as well as in prognosis.

Abstract P 011 Clinical and Immunophenotypic Characterisation of Early T Cell Precursor Acute Lymphoblastic Leukemia: a First Study from India G Smeeta1, Anita Chopra1, C Jagan1, Lalit Kumar2, Atul Sharma2, Sameer Bakhshi2, Rachna Seth3, Ajay Gogia2, RM Pandey4, Rajive Kumar1 Laboratory Oncology Unit, 2Department of Medical Oncology, Dr. B.R.A. Institute Rotary Cancer Hospital, 3Department of

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Abstract P 012 Biphenotypic Acute Leukemia: A Study of Clinical, Hematological & Immunophenotypic Profile Man Updesh Singh Sachdeva1, Manupriya1, Neelam Varma1, Subhash Varma2, RK Marwaha3 Department of Hematology, 2Department of Internal Medicine, Department of Paediatrics, Postgraduate Institute of Medical Education & Research, Chandigarh 1

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Background: Acute biphenotypic leukemia (BAL) is a rare neoplasm comprising of blasts showing more than one lineage on multi-colour flow cytometry. This prospective study was designed to identify cases of biphenotypic acute leukemia and study their clinical and hematological profiles. Methodology: EDTA anticoagulated bone marrow aspirate/peripheral blood samples of patients diagnosed as acute leukemia on the basis of morphology were utilized for immunophenotyping. A comprehensive panel of fluorochrome labeled monoclonal antibodies was used to identify the lineage of leukemic cells on flow cytometry. The patients diagnosed to have BAL, on basis of WHO 2008 classification, were selected for analyses of their clinical, hematological and immunophenotypic profile. Results: BAL represented 2.99 % (15/ 501) of all cases of acute leukemia over 2 years. 47 % (7/15) were children, all males, mean age of 5 years. 53 % (8/15) were adults, M:F = 6:2, mean age of 21.4 years. 53 % (8/15) were diagnosed as B/Myeloid and 47 % (7/15) were T/Myeloid. No correlation was observed between age and immunophenotype of BAL. Fever and

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lymphadenopathy were more frequently observed in BAL than ALL and AML, respectively. Thrombocytopenia was less frequent as compared to ALL or AML. On morphology, (73 %) 11/15 were diagnosed as AML and 27 % (4/15) were diagnosed as ALL. Conclusions: BAL is a rare type of acute leukemia (2.99 % of all our AL cases). Most of the clinical, hematological and morphological parameters fail to clearly predict its occurrence and a comprehensive panel of antibodies should be used to identify this neoplasm known to have a poor outcome.

6 weeks. The patient’s peripheral blood showed a leukoerythroblastic blood picture with Hb of 3.4 g/dL, TLC 6,400/mm3, and Platelet count of 89,000/mm3. Bone marrow aspirate and biopsy revealed a markedly hyper cellular bone marrow replaced by erythroid precursors, representing approximately 53 % of the marrow cells and Myeloblasts representing 49 % of non erythroid cells. The erythroid precursors displayed dysplastic morphology, including megaloblastic features, multinucleation, nuclear lobation and budding, and they were periodic acid-Schiff (PAS)–positive. So a diagnosis of acute erythroid leukemia (erythroid/myeloid) was made.

Abstract P 013

Keywords Erythroid leukemia, Erythroid/myeloid

Isolated CNS Relapse of in a Case of AML-M2 Mimicking Posterior Fossa Intracranial Haemorrhage—a Case Report and Brief Review of Literature K Kishore, UK Nath, SS Roy, P Chakrabarty, U Chaudhuri Institute of Hematology and Transfusion medicine, Kolkata Objective: This report is intended to stress the importance of looking for subtle clinical signs of CNS RELAPSE during follow-up visits even when the blood counts are normal in AML patients. Methods: This 17/f a case of AML-M2, 46XX, NPM1-FLT3-ITD/Asp835 Negative patient achieved CR after one 7 + 3 and was further treated with 3 cycles of HIDAC. She was in CR post consolidation. During her follow up visit, in spite of normal counts, she complained of mild headache and nausea. Her CT scan of the brain revealed increased attenuation in the midline posterior fossa with compressed IV ventricle and a provisional diagnosis of intracranial haemorrhage was given. In view of normal coagulation parameters we reviewed the plates again and went in for MRI and CSF analysis. Results: MRI showed an heterogeneous area in the posterior fossa in superior vermis region suggestive of a mitotic mass lesion. CSF also shows blasts confirming an isolated CNS relapse as bone marrow is also in remission now. Conclusion: The patients who were treated with only local therapy (intrathecal chemotherapy with or without radiation therapy) had an overall survival rate of 31.5 % compared to 21.4 % in patients treated with systemic therapy. The AML subtype, CNS-1 status at diagnosis, age at relapse, cytogenetic characteristics and the initial CT picture makes the present case unique.

Abstract P 014 Erythroleukemia (Erythroid/Myeloid): A Case Report T Santosh, RK Bhola, A Choudhary, AK Bal, MK Patro, J Naik, B behera, S Paradhan Department of Pathology, MKCG Medical College, Berhampur, Odisha Background: Acute erythroid leukemia is a rare form of acute myeloid leukemia (AML) with predominant erythroid lineage proliferation. It is a heterogeneous entity amongst AML that can occur at any age, including childhood, and comprises less than 5 % of AML. It’s defined as ‘‘a proliferation of more than 50 % erythroblast and [20 % myeloblasts within nonerythroid cells.’’ AML-M6 is a heterogeneous disease with poor response to standard chemotherapy that carries a poor prognosis. The new WHO classification subdivides acute erythroid leukemia into erythroleukemia (erythroid/myeloid) and pure erythroid leukemia. Allogeneic bone marrow transplant should be considered upfront for appropriate candidates once remission is achieved in AML-M6, as the risk of relapse and mortality is very high with this disease. Case Report: A 36 year Hindu female presented with fatigue, generalized body aches, pain since last

Abstract P 015 Study of Clinico-Hematological and Immunophenotypic Profile in Patients with Acute Lymphoblastic Leukemia in SCB Medical College & Hospital, Cuttack, Odisha Bhattacharyya Debmalya1, Das Sidhartha1, RK Jena2 1

Department of Medicine, SCBMCH, 2Department of Clinical Hematology, SCBMCH Aims and Objectives: To study clinical, hematological and immunophenotypic profile of patients with Acute Lymphoblastic Leukemia (ALL) and their association with mortality and remission. Methods: 60 naı¨ve consecutive cases of adult ALL (C15 years) were taken for study. Secondary and relapse ALL cases were excluded. Detailed history taking, clinical examination, hematological parameters, bone marrow study and Immunophenotyping using bone marrow sample or peripheral blood by Flow cytometer (BD FACS CALIBUR) were done in all cases. Patients who survived were treated with MCP-841 protocol and hematological remission was evaluated after completion of induction phase (1 month). Results: 68.3 % cases were suffering from B-ALL and 31.7 % from T-ALL. 58.3 % of cases were young adults (between 15 and 24 years of age). Bleeding manifestation was significantly associated with B-ALL (p \ 0.01) and lymphadenopathy was associated with T-ALL (p \ 0.01). FAB-L1 and FAB-L2 morphology in bone marrow was found in 38.3 and 61.7 % of cases respectively. In patients with B-ALL, cCD79a was positive in 100 % of cases, followed by CD19 (80.49 %), CD10 (85.37 %), CD34 (43.9 %). In patients with T-ALL, cCD3 was positive in 100 % cases, followed by CD7 (94.73 %), CD5 (63.16 %) and CD34 (21.05 %) 0.21.67 % of cases had myeloid co-expression, of which CD13 was the most common (53.84 %), followed by CD33 (38.46 %) and CD117 (7.69 %) 0.24.4 % of B-ALL and 15.8 % cases of T-ALL had myeloid co-expressions. 25 % of cases died before completion of induction phase and 46.7 % cases achieved complete remission (CR) after 4 weeks of induction phase. 21.7 % cases had partial remission and only 6.7 % cases had no remission. 60.97 % of B-ALL cases could achieve CR whereas only 15.78 % of T-ALL cases could achieve CR (p \ 0.002). Conclusion: B-ALL is more prevalent than T-ALL. For Immunophenotyping a panel of CD markers is required. Prognosis of B-ALL is better than T-ALL.

Abstract P 016 Pattern of Occurrence of Childhood Leukemias in a Tertiary Centre in Lucknow: A Eleven Year Study R Kushwaha, A Kumar, US Singh, K Archana King George Medical University. Lucknow Introduction: Pattern of childhood leukemia is known to vary through out the world. We present clinico-pathological profile of children

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196 presenting with Leukemia in our centre during a span of 10 years from January 2001 to August 2012. This reflects the leukemia pattern in Eastern U.P from where no such data is published till date. Materials and Methods: A retrospective and prospective study of children presenting with Leukemia was done from January 2001 to August 2012. Clinical details like demographic data, age, sex and presenting symptoms were noted. Peripheral smear was made and bone marrow aspiration was done. Smears were stained with Giemsa stain and were studied for morphology of cells. FAB classification was used to classify Leukemias based on morphological basis. Results: A total of 888 cases of Leukemia were studied. Distribution of cases males and females showed that it was more common in males (72.86 %). Youngest patient of the series was 2 month child presenting with Chronic Myeloproliferative Disorder, which is very rare in this age group. Acute Leukemia cases were 89.86 %. Chronic Leukemia was very rare (10.13 %). Among Acute Leukemia 64.53 % cases were of Acule Lymphoblastic Leukemia, 32.20 % cases were of Acute Myeloid Leukemia and 3.25 % case were of Acute Undifferentiated Leukemia. Cases of Acute Leukemia were equally distributed in two age groups, i.e. 0–5 years and 6–10 years. ALL-L2 was the commonest type of ALL (59.7 %). ALL-L3 was seen only in 3.37 % of cases. In acute myeloid leukemia AML-M2 was more common. Conclusion: There is geographic variation in the incidence of leukemia. In our series we have observed that ALL-L2 is the most common type of childhood leukemia. Cases were equally distributed from 0 to 10 years.

Abstract P 017 A Rare Clinical Antithesis of Hope and Despair: A Case Report P Pujari Ganesh, LS Raut, VV Bohara, GV Badarkhe, SS Ray Institute of Hematology & Transfusion Medicine, Kolkata Abstract: Myeloid sarcoma is an extramedullary tumour of immature cells of granulocytic series, generally occurring in approximately 2 % of patients with acute myeloid leukaemia. Myeloid sarcoma occurs mostly in adults aged 45–55 years, and it has a predilection for the bone, soft tissue, and skin. We here report a 28 year old female, presenting with fever, menorrhagia and gum hypertrophy. She was diagnosed as Acute Myeloid Leukaemia (M4) and was treated with 7 + 3 induction therapy. On day 14 post-induction, multiple nodular skin lesions developed gradually over both lower extremities. The biopsy from the lesions revealed Leukemia cutis. Post-induction day 14 marrow revealed no evidence of disease and medullary remission was documented on day 28. However, the skin lesions increased. With high dose Arabinoside and Mitoxantrone (HAM) the lesions completely disappeared after 10 days. However, patient succumbed to septic shock on day 18 of HAM. This case highlights the extremely unusual concurrent medullary remission and extramedullary relapse in a case of acute myeloid leukemia treated on induction therapy. Keywords Myeloid sarcoma, Induction, Failure, Leukemia cutis

Abstract P 018 Presence of FLAER Negative Population in Acute Leukemia Kotteeswari Kathirvel, Bargavi Balakrishnan, Harikrishnan Babu, Ansu Abu Alex, Rayaz Ahmed, Aby Abraham, Auro Viswabandya, Biju George, Vikram Mathews, Alok Srivastava Department of Haematology, Christian Medical College, Vellore Background: Fluorescent-labeled bacterial aerolysin (FLAER) is the single most reliable marker to monitor small PNH clones especially in

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 conditions such as myelodysplastic syndromes when traditional GPIlinked surface marker expression can be significantly altered. Studies with FLAER have described a sensitivity ranging between 0.5 and 1 % in identifying GPI-negative WBCs in samples from aplastic anemia patients (Cytometry B Clin Cyto, 2007). Also it has been shown that 5-15 % of PNH patients develop leukocyte dyscrasias which invariably are acute myelogenous leukemia (Leuk Lymphoma. 1999). Objective: To detect a FLAER negative population in acute leukemia samples by a single tube multiparameter flowcytometric assay. Methods: Peripheral blood and bone marrow samples of patients with newly diagnosed acute leukemia received for immunophenotyping are taken for this study. In a single tube assay, we have combined FLAER with CD33, CD14 and CD45 for the detection of PNH clone on blast and neutrophils of patient samples and neutrophils of healthy control samples simultaneously. 20,000 events were acquired and analyzed for all the cases using FACS Calibur. Blasts and neutrophils were gated by CD45 versus side scatter (R1) and CD33 versus side scatter (R2) as shown in Fig. 1a. Results: We have analyzed 31 newly diagnosed acute leukemia among which 13 cases of AML and ALL each (41.9 %) and 5 cases of APL (16.12 %). The median age of AML, APL and ALL cohorts are 29 years (range: 13–73), 27 years (range: 12–37) and 3 years (range: 0–28) respectively. The healthy control reference range for FLAER expression on neutrophils was 99.98 % (range: 99.79–100). The FLAER negative clone was detected on both blasts and neutrophils of patient samples. In AML the median range of FLAER-ve cells on blasts is 74.4 (range: 0.92–90.18) which is significantly higher than in ALL of 13.56 (range: 4.13–91.22) (p = 0.006) (Fig. 1b) whereas on neutrophils are 11.31 (range: 1–59.06) and 0.62 (range: 0–8.65) respectively (p = 0.0003) (Fig. 1c). Conclusion: In our study FLAER negative population were more common in acute leukemia and significantly higher in AML than ALL. This preliminary observation warrants a more detailed study on the biology, relevance and prognostic impact of this population on treatment outcomes.

Abstract P 019 Study of Incidence of Recurrent Genetic Translocations in Adult Acute Myeloid Leukemia Shano Naseem1, Neelam Varma1, Prateek Bhatia2, Jogeshwar Binota1, Subhash Varma3, Pankaj Malhotra3 1 Departments of Hematology, 2Paediatrics, 3Internal Medicine; PGIMER, Chandigarh

Background: World Health Organisation (WHO) classification of acute myeloid leukemia (AML) incorporates morphologic, immunophenotypic, genetic and clinical features in its classification scheme and is believed to be of more clinical relevance then the FAB classification as it defines entities that are biologically homogeneous and prognostically relevant. The latest 2008 WHO classification includes a separate category of AML with balanced translocation/ inversion, which comprises of 2 provisional and 7 categories of characteristic genetic abnormalities, including: (i) t(8;21);RUNX1RUNX1T1, (ii) t(15;17);PML-RARA, (iii) inv(16);CBFB-MYH11, (iv) t(9;11);MLLT3;-MLL, (v) t(6;9);DEK-NUP214, (vi) inv3;RPN1EVI1 and (vii) t(1;22);RBM15-MKL1. Of these, t(8;21);RUNX1RUNX1T1, t(15;17);PML-RARA and inv(16) CBFB-MYH11 have been reported in higher frequency than other translocations. Western literature quotes the incidence of fusion transcripts to be around 40–45 % in AML. However the data from Indian sub-continent is scarce. We, therefore planned this study to detect the incidence of common translocation/chimeric fusion transcripts in adult patients with AML for t(8;21);RUNX1-RUNX1T1, t(15;17);PML-RARA, and

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

Fig. 1 Analysis of FLAER-ve subset in newly diagnosed acute leukemias. a Representative gating strategy to quantify GPI deficient populations in AML. b FLAER-ve (%) on CD45dim population in newly diagnosed acute leukemias. c FLAER-ve (%) on neutrophils in newly diagnosed acute leukemias

(inv16); CBFB-MYH11 using a single multiplex RT-PCR assay. Materials and Methods: The present study was carried in the department of Hematology, PGIMER, Chandigarh from May 2010 to May 2012. Cases diagnosed as AML by bone marrow morphology, cytochemistry and flow cytometry immunophenotyping were enrolled in the study. For detection of fusion transcript, a single multiplex RT-PCR assay was carried out using primers specific to the transcript being tested. Results: During the study period 125 cases (male: female ratio = 1.2:1) of AML were tested for above mentioned fusion transcripts. Of these, 86 (68.8 %) cases were negative and 39 (31.2 %) cases were positive for any of the fusion transcript. t(15;17);PML-RARA was most common seen in 19/125 (15.2 %) cases, followed by t(8;21);RUNX1-RUNX1T1 in 17/125 (13.6 %) cases and inv(16);CBFB-MYH11 in 3/125 (2.4 %) cases. Conclusion: This study evaluated the incidence of common recurrent genetic translocations/chimeric fusion transcript in 125 cases of adult AML. The incidence of various fusion transcripts was 31.2 %, with t(15;17);PML-RARA and t(8;21);RUNX1-RUNX1T1 being more frequent, seen at a frequency of 15.2 and 13.6 % cases respectively. inv(16);CBFB-MYH11 was less common. Identification of these transcripts provides therapeutically and prognostically relevant clinical information.

Abstract P 020 A Study of Hematological and Genetic Profile of Precursor B Lymphoblastic Leukemia/Lymphoma Harbouring BCR-ABL Recurrent Genetic Abnormality Anita Tahlan1, N Varma1, S Naseem1, J Binota1, D Bansal2, P Malhotra3, RK Marwaha2, S Varma3 Departments of Hematology1, 2Pediatric Hemato-Oncology and 3 Internal Medicine, PGIMER, Chandigarh Introduction: Approximately 3 % of children and 25–30 % of adults with B lymphoblastic leukemia/lymphoma (B-ALL) are reported to harbour t(9;22)(q34;q11) and/or BCR-ABL positivity. This subset of

197 patients is considered a poor risk subgroup. Aim: To determine the hematological and genetic profile of precursor B-ALL with BCR-ABL positivity. Materials and Methods: The study was conducted from June 2010 to June 2012, at PGIMER, Chandigarh. Peripheral blood and bone marrow examination and immunophenotyping were done. Molecular analysis by reverse transcriptase PCR (RT-PCR) for BCR-ABL hybrid transcripts was performed on bone marrow or peripheral blood samples. Results: A total of 302 patients were diagnosed to have ALL, of which 257 were B-ALL. Seventeen patients (6.6 %) positive for BCR-ABL transcripts were included in the study. Median age was 26 years (range 1 year to 49 years). 13 out of 17 patients were males (76.4 %) and four females (23.5 %). Seven patients (41 %) were below 18 years of age. The total leucocyte count ranged from 38.6 to 404.9 9 109/L (median 49.5 9 109/L). Bone marrow morphology showed a heterogeneous population of immature cells in 13 cases with prominent nucleoli, irregular to indented nuclear membrane and cytoplasmic blebbing/vacuolations. The immunophenotypic profile was positive for the lymphoid markers in all cases. In addition five cases showed positivity for myeloid markers CD13 and CD33, One case showed positivity for CD13, CD33 and CD 117. RT-PCR for BCR-ABL transcript showed e1a2 in 12 cases (70.5 %), b2a2 in 3 cases and b3a2 in 2 cases (29.4 %). Conclusions: The overall prognosis improves with the addition of tyrosine kinase inhibitors, in the treatment protocols of BCR-ABL positive B-ALL. In a resource constraint setting, an attempt should be made to evaluate BCR-ABL if the morphology is heterogenous, cytoplasmic blebbing/ vacuolations are noted, along with aberrant positivity for myeloid markers CD13 and CD33. Keywords B-ALL. BCR-ABL transcripts

Abstract P 021 The Clinical Outcome of Children with Philadelphia chromosome (Ph) Positive Acute Lymphoblastic Leukemia (ALL) in a Tertiary Care Centre KG Srinivas, L Appaji, Aruna Kumari, KC Lakshmaiah Department of Medical and Pediatric Oncology, Kidwai Memorial Institute of Oncology, Bangalore, Karnataka Objective: To evaluate the clinical outcome of children with Ph positive ALL who received chemotherapy without tyrosine kinase inhibitors (TKIs) from 2004 to 2009. In the current era of TKIs this study serves as historical reference to evaluate the therapeutic impact of TKIs on the outcome of Ph positive ALL. Methods: 28 children, age less than 15 years with Ph positive ALL, registered at Kidwai Institute of Oncology, from 2004 to 2009, who received standard chemotherapy alone, according to MCP 841 protocol were analysed retrospectively. Outcomes were compared with 28 Ph negative ALL children who have taken same treatment during the same period. Matching was done for appropriate parameters. Philadelphia chromosome positivity was confirmed by translocation t(9:22) by cytogenetics. Results: In our study. 19 (67.8 %) were males and 9 (32.2 %) were females (M:F 2.1:1). 21 (75 %) were less than 9 years and 7 (25 %) were between 9 and 15 years. Complete remission was attained in 82.1 % of children after induction treatment (Day 28). 10 (35.7 %) had early relapse, among them 7 (25 %) had early bone marrow relapse, 2 (7.1 %) had early CNS relapse, 1 (3.5 %) had early testicular relapse. 3 (10.7 %) had late bone marrow relapse. There were three deaths during induction due to sepsis. The Event free survival (EFS) and Overall survival (OS) at 3 years was 42.8 and 52 % respectively. Median OS was 38 months. In Ph negative ALL, EFS and OS at 3 years was 73 and 80 % respectively. The median duration of follow up is 37 months (range 2 months to 90 months).

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Conclusion: Children with Ph positive ALL have inferior outcome with respect to EFS and OS when compared to Ph negative ALL when given standard chemotherapy alone. Due to limited resources, none of our children could afford transplantation. With the encouraging results of combining Imatinib (TKI) with standard chemotherapy in recent studies, these high risk children should be offered combined modality of treatment.

Abstract P 022 Novel NPM Mutation in the 30 -Untranslated Region Identified in a Patient with Acute Myeloid Leukemia Vinodhini Kumaraswamy, Ajay Abraham, Ashok Kumar Jeyavelu, M Sathya, Vivi M. Srivastava, Biju George, Alok Srivastava, Vikram Mathews, Poonkuzhali Balasubramanian Department of Haematology, Christian Medical College, Vellore Abstract: Mutations in nucleophosmin (NPM) gene are known to occur most frequently in normal karyotype acute myeloid leukemia (NK-AML). The frequency of exon 12 NPM mutations in adult AML patients’ ranges between 25 and 35 %, accounting for more than 50 % in adult NK-AML. NPM is a nucleo-cytoplasmic shuttling protein initially known to be involved in rearrangements in leukemia and lymphoma. The NPM exon 12 mutations are mostly 4 bp insertions, which disrupts the nucleolar localization signal (NoLS) at the C terminus of the NPM protein causing cytoplasmic accumulation of truncated NPM protein. The most common among NPM mutation is type A, with the insertion of tetranucleotide TCTG in the exon 12 seen in 75 to 80 % of cases. Type B and D mutations (CATG and CCTG insertions respectively) are observed in about 10 and 5 % of NPM mutated AML, other NPM mutations are very rare. We report here a novel deletion detected in exon 12 of NPM gene identified from a 41 year old male diagnosed with AML. Peripheral blood smear of this patient revealed total WBC count of 1,200/mm3 with 7 % blasts. There was anemia (Hb 8.5 mg%) and thrombocytopenia (Platelet counts 42,000/mm3). Bone marrow examination revealed FAB AML-M2 based on morphology and immunophenotyping. He had trisomy 8 on cytogenetic analysis which placed him in the intermediate risk category of AML. FLT3 ITD and NPM mutation detection was done using genomic DNA sample at diagnosis by PCR followed by GeneScan analysis. This patient was FLT3 ITD negative but there was a 5 bp deletion seen in the NPM exon 12 based on GeneScan electropherogram (Fig. 1). For further characterization of this mutation, the same sample was PCR amplified and subjected to automated sequencing using ABI genetic analyzer. DNA Sequencing revealed the presence of a novel 5 base pair (ATTTC) deletion in the exon 12 (Fig. 2). Unlike other exon 12 mutations, which are mostly insertions, this deletion is in the 30 un-translated region (30 UTR) region, 45 bp downstream to the stop codon (TAA) and hence will not result in the formation of a truncated protein. This mutation was further confirmed by cDNA sequencing as well and is in the position 930 from translational start site of the cDNA. NPM RNA expression for this patient was checked by RQPCR and was found not different from that of representative NPM type A, type B, type D and NPM wild type subjects. Flowcytometric evaluation of the diagnostic marrow showed 95 % positivity for CD34, which is against the previous reports suggesting NPM mutation is associated with low CD34 expression. Bioinformatic analysis (http://www.microrna.org/) of NPM 30 UTR region, where the mutation occurred, revealed potential loss of binding site for miR208 due this 5 bp deletion. This patient underwent conventional chemotherapy with daunorubicin and cytosine and achieved CR1, received consolidation and is in complete remission now. He did not develop any toxicity. This study reports a novel 30 UTR NPM mutation, which unlike the common NPM exon 12 mutations, does not disrupt open reading frame but possibly affects the stability of

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mRNA. Further characterization of this mutation is needed before we comment on its clinical significance. The study also highlights the importance of NPM gene sequencing in addition to GeneScan analysis in order to identify the specific NPM mutation.

Abstract P 023 Impact of Cytogenetics on Outcomes of Pediatric Acute Myeloid Leukemia: Our Experience from a Tertiary Care Cancer Centre in South India KS Rachan Shetty1, B Guruprasad1, L Appaji2, KC Lakshmaiah3 1

Department of Medical Oncology, 2Department of Paediatric Oncology, 3Department of Medical Oncology, Kidwai Memorial Institute of Oncology, Bangalore 560029, India Aim: To study the prognostic factors of paediatric Acute Myeloid Leukemia (AML) with chemotherapy. Materials and Methods: 37 patients who had pathologically proven diagnosis of AML and had received treatment at our institute from January 2007 to December 2010 were included for analysis. Patients were diagnosed based on morphology, cytochemistry, cytogenetics and immunophenotypic studies, molecular risk stratification was not done. AML patients received 7 + 3 induction with Cytarabine and Daunomycin, followed by 4 cycles of high dose Cytarabine consolidation, except for APML (acute promyelocytic leukaemia) patients who received All Trans retinoic acid (ATRA) with Daunomycin induction followed by 2 cycles of Daunomycin consolidation and maintenance with ATRA, Methotrexate and 6-mercaptopurine. All patients were analyzed for oncological outcome and these outcomes were correlated with initial total leukocyte count and cytogenetics. Results: Majority of patients had favourable cytogenetic abnormality. At the median follow-up at 24 months, (range 4–46 months), induction death rate was 27 and 73 % achieved complete remission. The relapse rate was 30 % and event free survival rate was 43 %. Two year survival was 66.7 % for favourable cytogenetics, 50 % for patients with intermediate risk, 0 % for those with unfavourable cytogenetics. High tumor burden with intermediate and unfavourable cytogenetics had significant correlation with outcome. Conclusion: Tumor burden measured by initial total count and WHO cytogenetic risk marker are the most important prognostic factors even in paediatric AML. Even with advent of molecular risk stratification cytogenetics remains the most important risk factor. Keywords Acute myeloid leukaemia (AML), APML (acute promyelocytic leukemia), world health organisation(WHO) cytogenetic risk matter

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

Abstract P 024 Coding Variants of DNA Repair Genes and Its Association with De Novo Acute Promyelocytic Leukemia GC Gaur1,2, SK Hasan1,2, T Ottone1,2, V Mantovani3, D Centonze4, F Lo-Coco1,2 1

Department of Biopathology, University of Rome ‘‘Tor Vergata’’, Rome, Italy; 2Laboratorio di Neuro-Oncoematologia, Fondazione Santa Lucia, Rome, Italy; 3Centre for applied Biomedical Research, St. Orsola-Malpighi University Hospital, Bologna, Italy; 4Clinica Neurologica, Department of Neuroscience, University of Rome ‘‘Tor Vergata’’, Rome, Italy Background: DNA is essential to life, but it is subject to damage from interaction with various endogenous and exogenous agents. Double-strand DNA breaks are arguably the most serious form of DNA damage and are predominantly repaired through either the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways. Single nucleotide polymorphisms (SNPs) in double-strand break repair genes may alter DNA repair capacity and, in turn, confer predisposition to leukemia. We analyzed polymorphic variants of DNA repair and detoxification genes in patients with acute promyelocytic leukemia (APL). Methods: Using MassARRAY highthroughput DNA analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we initially genotyped 26 APL patients and 561 healthy blood donors for 210 SNPs of 22 genes mostly involved in DNA repair and drug detoxification. Based on results of initial screening, we further analysed BRCA2, XRCC5, NBN (formerly NBS1) and LIG4 gene in another 40 de novo APL patients using DNA sequencing. Results: Based on our complete cohort analysis, we identified 2 genes which were significantly associated with risk of development of APL. We observed a difference in risk allele frequency between APL and Healthy controls for NBN (rs1063045, NG_008860.1:g.6881G[A): 32.5 % and 45.5 %, p = 0.003 and LIG4 (rs1805386, NG_007396.1:g.10970T[C): 12.1 % and 18.36 %, p = 0.07. The association of homozygous variants of NBN yielded higher risk of APL (OR: 3.042, p = 0.003), whereas T allele of LIG4 was found to be a susceptible allele in APL (OR: 1.630, p = 0.07). Conclusions: Susceptibility to develop APL may be linked to genetic variations in DNA repair genes that result in inefficient repair of chemical or radiation induced genetic damage. SNP rs1063045 of NBN gene could be an important factor which plays an important role in NHEJ pathway repair in case of APL.

Abstract P 025 Sub Categorisation of T Cell Acute Lymphoblastic Leukemia Using Who Criteria—A Retrospective Analysis

199 department of hematology were screened for T cell ALL cases. An attempt was made to subcategorize all T-ALL cases according to WHO criteria and also to assess expression of aberrant markers. Results: Out of 1740 acute leukemia cases diagnosed in 3 years, 29.1 % (507 cases) were of B-ALL and 3.1 % (54 cases) were of T-ALL. Among the 54 T-ALL cases, a complete T-cell panel was done in 33 cases. On applying stringent WHO criteria, 54 % (18 cases) were classifiable and 45 % (15 cases) were not classifiable. Among the classifiable cases, Pro T-ALL were 11.11 % (2/18), Pre and Medullary T-ALL were 5.5 % (1/18) each, and Cortical T-ALL were 77.77 % (14/18). In addition, aberrant expression of other lineage markers including CD79a (5.5 %), CD13 (7.4 %), CD33 (7.4 %), CD117 (11.11 %), CD 19 (5.5 %), CD10 (18.5 %) were seen in variable combinations. In addition, analysis of T Cell Receptor subtypes done in 36 cases, showed 13.8 % of cases expressing TCR alpha/beta and 50 % cases expressing TCR gamma/delta subtypes. Conclusion: Among the cases examined, only 54 % of cases could be subclassified following the present WHO criteria, indicating its limited utility in routine diagnostic use.

Abstract P 026 Clinicopathologic Profile of Leukemias in Infants—an Indian Scenario S Munot1, PG Subramanian1, S Gujral1, Y Badrinath1, A Kumar1, S Shinde1, S Mahadik1, P Amare-Kadam2, B Arora3, S Banavali3 Hematopathology Laboratory, 2Department of Cytogenetics, Department of Medical Oncology, Tata Memorial Centre, Mumbai

1 3

Introduction: Acute and chronic leukemias are rare in infants worldwide, with very limited Indian data on their clinical and hematological profile. We studied 50 consecutive cases of infant leukemias and the clinical, morphology, cytochemistry, immunophenotyping and cytogenetic data was correlated. Objective: To study the incidence and clinicopathological profile of leukemias in infants. Methods: A retrospective study was undertaken in our tertiary care cancer institute. The data of all newly diagnosed cases of leukemias in infants from the year 2005 to 2012 was obtained from electronic medical records. Cases were diagnosed either on peripheral blood, bone marrow aspirate or biopsy. Results: Of the total 8,223 cases of newly diagnosed acute leukemias in a 7 year period, 49 were diagnosed in infants (0.5 %) It included 26 cases of acute lymphoblastic leukemia, 10 cases of acute myeloid leukemia, 3 cases of juvenile myelomonocytic leukemia and 1 case of chronic myeloid leukemia. 9 cases could not be subtyped for want of additional samples. Only 2/49 cases occurred in neonates. There was a male preponderance (34/49). A detailed immunophenotypic and cytogenetic analysis will be presented in the study. Conclusion: This study provides important insights in the spectrum and biological behavior of infant leukemias.

BK Karthik Bommannan, MUS Sachdeva, Praveen Bose, Jasmina Ahluwali, Reena Das, Neelam Varma

Abstract P 027

Department of Hematology, Post Graduate Institute of Medical Education and Research, Chandigarh

Role of Nrf2 and its Downstream Target Genes in In Vitro Sensitivity to Arsenic Trioxide in non-M3 AML

Introduction: According to the 2008-WHO CLASSIFICATION, T-Acute Lymphoblastic Leukemia (T-ALL) has been subclassified into PRO T-, PRE T-, CORTICAL T- and MEDULLARY T-ALL based on immunophenotyping. Some studies indicate prognostic significance of these subcategories. Aim: This study attempts to subcategorize T cell acute lymphoblastic leukemia cases to assess utility of WHO criteria for the subcategorization. Methodology: All cases diagnosed as acute leukemia over a period of 3 years in the

Sreeja Karathedath, Ajay Abraham, Savitha Varatharajan, Biju George, Alok Srivastava, Vikram Mathews, Poonkuzhali Balasubramanian Department of Haematology, Christian Medical College, Vellore Background: Conventional chemotherapy with Cytarabine (Ara-C) and Daunorubicin (Dnr) can cure about 25 % of patients with

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 primary AML cells by qRT PCR. Patients with ATO IC50 more than the median (n = 16, IC 50 [ 2.37) had higher mRNA expression for the above mentioned Nrf2 target genes when compared to ATO sensitive patients (n = 16, IC 50 \ 2.37) though not reaching statistical significance, probably due to low sample number. Conclusion: This pilot study extends the possibility of using pharmacological inhibitors of Nrf2 to modulate ATO resistance in AML.

Abstract P 028 Synchronous Presentation of Squamous Cell Carcinoma with Acute Promyelocytic Leukemia: Report of a Rare Case Acute Myeloid Leukemia (AML) but majority fail to achieve long term remissions. Drug resistance and relapse are considered major causes of treatment failure. The toxic side effects of chemotherapeutic drugs make therapy intolerable and inefficacious especially in older patients. This emphasizes the need of developing new therapeutic strategies to improvise the treatment both in terms of cost and efficacy. Chemotherapeutic agents like Arsenic Trioxide (ATO) brings cell kill by inducing Reactive Oxygen Species (ROS). ATO has proved very effective in inducing remissions in de novo acute promyelocytic leukemia (AML M3), but has shown to have reduced activity in other AML subtypes. The redox sensitive transcription factor, Nrf2 (Nuclear Factor Erythroid 2 Related Factor) regulates oxidative stress in normal cells is an important candidate which determine ROS mediated apoptosis. NRF2 transcriptionally regulates the levels of antioxidant response genes ((NADPH Quinone Oxidoreductase (NQO1), Heme Oxygenase (HO-1), Glutamate Cysteine ligase (GCL) and drug efflux transporters like Multi drug resistance related protein-2 (MRP2)) and averts ROS mediated cytotoxic effects. Pharmacological modulation of Nrf2 by Nrf2 inhibitors could be well exploited to provide better treatment opportunities. We hypothesize that ATO insensitivity in non-M3 AML cells is due to increased Nrf2 levels which could be modulated. Materials and Methods: In vitro cytotoxicity to ATO was done in NB4 (AML-M3), U937 (non-AML-M3) and KASUMI1 (AML cell line with t(8;21)) cell line and primary AML cells (n = 32) using MTT Assay. This assay was extended to cell lines pretreated with pharmacological concentration (10 lM) of Nrf2 Inhibitor, Luteolin. Nuclear protein extraction was done using NE-PER kit (Pierce). Nuclear Nrf2 protein level was detected using Western blot and normalized to b-lamin. RNA extraction from AML cell lines and primary AML samples at diagnosis (n = 32) was done using Trizol method and the expression of the downstream target genes (NQO1, HO1, GCLC, GCLM, MRP2) of Nrf2 was done using qRT-PCR. Results and Discussion: Based on the MTT results, U937 was resistant compared to NB4 and Kasumi1 cell lines to ATO (IC 50 values; Table 1). Nuclear expression of Nrf2 protein by western blot showed U937 cell line to have higher nuclear Nrf2 compared to NB4 cell line (Fig. 1). To further identify the functional role of Nrf2, qRT-PCR analysis was done for the downstream Nrf2 target genes NQO1, HO1, GCLC and GCLM. ATO resistant U937 cell line showed 1.3, 3, 3.3 and 2.4 fold increase in the mRNA levels of Nrf2 downstream target genes NQO1, HO1, GCLC and GCLM respectively compared to ATO sensitive cell line NB4 (Fig. 2). Pharmacological inhibition of Nrf2 by Luteolin pretreatment efficiently reduced the IC50 of ATO resistant U937 cell line (Fig. 3). Nrf2 downstream target genesNQO1, HO1, GCLC and GCLM mRNA levels were analyzed in

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Ayushi Sahay, Meena Desai, Vijay Hirani2, Pankhi Dutta1 SevenHills Hospital, Mumbai, 2Modern Haematology and Chemotherapy Centre, Kolhapur, 1Kokilaben Dhirubhai Ambani Hospital, Mumbai Objective: Though rare, squamous cell carcinoma (SCC) has been seen to occur in acute promyelocytic leukemia (APML) patients as a consequence of treatment with arsenic trioxide. However, there is no case reported in literature of simultaneous occurrence of these two malignancies. We report a highly rare case. Method—Case Report: A 47 year old male presented with ulceration of the left inner cheek. There was no other significant past history or treatment history. There was history of regular tobacco chewing. Examination revealed a buccal ulcer which was non tender along with a palpable and hard, left submandibular lymph node. A biopsy done from the cheek ulcer revealed a moderately differentiated SCC with nests and sheets of moderately large malignant squamous cells irregularly infiltrating the submucosa and muscle layer. A routine hematological evaluation at this time revealed pancytopenia with Hb: 7.9 mg%; WBC: 660 cells/ mm3; platelet count: 53,000/mm3. A bone marrow (BM) examination was done and showed highly cellular aspirates with near total replacement of the marrow by abnormal, hypergranular promyelocytes along with classical faggot cells. The corresponding BM trephine biopsy showed near total replacement by sheets of immature myeloid cells. A diagnosis of APML was made. PML-RARA testing by gel PCR was positive for the bcr1 isoform. The patient refused any specific treatment and expired after 8 days at home. Conclusion: To the best of our knowledge, this is the first case in literature of synchronous presentation of SCC with APML and highlights the importance of bone marrow examination in carcinoma patients with pancytopenia.

Abstract P 029 Mixed Phenotype Acute Leukemia—a Growing Concern Shelly Poddar, SM Sethy, P Mohanty, RK Jena Department of clinical Hematology, Department of Pathology, SCB Medical College Aim of the study: To study the incidence of Mixed Phenotype Acute Leukemia (MPAL) out of 253 cases of Acute Leukemia studied. Objective: To correlate the haematoclinical presentation with cytomorphology, cytochemistry, immunophenotyping and treatment

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 outcome. Materials and Method: We evaluated 253 cases presenting as Acute Leukemia. Each of these were completely evaluated by a complete blood count, bone marrow aspiration, MPO &PAS positivity, multiparametric flow cytometry. Then they were evaluated for the treatment response. Observation: Out of 253 cases of Acute Leukemia 5 cases came out to be of Mixed Phenotype Acute Leukemia (MPAL). Of these 5 cases morphologically 3 were AML and 2 were ALL. They received a treatment plan of 3 + 7 CT followed by MCP841. In all of these cases outcome was poor, all patients died either before or after induction therapy. Discussion: MPAL accounts for about 2 % of acute leukemias and can occur in both children and adults but more common in adults. It can present morphologically as either AML or ALL. So to distinguish these group of patients we need to go for a immunophenotyping. In our study we have seen that there is 100 % mortality rate. We can conclude that this leukemia has a poor prognosis. Sl Age/ no. sex

PSC

BM

Immunophenotyping

1.

1.8 mn/ M

[80 %blast AML- MPAL M6

2.

27 years/ Ac ALL M leukemia

3.

Oral CT 70 years/ [50 %blast AML- (B + T) F M5 AntiMPOve

Expired during induction

4.

51 years/ Ac AML- MPAL M leukemia M1

Planned for 3 + 7 CT

Expired before induction

5.

32 years/ Ac ALL F leukemia

Received 3 + 7CT followed by MCP841

Cr after 2nd CT but expired due to infection after 4 months

MPAL

MPAL

Treatment

Outcome

MCP841

Expired during induction

3 + 7CT

Incomplete CR expired before next CT

Abstract P 030 Immunophenotypic Analysis of Transient Abnormal Myelopoiesis in 5 Children with Down Syndrome V Baloda1, S Gujral1, PG Subramanian1, Y Badrinath1, A Kumar1, P Amare Kadam2, S Banavali3, B Arora3 Department of Hematopathology, 2Cytogenetics Department, 3 Department of Medical Oncology, Tata Memorial Hospital, Mumbai 1

Objective: To study the immunophenotypic profile of transient abnormal myelopoiesis (TAM). Materials and Methods: Retrospective analysis of cases reported as TAM during 2007-2012. Results: 5 children (3 females, 2 males) with Down Syndrome with age between 5 and 45 days presented with a high WBC counts and high percentage of blasts in peripheral blood and/or bone marrow. Blasts were MPO negative. Immunophenotypic analysis using multi color flow cytometry was performed. Table 1 delineates the expression pattern of various markers on the blast population. The blasts did not express any of these B or T cell markers—CD19, CD10, CD20, CD3 and CD2 (2/2). Blasts expressed CD4 in one case. Conclusion: Transient abnormal myelopoiesis presented in Down syndrome children within 45 days of birth in our series. CD34, CD13, CD33, CD117, CD41, CD61, CD7 and HLA-DR are useful markers for characterization of blasts of TAM.

201

Abstract P 031 Volume, Conductivity, Scatter: Simply Ornaments or Diagnostic Tools of Acute Leukaemias: A Retrospective Analysis of 108 Cases of Acute Leukaemias in a Tertiary Oncology Centre Monali Gupta, DK Mishra, Mammen Chandy Department of Lab Hematology, Tata Medical Centre, Kolkata Introduction: The Automated Hematology analyser Coulter LH-780 uses a combination of three measurements Volume, Conductivity, Scatter (VCS) to identify WBCs in their near native state but it could take advantage of these parameters to evaluate their morphologic changes. Objectives: The aim of this study was to investigate whether VCS can become a new high-throughput screening method not only for the detection but also for the exact categorization of Acute Leukaemias. Materials and Methods: VCS parameters of 108 diagnosed cases of acute leukaemias analysed by Beckman Coulter LH 780 were studied retrospectively. The CBCs, scatterplots, flags were correlated with VCS to calculate their sensitivity and specificity alone and in combination. VCS trends and characteristic cluster patterns were identified in 67 cases of ALLs, 7 APMLs, 34 Non APMLs, 6 HLA-DR negative AMLs, 3 cases each of AML with myelodysplasia related changes and Acute leukaemia of ambiguous lineage. Results: High MLV, SD volume and high MLS and SD scatter was the characteristic VCS trend observed in 61 cases of ALLs while 5 cases deviated from the trend. AMLs showed high MNV and SD but low mean and SD scatter. APMLs exhibited least mean and SD scatter values in spite the hypergranularity of abnormal promyelocytes. Interestingly AML M5 cases exhibited high Monocyte mean scatter and normal SD scatter despite the fact that the monoblasts are agranular! A tight merging cluster pattern was seen in all 3 cases of HLA-DR negative AMLs. The current study also found ‘‘cluster patterns’’ in 66 cases of ALLs and their association with presence of aberrant myeloid antigen marker on immunophenotyping was statistically significant (p value \ 0.001). Conclusions: VCS alone proved to be a good diagnostic tool in ALLs as the sensitivity was 91.8 % and specificity was 80 % while in AMLs the sensitivity was 94.1 %. There were no AML cases wherein peripheral blood did not have blasts, so specificity could not be calculated. Keywords Beckman coulter LH 780, VCS, Mean neutrophil volume(MNV), Mean Lymphocyte volume (MLV), Mean lymphocyte scatter(MLS)

Abstract P 032 Minimal Residual Disease in Paediatric B Lineage Acute Lymphoblastic Leukemia: Comparison of Mid—Induction Peripheral Blood and Bone Marrow Samples Using Six Colour Flow cytometry BK Karthik Bommannan1, MUS Sachdeva1, Parveen Bose1, Neelam Varma1, Deepak Bansal2, RK Marwaha2 Department of Hematology, 2Department of Paediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh

1

Introduction: There is strong correlation between MRD levels and risk of relapse in childhood leukemias. Bone marrow aspirate sample on day 15 of induction is commonly used in paediatric cases to assess MRD levels for further management decisions. Peripheral blood sample might be used for the same if proven to yield similar MRD levels. Aim: To compare MRD levels in paired peripheral blood and bone marrow samples of paediatric B-ALL patients on day 15 of induction using six colour flow cytometry. Methodology: Five newly diagnosed, CD19 and CD10 dual positive, paediatric B lineage ALL patients were

123

202 analyzed on day 15 of chemotherapy for peripheral blood and bone marrow MRD levels. Lyse-stain-wash technique was used to process single 6 colour tube (CD19PECy7/CD10APC/CD20PerCP/CD34PE/ CD45APCH7/Syto13). One million events were acquired on BDFACS Canto II and analysed by BDFACS Diva software. Results: MRD levels of[0.01 % (range 0.02–0.81 %) were seen in all 5 bone marrow samples. All but one peripheral blood samples showed presence of MRD [ 0.01 % (range 0.04–0.36 %). Although, the levels of MRD vary slightly between bone marrow and peripheral blood samples, presence of peripheral blood MRD in all but one case, may favour using peripheral blood on day 15 for MRD detection instead of invasive bone marrow procedure. Conclusion: There is good concordance (4 out of 5 cases) of MRD level detection between mid-induction samples of peripheral blood and bone marrow aspirate for paediatric B-ALL cases. This result needs to be supported by more number of cases.

Abstract P 033 Monosomy 7 in Acute Myeloid Leukemia in India S Yuvarani1, Usha Sitaram3, Rayaz Ahmed2, Aby Abraham2, Auro Viswabandya2, Biju George2, Vikram Mathews2, Alok Srivastava2, Vivi M. Srivastava1 Cytogenetic Unit, 2Department of Haematology, 3Department of Transfusion Medicine and Immunohaematology 1

Background: Monosomy 7 is seen in 5–10 % of all acute myeloid leukemia (AML) and is associated with a poor prognosis. We describe the cytogenetic features of monosomy 7 in AML. Patients and Methods: G-banded karyotypes of all patients with AML and monosomy 7 seen in the Department of Haematology, Christian Medical College, Vellore between January 2003 and December 2009 were studied. Cytogenetic findings were correlated with clinical and laboratory features. Results: Monosomy 7 was seen in 71 patients (5.4 % of 1314 patients with AML), 52 of whom were males (73 %). There were 57 adults (median age: 39 years, range: 1–72). The median hemoglobin was 7.95 g/dl (range: 2.5–15.8); median WBC count, 6.4 9 109/L (range: 0.6 -271); median platelet count, 25.5 9 109/L (range: 3–401). The most common AML subtypes were AML-M2 (27 %) and AML, not otherwise specified (29 %). Dysplasia was seen in 46 % of cases while 10 % of these cases were of secondary AML. Monosomy 7 was the solitary abnormality in 19 patients (27 %). Complex karyotypes (two or more additional abnormalities) were seen in 38 (53 %). These included monosomy 5 (15 %), deletion 5q (8 %), trisomy 8 and monosomy 17 (7 %), and trisomy 21 and inversion 3 (6 %). Follow-up data was available for 20 of these patients. Only two of them survived more than 8 months. Conclusion: This is the first report of a large series of AML patients with monosomy 7 from India. While the incidence (5.4 vs. 3–10 %) and poor outcome are similar to other reports in the literature, these patients are younger (median age: 39 vs. 45–55 years) and show a higher male preponderance (2.3:1 vs. 1.5).

Myelo proliferative Neoplasms Abstract P 034 JAK2 (V617F) Negative Myeloproliferative Neoplasms— Prevalence and Points to Ponder Puttaiah Parimala, Karuna Rameshkumar and Cecil Ross1 1

Department of Clinical Pathology and Hematology, St John’s Medical College Hospital, Bangalore Introduction: Myeloproliferative neoplasms (MPN) are characterised by clonal proliferation of one or more of myeloid lineages and it is

123

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 known mutations in JAK 2 signaling contribute to pathogenesis. There is a significant group of patients who are JAK2 (V617F) negative. Aims and Objectives: To study prevalence, clinical and laboratory features of Philadelphia chromosome negative and JAK2 (V617F) negative myeloproliferative neoplasms. Materials and Methods: From January 2011 to July 2012, patients who fulfilled 2001 WHO criteria of MPN were included. Clinical features and laboratory profile including complete hemogram, bone marrow aspiration and biopsy were analysed. FISH for Philadelphia chromosome and JAK2 mutation by allele specific PCR were done for further characterization. Results: Among 44 patients diagnosed as MPN, 27 patients were diagnosed as JAK2 (V617F) negative. Polycythemia Vera was most frequent (59 %) followed by idiopathic myelofibrosis (26 %) and essential thrombocythemia (15 %). Male predominance was noted (92 %). JAK 2 positive patients were older and had higher frequency of splenomegaly. In comparison, JAK2 negative patients had a higher platelet count in ET (11.6 ± 2.4 9 109/L) and isolated erythrocytosis (19.4 ± 1.38 g/dl) in 94 % of patients with PV. Two of them had presented with thrombosis. Comment and Conclusions: Mutations at exon 12 associate with erythropoietin signaling have been reported in JAK 2 (V617F) negative PV in literature. The present study reinforces the findings of our previous study. In addition, it highlights need to look at other mutations among JAK2 (V617F) negative myeloproliferative neoplasms, which may be involved in activation of other signaling pathways.

Abstract P 035 Myelofibrosis—an Unusual Manifestation of HIV Infection in a Child Rohini Gupta2, Gunjan Mahajan1, Jagdish Chandra2, Sunita Sharma1, Anju Seth2, Bhavna Dhingra2, Praveen Kumar2 Department of Pathology, 2Department of Paediatrics, Lady Hardinge Medical College and associated Hospitals, Delhi

1

Introduction: Myelofibrosis is a myeloproliferative disorder in which abnormal type of bone marrow stem cells cause fibrosis. Secondary Myelofibrosis can be associated with haematological malignancies, infections, endocrine and connective tissue disorders. However, Myelofibrosis in HIV infected children is very rare. Case Details: A female child admitted postnatal for umbilical cord bleeding was given blood transfusion in 2004. Patient remained asymptomatic till 4 years of age when she presented with anasarca, acute gastroenteritis and sepsis. On examination, patient had severe anemia, hepatosplenomegaly and bilateral crepitations on auscultation. On investigations, patient had normocytic anemia (Hb-6.6 g/dl, MCV-84.6 fl), thrombocytopenia (platelet-61 9 103/ll) and normal total leucocyte count (TLC-6.7 9 103/ll). X-ray chest showed bilateral infiltrates in lungs. HIV serology was positive. A probable diagnosis of tuberculosis with HIV was made. CD4 counts were 602/mm3 (CD4 % = 22 %).Patient was put in HIV clinical stage 3 and started on ATT. On further follow up, the anemia and thrombocytopenia worsened, leucopenia developed and CD4 counts reduced to 255/mm3 (15 %). ART was thus started on 11/01/2011, but pancytopenia of patient did not improve. Serum vitamin B12 and folate levels were normal. Bone marrow aspiration was diluted. Bone marrow biopsy was hypocellular for age and showed focal islands of hematopoetic cells. Megakaryocytes were reduced in number. On reticulin stain bone marrow showed grade 3 fibrosis. Von Gieson stain showed focal areas of fibrosis. Thus a diagnosis of Myelofibrosis secondary to HIV infection was made. Patient was started on thalidomide and steroids for 3 months and then continued only on steroids. The peripheral blood count improved (Hb12.7 g/dl, TLC-5.8 9 103/ll, platelet-141 9 103/ll). She remained asymptomatic till date and did not require blood transfusion.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Table 1 Immunophenotypic profile of blasts Case 1 Case 2 Age at presentation

Case 3

Case 4

Case 5

5 days

45 days 11 days Neonate 40 days

Sex

M

M

Blast %

80

77 (PB)

59

F

F

M (BM) 57 (BM)

203 demonstrated conflicting results in regard to an overall survival (OS) benefit with the allogeneic approach. The role of allogeneic transplant remains under study especially in the high-risk population, which has high relapse rates with traditional autologous approaches. Future directions to reduce relapse include post-transplantation consolidation and maintenance therapy with either approved agents or new agents and immunotherapy, either vaccine based or natural killer (NK) and T-cell based.

20 (PB) (PB)

MPO

-

-

-

-

-

Abstract P 037

CD 34

+

+

+

+

+

CD 13

-

-

+

-

-

CD 33

+

+

+

+

+

CD 117

+

+

+

-

+

Anti MPO

ND

-

+

ND

-

CD 41

+

-

+

WK +

Inconclusive

CD 61

+

-

+

WK +

Inconclusive

Department of Haematology, Christian Medical College, Vellore

CD 14

-

ND

-

ND

ND

CD 235a

-

-

ND

ND

ND

CD 7

-

+

+

-

+

CD 56

+

-

+

-

-

HLA DR

-

+

-

-

-

Background: Busulfan in combination with cyclophosphamide is widely used conditioning regimen in Hematopoietic cell transplantation (HSCT) for various malignant and non-malignant disorders. Wide inter-patient variability in busulfan systemic exposure is observed with oral busulfan due to unpredictable intestinal absorption and differences in hepatic metabolism. To overcome the problems associated with oral administration, intravenous (i.v) busulfan was introduced in early 2000. Busulfan is mainly metabolized in the liver by glutathione S-transferases (GSTs). We have shown that GSTA1*B polymorphism explained the inter-patient variability in busulfan exposure and clearance in homozygous beta thalassemia patients receiving oral busulfan (ASH 2009, ASH 2010). There is no report on pharmacokinetics of i.v busulfan from Indian patients undergoing HSCT. The aim of the present study is to document the PK of i.v busulfan and to compare the first dose AUC and clearance in with the GSTA1*B polymorphism. Methods: Sixty patients receiving i.v busulfan once daily (n = 44) or four times daily (n = 16) as part of their conditioning regimen before transplantation between June 2010 and July 2012 at the department of hematology, Christian Medical College, Vellore were included in this study. Genomic DNA was isolated from the peripheral blood MNCs before transplantation and GSTA1*B polymorphism was analysed using PCR–RFLP method. Peripheral blood samples were collected at different time points (0, 3, 4, 5 and 7 h for Q24h and 0, 1, 2, 3 and 4 h for Q6h) after busulfan infusion. Busulfan plasma levels were measured using previously reported LC–MS/MS method. The area under the concentration versus time curve (AUC) and clearance of busulfan after the first dose was calculated as reported previously (Desire et al., IJMR, in press). Based on the AUC of the first day, subsequent doses were adjusted to achieve the target AUC. The AUC and clearance of busulfan were compared with the GSTA1*B genotype using Mann–Whitney U test. Results: Patient demographics and busulfan AUC and GSTA1*B genotypes are listed in Table. There are contradictory reports on the influence of GSTA1*B polymorphisms on i.v busulfan AUC and clearance in adult patients as well as in children. We report here that GST polymorphism does not significantly influence the first dose AUC or the clearance of i.v busulfan, though in a small number of patients. This is the first report from Indian population comparing GST polymorphism and pharmacokinetics of i.v busulfan. We conclude that, along with GST polymorphisms, there may be other genetic variants explaining the inter-individual variability in busulfan systemic exposure and clearance, which needs to be evaluated.

M male, F female, ND not done, WK+ weak positive, BM bone marrow, PB peripheral blood; +, positive; -, negative Conclusion: Myelofibrosis as presenting feature in HIV infection is rare. Although a few case reports of Myelofibrosis secondary to HIV infection in adult patients are present in Indian literature, none has been documented in children to the best of our knowledge.

Stem Cell Transplant Abstract P 036: Oral Presentation Stemming Role of Stem Cell Transplantation in Multiple Myeloma: Who, When, and What Type? Deepak Bansal Maharishi Markandeshhwar Institute of Medical Science & Research, Mullana Abstract: High dose chemotherapy with autologous stem cell transplantation has shown improved progression-free survival (PFS) over the conventional chemotherapy regimens. In the era of novel agents for myeloma in conjunction with the evolution of hematopoietic stem cell transplantation, many new questions arise. How to incorporate these novels into the transplant paradigm? Should transplant be delayed until relapse? In earlier eras, autologous trans- plantation was generally reserved for patients younger than 60 or 65 years. In this modern era, should there be an age limit for myeloma transplant? Now that medicine is individualized for every patient, chronologic age alone should not drive decisions regarding transplantation. What type of transplant (i.e., autologous compared with reduced intensity allogeneic transplant) is best? Several large international trials have

Effect of GSTA1*B Polymorphism on Pharmacokinetics of Intravenous Busulfanin Patients Undergoing Hematopoietic Stem Cell Transplantation S Gopinath, M Ezhil Pavai, Biju George, Vikram Mathews, Alok Srivastava, B Poonkuzhali

123

Severe AA PNH

1 2

Pre

1

Ph+ ALL

1

JMML SCID

1 1

JMML

1

SCID

1

THAL

16

Q6H (n = 16)

Variants— 07

Median = 875 (285–1,456)

Median—0.245 (0.163–0.444)

Median age— 4 years

Wild type— 09

Median = 670 (353–1,059)

Median—0.383 (0.189–1.869)

p value

0.4079

0.2105

(2–7 years)

Abstract P 038 Targeted Dose Adjustment of i.v Busulfan in Patients Undergoing Hematopoietic Cell Transplantation—CMC Vellore Experience Ezhilpavai Mohanan, Poonkuzhali Balasubramanian, Salamun Desire, S Gopinath, Mammen Chandy, Vikram Mathews, Alok Srivastava, Biju George Department of Haematology, Christian Medical College, Vellore Background: Busulfan is a bi-functional alkylating agent used in combination with cyclophosphamide or fludarabine as a conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Intravenous (i.v) busulfan is preferred over oral busulfan due to reduced inter-individual variation and better predictability in pharmacokinetics. Busulfan has a narrow therapeutic window, with toxic side effects such as sinusoidal obstruction syndrome (SOS) occurring at high systemic exposure and graft rejection or relapse at low exposure, thereby significantly influencing clinical outcome. We share our experience in targeted dose adjustment of i.v busulfan in patients undergoing HSCT for various hematological diseases in the past 2 years. Patients and Methods: Sixty patients diagnosed with various haematological disorders receiving high dose i.v busulfan as combination chemotherapy between June 2010 and July 2012, were included in this study. Forty three patients received single daily (Q24H) whereas 17 had 6 hourly dose of (Q6H) busulfan. Peripheral blood samples were collected at different time points and plasma were immediately separated. Busulfan levels were analyzed using a rapid and sensitive LC-ESI MS/MS method with deuterated internal standard (d8-Bu) and quantified using Multiple Reaction Monitoring (MRM). The results are interpreted as ng/ml. Inter and intra-day variability of the standards and controls was less than 10 %.An AUC of 5,000–5,500 lmol (cumulative AUC target: 20,000–22,000 lmol) in Q24H or 900–1,350 lmol in Q6H were targeted. Results: Diagnosis of the patients, busulfan dosing interval and plasma AUC on day 1 and 3 is listed in Table. Figure shows the first dose and dose adjusted AUCs after Q24H and Q6H respectively. Five patients (8 %) achieved targeted levels after first dose and required no further dose adjustment, while 39 (65 %) patients achieved targeted levels after first dose adjustment. However, in 16 (27 %) patients the dose had to be further adjusted on day 3. Eight (13 %) of these patients did not achieve the targeted AUC even after increasing the dose the second time and in 8 patients (13 %) the dose had to be reduced from the

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16

Day 1 Median = 671 (285-1456) Day 3 Median = 995 (557-1712)

Increase : 13 Median – 19% (2% to 39%) Decrease : 03 Median – 8% (4%-13%)

1000 500 0

C

1

THAL

Q6H (n= 16) Median age : 4 yrs (2-7 yrs)

U

Ph + ALL

0.2361

P = 0.0138

1500

A

2

0.3313

Q6H 2000

1

PNH

p value

No Change : 05

ay

1

Day 3 Median = 4986 (3220-7661)

D

Severe AA

Median = 3,554 Median— (1,990–10,450) 0.240 (0.097–0.494)

AUC (uMoles/min)

B-ALL

Wild type— 21

C

(1–58 years)

U

8

A

MDS

0 3

8 1

ay

MDS Pre B-ALL

5000

D

32 years

10000

C

1

Q24H (n= 44) Median age : 32 yrs (1-58 yrs)

Q24H P = 0.0397

15000

D

9

CMML

Day 1 Median = 3776 (2407-10456)

Increase : 30 Median – 15% (4% to 47%) Decrease : 09 Median – 25% (5%-38%)

U

1

Dose adjustment

A

9

AUC (µMoles)

3

CML CMML

CML

19

Bu dosing

ay

19

D

N=60

AML

Q24H (n = 44) Variants— 19 Median age—

AML

Median = 4,026 Median—0.186 (1,713–10,456) (0.108–0.510)

Diagnosis

C

Clearance (L/h/kg)

U

AUC (lmol)

A

GST A1*B

1

Diagnosis N = 60 Bu dosing

AUC (uMoles/min)

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

ay

204

initial rise. However, the dose was not increased beyond 20 % at any given time. When we analyzed the regimen related toxicity in these patients, 13 (22 %) developed mucositis Grade III-IV; 7 developed SOS. Three patients rejected the grafts and all of the three had primary graft failure. Conclusion: The target level busulfan for Indian patients undergoing HSCT with various hematological conditions has not been studied. There is also no documented pharmacokinetics (PK) of i.v busulfan in our population. From our experience with targeted dose adjustment of i.v busulfan, we observed that majority of these patients required dose escalation, which is very different from previous reports. The reason for this needs further PK and pharmacogenetic studies in our population.

Abstract P 039 Hematopoietic Stem Cell Transplantation in Acute Leukemias: Long Term Results, Impact of Conditioning Regimens and the Role of Prognostic Factors from a Tertiary Cancer Center in India Jayant Gawande1, Ravi Thippeswamy1, Bhausaheb Bagal1, Sadhana Kannan2, Manju Sengar1, Hari Menon1, Reena Nair1, Navin Khattry1 BMT Unit, Department of Medical Oncology, 2Department of Biostatistics, ACTREC, Tata Memorial Centre, Mumbai

1

Objectives: Hematopoietic Stem Cell Transplantation (HSCT) is the most curative option in acute leukemias. We retrospectively analyzed our data for survival outcomes, impact of conditioning regimens on outcomes and role of possible prognostic factors. Methods: One hundred and fourteen patients (101 allogeneic, 13 autologous) who underwent transplant for acute myeloid leukemia (AML-81) and acute lymphoblastic leukemia (ALL-33) between March 1983 and August 2011 were evaluated. Poor risk cytogenetics were seen in 84 and 38 % patients of ALL and AML respectively. Seventy nine patients (69 %) received full intensity (FI; cyclophosphamide-total body irradiation Cy-TBI and busulphancyclophosphamide Bu-Cy) and 21 patients (18 %) received fludarabine based reduced intensity (RI) conditioning. Prognostic factors evaluated for overall survival (OS) and relapse free survival (RFS) were WBC count at presentation, baseline cytogenetics, disease status at transplant, dose of cytarabine consolidation (18 vs. \18 g/ m2) in AML, conditioning regimen (Cy-TBI vs. Bu-Cy and FI vs. RI), time from diagnosis to transplant, duration of relapse free interval (RFI) pretransplant and chronic Graft versus Host disease (cGVHD). Univariate and multivariate comparisons of survival times for potential prognostic factors were made using log-rank test and cox regression analysis respectively. Results: Median age at

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 transplant was 26 years. Median time from diagnosis to transplant was 7 months. At the time of transplant, 59 % were in first remission (CR-1), 26 % in second remission (CR-2), 10 % in refractory state and in 5 % status at transplant was not known. Median follow up time was 46 months. The incidence of acute and chronic GVHD was 46 and 36 % respectively. The transplant related mortality (TRM) was 30 %. The TRM was significantly higher (P = 0.001) in the pre 2007 period (42 %) compared to the post 2007 period (10 %). The cumulative probabilities of OS and RFS at 8 years were 37 and 35 % for the whole group, 35 and 32 % for AML, 29 and 20 % for ALL respectively. On multivariate analysis, Cy-TBI conditioning (P = 0.01; P = 0.012)) and chronic GVHD (P = 0.009; P = 0.004) each were prognostic factors for both better OS and RFS. On univariate analysis, CR at transplant (P = 0.029) and RFI [ 9 months pre transplant (P = 0.017) were also associated with better OS and RFS respectively while RI conditioning showed a trend towards better OS (P = 0.087). Conclusion: Our long term results suggest that at least one-third of patients with acute leukemia are cured with HSCT. Patients with cGVHD and those receiving Cy-TBI had better survival. Reduced intensity conditioning is an option in patients with acute leukemias with at least comparable results.TRM has reduced significantly over recent years probably due to better supportive care.

Abstract P 040 Bacterial Contamination of Peripheral Blood Stem Cell Harvest: Incidence, Risk Factor and Clinical Outcomes Bhausaheb Bagal1, Sumathi SH2, Rajib De1, Vijay Patil1, Jayant Gawande1, Shashank Ojha2, Vivek Bhat3, Preeti Chavan4, Rohini Kelkar3, Navin Khattry1 BMT Unit, Department of Medical Oncology, 2Department of Transfusion Medicine, 3Department of Microbiology, 4Department of Laboratory Medicine, Tata Memorial Centre, Mumbai, India 1

Introduction: Microbial contamination is a potentially serious complication of peripheral blood stem cell (PBSC) harvest. Data on incidence, organism type, outcomes after infusion and risk factors is scarce. Methods: We studied medical records of 195 patients (autologous) and donors (allogeneic). Bacterial cultures of PBSC harvest were obtained immediately after PBSC collections, prior to freezing and after thawing of cryo-preserved PBSC. Risk factors analyzed for positive culture were type of transplant, method of mobilization, use of CVC versus peripheral venous access for apheresis, number of days of collections and total blood volume processed. Results: 155 subjects underwent apheresis yielding 334 PBSC bags (112 from healthy donors and 222 patients). Apheresis of adequate PBSC required median of 2 days (range 1–5). Median volume of blood processed per collection was 10.7 l. The incidence of microbial contamination was 9.8 %. Microorganism cultured were skin commensals (n = 19), gram negative pathogens (E. coli4, Kleibsiella-8 and Proteus mirabilis-2). Seven post thaw samples grew an organism, all of which were skin commensals. Only one patient receiving culture positive PBSC harvest developed fever with rigors during infusion. No microorganism present in the PBSC was recovered from blood cultures collected in post-transplantation period. Total blood volume processed greater than 10.7 liters was associated with higher likelihood of positive culture in PBSC harvest (p = 0.01). Conclusion: Our experience suggests infusion of contaminated PBSC graft does not play a significant role as a source for infections in the HSCT setting and higher blood volumes processed during apheresis increases the risk of bacterial contamination.

205

Abstract P 041 Breast Cancer Stem Cells (BCSCs): An Implication to Therapy in Chemoresistant Breast Cancer Preetha Bhadra, S Gangopadhyay, S Sengupta, A Mukhopadhyay Netaji Subhas Chandra Bose Cancer Research Institute, 16A Park Lane, Kolkata-700016, West Bengal, India Objective: Invasive and mesenchymal property of BCSCs with CD44+/CD24low/ALDH1+ phenotype has made them promising target for targeted treatment. Chemotherapy treatment uses medicine to weaken and destroy cancer cells in body, including cells at original cancer site and any cancer cells that may have spread to another part of body. Chemotherapeutic drugs for breast cancer are not well defined yet. Combination of drugs is also not fully known. Our objective is to define chemotherapeutic drugs and its action in breast cancer. Methods: Total of 20 early chemo-naı¨ve patients with biopsy proven triple-negative metastatic breast cancer in age group of 18–36 years were selected randomly and tested for CD44/CD24 cell analysis. Isolated BCSCs were cultured for in vitro drug sensitivity towards Platinum, Anthracyclin, Docetaxel, Rapamycin, Sunitinib, Sorafenib, Gefitinib. Correlation was drawn between cell differentiations, % of stem cells and drug response. Accordingly chemotherapy was designed for particular patient. % of BCSCs in pre and postchemotherapeutic condition was further compared. Results: We have detected BCSCs in 90 % of cases. Among positive sample shows in vitro drug sensitivity 4 (20 %) to Rapamycin, 1 (5 %) to Sunitinib, 1 (5 %) to Sorafenib, 1 (5 %) to Gefitinib, 3 (15 %) to Platinum, 1 (5 %) to Anthracyclin, 1 (5 %) to Docetaxel and rest shows no sensitivity to any drug. Conclusion: Thus primary aim to target BCSCs at onset of tumors in breast cancer patients to control metastasis and relapse of disease was somewhat obtained. We further plan to correlate ratio of selected markers present in pre and postchemotherapeutic condition with time to recurrence, mortality, morbidity and progression free survival. If no BCSCs prevail after chemotherapy, patients would be kept under observation and if traces are found, we would proceed to targeted therapy.

Abstract P 042 Infections in Hematopoietic Stem cell Transplant Recipients—an 12 Year Single Centre Experience of 207 Cases N Grover, Nair Velu, S Bhattacharya, A Sharma, AK Sahni, S Das, L Singh, GS Chopra, S Sharma, R Kapoor, V Behera AFMC, Pune Background: Infections are the main cause of mortality and morbidity in haematopoietic stem cell transplant (HSCT) patients despite prophylaxis and newer anti microbial drugs. Aim: To study the various infections in HSCT recipients at a large tertiary care hospital of India between Feb 1998 to March 2010. Study Design: Retrospective case series. Methods: Total 207 patients underwent HSCT with proper sterile precautions. Infections were diagnosed according to the results of clinical examination or specific diagnostic procedures and were defined based on CDC criteria. Bacterial infections were documented on positive cultures, fungal infections documented as per CDC criteria and viral infections diagnosed by RT-PCR results. Proper antibiotic, antiviral and antifungal therapy given whenever infection was suspected/documented. Results: 164/207 patients (79.2 %) had one episode of fever and 35/207

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206 (16.9 %) had second episode of fever. Second episode of fever was more frequent in patients who underwent allogenic (23.3 %) than autologous (4.2 %) and who nursed under clean side room (18/28) versus protected environment (17/179) with p \ 0.001. Total 267 documented infections occurred in 207 transplants in which 134 (48.9 %) were bacterial, 98 (35.7 %) viral, 38 (14 %) fungal and 1 had plasmodium falciparum. Pathogens identified were more in allograft than autograft transplants. Gram negative infections were identified more in which the most common was E. coli. CMV was the most common viral and candida the common fungal infection documented. Death occurred in 72 patients in which 50 were post allogenic. Conclusion: Overall incidence of all infections and febrile episodes was high and were comparable to both western and Indian studies. Infections were commoner in allograft than in autograft patients. Major causes of death included fungal pneumonia and bacterial pneumonia.

Abstract P 043 Long Term Results of Autologous Transplants for Hodgkin’s Lymphoma—a Retrospective Multi-Center Analysis from India Reetu Jain1, Navin Khattry2, Ravi Thippeswamy3, Adwaita Gore4, Bharat Bhosale5, Nandish Jeevangi6, Sadhana Kannan7, Anjana Sainani8, Ganapathy Bhat9, Reena Nair10, Tapan Saikia11, On Behalf of Indian Study Oncology Group Trust 1,8,9

Department of Medical Oncology, Jaslok Hospital and Research Centre, Mumbai; 2,3,5,6,7,10BMT Unit, Department of Medical Oncology, ACTREC, Tata Memorial Center, Mumbai; 4,11 Department of Medical Oncology, Prince Aly Khan Hospital, Mumbai; 1Dr.G.Deshmukh Marg, Mumbai 26, India Introduction: Autologous transplantation is the standard of care for patients of relapsed and refractory Hodgkin’s lymphoma (HL). We report the results of transplants from three centers in Mumbai and role of possible prognostic factors. Methods: One Hundred and three patients underwent transplant for HL from August 1994-May 2011. All patients received 2–3 cycles of DHAP, ESHAP, MINE, Mini-BEAM, GDP or ICE with or without rituximab as salvage chemotherapy. Cheson’s response criteria was applied for assessing response pre and post transplant at day 100. Forty-eight percent of patients received BEAM (carmustine, etoposide, ara-c and melphalan), while 52 % received LACE (lomustine, ara-c, cyclophosphamide and etoposide) regimen. Ninety-two percent received peripheral blood stem cells. Prognostic factors evaluated for progression free survival (PFS) and overall survival (OS) were stage at diagnosis, presence of B symptoms at diagnosis, time from diagnosis to transplant, number of lines of chemotherapy pretransplant, remission status at transplant, conditioning regimen, pretransplant salvage chemotherapy and serum albumin at transplant. Univariate and multivariate comparisons of survival times for potential prognostic factors were made using log-rank test and cox regression analysis respectively. Results: The median age at transplant was 23 years. B symptoms were present in 47 % at diagnosis. The median time from diagnosis to transplant was 1.9 years. Fifty-seven percent of patients had stage III and IV disease at diagnosis. At transplant, 41 % were in complete remission (CR) Sixty percent of patients who received platinum- based salvage achieved CR compared to 30 % of those who received ifosfamide (P = 0.012). Median number of lines of chemotherapy pretransplant was 2 and median serum albumin at time of transplant was 4 g/dl. Transplant related mortality was 11 %.The cumulative probability of OS and PFS at 3 years were 65 and 49 % respectively. Serum albumin [4.0 g/dl (P = 0.03) and CR at transplant (P = 0.025) were associated with better OS while CR at transplant was associated with improved PFS in multivariate analysis. Conclusion: Our data suggest that serum albumin and CR at transplant are important factors affecting

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 survival. Platinum based pre-transplant salvage regimens achieved better CR rates than predominantly ifosfamide based regimens.

Abstract P 044 Role of Reduced Intensity Conditioning Allogeneic Stem Cell Transplant for Young Adults with Acute Myeloid Leukemia in First Complete Remission Chepsy C Philip1, Abhijeet Ganapule1, Rayaz Ahmed1, Aby Abraham1, Biju George1, Kavitha M Lakshmi1, Alok Srivastava1, Vikram Mathews1 1

Department of Clinical Hematology; CMC Vellore

Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment for patients with Acute Myeloid Leukemia (AML). In AML, graft versus leukemia contributes significantly to durable remission. Reduced intensity conditioning (RIC) is frequently indicated in the elderly or those with co-morbidities. There is limited data on clinical outcomes in young adults with AML with a RIC allogeneic HSCT. Methods: From 2005 we have been using reduced intensity conditioning with Fludarabine and melphalan in patients with AML in CR1. The reason to change from myeloablative conditioning was our concern about the high treatment related mortality with our earlier myeloablative regime. We performed an analysis of 46 consecutive patients who received RIC. Kaplan Meir method was used to generate event free survival (EFS) and overall survival (OS). Results: The median age of the recipients was 34 (11–63). All patients engrafted. Median time to ANC C 500 and platelets C20,000 was 14 and 17 days respectively. Acute GVHD was seen in 24 patients [Grade 2–4: 20 (43.4 %); Grade 3–4: 09 (19.5 %)] and Chronic GVHD in 28 recipients [Limited: 21 (45.6 %); Extensive: 07 (15.2 %)] 0.4 recipients (8.7 %) relapsed. There were 12 deaths. Treatment related mortality was as follows (B100 days = 6.5 %, n = 03; B1 year = 17.3 %, n = 08). The estimated EFS was 68.6 % and OS was 71 % at 2 years with a mean follow up of 51 and 53 months respectively. Conclusion: The use of reduced intensity conditioning is a reasonable option to consider in young adults with AML in CR1.

Aplastic Anemia Abstract P 045: Oral presentation Pancytopenia: A Clinico Hematological Study Vijai Tilak1, Madhukar Rai2, Vikram Singh3 Department of Pathology, Institute of Medical Sciences, B.H.U., Varanasi; 2Department of Internal Medicine, Institute of Medical Sciences, B.H.U., Varanasi; 3Department of Internal Medicine, Vir Chandra Singh Garhwali Govt. Institute of Medical Sciences & Research, Srinagar, Pauri Garhwal, Uttarakhand Objectives: (1) To study the different causes of pancytopenia. (2) To evaluate the incidence of various disorders causing pancytopenia in children and adults. (3) To explore a cost effective and rapid test to differentiate between Megaloblastic anemia from megaloblastosis of myelodysplastic syndrome (MDS). (4) To compare our data with other studies on pancytopenia. Material and Methods: The present study was conducted in the Division of Hematology, Department of Pathology, Institute of Medical Sciences over a period of 45 months on 428 patients of pancytopenia. Results: The age of the patients ranged from 3 to 73 years. There was male

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 preponderance. The overall most common cause of pancytopenia was Aplastic anemia followed by Megaloblastic anemia. Aplastic anemia and acute Leukemia often bleed whereas Megaloblastic anemia rarely bleed. Patients with aplastic anemia and acute Leukemia were often children. Conclusions: Childhood pancytopenia is a distinct entity with a grave prognosis vis-a`-vis adult and elderly pancytopenia. Serum LDH is a cost effective and rapid test to differentiate megaloblastosis of vitamin B12 and/ or folate deficiency from MDS. Bone marrow examination can be deferred in pancytopenic patients with hypersegmented neutrophils; it can be performed later if there is no response to hematinics.

Abstract P 046: Oral Presentation Chronic Benign Neutropenia—a Case Report Rachana Garg KMC Manipal Introduction: Chronic benign neutropenia is defined as absolute neutrophil count (ANC)\1,000 cells/mm3 in infants and\1,500 cells/ mm3 in children and adults, persistently for more than 3 months. Case History: 1 year old male child presented with fever for 5 days and upper respiratory tract infection. Clinical examination was normal. His peripheral blood smear showed ANC of 120 cells/mm3 with eosinophilia and monocytosis. Bone marrow aspiration showed myeloid hyperplasia with increase in precursor cells. IgG was raised. Child was evaluated and no acquired immunodeficiency was seen. He was given 3 doses of G-CSF following which neutrophil counts improved, however counts done 4 days after G-CSF again showed ANC -252 cells/mm3. Conclusion: Chronic benign neutropenia is a rare disorder, but commonest cause of neutropenia in children in age group (3–30 months). Careful evaluation is required to avoid misdiagnosing it as leukemia or other hematological malignancies. In these cases close follow up of patient is needed to assess the course of the disease.

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Abstract P 048 Antilymphocyte (ALG)/Antithymocyte Globulin (ATG) for Severe Aplastic Anemia—Single Centre Experience Venkatesh S Ekbote, Biju George, Vikram Mathews, Aby Abraham, Riyaz Ahmed, M Kavitha, Alok Srivastava Department of Hematology, Christian Medical College, Vellore, Tamil Nadu Objective: To analyze response to immunosuppressive therapy with ATG/ALG in children and adults with severe and very severe aplastic anemia treated between 1985 and 2012. Methodology: We conducted a single institution retrospective analysis of severe and very severe aplastic anaemia patients treated with ATG-based IST from 1989 to 2012. Response, based on EBMTR guidelines, was determined at 3 and 6 months following ATG. Results: Two hundred ninety six patients with Severe and Very Severe Aplastic Anaemia with median age of 38 years (range 3–83 years) were studied, including 198 (66.9 %) males and 98 (33.2 %) females. The overall objective response (CR + PR) to ALG/ ATG CSA in this cohort was 50 %. Patients in younger age group (1–15 years) had poorer response rate—34 % compared to adults who had comparable response across all age groups; (16–29 years)—55 %, (30–50 years)—56 % and more than 50 years—59 %. Also, 32 (10.8 %) patients died of infection within first month of ATG/ALG treatment. On follow up, 14 patients (4.7 %) lost response while 13 (4.3 %) underwent clonal evolution to paroxysmal nocturnal haemoglobinuria, myelodysplastic syndrome or acute myeloid leukemia. Seven (2.4 %) patients underwent allogeneic stem cell transplantation. The overall survival in this cohort of patients was 57 %. It was 51, 52, 59 and 71 % in age groups: 1–15, 16–29, 30–50 and more than 50 years, respectively. Conclusion: This study suggests that ATG is a viable option in adult Indian patients even more than 50 year old. The poorer response seen in Indian children needs to be studied further.

Abstract P 047

Abstract P 049

A Clinico-Pathological Study of Cases of Pancytopenia—a Hospital Based Study

Spectrum of Chromosomal Breakage Syndromes Observed in Patients with and Without Bone Marrow Failure Syndromes (BMFS), at PGIMER, Chandigarh

Abhijit Phukan, Jina Bhattacharyya, DK Saikia, PK Gogoi Priti, Anil Sood, KS Rana, N Varma GMCH Objective: This study was conducted to evaluate. (1) The disease patter in different age and sexes. (2) Their clinical presentation. (3) Various causes of pancytopenia and their relative frequencies in this region. (4) Haematological parameters and bone marrow finding in various causes. Methods: The study was conducted in the Department of Pathology, GMCH, Guwahati, extending over a period of one year from 1st July 2011 to 30th June 2012. The criterion of selection of the patients was: (1) Hb \ 9 g. (2) Total leucocyte count \4,000/ mm3. (3) Platelet count \1 lakh/mm3. All the patients were subjected to PBS study, bone marrow aspiration and biopsy. We also did special stains, flow cytometry and cytogenetics as and when necessary. Results: From the study of 88 cases, various important results can be summarised as (1) Maximum cases Pancytopenia are observed in the age group of 31–40 years, with male predominance (61 %). (2) Common presenting features are generalized weakness (100 %), bleeding (23 %) etc. (3) Among the various causes, Megaloblastic anaemia is found to be the most prevalent (54 %). (4) Haematological parameters vary according the causes. (5) Hypercellularity of marrow is a feature in most of the cases (84 %). Conclusion: This study will help in finding the disease scenario in this part of the world.

Department of Hematology, Post Graduate Institute of Medical education and Research, Chandigarh Introduction: Fanconi Anemia (FA) is the most common chromosomal breakage (CB) syndrome and is the prototype of inherited bone marrow failure syndrome (BMFS). FA cases present during childhood or at times later in adulthood. Other chromosomal instability syndromes not characterized by BMFS include Nijmegen breakage syndrome (NBS), Ataxia telangiectasia (AT), Bloom’s syndrome and Xeroderma pigmentosa. Clinical heterogeneity of these disorders often leads to an incorrect or missed diagnosis. Although clinical suspicion index may be high for these disorders, definitive diagnoses (many times) based upon chromosomal breakage studies (CBS) are not well documented in India. This study was carried out to document the spectrum of these syndromes observed in patients with and without BMFS, at PGIMER, Chandigarh. Materials and Methods: The study included consecutive patients referred for work up of chromosomal breakage syndromes, between Jan 2011 and August 2012. In our department, appropriate phytohemagglutinin (PHA) stimulated peripheral blood lymphocyte cultures (PBLC) are set up for patients and controls, with and without clastogens [1. diepoxybutane (DEB) and 2. mitomycin C (MMC) for Fanconi Anemia; 1.

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208 DEB induced CBS, 2. X-irradiation and 3. treatment with bleomycin at G2 phase of cell cycle for Ataxia telangiectasia; and 1. PHA stimulated PBLC: (a) spontaneous breakage (b) G-banding for translocations, 2. DEB induced CBS, 3. MMC induced CBS 4. X-irradiation and 5. treatment with bleomycin at G2 phase of cell cycle for NBS]. Result: Out of 104 patients studied, 11 were diagnosed as Fanconi Anemia, 2 as Ataxia telangiectasia, whereas NBS was diagnosed in just 1 patient. Median age of FA patients was 8 years (range 5–32 years). Both the AT patients were males. Patient with Nijmegen breakage syndrome was an 18 months old boy. As highlighted earlier, work up for chromosomal breakage syndromes is extremely time consuming, though clinically very rewarding. Conclusion: Approximately 130 cases of Ataxia telangiectasia have been reported worldwide so far. Nijmegen breakage syndrome patient included in this study is the second case reported in India. The exact incidence of AT and NBS in India is not known. FA cases are being diagnosed at few centers in India (including our department), however the facilities to undertake specialized chromosomal breakage studies for patients without BMFS need to be widely established. This is required for appropriate genetic counseling and patient management. Unless specific confirmatory tests using a battery of clastogens, are performed, such cases will continue to remain under-diagnosed.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 lymphocytic leukemic. Among acute leukaemia, acute myeloid leukaemia outnumbered acute lymphoblastic leukaemia, (21.19 %against 7.2 %). Multiple myeloma was seen in 20.33 %. Non Hodgkin’s lymphoma was seen in 10.59 % while Hodgkin’s disease 6.6 %, MDS 1.69 %, MPN and LCH 1 %. Male to female ratio in haematological malignancies was 2.87:1. Maximum cases were observed in 51–60 years. Low grade fever, progressive pallor, weakness and body aches were the commonest symptoms (70 % cases) while pallor was the frequently observed sign. Conclusion: The prevalence of haematological malignancy is 52.44 %.This study may help in finding out their relative distribution in this region.

Abstract P 052 Monitoring of Response to Therapy in Chronic Myeloid Leukaemia Patients by Quantitative Real Time PCR on the International Scale: Our Experience in CML Patients in a Tertiary Care Centre in India J Kotwal, BK Chakrabarty, R Kapoor, V Nair, V Dutta Armed Forces Medical College, Pune

Chronic Leukemia Abstract P 050 A Case of Early Ocular Manifestation of Maculopathy in a 37 year Old Male Patient of CLL Sufia Khan, Syed Riaz Mehdi, Sharique Ahmad, Parul Gupta Era’s Lucknow Medical College, Lucknow, UP Abstract: We are presenting a case of 37 year old male patient referred from medicine OPD for routine Complete Blood Count (CBC). His Total Leukocyte Count (TLC) came out to be alarmingly high .His general blood picture (GBP) and bone marrow examination revealed it to be a case of Chronic Lymphocytic Leukemia (CLL). Later on immunophenotyping was also performed. His cervical lymph node biopsy was reported as a case of chronic lymphocytic leukemia/ small lymphocytic lymphoma. Subsequently, on his ophthalmic examination maculopathy was detected. CLL in young male has not yet been reported from India.

Abstract P 051 Clinico-Haematological Analysis of Haematological Malignancy, A Hospital Based Study Rahul Varshney, Muktanjali Deka, Jina Bhattacharya, PK Gogoi

Background: With advent of Tyrosine kinase inhibitors (TKIs), treatment and prognosis of Chronic myeloid leukaemia (CML) has been revolutionized. The patients go into complete haematological, cytologenetic (CCyR) and major molecular remission (MMR) with therapy. This is highly desirable monitoring reliability of RQ-PCR assay should be optimized in different laboratories. The IRIS group has advised cytogenetic remission but Adelaide group has favoured molecular monitoring to be more effective as cytogenetic remission corresponds to 3 log reduction at molecular level. In our laboratory in 2005 Bcr-Abl transcription was reported as copies/ml. Afterwards Bcr-Abl/Abl ratio expressed as percentage was started using the M BCR fusion Quant kit by Ipsogen in Rotor gene 3000. Base line value of Bcr-Abl/Abl ratio was calculated by taking median of 30 newly diagnosed cases of CML. With further improvements now Bcr-Abl Mbcr kit, compliant with updated international recommendations and contains an IS-MMR calibrator, aligned with international scale is used for converting NCN results to the International scale and report MMR. Methods: RQ-PCR on Light cycler 480 is used for monitoring of patients. WHO validated and accredited reference reagent and IS MMR calibrator is used in each run to calculate MMR on the IS scale. Results: Our experience related to 50 CML patients on therapy will be presented. Conclusion: Goals of therapy are to reach major molecular remission. It seems desirable that use of RQ-PCR for monitoring response to treatment for leukemia should follow the same pattern to satisfy criteria for clinical use as required by the appropriate international regulatory agencies. Keywords Chronic myeloid leukemia, Complete molecular response, Major molecular response, RQ-PCR, IS scale

GMCH Objective: Early diagnosis of haematological malignancy is important in clinical practice to prevent mortality. This study was conducted to evaluate their relative frequencies in different ages and sexes in this region. Methods: The present study was conducted in the Department of Pathology and Haematology, Gauhati Medical College & Hospital, Guwahati from 1st July 2011 to 30th June 2012. Patients were examined and blood tests including haemoglobin estimation, total and differential leukocyte count, platelets count, reticulocyte count and PBS study, Bone marrow aspiration and biopsy, special stains, flow cytometry and cytogenetics was done in each cases. Results: Out of 450 patient attending Haematology OPD 236 were having malignancy. About 29.66 % patient had chronic myeloid leukaemia while 1.69 % patient had chronic

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Abstract P 053 Incidence of Different BCR-ABL Transcript Variants Amongst CML Patients: Their Correlation with Presenting Features, Relative Risk and Response to Treatment with Imatinib Mesylate Pratik Deb, Prantar Chakrabarti, Utpal Chaudhuri, Shila Chakrabarti Institute of Hematology and Transfusion Medicine, Medical College, Kolkata

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Objective: To establish how different transcript variants of BCRABL play their role in the disease process of Chronic Myeloid Leukemia in our patient population. Method: RNA extraction, RTPCR, Spectrophotometry and agarose gel electrophoresis, Statistical analysis. Results: Amongst 80 patients (56 male, 24 female) 56.25 % patients showed the presence of b3a2 and 41.25 % patients showed the presence of b2a2 transcript variant. The rest 2.5 % (numerically, 2 cases) showed the expression of rare e19b2 variety. Patients with b2a2 transcript variant presented with higher Sokal, Hasford and EUTOS score than the patients with b3a2 transcripts. Difference between different parameters like Platelet count, TLC, Hemoglobin, Organomegaly was minor. After receiving almost 18 months of Imatinib therapy, patients with b2a2 reached hematological and cytological remission more than their counterpart. Conclusion: CML patients in our population with b2a2 variants tend to present with higher risk scores but respond better to Imatinib therapy.

Abstract P 054 Standardisation of RTPCR for Confirmatory Diagnosis of Chronic Myeloid Leukemia: First Time in a Tertiary Health Care and Teaching Hospital in West Bengal Rajarshi Aich, Pratik Deb, Utpal Choudhuri, Shila Chakrabarty, Prantar Chakrabarti

209 Road, Kolkata -700 032, West Bengal, India; 3Gupta College of Technological Sciences, Ashram More, Asansol-713301 Objective: Plants have been an immense source of therapeutics. Cancer, a dreadful disease is a leading cause of death worldwide. Synthetic anticancer agents used in chemotherapy are generally toxic or immunosuppressive. But compounds from natural sources tend to have more immunostimulants properties. We have studied the apoptosis on CML cell lines along with the immunomodulatory effects of the leaf extracts of Trichosanthes dioica. Method: We studied cell viability, cytotoxicity and apoptosis on two CML cell lines. Cell morphology was determined by Fluorescence and Confocal microscopy. DNA fragmentation was studied by gel electrophoresis. Effect of TDLE on NO production was studied in RAW264.7 cell line. Result: TDLE significantly inhibited the cell viability and cytotoxicity (MTT) in a time and concentration dependent manner. The fluorescence and Confocal study showed characteristic features of membrane blabbing, chromatin condensation the sign of early and late apoptotic changes in the cells after treatment with TDLE. Gel electrophoresis study showed fragmented DNA in the form of ladder. It was found that TDLE was able to induce NO production in resting RAW264.7 cell line. But when TDLE was used in combination with recombinant interferon-c, there was a marked cooperative induction of NO production in RAW264.7 cells. Conclusion: The present study reveals that the TDLE possesses potent anti-leukemic with immunomodulatory activity. Trichosanthes dioica constitute a major part of our every day diet. The compounds from TDL can be used as immunotherapeutic agents for cancer treatment. Studies are in progress to identify the mechanism of anti-leukemic activity of TDLE.

Institute of Haematology and Transfusion Medicine, Kolkata Objectives: Our challenge was to standardise a RTPCR protocol, for the first time in a tertiary health care system of West Bengal, for the confirmatory diagnosis of CML patients, and, at the same time, keeping the procedure as costeffective as possible. Methods: Total 80 patients were selected amongst which, 60 were diagnosed cases and 20 were suspected cases of CML. Total RNA extraction was done from the blood of all the patients and cDNA was made from it by reverse transcriptase PCR using random primers. PCR amplification of the BCR-ABL gene of cDNA was done using primers of standardized PCR protocol of BIOMED1. The PCR products were subjected to 2 % agarose gel electrophoresis and observed under UV light. Result: The PCR reaction was able to successfully amplify the target products of BCR-ABL. All 20 suspected cases of CML were diagnosed to be carrying BCR-ABL through our test and later 18 of those diagnosed cases were confirmed by bone marrow cytogenetics (BMC). The 2 failed cases were confirmed to be CML through FISH, thus establishing RTPCR’s higher sensitivity than BMC. All the established cases gave positive results. Conclusion: Our present study has led us to the development of a standardised RTPCR protocol for confirmatory diagnosis of CML. It had also affirmed that the primer pair combination used in Europe is properly working in our population.

Abstract P 055 Induction of Apoptosis in Chronic Myelogenous Human Leukemic Cells K562 and U937 by Trichosanthes dioica Leaf Extract Nilanjana Deb1, Moumita Ray1, Biswajit Chakraborty2, Chinmay Chowdhury2, Sugato Banerjee 3, Aparna Gomes1, Shila Elizabeth Besra1 1

Drug Development/Diagnostic & Biotechnology Division, Indian Institute of Chemical Biology (CSIR), 4 Raja S.C. Mullick Road, Kolkata-700032, West Bengal, India; 2Department of Chemistry, Indian Institute of Chemical Biology, (C.S.I.R), 4 Raja S. C. Mullick

Abstract P 056 Concordance Between ZAP-70 and IgVH Mutation Status in Chronic Lymphocytic Leukemia Ritu Gupta1, Lata Rani1, Nitin Mathur1, Atul Sharma2, Lalit Kumar2, Vinod Raina2 1 Department of Laboratory Oncology, Dr. B.R.A. IRCH, AIIMS, New Delhi, India-110029; 2Department of Medical Oncology, Dr. B.R.A. IRCH, AIIMS, New Delhi, India-110029

Objective: Following gene expression profiling, ZAP-70 has emerged as the most promising surrogate marker for the IgVH mutation status, a robust prognostic marker in CLL. Western studies using flow cytometry for ZAP-70 with 20 % cutoff value, showed an overall correlation between ZAP-70 expression and IgVH mutational status ranging from 77 to 90 % concordance. However, there are no such data from Indian population. We aimed to investigate the optimal cut off of ZAP-70 with IgVH mutational status and its concordance in CLL patients from India. Methods: Peripheral blood samples from 100 B-CLL patients were assessed for ZAP-70 and IgVH mutation status. ZAP-70 expression was analyzed on CD5+CD19+ CLL cells using multiparametric flow cytometry. For IgVH mutation status, RNA was extracted from peripheral blood MNC, transcribed to cDNA which was amplified by PCR according to BIO-MED-2 protocol, using VH1-VH6 family FR1 primers and 30 JH consensus primer. PCR products were purified and sequenced with an automated DNA sequencer. Ig-BLAST was used for comparing rearranged IgVH segments; cases with C98 % identity to germline were classified as unmutated (UM) while cases with\98 % identity were considered mutated (M). Results: Among a total of 100 patients, the number of mutated and unmutated cases were 62 and 38 respectively. Using ROC analysis, the optimal cut-off value for ZAP-70 was found to be C22 % (83 % sensitivity, 81 % specificity). Using this cut off value, 59 patients were ZAP-70 negative while 41 patients were positive for ZAP-70. Out of 59 ZAP-70 negative, 52 (88.1 %) were mutated and 7 (11.8 %) were unmutated. Out of 41 ZAP-70 positive patients, 31 (75.6 %) were unmutated and 10

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210 (24.3 %) were mutated. Therefore, a total of 83 % cases were concordant for ZAP-70 cut off and IgVH status (ZAP-70+UM, ZAP70-M) whereas 17 % cases were discordant (ZAP-70-UM, ZAP-70+ M). Conclusion: A cut off value of C22 % for ZAP-70 correctly predicted the IgVH mutation status in 83 % cases in Indian population.

Abstract P 057 VEGF in CML: significance and Correlation with Phase of Disease Purvi Mathur1, Usha Rusia1, Meera Sikka1, RK Grover1 1 Department of Pathology, University College of Medical Sciences & GTB Hospital, Delhi-110095, India; 2Delhi State Cancer Institute

Background: Current data suggests that angiogenesis plays a significant role in the progressive growth and metastasis of many tumors, including hematological malignancies. Vascular endothelial growth factor (VEGF) is a key cytokine involved in the angiogenic process in tumors leading to acceleration of tumor growth rate, invasion and metastasis. The current study focused on evaluating the serum levels and bone marrow expression of this cytokine in Chronic myeloid leukemia. Aims and Objectives: The present study aimed to document the serum level of vascular endothelial growth factor (VEGF) in different stages of Chronic myeloid leukemia (CML), to correlate the levels with clinicopathological factors and to study the immunohistochemical expression of VEGF in bone marrow in these patients. Materials and Methods: Serum levels of VEGF were evaluated in 40 Philadelphia chromosome positive patients of Chronic myeloid leukemia (CML) and 40 age and sex matched healthy controls by using commercially available ELISA kits. Bone marrow biopsies were obtained from 50 patients of CML. Biopsies from 10 patients suspected of lymphoma which were morphologically normal were used as controls. Immunohistochemical staining of VEGF was performed by the labeled streptavidin avidin biotin method to establish it’s cellular distribution in bone marrow. Results: Serum VEGF levels were found to be significantly (p \ 0.0001) elevated in patients as compared to controls and in patients in blast and accelerated phase of CML as compared to those in the chronic phase (p = 0.02).Correlation of serum VEGF levels with established prognostic factors showed a positive correlation between serum VEGF levels and total leucocyte counts (p = 0.04), blast percentage (p = 0.006), Hasford score (p = 0.02) and a positive trend with splenomegaly (p = 0.06). The cellular distribution of VEGF in the bone marrow was found to be similar in both patients and controls, being positive in megakaryocytes and negative in erythroid cells and neutrophils. The myeloid precursors showed a variable positivity for VEGF. Conclusion: Serum VEGF levels may play a role as an important independent prognostic factor in patients of CML. The marked elevation of VEGF levels in patients of blast crisis as compared to the chronic phase indicates that raised VEGF levels could signify advanced disease stage. The findings demonstrated in the study suggest that antiangiogenic therapy could potentially be successful in treatment of these patients.

Abstract P 058: Oral Presentation The Effect of Imatinib Mesylate Therapy on Bone Marrow Morphology in Chronic Myeloid Leukemia Patients—an Immunohistochemical Study Kamal Preet1, Usha Rusia1, Meera Sikka1, RK Grover2 1

Department of Pathology, University College of Medical Sciences & GTB Hospital, Delhi, India; 2Delhi State Cancer Institute

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Background: Chronic myeloid leukemia (CML) is the commonest leukemia occurring in Indian adults. Imatinib Mesylate is now being used in these patients as first line of treatment. There is scarcity of data on the effect of this drug on bone marrow morphology from Indian subcontinent. Moreover there is no study which has objectively tried to measure the effect of Imatinib Mesylate on stem cells and proliferation in Indian patients of CML. Aims: To evaluate the morphological changes in the peripheral blood, bone marrow aspirates and biopsies in patients of CML before and after treatment (6 months) with Imatinib Mesylate and to study the effect of Imatinib on proliferating marrow stem cells using CD34 and PCNA. Material and Methods: Morphological features were studied on peripheral blood, bone marrow aspirates and biopsies in 30 patients of CML at presentation and at the end of six months of Imatinib therapy. Results: The M:F ratio was 2.2:1 and the median age was 30 years. Common complaints at presentation were splenomegaly (97.7 %), anaemia (82 %), weakness (51 %), pain abdomen (53 %) and fever (40 %). At the end of 6 months of therapy, 96 % of the patients achieved complete hematological remission. The bone marrow aspirates and biopsies showed reduction in overall cellularity, reduction in the M:E ratio, reappearance of erythroid colonies and relative decrease in myeloid precursors in all patients except one. Megakaryocytes showed normalization of morphology and numbers. Myelofibrosis post therapy was seen in 48 %patients. Only one patient had significant myelofibrosis (grade 2-3). A statistically significant reduction in the number of CD34+ progenitors from 20 to 50 cells/hpf pre therapy to 3-35 cells/hpf post therapy was observed. Post therapy PCNA counts were 42-126 cells/mm2 which were markedly lower than the initial counts. Conclusion: Imatinib mesylate is an effective drug as first line treatment of CML patients. Apart from causing haematological remission, it also effectively acts on the CD34+ stem cells and proliferating fraction in CML. Sequential bone marrow biopsies can be useful in monitoring disease remission and progression in CML patients, hence bone marrow biopsies need to be an integral part in the follow up and management of CML patients.

Abstract P 059 Different Clinical and Haematological Features in CML Patients During First Presentation—a Study of 166 Patients Seen at Referral Centre Chetan Jain, Palash Kumar Mandal, Nirmal Kumar Bhattacharya, Swapan Kumar Sinha Medical College, Kolkata Introduction: Chronic myelogenous leukaemia (CML) is a common myeloproliferative disorder (MPD) seen in our country and world over. The condition has varied clinical presentations and haematological features but demonstration of Philadelphia chromosome is the surest test for a confirmation at present.CML may be seen in one of three stages-chronic phase (CP) or accelerated phase (AP) or blast phase (BP), each having different features. Objectives: To analyze the different clinical and hematological features at presentation of patients diagnosed as chronic myelogenous leukaemia (CML). This study was conducted in Medical College, Kolkata from July 2009 to June 2012. A total of 166 patients of chronic myeloid leukaemia were included in the study. The diagnosis was made on clinical history, physical examination, complete haemogram, bone marrow examination and cytogenetic study. Results: Out of 166 CML patients, males predominated, with a M:F ratio of 3:2. The youngest patient was 8 year old and the oldest patient was of 77 years old with a mean age of 43 years. The peak age incidence was 53 years. Cytogenetic study showed Ph chromosome to be positive in 160 cases while 6 patients

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showed BCR-ABL1 positivity by RT-PCR. Symptoms of patients included fatigue (79 %), low grade fever (18 %), body ache (38 %), weight loss (40 %), arthralgia (17 %), bleeding manifestations (10 %), dragging sensation in left loin (15 %) etc. Common signs were splenomegaly (98 %), sternal tenderness (71 %), lymphadenopathy (42 %), hepatomegaly (40 %) and purpura (12 %).Haematological features were marked leucocytosis with basophilia (2–17 %), thrombocytosis/thrombocytopenia (40,000– 8,00,000/lL), anemia (Hb6.1–10.4 g/dl). Marrow was hypercellular in all, blasts varied from 1 to 7 %. Conclusion: Though CML may have variety of clinical findings a correct approach to diagnosis with proper management may prolong the life of these patients.

Response) prior to developing tuberculosis. Four drug ATT was started under RNTCP. Result: Four out of five (80 %) patients progressed to Accelerated Phase/Blast Crisis while on antitubercular therapy, while IRIS data has showed that only 7 % progressed to Accelerated Phase/ Blast Crisis. The exact cause is not known, but drug interaction might have played an important role in reducing the therapeutic efficacy of Imatinib as the interaction of Imatinib with Rifampicin is well known. Conclusion: The concomitant use of Imatinib and antitubercular therapy may lead to disease progression in CML. A systematic registry should be maintained to document the outcome of CML patients on antitubercular therapy.

Keywords Chronicmyelogenousleukaemin, Clinical features, Haematological features

Abstract P 062

Abstract P 060 Chronic Myeloid Leukemia in Children & Adolescents: An Experience from a Tertiary Centre

Clinical and Biological Features of Chronic Lymphocytic Leukemia: A Single Tertiary Centre Experience Punit Jain, Bargavi Balakrishnan, Ansu Abu Alex, Biju George, Aby Abraham, Rayaz Ahmed, Alok Srivastava, Vikram Mathews Department of hematology, Christian Medical College, Vellore

Lalit Raut, Vinay Bohara, Girish Badarkhe, Ganesh Pujari, Uttam Kumar, SS Ray, Prantar Chakrbarti, Utpal Chaudhuri Institute of Haematology & Transfusion Medicine, Kolkata 700073 Aim: The aim of the study was to evaluate the characteristics at presentation and the treatment outcome in CML in children and adolescents age group. Methods: Retrospective analysis was carried out at a single centre in India. Thirteen patients (B17 years) attending CML outdoor from April 2008 to August 2012 were included in the analysis. Results: CML-CP was the most common phase at presentation. Maximum patients belonged to the 14–17 years age group. Disease was common in male sex. Splenic discomfort and asthenia were the most common symptoms and splenomegaly was the most common sign. The median WBC count at presentation was 65 9 109/ L. In majority (69.23 %) of patients the WBC count was between 20 9 109/L to 99 9 109/L. The median hemoglobin at presentation was 9.5 g/dL. The median platelet count at presentation was 462 9 109/L. CHR was documented in 11 out of 12 (91.66 %) evaluable responses. At 18 months of treatment MMR was achieved in 3 out of 5 evaluable patients. In one of the remaining two patients MMR was documented at 24 months while in the other patient less than MMR was documented at 12 months. Conclusion: The presenting features of CML in the children and adolescent age group are similar to that shown in other studies. The treatment with Imatinib was effective and well tolerated.

Abstract P 061 Does the Concomitant Use of Antitubercular Drugs Alter the Natural History of CML? V Badarkhe Girish, Lalit Raut, Vinaykumar Bohra, Ganesh Pujari, Prantar Chakrabarti, SS Ray, Uttamkumar Nath, Utpal Chaudhuri Institute of Haematology & Transfusion Medicine Kolkata-73 Objective: We report the outcome of CML in five patients receiving Imatinib and anti tubercular therapy. Methods: Four CML-CP patients (C17 years) attending CML clinic developed tuberculosis (2 pulmonary and 2 extra pulmonary) while on Imatinib and another one was diagnosed with Pulmonary tuberculosis and CML upfront and started on ATD and Imatinib concomitantly. All the four patients who developed tuberculosis later had achieved CCyR (Complete Cytogenetic

Objective: To evaluate the clinical and biological features of chronic lymphocytic leukemia (CLL) at presentation, indication for treatment and comparison with reported data. Methods: We undertook a descriptive retrospective study to evaluate cases diagnosed to have CLL over the last 5 years at a tertiary care centre in India and review its literature. Results: 125 cases were diagnosed to have CLL in this period. The median age was 60 years (range; 34–85). 45 (36 %) patients were \55 years (young CLL), while 3.2 % were \40 years. We found a male to female ratio of 3:1. 52 % of the cases were incidentally detected, while 29 % presented with swellings, followed by anemia requiring transfusions in 5 % and fever in 1.6 % of cases. Hepatomegaly was seen in 47.2 % and splenomegaly was seen in 40.8 %. 46.4 % patients had anaemia with autoimmune hemolytic anaemia being seen in 3.2 % patients. Thrombocytopenia (\1 9 109/ L) was seen in 21.6 %. The median white cell count and the absolute lymphocyte count were 40 9 109/L and 33 9 109/L, respectively. Majority of patients presented in Rai stage II (25.6 %) followed by stage IV (24 %), stage I (19.25 %), stage III (18.4 %) and lastly stage 0 (11.2 %). 68 (54 %) patients received treatment, out of which follow up was available in 53 patients (range 3–45 months). Out of these, 16 % had stable disease, 13.6 % had partial remission, 7.2 % had progressive disease, while 5.6 % reached complete remission. In our series, we also found CD 38 positivity in 42.4 % of the patients. Of the patients treated, progressive disease was noted in 2.7 % patients in CD38 negative patients as compared to 13.2 % patients in CD 38 positive patients. Conclusions: Our findings are similar to data reported internationally, except that we had a larger number of younger patients. Most of our patients presented in an advanced stage. Chlorambucil continues to be a widely used therapeutic option.

Abstract P 063 Profile of Chronic Lymphocytic Leukemia (CLL)—Single Centre Study from Eastern India Sandeep Saha, Basab Bagchi, Tuphan Kanti Dolai, Prakas KR Mandal, Ashutosh Panigrahi, Meet Kumar, Maitreyee Bhattacharyya, Shyamali Dutta, Rajiv De, Malay Ghosh Hematology Department, NRS Medical College and Hospital, Kolkata Objective: To determined the demographic and clinical profile of CLL patients from Eastern India. Methodology: Prospective demographic,

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Abstract P 064 Spectrum of BCR-ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia Receiving Imatinib Mesylate Therapy

DISTRIBUTION OF ABL KINASE MUTATION 12 10

No. of patients

8 6 4 2

mutation (L384M) and mutations in STI 571 contact site (F317L) were also observed. In addition, one novel mutation (codon 246–273 indel) in p-loop was identified in two cases. The functional consequence remains to be characterized. The median BCR-ABL IS ratio in 54 patients who had a mutation was 21.43 [0.57–82.42] compared to 16.32 [0.156–74.99] in patients who did not have a mutation which was not statistically significant (p = 0.2). The frequency of ABL kinase domain mutations seen in this study suggests that the emergence of a mutation may not be the major mechanism of imatinib resistance in this cohort. Other mechanisms of resistance including pharmacogenetic variations, plasma sequestration of the drug or over expression of BCR-ABL are being evaluated in an ongoing study.

Abstract P 065

Senthamizh Selvi, Preetha Markose, C Ezhilarasi, Eunice S Edison, Sachin Jain, Biju George, Alok Srivastava, Vikram Mathews, B Poonkuzhali

A Study to Analyze the Hematological Parameters in Newly Diagnosed CML Patients with b2a2 Type of BCR-ABL Transcripts

Department of Haematology, Christian Medical College, Vellore

Jogeshwar Binota1, Shano Naseem1, Neelam Varma1, Prateek Bhatia1, Deepak Bansal1, S Varma2

Abstract: Imatinib mesylate is the first line therapy for chronic myeloid leukemia (CML) and induces durable complete haematological, cytogenetic responses. Despite the impressive responses, a proportion of patients show resistance to imatinib. This has been attributed to several mechanisms of which point mutations in the BCR-ABL kinase domain is the most common mechanism. To date, approximately 90 mutants in the BCR-ABL kinase domain conferring different levels of resistance to imatinib have been identified. The aim of the present study was to analyze the spectrum of BCR-ABL kinase domain mutations in patients with clinical resistance to imatinib. A total of 162 CML patients who failed to achieve major molecular response (in the international scale—IS \ 0.1 %) were included in the study. RNA was extracted from peripheral blood leucocytes, cDNA was synthesized. BCR-ABL transcripts were quantified using real time quantitative PCR (RQ-PCR) using ABL as control gene. Mutations in BCR-ABL kinase domain was evaluated by nested PCR followed by direct sequencing. Mutations were identified in 54 of 162 (33.33 %) cases. Nineteen were in chronic phase (CP), 26 in accelerated phase (AP) and 9 in blast crisis (BC). Twenty two different mutations were identified and the distribution of these mutations is shown in Figure. T315I mutation, which is associated with resistance to first and second generation tyrosine kinase inhibitors, was the most common mutation seen [11/54; 20.37 %] followed by the p-loop mutation M244V [12.96 %]. Among the other reported mutations, p-loop mutations (Q252H, G250E and Y253H), activation loop

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Novel indel

G 250E

T 315I and Y 253F

E 255K

Mutations in ABL Kinase Domain

D295G

E 255V

F 311I

E 355G

F 359I

F 317L

H396R

L 248V

L 387F

L 273M

L 384M

L 387M

M351T

M244V

T 315I

Q252H

Y 253H

0

Y 253F

clinical, investigations including immunophenotype (IPT) data were collected from patients attending hematology services of NRS Medical College, Kolkata. Period: October 2009 to March 2012. Results: Among the 38 CLL, 29 were male and 9 female. Median age of presentation were 57 (range 37–78) years. Eight (21.05 %) patients were asymptomatic at presentation and 73.68 % (28/38) with fever, 78.94 % (30/38) with pallor and 57.89 % (22/38) with infection (RTI). On examination 28 (73.68 %) had generalized nontender lymphadenopathy, lymph node size average 2–4 cm and maximum 6 cm. Ten (26.32 %) had no peripheral lymphadenopathy (clinically) which were revealed by CECT or USG. Twenty six (68.42 %) patients presents with both splenomegaly and hepatomegaly. One had extra nodal involvement over skin (nodular lesion) below the right eye. Fifteen (39.47 %) presents with TLC [50,000, 30 (78.94 %) with anemia (normocytic normochromic anemia) and 23 (60.52 %) thrombocytopenia. Six (15.78 %) had positive Coombs test. BM involvement with interstitial pattern was found in 17 (44.73 %), nodular in 8 (21.05 %), mixed in 6 (15.78 %) and diffuse pattern in 7 (18.42 %). Myelofibrosis was found in two cases. Immunophenotyping revealed CD5+ in all cases, CD23+ and CD20+ in 36 (94.73 %), FMC7 negative in 21 (55.26 %) and sIgM positive in 23 (60.52 %) cases. Thirteen (34.21 %) were CD 38 positive. Twenty two (57.89 %) patients had advanced disease (Binet C) at diagnosis. Conclusions: (1) Majority of CLL presents at fifth decades. (2) Poor prognostic feature (CD 38 positive) were found in 34.21 % subjects. (3) One third patients had positive FMC7 and negative sIgM. (4) 57.89 % presents with advanced stage of disease.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

Department of Pediatrics, 2Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh 1

Introduction: Chronic myelogenous leukaemia (CML) is the commonest leukemia in Asia. However very few studies have been published from India, documenting the frequency of BCR-ABL fusion transcripts, possibly due to diagnostic techniques not being widely available in our country. The sine qua non of CML, t(9;22)(q34;q11) and/or BCR-ABL positivity must be detected in all cases of CML. Major bcr (M-bcr) is almost always involved in CML patients, resulting in b2a2 and b3a2 mRNA transcripts, p210 chimeric protein, and classical CML phenotype. Variable frequencies of BCR-ABL fusion transcripts have been reported from different parts of the world. b3a2 and b2a2 type of BCR-ABL transcripts are detected in approximately 95 % of CML cases, b3a2 transcript being more common than b2a2 transcript. The aim of the present study was to analyze the hematological parameters at presentation in CML patients with b2a2 type of transcripts. Materials and Method: A retrospective and prospective analysis of CML cases was undertaken over a period of 3 years (Sep 2009–August 2012). Multiplex RT-PCR was performed to determine the BCR-ABL transcripts in all the newly diagnosed CML cases. The hematological parameters studied in CML cases with b2a2 transcript were total leucocyte count (TLC), platelet count, absolute basophil count and LAP score. Results: A total of 410 new CML cases were

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 diagnosed over the study period. The incidence of b2a2 transcript was 26.3 % (108/410). The age ranged from 3.5 to 72 years with median age of 36.9 years and M:F ratio was 1.6:1. The TLC ranged from 5.8 9 109 to 924.4 9 109/L with median of 160.2 9 109/L and 26 % of the cases had a very high TLC of more than 200 9 109/L at presentation. Platelet count ranged from 7 9 109 to 1644 9 109/L with a median 370.3 9 109/L. 5.6 % cases had a platelet count \100 9 109/L and 28.3 % [450 9 109/L. Absolute Basophil count ranged from 0.069 9 109 to 57.05 9 109/L. Severe basophilia ([1000 9 109/L) was noted in 74 % cases. Discussion and Conclusion: The incidence of b2a2 transcript in India has been reported to be 28–30 %. The present study shows a slightly lower incidence of b2a2 transcript in CML cases in our region. The median ranges for all the hematological parameters are comparable to that described in western literature and slightly higher than few of the studies reported from Indian subcontinent. Keywords BCR-ABL transcripts, CML, Hematological parameters

Abstract P 066 Isolated Central Nervous System Blast Crisis in a Patient of Chronic Myeloid Leukemia on Imatinib Mesylate Therapy

213 Introduction: Chronic myeloid leukaemia (CML) is one of the commonest leukemias in India. Patients of CML are subjected to risk stratification at the time of diagnosis according to the available scoring systems, namely the Sokal and Hasford scores. Objective: Comparison of the Sokal and Hasford scoring systems for predicting progression free survival and overall survival in patients with CML. Methods: The data of 52 consecutive patients (n = 52) with diagnosis of chronic myeloid leukemia were analyzed retrospectively. The Sokal score and Hasford score at diagnosis were compared in terms of prediction of progression-free survival (PFS) and overall survival (OS) at one year after diagnosis. Results: The median patient age was 38 years (range 11–72). Thirty percent, 38 and 32 % patients belonged to the Sokal low, intermediate and high-risk groups respectively. Forty two percent, 35 and 23 % had low, intermediate and high-risk Hasford scores, respectively. The PFS was 90 % and OS was 94 % at one year after diagnosis. Both the Sokal and Hasford scores predicted better PFS for low-risk patients. However, there was no significant difference between the two scores in predicting PFS or OS. Conclusion: In our study there was no significant difference between the Sokal and Hasford scores in predicting survival in CML. Late presentation of patients may be one of the reasons for the higher prevalence of intermediate and high-risk groups in the study.

Sanjeev, Ruchi Gupta, Soniya Nityanand Department of hematology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow Objective: Imatinib mesylate is the treatment of choice in all phases of chronic myeloid leukemia (CML). However, the drug or its active metabolites have a poor penetration in the central nervous system (CNS). Thus CNS acts as a sanctuary site for malignant cells in these patients. We report an unusual case of CML on Imatinib therapy who presented with an isolated CNS blast crisis and peripheral neuropathy. Case-Report: A 25 year old male patient, a known case of CML, presented to the Department of Hematology, SGPGI, in Aug 2011 with complaints of headache, double vision, vomiting and tingling sensation of both lower limbs for past 3 months. He was on Imatinib therapy since February 2011. Salient examination findings were neck rigidity, bilateral flexor plantar reflexes and absent deep tendon reflexes. The patient was in hematological remission as confirmed by peripheral blood and bone marrow examination. Nerve conduction study of lower limbs was suggestive of axonal neuropathy. The CSF showed a turbid appearance with protein—153 mg/dl, sugar—8 mg/dl, chloride—690 meq/l and TLC12,800/cmm. Giemsa staining of CSF smears showed 98 % blasts and flow cytometry revealed a mixed phenotype blast crisis (B-Myeloid). The BCR-ABL transcript was 51.4 %. Patient was started on Dasatinib. Triple drug intra-thecal chemotherapy was given till three samples of CSF were negative for blasts. The patient 4 months later developed a systemic blast crisis and expired. Conclusion: Isolated CNS blast crisis has been rarely reported in CML. Thus in a CML patient, who presents with CNS symptoms like headache, vomiting, diplopia, etc., even if in hematological remission, a suspicion of isolated CNS blast crisis should arise and the patient should be investigated accordingly.

Abstract P 067 A Comparative Study of Sokal Versus Hasford Scoring Systems in Chronic Myeloid Leukemia Patients in a Tertiary Care Hospital in Eastern India Lopamudra Chakravarty1, Prantar Chakrabarti1, Uttam Kumar Nath1, Siddhartha Sankar Ray1, Utpal Chaudhuri1 1

Institute of Haematology & Transfusion Medicine (IHTM), 3rd floor, MCH Building, Medical College, 88 College Street, Kolkata 700073, India

Abstract P 068 Characterization of Genes Associated with Disease Progression in Chronic Myeloid Leukemia by DNA Microarray: AIIMS Experience Sudha Sazawal, Sunita Chhikara, Rekha Chaubey, Rekha Kataria, Manoranjan Mahapatra, Pravas Mishra, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi-110029 Background: Imatinib resistance is associated with point mutation in tyrosine kinase domain in 20–40 % of CML patients. The studies on genetic events that cause the progression of chronic phase to blast crisis CML in the remaining patients are very limited. In view of this, we explored gene expression profiling by DNA microarray in CML patients. Objectives: To identify predictive genes that might prove informative for stage progression in CML. Methods: Patients of CML in chronic, accelerated and blast phase were the subjects of this study. RNA was isolated from peripheral blood and QC checked using Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). The samples were labelled, hybridized, scanned and the data was extracted according to the manufacturer protocol (Agilent Technologies). The data was analyzed using Agilent Genespring 12.0 software. Results: A total of 14 CML patients (M::8, F::6), median 40 years (range 20–60 years), chronic phase (n = 6), accelerated phase (n = 3), blast crisis (n = 5) were analysed for changes in gene expression. A set of genes involved in apoptosis (DAPK1, STAT protein, TNF and PDCD1), cell adhesion (TNXB, VWF, CD6) and MAPK pathway were significantly (p B 0.05) under expressed in all the accelerated phase CML patients. Patients with blast crisis (n = 5) showed significantly elevated expression of all the genes except DAPK1. SOCS-2 showed elevated expression both in accelerated (2/3) and blast phase (3/5) CML. The above genes showed no expression in the chronic phase patients. The reported mutations were not observed in any of our patients. Conclusion: Thus, under expression or elevated expression of the above genes is associated with the disease progression in CML.

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Abstract P 069 Correlation of Multidrug Resistance Gene Polymorphism (T1236C) with Imatinib Response in Chronic Myeloid Leukemia Patients: AIIMS Experience Sunita Chhikara, Sudha Sazawal, Rekha Chaubey, Rekha Kataria, Manoranjan Mahapatra, Pravas Mishra, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi-110029 Background: Imatinib mesylate (IM) is a selective tyrosine kinase inhibitor used for treatment of patients with chronic myeloid leukemia (CML), however; some patients develop resistance to it. Several gene polymorphisms have been associated with the resistance and one such polymorphism is MDR1. Its role has been reported differently among different populations and different ethnic groups. So far there are no reported studies from India. Aim of the study: To study the association of MDR1 (T1236C) polymorphism in Indian CML patients and to correlate with response to imatinib. Material and Method: CML, chronic phase, patients treated with imatinib (400 mg/day) for 24 months were analyzed for T1236C polymorphism. DNA was extracted from peripheral blood and subjected to PCR–RFLP using restriction enzyme HaeIII. Results: A total of 100 CML chronic phase, patients at presentation were included (M:F ratio 2:1) mean age 40 (range 20–60 years). Of these, 66 patients showed response to imatinib and 34 patients showed resistance. Among 66 responders the frequency of three different alleles of T1236C locus was as follows; CC-43 (86 %), CT-18 (69.3 %), TT-5 (21 %) and in non responders (n = 34): CC-7 (14 %), CT-8 (30.7 %), and TT-19 (79 %). Resistance to imatinib was higher in patients who were homozygous for the TT allele in contrast to patients with CT/CC genotype (79 vs. 19.7 %, P B 0.001). Thus, prevalence of TT genotype seems to be higher in imatinib resistant patients compared to those who responded to the drug. Conclusion: It was observed that homozygous TT allele was associated with higher risk of imatinib resistance.

Abstract P 070 Effect of Long-Term Treatment with Imatinib on Bone Health: Single Institution Experience from Jaipur Hemant Malhotra1, Bharti Malhotra2, Om Singh Rathore2, Ajay Yadav1, Shipra Bhargava2, Pratibha Sharma2, Ashwin Mathur1, Sandeep Jasuja1 Division of Medical Oncology, Department of Medicine, 2Advanced Research Laboratory, SMS Medical College & Hospital, Jaipur, India 1

Background: Standard treatment for newly diagnosed patients of Chronic Myeloid Leukemia (CML) today is the bcr/abl tyrosine kinase inhibitor, Imatinib. The drug has to be continued for several years, possibly life-long. Long-term toxicity of the drug is not established yet. In the present study, we report the effects of the Imatinib on bone health as assessed by serum bone markers, serum vitamin D3 levels and bone mineral density by the DEXA scan. Design and Methods: We evaluated 104 CML patients who had received Imatinib for more than 1 year at the Birla Cancer Center, SMS Medical College Hospital, Jaipur. The serum calcium, magnesium and 25-hydroxyvitamin D3 levels were analyzed. The Bone Mineralization Density (BMD) of AP spine in these patients was measured using DEXA scan. Results: Eighty six percent (86 %, n = 89) of the total patients (n = 104) received 400 mg IM daily

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 while 15 % (n = 11) of the patients received 600 mg IM. Eighty percent (80 %) (n = 82) of the patients received IM for more than 3 year, 12 % (n = 13) received for more than 2 years while 8 % (n = 9) received IM for more than one year. Eleven out of the 104 patients (11/104) (10 %) and 7/104 (6 %) patients were found to have low serum calcium and low serum magnesium levels respectively. The levels of 25-hydroxyvitamin D3 were found to be deficient in 24 % (n = 26) and insufficient in 39 % (n = 40) of the total patients. Twenty three percent (23 %, n = 30) of the patients were found to be osteopenic while 8 % (n = 11) were found to have osteoporosis according to their T score values of AP spine as assessed by DEXA scan. Conclusion: Our findings suggest that long-term Imatinib may have an effect on bone health. Periodic monitoring for serum markers of bone health (vitamin D3, calcium and magnesium) and for osteoporosis (by DEXA scan) should be considered in all patients on long-term Imatinib, especially younger patients. Appropriate supplementation with Vitamin D and/or calcium should be considered for the deficient patient.

Abstract P 071 Detection of bcr/abl Kinase Domain Mutations by PCR–RFLP and Sequencing in Imatinib Resistant CML Patients Hemant Malhotra1, Bharti Malhotra2, Shipra Bhargava2, Pratibha Sharma2, Om Singh Rathode2, Ashwin Mathur1, Sandeep Jasuja1 1 Division of Medical Oncology, Department of Medicine, 2Advanced Research Laboratory, SMS Medical College & Hospital, Jaipur, India

Background and Objective: BCR-ABL oncogene mutations are responsible for the failure of Imatinib (bcr/abl tyrosine kinase inhibitor (TKI)) treatment in patients with chronic myeloid leukemia (CML). Moreover, the second generation TKI to be given after Imatinib resistance depends on the type of mutation present. Patients with the T315I mutation are resistant to second generation TKIs drugs like Dasatinib and Nilotinib. Therefore it is important to routinely test mutation in CML patients who have relapsed or have a sub-optimal response to Imatinib. Sequencing has been widely used to screen for bcr/abl kinase domain (KD) mutations in CML patient as a gold standard but it is expensive and time consuming. PCR RFLP can be used for early detection of the critical mutations like the T315I and the technique can provide rapid and inexpensive detection of important specific mutations. Our study was planned to compare PCR RFLP and sequencing in CML patients who have relapsed after or are resistant to Imatinib treatment. Methods: Forty CML patients who have relapsed after or are resistant to standard Imatinib therapy after one year were included in the study. PCR–RFLP of Thr315Ile mutation was done using Dde1 restriction enzyme and ABL KD mutations were screened by semi-nested reverse transcription PCR followed by sequencing on ABI 3500 Dx Genetic Analyzer. Results: Seven different point mutations were detected in the 7/40 (17.50 %) TKI resistant patients. These mutations were located at the four hot spots of ABL kinase domain: one at the P-loop (Q260H), two at the imatinib binding site (T315I, T317I), two at the catalytic domain (M351, E355G) and two at the active A loop (V379E, D381N). Dde1 RFLP showed wild-type pattern characterized by 2 fragments of 171 and 36 bp in thirty nine patients and one patient had T315I mutation which showed 207 bp uncut band. Interpretation and Conclusion: Screening of mutation by PCR RFLP helps in identifying T315I mutation in non responders at an early stage; this is one of the most important mutation as these patients do not respond to dasatinib and nilotinib. However sequencing needs to be done in

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 patients not having this mutation to help in therapeutic decisions regarding the choice of the second generation TKI.

MDS

215 transforms to AML, the biologic course in RA, RARS is prolonged and indolent with a very low incidence of evolution to AML. A 50 year old female patient presented with Bicytopenia (anemia, thrombocytopenia) and diagnosed to be a case of RCMD. After a span of 4 months she transformed into AML which is quite rare. Keywords MDS, AML, Myelodysplasia

Abstract P 072 Methylation Status of Fragile Histidine Triad (FHIT) Gene and its Impact on Prognosis of Patients with Myelodysplastic Syndrome-AIIMS Experience Rekha Chaubey, Sudha Sazawal, Sunita Chikkara, Manoranjan Mahapatra, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi-29 Background: Myelodysplastic syndromes (MDSs) are clonal hematologic disorders that frequently represent an intermediate disease stage before progression to acute myeloid leukemia (AML). Although several cytogenetic abnormalities have been associated with this heterogenous disorder little is known about the genetic changes responsible for the disease. Deregulated epigenetic mechanisms are likely involved in the pathogenesis of MDS. Thus, we evaluated the FHIT methylation in MDS patients and correlated with the prognosis. Aim of the Study: To investigate the frequency of FHIT gene methylation and its correlation with the prognosis in MDS patients. Methodology: DNA was extracted from bone marrow/peripheral blood leucocytes of MDS patients. Bisulphite modified DNA was amplified by methylation specific PCR. All the baseline hematological and clinical parameters were correlated with the FHIT methylation status. Results: A total of 100 MDS patients were analyzed (M:F ratio 2:1) mean age 46 years (range 9–84 years).Forty three patients (43 %) showed methylation with significantly high frequency in RAEB-2 subtypes (P \ 0.02). On comparing the methylation status with other prognostic factors, FHIT gene methylation was inversely associated with low WHO risk (P \ 0.006). The overall survival (4 years) was significantly shorter in patients with methylated FHIT gene when compared to the patients with unmethylated FHIT gene (P \ 0.005). Conclusion: These results suggested that aberrant methylation of the FHIT gene might be involved in the disease progression and be an adverse prognostic factor in MDS.

Abstract P 073

Coagulation Abnormalities Abstract P 074 Type 2 B Von Willebrand Disease—A Case Report Sushma Belurkar, Chethan Manohar KMC, Manipal Introduction: Von Willebrand Disease (VWD) is the most common human bleeding disorder. Most of the patients have simple quantitative deficiency of von Willebrand factor (VWF) but around 20 % can have qualitative defects in the VWF and are referred to as VWD type II. Type II is further classified into IIA, B, M and N types. Type IIBVWD is a rare type of VWD that is characterized by enhanced affinity of VWF to aspecific platelet receptor, glycoprotein Ib-IX complex. The characteristic functional abnormality in this variant is demonstrated by ristocetin induced platelet aggregation (RIPA). In type II B VWD very low doses of ristocetin, which have no effect on normal platelet rich plasma result in full aggregation. Case report: A 7 year old child presented with maculopapular rash on the body with bleeding tendency. Patient also had history of prolonged bleeding following tooth fall. There was no history of bleeding from any other sites. There was no positive family or sibling history. Laboratory investigations revealed a prolonged bleeding time ([15 min) and mildly prolonged APTT (40.4 s). Platelets were mildly reduced in number. RIPA showed [50 % aggregation with low dose ristocetin (0.5 mg/ml) and VWF assay was also mildly reduced (38 %). Hence a diagnosis of type IIbVWD was considered. Hyperaggregation with low dose ristocetin was not corrected by adding normal platelets but was corrected by adding normal plasma, hence platelet type of VWD was ruled out. Conclusion: Type IIBVWD is a rare subtype of VWD which can be easily distinguished from other subtypes of VWD due to its characteristic feature of hyperaggregation with low dose ristocetin however it needs to be differentiated from platelet type pfVWD by doing proper mixing studies.

A Rare Case of Transformation of MDS to AML Abhisek Saini, Sima Chauhan, Sukumar Chakravarty, Raghumani Mohanty Department of Pathology, Institute of Medical Science & SUM Hospital, Bhubaneswar Abstract: The MDS, represent a spectrum of stem cell malignancies that manifest dysplastic and ineffective blood cell production in one or more of the major myeloid cell lines compounded by a variable risk of transformation to Acute Myeloid Leukemia (AML). MDS occurs principally in older adults with a median age of 70 years, with a nonage corrected annual incidence of 3–5/100,000 persons but rising to [20/100,000 among those over the age of 70 years. MDS usually arises denovo with the risk increasing proportionate to age. Although progression to AML is the natural course in MDS, only 10–15 % of MDS develop AML, and rest progress to BM failure. The percentage of patient who progress to AML varies substantially in various subtypes. A higher percentage of MDS with increased myeloblasts

Abstract P 075 Clinical and Haemostatic Profile in Suspected Female Haemophilia—A Carriers S Kate1, A Saxena2, S Aggarwal1, N Gupta1 1 Department of Medicine, MAMC, New Delhi; 2Department of Biochemistry, MAMC, New Delhi

Background: Haemophilia A is an X-linked recessive bleeding disorder resulting from deficiency of Factor VIII. Hence screening asymptomatic women carriers is important for an effective informed decision at their pregnancies. Objective: To study the hereditary transmission in families with hemophilia A and to conduct screening coagulation tests amongst the suspected carriers. Methods: Sixty one suspected carriers in 25 families of haemophilia A were subjected to pedigree analysis and coagulation studies including prothrombin time, activated partial thromboplastin time and

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216 Factor VIIIc levels. Ten families (10 patients and 31 suspected carriers) were subjected to RFLP linkage analysis using bcl1 (Intron 18) and HindIII (Intron 19) markers. Results: On the basis of pedigree analysis, Fourteen suspected carriers could be classified as Obligate (22.9 %) and 47 as Possible (77.0 %). Six out of 61 suspected carriers (10 %) had an abnormal APTT correcting with normal plasma whereas 53 (86.8 %) had an abnormal mean Factor VIIIc activity with an average of 25.47 ± 16.3 %. None of the 8 suspected carriers with normal FVIIIc was obligate carrier or had a history of bleeding. Eight out of 25 families (32 %) had a family history of haemophilia. The suspected carriers with a positive family history (inherited case) had a significantly lower Factor VIIIc levels as compared to those without, i.e. sporadic case (p = 0.04). Molecular genetic analysis in 10 families using bcl1 and hind III RFLP markers gave noninformative results. Conclusions: Suspected women carriers of hemophilia are more likely to have lower Factor VIIIc levels where there is a positive family history.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 This nonsense mutation predicted premature termination of translation (Lys350Stop) in the catalytic domain of PROC to result in a severe phenotype. Further studies confirmed that both the parents were heterozygous carriers of Lys350Stop mutation. Subsequently, the mother presented to us for a prenatal diagnosis during her next pregnancy. The foetus was homozygous for the Lys350Stop mutation in the PROC gene and thus affected by this disorder. Conclusions: This is the first report describing mutations in PROC gene from southern India and the mutation data was helpful in offering genetic diagnosis to families affected by this disorder.

Abstract P 077 Diagnostic Value of a Quantitative Immunoturbidimetric d-Dimer Assay in the Evaluation of Oncology Patients who have a Moderate Pretest Probability of Thrombosis

Abstract P 076 J Kotwal, MK Patra, A Kotwal, BN Kapoor, V Dutta, V Nair Genetic Diagnosis of Protein C Deficiency in Two Unrelated Families from India G Sankari Devi1, S Rajkumar 1, E Sumitha 1, P Shenbagapriya2, SC Nair#, V Mathews 1, GR Jayandharan1, A Srivastava1 Departments of 1Hematology, 2Immunohematology and Transfusion Medicine, Christian Medical College, Vellore, Tamil Nadu, India Introduction: Protein C (PC) is a vitamin K dependent coagulation factor which regulates the activity of factor (F) V and FVIII. The incidence of clinically significant protein C deficiency has been estimated to be 1 in 20,000. Only *322 mutations in the protein C gene (PROC) have been reported as being disease-causative in the literature. Identification of additional mutations is important for understanding the biology of protein C deficiency as well as offering genetic testing to families affected by this disorder. Material and Methods: Two unrelated families diagnosed with hereditary PC deficiency at Christian Medical College, Vellore were investigated. PC activity and antigen levels were measured. Mutation analysis was performed by amplification of the PROC gene using 9 pairs of primers designed by Primer3 software. A direct DNA sequencing analysis of the amplicons was carried out in an ABI 3130 genetic analyzer. In silico analyses, including PolyPhen-2, SIFT, multiple sequence alignment, splicing prediction were performed to predict the consequences of each variant identified. Results and Discussion: In family 1 the proband, a 21 year old female had lost two children due to purpura fulminans and a spontaneous abortion. The Protein C levels was 19.2 % (proband/mother) and 24.2 % (husband) reduced in both the parents, consistent with a heterozygous phenotype and suggesting a autosomal recessive pattern of inheritance. Mutation analysis identified a p.Cys160Arg missense substitution in the epidermal growth factor domain of Protein C n a heterozygous state in both the parents. Cys160 is conserved across various species (Homo sapiens, Bos Taurus, Canis lupus familiaris, Gallus gallus, Mus musculus, Pan troglodytes, Rattus norvegicus) and a mutation at this site is predicted to affect the disulphide linkage between Cys160– Cys147 and thus affect the tertiary structure of Protein C. This family subsequently underwent antenatal diagnosis. The foetus was found to be only a carrier of this mutation and the family continued with this pregnancy. In the second family, a 6 day old, full-term neonate born from a consanguineous marriage presented with progressive ischemia and gangrene of multiple body parts. Excluding the common infective causes, purpura fulminans secondary to a thrombotic cause or sepsis was suspected. Protein C level in this patient was found to be 1 %. On genetic analysis, a novel c.1048 A ? T transversion at codon 350 of the PROC gene was identified in the proband in a homozygous state.

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Armed Forces Medical College, Pune Objective: Malignancies may present with life-threatening complications like Deep vein thrombosis (DVT) and consequent pulmonary embolism (PE). Unfortunately, diagnosis based on history and examination alone is often unreliable. The d-dimer test has a high sensitivity and low specificity in diagnosis of thrombosis. Earlier evaluations studies were based on ELISA for d-dimer which is not easily applicable for emergency evaluation of patients. The goal of this study was to test the efficacy of automated quantitative immunoturbidimetric d-dimer assay on Coagulometer. Subjects and Methods: Patients at the oncology centre at a tertiary care centre with moderate probability of DVT for 3 years were evaluated. 210 such cases were studied. All (210) were in intermediate risk for DVT as per Well’s score (score 2–4) and 105/210 (50 %) were in intermediate score for PE as per the revised Geneva score. D-dimer was evaluated by the automated immunoturbidimetric d-dimer assay (Lia test D-Di from Stago diagnostica) on the STA compact. Results: 210 cancer patients were evaluated. Taking the value of d-dimer \0.5 ml (FEU) as negative, 50 patients were negative and 160 were positive. However, out of the 160 d-dimer positive cases only 16 were detected to have DVT/PE. ROC curve was drawn and the max sensitivity and specificity was at a cut off of 0.80 ml (Sens 75.4 %. Spec 73.8 % and AUC 0.815). We observed that all the 16 cases of DVT/PE had d-dimer [ 1.0 |ag/ml. Thus as per our study a d-dimer \1.0 fj.g/ml would rule out DVT/PE in intermediate risk cancer patients. Conclusion: The results seem to support the use of a quantitative d-dimer assay on a coagulometer as a first-line test in evaluation for DVT/ pulmonary embolism in cancer patients when the clinical probability of the presence of DVT/PE is intermediate. Keywords d-dimer, Immunoturbidimetric, Coagulometry

Abstract P 078 Assessment of Protein C, Protein S and Prothrombin Level in Gastrointestinal Carcinoma Patients with and Without Metastasis Ashutosh Kumar1, Mohd. Yusuf1, MLB Bhatt2, Surya Kant3, Abhijit Chandra4 Department of Pathology, 2Department of Radiation Oncology, Department of Pulmonary Medicine, 4Department of Surgical Gastroenterology, King Georg’s Medical University, Lucknow, Uttar Pradesh, India 1

3

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Background: A hypercoagulable or prothrombotic state of malignancy with metastasis occurs due to the ability of tumor cells to activate the coagulation system. Material and Method: A total of 80 Patients with gastrointestinal carcinoma such as malignant carcinoma rectum, malignant carcinoma oesophagus and malignant carcinoma colon with and without metastasis were studied in order to evaluate the presence and extent of hemostatic abnormalities in case of gastrointestinal carcinoma. Results: The mean average prothrombin time in patients of gastrointestinal carcinoma was less than normal value. Similarly, the average activated partial prothrombin time was also less than the normal value. The mean level of Protein C ranged from 55 to 90 %. The mean level of Protein S ranged from 48 to 95 %.Out of the total 80 patients, 7 were Protein C deficient and 5 were Protein S deficient. However, 3 were Protein C and S both deficient. The Protein C level was significantly lower (p \ 0.0001) in Protein C deficient patients with metastasis compared to without metastasis. Similarly, the Protein S level was significantly lower (p \ 0.0001) in protein S deficient patients with metastasis as compared to without metastasis. The Protein C and S levels were also lower in those who were deficient with metastasis. Conclusion: Our study conclude that APC resistance in Gastrointestinal Carcinoma with metastasis may contribute thrombotic episodes in these patients. Cancer patients including GI malignancy are at increased risk for the development of thrombotic events that contribute significantly to the morbidity and mortality of malignancy in these patients.

Abstract P 079 Changes in the Coagulation Profile During Induction Chemotherapy in Childhood Acute Lymphoblastic Leukemia Shivali Sehgal, Sunita Sharma, Jagdish Chandra1, Anita Nangia Department of Pathology and Paediatrics1, Lady Hardinge Medical College and Kalawati Saran Children’s Hospital Introduction: Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. The risk of thrombosis in children with ALL varied from 1.1 to 36.7 % in literature. Two important factors triggering hypercoagulability are tumor cells themselves and chemotherapeutic drugs, primarily l-asparaginase. Aims and Objectives: To study the effect of induction chemotherapy on coagulation parameters in paediatric ALL patients. Materials and Methods: Thirty seven newly diagnosed patients of ALL up to 18 years of age were evaluated along with 30 age and sex matched controls. At the time of diagnosis (day 0), various coagulation parameters (PT, APTT, fibrinogen, d-dimer, Protein C, Protein S, Antithrombin, tPA, PAI-1) were tested. These were sequentially analysed on day 14 (after the completion of l-asparaginase doses) and on day 28 of therapy (after the completion of induction). Results: No major change in PT and APTT was observed during chemotherapy, however, fibrinogen levels declined significantly (p = 0.04) following l-asparaginase treatment. D-dimer levels were significantly raised at diagnosis and throughout induction therapy. PC, PS and AT were reduced in the initial part of induction followed by a rise in the second half of therapy, reaching their respective baseline levels (p \ 0.05). The tPA levels were significantly reduced in the patients at diagnosis and throughout therapy (p \ 0.001). PAI-1 levels were comparable to controls at presentation and showed a rising trend during therapy. Conclusion: The coagulopathy in ALL is multifactorial. The thrombin activation during induction therapy coupled with deficiency of anticoagulants and decreased fibrinolysis shifts the balance towards hypercoagulability. This may enhance the risk of thrombosis in these patients.

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Abstract P 080 Hypobaric Hypoxia Alters Coagulation and Fibrinolysis Pathways Neha Gupta1, Tathagata Chatterjee2, Srishti Gupta2, Mohammad Z Ashraf1 1

Genomics Group, Defence Institute of Physiology & Allied Sciences, Lucknow Road, Timarpur, Delhi, 110054 India; 2 Department of Pathology, Army Hospital (R&R), Delhi Cantt, New Delhi-110010 India Introduction: Studies have indicated that high altitude facilitates the occurrence of thromboembolic disorders (TED). However, the exact mechanism still remains to be understood. The present study was designed to understand the role of HH in TED using rat model of deep vein thrombosis. Methods: Deep Vein thrombosis was induced by flow restriction model via ligation of inferior vena cava just below the renal veins. Hypoxic animals were exposed to simulated altitude of 15,000 ft (429 mmHg). Histopathological studies were conducted in thrombus, lungs and heart tissue. Expression levels of fibrinolytic phase protein including PAI-1, tPA were detected by western blotting. Results: 24 h of hypobaric hypoxic exposure resulted in animal death and there were indications of Disseminated Intravascular coagulation (DIC) in HH exposed animals which led to excessive bleeding resulting in animal death. Histopathological studies revealed the presence of fibrin thrombi in lungs tissue of thrombotic animals on exposure to HH. At a HH exposure of lower time point (6 h) thrombus formation could be captured in IVC of rats. Moreover, the presence of fibrin thrombi in coronary heart vessels suggests that thrombus formed in the IVC would have detached and migrated to the lungs via pulmonary circulation. Levels of fibrinolytic phase proteins; PAI-1, tpA were found to be altered under hypoxic conditions. Conclusion: The in vivo experiments conducted so far have suggested that hypobaric hypoxia leads to the development of hypercoagulation followed by hypofibrinolytic state which leads to thrombo embolism and ultimately resulting in animal mortality.

Abstract P 081: Oral Presentation Screening for Lupus Anticoagulant in Women with Spontaneous Fetal Loss and its Association with Anticardiolipin Antibodies Akanksha Rawat1, Meera Sikka1, Usha Rusia1, Kiran Guleria2 1

Department of Pathology, 2Department of OBG, University College of Medical Sciences and GTB Hospital, Delhi Introduction: Recurrent pregnancy loss affects 1 % of pregnant women. Several causes of RPL have been observed. However, the potential cause remains unexplained in several cases which must be accurately identified for appropriate treatment. Aims: To study the prevalence of lupus anticoagulants (LA) and anticardiolipin (aCL) antibodies in women with spontaneous fetal loss using a combination of tests, and to confirm presence of LA by using a platelet neutralization procedure. Methods: Hundred women with spontaneous/recurrent fetal loss and fifty healthy controls were included in the study. The following tests were done: complete blood counts (LH 500), PT, APTT, APTT using LA sensitive reagent, TT, KCT, dRVVT (screening and confirmatory) for LA detection and ELISA for IgG and IgMaCL antibodies. Results: PT and TT were not prolonged in any patient or control. APTT of patients was prolonged in 8 (8 %) patients. Use of LA sensitive reagent identified 13 additional patients suspected of having LA. dRVVT screening time was found to be prolonged in 16 (16 %) patients and 15/16 patients showed

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218 confirmed positivity. Thus, the prevalence of LA in women with recurrent fetal loss was 15 %.A combination of LA sensitive APTT and LA screening test was found to be most sensitive and specific in identifying LA. IgG and IgM anticardiolipin antibodies were estimated in 78 patients and all controls. One (1.2 %) patient was positive for aCLIgG antibodies and 3 (3.8 %) patients were positive for aCLIgMantibodies. Thus anticardiolipin antibodies were detected in 4 (5.1 %) patients. Conclusions: Screening for antiphospholipid antibodies must be done in women with spontaneous/recurrent fetal loss even in absence of other clinical manifestations. The use of sensitive tests like LA sensitive APTT and confirmatory and specific test like dRVVT can diagnose a significant number of these underdiagnosed patients.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 donor splice site, 10 deletions and 6 insertional mutations were detected in the present study. A homozygous mutation in a classical female hemophilic and heterozygous mutation in another female bleeder was detected. Double mutations were detected in 2 cases (1 familial and 1 unrelated). 8 CRM (+) and 6 CRM reduced cases were identified, p.R531C was observed to be associated with both CRM (+) and reduced cases, whereas p.F309L was observed in 2 unrelated CRM (+) cases. Conclusion: High heterogeneity of F8 gene mutations, with unique mutations in most of the patients analyzed, suggests the need for maintaining a mutation database in India. Presence of double mutations gives an indication for a total gene scan, even when one mutation is identified. Presence of a female bleeder suggests that all carriers should be screened for FVIII levels, as; occasionally the heterozygote carriers may have very low levels of FVIII, most probably due to extreme lyonization.

Abstract P 082 Coagulation Aberration in HIV Infected Patients Swapan Kumar Sinha, Soumit Mondal Department of Pathology, Medical College, Kolkata Objective: To study disorders of haemostasis in untreated cases of HIV/AIDS. Total Platelet Count, prothrombin time (PT), activated partial thromboplastin time (APTT) was performed on 53 diagnosed patients of HIV. Methods: Citrated blood (1:9) samples were collected for P.Time, APTT, FDPs and EDTA blood for Complete Blood Count ART Centre of Medical College and School of Tropical Medicine. PTimre and APPT was performed as per validated methods with Internal Quality Control. Platelet count was done by making blood smears of individual samples and counting under light microscope. Results: Among them 19/53 pts. (35.84 %) had decreased plt. count. Only 7/53 pts. (13.2 %) had elevated PTimer, 6/53 (11.32 %) had elevated APTT and 9/53 (16.98 %) had elevated PT, APTT and Low platelet. Conclusion: Thrombocytopenia may be due to increased destruction commonly immune mediated, increased consumption due to microangiopathy, TTP or decreased production. Thromboplastin like substances, Factor X activation by angiopathy or due to CMV infection may lead to activation of coagulation system. Acquired Factor V Leiden may lead to hypercoagulable states including APLAS. A detail study in HIV patients for coagulation disorders will be more informative.

Abstract P 083 F8 Gene Mutation Analysis in Inversion Negative Haemophilia A Patients—Identification of 29 Novel Mutations

Abstract P 084 Procoagulant Microparticles and Their Association with Pregnancy Loss Rucha Patil, Kanjaksha Ghosh, Shrimati Shetty National Institute of Immunohaematology (ICMR), Mumbai Objective: To study and analyze the role played by the circulating microparticles in women suffering from pregnancy loss (PL) with both recurrent miscarriage (RM) and unexplained fetal loss (UFL) and characterize their cellular origin. Methods: Twenty six women with RM and 16 normal healthy control women were analyzed in the present study. Methodology for analysis of microparticles has been standardized on BD FACS Aria flow cytometer, using the ‘Megamix beads’ by participating in the ‘‘Vascular Biology SSC workshop: standardization of FCM-based PMP enumeration’’. Results: The total annexin MP and activated endothelial (CD 62e) in patients with recurrent miscarriages of all categories (early, late and early and late) were higher than in women in the control group (P \ 0.05). Differences in platelet (CD41a), non activated endothelial (CD 146), leukocyte (CD45) and erythrocyte (CD 235a) MP levels were not statistically significant. Conclusion: Our findings suggest that microparticles may have a role in the pathogenesis of recurrent miscarriages. One of the proposed causes of recurrent miscarriages is uteroplacental thrombosis, and MPs may be associated with this. In addition, presence of elevated total annexin and endothelial microparticles at a distance from the adverse pregnancy event suggest a continued chronic endothelial damage or activation which might be one of the causes of recurrent pregnancy loss. Although none of the patients in this study had any thrombotic episode, it is possible that this might become apparent in pregnancy.

Preethi Satish Nair, Shrimati Shetty, Kanjaksha Ghosh

Abstract P 085

Department of Haemostasis, National Institute of Immunohaematology (I.C.M.R), K.E.M Hospital campus, Parel, Mumbai-400012, India

A Study on the Environmental and Genetic Factors Affecting Inhibitor Formation in Severe Hemophilia A Patients

Objective: Identification of causative mutations in HA is very important for genetic diagnoses, assessing clinical manifestations and establishing a mutation database, enabling an accurate genetic diagnosis in all affected families. Method: We analyzed 108 inversion negative cases (37 severe, 36 moderate and 35 mild), by multiplex PCRs and CSGE technique followed by DNA sequencing. The pathogenicity of these mutations was assessed using various prediction softwares and genotypic–phenotypic correlation was studied. Results: We identified mutations in 82 patients with 56 unique mutations (29 novel and 27 recurrent). 35 missense, 3 nonsense, 1

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Sachin David1, Vikram Mathews1, Shashikant Apte3, Giridhara R. Giridhara R. Jayandharan1, Sukesh C. Nair2, Dolly Daniel2, Suresh Kumar1, Ansu Abu Alex1, Saravanan Ganesan1, Kannan Subramanian3, Rayaz Ahmed1, Aby Abraham1, Auro Viswabandya1, Biju George1, Alok Srivastava1 Departments of 1Haematology, 2Immunohaematology and Transfusion Medicine, Christian Medical College, Vellore, Tamil Nadu; 3Department of Haematology, Sahyadri Speciality Hospitals, Pune

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Introduction: Inhibitors or neutralizing antibodies against therapeutic FVIII is a severe complication affecting the treatment of hemophilia. A number of environmental and genetic factors have been reported to be associated with the risk of inhibitor development. We undertook a community based study to evaluate the prevalence of inhibitors among patients with severe hemophilia A and to study the predisposing environmental and genetic factors. Material and Methods: Samples from 312 patients were collected which fulfilled the inclusion criteria (severe hemophilia A and at least 5 life time exposures to factor replacement). A detailed questionnaire was administered to all subjects to obtain demographic, clinical and treatment details. Genomic DNA was used for PCR–RFLP’s (IL4Ra Ile50Val; Arg551Gln, IL4590C/T, IL5 746T[C, CTLA4 49G/A), allele specific PCR’s for identifying cytokine polymorphisms (TNF a-308G/A, TGF b 1 codon 10T/C; codon 25C/G, IL6 -174C/G, IL10 -592A/C; -819C/T; 1082A/G, IFNc +874T/A) was done using a CYTGEN cytokine genotyping trays from One Lambda (Canoga Park, CA, USA), VNTR’s in IL5Ra 30 UTR by gene scan. HLA Class II typing was done using standard kit (AllSet+ SSP PCR kits. Dynal, Merseyside, UK). The immunophenotype of the Treg population was analyzed by flow cytometry using co-expression of the CD4+, CD25+ markers. Results: Sixty patients (19.2 %) had evidence of inhibitors. Of these 22 patients (36.6 %) had high titre inhibitors (C5 BU). Table 1 compares the demographic and laboratory variables among cases that had inhibitors versus those that did not. We could not find a correlation with environmental factors. While all patients in this study had FVIII levels \1 %, patients with inhibitors had a significantly lower FVIII level than those that did not (Table 1). Cases with inhibitors were significantly more likely to have siblings with inhibitors (Table 1). Of the evaluated immune-regulatory gene polymorphisms a significant protective association was noted with the heterozygous IL4-590 C/T allele and development of inhibitors (RR = 0.22; 95 % CI 0.108–0.442: P = 0.000). This protective effect was retained in the high titre subset when low titre inhibitor positive cases were excluded from the analysis. There was also a trend to decreased risk of inhibitor formation among those that were HLA-DRB1-07 positive and this achieved statistical significance among the inhibitor cases that were high titre positive (RR = 0.24; 95 % CI 0.055–1.05: P = 0.047). It is recognized that there is significant heterogeneity in allele distribution of DRB1-07 and DRB1-13 in the Indian population (www.ebi.ac.uk/imgt/hla). There was no significant association with T-reg levels and inhibitors. Conclusions: In this series there was no apparent association with any of the environmental parameters. There was a significant association with IL4-590 C/T polymorphism, HLA-DRB1-13 and a trend with HLADRB1-07. This along with the increased incidence of inhibitors in siblings with hemophilia suggests that inherited parameters may have a more significant pre-disposing effect than environmental factors in inhibitor formation in this population.

Abstract P 086 Hematological Parameters with Perinatal Outcome in Preeclamptic Toxaemia—a Study in a Tertiary Care Hospital of Eastern India Koyela Mondal1, Swapan Kumar Sinha1, Palash Kumar Mandal1, Bandana Biswas2, Swati Bhattacharyaa3, Sanghamitra Chakraborty3 1 Department of Pathology, Medical College, Kolkatta, Rabindra nath Mandal, Dehanadapur, Hooghly. 2Department of Gynaecology & Obstetrics, Medical College, Kolkatta, Rabindra nath Mandal, Dehanadapur, Hooghly. 3Department of Biochemistry, Medical College, Kolkatta, Rabindra nath Mandal, Dehanadapur, Hooghly.

219 Introduction: Preeclampsia and eclampsia are important causes of morbidity and mortality in pregnant women in developing countries. Hemolytic anaemia and related disorders like TTP, HELLP and HUS are commonly seen during pregnancy associated with preeclampsia and eclampsia and may cause further complications. Objectives: To study haematological parameters, coagulation profile and liver enzymes with perinatal outcomes in preeclasmpsia and eclampsia. Methods: Out of 10,782 number of antenatal mothers attending the labour room during the period of August 2011 to July 2012, 636 were found to have severe preeclampsia and eclampsia. Physical examination of patients with routine urine and blood examination was done in all the patients. Complete hemogram with coagulation profile (PT, APTT, FDP) and liver function tests of all 636 mothers were noted. Results: Patients were in age range of 21–38 years. The criteria for severe preeclampsia included sustained blood pressure of at least 160/110 mmHg or higher with persistent proteinuria on urine dipstick or elevated creatinine ([1.2 mg/dL). Out of 636, 305 (48 %) mothers were anaemic, fifty cases (8 %) of thrombocytopenia alone. Out of 305 anaemic mothers, 57 (19 %) presented with HELLP syndrome. PT, APTT, FDP was raised in 19, 45 and 9 cases respectively while combined PT and APTT in 50 cases. Still birth was found in 31 cases (5 %) and 190 cases (30 %) were of small for gestational age. Conclusion: Early detection of toxaemia and also hemolytic disorders in antenatal patients may in preventing morbidity and mortality of mothers and newborns.

Abstract P 087 Genetic Determinants of Warfarin Dosage in Indian Patients Tejasvita Gaikwad, Shrimati Shetty, Vrinda Kulkarni1, Cecil Ross2, Shanaz Khodaiji3, Kanjaksha Ghosh National Institute of Immunohematology (ICMR), KEM Hospital, Mumbai; 1Nair Hospital, Mumbai; 2St.John’s Hospital, Bangalore; 3 P.D Hinduja Hospital, Mahim Introduction: Warfarin, a widely prescribed anticoagulant for prevention of thrombosis, is associated with narrow therapeutic window where even small variations in dosing may result in hemorrhagic or thrombotic complications. Several polymorphisms in VKORC1, CYP2C9, CYP4F2, EPHX1, GGCX, factor IX, factor II and factor VII have been shown to be associated with warfarin dose requirement. Material and Methods: 120 warfarin treated patients were screened for CYP2C9*2, CYP2C9*3, VKORC11639G/A, EPHX1 T113C (rs1051740) by PCR–RFLP and allele specific PCR method. Results: See Table. (1) CYP2C9 and VKORC1 promoter region polymorphisms: Overall 44.4 % studied patient population was found to be carriers for any of the three warfarin sensitive alleles, requiring low warfarin dose as compared to wild type, i.e. 3.1 mg versus 6.25 mg (±1.3 mg) OD. Of this, approximately 62 % patients manifested with bleeding problems while on warfarin. (2) EPHX1 gene polymorphisms: Patients with CC genotype require approximately 1 mg/day less warfarin than patients with TT genotype. Conclusions: Approximately 45 % of the patients carry any one of other 3 strongly linked warfarin sensitive polymorphisms, suggesting the need for identifying the warfarin genotype before starting the dose. The strongest determinant of warfarin dosage was shown to be VKORC1 AA, followed by cytochrome 3 and cytochrome 2 polymorphisms. EPHX1 polymorphism showed a minor effect on warfarin dosage. Keywords Warfarin genotype, VKORC1, CYP2C9, EPHX1, Warfarin adverse effect

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220

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

Association of different variants with warfarin dose requirement and over anticoagulation No. Mean warfa- Patients rin with dose INR \ 3 Wild type allele carriers VKORC1 GG + CYP2C9*1/ *1 + EPHX1 TT

31

6.2

7 (22.5 %)

Abstract P 088 FXI Deficiency—a Rare Cause for APTT Prolongation Archana Suman, SK Bose, Joseph, Alaknanda, C Batra, J Ahluwalia, S Varma1, N Varma Department of Hematology and Internal Medicine1, Postgraduate Institute of Medical Education and Research, Chandigarh

VKORC1 GA + CYP2C9*1/ *1 + EPHX1 TT

9

4.0

6 (66.6 %)

VKORC1 AA + CYP2C9*1/ *1 + EPHX1 TT

1

2.0

1 (100 %)

VKORC1 GG + CYP2C9*1/ *2 + EPHX1 TT

3

4.41

1 (33.3 %)

VKORC1 GG + CYP2C9*1/ *3 + EPHX1 TT

5

3.36

3 (60 %)

VKORC1 GG + CYP2C9*3/ *3 + EPHX1 TT

1

2

1 (100 %)

Introduction: FXI deficiency is a rare bleeding disorder characterized by a hemorrhagic syndrome of variable degree of severity. FXI is involved in intrinsic coagulation pathway. Here we present a case report of patient with FXI deficiency. Case Report: A 16 year old Muslim girl, presented with an episode of hemochezia 2 years back and epistaxis since 2 years. She had no family history of bleeding or previous transfusion. She was born of a consanguineous marriage. Laboratory tests revealed normal PT and prolonged APTT which was corrected by normal pooled plasma. Mixing studies showed correction of APTT with Factor VIII and IX deficient plasma and no correction with FXI deficient plasma. The platelet count was normal. Factor XI assay revealed marked deficiency (1 %) in patient. Family screening revealed low FXI activity in the mother and father, 40 and 39 % respectively. Her sister was asymptomatic and had normal FXI levels. Conclusion: FXI deficiency is a rare cause of bleeding and must be looked for in patients with isolated APTT prolongation. The diagnosis is usually delayed in females with prolonged APTT, since a commoner cause—Von Willebrand disease needs to be excluded. This case reiterates the fact that as has been described earlier, the factor levels do not coincide with symptom severity. Less than 150 cases of FXI deficiency have been reported so far in India.

VKORC1 GG + CYP2C9*2/ *3 + EPHX1 TT

1

3.5

1 (100 %)

Abstract P 089

One variant carriers VKORC1 variant carriers

Cytochrome variant carriers

The Laboratory Profile of the Thrombotic Antiphospholipid Syndrome

EPHX1 variant carriers VKORC1 GG + CYP2C9*1/ *1 + EPHX1 TC

30

5.19

8 (26.6 %)

VKORC1 GG + CYP2C9*1/ *1 + EPHX1 CC

10

5.02

3 (30 %)

VKORC1 GG + CYP2C9*1/ *3 + EPHX1 TC

9

2.94

4 (44.4 %)

VKORC1 GG + CYP2C9*1/ *3 + EPHX1 CC

3

3.16

2 (66.6 %)

VKORC1 GA + CYP2C9*1/ *2 + EPHX1 TT VKORC1 GA + CYP2C9*1/ *3 + EPHX1 TT

1

3.5

1 (100 %)

1

2.5

1 (100 %)

VKORC1 GA + CYP2C9*1/ *1 + EPHX1 CC

1

2.5

1 (100 %)

VKORC1 GA + CYP2C9*1/ *1 + EPHX1 TC

14

2.98

8 (57.14 %)

Department of Hematology, PGIMER Chandigarh

Combined variant carriers

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J Ahluwalia, Joseph, SK Bose

Objective: The antiphospholipid antibody syndrome (APS) is the commonest acquired cause of thrombosis). We attempted to determine the prevalence of APS in patients with thrombosis and to study the pattern of positivity in the laboratory tests. Methods and Patients: Patients with thrombosis undergo 3 tests viz Lupus anticoagulant testing (clot based screening and confirmation) and anticardiolipin and anti beta 2 Glycoprotein 1 antibody assay (both by EIA) as a part of workup of the APS. Laboratory data of patients reporting for the investigation for thrombophilia for a thrombotic event was reviewed and examined for the persistent positivity of these tests at an interval of at least 3 months. Results: In the period of 3 years, 991 cases were tested for the APS. 889 cases were available for analysis. In 186 (20 %) of these any one of the APS antibodies were positive at least once. In 30 (3.3 %) the antibodies persisted for at least 12 weeks and they qualified for the APS. Only 2 cases had persistent positivity for all 3 antibodies. In 22 cases only 1 antibody was positive. Anti beta 2 GP 1, Anticardiolipin and LA antibodies were positive in 28, 9 and 4 cases respectively. Fifteen of the 30 cases had CNS vascular obstruction, whereas 6 had limb DVT. Conclusions: Positivity for all 3 antibodies is rare. Anti beta2 GP 1 antibodies were the commonest ones encountered. Testing for LA is less frequently positive possibly because of inability to reconfirm the initial positive test due to start of anticoagulation.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Table 1 Comparison of cases with and without inhibitors Demographic parameters Inhibitor positive: N = 60 N (%)/median (range)

Inhibitor neg- P value ative: n = 252 N (%)/median (range)

Age (years)

18,.5 (3–59)

18 (2–75)

0.647

Age at diagnosis of hemophilia (years)

1.5 (0–55)

1.5 (0–32)

0.718

FVIII:C (%)

0.25 (0.04–0.99) 0.35 0.007 (0.10–0.60)

No: of exposures in first year of life

0 (0–4)

0 (0–25)

Life time number of exposures

30 (2–336)

23.5 (5–1,000) 0.164

FVIII (life time) total factor replacement used (IU)

13,200

(1,000–241,000)

9,200

221 obtained would assist in establishing a National Mutation Database, and enable an accurate carrier and antenatal diagnosis in affected families by direct mutation analysis.

Abstract P 091

0.696

(500–425,600) 0.133 Number of joints affected 2 (0–7)

3 (0–7)

0.505

Parental consanguinity

18 (30)

56 (22.2)

0.237

Familial hemophilia

38 (63)

152 (60.3)

0.769

Sporadic hemophilia

22 (36.7)

100 (39.7)

0.769

Siblings with inhibitors

4 (6.6)

5 (1.96)

0.016

Discordance between siblings for inhibitors

7 (11.6)

12 (4.76)

0.044

Chronic Illness

6 (10)

19 (8)

0.596

Past history of surgical procedure

7 (11.7)

52 (20.6)

0.142

Abstract P 090 Molecular Basis of 19 Severe Factor XIII Deficient Cases

Variations of PT Reagent to FX Deficiency Shenbagapriya, Ramya, Jennifer, Suresh, Vandana Kamath, Usha Sitaram, Sukesh C Nair Department of Transfusion Medicine & Immunohaematology, Christian Medical College, Vellore, India Abstract: FX deficiency is a rare bleeding disorder which results in variable bleeding tendency, but patients with severe FX deficiency tend to have severe bleeding manifestations among the rare bleeding disorders. Prothrombin time reagents may vary in sensitivity to FX deficiency and congenital variants have been identified in which PT or APTT being normal. We have a case of 31 year old female with complaints of menorrhagia for the past 3 months, easy bruisability and prolonged bleeding. Laboratory Findings: Plasma clotting test and Global haemostatic test were done and the results are as follows: Plasma Clotting test: PT PT (Innovin:Recombinant Thromboplastin): Pt: [ 2 min Normal range: 10.0–12.5 s INR: [10.0;PT (Thromborel S:Human Placental Thromboplastin): Pt: 28 s;aPTT: Pt:52.4 s Normal range: 25.0–34.8 s;‘ PT with ‘ control plasma: 11.4 s;‘ PT with ‘ adsorbed plasma :23.1 s;‘ PT with ‘ aged serum: 11.0 s;TT: Pt:13.8 s Normal range: 12–16 s;FVIII: 367.6 % FIX:315.4 % FXI: 195.8 % FV: 167.0 %; FX: \1 % (Innovin:Recombinant Thromboplastin); FX: 5.7 % (Thromborel S:Human Placental Thromboplastin); Lupus Anticoagulant (DRVVT): Negative Pt: Screen: 64.5 s Screen Mix: 40.2 s Control Screen: 37.2 s. Global Haemostatic test also showed normal clotting parameters. Marked prolongation of PT ([2 min–132.5 s) to recombinant Thromboplastin which also resulted in its assay system revealing severe deficiency of FX. This could be either a laboratory phenomenon or FX variant.

Abstract P 092

Sharda Shanbhag, Shrimati Shetty, Kanjaksha Ghosh National Institute of Immunohaematology (ICMR), Mumbai Introduction: Congenital Factor XIII (FXIII) deficiency is a rare autosomal recessive disorder affecting 1 in 1–5 million individuals, with a higher prevalence in countries where consanguineous marriages are common. It is a serious bleeding diathesis, generally manifested as umbilical stump bleeding, post-injury prolonged bleeding, intra cranial bleeding, and spontaneous abortions in women. FXIII deficiency is usually attributed to mutations in F13A gene, on chromosome 6. Material and Methods: We analyzed 19 FXIII deficient patients, diagnosed on the basis of their clinical history, normal screening coagulation and clot solubility assay. Genomic DNA was extracted by the phenol chloroform method, and mutations were detected by direct DNA sequencing of F13A gene. 14 of 19 patients had history of primary consanguinity. Results: 17 mutations were detected in 19 FXIII deficient patients, of which 8 were missense (5 novel, 3 recurrent), 6 were nonsense (3 novel, 3 recurrent), and 2 patients showed a novel single base pair deletion; one patient showed a splice site mutation in exon 14. A large deletion in exon 3 is suspected in 2 unrelated patients because of repetitive failure of PCR amplification of this exon. Seven polymorphisms were detected in these patients, of which one is novel. Conclusions: We have identified F13A gene mutations in all the 19 FXIII deficient patients, and observed a high heterogeneity in the mutation profile. The data

The Epidemiology of FVIII Inhibitors in Indian Haemophilia A Patients Patricia Pinto, Preethi Nair, Priyanka Kasatkar, Tejasvita Gaikwad, Shahnaz Ali, Anshul Jadli, Rucha Patil, Bipin Kulkarni, Kanjaksha Ghosh, Shrimati Shetty Department of Haemostasis & Thrombosis, National Institute of Immunohaematology, (Indian Council of Medical Research), Mumbai, India Objective: A serious complication of replacement therapy in patients with bleeding disorders, is the development of specific antibodies or ‘inhibitors’ to the deficient coagulation factors, particularly in haemophilia A patients. This leads to an increase in the management cost, morbidity and mortality, especially post-operatively in case of FVIII inhibitors. The mechanism of FVIII inhibitor development is quite complex and it is difficult to predict their development, but a prompt and accurate diagnosis is critical as early therapy can save lives. The aim of this study was to screen patients with bleeding disorders for inhibitors, and analyse the incidence of inhibitors in different regions in India. Methods: Samples were collected in sodium citrate vacutainers from patients in different cities in India for coagulation and inhibitor screening assays. The Bethesda assay was performed in inhibitor positive samples for confirmation and quantification of the FVIII Inhibitor titre. Patient

123

222 details were recorded in a clinical proforma. A total of 800 patients with bleeding disorders from different cities of India were screened for ‘Inhibitors’. Results: Out of the 800 samples screened, 710 were Haemophilia A patients, out of which 51 were positive for ‘FVIII Inhibitors’. Conclusion: The highest incidence of FVIII Inhibitors, i.e. 20.48 % was seen among the Chennai samples, followed by Hyderabad, Mumbai and Guwahati, which showed an incidence of 12.5, 11.02, and 8.51 % FVIII Inhibitors respectively, with respect to the samples analysed. The other regions showed an Inhibitor incidence\7 %. The overall FVIII Inhibitor incidence in the haemophilia A samples studied was 7.18 %. Keywords FVIII Inhibitors, epidemiology, haemophilia A

Abstract P 093 Analysis of FVIII Polymorphism Haplotypes as Risk Factors for FVIII Inhibitor Development in Indian Severe Haemophilia A Patients Patricia Pinto, Kanjaksha Ghosh, Shrimati Shetty Department of Haemostasis & Thrombosis, National Institute of Immunohaematology, (Indian Council of Medical Research), Mumbai, India Objective: ‘Factor VIII (FVIII) inhibitor’ development, a serious complication of FVIII replacement therapy in haemophilia A patients, leads to an increase in management cost, morbidity and mortality, especially post-operatively. Recently, four non-synonymous singlenucleotide polymorphisms (SNPs) in the F8 gene, G1679A [R484H], A2554G [R776G], C3951G [D1241E], and A6940G [M2238V], whose haplotypes encode six wild-type FVIII proteins (H1–H6), have been implicated as risk factors for FVIII inhibitor development due to mismatched FVIII transfusions. The aim of the study was to determine if mismatched FVIII transfusions could significantly contribute to FVIII inhibitor development in Indian haemophiliacs. Methods: We analyzed the above four SNPs, in 60 Indian severe hemophilia A patients, i.e. 30 inhibitor positive and 30 inhibitor negative control patients, by direct DNA sequencing of the relevant regions of the F8 gene. Results: The prevalence rates of the H1 and H2 haplotypes, were found to be 1.00 and 0.00 among the inhibitor positive patients, and 0.94 and 0.06 among the inhibitor negative patients, respectively. These two haplotypes (H1 and H2) match the recombinant FVIII products Kogenate (Bayer) and Recombinate (Baxter), used clinically. Conclusion: The F8 polymorphism haplotypes are probably not a risk factor for FVIII inhibitor development in Indian haemophiliacs, but could explain the lower inhibitor incidence. Further studies, in a larger patient cohort, with regard to association with F8 mutations and other genetic risk factors of inhibitor development, could provide useful insights into the FVIII immune response. Keywords FVIII Inhibitors, F8polymorphisms, Haemophilia A

Abstract P 094 Effect of Antitubercular Therapy on Haemostasis Including Coagulation Factor VIII in Patients with Tuberculosis Aditya Kutiyal, Naresh Gupta, Sandeep Garg Department of Medicine, Maulana Azad Medical College, New Delhi-110002, India Objective: To study the effect of antitubercular therapy on haemostatic parameters including coagulation Factor VIII in patients with

123

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 different types of tuberculosis. Methodology: Adult patients over 12 years of either sex with newly diagnosed tuberculosis of different sites categorized as pulmonary, CNS, disseminated and others were included. Detailed clinical assessment included the haemostatic parameters namely Prothrombin Time, Activated Partial Prothrombin Time, Factor VIII, fibrinogen and D-dimers were done in all, before and after 2 months of intensive standard antitubercular therapy. Results: Mean age of the 128 subjects was 31.55 ± 15.03 years, ranging 12–75 years. Pulmonary, CNS, disseminated and other types of tuberculosis comprised 30.5, 28.9, 17.2, and 23.4 % patients respectively. Abnormal PT and APPT at baseline was seen in 64 (50 %) and 23 (18 %) cases respectively whereas the mean coagulation Factor VIII level was 125.08 % ± 72.83. Factor VIII was deranged in 45 (35.15 %) cases, being high in 28.12 % and low in 7.03 %. Baseline fibrinogen levels were altered in 73 (57 %), with 62 being elevated beyond 400 mg%. D-dimers were high in 57.8 % patients. Platelets were disturbed in 66 (51.5 %), with thrombocytopaenia in 47 and thrombocytosis exceeding 400 9 109/l in 19 cases. Following 2 months ATT, the PT became normal in statistically significant 32 (50 %) of 64 abnormal at baseline. Factor VIII levels did not change significantly after ATT (125.08 ± 72.83 vs. 126.21 ± 37.65) whereas fibrinogen decreased significantly from 391.43 ± 167.34 to 272.33 ± 92.66 mg% (p \ 0.01). Cases with elevated D-dimers came down to 12.5 %. Interestingly, all the platelet disturbances reverted to normal within 2 months of ATT. Subgroup analysis of different categories of tuberculosis mirrored these results except the PT in disseminated group. Conclusions: Elevated fibrinogen, D-dimers, and Factor VIII levels were observed in patients with different types of tuberculosis, favouring a state of hypercoagulability which improved with anti-tubercular therapy. Keywords Tuberculosis, Haemostasis, Coagulation factor VIII, Hypercoagulability

Hemoglobino Pathies Abstract P 095 Evaluation of D-10 Hemoglobin Testing System for Diagnosis of Beta Thalassemia Carrier State Debajyoti Singha Roy Ramakrishna Mission Sevapratisthan and Vivekananda Institute of Medical Science, 99 Sarat Bose Road, Kolkata-26 Objective: Prevention is the only way to reduce the burden of thalassemia through beta thalassemia carrier state detection. The most reliable method is High Performance Liquid Chromatography (HPLC). The conventional HPLC instrument (VARIANT, BIO-RAD) is very expensive ([3 million).The present study examines the feasibility of using a relatively less expensive HPLC instrument D-10 HEMOGLOBIN TESTING SYSTEM, BIO-RAD which was initially launched for HbA1c estimation by HPLC. After a thorough search we could not find any comparative study between the VARIANT and D-10. Methods: Study population- Patients who are diagnosed as beta thalassemia carrier by D-10 in RKMSP and VIMS. Exclusion criteria: 1. Children \ 6 months; 2. Patients with prior blood transfusion; 3. Co existing hematinic deficiency and borderline HbA2 value. Data collection: same blood samples of the cases diagnosed as beta thalassemia carrier by D-10 are tested in SYSMEX KX21 cell counter for red cell indices and also in VARIANT, BIO-RAD. Some retained samples are also run in D-10 on day 7. Analysis: statistical analysis done to establish correlation between results obtained from D-10 and VARIANT. Results: Sample size 96. (1) Between D-10 and

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 VARIANT. (2) HbF value- 99 % correlation. (3) HbA2 value—83 % correlation. (4) Not a single case misdiagnosed. (5) 16 samples run on day 1 and 7 in D-10. (6) HbF—95 % correlation. (7) HbA2—97 % correlation. Conclusions: After obtaining a good correlation value it can be concluded that D-10 HEMOGLOBIN TESTING SYSTEM is a reliable, low cost instrument for beta thalassemia carrier state detection.

Abstract P 096 Detection of Pattern of Hemoglobin in Patients Advised HPLC—A Hospital Based Study Aishwarya Raj, Jina Bhattacharya, UC Dutta, PK Gogoi Department of Hematology, Gauhati Medical College and Hospital, Guwahati, Assam Objective: Study was conducted to detect the pattern of hemoglobin and relative distribution of different variants in patients attending the Hematology department, GMCH. Methods: Present study was conducted in Department of Haematology, Gauhati medical college and hospital from 1st December 2010 to 31st July 2012. Geographical distribution of cases predominantly included parts of Assam. Patients presenting with clinical features and blood examinations indicating haemoglobinopathies were subjected to HPLC. Further tests were carried out in selected cases (sickling test, acid elution test, osmotic fragility test etc.). Result: Out of total 783 patients subjected to HPLC, 394 cases (50.38 %) were having normal Hb A. Among the 49.62% cases with abnormal hemoglobin, the most prevalent pattern was heterozygous Hb E (21.18 %) followed by homozygous Hb E (15.07 %). Compound heterozygous Hb E-b was the next commonly observed variant (6.13 %). 2.29 % had sickle cell disease; 0.26 % had sickle cell trait, 0.38 % had Hb S-b trait, while two cases (0.24 %) with double heterozygous Hb S-Hb E were observed. A single case (0.12 %) of hereditary persistence of fetal hemoglobin was noted. 0.89 % cases suffered from beta thalassemia major, while 3.06 % cases were heterozygous for beta thalassemia. Conclusion: Study shows that the prevalence of abnormal hemoglobin pattern is 49.62 %, of which the most common is noted in Hb E (36.25 %) in this part of India.

Abstract P 097 Trials of Hydroxyurea in Sickle Cell Hemoglobinopathies Patients of Eastern India Prasanta Purohit, Dilip Kumar Patel1, Siris Patel, Snehadhini Dehury, Subasa C Bishwal, Satyabrata Meher, Bidyapati Pradhan, Kishalaya Das Sickle Cell Clinic & Molecular Biology Laboratory, VSS Medical College, Burla, Sambalpur, Odisha; 1Department of Medicine, OSCP, NRHM, V.S.S. Medical College, Burla, Odish Introduction: Increased level of HbF (22.3 ± 6.9) was found to have protective effect against painful crisis, osteo-necrosis, ACS and splenic dysfunction in Sickle cell disease patients of Odisha (Mashon RS, 2009). Low dose Hydroxyurea (10 mg/kg body-wt/day) effectively increased the level of HbF in this patients (Patel DK, 2012). Unfortunately, the use of HU is extremely limited in India, because of its high cost and the apprehension of its toxicities. Objective: To assess the clinical and haematological response of low dose Hydroxyurea in patients of sickle cell hemoglobinopathies in eastern India. Materials and Method: The study was undertaken at Sickle Cell Clinic & Molecular Biology Laboratory, V.S.S. Medical College,

223 Burla, Odisha, India, from 2006 to 2012. 1887 patients were enrolled including 1738 HbSS (35.7 % paediatrics and 64.3 % adults), 125 HbS-b thalassemia (33.6 % paediatrics and 66.4 % adults), 18 HbSD and 6 HbSE. The indication were [3 VOC or [2 blood transfusion in last 12 months of presentation. Detailed baseline studies, i.e. CBC, HPLC, Bio-chemical, liver function test were done and cases were followed up at three month intervals. Here we analysed the data of 364 cases under regular follow-up for more than 2 years of HU therapy. Result: After 2 years HU therapy %HbF increased significantly from 18.6 ± 6.9 to 22.5 ± 7.3 but the proportionate rise of HbF varied from case to case. MCV, MCH and MCHC level also increased significantly in all cases. The frequency of painful crises reduced significantly after HU therapy. In paediatric cases response rate was 71.5 % where as in adults it was 91.2 %. Following HU therapy, about 95.0 % patients became transfusion independent. Transient bone marrow suppression (Absolute Neutrophil Count \2,500/lL, platelet count \80,000/lL) was occurs in 6.96 % cases. Discussion: With a minimal dose (10 mg/kg body-wt/day) of HU, most of the patients showed an impressive improvement in clinical and haematological parameters. In resource poor country like India, low dose HU therapy provides a suitable therapeutic option for a vast number of untreated sickle cell hemoglobinopathies patients.

Abstract P 098 Clinical Hematological and Molecular Characterization of Sickle Cell HbDPunjab (HbSD) in Eastern India: The Largest series in world Subasa C Bishwal, Dilip Kumar Patel1, Siris Patel, Snehadhini Dehury, Prasanta Purohit, Saytabrata Meher, Bidyapati Padhan, Kishalaya Das Sickle Cell Clinic, VSS Medical College & Hospital, Burla, Sambalpur, Odisha-768 017; 1Department of Medicine, V.S.S. Medical College, Burla, Odisha Introduction: Although HbDPunjab[b-121(GH4)Glu ? Gln] is the commonest haemoglobinopathy in northern India, HbSD, the compound heterozygote state with sickle gene is rare. Hereby we report detailed profile of 36 cases Hb SD, the largest number hitherto reported in world literature. Objectives: To describe the clinical, hematological and molecular profile of 36 HbSD patients from Eastern India. Materials and Methods: HbSD disease was diagnosed by HPLC. All cases were subjected to detailed clinical, radiological, biochemical examination and CBC. HbS mutation was confirmed by ARMS-PCR, HbDPunjab mutation was confirmed by EcoR1-RE digestion following PCR, RFLP and Xmnl polymorphism were studied by analyzing six RE sites as described by Orkin et.al (1982). a-Thalassaemia was diagnosed by GAP-PCR. Results: The mean age of patients was 21.63 ± 11.7 years and majority belonged to a particular caste namely the Agharia, which is a predominant caste of Western Odisha. Highest number (30.55 %) of patients was from Sundergarh district. The commonest clinical presentation was blood transfusion (in 69.44 % of patients) followed by painful crisis (66.67 % of patients). 32.0 % of patients had splenomegaly and hepatomegaly. 18.18 % had AVN of the femur head, 12.0 % had cholelithiasis, whereas 8.0 % had splenic atrophy. The mean Hb, MCV and MCH were 8.4 ± 2.59 g/dL, 88.54 ± 11.0 fl and 29.4 ± 4.34 pg respectively in the HbSD patients. The mean HbF concentration was 19.05 ± 9.95 % and HbD was 41.46 ± 3.89 %. 81.18 % were heterozygote for Xmnl polymorphism whereas 18.82 % were homozygotes and 5 cases (15.0 %) had deletional a-thalassaemia. Conclusion: We report the detail profile of 36 cases of HbSD patients who were thoroughly investigated and their detailed molecular characterization was accomplished.

123

224

Abstract P 99 Epidemiology of Beta Thalassemia Trait in Western Odisha Satyabrata Meher, Dilip Kumar Patel1, Siris Patel, Prasanta Purohit, Snehadhini Dehury, Subasa C Bishwal, Bidyapati Padhan, Kishalaya Das Sickle Cell Clinic, VSS Medical College & Hospital, Burla, Sambalpur, Odisha-768 017; 1Department of Medicine & Project coordinator, OSCP, NRHM, V.S.S. Medical College, Burla, Odisha Introduction: Heterozygote state of Beta thalassaemia (Beta thalassaemia trait or BTT) is benign and asymptomatic. Detection of BTT has been very important in the effort of prevention of the disease by prenatal diagnosis. Microcytic hypochromic red cells and an elevated HbA2 fraction is hallmark to detect BTT. Objective: To study the epidemiology of BTT in Western Odisha from our Centre. Material and Method: Total of 5690 blood samples were collected from cross sectional study from different parts of Western Odisha were studied at the Sickle Cell Clinic and Molecular Biology Laboratory, V.S.S. Medical College, Burla, Odisha, India. Samples were subjected to CBC and CE-HPLC to quantify haemoglobin fractions. Cases with MCV \ 80 fl and MCH \ 27 pg were suspected for BTT (Colah et al. 2007). Cases with HbA2 [ 3.5 % (Madan et al. 2010) and HbF \ 3.0 % were considered as BTT. Results: From a total of 5690 cases, 213 (3.7 %) individuals were found with BTT. The mean age of males and females were 32.1 ± 14.1 and 31.7 ± 14.6 years respectively. 3 (1.16 %) cases of BTT were found among a total of 259 ANC cases screened. 97.65 % of BTT were Hindu by religion and only 2.35 % were from other religions. Among Hindus, 44.6 % fall under OBC and 24.9 % under SC category, whereas 30 cases each were from ST and general category. While BTT was found preponderant among individuals from various districts of Odisha; Cases of BTT were also detected in individuals from West Bengal, Chhattisgarh and Andhra Pradesh. Conclusion: The prevalence of beta thalassemia gene in Western Odisha was 3.7 %. The finding that the religious groups like Muslims and Christians have lower frequency of BTT differs from the observation (Sur and Mukhopadhyay 2006) from West Bengal. The trend observed in this study though is unique in its representation of epidemiology of BTT from Eastern India; a larger population-based investigation is required to map the spread of b-thalassaemia gene in Eastern India.

Abstract P 100 Clinical and Molecular Characterization of 194 cases of Sicklebeta Thalassaemia in Western Odisha and Their Response to Hydroxyurea Therapy Snehadhini Dehury, Dilip Kumar Patel, Siris Patel, Prasanta Purohit, Subasa C Bishwal, Satyabrata Meher, Bidyapati Padhan, Kishalaya Das Sickle Cell Clinic, VSS Medical College & Hospital, Burla, Sambalpur, Odisha-768 017; 1 Department of Medicine, OSCP, NRHM, V.S.S. Medical College, Burla, Odisha-768017 Introduction: Hemoglobin Sickle-b-thalassaemia is a variant form of sickle cell disease, resulting from the inheritance of HbS and b-thalassemia genes. Objective: To study the clinical, hematological and molecular profile of sickle-b-thalassaemia double heterozygotes in western Odisha. Materials and Methods: 194 cases of HbS-b-thalassemia were studied at Sickle Cell Clinic, VSS Medical College Hospital, Burla, Odisha, India.

123

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Cases were diagnosed by CE-HPLC (BioRad Variant II) and family study and confirmation was done by ARMS-PCR. a-Thalassaemia was studied by GAP-PCR. Cases with severe clinical features were treated with Hydroxyurea at a dose of 10 mg/kg body wt/day orally. Results: Three bthalassaemia mutations [IVS1-5(G-C), FS41/42(-CTTT), codon 15 (G [ A)] accounted for 97 % of all HbS-b-thalassaemia cases. IVS1-5(GC) was the commonest mutation (184/194, 94.85 %). The mean %HbA2, %HbF and %HbS were 5.02 ± 0.86, 19.04 ± 8.89 and 69.84 ± 9.91 respectively, whereas the mean RBC, MCV and MCH were found to be 3.90 ± 0.98 9 103/ll, 71.68 ± 9.48 fl and 23.72 ± 8.94 pg respectively. Commonest clinical presentation was splenomegaly (39.69 %) followed by hepatomegaly and AVN (23 and 7.2 % respectively). 57.21 % had severe anaemia (Hb below 9 g/dL). Deletional a-thalassemia was found in 25 % of cases. 64.43 % patients with VOC more than 3/year and BT more than 2/year were treated with hydroxyurea.Conclusion: IVS1-5(G-C) mutation was the commonest and found in 94.85 % of cases. FS41/42(-CTTT) and codon 15 (G[A) mutations were reported for the first time from this region. a-Thalassaemia had no significant effect on the clinical and hematological features except that HbF was significantly high in a-thalassaemia group. Low dose Hydroxyurea had an ameliorating affect on the clinical and hematological profile of HbS-b-thalassaemia patients in western Odisha.

Abstract P 101 The ‘Odisha Sickle Cell Project’—a New Horizon of Hope for Sickle Cell Aggrieved Patients in Odisha Siris Patel1, Dilip Kumar Patel, Prasanta Purohit, Snehadhini Dehury, Subasa C Bishwal, Satyabrata Meher, Bidyapati Pradhan, Kishalaya Das Sickle Cell Clinic & Molecular Biology Laboratory, VSS Medical College, Burla, Sambalpur, Odisha; 1V.S.S. Medical College, Burla, Odisha Introduction: Sickle cell disease (SCD) is a serious health problem in Odisha with significant morbidity and mortality. It is a huge burden for the patients and his families and a serious challenge to the medical fraternity. However the major constraint for management of this problem is non-availability of health facility in remote areas and lack of awareness amongst health care professionals about the various treatment of this disease. In view of this, National Rural Health Mission (NRHM), Govt. of Odisha sponsored Odisha Sickle Cell Project. Objective: Screening of population for sickle cell haemoglobinopathies and develop an infrastructure for investigation, registration, treatment, follow-up and counselling of all patients with SCD of Odisha. Materials and Method: The Odisha Sickle cell Project (OSCP) was started in April-2010, under this project Sickle cell unit were constructed at six DHHs of Western Odisha. Counselling and diagnosis of sickle cell patients at peripheral hospitals is done by a field worker and trained Laboratory technician. A state-of-art Molecular Biology & Haematology Laboratory has been developed at the referral centre, V.S.S. Medical College & Hospital, B urla. Cases referred from periphery are examined, counselled, registered here. This laboratory has facility for Hb electrophoresis, CBC, Biochemical test, CE-HPLC, PCR & Flow Cytometry on a routine basis. Severe cases of SCD are started Hydroxyurea therapy at a low dose (10 mg/kg body wt/day). 2073 individuals have been screened for sickle cell haemoglobinopathies in 14 health camps organised in various districts of western Odisha. Simultaneously 10 CMEs have been coordinated for creating awareness among the doctors. Results: Till date the number of patients examined, counselled, diagnosed to have sickle cell haemoglobinopathies at VSS Medical College & Hospital, Burla are 28321, 13720 and

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

225

Table 1

Table 2

Cell

Rh-hr

Kell

Duffy

Cell

Kidd

Rh-hr

Kell

Duffy

Kidd JKa

JKb

1

CCDee

R1R1

+

+

0

0

+

0

+

0

+

+

+

+

+

1

CCDee

R1R1

+

+

0

0

+

0

+

0

+

+

0

+

+

2

ccDEE

R2R2

+

0

+

+

0

+

+

0

+

0

+

0

+

2

CCDee

R1R1

+

+

0

0

+

+

+

0

+

0

+

+

0

3

ccddee

rr

0

0

0

+

+

+

+

0

+

+

0

+

0

3

ccDEE

R2R2

+

0

+

+

0

0

+

0

+

+

0

0

+

4

Ccddee

rIr

0

+

0

+

+

0

+

0

+

+

0

+

0

5

ccddEe

II

r r

0

0

+

+

+

0

+

0

+

+

0

0

+

6

ccddee

rr

0

0

0

+

+

+

+

0

+

0

+

+

0

Cell

D

C

E

c

e

K

k

Kpa

Kpb

Fya

Fyb

JKa

JKb

Lewis Le

a

P Le

b

MNS

LUTH

P1

M

N

S

s

Lu

a

Result Lu

b

IS

37°C

AHG

D

C

E

c

e

K

k

Kpa

Kpb

Fya

Fyb

7

ccddee

rr

0

0

0

+

+

0

+

+

+

0

+

0

+

1

CCDee

R1R1

0

+

+

+

0

+

0

0

+

–

–

–

8

ccD.ee

R0r

+

0

0

+

+

0

+

0

+

0

0

+

0

2

ccDEE

R2R2

+

0

+

+

+

+

+

+

+

–

4+

4+

9

ccddee

rr

0

0

0

+

+

0

+

0

+

0

+

+

+

3

ccddee

rr

0

+

+

0

+

0

+

0

+

–

4+

4+

10

ccddee

rr

0

0

0

+

+

0

+

0

+

0

+

+

0

11

ccddee

rr

0

0

0

+

+

0

+

0

+

+

0

0

+

Screening cell panel positive in cell no. 2 and 3 and was suggestive of anti-c, anti-K, anti-N and anti-s. (BIORAD, ID Diacell I,II,III.LOT NO: 06084.71x,06094.71x,06104.71x)

5840 respectively. At six DHHs the number of new cases of sickle cell haemoglobinopathies detected are 1381, 737, 568, 1005, 578 and 1020 at Bolangir, Bargarh, Sambalpur, Jharsuguda, Deogarh and Sundergarh, respectively. Of the 5840 cases registered 5335 HbSS, 194 HbS-b thalassemia, 36 HbSD, 56 HbAD, 13 HbSE, 18 HbAE, 2 HbSC, 16 bthalassemia major, 160 b thalassemia trait, 5 Gc(Acdb)0 thalassemia and 5 HPFH. 1997 no. of cases has been started Hydroxyurea (10 mg/kg body wt/day). It has been observed that low dose Hydroxyurea is effectively reducing the frequency of VOC and blood transfusion. Conclusion: Under OSCP we have diagnosed thousands of new cases of Sickle cell haemoglobinopathies, counselled them and have created awareness among the doctors of Western Odisha for better management and treatment of this Sickle Cell Disease.

Cell

Lewis

P

MNS

Lea

Leb

P1

M

LUTH. N

S

s

Lua

Result Lub

IS

37°C

AHG

1

CCDee

R1R1

0

0

+

0

+

+

+

0

+

–

–

–

2

CCDee

R1R1

0

+

+

+

0

+

0

+

+

–

–

–

3

ccDEE

R2R2

+

0

+

+

0

0

+

0

+

–

4+

4+

4

Ccddee

rIr

+

0

+

+

+

0

+

0

+

–

4+

4+

5

ccddEe

rIIr

+

0

+

+

+

0

+

0

+

–

4+

4+

6

ccddee

rr

0

+

0

0

+

0

+

0

+

–

4+

4+

7

ccddee

rr

+

0

+

+

+

+

+

0

+

–

4+

4+

8

ccD.ee

R0r

+

0

+

+

+

+

0

0

+

–

4+

4+

9

ccddee

rr

0

0

0

0

+

+

0

0

+

–

4+

4+

10

ccddee

rr

0

+

0

+

+

+

+

+

+

–

4+

4+

11

ccddee

rr

0

+

+

+

0

+

0

0

+

–

4+

4+

Above result shows positive agglutination in Panel cell no. 3 to 11 except no. 1 and 2. So the alloantibody to c antigen is confirmed. (BIORAD, ID DiaPanel.LOT NO: 06171.52.x-06271.52.x & 05361.52.x-05461.52.x)

Abstract P 103 Abstract P 102

Hemoglobin J: A Case Report

Delayed Hemolytic Transfusion Reaction Caused by Anti-C Antibody in A E-Beta Thalassemic Patient

Anurag Sharma1, Amit Kumar Adhya1, Santosh Kumar Panda2, Manas Nayak3 Department of Pathology, KIMS, Bhubaneswar, Odisha; 2KIMS, Bhubaneswar, Odisha; 3Department of Paediatrics, KIMS, Odisha 1

Ritam Chakrabarty, Suvro Sankha Datta, Biplabendu Talukder, Somnath Mukherjee, Prasun Bhattacharya, Krishnendu Mukherjee Department of Immunohematology & Blood Transfusion, Medical College Hospital, Kolkata-73 Objective: Delayed hemolytic transfusion reaction (DHTR) occurs 3–21 days after blood transfusion. It usually presents as fever, anaemia with mild jaundice and sudden drop in haemoglobin level few days after transfusion. Here we present a case of DHTR in a 26 years old E-Beta thalassemic female, with blood group of O positive, caused by the alloantibody to c antigen. Methods: The patient was transfusion dependent since 2006. Her haemoglobin dropped to 4.6 g/dl, from 6.4 g/dl, within 2 weeks after two units of O positive packed red cell transfusion. A serological work-up was performed to detect the cause of this sudden drop in haemoglobin level. The direct antiglobulin test (DAT), autocontrol and antibody screening by three and eleven cell panel were performed in polyspecific coombs card (anti IgG + C3d). Results: DAT and autocontrol showed no agglutination, but antibody screen with commercial cells (BIORAD, ID, Diacell) showed agglutination (4 +) in Nos. 2 and 3 of the three cell panel and in Nos. 3–11 of the 11 cell panel (Tables 1, 2). Conclusion: The alloantibody to ‘c’ antigen was detected and c antigen negative O positive packed red cell was given for safe transfusion. Alloantibody is the major cause of DHTR in transfusion dependent patients. So an extended Rh (C,E,c,e) and Kell phenotyping should be performed in these patients before the first episode of blood transfusion and antibody screening should be considered routinely if there is any evidence of DHTR.

Introduction: Haemoglobin J is a very uncommon hemoglobinopathy. The clinical presentation and follow up of such a case is presented here. Case: A term baby boy, birth wt 2.2 kg delivered to G5L1A3 mother by LSCS. Baby looked pale and had abdominal distension due to hepatosplenomegaly. His hemogram revealed Hb9.6 g/dl, TWBC-12000/CC, TPC-60,000/CC. Peripheral smear was suggestive of hemolysis. Reticulocyte count was 10 % and DCT was negative. Baby developed jaundice on day one of life and Liver function test showed conjugated hyperbilirubinemia with elevated transaminase and mild raised ALP. On D2, baby had respiratory distress secondary to PDA, managed conservatively. TORCH antibody screening was negative. During NICU stay baby never looked sick looking. USG Abdomen showed severe hepatosplenomegaly and no other obstructive pathology in biliary tract and Neurosonogram was normal. Hb electrophoresis revealed fast moving Hb variant (Hb J variant). For severe anaemia packed cell transfusion was given. On 10 week follow up, Baby is gaining weight and cholestasis is decreased. Discussion and Conclusions: There are more than 50 hemoglobin J variants described in the literature. They all have an electrophoretic mobility ‘‘faster’’ than ‘‘A’’ on cellulose acetate, in common. All are classified under ‘‘variants of the alpha- or betachains’’ or ‘‘hemoglobins with more than one amino acid substitution in the alpha chain’’. The clinical presentation and prognosis of the patients is variable and is determined by the presence or absence of other beta chain variants. Further follow up is required in our case to fully delineate the course of the disease.

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Abstract P 104 A Novel 9 bp Denovo Deletion [HBB: c.132_141del9bp] in b-Globin Gene Causing Heinz Body K Neelakandan, Neeraj Arora, Sathish Kumar1, Usha Sitaram2, Alok Srivastava, Eunice Sindhuvi Edison, RV Shaji Department of Haematology, Christian Medical College, Vellore; Department of Child Health, Christian Medical College, Vellore; 2 Department of Transfusion Medicine & Immunohaematology, Christian Medical College, Vellore 1

Abstract: The ‘‘Heinz-body anaemias’’ are a group of hemolytic syndromes of diverse etiology with common morphologic characteristics. One of the rare causes for Heinz-body anaemias is dominant b-thalassaemia (b-thal). Dominant b-thal presents with moderate to severe haemolytic anaemia with jaundice and splenomegaly and is associated with a thalassemia intermedia phenotype in the heterozygous state. We describe a case of dominant b-thal with severe hemolytic anaemia caused by a novel denovo deletion in b-globin gene. The patient was a 3 year old girl who presented with transfusion dependent anaemia and hepatosplenomegaly (liver-4 cm; spleen8 cm). She had marked reticulocytosis (19 %) and elevated LDH levels. Both direct and indirect coombs test were negative. The blood picture and other common tests (G6PDH, autohaemolysis, sickling preparation, Hams and sucrose lysis test, osmotic fragility) done for haemolytic anaemia were unremarkable, but Heinz bodies test was positive. Interestingly, peripheral blood smear stained with brilliant cresyl blue showed numerous RBCs containing hemoglobin H (HbH)like inclusions. However, the eight common alpha (a)-globin deletions prevalent in our population screened by a multiplex PCR were absent. We performed sequencing of globin genes to identify possible mutations that produce unstable haemoglobin causing haemolytic anaemia. We identified a heterozygous state of a novel 9 bp deletion (TCC TTT GGG) in the exon 2 of the beta globin gene (HBB: c.132_141del9bp). This region of the b globin is situated in or around the heme pocket and the mutations that destabilize heme binding has been found to be associated with extremely unstable hemoglobins. Both parents had normal HbF, HbA2, MCV and MCH values and had normal b globin gene sequences. Most of the mutations that cause dominant b-thalassaemia reside in the exon 3 of the b globin gene, although rare cases with mutations in other exons 1 and 2 have also been reported. Our case serves to underscore the importance of considering dominant b thalassaemia as a cause of moderate to severe hemolytic anaemia in children even without a significant family history. DNA based diagnostics can readily detect and confirm these uncommon b globin gene mutations, and help implement appropriate treatment planning and genetic counselling.

Abstract P 105 Profile of Liver Fucntion Test in Sickle Cell Patients Mitali Madhumita Rath, MK Panigrahi, Sudha Sethy, Rajib Nayak, Pranati Mohanty, Kalyani Prava Gouda, Sujata Pujari, SR Mohapatra, RK Jena Department of Pathology & Clinical Hematology, S.C.B. Medical College & Hospital, Cuttack Introduction: Abnormal liver function tests are common in patients with sickle cell anaemia, even in the absence of liver disease.It may be

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 hemolytic, cholestatic, hepatocellular and mixed pattern. There is a paucity of data on liver function in sickle cell disease (SCD) from this region. Aims and Objective: The present study aims at determinig the prevalance and the profile of abnormal liver fuction test in sickle cell patients. Material and Mathods: One hundred consucutive patients of sickle cell disease (homozygous)attainding the out patients department of hematology, S.C.B. Medical College from January 2012 to July 2012 were included in this study. Detailed clinical history was taken and subjected for complete blood count, hemoglobin profile, liver function tests, viral markers, ultrasonography of abdomen and pelvis. Results: The 100 patients included in the study ranged in age from 8 to 62 years (mean 30.8 ± 9.11 years) and included 64 males and 36 females. Mean episode of vaso-oclusive crisis was 2.65 ± 1.82 times and mean units of blood transfusion was 3.74 ± 1.75. Mean hemoglobin concentration was 8.2 ± 2.3 g/dl. Abnormality in liverfuction test was found in 86 % of the study participants. The mean serum total bilirubin, AST, ALT, ALP were 8.4 ± 6.5 mg/dl, 104 ± 43 U/L, 68 ± 27 U/L, 258 ± 76 U/L respectively. 49 % had hemolytic, 12 % had obstructive 25 % had mixed pattern of LFTs. On ultrasonography 24 % had hepatomegaly, 46 % had spleenomegaly, 11 % cholelithiasis and 2 % had both cholelithiasis and choledocholithiasis. Conclusion: Majority of sickle cell homozygous patients had abnormal liver fuction. One fourth of the patients had mixed pattern of LFTs that can not be explained by either hemolytic or obstructive component.

Abstract P 106 Profile of Hb E/beta Thalassaemia Patients After Splenectomy— Experience of a Thalassemia Care Centre in Eastern India Prakas Kumar Mandal, Basab Bagchi, Sandeep Saha, Tuphan Kanti Dolai, Sanjay Misra, Malay Kumar Ghosh, Maitreyee Bhattacharrya Department of Hematology, N.R.S. Medical College and Hospital, Kolkata-700014 Objective: The study was done to evaluate various complications following splenectomy in HbE/beta thalassaemia (EBT). Methods: A total of 72 splenectomised EBT were studied retrospectively. Detail history including age of splenectomy, transfusion requirement (before and after splenectomy) was taken. Physical examination was done for facial deformities, pubertal growth. Baseline Hb %, serum ferritin level, evidence of pulmonary hypertension (PHTN), thrombo-embolic manifestations (VTE), extramedullary hematopoiesis were noted. Results: Out of 1380 registered EBT patients, 72 (5.22 %) underwent splenectomy. Ten (13.9 %) were diagnosed \1 year, 24 (33.3 %) 1–5 years, 23 (31.87 %) 5–15 years and 15 (20.83 %) [ 15 years. Thirty five (48.57 %) started transfusion \ 5 years and one at 39 years. 19 (26.4 %) underwent splenectomy \ 10 years, 38 (52.7 %) between 10–20 years and 15 (20.8 %) [ 20 years of age. Mechanical discomfort was the leading cause for splenectomy in 37 (51.39 %) followed by increased transfusion requirement in 15 (20.83 %). Mean baseline Hb level of 5.43 g/dl with 48 (66.6 %) pre-splenectomy and majority had Hb % of 6.8 g/dl Post-splenectomy. Mean transfusion requirement was remarkably reduced from 18.1 unit/year to 7.8 unit/year after splenectomy. Four (5.5 %) received single transfusion till date. Mean serum ferritin level was increased from 907.58 ng/ml (before) to 1091.6 ng/ml (after) ins pite of reduced transfusion requirement after splenectomy. 48

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 (66.6 %) and 31 (43.05 %) patients had facial deformities and delayed pubertal growth respectively. Four (5.5 %) had evidence of extramedullary hematopoiesis, 15 (20.83 %) had VTE and 7 (9.7 %) developed PHTN. Though 48 (66.67 %) was started with iron chelation therapy, majority 38 (52.7 %) stopped and 10 (13.89 %) are continuing till date. Conclusions: Though splenectomy reduces requirement of transfusion; it does not prevent skeletal abnormalities and delayed puberty. VTE are increased. Patients should be encouraged for effective Iron chelation therapy.

Abstract P 107 Optimizing the Dose of Hydroxyurea in Thalassemia Intermedia Vinaykumar V Bohara, Lalit Raut, Girish Badarkhe, Ganesh Pujari, P Chakrabarti, S Ray, U Nath, Utpal Chaudhuri Institute of Hematology and Transfusion Medicine (IHTM), Kolkata Introduction: The optimum dose of hydroxyurea in the management of thalassemia patients is an area of gray zone. We report the results of study carried out on patients of E-b thalassemia. Aims and objectives: To determine the optimum dose of hydroxyurea in E-b thalassemia patients and to assess its efficacy and safety. Methods: This was a prospective, single center study in which E-b thalassemia patients with no or minimum transfusion needs were enrolled and randomised to receive 10 (group A) and 20 mg/kg/day (group B) hydroxyurea. Complete response (CR) was defined as transfusion independence or rise of haemoglobin (Hb) by [1.5 g%. Partial response (PR) was rise in Hb by 0.5 to 1.5 g% or reduction in transfusion needs. Anything less was defined as no response (NR). Results: Median age in group A (n = 32) and B (n = 31) was 12.5 and 12 years, respectively (p [ 0.05). The median follow up was 9 and 6 months in groups A and B respectively (range 3–9 months). In group A response was seen in 65.7 % (20 out of 32). Of this CR and PR was seen in 32.3 % (n = 10) and 33.4 % (n = 11). The rise in Hb in group A was statistically significant at 3 and 6 months. In group B there was NR in 83.9 % (26 out of 31) of patients. There was no CR and eight (25.8 %) patients discontinued hydroxyurea at 6 months due to adverse effects requiring intervention. High baseline HbF was associated with higher response in group A but not in group B. In Complete responders IVS 1-5(G-C) was the commonest mutation. Conclusion: Hydoxyurea is effective and well tolerated at 10 mg/kg/d dose in E-b thalassemia (Thalassemia Intermedia) in Eastern India.

Abstract P 108 Association of Genetic Modulators of b Thalassemia Linked to Raised Fetal Hb and its Effect on Clinical Severity Pooja Dabke, Roshan Colah, K.Ghosh, Anita Nadkarni National institute of Immunohematology, 13th Floor NMS Building, KEM hospital Campus, Parel, Mumbai-12 Objective: Study of genetic modifiers linked to raised HbF and its effect on clinical severity of b thalassemia. Methods: Study group consisted of 104 patients of hemoglobinopathies and 50 healthy

227 individuals. HbA2 and HbF levels were measured using HPLC System. XmnI polymorphism, Ac-d intergenic haplotypes, (AT)x(T)y motif, Pre G c haplotype, b globin LCR motifs were studied by PCR–RFLP, Gene scan analysis and DNA sequencing. Results: Clinically 40 patients were classified as thalassemia major and 39 as thalassemia intermedia. 13 different b thalassemia mutations were encountered. IVS1nt5 (G ? C) was the prevalent mutation (56.96 %). 25 % of the thalassemia intermedia chromosomes showed presence of (AT)9(T)5 motif. The Pre G c TAG haplotype associated with higher HbF levels was present in homozygosity in 48.57 % of the thalassemia intermedia cases as against 12.82 % of the thalassemia major cases. 80.55 % of the milder patients showed presence of the Ac-d intergenic haplotype T. All the 11 sickle cell anemia patients showed homozygosity for Pre G c TAG and Ac-d intergenic T haplotypes showing their linkage with Arab-Indian haplotype. In b LCR region the HS2 motif were well spread among the patients. The HS3 motif did not show any change with respect to the reference sequence. Conclusion: As 33.33 % of our milder cases showed presence of 4 modifiers linked to higher HbF (XmnI, (AT)9(T)5, TAG Pre G c, Ac-d intergenic T haplotype), it appears that in the Indian scenario these secondary modifiers of b thalassemia seem to contribute to ameliorate the disease severity.

Abstract P 109 A Hospital Based Study of Thalassemia from Kolkata Deboshree M. Bhattacharyya, Jayasri Basak, Soma Mukhopadhyay, Ashis Mukhopadhyay Netaji Subhas Chandra Bose Cancer Research Institute, 16A Park Lane, Kolkata-700016, West Bengal, India Objective: The present study represents a population of Eastern Indian state of West Bengal. This is a hospital based study where in the last 3 years 242 individuals have been screened for their carrier status. The main objective was to study the genotype-phenotype correlation of b thalassemia cases, and finally to identify different b thalassemia mutations prevalent in West Bengal. Method: After obtaining written consent from each individual or parents of individuals under the age of 18 years, 3 ml of peripheral blood was drawn from each subject and stored in EDTA vials. At first complete blood count was done followed by High Performance Liquid Chromatography. DNA is then extracted from the remaining blood following Millers method (1988). ARMS PCR and GAP PCR were done to detect beta and alpha mutations respectively. Results: Total of 242 subjects were studied which included 22.31 % b thalassemia carriers, 2.1 % of b thalassemia major 1.24 % sickle beta thalassemia, 6.2 % Eb thalassemia, 8.3 % HbE carriers and HbE 2.1 % homozygous. 10 Eb patients responded to hydroxyurea treatment and required less blood transfusion whereas 5 Eb patients did not respond to the treatment and required regular blood transfusion. The severity of the Eb mutations depends on the genotype of the beta mutation. Apart from these we have identified families having mutations for HPFH, Fanning Lubbock and a alpha thalassemia. Conclusion: Genotype phenotype correlation plays a significant role in determining the severity of thalassemia. Among the studied samples the prevailing beta mutations are IVS 1-5, codon 15 and codon 30. Compound heterozygosity of HbE with the above mentioned beta mutation produces mild to severe anemia. The above study gives an idea of the thalassemia status of Kolkata.

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Abstract P 110 Role of NESTROFT in Mass Screening of Thalassemia Nabamita Pal, Deboshree M. Battacharyya, Ashis Mukhopadhyay, Abhijit Chakraborty, Swati Dasgupta, Priyabrata Das, Soma Mukhopadhyay, Jayasri Basak Department of Molecular Biology, Netaji Subhas Chandra Bose Cancer Research Institute, 16 A, Park Lane, Kolkata-700016, West Bengal, India Objective: Thalassemia represents a major health problem worldwide. Present carrier status of Thalassemia in West Bengal according to Sur D et al. is 20.47 %, which is comparatively higher than other states of this country. Our main aim is to arrange Mass Awareness and Screening camps among both urban and rural West Bengal and screen people to know their thalassemia carrier status. Method: In the present study from January 2009 to January 2011 screening was carried out among various populations of West Bengal including tribal. With their individual consent 3 ml of peripheral blood was collected in EDTA vials. NESTROFT was done on spot using 0.36 % Saline Buffer solution (Sodium chloride, Sodium dihydrogen phosphate, Disodium hydrogen Phosphate). Complete Blood Count was performed within 24 h of collection. HPLC (High Performance Liquid Chromatography) was done to identify the beta samples. Result: In this period total 6482 individuals have been screened. Among them 25 % were NESTROFT positive and 50 % are negative. Rest of them were doubtful cases. NESTROFT result was compatible with the respective HPLC reports. Most of the NESTROFT positive were either carriers of beta thalassemia, sickle cell anemia, carrier of alpha thalassemia and victims of Eb thalassemia. The doubtful cases of the test contributed to mostly E carriers and individuals suffering from iron deficiency anemia. The efficiency of the NESTROFT test is 71.29 % among the general population of West Bengal. The specificity and sensitivity of the test are 61.1 and 72 % respectively. Conclusion: Hence NESTROFT is both cost-effective and cost-efficient test in Thalassemia Mass Screening. It plays a significant role in thalassemia carrier detection but HPLC is mandatory in the area where HbE is prevalent.

Abstract P 111 Role of Wheat Grass Juice as an Iron Chelator in Intermediate Thalassemia Patients Abhijit Chakraborty1, Priyabrata Das1, Manoj Kar2, Soma Mukhopadhyay1, Suvra Mondol3, Ashis Mukhopadhyay1 1

Netaji Subhas Chandra Bose Cancer Research Institute, 16A Park Lane, Kolkata-700016, West Bengal, India; 2N.R.S Medical College & Hospital, Kolkata-700014, West Bengal, India; 3National Research Institute for Ayurvedic Drug Development, Kolkata-700091, West Bengal, India Objective: The aim of the study is to observe the effect of wheat grass juice in reducing ferritin level and increasing the hemoglobin level in intermediate Thalassemia patients. We will also study the biochemical properties of wheat grass juice. Methods: We performed a study on 236 patients of intermediate thalassemia during the period from March 2009 to March 2012. Of these, 48 transfusion dependent intermediate thalassemia patients whose mean age is 6.42 ± 1.71 were selected.

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 30 ml of fresh juice from 5-7-day-old wheat grass leaves including stems was given daily to all 48 patients regularly for 6 months. Wheat grass juice was analyzed by cation exchange column chromatography. Deoxyribose degradation assay was performed to study the iron chelating activity of wheat grass. Result: Wheat grass juice is found to be rich in active ingredients which play an important role in dietary absorption of iron from intestine. It is also found to contain unique active ingredients with iron chelating property. The mean serum ferritin level of the patients before treatment was 2,250 (range 650–3,100), which reduced to 950 (range 68–1680) (p \ 0.0001) after treatment. The mean levels of hemoglobin before starting wheat grass juice were 6.2 g%. After 6 months of wheat grass therapy the mean value for hemoglobin was 7.8 g% (p \ 0.005). The performance status was improved from 60 to 80 % (Karnofsky) after wheat grass treatment. The mean interval between transfusions was also found to increase. Conclusions: Hence, wheat grass juice is an effective iron chelator and it is an effective alternative of blood transfusion. Its use in intermediate thalassemia patients should be encouraged.

Abstract P 112 Prevalence of Thalassemia in West Bengal with Special Reference to Different Tribes Jayasri Basak, Deboshree M Bhattacharyya, Soma Mukhopadhyay, Swati Dasgupta, Abhijit Chakraborty, Priyabrata Das, Nabamita Pal, Sukanta Koner, Ashis Mukhopadhyay Department of Molecular Biology, Netaji Subhash Chandra Bose Cancer Research Institute, Kolkata Introduction: Thalassemia is a hereditary anemia resulting from defect in hemoglobin production and it is the most common genetic disorder worldwide. Prevalence of Thalassemia (both alpha and beta) is high in West Bengal, especially among the scheduled tribes. To reduce the Thalassemia burden from West Bengal we have taken a project for Thalassemia carrier screening through awareness and screening program among the rural and urban populations. Our target population were tribes, e.g., Toto, Rabha, Munda, Oraon, Kerketa, Sardar etc. residing in remote rural areas. Materials and Methods: From January 2009 to December 2011, we have organized 73 camps in different districts of West Bengal. After taking written consent, 2–3 ml peripheral blood samples were collected from each interested people attending the mass awareness camp. NESTROF, CBC, HPLC were done for all samples. To detect alpha and beta mutations GAPPCR and ARMS-PCR were performed following the standard protocol. Results: During the above mentioned period, we have screened on total 8387 individuals (4,869 male; 3,518 female) for Thalassemia carrier detection through camps, organized in 14 districts of West Bengal. Out of 8,387 screened individuals, tribes were 2,885 and general caste including scheduled castes was 5502. Among tribes HbE carrier and homozygous percentage are 14.6 and 4.01 respectively while percentage of sickle cell anemia and beta Thalassemia carriers are 1.25 and 4.19 respectively. Total beta carrier percentage among general caste and scheduled caste is 6.94. Our result also revealed that alpha Thalassemia carrier among Oraon and Sardar tribes is very high. Conclusion: Incidence of Thalassemia is high in different rural populations especially among tribes. So, large scale awareness and preventive program should be taken.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

Abstract P 113 Spectrum of Beta Globin Gene Mutations in North Indian Patients with Thalassemia Major: Application in Prenatal Diagnosis Reena Das, Jasbir Kaur, Sanjeev Chabra, Jasmina Ahluwalia, Amita Trehan1, Subhash Saha2, Rashmi Bagga2, Deepak Bansal1, Inusha Panigrahi1, RK Marwaha1, G Garewal Departments of Hematology, Pediatrics1 and Obstetrics & Gynaecology2, PGIMER, Chandigarh 160 012, India Objectives: Thalassemia major (TM) is an autosomal recessive disorder where the parents are asymptomatic beta thalassemia carriers (bTT). The prevalence of bTT is 3.5 % in north Indians. We analyzed the spectrum of mutations in 1500 TM alleles and its application to offer prenatal diagnosis to couples who were both bTT. Method: ARMSPCR and beta globin gene sequencing from genomic DNA from the peripheral blood was carried out. Prenatal diagnosis was offered to 570 pregnancies in the last 14 years using molecular analysis. Results: Common five Indian mutations were found in 86.8 % alleles; commonest being IVS 1,5 (G-C) [29.4 %], followed by Fr 8/9 (+G) [21 %], 619 bp deletion [17 %], IVS 1,1 (G-T) [8.5 %] and Fr 41/42 (-TTCT) [10.8 %]. Codon 16 (-C) was seen in 4 % and uncommon b++ mutations -88 (C-T) and Cap + 1 (A-C) was 2.6 and 2 % respectively. Rare mutations constituted 4.5 % alleles and many were identified by beta globin gene sequencing. Only 0.1 % alleles remained uncharacterized. Results of the prenatal diagnosis of 570 pregnancies showed that in 25.7 % cases the fetuses were normal, 49.8 % cases were bTT, 23.2 % were TM and in 1 % cases we were unable to distinguish between bTT and TM. Seven cases had twin pregnancies and all except one case showed similar normal/bTT/TM status. Conclusion: This application has helped to prevent the birth of 132 children with thalassemia major in the region and alleviated the anxiety of 430 pregnancies to have children who were either normal or carriers.

Abstract P 114: Oral Presentation Clinico-Hematological Analysis of Hb Variants and Spectrum of Hemoglobinopathies on HPLC: a 3 Year Study from a Tertiary Care Centre of North India

229 peak in the C window. 2 cases (1.6 %) were each of Hb Q India, Hb Lepore trait and beta thal major. Conclusion: Our study highlights that high performance liquid chromatography (HPLC) is a simple yet rapid and reliable tool for the detection of haemoglobin variants.

Abstract P 115 Determination of Allelic Frequency of SNPs at Quantitative Trait Loci (QTL)s of Fetal Hemoglobin Charu Batra1, Jasbir Kaur1, Amita Trehan2, Jasmina Ahluwalia1, Reena Das1 Department of Hematology, 2Department of Pediatrics, Postgraduate Institute of Medical Education and Research, Chandigarh

1

Introduction: Fetal Hemoglobin (HbF) is considered a ‘quantitative trait’ (QT) wherein multiple genes together with a small environmental component determine the value measured in any given individual. High HbF levels are associated with milder disease progression and fewer complications in patients with Sickle cell Disease (SCD) and b thalassemia. Recently conducted Genome-Wide Association Studies (GWAS) in European and African American populations and patients with hemoglobinopathies, have identified single-nucleotide polymorphisms (SNPs) from chromosomal loci that contribute to varying expression levels of HbF and other clinical traits. They include the Xmn1-Gc polymorphism and SNPs at BCL11A and HBSB1L-cMYB inter-region loci. To the best of our knowledge, the allelic frequency of SNPs at BCL11A and HBS1L-MYB intergenic region in normal North Indian population and their association with thalassemia in India has not been studied. Objective: Determination of prevalence of polymorphism at SNP rs11886868 in BCL11A exon 2, SNP rs4895441 in the HBSB1L-cMYB inter-region and SNP rs74822144 in b globin gene cluster (The Xmn1-Gc polymorphism) in normal North Indian population. Methods: Single-nucleotide polymorphism (SNP) analysis was performed by using polymerase chain reaction (PCR)/restriction enzymes on genomic DNA extracted from peripheral blood leukocytes of fifty healthy normal children [3 years of age. Enzymatic digestion was performed by XmnI, MboII, and RsaI for, rs74822144, rs11886868 and rs4895441 respectively. Results: Minor allele frequencies for rs11886868 and rs4895441, rs74822144 were 0.35, 0.13 and 0.26 in normal North Indian children. We plan to determine the allelic frequency at these loci in children with thalassemia intermedia and to correlate the SNPs with the clinical phenotypes

Poojan Agarwal, Snigdha Goyal, Aneesha Mohanpuria, Vijay Kumar, Sadhna Marwah, A.S.Nigam, Gurdeep Buxi Department of Pathology, PGIMER, Dr. Ram Manohar Lohia Hospital, New Delhi

Abstract P 116

Objective: To analyse and characterize various Hb variants and hemoglobinopathies using high performance liquid chromatography (HPLC). Methods: The present retrospective study was carried out at PGIMER, Dr. RML Hospital, by analysing the data obtained from September 2009 to August 2012. The clinical and haematological records of patients suspected of hemoglobinopathies were retrieved and studied with the HPLC pattern. Family studies carried out in indeterminate cases were also reviewed. Results: Abnormal haemoglobin fractions on HPLC were seen in 121 of the 1846 cases studied. Of these, beta thalassemia trait was the predominant abnormality with a total of 69 cases (57.02 %). HbS was the next most common Hb variant, observed in 18 cases (14.8 %). Of these, four cases were homozygous for sickle cell disease. Other hemoglobins observed were Hb D Punjab (7 cases; 5.78 %), alpha thal (7 cases; 5.78 %) and Hb E (5 cases; 4.13 %). Hb J Meerut, HPFH and combined Hb E/beta thal inheritance had 3 cases each (i.e. 2.3 % each). 5 cases also showed

Non-Transfusion Dependent Thalassemia; Management Issues Maitreyee Bhattacharyya, Asutosh Panigrahi N.R.S Medical College, 138, A.J.C Bose Road, Kolkata 700014 Objectives: Non transfusion dependent thalassaemia (NTDT) usually do not require regular RBC transfusions for survival but associated with a variety of serious clinical complications that require proactive and comprehensive management. Methods: We investigated clinical and biochemical profile of non-transfusion dependent thalassemia patients attending thalassemia clinic of N.R.S medical College, Kolkata. Results: Out of 179 patients studied, 158 were HbE b thalassemia and rest Sb or Hb E disease or b thal intermedia. Mean age of diagnosis was 15 year (range 1–48 years). Mean Hb % was 7.4 g/dl (range 4–10 g/dl). Mean age of 1st BT was 17 years 20 (11 %) patients didn’t have splenomegaly, 47 (26 %) without skeletal deformity (ineffective erythropoiesis

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230 indicator). Growth failure observed in 87 patients, 95 were with jaundice, 5 had delayed puberty. Predominant complications observed was Gall stone in 1 patient, thyroid dysfunction in 5 patients VTE in 1. We didn’t observe diabetes, pulmonary hypertension, in any patients. Serum ferritin was above 1000 in 16 [with normal development in 4 (2 %) and normal puberty in 6 (3 %)] and 122 patients had s.ferritin between 300 and 1,000. Management provided were transfusion [required 98 (55 %), frequent in 7 (4 %) and not required in 81 (45 %)], splenectomy in 3 (1.6 %), hydroxylurea 2 (1 %), thromboprophylaxis in 1 (0.5 %) iron chelation in 17 (9 %) and monitoring. Conclusion: Results from our study shows that effective management of NTDT patients in our genetic background can give a far better quality of life.

Abstract P 117 Profile of Hemoglobinopathies in the State of Odisha Anasuya Lenka, Sudha Sethy, Rajeeb Nayak, RK Jena, Pranati Mohanty Department of pathology and department of clinical hematology, S.C.B. Medical College and Hospital, Cuttack Objectives: To determine the clinical and hematological profile of different hemoglobinopathies. Materials and Methods: Patients with clinical features of chronic hemolytic anemia without history of blood transfusion in last three months attending the department of clinical hematology of SCB Medical College & Hospital, Cuttack from November 2007 to February 2012 are included in the study. About 2 ml of blood was collected from each patient which was investigated in fully automated capillary zone electrophoresis. Screening tests like sickling test and complete blood count by five part cell counter (sysmax 2000I) were done in all cases. Results: Out of total 6,195 cases hemoglobinopathy was found in 3,263 cases. Beta-thalassemia constitutes 28.1 % cases with 21.02 % of trait, 4.51 % thalassemia intermedia and 2.57 % of thalassemia major cases. Sickle cell disorder was found in 27.03 % cases with sickle cell trait in 23.17 % and sickle cell disease in 3.86 % cases. Sicke-beta thal was 21.88 %. Hb E was found in 2.69 % cases and E-beta thal in 9.65 % cases. Conclusion: Hemoglobinopathy is common in almost all parts of Odisha. They cause high degree of morbidity and mortality along with socio- economic burden. In this study diagnosis could not be confirmed in 8.8 % of cases which needs further investigations like parental evaluation and globin chain mutation analysis. Keywords Hemoglobinopathies, Sickle cell, Thalassemia, Hb-E, Odisha state

Lymphomas Abstract P 118 Plasmablastic Lymphoma of the Retroperitoneum in an HIV-Negative Patient Nishad Dhakate1, Soniya Nityanand2 1

S.G.P.G.I, Lucknow; 2Department of Hematology, SGPGI, Lucknow

Abstract: Plasmablastic lymphoma (PBL) is an aggressive nonHodgkin lymphoma classically occurring in individuals infected with HIV. Plasmablastic lymphoma has a predilection for the oral cavity and jaw. However, recent case reports have shown plasmablastic lymphoma in the stomach, lung, nasal cavity, cervical lymph nodes and jejunum in HIV negative individuals. We report what is, to the

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 best of our knowledge, the second case of plasmablastic lymphoma occurring in the retroperitoneum of a HIV-negative man. Case Report: A 57 years old male, known case of hypertension presented with chief complaints of swelling in the right thigh since 3.5 months. MRI pelvis revealed a soft tissue lesion in adductor compartment of right thigh. FDG PET scan showed a soft tissue swelling in retroperitoneum extending into the pelvis along bilateral psoas muscle, on the right extending into right inguinoscrotal region, inguinofemoral region and in adductor compartment. Pathological examination showed tumour cells with eccentrically placed nucleus and moderate amount of eosinophilic cytoplasm consistent with plasmablastic lymphoma. Immunohistochemistry was diffusely and strongly positive for vimentin and CD 138 and focally and weakly positive for LCA, CD 38 and CD 79a consistent with plasmablastic lymphoma. His LDH was 5864, creatinine 2.8 mg%, Na 132 meq, K 6.5 meq, uric acid: [ 20.0 mg, phosphorus: 5.9 meq and calcium 8.8 mg% suggestive of tumour lysis syndrome which required dialysis. He was HIV and EBV negative. Serum protein electrophoresis was normal. Serum free kappa light chains of 42.8 mg/L (3.3–19.40) and serum free lambda light chains of 760 mg/L (5.71–26.30). Bone marrow Aspiration and Biopsy showed no e/o myeloma or lymphoma. Our case was started on standard CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy. In view of minimal response he was shifted to salvage chemotherapy (ESHAP). But patient expired due to neutropenic sepsis.

Abstract P 119 Primary Bone Marrow Lymphoma: A Diagnostic Dilemma M Deepak Nayak1, Lakshmi Rao, Sushma V. Belurkar, Chethan Manohar 1

Department of Pathology, Melaka Manipal Medical College, Manipal University, Manipal; Department of Pathology, Kasturba Medical College, Manipal University, Manipal

Introduction: Bone marrow involvement by non Hodgkin Lymphoma (nHL) has conventionally been considered as a systemic dissemination of a nodal malignancy. Primary Bone Marrow Lymphoma (PBML) is a rare exception to this rule. This entity accounts for less than 50 cases in published literature; thereby highlighting its diagnostic novelty and clinical significance. Apart from the exclusion of a leukemia originating in the bone marrow, the current criteria include ruling out a nodal or an extranodal involvement (including the bone). We present one such unique case in a 41 year old male patient presenting with isolated thrombocytopenia. Case Report: The peripheral smear showed abnormal large lymphoid cells; also reflecting in the bone marrow aspirate. This led to consideration of alternative diagnoses such as acute lymphoblastic leukemia and lymphoma. The clinico-radiological work up showed an absence of organomegaly and lymphadenopathy; thus necessitating a review of the bone marrow study. The trephine biopsy revealed interstitial and paratrabecular infiltrates of abnormal lymphoid cells with brisk mitotic figures. The immunohistochemistry panel for lymphoma showed a strong positivity for CD10, CD 20 and BCL 2 in the lymphoid cells. The other markers such as CD34, CD99 and BCL6 were negative. Considering all the above information, a diagnosis of PBML was rendered. A 5 month follow up has thus far showed a favourable response to chemotherapy. Conclusion: Primary Bone Marrow Lymphoma is an uncommon lymphoid malignancy, often overshadowed by its more common neoplastic counterparts. An awareness of this entity is essential to establish an accurate diagnosis and adequate therapy.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

Abstract P 120

231 judgment necessitating a high index of clinical suspicion and a good clinicopathological correlation to arrive at a final diagnosis.

Gastric Outlet Obstruction in a Child—a Rare Manifestation of Burkitts Lymphoma Anand Prakash1, Kanishka Das2, Usha Kini3 1 St Johns Medical College Hospital, Department of Pediatrics, Bangalore, India; 2St Johns Medical College Hospital, Department of Pediatric Surgery, Bangalore, India; 3St Johns Medical College Hospital, Department of Pathology, Bangalore, India

Purpose: Burkitt’s lymphoma may arise in many atypical locations which on rare occasions can include stomach. A case of one such primary non-endemic gastric Burkitt’s lymphoma is described here in a non-HIV child who presented with features of gastric outlet obstruction and had a dramatic resolution of symptoms with chemotherapy. Method: Case report: A 6 year old undernourished girl with history of abdominal pain and persistent vomiting of 2 months duration presented with a large, firm, irregular epigastric mass. The CT-scan revealed a gastric mass with diffuse uniform thickening of its wall involving the body and antrum with near complete obliteration of the pyloric orifice. A pelvic lymphnodal mass lesion measuring 5.0 9 4.5 cm along with multiple perigastric and mesenteric lymph nodes, were also seen. A tru-cut biopsy of the gastric mass showed a high grade diffuse mature B cell lymphoma with positivity for CD10, CD 20, CD 79a and Ki67 index of 100 % and negative expression for Tdt, diagnostic of Burkitt’s lymphoma and Stage III as bone marrow aspiration biopsy and CSF were negative for lymphoma cells. Results: The patient was started on NHL-902 protocol. Within a week of commencing therapy of cytoreduction with vincristine, cyclophosphamide and prednisolone her symptoms of gastric outlet obstruction resolved. She continued therapy as per protocol with COPAD and COPADM and is currently well. Conclusion: The case presented here highlights an uncommon presentation of a common pediatric lymphoma and demonstrates the prompt resolution of gastric outlet obstruction with chemotherapy.

Abstract P 121: Poster Presentation A Case Series of Three Patients of Hematolymphoid Malignancy with Primary Presentation of Renomegaly

Abstract P 122: Oral Presentation Hodgkin Lymphoma: Immunohistochemical Features and Association with Epstein-Barr Virus Debdatta Basu, S Muthu Department of Pathology, JIPMER, Pondicherry Background and Objectives: Hodgkin Lymphoma (HL) shows variation in epidemiological, immunohistochemical features and association with Epstein-Barr virus (EBV). Immunohistochemical features as well as the EBV status of HL patients have an impact on the prognosis. The objectives of this study were to classify HL based on histomorphology and immunophenotype, to determine the association of EBV with HL and to compare the histological subtype, proliferation index, and clinical features between EBV positive and EBV negative cases. Materials and Methods: There were 82 patients of HL diagnosed over a period of 5 years. The histomorphological features were analyzed. Immunohistochemistry was done with a panel of markers including CD3, CD20, CD15, CD30, LCA, EBV LMP-1 and PCNA. The clinical, histomorphological and immunohistochemical parameters were correlated and statistical analysis were performed using Fischers exact test and Chi-square test. Results: There were 80 cases of classical Hodgkin Lymphoma (CHL) and two cases of Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL). Nodular sclerosis was the most common subtype of CHL (48 cases—60 %). CD30, CD15, CD20 were positive in 100, 83.8 and 10 % cases respectively. Group A immunophenotype (CD30+, CD15+, CD20-) was the most common (75 %) followed by Group B (CD30+, CD15-, CD20-). Both cases of NLPHL were CD20+, CD45+, CD15-, CD30- and negative for EBV. EBV LMP-1 was positive in 46 CHL cases (57.5 %). EBV association was more common in children, males, mixed cellularity and lymphocyte depleted subtypes, and in patients with bone marrow infiltration, high clinical stage and B-symptoms. PCNA expression was high in almost all cases irrespective of their EBV status. Conclusion: The immunohistochemical features of HL in our study population was similar to that of the Western countries. EBV positivity in HL was more often associated with adverse prognostic factors. However the overall impact of these differences on the clinical outcome of these patients need to be studied in large scale clinical studies.

Bidish Kumar Patel, Pritinanda Mishra, Debdatta Basu, Rakhee Kar Department of Pathology, JIPMER, Puducherry-6 Introduction: Extramedullary renal involvement in hematological malignancies is a rare occurrence and may lead to diagnostic dilemmas. Report: We report a series of three patients, two children, a girl aged seven and a boy of 11 years and an adult male aged 37 years, all of whom on initial presentation had renomegaly and were investigated for a suspected renal disease. Two of them underwent a renal biopsy. Subsequently, based on further clinical evaluation, peripheral blood and bone marrow examination and lymph node biopsy in one; all the three patients were diagnosed with high-grade hematolymphoid malignancies. Both the male patients had precursor B cell Acute Lymphoblastic Leukemia with Renal infiltration while the girl was diagnosed as a primary Renal T cell Acute Lymphoblastic Lymphoma with infiltration of the bone marrow. Conclusion: This series of three cases highlight the fact that hematolymphoid malignancies can at times masquerade as renal disease. Leukemias and lymphomas can have myriad clinical manifestations which can mislead the clinical

Abstract P 123: Poster Presentation Diagnosis of Hematolymphoid Malignancies in Serous Fluids—a Spectrum of Three Cases S Prasath, NG Rajesh, Debdatta Basu Department of Pathology, JIPMER, Pondicherry Background: Involvement of serous fluids by hematolymphoid malignancies is a common manifestation across the spectrum of indolent as well as high grade lesions. At times, involvement of serous cavities is the primary manifestation of lymphoma/leukemia thus presenting a challenge in accurate diagnosis. Case Series: We present a series of three cases presenting with pericardial, pleural and peritoneal effusions. Our first patient, a 25 year old male presented with massive pericardial effusion to the casualty. His pericardial fluid cytology, revealed numerous atypical lymphoid

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232 cells which were positive for PAS stain. His peripheral smear, bone marrow aspirate and biopsy with immunohistochemical work up of CD3, Tdt and CD 10 positive lymphoid cells confirmed the diagnosis of acute lymphoblastic leukemia. Our second patient presented with history of ischemic heart disease and bilateral pleural effusion and was suspected to have congestive cardiac failure. His pleural fluid cytology revealed sheets of mature lymphoid cells and scattered mesothelial cells. His peripheral smear and bone marrow aspirate showed lymphocytosis (80 %) with CD20+, CD5+ and CD23+ nodular to interstitial deposits of lymphoid cells in biopsy. Our third patient a 5-year-old child presented with ascites and ultrasound revealed retroperitoneal nodes. Ascitic fluid cytology showed CD 10+, Tdt-, CD20+ lymphoid cells with basophilic cytoplasm, prominent vacuolation and high mitotic activity. We diagnosed Burkitt lymphoma and child is on treatment. Conclusion: Knowledge of various manifestations of hematolymphoid malignancies in serous fluids is crucial in accurate diagnosis and timely management.

Abstract P 124: Oral Presentation

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 It is considered to be a well-tolerated drug with little known side effects. The reported toxic effects of gemcitabine include myelosuppression, altered liver function tests, flu-like syndrome, bronchospasm, rash, itching, and fever. Gemcitabine has not been frequently used in pediatric malignancies and to our knowledge cutaneous reaction has never been reported in children. Method: A 8-year-old boy was admitted in our hospital because of fever and multiple swellings on both sides of his neck in March 2012. On examination multiple bilateral cervical lymph nodes were palpable. Abdominal examination showed hepatosplenomegaly. Biopsy of cervical lymph node suggested Hodgkin’s disease. Diagnosed as stage III B Hodgkin’s disease, he was treated with ABVD based chemotherapy. Re evaluation following four cycles of chemotherapy revealed progressive disease, so patient was put on ifosfamide, gemcitabine, vinorelabine and prednisolone (IGV) based chemotherapy. Results: On Day 3 of treatment child developed a maculopapular, itchy skin rash. The rashes involved the neck, chest, back, upper arms and abdominal wall. The rash subsided in severity within 4-5 days with the use of oral antihistamine. However, it reappeared again on Day 5 on repeat challenge with gemcitabine during second cycle of chemotherapy. The skin lesions were again easily managed with oral antihistamines. Conclusion: Doctors dealing with chemotherapy drugs should be aware of this kind of side effect in order to avoid a misdiagnosis and identify the cause of rash.

Primary Extranodal non Hodgkin’s Lymphoma of the Head and Neck—a Study from a Tertiary Care Centre in South India Pritinanda Mishra, Sreeya Das, Debdatta Basu, Rakhee Kar, Bhawana Badhe, Sajini E Jacob Department of Pathology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry Introduction: Primary extranodal non Hodgkin’s lymphoma (NHL) of the head and neck accounts for 10–20 % of all cases of NHL. This study was undertaken to ascertain the anatomic distribution, histological subtypes and sites of extranodal NHL presenting in the head and neck region. Materials and Methods: This was a descriptive study which included record review of the departmental archives over a period of 3 years (2010—2012). Clinicopathologic features of 25 cases were studied in detail. Results: Of the 25 cases involving head and neck 16 were males and nine were females. Mean age was 50 years. The most common sites of involvement were Waldeyer’s ring, followed by nose, brain, orbit, palate and thyroid. Histologically majority of the cases (80 %) were B cell origin of which diffuse large B cell lymphoma was the commonest subtype (15 cases), followed by plasmablastic lymphoma (2 cases) and a single case each of small lymphocytic lymphoma, lymphoblastic lymphoma and low grade marginal zone lymphoma of the mucosa associated lymphoid tissue (MALT) type. T cell lymphomas constituted 20 % of the cases. Conclusion: This retrospective study in a tertiary care centre in South India illustrates the pattern of clinical and histological distribution of various common and uncommon subtypes of extranodal NHL in the head and neck region.

Abstract P 125 Gemcitabine Induced Skin Rash in a Boy with Hodgkin Disease Vikas Dua1, Jai Bhagwan Sharma2 1 Department of Pediatric Hematology Oncology, Action Cancer Hospital, Delhi, India; 2Department of Medical Oncology, Action Cancer Hospital, Delhi, India

Objective: Gemcitabine is used in various carcinomas like lung cancer, pancreatic cancer, bladder cancer and breast cancer in adults.

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Abstract P 126 A Retrospective Analysis of Patterns of Distribution of Lymphomas in Elderly Patients in a Tertiary Care Hospital in South India Over a Period of 5 years Parimal Sarda1, Marie Therese Manipadam1, Sheila Nair1, Auro Viswabandya2 1

Department of Pathology, Christian Medical College, Vellore; Department of Hematology, Christian Medical College, Vellore

2

Introduction: This study aims to analyse the pattern of distribution of lymphoid neoplasms in elderly patients (age more than 60 years) in a single tertiary care centre in South India using WHO 2008 classification. Material and Methods: This is a retrospective analysis of 359 cases diagnosed as lymphoma in elderly patients during the period of Jan 2007 to Dec 2011. Results: A total number of 359 elderly patients with diagnosis of lymphoma were included in this study. There were 245 males and 114 female patients. The mean age was 66.72 years. Of these, non Hodgkin lymphomas (NHL) accounted for 89.97 % (n = 323), Hodgkin lymphoma (HL) 9.75 % (n = 35) and post transplant lymphoproliferative disorder 0.28 % (n = 1) respectively. In NHLs, B cell neoplasms accounted for 90.09 % (n = 291) and T cell neoplasms 9.91 % (n = 32). In B cell neoplasms, mature B cell NHL accounted for 99.65 %. In mature B-NHL, indolent lymphomas accounted for 43.10 % (n = 125) and aggressive lymphomas 56.90 % (n = 165). The subtypes of non Hodgkin lymphomas were as follow: diffuse large B cell lymphoma (n = 161, 50 %), follicular lymphoma (n = 33, 10 %), chronic lymphocytic leukemia/small lymphocytic lymphoma (n = 17, 5.26 %). In T cell neoplasms, mature T cell NHL accounted for 96.7 % and precursors T-LBL 3 %. Among the mature T cell NHLs, the pattern of distribution was; peripheral T cell lymphoma, not otherwise specified in 14 (4.34 %) patients, angioimmunoblastic T cell lymphoma in 9 (2.78 %) and anaplastic large cell lymphoma in 4 (1.23 %). Mixed cellularity was the major subtype of classical Hodgkin lymphoma found in 13 (38 %) where as Nodular lymphocyte predominant HL constituted 3 % (n = 1). As compared to the distribution pattern of lymphomas including all age groups in our institution the striking feature was that

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Table 1 Immunophenotypic patterns in Mature T/NK cell lymphoproliferative disorders HSL (%)

T-LGL (%)

MF/SS (%)

ATLL (%)

CD2

10 (100)

3/3 (100)

3 (100)

3 (100)

CD3+

10 (100)

4 (100)

2 (66)

3 (100)

CD5+

1 (10)

2/3 (67)

3 (100)

3 (100)

CD4+ CD8-

0

0

2 (66)

3 (100)

CD4- CD8+

4 (40)

4 (100)

0

0

CD4- CD8-

6 (60)

0

0

0 0

CD4+ CD8+

0

0

0

CD7+

10 (100)

3/3 (100)

1 (33)

0

CD16+

6 (85)

2 (50)

0

0

CD25+

0

0

1 (33)

3 (100)

CD56+

3 (30)

2/4 (50)

0/1

0

CD57

0/8

1/3 (33)

ND

0

TCRab+

1 (10)

3/3 (100)

1/1 (100)

2 (66)

TCRcd+

9 (90)

0

0/1

0

Total cases

10

4

3

3

T-PLL (%)

Aggressive NK- cell leukaemia (%)

NK CLPD (%)

T-CLPDu (%)

CD2

1 (100)

1 (100)

2

3 (100)

CD3+

0

1 (100)

0

3 (100)

CD5+

0

1 (100)

0

3 (100)

CD4+ CD8-

0

1 (100)

0

2 (67)

CD4- CD8+

1 (100)

0

1 (50)

1 (33)

CD4- CD8-

1 (100)

0

1 (50)

0

CD4+ CD8+

0

0

0

0

CD7+

1 (100)

1 (100)

1 (50)

2 (67)

CD16+

1 (100)

0

2 (100)

0

CD25+

0

0

0

1 (33)

CD56+

0

1 (100)

2 (100)

0

CD57

0

0

1 (50)

0

TCRab+

0

0

1 (50)

3 (100)

TCRcd+

0

0

0

0

Total cases

1

1

2

3

B cell NHLs are more common in elderly patients. T-LBL and ALCL are more common in the former group, but this included childhood lymphoma also, hence when these are excluded overall frequency becomes similar. Frequency of EBV + DLBCL of elderly will be also presented. Conclusions: This is the first study from India showing the pattern of distribution of lymphoid neoplasms in the elderly patients.

Abstract P 127 Lymphoma Involving Spleen and its Histopathological Pattern in a Tertiary Care Hospital in South India: A Five Year Study Subramaniam Kandasamy, Marie Therese Manipadam, Sheila Nair Department of Pathology, Christian Medical College, Vellore Introduction: Lymphoma involving spleen is heterogeneous array of disease whose accurate diagnosis is essential in further management. The aim of this study is to analyze the lymphoma involving spleen and its histopathological pattern in a South India tertiary care centre using WHO 2008 classification. Material and Methods: A retrospective study of all splenic lymphomas diagnosed in Christian Medical College Hospital, Vellore during a 5 year period (Jan 2007 to Dec 2011). Results: This study includes 69 cases diagnosed as splenic lymphoma

233 from January 2007 to December 2011. This includes 50 males and 19 females. The mean age was 47 years (range from 7 to 74 years). NonHodgkin lymphoma (NHL) was more common 88.4 % (n-69), than Hodgkin lymphoma (HL) 11.6 % (n = 69). In NHL, B cell neoplasms accounted for 73.8 % (n = 61) and T cell neoplasms 26.2 % (n = 61). The subtypes of NHL were as follow; splenic marginal zone lymphoma (n = 13; 28.8 %), diffuse large B cell lymphoma (n = 12; 26.6 %), hairy cell leukemia (n = 6; 13.3 %), follicular lymphoma (n = 4; 8.8 %), T cell/histiocyte-rich large B cell lymphoma (n = 2; 4.4 %) and one each of mantle cell lymphoma (2.2 %), lymphoplasmacytic lymphoma (2.2 %) and chronic lymphocytic leukemia/small lymphocyte lymphoma (2.2 %). 3 cases of low grade B cell lymphoma, unclassifiable. Among T cell NHL NK/T cell lymphoma accounts for 37.5 % (n = 6/16) followed by hepatosplenic T cell lymphoma (n = 5/16; 31.25), Peripheral T cell lymphoma (PTCL) (n = 3/16; 18.25 %) and one case of T cell large granular lymphoma. Splenic marginal zone and diffuse large B cell lymphomas were the commonest types. As compared to western literature, follicular lymphomas were infrequent and peripheral T cell lymphomas including Hepatosplenic T cell lymphomas and NK/T cell lymphomas were more common. Conclusions: This study shows the frequency and pattern of lymphomas involving spleen in a tertiary care centre.

Abstract P 128 Mature T/NK-Cell Lymphoproliferative Disorders: A Tertiary Centre Flow Cytometry Experience Neeraj Arora, Bhargavi Balakrishnan, Ansu Abu Alex, Rayaz Ahmed, Aby Abraham, Biju George, Alok Srivastava, Vikram Mathews Department of Haematology, Christian Medical College and Hospital, Vellore 632004 Abstract: The classification of the T cell malignancies is complex and based on clinical features and laboratory investigations, which include morphology, histology, immunophenotype, HTLV-I serology and molecular genetics. Mature T/NK-cell lymphoproliferative disorders are rare disorders with morphologic heterogeneity, and lack of specific immunophenotypic markers for clonality. We retrospectively analyzed immunophenotypic data from consecutive cases of mature T/NK cell lymphoproliferative disorders diagnosed in our flow cytometry laboratory from 2009 to August 2012. These cases were diagnosed based on immunophenotyping of PB and BM aspirates and morphology as per WHO 2008 guideline. The gating strategy used included FSC/SSC, CD45/side scatter and CD3 gating. The initial panels evaluated for acute leukemia or mature lymphoma depending on the morphology. In all patients a T cell specific secondary panel was used for further characterization using CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD16, CD19, CD25, CD34, CD56, CD57, TCRab, TCR-cd, CD1a, HLA-DR and TdT (BD Biosciences, USA) in most cases. Correlation was carried out with immunohistochemistry on tissue (wherever available) such as BM, lymph node and skin biopsies, serological tests for HTLV1 and other laboratory investigations. Of the total 285 mature lymphoid neoplasms, T/NK-cell lymphoproliferative disorders constituted 9.5 % (n = 27) and B cell lymphoproliferative disorders including 12 cases of Burkitts lymphoma constituted 90.5 % (n = 258). Plasma cell disorders were not included in the study. The commonest mature T/NK-cell lymphoproliferative disorder in this study was hepatosplenic lymphoma (HSL) (n = 10, 37.1 %) followed by T-large granular leukaemia (TLGL) (n = 4, 14.8 %), Adult T cell leukaemia/lymphoma (ATLL) (n = 3, 11.1 %), Mycosis/Sezary syndrome (MF/SS) (n = 3, 11.1 %) and T cell prolymphocytic leukaemia (T-PLL) (n = 1, 3.7 %).

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234 Mature NK-cell disorders (n = 3, 11.1 %) included 2 cases of aggressive NK-cell leukaemia and a case of chronic NK cell lymphoproliferative disorder (NK-CLPD). Because of the lack of clinical details a definite diagnosis could not be made in 3 cases and these cases were labeled as T cell lymphoproliferative disorders unclassifiable (T-CLPD u).The different expression patterns of T cell markers in various subtypes are summarized in Table 1. To conclude with an appropriately selected panel, phenotypic analysis using FCM is very useful for the diagnosis of T cell and NK-cell lymphoproliferative disorders.

Abstract P 129 Result of Paediatric Non Hodgkin’s Lymphoma with Intensified Short Duration Chemotherapy

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 CNS multiple times with complete response to conventional therapy each time is very rare. We report a case of multiple CNS (Only) relapse of a case of extra nodal Non Hodgkin’s Lymphoma with repeated complete remissions. A 41 years male patient, who was a known case of NHL, DLBCL, CD20 Positive, of Maxilla and Ethmoid with mild extension to intracranial, but not involving the brain parenchyma presented to TMH, Mumbai in 2003. This was an extra nodal NHL in the face around nose, nearer to cranium. Following 6 cycles of CHOP chemotherapy and local Radiotherapy, he showed complete response. About 3 years later again presented to the same hospital with first time brain relapse only (ataxia, brain lesions on MRI), received treatment as per CNS protocol. Post treatment scan showed complete response and disappearance of lesion. Two years later follow up MRI showed normal brain. Later he presented to our hospital with multiple (two times) only CNS relapses at a gap of 4 years and 10 months respectively with complete response to treatment.

Prattusha Sengupta, P Gupta, S Biswas, A Mukhopadhyay Netaji Subhas Chandra Bose Cancer Research Institute, 16A Park Lane, Kolkata-700016, West Bengal, India Objective:Aim of our study was to observe result of intensified short duration chemotherapy in pediatric Non-Hodgkin’s Lymphomas (NHL). Methods: We included consecutive 240 pediatric NHL patients in pediatric haematooncology department of Netaji Subhash Chandra Bose Cancer Research Institute during period from 2000 to 2011. Inclusion criteria: patients \18 years of age with a diagnosis of NHL. Exclusion criteria: Patients with [25 % blasts in the bone marrow. Each patient received three cycles A and three cycles B of MCP 842 protocol of INCTR. Response was assessed at the completion of 2 cycles of chemotherapy (1 each of A and B) and 6 cycles of chemotherapy. Result: 70 (29.17 %) patientsLymphoblastic Lymphoma (LL), 98 (40.83 %)-Burkitt Lymphoma. 60 (25 %)-diffuse Large B Cell Lymphoma (DLCL), 12 (5 %)Anaplastic Large Cell. The abdomen was the most common site in 80 (33.3 %) of involvement followed by mediastinum in 38 (15.83 %). 202 (84.17 %) patients achieved complete response after 2 cycles of therapy. 20 (8.33 %) achieved partial response, 10 (4.17 %) had no response, 10 (4.17 %) were not evaluable. With median follow up of 4 years a total of 64 (26.67 %) patients (28 LL, 24 Burkitt Lymphoma, 8 DLCL and 4 ALCL) had died. Causes of death were progressive disease in 48, infection in 12, hepatitis in 2 and unknown 2. 176 (73.33 %) patients are alive and disease free. Patients tolerated chemotherapy well. Grade-IV febrile Neutropenia was seen in 52 patients. Conclusion: Result of short duration chemotherapy MCP842 is promising. We intend to continue the same protocol for next few years.

Abstract P 130 Multiple CNS (Only) Relapse of a Case of Extra Nodal Non Hodgkin’s Lymphoma with Repeated Complete Remissions Deba Dulal Biswal, Dinesh Pendharkar, Akshay D. Shah, Suresh H. Advani Asian Institute of Oncology, Mumbai Abstract: Relapse of Non Hodgkin’s Lymphoma (NHL) in CNS though occurs, is a rare phenomena. Relapse of extra nodal NHL in

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Abstract P 131 Comparison of Efficacy and Safety of Rituximab (MabtheraÒ) and its Biosimilar (RedituxÒ) in Diffuse Large B Cell Lymphoma (DLBCL) Patients Treated with Chemo-Immunotherapy: A Retrospective Analysis Partha Sarathi Roy, Shiji John, Sadhana Kannan, Sumeet Gujral, Jayant Gawande, Bhausaheb Bagal, Navin Khattry, Manju Sengar, Hari Menon, Reena Nair Hematolymphoid Disease Management Group, Department of Medical Oncology, Tata Memorial Centre, Mumbai, India Objective: Rituximab with CHOP regimen (R-CHOP) has been the standard of care for DLBCL. RedituxÒ, a biosimilar molecule of Rituximab, was in use in India since 2007. The objective of this retrospective audit is to compare the efficacy, safety and toxicity of MabtheraÒ with RedituxÒ. Methods: Two hundred twenty-three patients aged C18 years who received 4-8 cycles of R-CHOP regimen between January 2004 to June 2010 were included. Complete response (CR), Partial response (PR)] in both groups were evaluated as per Cheson’s criteria and compared by ChiSquare test. Overall survival (OS) and progression-free survival (PFS) were estimated by Kaplan–Meier method and compared by two-sided log rank test. All grade 3–4 hematological and nonhematological toxicities were recorded. Results: One hundred one patients received MabtheraÒ, 72 received RedituxÒ. In 17 patients, the brand used was unknown and 33 patients received both. Baseline characteristics at presentation were similar in both groups. Median number of treatment cycle received was 6 in each group. Observed CR rate was 75 % in MabtheraÒ group and 81 % in RedituxÒ group. OS at 5 years was 84 % (MabtheraÒ 83 % and in RedituxÒ 90 %; P = 0.214). PFS at 5 years was 67 % (MabtheraÒ 71 % and RedituxÒ 78 %; P = 0.579). We also evaluated patients who had received C4 cycles of MabtheraÒ or RedituxÒ in their respective groups, and found no differences in efficacy and outcomes. Three patients died during treatment in each group. Grade 3–4 febrile neutropenia was observed in 23 % (MabtheraÒ) and 20 % (RedituxÒ); grade 3–4 oral mucositis and diarrhea was seen in 27 % (MabtheraÒ) and 20 % (RedituxÒ). No difference in infusional reactions was observed. Conclusion: In this retrospective analysis we found RedituxÒ is as efficacious and safe as MabtheraÒ.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

Abstract P 132 Utility of Flow Cytometry in Bone Marrow Staging of Lymphoma K Ghodke, T Shet1, E Sridhar1, S Ghogale, Y Badrinath, S More, PG Subramanian, S Gujral

235 CD7. Variable expression of CD1a, CD2, CD4, CD8 and CD5 was seen. 2 cases were classified as peripheral (mature) T cell leukemia/lymphoma. These cases showed bright positivity for CD45 and T lineage markers with aberrant loss of some markers. Conclusion: Flowcytometric immunophenotyping is a useful tool in identifying and classifying T cell leukemia/lymphoma.

Department of Hematopathology, 1Department of Histotopathology, Tata Memorial Centre, Mumbai Introduction: Immunophenotypic (IPT) analysis is an established tool in the diagnosis and classification of many hematolymphoid disorders; however, the role of flow cytometry (FC) in detecting bone marrow (BM) involvement for the staging of lymphoma is yet to be defined. Objectives: To evaluate the role of FC immunophenotyping of BM in the staging of lymphoma and its correlation with morphology on bone marrow aspirates (BMA) and trephine biopsy (BM Bx) with immunohistochemistry. Materials and Methods: A retrospective review was done of 193 consecutive staging BM over a period of 2 month (June–July 2012). Of these, FC analysis was asked for in 60 cases (as per clinicians request). Morphologic and FC IPT analyses were performed and results were compared with BM Bx findings. FC IPT analysis was done by six color antibody panel on FACSCANTO II. Results: As seen in following table, FC helped in detection of BM involvement in 5 % (3/60) cases in which morphological analyses on BM failed to detect the involvement. Conclusion: FC IPT analysis on staging marrow contributed upstaging in 5 % of cases.

Comparison of FC IPT analysis and morphological analysis in 60 cases FC analysis

IPT Morphological analysis BMA (i) BMA (i) BMBx BMBx (i) (ui)

Total

BMA (ui) BMBx (i)

BMA (ui) BMBx (ui)

i

14

0

7

3

24

ui

0

0

4

32

36

14

0

11

35

60

Total

i involved, ui uninvolved)

Plasma Cell Dyscrasias Abstract P 134 sFLC Assay: Establishing National Reference Range— A Single Tertiary Care Centre Experience Sarika Singh, Rajive Kumar, Vinod Raina, Lalit Kumar All India Institute of Medical Sciences, New Delhi Introduction: Serum free light chain (sFLC) assay measures levels of free j&k immunoglobulin light chains. It is recommended to be used as a screening test for all plasma cell dyscrasias (PCD).Therefore, included in both the guidelines for diagnosis as well as uniform response criteria during therapy by International myeloma working group. To conduct any study on sFLC we take a cue from reference range data provided by west, due to non availability of Indian data. Aims and Objectives: This study was aimed to establish normal reference range of sFLC in healthy donors and its comparison with other non myeloma cohorts to validate the hypothesis that prevalent subclinical infections and inflammation can result in higher j/k and to establish if any ethnic variations exist in these cohorts compared to west. Materials and Methods: We studied 135 cases of healthy blood donors, 25 cases of tuberculosis, autoimmune disorders, chronic renal failure (CRF) and MM each during 2009–2010. sFLC assay was performed by nephelometric bioassay. Results: j levels ranged from 6.8–70.7, 8.9–77.7, and 5.9–5420 mg/L in healthy donors, non myeloma cohort and MM cases respectively. k levels were 3.3–66.7, 4.3–125, and 0.002–669.1 mg/L levels in healthy donors, non myeloma and myeloma cohort respectively. The j/k was 0.6–3.1 mg/L in healthy donors while was 0.4–1.1 mg/L in autoimmune disorder, 0.6–6.9 mg/L in CRF, 0.61–1.66 in TB patients. Conclusion: In contrast to west, all the parameters were higher in Indian population, more so was the j/k. Since this deviation is not observed in other non myeloma cohorts, this has a bearing on the ethnicity rather than subclinical infections/inflammation which was hypothesised to be the cause.

Abstract P 133: Poster Presentation Flowcytometric Immunophenotyping of T Cell Leukemia/Lymphoma Jyoti Garg, Poojan Agarwal, Vijay Kumar, Sadhna Marwah, AS Nigam, G Buxi Department of Pathology, PGIMER & Dr. Ram Manohar Lohia Hospital, New Delhi Objective: To evaluate the role of flowcytometric immunophenotyping in T cell leukemia/lymphoma. Methods: Flowcytometric analysis was carried out on peripheral blood samples of 46 patients suspicious of acute leukemia. Cases expressing T cell lineage markers were further characterized with the help of a panel of antibodies comprising of TdT, CD3, CD4, CD8, CD7, CD5, CD2, CD1a, CD56 and CD45 using BD FACS Calibur. Results: Of the 10 cases expressing T cell lineage markers, 8 cases were classified as T-lymphoblastic leukemia/lymphoma. All of these cases showed bright positivity for TdT, cCD3 and

Abstract P 135: Oral Presentation Immunohistochemical Profile and Bone Marrow Angiogenesis in Multiple Myeloma with Reference to its Clinical Significance Sarah Grace Priyadarshini, Debdatta Basu, Rakhee Kar, TK Dutta1 Departments of Pathology and Medicine1, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry Aims of the Study: To study the histomorphological features of bone marrow in multiple myeloma and to evaluate the immunohistochemical profile and angiogenesis using a panel of markers including CD38, kappa and lambda light chain, CD56, Cyclin D1, Ki67 and CD 34 and to correlate immuno-hematological profile with various clinical parameters. Materials and Methods: The study includes 48 cases of myeloma diagnosed over the period of 5 years. The

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236 histomorphological features like plasma cell morphology, percentage of plasma cells and pattern of infiltration were studied. Angiogenesis was assessed by calculating the microvessel density (MVD) using immunohistochemistry for CD34. Proliferation was assessed using both Ki67 and CD38 highlighted cells. Immunohistochemistry for CD56 and Cyclin D1 was also done. Results: A significant association was seen between the plasma cell morphology and the pattern of infiltration (p = 0.0067). The percentage of plasma cells showed a significant association with the clinical staging. The MVD was significantly associated with plasma cell morphology (p = 0.04) as well as pattern of infiltration (p \ 0.001). The proliferation index was also significantly associated with the plasma cell morphology (p = 0.003). Both the Ki67 and MVD showed an increasing trend with clinical staging and MVD was significantly associated with serum albumin (p = 0.02). CD56 negativity was associated with circulating plasma cells and also with higher clinical stage. Cyclin D1 positivity was not seen in poorly differentiated morphology and the mean Ki67 was lower in the cyclin D1 positive group. It was also found that the cases with kappa light chain restriction had significantly less of poorly differentiated morphology, diffuse pattern of infiltration, lower MVD and Ki67. Conclusion: The study of bone marrow morphology and angiogenesis with the aid of immunohistochemistry is useful for prognosticating patients with multiple myeloma.

Abstract P 136 A Case of Hemophagocytic Syndrome with Reactive Plasmacytosis in a 16 Years Old Immunocompromised Patient

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 malignant lymphoma often demonstrate a positive antiglobulin test, but they rarely develop overt clinical symptoms of AIHA. AIHA has rarely been documented in patients with multiple myeloma. We conducted a prospective study to detect the presence of AIHA in patients with multiple myeloma and its impact on clinical presentation and outcome of disease. Study Design and Method: A total of 66 patients diagnosed to have MM over a period of 18 months were the subjects of the study. Patients with (a) severe anemia (hemoglobin \ 6 g/dl), (b) requiring frequent blood transfusions and (c) having clinical and laboratory features of hemolysis, were screened for AIHA, by performing direct and indirect antiglobulin (Coomb’s) test. Results: Of the total of 66 patients of multiple myeloma, seventeen patients were screened for AIHA. Seven (10.6 %) were found to be complicated with AIHA and carried auto-antibodies in their sera. Five of these patients had denovo multiple myeloma and two had multiple myeloma relapse. The IgG subclass of the antibody binding to red cell membrane was compared with that of M-protein and these findings were showing full coincidence in all the seven patients. Six patients (85.7 %) had stage IIIA and one patient (14.3 %) having stage IIIB disease. All of these patients were positive for subtypes of IgG with IgG1 (n = 2), IgG2 (n = 4), IgG3 (n = 1) and IgG4 (n = 1). One patient had simultaneous positivity for IgA and IgG2, with presence of cold antibodies in the serum. Conclusion: We found that autoimmune hemolysis is a frequent finding in patients with multiple myeloma and failing to rule out this phenomenon may overburden the management protocol in form of multiple blood transfusion and related complications.

Prakriti Shukla, Syed Riaz Mehdi, Sharique Ahmad, Nishi Tandon Era’s Lucknow Medical College and Hospital, Lucknow Abstract: Hereditary and sporadic cases of hemophagocytic syndrome (HPS) are primarily reported in children. We present a case of hemophagocytic syndrome with reactive plasmacytosis in a 16 years young male. The patient presented to us with persistent fever, hepatosplenomegaly, lymphadenopathy, anaemia, leucopoenia and marked thrombocytopenia with elevated triglycerides level. For all the above complaints he was advised bone marrow examination which revealed hypocellular smears with marked erythrophagocytosis by histiocytes and his NK cell activity was reduced which was determined by Flow cytometry. He was diagnosed as a case of hemophagocytic syndrome but before any treatment could have been started, he expired. This experience showed that HPS may be a life-threatening condition. Its rarity combined with non-specific clinical features, such as fever and lymphadenopathy, makes it a diagnosis that can be easily missed. Keywords Hemophagocytosis, Plasma-cytosis, Immunocompromised

Abstract P 137 Prevalence of Autoimmune Hemolytic Anemia in Multiple Myeloma: A prospective study Rajesh Kashyap1, Abhay Singh2, Pradeep Kumar1 1 Department of Hematology, Sanjay Gandhi Postgraduate Institute of medical Sciences, Lucknow, Uttar Pradesh, India; 2Department of Transfusion Medicine, Sanjay Gandhi Postgraduate Institute of medical Sciences, Lucknow, Uttar Pradesh, India

Objective: Autoimmune hemolytic anemia (AIHA) is often associated with B cell lymphoproliferative disorders. Patients with

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Abstract P 138 A Case of Waldenstrom Macroglobulinemia Treated with Plasmapheresis Suvro Sankha Datta, Biplabendu Talukder, Ritam Chakrabarty, Somnath Mukherjee, Prasun Bhattacharya, Krishnendu Mukherjee Department of Immunohematology & Blood transfusion, Medical College Hospital, Kolkata-73 Objective: Therapeutic plasma exchange is a conjunctive modality of treatment to decrease paraproteinemias associated with hyperviscosity. Here we narrate our experience in treating a diagnosed case of Waldenstrom macroglobulinemia in 70 years old man with moderate anaemia and severe features of hyperviscosity syndrome by serial TPE and chemotherapy. Methods: Three therapeutic plasma exchanges (TPE) were performed by intermittent cell separator (Hemonetics, MCS+, USA). His first two exchanges were done on alternate day followed by chemotherapy and third TPE was done six weeks after the second TPE. Results: Initially his serum IgM was 11.3 g/dl on serum protein electrophoresis. At the end of second TPE the patient was relieved of his weakness and syncope. Before third TPE his serum IgM again became 9.901 g/dl. After twenty four hours of third TPE, his serum IgM level came down to 3.13 g/dl and the patient became asymptomatic. Conclusion: The relationship between blood viscosity and abnormal immunoglobulin concentration is exponential, such that removal of relatively small amount of immunoglobulin by TPE will result in a large reduction in serum viscosity. So TPE must be used in conjunction with chemotherapy for symptomatic relief in case of Waldenstrom macroglobulinemia.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

Abstract P 139 Crystalline Inclusions in Myeloma Cells: A Case Report

237

Serial no.

Pre-exchange hematocrit

Total blood volume processed

Replacement fluid

1

37.10 %

2,139 ml

*5 % albumin:850 ml + FFP:900 ml + ACD:220 ml

2

36.80 %

2,040 ml

*5 % albumin:800 ml + FFP:750 ml + ACD:187 ml

3

35.30

2,087 ml

*5 % albumin:900 ml + FFP:600 ml + ACD:260 ml

Shweta Sushmita, Amit Kumar Adhya Department of Pathology, KIMS, Bhubaneswar, Odisha, 751024 Introduction: The presence of crystalline inclusions in plasma cell myeloma is a rare phenomenon and cases have been reported with rod, needle, and rectangular shaped crystals. Here, we present a case of plasma cell myeloma with needle and rhomboid shaped intracellular crystalline inclusions and extracellular crystal depositions in the bone marrow. Case: A 56 year old male patient presented with low back pain. On investigation he was found to have multiple lytic bone lesions. Serum M band was positive. Bone marrow study revealed 90 % myeloma cells with numerous intracellular and extracellular needle and rhomboid shaped crystalline inclusions. The inclusions were negative for MPO, SBB but positive for NSE. Discussion: Crystalline inclusions are believed to be due to accumulation of cytoplasmic immunoglobulins secondary to a block in the protein synthetic pathway Since rhomboid crystal depositions can be seen in other clinical conditions such as pseudogout, this case invites consideration of plasma cell myeloma in the differential diagnosis of patients with rhomboid crystalline deposition in the bone marrow and in sites/organs other than the bone marrow. This case and the review of similar cases presented in the literature suggest that extracellular rhomboid crystals in the bone marrow and extramedullary sites such as articular spaces or renal parenchyma should raise suspicion of a plasma cell neoplasm, yet other differential diagnoses such as granulocytic sarcoma or reactive processes should also be considered.

Abstract P 140 Multiple Myeloma Presenting as Proptosis: A Diagnostic Dilemma Pattanayak Lucy1, Padhi Sanjukta1, SN Senapati1, Samantaray Sagarika2, Rout Niranjan2 Department of Radiation Oncology, 2Oncopathology, AHRCC, Cuttack

1

Background: Plasma cell neoplasms are characterized by monoclonal proliferation of plasma cells or terminally differentiated B cells. They may present as solitary plasmacytoma, which can affect bone or soft tissues, or they can manifest with systemic involvement as multiple myeloma. Extramedullary presentation of multiple myeloma is uncommon and the orbit is involved in less than 0.25 % cases till date. We describe an unusual case of multiple myeloma that presented to us with proptosis. Case: A 45-year-old male presented with gradual proptosis of the right eye for a period of 15 days. Local examination revealed a forward and outward protrusion of the eyeball, restricted movements and loss of vision in the right eye. On palpation there was a firm mass 2.5 9 2.5 cm in size, nontender in the superior orbit of the eye. Hb was 7 g% and ESR was 110 mm in the 1st hour. Scrape cytology from the mass showed sheets of binucleate and multinucleate plasma cells suggestive of plasmacytoma. Bone marrow aspiration and cytology showed oval cells with pale basophilic cytoplasm, perinuclear halo, nuclei with cartwheel chromatin suggesting plasma cells which were [30 %. Serum Bens Jones protein was positive and electrophoresis showed a sharp M band in the gamma region confirming the diagnosis of Ig G multiple myeloma. Skeletal survey was normal. Conclusion: Orbital myeloma with negative skeletal survey is infrequently encountered. Cytological picture from the lesion as well as bone marrow aspiration cytology is

*Albumin is reconstituted with normal saline, FFP: Fresh Frozen Plasma, ACD: Acid citrate dextrose confirmatory in the absence of bony lesions. Associated features like anemia help in staging and prognostication. Keywords Plasmacytoma, Multiple Myeloma, Orbit, Proptosis,

Abstract P 141 Primary Amyloidosis Associated with IgM k Proteinemia Presented as Nephrotic Syndrome Upasana Das, SM Sethy, RK Jena, P Mohanty Department of Clinical haematology, Department of Pathology, S.C.B. Medical College, Cuttack, Odisha Introduction: Primary amyloidosis presents with systemic or localised form without any evidence of preceding or coexisting disease, paraproteinemia or plasma cell dyscrasias. The amyloid of primary amyloidosis is made by plasma cells in bone marrow. The L chains are secreted into the serum and they undergo partial lysosomal proteolysis and get deposited as insoluble amyloid filament. Three fourth of immunoglobulin light chains are of the k group. The organs most commonly involved are the kidneys, heart, blood vessel walls. Renal amyloidosis may manifests in many forms. Out of all presentations nephritic syndrome is one of them. Usually the mean age is 43.9 years. This is a case of presented in young male. Case Report: A 20 year male boy presented with bilateral pitting pedal edema, puffiness of face and loss of weight for last 6 months. Serum albumin was 1.5 g/dl, s. Protein 2.9 g/dl, urine albumin +++. The case was diagnosed to be nephrotic syndrome. The capillary zone electrophoresis showed the presence of M band, serum immunofixation revealed monoclonal IgG k type. Bone marrow aspiration 5 % marrow plasma cells. The core needle biopsy of kidney showed presence of glomerular and vascular deposition of amyloid. Renal manifestation in amyloidosis carry a grave prognosis. Keywords Primary Amycoidoses, Nephrotic Syndrome, 1Gms 2chain

Abstract P 142 Secondary Malignancy (AML) in Case of Post Chemotherapy (MPV) Multiple Myeloma S Burma, P Bhuyan, S Sethi, RK Jena Department of Pathology & Department of Clinical Hematology, S C B Medical College, Cuttack, Odisha Introduction: The multiple myeloma patients may develop AML or MDS as a secondary malignancy either due to a complication of

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238 chemotherapy or may be in the absence of any chemotherapy. We reported only two such cases out of 145 multiple myeloma receiving MPV therapy from July 2010 to July 2012. Case Report: We have reported two cases of multiple myeloma who developed AML 2 years after the diagnosis. They had been treated with oral melphalan, prednisone and vincristine (MPV) for MM. The patients were achieved complete remission. But after 2 years later, one case presented with fever, generalized weakness. BM examination reveals [50 % myeloblasts with [10 % maturation and diagnosed as AMLM2. But the patient was died after 2 days of initiation of CT. Another case, 1‘ years later, presented with fever, sternal tenderness and mucocutaneous bleeding and diagnosed as AML-M4. The patient expired due to thrombocytopenia before initiation of any chemotherapy. Conclusion: Incidence of secondary myeloid leukemia in the setting of multiple myeloma is very uncommon. In our series it is \2 %. Such leukemias are typically acute myeloid leukemia (AML). The myelomonocytic subtype is particularly common.

Abstract P 143 Stomach Involvement in a Relapse Case of Multiple Myeloma Priyadarshini Dehuri, Sudha Sethy, Rabindra Kumar Jena, Pranati Mohanty Department of Clinical Haematology and Department of Pathology, S.C.B Medical College, Cuttack Introduction: Multiple myeloma is a neoplastic proliferation of monoclonal plasma cells usually seen in the bone marrow. Relapse cases with extramedullary presentation in the form of stomach lesion is only rarely reported. Case Report: A 60 year female post chemotherapy for multiple myeloma presented with painful swelling in abdomen, fever and anaemia. The USG revealed a gastric mass involving fundus and body. With a clinical suspicion of adenocarcinoma, biopsy was advised. The biopsy reports were suggestive of either high grade lymphoma or undifferentiated carcinoma. Immunohistochemistry was suggested for definite diagnosis. The IHC stains proved all lymphoma markers to be negative. There was pan cytokeratin positivity. Most importantly CD 56 positivity was noted. In the light chain analysis, kappa to lambda ratio was 3:1. Thus all the investigations favoured a diagnosis of a monoclonal plasma cell expansion. Conclusion: Relapse cases of multiple myeloma with extramedullary presentation being very rare, need attention for their further management.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 study showed features of Plasmacytoma. On bone-marrow aspiration, the case was diagnosed to be multiple myeloma supported by M-Band in serum electrophoresis.

Platelet Disorders Abstract P 145 Profile of Vascular Thrombotic Events in Essential Thrombocythemia A Krishna Prasad, M Shetty, AMVR Narendra, VR Srinivasan Nizam’s Institute of Medical Sciences, Hyderabad, AP Introduction: Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by the clonal proliferation of primarily megakaryocytes, resulting in thrombocythemia. Complications, often thrombotic, are more common in patients with high platelet counts with an incidence of 84 %. Profile of vascular thrombotic events in ET both at presentation and on follow up was reported below. Aim: Study of vascular thrombotic events in ET patients. Materials and Methods: Vascular thrombotic events information, from the case records of ET patients attending General Medicine department, was retrospectively analyzed. Period of collection was 14 years. All cases satisfied the 2008 WHO criteria for diagnosis of ET. Results: Total cases were 15. Males were 8 and females 7. Mean age was 49 years (range of 24–75 years). Mean platelet count at presentation 13.7 l/ll (range of 5–40 l/ll). All cases presented with vascular events. Total thrombotic events were 25 (arterial 17 and venous 9). Mean platelet count in arterial thrombosis was 11.1 l/ll. Arterial thrombosis noted in cerebral vessels (6), coronaries (4), digital arteries (2), renal artery (1), anterior spinal artery (1), aorto-iliac vessels (1), ulnar artery (1) and tibial artery (1). Mean platelet count in venous thrombosis was 8.6 l/ll. Venous thrombosis noted in portal veins (6), deep venous system of lower limbs (4), spleenic vein (2) and superior mesenteric vein (2). Surgeries were done in 7 cases [CABG (1), amputation of limbs (2), splenectomy (4)]. JAK2 positivity was noted in 3 out of 5 cases. Conclusions: Arterial events were more than venous events. Recurrent vascular thrombosis was noted in 11 cases due to poor control of the disease (twice in 9 and thrice in 2 cases). Thrombotic complications affect the quality of life.

Keywords Multiple Myeloma, Extramedullary, Stomach, CD56

Abstract P 144 Multiple Myeloma with CNS Involvement: A Rare Case Report Surabhi, KL Tripathy, P Bhuyan, A Mishra, BP Das Background: Involvement of CNS is extremely rare in multiple myeloma, observed in approximately 1 % of the patients. Case Report: A 35 year old female presented in Neurosurgery OPD with rapidly progressive scalp swelling over left side of head since 4 months and weakness of right upper and lower limbs for last 1 month. On examination, the swelling measured 10 9 10 9 6 cm and was tense with variegated consistency. X-ray skull showed a bony erosion in left frontoparietal region with soft tissue swelling. CT scan of brain showed a solid SOL in frontal lobe, 8 9 7 cm, with erosion of left frontal bone and involvement of scalp. With differential diagnosis of meningioma with malignant transformation, lymphoma the patient underwent debulking surgery. Result: The histopathological

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Abstract P 146 Study of the Cytokine Profile of Type 1 and Type 2 T-Cells in Children with Acute Immune Thrombocytopenia (ITP) Vanita Bhaskar1, Anita Nangia1, Sunita Sharma1, Jagdish Chandra2, Anju Seth2 Department of 1Pathology and 2Paediatrics, Lady Hardinge Medical College and Associated Kalawati Saran Children’s Hospital, New Delhi Objectives: To study pretreatment and post treatment cytokine profile of Type 1 (Th1 = IFNc, IL-2) and Type 2 (T2 = IL-4, IL-10) T-cells in children with acute ITP after immunomodulatory treatment. Method: 30 newly diagnosed ITP patients below 18 years of age, were subjected to Complete Haemogram with platelet count, Peripheral smear and bone marrow aspirate (BMA). Serum cytokine level for IFNc, IL-2 (Th1 subset) and IL-4, IL-10 (Th2 subset) were performed

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 in all using ELISA. Results: The mean values of Th1 cytokines (IFNy, IL-2) were higher and Th2 cytokines (IL-4, IL-10) were lower in all the patients pre treatment. Decrease in mean levels of Th1 and an increase in mean values of Th2 cytokines post treatment were seen with p value statistically significant. Conclusion: The present study found an increase in Th1 cytokines (IFNc and IL-2) and decrease in Th2 cytokines (IL-4 and IL-10), in all paediatric patients of acute ITP at time of diagnosis emphasising the T cell dysregulation as the early event in pathophysiology of acute ITP. The decrease in Th1 cytokines and increase in Th2 cytokine post treatment with normalisation of Th1/ Th2 ratio is an excellent indicator of response to treatment.

Abstract P 147 T Regulatory Cells in Acute Immune Thrombocytopenic Purpura—Study from a Tertiary Centre Pratibha Dhiman, Arnab Chattopadhyay, Utpal Chaudhuri Institute of Haematology & Transfusion medicine (IHTM), Medical College Kolkata Introduction: Immune thrombocytopenic purpura (ITP) is characterised by enhanced platelet destruction due to platelet autoantibodies. There is an increasing evidence that regulatory T lymphocytes play an important role in pathogenesis. Objective: To determine the frequency of CD4+ C425+ CD127low T regulatory cells by flow cytometry and to investigate the differences in their expression during periods of disease activity and remission in acute ITP. Materials and Methods: A total of 30 patients with acute ITP (12 males and 18 females), aged 5–40 years were enrolled in the study. Diagnosis of ITP was based on presence of thrombocytopenia only with no evidence of red cell and white blood cell abnormalities, normal or increased number of megakaryocytes in bone marrow smears and exclusion of other causes of thrombocytopenia. Age and sex matched 30 control are also enrolled. All patients and controls are subjected to clinical evaluation, complete blood count, direct coombs’ test, antinuclear antibody detection, serology testing and morphological examination of bone marrow samples (in case of patients only). T regulatory cells were identified by flow cytometry after separation of peripheral blood mononuclear cells using cell preparation tubes. T regulatory cells are defined as CD4+ CD25+ CD127low lymphocytes. Results: The frequency of T regulatory cells ranged from 1.4 to 3.4 % in acute ITP patients at the time of diagnosis. Patients who achieved remission, the T regulatory cells were increased ranging from 6.5 to 9 %. Among controls, the levels range from 7.2 to 10.5 %. Conclusion: The study imply one of the possible fundamental role of T regulatory cells in pathogenesis of ITP patients.

Abstract P 148 A-3 Haplotype of EPCR Gene: Is it a Risk Factor for Recurrent Miscarriage in Indian Population Amit Sharma, Ravi Ranjan, Suhail Akhter, Kamal Kishor, Hareram Panday, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi, India Background: Recurrent miscarriage affects 1–5 % pregnancies and in 50 % of these women, the cause of the preceding miscarriages is

239 unknown. It is multi-factorial in origin and the role of inherited thrombophilia and gene polymorphisms of coagulation and anticoagulation factors such as-thrombomodulin and endothelial protein C receptor (EPCR) are involved in the pathogenesis of recurrent miscarriage. The EPCR facilitates PC activation by the thrombin-thrombomodulin complex. In plasma soluble form of EPCR (sEPCR) inhibits both anticoagulant activity of activated protein C (APC) by blocking its binding to phospholipids and protein C (PC) activation by competing for PC with membrane-associated EPCR. sEPCR levels suggested under genetic control. There are 13 known polymorphisms in EPCR gene which are in complete linkage disequilibrium; these defined three haplotypes: A-1, A-2, A-3. Out of them, A-3 was most studied and found strongly associated with high sEPCR levels in deep vein thrombotic patients in different population. However role of A3 haplotype of EPCR is a risk factor for recurrent miscarriage remains controversial. Objective: The aim of this study is to identify the role of EPCR haplotype (A3) in the pathogenesis of recurrent miscarriage. Methods: 100 consecutive patients with recurrent miscarriage and equal number of age and sex matched healthy controls were the study subjects. All study subjects were genotyped for A3 haplotype tagging SNP (A6936G) using PCR–RFLP. Results: Homozygosity of A-3 genotype did not show any significant association (P = 0.21, c2 = 1.55) while the allelic frequency of the tagging SNP (A6936G) showed a trend of association (P = 0.07, c2 = 3.10) with the recurrent miscarriage. Conclusion: A trend of association in the allelic frequency of the tagging SNP (A6936G) reveals that in a large sample size this A-3 Haplotype may be found associated with the recurrent miscarriage in Indian population.

Abstract P 149 Immune Thrombocytopenic Purpura in a Girl with Acute Lymphoblastic Leukemia—an Unusual Association Vikas Dua1, Jai Bhagwan Sharma2 1 Department of Pediatric Hematology Oncology, Action Cancer Hospital, Delhi, India; 2Department of Medical Oncology, Action Cancer Hospital, Delhi, India

Objective: Lymphomas have been associated with Immune thrombocytopenic purpura (ITP) but it is rare to see ITP in patient with acute lymphoblastic leukemia (ALL). Nine cases of ITP in children with ALL have been reported so far, three of them were acute ITP, and rest were chronic ITP. Only one such case has been reported from India. Method: A 15-year-old girl was admitted in our hospital because of fever, increasing pallor and bleeding spots over the body in July 2010. Peripheral blood smear showed 54 % blasts. The bone marrow was consistent with pre-B ALL. Patient was started on chemotherapy using BFM-95 regimen. Bone marrow showed remission on the 33rd day of treatment. After phases of Protocol-I, Protocol-M, and Protocol-II, patient was started on maintenance therapy including 6-mercaptopurine and methotrexate. After 20 months into therapy, the patient was noted to have a platelet count of 38,000/mm3. Results: Therapy was stopped and a suspicion of relapse was kept and bone marrow was performed. An adequate number of megakaryocytes with findings of ALL in remission were detected. The patient was started on prednisone at 2 mg/kg orally daily. After 3 weeks of prednisolone, she had improvement of her platelet count to 78,000/mm3. Her last platelet count in July 2012 was 88,000/mm3.Secondary causes of ITP were ruled out. Conclusion: The presence of thrombocytopenia in patient of ALL does not always means relapse of ALL or as a result of chemotherapy but the possibility of ITP should also be considered.

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Abstract P 150 Comparison of Platelet Counts by Sysmex XE 2100 and LH 750 with the International Flow Reference Method in Thrombocytopenic Patients Tina Dadu, Sehgal Kunal, Shaikh Anjum, Khodaiji Shanaz Department of Hematology, P.D.Hinduja Hospital & Medical Research Centre, Mumbai Background: There are several methods for counting platelets, of which The International Flow Reference method is considered to be the gold standard. We compared the platelet count given by this method to the count given by automated analyzers using other methods. Aims: To compare the platelet counts obtained by XE2100 (Impedance, optical florescence and reported based on switching algorithm) and LH 750 (Impedance) with the international flow reference method in thrombocytopenic blood samples. To calculate the sensitivity, specificity, PPV and NPV of various technologies at the clinically relevant transfusion thresholds of 10,000 and 20,000/ll. Materials and Methods: A total of 118 blood samples with platelet count of\50,000/ll were selected for the study. Platelet counts of all samples were analyzed by all methods using the Sysmex analyzer, LH750 and IRM in parallel within 6 h of collection. Results and Conclusions: Sysmex-R had the least Bias and 95 %LA range and thus correlated best with IRM values. LH-750 had a higher Bias compared to Sysmex-O and Sysmex-R but a strikingly similar 95 %LA ensures similar results in all three methods. In fact, in the oncology subset, it had the narrowest 95 % LA which made it the best performer in this subgroup. Of the three Sysmex results, Sysmex-I had the highest Bias, widest 95 % LA, and highest potential risk of over transfusion. Hence Sysmex-R and LH750 were found to be reliable tools for estimation of platelet count in thrombocytopenic patients.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 composite gold standard of SRA and clinical probability. The incidence of SRA positive HIT was 0.46 % (1/217).

Abstract P 152 Coexistence of Macro Thrombocytopenia and Glansmann Thrombasthenia—a Case Report Shenbagapriya, Ramya, Jennifer, Suresh, Vandana Kamath, Usha Sitaram Sukesh C. Nair Department of Transfusion Medicine & Immunohaematology, Christian Medical College, Vellore, India Abstract: Congenital platelet disorders associated with thrombocytopenia often pose special diagnostic challenges to clinicians and diagnostic laboratories. Glansmann thrombasthenia (GT) is a rare autosomal recessive disorder characterized by mucocutaneous bleeding, normal platelet counts, morphology, and prolonged bleeding time and absent clot retraction. Here we present a case of 10 year old female who presented to us with the history of mucocutaneous bleeds requiring transfusions. Laboratory investigations revealed mild thrombocytopenia with giant platelets (MPV: 15fL), prolonged bleeding time, good clot retraction, platelets failed to aggregate with ADP, Collagen, AA and Epinephrine and aggregation seen with Ristocetin. Flow cytometric analysis of GpII bIIIa also showed findings consistent with GT. An unusual finding of macrothrombocytopenia with Glansmann thrombasthenia was identified.

Abstract P 153 Spectrum of Platelet Function Disorders Presenting with Bleeding Manifestations to a Tertiary Care Centre

Abstract P 151

Navanila Samanta, Prantar Chakrabarti, Siddhartha Sankar Ray, Uttam Kr. Nath, Utpal Chaudhuri

Evaluation of Various Laboratory Assays in Detection of Heparin Induced Thrombocytopenia in an Adult General ICU

Institute of Haematology and Transfusion Medicine (IHTM), Medical College, Kolkata

Farhad N Kapadia1, Amruta S Ketkar2, Anand S Deshpande3, Vipla C Puri3, Shanaz J Khodaiji3

Introduction: Platelet aggregometry serves as an important tool for evaluation of the patient with bleeding manifestations especially when coagulation profile is normal. IHTM has been performing platelet aggregometry as a clinical service for the last 10 years. Here, we are presenting the data of 82 bleeders referred in the last 1 year for platelet aggregometry using common platelet agonists. Aim: (1) Determination of type of platelet dysfunction among patients with bleeding symptoms. (2) To understand the correlation between bleeding time and platelet aggregation. Method: The detailed clinical notes of the patients referred for platelet aggregometry were analyzed. Tests were performed using ADP, Collagen, Arachidonic acid and Ristocetin (standard and low dose) by Chrono log Model-700. Results: Patients were referred because of more skin-related bleeding symptoms rather than wet purpura. Using platelet aggregometry, only 33 % demonstrated characteristic platelet dysfunction. 7 patients had Glanzmann thrombaesthenia. Bleeding time was prolonged ([7 min) in only 15 % patients who had platelet dysfunction on aggregometry. Impairment of aggregation with collagen and arachidonic acid translates into prolonged bleeding time (p \ 0.05). 8.5 % patients showed hyperaggregation though they had bleeding manifestations. Conclusion: Platelet aggregometry is a useful tool for evaluation of a bleeding disorder but may remain inconclusive in 67 % cases if not supplemented by other tests. Bleeding time is not a sensitive test for detecting platelet dysfunction.

1 Department of Intensive Care, P. D. Hinduja National Hospital and MRC; 2Department of Research, P. D. Hinduja National Hospital and MRC; 3Department of Laboratory Medicine, P. D. Hinduja National Hospital and MRC

Objective: Heparin Induced Thrombocytopenia (HIT) is an immune mediated complication of heparin therapy. Our objective was (1) to compare various laboratory assays for HIT against clinical probability (4-T score) and 14C-Serotonin Release Assay (SRA) which was the composite gold standard and (2) to determine the incidence of HIT in the ICU. Methods: The study Group (n = 217) consisted of consecutive ICU patients with heparin exposure followed by thrombocytopenia. The clinical probability (4-T score) was applied to the study group. Enzyme Linked Immunosorbent Assay (ELISA), Particle Gel Immunoassay (PGIA), SRA and Platelet Aggregation Assay (PAA) were performed. Results: The 4-T score showed that 1/217 patients had high probability, 48 had intermediate probability and 168 had low probability for HIT. One patient was positive by SRA, 3 by PGIA and 33 by ELISA. The incidence based on a combination of clinical features and laboratory findings was 1.8 %. Conclusions: A greater number of false positives were observed by ELISA than by PGIA when compared to a

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Others Abstract P 154: Poster Presentation

241 turnaround time for report preparation by 58 % and increased the capacity of the lab by 30 %. Conclusion: A template based reporting system for reporting peripheral smear interpretations is an effective tool in reducing the turnaround time and the error rate of the lab for reporting peripheral smears.

Systemic Mastocytosis—A Rare Entity Ketan Mallya2, Bikash Singhania1, K Annamma2, B Sushma1, R Lakshmi1

Abstract P 156

Kasturba Medical College, Manipal University, Manipal; 2Melaka Manipal Medical College, Manipal

A Case of Hemophagocytic Syndrome with Reactive Plasmacytosis in a 16 years Old Immunocompromised Patient

Introduction: Mastocytosis encompasses a rare and heterogeneous group of clinical disorders characterised by abnormal growth and accumulation of mast cells in various organs, commonly skin and bone marrow. Here we report a case of systemic mastocytosis with bone marrow involvement as the first manifestation. Specific histocytological techniques were needed to establish the diagnosis. Case Report: A 52 year old lady presented with severe lower and upper back pain of 8 months duration. On local examination severe tenderness was elicited over dorsal and lumbar spine. X-ray and MRI for the spine showed lytic lesions at the L1 to L5 vertebrae. Bone scan was suggestive of osteoblastic changes. Gastroscopy demonstrated pangastritis. Metabolic and endocrinological etiologies were excluded because laboratory evaluation was unremarkable. Iliac crest bone marrow biopsy demonstrated infiltration by mast cells, arranged in sheets and whorls. Extensive areas of marrow fibrosis was seen along with bone destruction. Special stains: AFB—Negative. Toluidine blue—positive in mast cells. Immunohistochemistry: CD117—positive in mast cells. Conclusion: The diagnosis of mastocytosis requires a great degree of clinical suspicion as it is a rare disease that may go unrecognized or misdiagnosed because of the non specificity of many of its symptoms, particularly in cases with visceral involvement and absence of characteristic skin lesions of urticaria pigmentosa. Bone involvement in mastocytosis is related to infiltration of bone marrow by mast cells. Bone marrow biopsy and histocytological techniques are the key for diagnosis.

Prakriti Shukla, Syed Riaz Mehdi, Sharique Ahmad, Nishi Tandon

1

Abstract P 155

Era’s Lucknow Medical College and Hospital, Lucknow Abstract: Hereditary and sporadic cases of hemophagocytic syndrome (HPS) are primarily reported in children. We present a case of hemophagocytic syndrome with reactive plasmacytosis in a 16 years young male. The patient presented to us with persistent fever, hepatosplenomegaly, lymphadenopathy, anaemia, leucopoenia and marked thrombocytopenia with elevated triglycerides level. For all the above complaints he was advised bone marrow examination which revealed hypocellular smears with marked erythrophagocytosis by histiocytes and his NK cell activity was reduced which was determined by Flow cytometry. He was diagnosed as a case of hemophagocytic syndrome but before any treatment could have been started, he expired. This experience showed that HPS may be a life-threatening condition. Its rarity combined with non-specific clinical features, such as fever and lymphadenopathy, makes it a diagnosis that can be easily missed. Keywords Hemophagocytosis, Plasmacytosis, Immunocompromised

Abstract P 157: Oral Presentation VCS Parameters in Viral Infections Mohammed Musheb, Chethan Manohar

A Template Based Reporting System for Peripheral Smear Interpretation Shabnam Roohi1, Venkatesha Joshi2, Devendra Prasad2, Padam Kafle3 1

Department of Haematology and Clinical Pathology, Apollo Hospitals Bangalore; 1Apollo Hospitals Bangalore; 3 Akhil systems Pvt.Ltd Objective: The peripheral smear examination and interpretation is an important part of a pathologist’s day to day activity. A template based reporting system was developed to help reduce the turnaround time for preparation of an accurate, comprehensive and concise peripheral smear report. Methods: The knowledge base of previously reported cases, references from text books and the internet were used to develop these templates. These templates were then uploaded onto to the hospital information system using Microsoft visual studio 2005 and visual basic 6.0. A guide for appropriate template to be uploaded based on the haematology analyzer output was developed. The technicians were trained to upload the appropriate template based on the analyzer output. The pathologist reviewed these for accuracy and completeness after slide review. Results: Of 504 uploaded peripheral smear templates screened for accuracy 242 were normal studies, 262 were abnormal, out of the abnormal studies 25 required to be modified and there was no change in the normal study templates. Therefore the error rate was 4.96 %. Implementation of this system reduced the

Kasturba Medical College, Manipal, Udupi-576104 Objectives: To study the utility of WBC research population data (RPD) provided as VCS parameters on the Coulter LH750 Hematology analyzer for the screening of viral blood samples against normal controls. Materials and Methods: The WBC differential system of Coulter LH750 consists of a dedicated flow channel based on the ‘Coulter principle’ & identifies the WBC types according to three physical measurements: (1) Impedance method applied to direct current-Cell volume (V). (2) Radiofrequency method—nuclear size and density-conductivity (C). (3) Light scatter of a laser beam– cytoplasmic granularity (S). Study groups: 1. 106 healthy blood donors (controls). 2. 118 presumed viral infections—cases (with bacterial cultures and serology negative, autoimmune serology negative). Samples were run on the Coulter LH750 on DC mode and their VCS parameters, i.e. mean (M) and standard deviation (SD) of volume (V), conductivity (C) and scatter (S) for the different leukocyte populations (N,L,M,E) were studied. Results: Studying the various VCS parameters between 2 groups, it was found that mean and standard deviation parameters for volume, conductivity and scatter for neutrophils, lymphocytes and monocytes were significantly different between the controls and cases. LSdV, NSdS in group 2 showed significant difference and ROC curve analysis showed best AUCs for mean monocyte volume-MMC (sensitivity 80% -specificity 57% increasing to 74% for a sensitivity of 72% at a value of 166.3). The scatter parameters proved to be non predictive. Conclusion: In

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242 conclusion, the leukocyte VCS parameters is moderately predictive in the detection of viral infections and further studies with serologically proved viral infections would be useful.

Abstract P 158 Anti M Antibodies Complicating Transfusion Therapy: A Report of Two Cases Meenakshi Rao, Sangeeta Pahuja, P Lalita Jyotsna, Manjula Jain Department of Pathology & Blood Bank, Lady Hardinge Medical College, Delhi Introduction: Anti M is a naturally occurring antibody usually active at temperatures below 37°C and thus is of no clinical significance. However, rarely the antibody agglutinates red cells at 37°C or at the antiglobulin phase of testing and can lead to haemolytic transfusion reactions and haemolytic disease of newborn. Case 1: A 3 year/F with no history of blood transfusion (BT) presented with severe infection and anemia. Forward grouping showed AB positive; reverse grouping was inconclusive. Blood grouping at different temperatures showed panagglutination at 22°C which enhanced at 4°C and resolved at 37°C. DAT was negative. Antibody identification using 11 cell panel suggested presence of anti-M antibodies. DTT treatment of serum suggested presence of IgG + IgM type of anti-M antibodies. Case 2: A 7 year/M with no history of BT presented with severe anemia with impending CHF. Blood group (BG) was A positive. Antibody screen was positive. Reverse BG at different temperatures showed agglutination with all 3 cells at 22°C which enhanced at 4°C. Only B cells showed reaction at 37°C. DAT was negative. Antibody identification with 11 cell panel suggested presence of anti-M antibodies. DTT treatment of serum suggested presence of IgM antibodies. Both children were successfully transfused with M-negative compatible blood units according to their antigen profile. Conclusion: Although considered clinically insignificant, anti-M antibodies have been implicated in HDN and delayed haemolytic transfusion reactions. They can assume significance in cases of clinically induced hypothermia. Also, these are the cause of incompatible cross-match results causing delay in blood transfusion. Hence, it is extremely important to carefully interpret the results of blood grouping and antibody screening.

Abstract P 159 Acute Megaloblastic Anemia Presenting as a Severe Fulminating Pyrexial Illness. A Case Series Vineet Behera1, Velu Nair2, Rajan Kapoor2, C Agrawal1 Department of Medicine, AFMC Pune; 2Medicine & Hematology, AFMC, Pune

1

Background: Megaloblastic anemia is a common illness known to have diverse and protean manifestations. But megaloblastic anemia presenting as a severe fulminating pyrexial illness is rarely reported. Objective: To study the clinicopathological profile of megaloblastic anemia presenting as an acute pyrexial illness. Materials and Methods: This is a retrospective study in which the clinical, laboratory and therapeutic profile of patients of megaloblastic anemia presenting as a acute febrile illness was studied. Results: A total of 14 cases were included. The average age of patients was 31.8 years with 09 (64.28 %) vegetarians. All patients presented with an acute febrile illness (mean duration 9.1 days) with 71 % having high grade fever with chills or rigors. All had features of symptomatic anemia. Jaundice was seen in 12 patients (85.71 %), splenomegaly in 10 (71.42 %), hepatomegaly in 8 (57.14 %) and features of peripheral neuropathy in 4 (28.47 %) patients. Investigations revealed mean hemoglobin of 4.1 g/dl (range 2.6–7.4), leucopenia and thrombocytopenia in all, mean ANC

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 1100/mm3 (ANC \ 500 in 4 patients) with mean MCV being 113 (maximum 132) with macrocytes and hypersegmented neutrophils seen in all. The mean serum bilirubin was 3.88 mg/dl (unconjugated) with normal enzymes, mean LDH 1733.42. Serum B12 levels were low (\140 pg/ml) (mean—112) in all patients with folic acid levels low in 10 patients (71 %). Bone marrow examination showed cellular reactive bone marrow with megaloblastoid changes in all. All patients responded dramatically to therapy with parenteral vitamin B12, folic acid and other supportive therapy with gradual recovery and normalization of investigations. Conclusion: Megaloblastic anemia should be considered as a differential diagnosis of acute pyrexial illness in all patients having a similar presentation to enable early diagnosis and treatment of this easily treatable disorder.

Abstract P 160 High Incidence of Zidovudine Induced Anaemia in HIV Infected Patients in Southern Odisha Kaibalya Ranjan Dash, Lalit Kumar Meher, PK Hui, SP Mohanty, SN Nayak ART Center, Department of General Medicine, MKCG Medical College & Hospital, Berhampur, Odisha Aims and Objectives: This study was conducted with an aim to determine prevalence of ZDV induced anaemia in HIV infected patients initiated on ZDV containing ART regimen and also to find out correlation with any factor for causing ZDV induced anaemia. Materials and Methods: This is a retrospective study carried out in ART center, MKCG MCH, Berhampur between 2009 Jan to 2011 Dec. HIV infected patients registered at ART center were treated according to NACO guidelines. Patients (n = 1221) with Hb [ 8 g/dl were prescribed ZDV based ART regimen. Patients having anaemia (\8 g/dl) were excluded from the study. Correlation of baseline characteristics (age, sex, weight, Hb lebel, CD4 count, WHO clinical stage) with risk of developing anaemia was also calculated. Results: 178 (14.6 %) patients on ZDV regimen developed anaemia and 6.8 % (n = 83) of these developed severe Anaemia (\6.5 g%). Patients with low CD4 count were more prone to develop Anaemia. Age, sex, weight, WHO clinical stage had no relation to development of Anaemia. Interpretation and Conclusion: Incidence of ZDV induced Anaemia is very High and patients having low CD4 count were more susceptible to develop anaemia.

Abstract P 161: Poster Presentation Validation of Automated ESR Machines with Modified Westergren Method Asha Patil1, M Deepak Nayak2, Sushma V. Belurkar3, Seemitr Verma3 Manipal College of Allied Health Sciences, Manipal; 2Melaka Manipal Medical College, Manipal; 3Kasturba Medical College, Manipal Aims and objectives: The study was conducted with the primary objectives of comparing the following methods of determining ESR. The new Ves MATIC cube 80TM instrument with modified Westergren method. Ves MATIC cube 80TM with the Ves matic 20TM instrument, which is currently used in our laboratory. Ves matic 20TM with modified Westergren method. Materials and Methods: The Ves MATIC cube 80TM and Ves matic 20TM ESR analyzer were compared with modified Westergren method. A paired, simultaneously collected venous blood sample was obtained from 200 random patients who had a request for both CBC and ESR; that arrived at the sample collection center, Kasturba Hospital, Manipal. All the samples were evaluated

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 using the three methods in our clinical laboratory as per the methodology for each of the individual methods. Statistical Analysis: Bland and Altman statistical analysis was applied for evaluating the automated ESR analyser against the modified Westergren method. Results: The analysis revealed a low degree of agreement between manual and automated method especially for higher ESR values ([60 mm/h); whereas a reasonable concordance was observed between values of 20–60 mm/h. When ESR values were \20 mm/h a good concordance was seen with all the three methods. We went a step further comparing Ves MATIC cube 80TM with Ves matic 20TM and found that the discrepancy existed for higher values of ESR. Conclusion: The fully automated Ves MATIC cube 80TM and Ves matic 20TM for ESR measurement tend to underestimate the manual ESR reading. Hence it is recommended that a correction factor should be applied for a range of ESR values while using these machines. Further studies & validation are needed.

Abstract P 162 Primary Diagnosis of an Unsuspected non-Haematologic Malignancy by Bone Marrow Biopsy Examination Krishnendu Halder1, Palash Kumar Mandal1, Swapan Kumar Sinha1, Debdas Bose1, Debasis Banerjee2

243 to be mucopolysaccharides rich in hyaluronic acid. Objective: To study the spectrum of causes of gelatinous transformation in bone marrow. Materials and Methods: Bone marrow aspirates were examined for a period of 1 year (1.09.2010 to 31.08.2012). Gelatinous transformation was confirmed by Alcian blue stain (pH 2.5).This was correlated with the clinico-hematological profile of the patient. Bone marrow cellularity and ratio of hematopoietic cells: non hematopoietic cells (plasma cells, mast cells and stromal cells), increased vascularity (capillary cores) was assessed. Results: A total of 732 samples of bone marrow aspirate were received 1 year period of which 35 showed gelatinous bone marrow transformation (GBMT). Incidence was nearly 4.8 % Male:Female = 2:1 (23:12). Fourteen patients (40 %) were in pediatric age group (\15 years), 13 (37 %) were adolescents and young adults (15 to 40 years) 4 each (23 %) were in 5th and 6th decades respectively. The bone marrow was hypocellular in 23 (66 %), normocellular in 9 (25 %) and hypercellular in 3 (9 %) cases. Conclusion: Capillary fronds in the bone marrow aspirates, irrespective of an increased number of lymphocytes, plasma cells, mast cells and stromal cells was associated with specific infections like TB, Leptospirosis and non-specific infectious conditions. 5 study cases (2 children) progressed to aplastic anaemia. The presence of gelatinous transformation in bone marrow along with relative increase in lymphoid, plasma, mast and stromal cells and the paucity of capillary fronds could be a sign of impending aplastic anaemia, moresoever in children.

1

Department of Pathology Medical College; 2Department of Pathology, Vivenkanda Institute of Medical Sciences, Kolkata Objective: To diagnose the primary site of metastatic non-haematologic malignancies by bone marrow trephine biopsy. Methods: A prospective observational study was done at a tertiary care hospital of eastern India from august 2009 to july 2012. Patients advised bone marrow biopsy examination for causes like pancytopenia, anemia, leucoerythroblastic blood picture on peripheral blood smears were reviewed during this period. Patients with metastatic non-haematologic malignancies were selected out of which only unsuspected malignancy with metastasis to bone marrow formed the study group. Results: Sixteen cases of metastatic malignancy with unknown primary site were noted. The age range of the patients was 10–65 years. The most common histologic subtype was adenocarcinoma and the commonest site identified later was colon. Four cases remained undiagnosed till the end. Conclusion: Bone marrow biopsy was fruitful for initial diagnosis of a fair number of patients with occult malignancy. A detailed history with ancillary investigations like endoscopy, advanced imaging and PET scan could diagnose the primary site in most of the cases. However prognosis remained poor due to disseminated malignancy.

Abstract P 163 Gelatinous Transformation of Bone Marrow: A Prospective Tertiary Center Study, Indicating Varying Trends in Epidemiology and Pathogenesis S Singh1, R Sen1, M Gupta1, G Singh1, S Chabbra1, R Verma1, Abhinav2 1 Department of Pathology, Pt.Bhagwat Dayal Sharma P.G.I.M.S, Rohtak; 2Department of Hospital Administration, DCRUST, Murthal, Sonipat

Introduction: Gelatinous transformation of bone marrow characterized by fat cell atrophy, focal loss of hematopoietic cells and deposition extracellular gelatinous material histochemically proven

Abstract P 164 Follicular Dendritic Cell Tumour in a Patient of Chronic Myeloid Leukemia: A Rare Association Ragini Singh1, Rajeev Sen1, Nisha Marwah1, PS Gahlaut2, Soumik Chaudhuri3, Nisha Sharma1, Jatin Ahuja4, Kanika1 Department of Pathology, PGIMS, Rohtak; 2Department of Medicine & Haematology, PGIMS, Rohtak; 3Department of Haematology, PGIMS, Rohtak; 4Department of Medicine, PGIMS, Rohtak 1

Abstract: Follicular dendritic cells, also known as dendritic reticulum cells, are important components of the immune system and are essential for functions of antigen presentation. Malignancies arising from these cells are rare and the first such primary neoplasm arising from follicular dendritic cells was reported only in 1986. The commonest presence of these follicular dendritic cell sarcomas are in the lymph nodes, especially of the cervical, axillary and mediastinal region but extra-nodal sites such as head and neck and gastro-intestinal tract may be present in up to a third of patients. The etiology remains an enigma with a possible link to the Epstein-Barr virus. Immunohistochemical studies assume great importance in differentiating this from its close congeners. We describe a case of Follicular Dendritic Cell Sarcoma, arising in a patient of Chronic Myeloid Leukemia (CML) on treatment with Imatinib mesylate for the past 6 years. The Histopathological features of diffuse effacement of the lymph nodes with spindle cells which were positive for CD 23, CD 10, LCA and vimentin, showed the closest resemblance to follicular dendritic cell sarcoma. This case deserves reporting due to the rarity of the disease and hitherto unreported association with CML. Furthermore, the pathological diagnosis is challenging and requires a close-knit effort between the pathologist and the haematologist.

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Abstract P 165 Myelonecrosis—a Clinicopathological Study from a Tertiary Care Centre in South India

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 was optically clear. Oil red O staining shows reddish granules confirming Jordan anomaly. This finally was a case of Dorfman Chanarin syndrome. Conclusion: Detailed study of neutrophils in an optimally stained peripheral smear is a simple, though valuable diagnostic procedure.

J Sree Rekha, Debdatta Basu, Rakhee Kar Department of Pathology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry Introduction: Myelonecrosis is a relatively uncommon clinicopathologic entity. The etiology of myelonecrosis is diverse, and malignancy, especially hematopoietic in origin, is the most common underlying disease. Objective: To describe the causes, diagnostic features and clinical significance of myelonecrosis. Methods: 21/5679 bone marrow biopsies showing myelonecrosis were identified from a retrospective review of trephine biopsies done between January 2000 to July 2012. Clinical findings, peripheral smear and bone marrow findings of these were reviewed. Results: Among 21 patients, 16 (76.2 %) were males and five (23.8 %) were female. Fever was the most common presenting complaint seen in 12 cases (57.1 %). Pallor was the most common clinical sign seen in 8 cases (38.1 %). Five patients were retropositive. Peripheral smear examination showed pancytopenia in six cases (28.5 %), leucoerythroblastic blood picture in six (28.5 %) and leukaemia in four (19.0 %). Sickle cells were seen in peripheral smear in one case. Myelonecrosis was identified on bone marrow aspiration smears in five cases. Among 21 cases 12 cases (57.1 %) were due to a neoplastic cause, four due to tuberculous infection (three in retopositive patients), one due to sickle cell disease. In three cases the cause could not be identified. Among the neoplastic causes, acute leukemia was seen in 6 cases (4—ALL, 2— AML), non-hodgkin lymphoma in two, metastatic adenocarcinoma in two. Hodgkins disease and malignant melanoma accounted for one case each. Conclusion: Though conditions associated with myelonecrosis are varied, malignancy is the commonest cause. Haematological findings are often present and may give clue to the underlying disorder. Keywords Myelonecrosis, Bone marrow malignancy

Abstract P 166: Poster Presentation Neutrophilic Cytoplasmic Inclusions and Their Utility in Diagnosis of Diseases Y Sharmila, Sajini Elizabeth Jacob, Debdatta Basu Department of Pathology, JIPMER Introduction: Qualitative changes in white blood cells have diagnostic significance in certain situations. These include changes like cytoplasmic vacuolation, abnormal granulation, abnormalities in nuclear segmentation etc. A detailed study of a peripheral smear, stained by a standardized Romanowsky method can provide diagnostic clues which when correlated with the clinical findings can clinch the diagnosis. Report: We report a series of eleven cases where peripheral smear examination of neutrophils helped us to diagnose varied clinical entities. There were five cases where the leucocytes showed abnormal giant cytoplasmic granules. Correlating with the clinical findings, a diagnosis of Chediak Higashi syndrome was made. Examination of the peripheral smear in four cases of neonatal sepsis showed neutrophils with golden yellow crystals in the cytoplasm which are indicative of bilirubin crystals. One patient with history of fever showed brownish pigmentation in neutrophils. Detailed search of the smear picked up an occasional gametocyte of Plasmodium falciparun. The last case was of a 2 year old male child who presented with hepatomegaly and icthyosis. Peripheral smear shows vacuolation in the neutrophils which on Sudan black staining

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Abstract P 167: Oral Presentation Tumors of non Haematopoietic Malignancies of Unknown Origin in the Bone Marrow: A 5 Year Study from a Tertiary Care Centre in South India Sreeya Das, Pritinanda Mishra, Rakhee Kar, Debdatta Basu, Sajini E Jacob Department of Pathology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry Introduction: Tumors of unknown origin represent a common presentation of malignancy and account for 3 to 10 % of cancers. They are infrequently reported and account for up to 12 % of all cancers detected by bone marrow biopsy. The study was undertaken to assess the involvement of bone marrow with tumors of unknown origin and its significance in establishing primary diagnosis. Materials and Methods: This was a descriptive study which included record review of the departmental archives for the last 5 years (January 2007 to December 2011).A bone marrow tumor of unknown origin was defined as a metastatic cancer in the bone marrow in which a primary site was not apparent at the time of clinical evaluation. A total 2,426 of bone marrow examination was done in this period of time, of which 15 cases were non haematological malignancy of unknown malignancy. Results: Bone marrow metastases of solid tumors were identified in 15 cases (0.6 %). Of the 15 cases in 13 cases a definitive primary site could be identified by correlating clinicoradiological findings with morphology and immunohistochemistry. In the pediatric population there was one case each of neuroblastoma, rhabdomyosarcoma and metastatic small round cell tumor. In the adult population adenocarcinoma of gastrointestinal tract (41.7 %) was the commonest. Conclusion: Bone marrow metastasis can masquerade a primary haematopoietic disorder; however its detection has both therapeutic and prognostic significance. Immunohistochemistry is an useful adjunct to morphology in reaching a definitive diagnosis.

Abstract P 168 Haematological Profile in HIV Patients Before and After Haart PS Ghalaut, P Mehta, R Singh, S Dash, V Singh Department of Clinical Haematology, Pathology & Biochemistry, Pt. B.D. Shrama PGIMS, Rohtak Introduction: A number of haematological abnormalities are reported in HIV patients such as anemia, neutropenia, thrombocytopenia. Some of these are reversible with Highly active antiretroviral therapy (HAART).The present study was undertaken to see the effect of HAART in patients of HIV admitted at PGIMS, ROHTAK. Aims and Objectives: The present study was to evaluate the haematological profile in HIV patients before and after HAART. Methods: A Total newly diagnosed (by ELISA) 100 HIV positive patients, in age group 16-64 were taken for the study. 50 patients were put on HAART and other 50 patients were not on HAART. Various haematological parameters like Hb, TLC, DLC, APC, BT, CT, PTI, APTT, serum ferritin, B12 level and serum folate level, CD4 levels and bone marrow was done base line and after 3, 6 months post HAART level. Statistical analysis was done by Chi square test. Observation: The

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 most frequent haematological abnormality was found to be normocytic normochromic anaemia. (80 %) with mostly normo cellular bone marrow. The observed value of mean Hb on HAART in study group showed rise from 10.16 ± 2.30 g/dl to 10.96 ± 1.47 g/dl after 6 months which was statistically significant (p \ 0.01).in non HAART group it show a fall in mean Hb from 9.84 ± 3.03, and to 9.42 ± 2.28 after 6 month follow up. The WBC count increased in study group from 5110.64 ± 4401 to 6220 = -140 which was significant (p \ 0.05), as compared to non significant rise from 5433 ± 4408 to 5888 ± 1547 in control group. The mean platelet count in study group shows raised from 2.25 ± 0.737 to 2.52 ± 0.433 which was significant than the controlled group with non significant rise from 2.19 ± 84 to 2.35 ± 6111. There were no significant findings in other parameters. Conclusion: The study demonstrates that normocytic normochromic anaemia was most common haematological abnormality in HIV patient’s. There was significant improvement in haemoglobin level, white blood cell level, platelet level, absolute neutrophil count, and bone marrow picture in the patients who were on HAART and rise was statistically significant.

245 diagnosed in the Haematology section of Pathology Department, GMCH-32B, Chandigarh over a seven year period (2004–2011). The detailed clinical data, MGG stained slides of bone marrow aspirate and H&E stained slides of trephine biopsies done in all the cases were retrieved from archival material. Results: 15 cases diagnosed with visceral leishmaniasis were in the age group of 5–50 years with M:Fratio of 1.1:1. Sign and symptoms included fever (100 %), splenomegaly (93.3 %), hepatomegaly (86.7 %) and lymphadenopathy (6.7 %). Pancytopenia was noted in 73.3 % of cases while the remaining cases had bicytopenia. Bone marrow hypercellularity was noticed in 53.3 % cases. Main bone marrow aspirate findings were increase in plasma cells (66.7 %), increase in histiocytes (66.7 %), increase in eosinophils with precursors (40 %), hemophagocytosis (33.3 %), increased bone marrow iron [ 2 + (40 %) and PelgerHuet cells (13.3 %) of cases. Additional features noted in trephine biopsy were focal benign lymphoid aggregate (6.7 %), gelatinous transformation (6.7 %), marrow fibrosis (13.3 %) of cases.. The average parasite density in smear was 3+ and range of positivity was 1+ to 5+. Conclusions: A note of such aforementioned observations together or individually should warn a pathologist to do more vigorous search for L.D. bodies.

Abstract P 169: Paper Presentation Clinico-Haematological Analysis of Haematological Malignancy, a Hospital Based Study Rahul Varshney, Muktanjali Deka, Jina Bhattacharya, PK Gogoi Department of Haematology, GMCH Objective: Early diagnosis of haematological malignancy is important in clinical practice to prevent mortality. This study was conducted to evaluate their relative frequencies in different ages and sexes in this region. Methods: The present study was conducted in the Department of Pathology and Haematology, Gauhati Medical College & Hospital, Guwahati from 1st July 2011 to 30th June 2012. Patients were examined and blood tests including haemoglobin estimation, total and differential leukocyte count, platelets count, reticulocyte count and PBS study, Bone marrow aspiration and biopsy, special stains, flow cytometry and cytogenetics was done in each cases. Results: Out of 450 patient attending Haematology OPD 236 were having malignancy. About 29.66 % patient had chronic myeloid leukaemia while 1.69 % patient had chronic lymphocytic leukemic. Among acute leukaemia, acute myeloid leukaemia outnumbered acute lymphoblastic leukaemia, (21.19 %against 7.2 %). Multiple myeloma was seen in 20.33 %. Non Hodgkin’s lymphoma was seen in 10.59 % while Hodgkin’s disease 6.6 %, MDS 1.69 %, MPN and LCH 1 %. Male to female ratio in haematological malignancies was 2.87:1. Maximum cases were observed in 51-60 years. Low grade fever, progressive pallor, weakness and body aches were the commonest symptoms (70 % cases) while pallor was the frequently observed sign. Conclusion: The prevalence of haematological malignancy is 52.44 %.This study may help in finding out their relative distribution in this region.

Abstract P 170 To Study Spectrum of Morphological Findings on Bone Marrow Examination in Cases of Visceral Leishmaniasis Anshu Palta1, Sanjeev Garg1, Anita Tahlan1, Atul Sachdev2 1 Department of Pathology, 2Department of Medicine, Govt. Medical College and Hospital, Sector 32, Chandigarh

Objective: To evaluate various morphological findings on bone marrow aspirate and trephine biopsies in cases of visceral leishmaniasis. Method: This is a retrospective analysis of 15 cases of visceral leishmaniasis

Abstract P 171: Oral Presentation Endothelial Dysfunction in Young Patients of Coronary Artery Disease in a Tertiary Care Setup Shipra Garg1, Satendra Sharma1, Meera Sikka1, Rajnish Avasthi2, Neelam Wadhwa1, Gopesh Mehrotra3 1 Department of Pathology, University College of Medical Sciences & Guru TegBahadur Hospital, Delhi; 2Department of Medicine, University College of Medical Sciences & Guru TegBahadur Hospital, Delhi; 3Department of Radiology & Imaging, University College of Medical Sciences & Guru TegBahadur Hospital, Delhi

Aims and Objectives: A case-control study was planned to determine the association between occurrence of CAD in young patients with endothelial nitric oxide synthase polymorphism (eNOS) Glu298Asp alone and in combination with TNF-a activity and Flow mediated dilatation (FMD) of brachial artery. Methods: The study enrolled 30 diagnosed young male patients of CAD below 40 years as cases and 30 healthy age matched male individuals without history of CAD as the control group. Polymerase chain reaction- restriction fragment length polymorphism (PCR–RFLP) analysis for detection of the eNOSGlu298 ? Asp (G894T) variant was carried out along with TNF-a activity by ELISA technique in cases and controls. Endothelial dysfunction was also assessed by non-invasive brachial artery FMD through ultrasonography. Results: The distribution of GG, GT genotypes in young CAD was observed to be 73, 27 % and 80, 20 % in cases and control subjects respectively with no mutant TT found in both study groups. There was lack of significant association between T allele inheritance (GT + TT vs GG) in young CAD patients (p = 0.76) with similar ‘‘T’’ allele frequency in cases and controls. In premature CAD patients increased TNF-a activity was found to be statistically significant (p = 0.008) with strong association in young patients with eNOS Glu298Asp polymorphism GT + TT (p = 0.004). Two-third of cases with reduction in FMD brachial artery showed significant association with eNOS Glu298Asp GT + TT genotype than GG (p = 0.02). Conclusion: Although no significant association of eNOS Glu298Asp polymorphism was found in young CAD, the observation of GT genotype in almost one fourth cases of CAD may indicate a need to demonstrate this polymorphism as a useful genetic marker with it’s possible role to augment the risk of premature atherosclerosis and subsequent events in younger patients with few conventional risk factors. The association of elevated TNF-a activity and decreased FMD brachial artery in young

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246 CAD with eNOS Glu298Asp polymorphism may indicate the probable role of endothelial dysfunction and inflammation in pathophysiology of atherosclerosis in young patients.

Abstract P 172 Role of Radiation in the management of Mycosis Fungoides DK Parida Department of Radiation Oncology, North Eastern Indira Gandhi Regional Institute of Health & Medical Sciences (NEIGRIHMS), Shillong Abstract: Mycosis Fungoides is a low grade, chronic lymphoproliferative disorder of the skin caused by abnormal proliferation of CD4+ T cells. The clinical manifestation ranges from Premycotic, Macule, Plaques, Tumos and Erythrodermic phase/stage. The leukemic variant is known as Sezary Syndrome. In order to bring all the lymphoproliferative disorders of skin the term ‘‘Cutaneous T-Cell Lymphoma’’ was introduced by Eldelson. The overall incidence of mycosis fungoides is about 4 per 100,000 population. Because of unavailability of diagnostic facilities many times there is underreporting of the disease. Majority of the patients present between 40 and 60 years of age. From among the spectrum of treatment modalities, radiotherapy treatment produces the best therapeutic response in terms of cure and palliation. The important prognostic factors were (1) type of the lesions, (2) extent of cutaneous involvement, (3) Involvement of lymph node at the time of presentation and (4) Presence of lymphoma cells in peripheral circulation. We treated 14 patients of mycosis fungoides patients between the age of 27–82 years between 1985 and 1998. Total Skin Electron Irradiation Treatment was given to the patients by Linear Accelerator. The dose range was between 8 and 36 Gy. It was also observed that the end response was directly proportional to the total dose of radiation treatment delivered to the patient. Subsequently the treatment techniques were innovated and modified. The further radiation treatment was delivered by high dose rate mode. This innovation brought down the treatment time from 2 h to 15 min, hence became very much patient as well as machine compliant. By this method we could deliver total dose of 36 Gy to all the patients. Seven patients were treated between 1998 and 2000. The major radiation related toxicities were skin blisters, swelling of joints, desquamation of skin etc. These adverse effects were also dose dependant. Because of these problems the treatment has to be interrupted and the total duration of treatment was stretching beyond 10-12 weeks of time. The ultimate response of radiation treatment depends upon the (1) total radiation dose and (2) total treatment duration. If the treatment duration is unnecessarily prolonged, the effect of radiation on the disease becomes poor resulting in either residual or quick recurrence of the disease. We modified the treatment schedule from daily to alternate day radiation treatment schedule beyond third week, which produced the desired result. Four patients were treated by this method between 2000 and 2004. Skin reactions were visibly less for which no treatment interruptions were warranted. The total dose of 36 Gy could be delivered within a stipulated time period which produced best of disease control. Out of 25 patients, 20 patients remained disease free at the end of 5 years, 5 patients had recurrent disease. Two patients had progressive disease and 3 patients died.

Abstract P 173: Oral Presentation Eosinophilia: An Approach to Management Maya Gopalakrishnan, Karyampudi Arun, Kiran Kumar Matta, KK Nisar, TK Dutta Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 Background: Peripheral blood eosinophilia is a common and interesting presentation of various hematological and non-hematological disorders. Despite extensive evaluation, up to 50 % of patients with eosinophilia remain undiagnosed. Objective: To evaluate patients presenting with peripheral blood eosinophilia using a diagnostic algorithm. Methods: We report a prospective case series of patients presenting with peripheral blood eosinophilia. A structured diagnostic algorithm was used, focusing on empiric treatment of filarial and helminthic infections even after negative test results owing to their high prevalence in south India. Results: Five patients presenting with different clinical features and absolute eosinophil count ranging from 2,530 to 32,000 per mm3 were diagnosed using the algorithm. A 47 year old businessman presenting with low backache was diagnosed to have indolent systemic mastocytosis. Another 54 year old shopkeeper with bleeding gums was diagnosed as having myeloproliferative disorder. An asymptomatic agricultural labourer aged 50 years who was referred for eosinophilia improved with empiric antihelminthic therapy. A 48 year old labourer with wheeze for 2 months was found to have tropical pulmonary eosinophilia. Whereas another 55 year old driver presenting with dry cough and reticulonodular shadowing on X-ray was ultimately diagnosed to have allergic bronchopulmonary aspergillosis even after positive filarial serology. Conclusion: A finding of eosinophilia is related to a wide range of disorders. Therefore, a structured diagnostic algorithm suited to local prevalence of diseases may be useful in effective diagnosis and treatment of patients with eosinophilia.

Abstract P 174 Anemia in Zidovudine Treated Patients in HIV/AIDS Parna Bhaumik1, Swapan Kumar Sinha1, Pramit Ghosh1, Indranil Dhar2, Bibhuti Saha2, Mimi Gangopadhyay3, Jyotirmoy Pal3 1

Medical College, Kolkata; 2School of Tropical Medicine, Kolkata; North Bengal Medical College, SusrutaNagar, Darjeeling

3

Introduction: Anemia is common in HIV/AIDS patients primarily due to HIV infection as well as due to secondary infections (CMV, Parvo virus B19). Hemolytic anemia, anemia due to GI bleeding, Vitamin B12 deficiency due to malabsorption, deranged Iron metabolism anemia of chronic disease etc. are different types of anaemia encountered in HIV/AIDS patients. HAART particularly Zidovine (AZT) induced secondary anemia is commonly associated with marrow toxicity. Inhibition of thymidine kinase and DNA chain translocation may lead to anemia. However other lineages of cells like neutrophils and platelets are affected. Aims and Objectives: The aim of the study was to find significant relation between incidence of anemia and AZT therapy in Aids patients. Materials and Method: 930 patients of HIV/AIDS were registered in Antiretroviral Centre, School of Tropical Medicine, Kolkata from July 2011 to June 2012 and 780 patients were registered in North Bengal Medical college ART Centre. Out of these 1083 patients were initiated with AZT and 325 patients developed anemia (Hb \ 8.00 g/dl). EDTA blood samples were collected and CBC was performed. Conclusion: The present study with limited number of patients show anaemia is commonly associated with AZT HAART. Further study on pathophysiology of anemia including types of anaemia e.g. Immunohaemolytic, Microangiopathic, Nutritional, due to Marrow Suppression/dyserythropoietic anemia may be necessary.

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Abstract P 175 Bacterial Profile in Haematological Malignancies Anjali Akhuj1, Lalit S. Raut2, Swagnik Roy1, Kalpana Karak1, Subrata Bhattacharya1, Prantar Chakrabarti2, Utpal Chaudhuri2, Barun Saha Dalal1 1

Department of Microbiology, KPC Medical College & Hospital, Jadavpur, Kolkata; 2Institute of Haematology and Transfusion Medicine, Medical College & Hospital, Kolkata Introduction: The choice of empiric antimicrobial therapy in febrile patients with haematological malignancies is based on susceptibility pattern of locally prevalent pathogens. Aim: To review the bacterial etiology and antimicrobial susceptibility pattern of blood culture isolates in these patients. Method: In this one year retrospective study, blood samples were processed by Rapid culture automated Bac T/Alert 3D systems (Biomerieux). Results: Out of 458 blood samples, 18.34 % were positive; gram negative organisms constituted 57.14 % and gram positive organisms 42.87 %. Gram-positive isolates were coagulase negative Staphylococcus (CoNS) 77.77 %, Staphylococcus aureus 13.88 % and Enterococci 8.33 %. There were 20 % Methicillin Resistant Staphylococcus aureus (MRSA). Methicillin sensitive CoNS were 64.28 %. All gram positive isolates were sensitive to glycopeptides and linezolid. Most common gram negative isolates were Pseudomonas aeruginosa 39.58 % followed by Escherichia coli 22.99 %, Klebsiella pneumoniae 16.66 %, Nonfermenting gram negative bacilli 8.33 % and Acinetobacter sp. 4.16 %. Ralstonia paucula, Providencea sp. and Cedeceae neteri were isolated in 2, 1 and 1 cultures, respectively. In Enterobacteriaceae, 71.42 % were ESBL producers. Amongst the gram negative isolates Fluoroquinolone resistance was noticed in 66.67 %. All gram negative isolates were sensitive to Polymyxin and Colistin. Multidrug resistant Pseudomonas aeruginosa sensitive only to Polymyxin and Colistin were 15.78 %. Conclusion: Due to emergence of multidrug resistant pathogens, monitoring antimicrobial resistance pattern locally is crucial in making decisions of empirical therapy.

247 Conclusion: No statistical significant ‘p’ value was obtained from the study. So there was no significant correlation between the cord blood hemoglobin and iron profile with the APGAR score of the newborns.

Abstract P 177 Plasmapheresis in a Case of Severe Acute Hemolysis Meenu Bajpai, Suman Lata1, Raju Singh Department of Transfusion Medicine, 1Department of Nephrology, Institute of Liver and Biliary Sciences, New Delhi Introduction: Acute hemolysis is a difficult situation to treat and usually leads to acute renal failure due to acute tubular necrosis. The patient’s vascular system is loaded with free haemoglobin and toxic effects of the same are seen. Here we present a case of acute hemolysis of unknown origin treated successfully with plasmapheresis. Case: A 22 year old male presented at the emergency unit with fever, jaundice for 2 days associated with progressive shortness of breath and altered mutation for one day. The patient was labelled as Acute severe hemolysis with jaundice and put on symptomatic therapy while awaiting investigations. The patient was unresponsive to symptomatic therapy and plasmapheresis was suggested as a therapeutic modality in consultation with nephrology and transfusion medicine. Two sessions of plasmapheresis were done on alternate days starting from day 2 of admission in which one volume of plasma was exchanged using fresh frozen plasma as replacement. Post plasmapheresis there was improvement in the patient’s parameters. Investigation for all possible causes of hemolysis was done. The patient was negative for all viral hepatitis markers, leptospira, HIV, mycoplasma. Blood and urine culture were negative as were Indirect and direct Coombs test. The patient gave no history of intake of any poisonous substance or animal bite. The cause for the acute hemolysis remained an enigma. The patient suffered from acute tubular necrosis of the kidneys from which he recovered and was discharged. Conclusion: Therapeutic plasmapheresis was a life saving intervention in the above case and should be considered when routine therapies fail to bring about the desired outcome.

Abstract P 176 A Comparative Study of Cord Blood Hemoglobin, Iron, Ferritin and TIBC Level in Babies of Primigravida and Multigravida with Relation to APGAR Score in a Tertiary Care Hospital Madhumita Mondal1, Ranapratap Ojha1, Swapan Kumar Sinha1, Utpal Chowdhury2 1

Department of Pathology, Medical College Kolkata; 1I.H.T.M., Medical College Kolkata Objectives: (1) To compare the values of hemoglobin and iron profile of newborns with APGAR score. (2)To establish relation between iron profile and level of hemoglobin of newborn. Methods: We conducted a prospective observational study over a period of 1 year. The study population consisted of 48 pregnant women and their single live-birth baby. Among the mothers 19 were primigravida and 29 were multigravida. All the babies were delivered normally. Cord blood was collected from all the cases in both EDTA and clotted vials. EDTA blood was used for hemoglobin estimation and serum was used for estimation of iron, ferritin and TIBC. APGAR score of all the newborns at 5 min was measured. All the results were noted and analyzed by slandered statistical methods. Results: APGAR score results showed 16 babies were in moderate to severe depression. Preterm babies were proportionately more depressed than term ones. Hb concentration varied from 13 to 18.7 g/dl. The iron profile of all the newborns were within normal limit though case to case variation was present.

Abstract P 178 ABO Blood Groups Differentially Allow Plasmodium falciparum Growth Vrushali Pathak, Roshan Colah, Kanjaksha Ghosh Objective: Plasmodium falciparum malaria has an impact on the distribution of ABO blood groups in humans. The ratio of group O to A is higher in geographic regions where malaria is currently or was previously endemic. It is known that, during infection with P. falciparum, group O offers a survival advantage, however the mechanism by which the survival advantage is translated is not known. This study was undertaken to correlate the P. falciparum parasitemia with the ABO blood groups in an in vitro culture system. Methods: We used an in vitro culture system that involved co-culturing of P. falciparum (strain 3D7) infected erythrocytes with uninfected different blood group erythrocytes. A fixed number of parasites were allowed to grow in fixed number of erythrocytes without addition of fresh cells for up to 5 days. Mean percent parasitemias (Percent parasite-infected erythrocytes) were calculated after every 24 h. Results: Percent parasitemia (mean ± SD) in A, B and O group cultures on fourth day were 15.4 ± 0.7, 13.3 ± 1.3 and 19.9 ± 3.2 respectively. Parasitemia was found to be significantly higher in O blood group when compared to A and B blood groups (P \ 0.01). Conclusion: This work demonstrated that blood groups differentially allow

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248 P. falciparum growth. The resistance of O group hosts in vivo is not due to low parasitemia some other mechanisms need to be explored.

Abstract P 179 Diagnosis of Malaria on the XT 2000i Pankhi Dutta, Chitrangi Navadkar, Sharyu Awate1, Sandhya Bastian2 Kokilaben Dhirubhai Ambani Hospital, Mumbai; 1SevenHills Hospital Mumbai; 2Sysmex India Pvt Ltd Objective: Detection of malaria on different fully automated 5-part cell counters have been reported with varying sensitivities and specificities. This study was conducted to evaluate the efficiency of the XT 2000i (Sysmex) in diagnosing malaria. Method: The complete blood counts including the various scattergrams of 446 febrile patients suspected to have malaria were analyzed. Thick and thin blood smears were examined by two observers and the cases were divided into malaria positives and malaria negatives. In both groups, abnormal scatter patterns were looked for in the WBC, WBC/Baso and reticulocyte scattergrams respectively. Results: Malaria positive cases were 276 while 170 were negative. In the malaria positive group, additional cluster on the WBC scatter, complete voting out of the differential, flag for ‘abnormal WBC scatter’, extended ‘ghost’ cluster in the WBC/Baso scatter and abnormal events in the reticulocyte scatter were seen in 93, 39.9, 27.5, 78.3 and 93.9 % of the cases respectively while the same were seen in only 4.12, 0.6, 0.6, 0.6 and 7.6 % respectively, in the malaria negative group. In predicting malaria positivity, the sensitivity and specificity of presence of an additional cluster on WBC diff scatter were 92 and 96 % respectively while it was 78 and 99 % respectively for an extended ghost cluster on the WBC/Baso scatter. Presence of abnormal events in the retic scatter had a sensitivity and specificity of 93.6 and 94 % respectively. Conclusion: It is possible to detect malaria on the XT 2000i in an automated manner with reasonable sensitivity and specificity. Familiarization with the various abnormal scatter patterns will be helpful in increasing the overall sensitivity of malaria diagnosis.

Abstract P 180 Haematological and Inflammatory Parameters in Patients with Different Types of Tuberculosis and Their Response to Antitubercular Therapy Aditya Kutiyal, Sandeep Garg, and Naresh Gupta Department of Medicine, Maulana Azad Medical College, New Delhi Aim: To study the haematological parameters and markers of inflammation in patients with tuberculosis involving different sites/ organs and to correlate these abnormalities following antitubercular treatment. Methodology: Adult patients [12 years age of either sex, newly diagnosed tuberculosis ATT naı¨ve were studied. Four subgroups were analyzed stratified on the site of tuberculosis namely pulmonary, CNS, disseminated and others. Haematological parameters like Hb, WBC, platelet count, ESR, LDH, CRP and albumin were assayed at baseline and 2 months after ATT. Results: Mean age amongst the 128 patients was 31.55 years. Pulmonary, CNS, disseminated and other types of tuberculosis comprised 30.5, 28.9, 17.2, and 23.4 % patients respectively. At baseline, mean Hb, WBC, platelet, ESR, LDH and albumin was 10.87 g% ± 2.17, 9.24 9 109/ l ± 4.27, 256 9 109/l ± 1.38, 61.43 mm ± 21.10, 753.92 iu ± 183.39 and 3.45 g% ± 0.65 respectively. Anaemia was present in

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 75.78 %, leukocytosis in 49.21 % and leukopenia in 3.9 %. Thrombocytosis and thrombocytopenia was encountered in 12.5 and 38.28 % respectively. ESR levels were raised in 98.43 %. Hypoalbuminemia was seen in 75 % patients. CRP was elevated in 13 patients. Follow-up comparison after 2 months ATT revealed mean Hb, WBC, platelet, ESR, LDH and albumin to be 11.72 g% ± 1.79, 7.93 v ± 1.4, 207 9 109/l ± 0.33, 8.34 mm ± 4.29, 205.70 iu ± 42.11 and 3.83 g% ± 0.38 respectively. The p values for change in all the parameters were highly significant (\0.01). This was true across the four subgroups of different tuberculosis. CRP was no longer elevated in any patient after ATT. Conclusion: Tuberculosis presents with varied haematological and inflammatory abnormalities in majority which revert with 2 months of ATT. A normal ESR is highly unlikely and anemia is very likely in tuberculosis at presentation. Leucocytosis may occur in up to half of the cases. Keywords Tuberculosis, Haematology, Inflammation, Acid phase reactarts

Abstract P 181 Immature Granulocyte Percentage (IG%) in the Diagnosis of Sepsis Pankhi Dutta1, Shashikala Shivaprakasha, Sharyu Awate, Monisha Sethi2 1

Kokilaben Dhirubhai Ambani Hospital, Mumbai; SevenHills Hospital, Mumbai; 2Sysmex India Pvt Ltd Objective: Immature Granulocytes is a new, fully automated, FDA approved parameter available on some cell counters from Sysmex, Japan. We evaluated the role of IG% as an aid in the diagnosis of sepsis as compared to the traditional parameters like absolute neutrophil count (ANC) and total leukocyte count (TLC). Method- The complete blood count (CBC) of 166 consecutive patients in whom sepsis was suspected and for whom blood cultures were ordered, were analyzed on the XT 2000i for the IG%, TLC and ANC. Blood culture (BC) positivity was considered diagnostic of sepsis. The counts in the BC positive group versus the culture negative group were compared. The Mann–Whitney test was applied to find the ‘p value’. The sensitivity and specificity of these parameters in predicting sepsis were also calculated. Results: Of 166 patients (107-males, 59 females, aged 9–98 years), 62 were BC positive and 104 were negative. In the BC positive group, the mean IG% was 2.55 (range: 0–6.2 %) versus a mean of 1.46 (range: 0–3.35) in the BC negative group (p = 0.0025). The BC positive group had higher TLC and ANC values as compared to the negative group. The sensitivity and specificity of IG% in predicting BC positivity was 61 and 59 % respectively while it was 87 and 29 % respectively for ANC and 84 and 38 % respectively for TLC. Conclusions: The IG % had a lesser sensitivity but higher specificity versus ANC & TLC in predicting BC positivity. It is automated, available at the time and cost of a routine CBC, and can be a useful parameter in the diagnostic workup of sepsis.

Abstract P 182 Haematological Profile, Serum Iron and Ferritin Level in Anemia of Inflammation: A Prospective study Nibedita Sahoo, AK Dash, P Sahu, B Mishra, G Rath, SP Pradhan Department of Pathology, MKCG Medical College, Berhampur Introduction: Anemia of Chronic Disease (ACD) is the most common anemia found in hospitalised patients and it is the SECOND most

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prevalent after Iron Deficiency Anemia (IDA). ACD seen in patients with chronic infections, malignancy, autoimmune disorders. Condition also termed as ‘‘Anemia of Inflammation’’. Aim and Objective: Combined clinical history, haematological evaluation, serum iron and ferritin estimation to reach diagnosis of ACD. Material and Methods: Patients of chronic infection, Autoimmune disorder or malignancy admitted to different Department s of M.K.C.G.M.C & Hospital are included in the study during period of 2010–2012. Routine haematological profile, ESR, serum iron, serum ferritin and bone marrow aspiration with special emphasis to iron study was performed in 70 patients. Results: Prevalence of ACD was 71 %, IDA 13 % and non ACD 16 %.Among the patients with ACD mild to moderate anemia present in 74 % cases. On peripheral smear examination normochromic normocytic anemia in 46 %, normocytic hypochromic 12 %, dimorphic 16 %, microcytic hypochromic 26 % cases observed. Significant correlation of degree of anemia with ESR was obtained. All cases showed either increase or normal bone marrow iron store. Conclusion: An increase serum Ferritin, increase Erythrocyte Sedimentation Rate (ESR) and marrow iron staining results indicating plentiful iron store distinguish people with ACD from IDA.

diagnosed with ALL and being treated at department of haematology, SGPGIMS were subject of the study. The diagnosis of ALL was made based on complete hemogram, bone marrow examination with cytochemistry and immunophenotyping by flow cytometry. The clinical records of the patients were analysed for occurrence of non-haematological complications occurring 2 weeks after the start of therapy. The patients were treated as per the BFM protocol (BFM 90 of BFM 95 protocol). Result: One hundred and one patients had 147 events of nonhematological complications. Gastrointestinal toxicity was the most frequent complication and occurred predominantly as hepatitis. Hyperglycemia was seen in 25 cases (24.7 %) and was most frequent in patients above the age of 20 years. Neurological complication was predominantly seen in patients below the age of 20 years (76.2 %) and seizures was the most common presentation. Osteonecrosis involving the hip joints was seen two adult young males. Conclusion: Non-hematological complications are quite frequent during the late phase of treatment of ALL patients. The Gastrointestinal tract, nervous system and endocrine system most frequently affected. Many of these events are missed because of low levels of clinical suspicion. This study highlights the incidence of these complications as they are associated with high morbidity and mortality.

Keywords Anemia of inflammation (A1), Anemia of chronic disease (ACD), Irondeficiency anemia (IDA)

Abstract P 185

Abstract P 183: Poster Presentation

Diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH) by Flow Cytometry, and Study of its Association with Aplastic Anemia and MDS

Richter’s Transformation : A Case Report Ipsita Dhal, Sudha Sethy, Rajeeb Nayak, Rabindra Kumar Jena Department of pathology and Department of clinical haematology, SCB Medical College, Cuttack Introduction: Richter’s transformation or Richter’s syndrome is a complication of B cell chronic lymphocytic leukemia (CLL) in which the leukemia changes into a fast growing diffuse large Bcell lymphoma (DLBCL). Richter’s syndrome affects nearly about 5 % of all CLL patients at some point during their lives. Case History: A 56 year old male presented with generalised lymphadenopathy and splenomegaly. His haematological parameters were haemoglobin 7.5 g/dl, total leucocyte count 50,000/cmm with 80 % atypical lymphocytes. He was diagnosed as a case of CLL/Small lymphocytic leukemia and was put on CVP regimen (4 cycles). He attended remission. After one and half years he came with complaints of fever, myalgia and generalised lymphadenopathy. His peripheral blood smear showed features of CLL. Biopsy from cervical lymph nodes showed features of non Hodgkin’s lymphoma (NHL), which was subsequently confirmed by immunohistochemical stains showing positivity for B lineage markers (CD 20, CD 23). Thus the final impression was DLBCL. Conclusion: we report this case because of its rarity.

Abstract P 184 Treatment Related Complications in Patients with Acute Lymphoblastic Leukemia Rajesh Kashyap, Mukul Agarwal, Pradeep Kumar, Garima Agarwal Department of Hematology, Sanjay Gandhi Postgraduate Institute of medical Sciences, Lucknow, Uttar Pradesh, India Objective: To study the different non-haematological complications occurring during the late phase (2 weeks of induction therapy to remission) treatment of ALL patients. Material and Methods: Patients

Shivangi J. Harankhedkar, Sudha Sethy, Pranati Mahanty, BP Das, RK Jena, SR Mahapatra SCB Medical College and Hospital, Cuttack Background: PNH is a consequence of non malignant clonal expansion of one or several hematopoietic stem cells due to a somatic mutation in PIG-A gene. Resultant deficiency of glycosyl phosphatidyl inositol-anchored proteins (GPI-APs), CD55 and CD59, accounts for the primary manifestation of intravascular hemolysis. PNH may also arise in association with aplastic anemias and MDS. Aims and Objectives: (1) Diagnosis of PNH by using multiparameter flow cytometry with antibodies specific for GPI-APs, CD55 and CD59, and assess severity of hemolysis. (2) Detect PNH clones in patient of aplastic anemia and MDS, and correlate with therapy and prognosis. Materials and Methods: 253 patients of pancytopenia and refractory anemia (Coombs negative) were screened for PNH clones. Using 2 ml of peripheral blood (with EDTA), CD55 (DAF) and CD59 (MIRL) deficient clones were detected on granulocytes by multiparameter flow cytometry. Other supportive routine investigations were done. Results: Out of 253 patients, classical PNH: 18. PNH clones in 50 aplastic anemia patients: 17. PNH clones in 5 MDS patients: 2. All cases presented with anemia. Pancytopenia (80 %), hyperbilirubinemia (20 %), reticulocytosis and evidence of hemolysis (75 %). Conclusion: (1) Flow cytometry is considered to be gold standard for diagnosis of PNH. (2) Association of PNH with aplastic anemia and MDS is identified as better prognostic indicator.

Abstract P 186 ‘Bleeding’ and ‘transfusion Support’ in Acute Myeloid Leukemia Patients: The Challenges Rahul Chaurasia, Priti Elhence, Sonia Nityanand, Anupam Verma Department of Hematology, Others Department of Transfusion Medicine, SaNjay Gandhi PGIMS, Lucknow

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250 Background: Bleeding due to severe thrombocytopenia in acute myeloid leukemia (AML), may occur due to the disease process itself or because of the chemotherapy. Prophylactic platelet transfusions are given to these patients to prevent the occurrence of bleeding. Occurrence of bleeding in these patients is therefore an indicator of presence of factors whereby patient’s response to transfusion is suboptimal. Bleeding may be classified according to WHO grading system and management of bleeding is done according to bleeding grade. There is however insufficient data to validate this approach. Objectives: To study the WHO bleeding grades and therapeutic transfusion support in AML patients undergoing chemotherapy and prophylactic platelet transfusions. Materials and Methods: A prospective study of 19 patients suffering from AML was carried out at our center. Patient details, clinical details, bleeding grades as per WHO, the platelet transfusion data including the indication, trigger dose and the response to the platelet transfusion were recorded and analyzed. Results: 256 bleeding days was observed of the total 1302 patient days. WHO Bleeding grades observed were Grade 1 = 115 days, Grade 2 = 86 days, and Grade 3 = 55 days, no grade 4 bleeding was observed. GI bleeding (28.5 %) was the most common site of the bleed followed by oropharyngeal bleed (19.1 %), epistaxis (17.1 %), mucocutaneous bleeding (16.4 %), genitourinary (14.4 %) and others (4.3 %). Mean pre transfusion Hb was 7.82 ± 1.32 (4.3–12.3) and platelet count was 17.54 ± 12.24 (6–46) for which total 135 LPRBC units (mean 0.53 per episode) and 835 RDP units (mean 3.26 per episode) was transfused. Clinically bleeding was stopped after platelet transfusion. No bleeding related mortality was noted. Conclusion: Despite the prophylactic platelet transfusion 19.6 % of observation days, majority of III bleeding episodes were in patients who started as grade I or Grade II bleedings therefore this management approach needs further improvement.

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 accumulation of mast cells in various organs, commonly skin and bone marrow. Here we report a case of systemic mastocytosis with bone marrow involvement as the first manifestation. Specific histocytological techniques were needed to establish the diagnosis. Case Report: A 52 years old lady presented with severe lower and upper back pain of 8 months duration. On local examination severe tenderness was elicited over dorsal and lumbar spine. X-Ray and MRI for the spine showed lytic lesions at the L1 to L5 vertebrae. Bone scan was suggestive of osteoblastic changes. Gastroscopy demonstrated pangastritis. Metabolic and endocrinological etiologies were excluded because laboratory evaluation was unremarkable. Iliac crest bone marrow biopsy demonstrated infiltration by mast cells, arranged in sheets and whorls. Extensive areas of marrow fibrosis was seen along with bone destruction. Special stains: AFB—negative; Toluidine blue—positive in mast cells. Immunohistochemistry: CD117—positive in mast cells. Conclusion: The diagnosis of mastocytosis requires a great degree of clinical suspicion as it is a rare disease that may go unrecognized or misdiagnosed because of the non specificity of many of its symptoms, particularly in cases with visceral involvement and absence of characteristic skin lesions of urticaria pigmentosa. Bone involvement in mastocytosis is related to infiltration of bone marrow by mast cells. Bone marrow biopsy and histocytological techniques are the key for diagnosis.

Abstract P 189 Flowcytometric Characterization of B-ALL: A 3 Year Study from a Tertiary Care Centre Nabhajit Mallik, Vijay Kumar, Anjali Sharma, Gurdeep Buxi Department of Pathology, PGIMER & Dr. Ram Manohar Lohia Hospital, New Delhi

Abstract P 187 Sickle Cell Anaemia—Community Control Programme Amongst Tribal Groups from Satpuda Hilly Ranges in Maharashtra, India SL Kate, Gunvant H. Yeola, Prashant N. Dalvi, Girish T. Kulkarni, Yogesh S. Prabhune Maharashtra Arogya Mandal, Hadapsar, Pune-411028 Abstract: Amongst all the tribal area of Maharashtra Sickle Cell Disorder is very common amongst Bhill and Pawara tribal population groups residing in the Satpuda hilly ranges (trait prevalence 1:5). Considering counselling is the only alternative, to control the disease, we have established a community control centre in high risk area located between 3rd and 4th hilly ranges of Satpuda hills (at Roshmal Budruk, Tal. Dhadgaon, Dist. Nandurbar Maharashtra state. We provided accurate diagnosis, possible treatment, follow-up counselling. We have now 1501 sickle cell patients under our medical supervision. Counselling is provided to patients, parents and public from this area. Details will be described in the presentation.

Objective: To evaluate the expression of Common Acute Lymphoblastic Leukemia Antigen (CD10), CD34, HLA-DR and myeloid antigens in all cases of B-ALL over a period of 3 years in a tertiary care centre in North India. Methods: The study was carried out at the Department of Pathology, PGIMER & Dr. RML hospital, by analysing the flow cytometry data over a period of 3 years, from September 2009 to August 2012. All cases of B-ALL were studied to look for the expression of CD10, CD34, HLA-DR and myeloid antigens (CD13, CD33). Results: A total of 36 cases of B-ALL were retrieved, of which 27 (75 %) patients were male, and 9 (25 %) were female. The youngest patient was 3 months old, while the oldest was 48 years. 29 cases (80.5 %) showed CALLA positivity, while 7 (19.5 %) were CALLA negative. CD34 positivity was seen in 32 cases (88.9 %) while 4 cases (11.1 %) were negative for CD34. Only 1 case (2.7 %) was negative for HLA-DR, and the remaining 35 cases (97.3 %) were positive. A total of 13 cases (36.1 %) showed aberrant expression of myeloid markers in the form of CD13 positivity. Conclusion: The present study illustrates the role of flow cytometry in immunophenotypic characterization of B-ALL, especially with respect to markers that carry prognostic significance for the patient.

Abstract P 190: Poster Presentation Abstract P 188 Hairy Cell Leukemia—A Rare Case Report Systemic Mastocytosis: A Rare Entity Sugatha Sahu, S Behera, SP Pradhan M Ketan, Bikash Singhania, K Annamma, B Sushma, R Lakshmi 1

Kasturba Medical College, Manipal University, Manipal; 2Melaka Manipal Medical College, Manipal Introduction: Mastocytosis encompasses a rare and heterogeneous group of clinical disorders characterised by abnormal growth and

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Department of pathology, MKCG Medical college, Berhampur, Odisha, India Introduction: Hairy cell leukaemia (HCL) is a rare, clonal, chronic lymphoproliferative disorder commonly seen in males in the middle years of life. It represents 2 % of all leukemia’s. Pancytopaenia with moderate

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 to massive splenomegaly is the most common clinical presentation. The diagnosis is based upon the recognition of the characteristic ‘‘hairy’’ nature of the leukemic lymphoid cells in the peripheral blood smears and the typical appearance and pattern of infiltration in the bone marrow biopsies in association with increased reticulin and eventual fibrosis of the marrow. Case Report: We report a case of 54 year male who presented with pallor, fever, abdominal distension. On physical examination splenomegaly was detected. Peripheral blood examination showed pancytopenia and lymphocytes with abundant cytoplasm which spread into hair-like processes. The bone marrow aspirate was a dry tap. The trephine biopsy has the characteristic features of a honey comb appearance. The cytochemistry was positive with tartrate resistant acid phosphatase (TRAP).Hence diagnosis of hairy cell leukemia was made. Conclusion: HCL is an easy diagnosis to make on the morphology of well made blood and bone marrow smears. Although there are no specific markers for HCL, cytochemistry and flow cytometry readily confirm the diagnosis.

Abstract P 191: Poster Presentation Niemann-Pick Disease Presenting with Hepatosplenomegaly and Thrombocytopenia

251 expression of surface antigens that indicated myeloid phenotype of blast cells because of their strong association with typical cases of AML, e.g. CD5 negativity: score 2; CD10 negativity: 1; CD13 positivity: 1; CD117 positivity: 0.5 and HLA DR positivity: 1. Cases of T-ALL(M) had a score \2.5 and those of AML(T) [ 3.5. In this prospective study we tested the sensitivity and specificity of this scoring system. Materials and Methods: Immunophenotyping was performed in 1574 additional cases of acute leukemia from May 2010 to July 2012 using a primary panel consisting of antibodies against CD 3, 5, 7, 10, 13, 19, 20, 22, 33, 34, 45, 117 and HLA-DR. Additional markers such as CD2, 4, 8, 14, 15, 41, 61, 64, 99 and glycophorin were tested as indicated. The presence of cytoplasmic (c) myeloperoxidase, cCD79a and cCD3 was examined in all cases with aberrant expression of CD markers by blast cells. Results: 126/130 cases of AML(T) had a score of C3.5 (sensitivity and specificity = 97 %) while 71/73 cases of T-ALL(M) had a score B2.5 (sensitivity 97; specificity 98.5 %). There was no case of true mixed (T and myeloid) leukemia. Conclusions: The scoring system proposed by us allows accurate distinction between AML(T) and T-ALL(M) in most cases at a much lower cost than that involved in the use of expensive reagent panels, including cytoplasmic markers, and advanced flow cytometry software.

Abstract P 193 Mitali Swain, SK Behera, SP Pradhan Department of Pathology, MKCG Medical College, Berhampur, Odisha, India Introduction: Niemann-Pick disease (NPD) is an autosomal recessive genetic disorder resulting in abnormal lipid metabolism. NPD is a clinically and biochemically heterogeneous disorder, with four variants. Type A and B have a generalized sphingomyelinase deficiency, whereas types C and D have normal sphingomyelinase levels. Case Report: We report two cases of Niemann-Pick disease in children, one aged 9 months and the other of 2 years, complaining of abdominal distension and recurrent fever. The older child also complained of epistaxis and melena. Both the children had coarse facial features and growth retardation. The abdominal ultrasound confirmed the clinical findings of hepatosplenomegaly. Bone marrow and liver biopsy examination revealed presence of foamy histiocytes, suggestive of Gauchers or Niemann-Pick. Confirmatory study by lysosomal enzyme from leucocytes was normal for b-Glucosidase and sphingomyelinase specific for Gauchers and Niemann Pick type A or B respectively. So, a diagnosis of Niemann-Pick disease (types C or D) was made. Conclusion: Niemann-Pick disease in children is very rare, with a reported incidence of 1:1, 20,000 to 1:1, 50,000 live births. Prenatal diagnosis with amniocentesis or chorionic villus sampling is available. Keywords Niemann-Pick disease, Sphingomyelinase, Hepatosplenomegaly

Abstract P 192 A Prospective Study to Test the Usefulness of an Immunophenotype-Based Scoring System to Distinguish Between T-ALL with Myeloid Markers [T-ALL(M)] and AML With T-Lymphoid Markers [AML(T)] Vishal Mehrotra, Manisha Ramani, Janmejay Yadav, Pallavi Gujarathi, Santosh Parab, Archana Vazifdar and Amar Das Gupta Sections of Hematology and Flow Cytometry, SRL Limited, Mumbai Introduction: From a retrospective analysis of 1874 cases of acute leukemia we proposed a scoring system in 2010 to distinguish between cases of AML(T) and T-ALL(M) on the basis of scores assigned for

CD200 Expression in B-Chronic Lymphocytic Leukemia (B-CLL)—it is not an all or None Phenomenon Manisha Ramani, Vishal Mehrotra, Janmejay Yadav, Pallavi Gujarathi, Santosh Parab, Amar Das Gupta Hematology & Flow Cytometry Sections, SRL Limited, Mumbai Introduction: Immunophenotypic distinction between B-CLL and nonCLL B-chronic lymphoproliferative disorders (B-CLPD) can be challenging and is sometimes impossible due to overlapping features among these entities. However, a firm diagnosis is necessary for proper management of different types of CLPD. Recently CD200, a B-lymphoid antigen has been shown to be useful in this regard by virtue of its close (100 %) association with B-CLL and its absence in the lymphoma cells in non-CLL B-CLPD, except hairy cell leukemia and B-lymphoblastic leukemia/lymphoma. Hence CD200 has been proposed in recent studies as a diagnostic marker for B-CLL. We wanted to verify this claim by checking the specificity and sensitivity of this marker for the diagnosis of B-CLL. Materials and Methods: Twenty eight cases of B-CLL and 28 cases of other types of B-CLPD (splenic marginal zone lymphoma 7; mantle cell lymphoma 10; ‘hairy cell’ leukemia 3; others 8) were immunophenotyped between April and August 2012 using a large panel of antibodies against CD3, 5, 10, 11c, 19, 20, 22, 23, 25, 38, 103, 200, FMC7, HLA-DR, immunoglobulin light chains and surface immunoglobulins. Results: Lymphoma cells in all 28 cases of B-CLL were CD200 positive (sensitivity 100 %), while 9/28 cases of non-CLL B-CLPD (‘hairy cell leukemia: 3/3; mantle cell lymphoma: 2/10; others: 4/8) also were CD200 positive indicating a relatively low specificity (68 %). Conclusions: Our study suggests that although CD200 is a highly sensitive marker for B-CLL, its low specificity would limit its utility in the differential diagnosis between this entity and the other types of B-CLPD.

Abstract P 194 Study of Treatment Outcome of Different Art Regimen Basing on CD4 Count Vandana Raut, P Kote, RK Bhola, SK Behera, S Pradhan Vandana Raut, MKCG Medical College, Brahmapur

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252 Introduction: HIV is the leading infection in southern Orissa. 37.8 % of total HIV Population of Orissa is contributed by Ganjam district alone, and the most probable reason being; unemployed people from coastal districts like Ganjam migrate to industrial areas where HIV cases are more. We have a functional ART centre since 2006 and mainly four different regimens are followed consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) and one either a non-nucleoside reverse transcriptase inhibitor (NNRTI), or a protease inhibitor (PI). Aims and Objectives: (1) To know the efficacy of each of these regimen to restore the immune function (as indicated by the CD4 cell count). (2) Compare the treatment outcome of patients in relation to their base line cd4 count at onset of ART. (3) Response to ART in HIV patient’s co infected with TB. Materials and Method: The study included 464 HIV cases which were followed over a period of 1 year. Efficacy of different ART regimens is studied in terms of CD4 count. Result: Patients age ranges from 15 to 60 years with mean age of 37.4 years and M:F is 1.4:1. Among 464 cases 220 patients are in regimen I(d4T, 3TC, NVP), 60 patients are in regimen II(d4T, 3TC, EFV), 100 patients are in regimen III(AZT, 3TC, NVP) and 84 patients are in regimen IV(AZT, 3TC, EFV). % increase in CD4 count is maximum with regimen I. Conclusion: From this we conclude that increase in CD4 count is best achieved with regimen I and presence of TB impairs the response to ART.

Abstract P 195 Essential Thrombocythemia Transforming into Acute Leukemia: A Case Report Shabnam Roohi1, Jyothsna Krishnappa2, PP Bapsy3, CN Patil3, Manish Chugh1

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256

Abstract P 196 Evaluation of ADVIA 2120 Haematology Analyzer Shabnam Roohi, BC Sangamesh, LB Manigantan Department of Haematology and Clinical Pathology, Apollo Hospitals, Bangalore, Karnataka, India Objective: To evaluate performance of Siemens ADVIA 2120 fully automated haematology analyzer, compare it with the Beckman Coulter ACT i 5 Diff and manual reference methods. Methods: The precision, accuracy and Bias of the equipment were evaluated using standard methods. The Linearity and Carryover of the equipment was validated. The routine haematological parameters generated by the ADVIA 2120 were compared to those obtained from ACTi 5 Diff. The automated differential counts, platelet counts, n RBC and reticulocyte percent were also compared to manual methods. Results: The precision, accuracy and Bias of the equipment of the equipment were within acceptable limits. Linearity and carryover were within the limits established by the manufacturer. There was statistically significant difference between the two equipments for platelet count and eosinophil count, the difference for the other routine parameters was not statistically significant. The leukocyte differential counts indicated an excellent correlation with the manual reference method for Neutrophils, Lymphocytes and Eosinophils, a good correlation for Monocytes and a poor correlation for Basophils. The platelet count and reticulocyte count indicated an excellent correlation with the manual reference method, the enumeration of nucleated red blood cells by the automated method showed moderate correlation with the manual reference method. Conclusion: The results generated by the ADVIA 2120 analyzer are comparable to the existing equipment and the reference methods. The potential of new parameters generated by the analyzer in extending the clinical role of haematological studies needs to be explored.

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Department of Haematology and Clinical Pathology; 2Department of Internal Medicine; 3Department of Medical Oncology, Apollo Hospitals, Bangalore Introduction: The transformation of essential thrombocythemia into myelofibrosis is a rare and late complication. The recent advances in the management of this disease and its complications have raised a hope for better control. In this case report we present a case of an elderly patient having essential thrombocythemia with rapid transformation to myelofibrosis and leukemia. Case Description: A 79 year old female case of essential thrombocythemia detected in 2007, on symptomatic treatment, presented to Apollo hospitals in April 2012 with fever, low back ache, easy fatigability and loss of appetite. She was thin, pale and had massive splenomegaly on examination. Peripheral smear examination revealed a leukoerythroblastic blood picture with severe thrombocytopenia. Bone marrow aspirate was aparticulate with scattered immature myeloid cells. The bone marrow biopsy revealed grade four fibrosis on a scale of 0–4. A diagnosis of Essential Thrombocythemia transforming to Myelofibrosis was made. Considering her high risk status, regular follow-up with blood transfusion was initiated. In august 2012 she presented with worsening general weakness and breathlessness. Peripheral smear showed 24% blasts, suggestive of leukemic transformation probably myeloid. Flow cytometry report is awaited. Discussion: Progression of essential thrombocythemia to Myelofibrosis is 2.8 and 2.3% for acute leukemia with a median of 11 years. In this case the progression to acute leukemia was within 5 years of the initial diagnosis. Conclusion: Transformation of essential thrombocythemia to Myelofibrosis and acute leukemia can be rapid. Therefore the use of targeted therapy even for high risk patients needs to be explored.

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Abstract P 197 Cytogenetic Abnormalities in Acute Leukemia Cases—Single Centre Study from Eastern India Basab Bagchi1,Tuphan Kanti Dolai1, Prakash KR Mondal1, Ashutosh Panigrahi1, Sandeep Saha1, Meet Kumar1, Maitreyee Bhattacharyya1, Shyamali Dutta1, Rajiv De1, Malay Ghosh1, Bani B Ganguli2 1

Hematology Department, NRS Medical College and Hospital, Kolkata; 2Genetics Centre, Navi Mumbai, Maharashtra Objective: Cytogenetics has emerged as an effective tool in diagnosis, classification, risk stratification, prognostication and as a guide for providing appropriate therapy in acute leukemia cases. However there is not enough data from India. This study was conducted to determine the incidence and common cytogenetic abnormalities in AML and ALL. Methodology: Prospective collection of cytogenetics data from patients attending hematology services of NRS Medical College, Kolkata. Demographic data, diagnosis data collected, cytogenetics analysis done in all patients. Period: July 2009 to July 2012. Results: Of the 235 patients 152 were males and 83 were females. 118 had ALL and 117 had AML. Among ALL(118) cases 86 patients were \18 years of age and 32 patients were C18 years of age. Normal Karyotype was observed in 31/118 (26.3 %) patients. Hyperdiploidy was the commonest cytogenetic abnormality observed in 38/118 (32.2 %) of ALL patients, followed by t(9;22) detected in 15/118 (12.7 %) of patients. The next common abnormality was t(4;11), detected in 9/118 (7.6 %) of ALL patients. Among the ph

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 positive patients 12/15 (80 %) were C18 years. Among AML 38/117 (32.5 %) had normal karyotype. t(15;17) was the commonest cytogenetic abnormality [13/117 (11.1 %)] followed by trisomy 8 (10.25 %) and t(8;21)9.4 %. Conclusions: 1. Cytogenetic abnormalities were present in 73.7 % of ALL and 67.5 % of AML. 2. Hyperdiploidy was the most common cytogenetic abnormality in ALL followed by t(9;22). 3. Among the Philadelphia positive patients 80 % were C18 years. 4. t(15;17) was the most common cytogenetic abnormality in AML followed by trisomy 8 and t(8;21).

Abstract P 198 Coagulation Aberration in HIV Infected Patients Swapan Kumar Sinha, Soumit Mondal Department of Pathology, Medical College, Kolkata Objective: To study disorders of haemostasis in untreated cases of HIV/AIDS. Total Platelet Count, prothrombin time (PT), activated partial thromboplastin time (APTT) was performed on 53 diagnosed patients of HIV. Methods: Citrated blood (1:9) samples were collected for P.Time, APTT, FDPs and EDTA blood for Complete Blood Count ART Centre of Medical College and School of Tropical Medicine. PTimre & APPT was performed as per validated methods with Internal Quality Control. Platelet count was done by making blood smears of individual samples and counting under light microscope. Results: Among them 19/53 pts. (35.84 %) had decreased plt. count. Only 7/53 pts. (13.2 %) had elevated PTimer, 6/53 (11.32 %) had elevated APTT and 9/53 (16.98 %) had elevated PT, APTT and Low platelet. Conclusion: Thrombocytopenia may be due to increased destruction commonly immune mediated, increased consumption due to microangiopathy, TTP or decreased production. Thromboplastin like substances, Factor X activation by angiopathy or due to CMV infection may lead to activation of coagulation system. Acquired Factor V Leiden may lead to hypercoagulable states including APLAS. A detail study in HIV patients for coagulation disorders will be more informative.

Abstract P 199 Genotypic and Phenotypic Characterisation Bernard-Soulier Syndrome Patients from India Objective: Molecular characterization of BSS patients from India. Introduction: Bernard Soulier Syndrome (BSS) is a rare autosomal recessive bleeding disorder caused by an absence of or defects in the platelet membrane glycoprotein GP/IX/V complex. Methods: The diagnosis of 10 patients was based on low platelet count, presence of giant platelets in the peripheral smear, platelet function and platelet receptor study, flow cytometry study to assess the surface expression of GP1b/IX/V complex showed reduced expression, genomic DNA was screened for mutations in GP1BA, GP1BB and GP9 through direct sequencing. Results: We studied 10 patients diagnosed with BSS with classical bleeding symptoms. Bleeding diathesis measured by the World Health Organisation bleeding scale (grade 0, no bleeding; grade 1, petechiae; grade 2, mild blood loss; grade 3, gross blood loss; grade 4, debilitating blood loss) ranged from 1 to 4. All subjects suffered from frequent ecchymoses, epistaxis and gum bleeding, the three menstruated females had severe menorrhagia that required treatment. All the patients had low platelet count ranging from 6 9 109/L to 55 9 109/L determined automated cell counter (XT-2000i, Transasia Bio-Medical Ltd,

253 India).Thrombocytopenia is always more severe when platelets were measured by a cell counter, because it does not recognises and enumerate large platelets, as to mean platelet volume (MPV)/platelet size, in none of the patients the instrument could report the MPV due to its platelet abnormality. Ristocetin (normal and low dose) induced platelet aggregation was absent in all patients except one, which was unavailable for study (BSS1.01), whereas it was normal in controls. Sequencing analysis of the GPIba, GP1bb and GP9 gene revealed homozygous changes in the probands. Of the 6 mutations; 4 were found out to be novel mutations including one nonsense changes in GP1bb (p.Cys32X) and two missense changes in GP9 (p.Thr95Asp) and (p.Cys135Tyr) and one frameshift change in GP1ba (p.Met338AsnfsX13). The known mutations found out to be one missense change in GP9 (p.Cys24Arg) in one patient with severe bleeding diathesis and one frameshift change in GP1ba (p.Val485ValfsX12). Conclusion: The molecular data presented here is a new data on BSS patients from India apart from the existing data, adding significantly to the mutation database of this condition and also useful for its genetic diagnosis in India.

Abstract P 200 Comparative Evaluation of Bone Marrow Trephine Biopsies with Bone Marrow Aspirates: A Tertiary Care Institutional Experience Usha R Singh, Surbhi Goyal, Usha Rusia University College of Medical Sciences & Guru Teg bahadur Hospital, Delhi Introduction: Bone marrow examination is an important diagnostic tool to evaluate various neoplastic and non neoplastic hematological diseases. Aims: To assess correlation between bone marrow biopsy and aspirate. Correlation of lymphoma positivity with trephine biopsy length. Methods: Diagnostic correlation was done between bone marrow aspirates & bone marrow trephine biopsies in 518 cases received in the department of pathology from Jan 2011 to Feb. 2012. Proportion of biopsy showing marrow infiltration by lymphoma cells was studied in relation to total trephine length. Biopsies were fixed in formalin & length measured. After EDTA decalcification they were processed routinely and H&E stained. Special stain & immunohistochemistry was done if needed. Results: We found a positive correlation of 88.6 % for acute leukemias and plasma cell dyscrasias, 60% for nonhematopoietic metastases, 53.7 % for NHL, 38.5 % for aplastic anemia, 5% for Hodgkin’s lymphoma, and 0 % for granulomatous infections and myelofibrosis. The maximum proportion of positivity in lymphoma was attained at a length of 1.3 cm. Conclusion: Granulomatous infections and myelofibrosis could only be diagnosed on biopsy. Though both are complimentary, biopsy is more sensitive and informative in detection of nonhematopoietic metastases, assessing marrow involvement in lymphomas and plasma cell deposits in multiple myeloma. In acute and chronic leukemias, immune thrombocytopenic purpuras and nutritional anemias biopsy is a useful adjunct to aspirate in diagnosis and follow up. A biopsy length of C1.3 cm was adequate for assessing lymphoma infiltration.

Abstract P 201: Poster Presentation Anticardiolipin Antibodies in Women with Spontaneous Fetal Loss Meera Sikka1, Akansha Rawat1, Usha Rusia1, Kiran Gulleria2 1

Department of Pathology; 2Department of OBG, University College of Medical Sciences & GTB Hospital, Delhi, India

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254 Introduction: Spontaneous fetal loss is a frequent complication of pregnancy. Recurrent pregnancy loss (RPL) i.e. occurrence of three consecutive pregnancy losses during the first trimester affects 1 % of pregnant women. Several causes of RPL have been reported . Antiphospholipid syndrome, an autoimmune disorder characterized by RPL, arterial and/or venous thrombosis and elevated titers of antiphospholipid antibodies is a frequent cause of RPL. Anticardiolipin antibodies (ACA) are one of the significant antibodies detected in this syndrome. Aim: To ascertain the prevalence of anticardiolipin antibodies in Indian women with spontaneous/recurrent fetal loss. Methods: Seventy eight women with spontaneous/ recurrent fetal loss in the first trimester were included in the study. A detailed history and physical examination was done. Anticardiolipin antibodies, IgG and IgM were determined by ELISA using commercially available kits. Results: IgG antibodies were detected in 1 (1.2 %) woman and IgM in 3 (3.8 %). These women had no other clinical manifestation. Other pregnancy related complications were significantly more in ACA positive women as compared to ACA negative women. Conclusions: Presence of ACA defines a population at risk for fetal loss and other pregnancy related complications. Women with RPL must be screened for these antibodies even in the absence of other clinical symptoms as on treatment these women have a better chance of a subsequent successful pregnancy.

Abstract P 202 Non Immunoglobulin CMYC Translocation in High Grade B Cell NHL: The Gray Zone Between Diffuse Large B Cell Lymphoma and Burkitt’s Lymphoma M. Parihar1, A. Gupta1, A. Yadav1, S. R. Arun1, S. Bhave2,V. Radhakrishnan2, A. Chakrapani2, D. K. Mishra1, M. Chandy2 1 Departments of Cytogenetics and Lab Hematology; 2Clinical Hematology, Tata Medical Center, Kolkata, India

Background: High grade B cell non Hodgkins lymphoma (NHL) with features between diffuse large B cell lymphoma (DLBCL) and Burkitt’s lymphoma (BL) have been in the grey zone of diagnostic uncertainty. Although immunoglobulin gene CMYC translocations define BL, the CMYC gene may be involved in DLBCL as a secondary hit to the BCL6 or the BCL2 gene translocations. We present a case of non immunoglobulin gene (Ig) CMYC translocation,t(3;8)(q27;q24) juxtaposing the CMYC gene to the BCL6 gene in a patient with features of Burkitts lymphoma . Case report: Our patient a 29 year old male presented with weakness and low backache following surgery for an ileocaecal mass operated outside and diagnosed as DLBCL. Clinical examination revealed no lymphadenopathy or hepatosplenomegaly. A complete blood count showed low platelet counts with other parameters being normal. The bone marrow aspirate showed 90 % L3 lymphoblasts which on flowcytometry expressed CD43, CD10, CD19, CD38, cCD79a and CD22 with surface immunoglobulin expression. The trephine biopsy showed infiltration by sheets of abnormal cells with grade 2 fibrosis. Immunohistochemistry showed positivity for CD20,BCL6 and negativity for MUM-1 and BCL2 with 100 % Ki67 staining. Karyotyping showed 46,XY,t(3;8)(q27;q24)+2mar[20]. Metaphase FISH using breakapart CMYC probe confirmed the CMYC gene rearrangement with the partner chromosome being 3q27. Discussion: The non-IG-MYC translocations have been said to be almost exclusively secondary events and rarely occur in typical BL. From clinical point of view these cases with MYC rearrangements but without an mBL signature are associated with a poor clinical outcome and classified as intermediate BL/DLBCL by the WHO.

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Abstract P 203 A Novel t(2;12) in Precursor T Cell ALL Involving the ETV6 Gene M. Parihar1, A. Gupta1, A. Yadav A1, S. R. Arun1, A. Bhattacharyya2, D. K. Mishra1, M. Chandy3 Departments of Cytogenetics and Lab Hematology; 2 Paediatric Oncology; 3Clinical Hematology, Tata Medical Center, Kolkata, India 1

Background: Rearrangements of the short arm of chromosome 12 (12p), that harbors the ETV6 and the CCND genes have been reported in acute lymphoblastic leukemia (ALL) .Although the role of ETV6 in precursor B cell ALL has been extensively studied only rare cases of ETV6 gene involvement in paediatric T cell ALL have been described where the CCND gene is more commonly involved. We report a case of early precursor T cell ALL in a 13 year old male with t(2;12)(p13;q31) involving the ETV6 gene. There have been no reports to date on t(2;12) in ALL. Case Report: The patient presented with history of fatigue and irregular fever for the past 6 months. On examination severe pallor, generalized lymphadenopathy and hepatosplenomegaly were present. Investigations revealed low hemoglobin, normal white blood cell count and low platelet counts. Peripheral blood showed 14 % blasts. The bone marrow showed 90 % L2 lymphoblasts with bright expression of cCD3 and CD7, and moderate expression of CD2, CD5, HLA DR and Tdt. The karyotype showed 46,XY,t(2;12)(q31;p13) [20]. The involvement of ETV6 gene was confirmed by metaphase FISH. The patient received the UKALL 2003 Regimen B protocol. He was in complete remission (CR) with negative MRD by flowcytometry on day 29 and remains to be in CR post consolidation therapy. Discussion: The t(2;12)(p13;q31) results in the relocation of the ETV6 present on 12p13 to 2q31 locus that harbors the class 1 homeobox gene (HOX) cluster D. The homeobox genes are expressed during early stages of T cell development and the exact mechanism of activation of these HOX genes remains to be explored. Our report highlights the importance of these genes in leukemegenesis of T cell ALL by probable developmental arrest at specific T cell stages.

Abstract P 204 Cytogenetic Profile of Hematolymphoid Neoplasms in a Newly Opened Tertiary Care Center: The First Year Experience from East India M. Parihar1, A. Gupta1, A. Yadav1, S. R. Arun1, S. Bhave2, V. Radhakrishnan2, A. Chakrapani2, A. Bhattacharyya3, D. K. Mishra1, M. Chandy2 1 Departments of Cytogenetics and Lab Hematology; 2Clinical Hematology; 3Paediatric Oncology, Tata Medical Center, Kolkata, India

Introduction: Conventional cytogenetic analysis of hematolymphoid neoplasms provides valuable diagnostic and prognostic information. We describe the cytogenetic profile of hematolymphoid neoplasms from East India. Material and methods: All patients that were referred for karyotyping at Tata Medical Center, from August 2011 to July 2012 were studied. The bone marrow findings and diagnosis was recorded. G banded karyotypes were reported as per the ISCN. Results: A total 221 bone marrow samples were studied. The turnover time ranged from 2 to 10 days with a median TAT of 5 days. The disease distribution was as follows 108 patients with Acute Lymphoblastic leukemia (ALL),53 with acute myeloid leukemia (AML),22 with myelodysplastic syndrome (MDS),16 with Non hodgkins lymphomas(NHL), 10 with myeloproliferative neoplasms (MPD), 9 with chronic myeloid lelukemia (CML), and 3 with

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 multiple myeloma(MM). Metaphases were available in 212 cases with a success rate of 96.5 %. Among the ALL patients 65 % had abnormal karyotypes, hyperdiploidy being the commonest abnormality in children (21/72, 30 %) and t(9;22) in adults(9/36, 25 %). The other cytogenetic abnormalities seen were MLL gene rearrangements (3 %), hypodiploidy (2 %),t(1;19)(3 %), del6q(6 %) and t(12;21)(2 %). The following cytogenetic abnormalities were seen in patients with AML (58 % abnormal Karyotypes): t(15;17) in 7 patients, (8;21) in 5,trisomy 8 in 3,3q abnormalities in 3,monosomy 7 in 4,del 9q in 2, MLL gene rearrangements in 1 and complex karyotype in three. Abnormal karyotypes were seen in 32, 62 and 30 % of patients with MDS,NHL and MM. Conclusion: The incidence of hyperdiploidy in paediatric ALL patients is similar to what has been reported in western literature and higher than some reports from India. The incidence of t(9;22) in adult ALL,t(8;21) and t(15;17) in AML is similar to what has been reported from India and west.

Abstract P 205 Jumping Translocations in AML: A Role in Leukemogenesis Along with Tumor Progression M. Parihar1, A. Gupta1, A. Yadav1, S. R. Arun1, A. Bhattacharyya2, D. K. Mishra1, M. Chandy3 1

Departments of Cytogenetics and Lab Hematology; 2Paediatric Oncology; 3Clinical Hematology, Tata Medical Center, Kolkata, India Background: Jumping translocations (JT) are rare cytogenetic phenomenon resulting from translocation of the same segment of the donor chromosome on to various recipient chromosomes creating multiple related clones. Previous reports of JT in myeloid malignancies have implicated its role in disease progression in patients with myelodysplastic syndrome or myeloproliferative disorders that evolve into acute myeloid leukemia (AML). We present a rare case of JT in a 1 year old female with de novo AML M5. Case report: The patient presented with fever and purulent discharge from the left ear. On examination pallor, facial edema, proptosis of the right eye, mass in the maxillary region and hepatosplenomegaly were seen. Investigations revealed low hemoglobin, normal white blood cell count and low platelet counts. There were no blasts in the peripheral blood. The bone marrow was completely replaced by blasts having morphology akin to monoblasts with expression of CD13, CD33, CD34, HLA-DR, CD117, CD64, CD4 and MPO negativity on immunophenotyping. Karytotyping showed a JT with the long arm of chromosome 1 (1q12) as the donor fragment and short arms of chromosome 12 (12p13) and 13 (13p12) as the recipient chromosomes in 24 and 5 metaphases respectively. The patient died within few days of admission. Discussion: The most commonly involved chromosomal segment in JT in myeloid malignancies is 1q and is associated with a poor prognosis. However its presence here in a de novo pediatric AML indicates its role in leukemogenesis unlike previous reports that have implicated its role only in tumor progression.

Abstract P 206 Loss of Y Chromosome in Acute Lymphoblastic Leukemia: Constitutional or Neoplastic S. R. Arun1, M. Parihar1, A. Gupta1, A. Yadav1, A. Bhattacharyya2, D. K. Mishra1, M. Chandy3 Departments of Cytogenetics and Lab Hematology; 2Paediatric Oncology; 3Clinical Hematology, Tata Medical Center, Kolkata, India 1

255 Introduction: Loss of chromosome Y chromosome is a well established cytogenetic abnormality in myeloid neoplasms and has been reported with varying frequency ranging from 6.3 to 16.4 % in various hematological disorders. There have been no reports of a neoplasia associated loss of Y in acute lymphoblastic leukemia (ALL) and occasional reports in adult ALL have suggested it to be an age related phenomenon rather than a neoplastic one. We report a case of Philadelphia positive mixed phenotype ALL with loss of Y as a secondary associated abnormality. Case Report: The patient a 4 year old male child presented with a history of low back ache, bone pain, and inability to walk. Systemic examination showed hepatosplenomegaly. His blood counts revealed low hemoglobin, raised total leucocyte count with 57 % blasts and low platelets. Bone marrow examination showed 72 % L1 lymphoblasts. Immunophenotyping showed mixed lineage phenotype with bright expression of CD10, CD19, CD34, cCD22, and cCD3, TdT, HLA-DR and aberrant CD13. Cytogenetic analysis (karyotype and FISH) showed two clones,one showing t(9;22) with additional philadelphia chromosome (ph) and loss of Y chromosome in 16 metaphases and the other showing t(9;22) with t(1;19) in 4 metaphases. Phytohaemagglutinin stimulated peripheral blood cultures showed a normal karyotype ruling out constitutional loss of Y chromosome. Conclusion: Loss of Y chromosome can be seen in ALL as a part of genetic evolution. The other interesting findings were presence of additional Philadelphia chromosome and t(1;19) as secondary abnormalities in genetic evolution in a case of t(9;22) ALL.

Dr. J.C. Patel Award Session Abstract P 207 RNA Expression of Genes Involved in Cytarabine Metabolism and Transport is an Independent Predictor of Ex-vivo Cytarabine Response in Acute Myeloid Leukemia Ajay Abraham, Savitha Varatharajan, J Ashok Kumar, K Sreeja, Vivi M Srivastava, RV Shaji, Rayaz Ahmed, Aby Abraham, Biju George, Mammen Chandy, Alok Srivastava, Vikram Mathews and Poonkuzhali Balasubramanian Department of Haematology, Christian Medical College, Vellore Abstract: Wide inter-individual variation in terms of treatment outcome exists among AML patients receiving chemotherapy with cytarabine (Ara-C) and daunorubicin. The pre-requisite for the cytotoxic action of Ara-C is its enzymatic conversion to active triphosphorylated form Ara-CTP. The ex vivo cytotoxicity to Ara-C in AML patients exhibited a wide inter individual variation, and were stratified into 3 groups based on IC-50 values (\5.4 lM, [5.4 lM and [80 lM of Ara-C respectively). DCK and hENT1 RNA levels were significantly higher in Ara-C ex vivo sensitive samples compared to those with intermediate sensitivity and resistant patients. Ara-C resistance index (RI) was proposed based on candidate gene expression data where, RI = DCT (dCK 9 ENT1)/DCT CDA. RI values were significantly higher in resistant and intermediately sensitive compared to sensitive samples (median 5.428 and 5.278 vs. 3.475; p \ 0.0001). The median RI for patients who presented at relapse was significantly higher compared to that of ex vivo sensitive and resistant samples at diagnosis. Molecular markers including FLT3-ITD, NPM mutation, AML-ETO and inv16, as well as MN1, BAALC, ERG1 and CD34 expression, did not show any significant association with Ara-C RI or IC50. This study suggests RI as an independent predictor of ex vivo Ara-C sensitivity irrespective of cytogenetic and molecular risk factors.

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Abstract P 208 Comparative Evaluation of the Coagulation Parameters Before and After Chemotherapy and/Radiotherapy in Solid Tumours Supriya Dhar1, Swapan Kumar Sinha2, Pranab Kumar Biswas2, Shibashish Bhattacharya3 1

Department of Transfusion Medicine, Tata Medical center, Kolkata; Department of Pathology, Medical College, Kolkata; 3Department of Oncology, Medical College, Kolkata

2

Objective: To evaluate the coagulation abnormalities in patients with solid tumors and changes in their coagulation profile following chemotherapy and or radiotherapy. Materials and Methods: A prospective observational study was carried out in the Department of Pathology, Medical College, and Kolkata. Forty one patients between 15 and 70 years with solid neoplasm were evaluated prior to therapy and following treatment with radiotherapy and or chemotherapy during the period between November 2010–November 2011. Results: In our study 14 % of cervical cancer patients showed prolonged PT and APTT before therapy which normalized after therapy. Around 62 % of breast cancer patients showed prolonged PT and APTT before therapy. After therapy, 37.5 % showed prolonged PT and 12 % show shortening of APTT. Around 44 % lung cancer patients showed pre therapy increased PT and APTT. After therapy both PT and APTT were shortened in 33.3 and 44 % cases respectively. No coagulation abnormality was seen in gastric cancer patients. Around 44 % lung cancer patients had increased FDP levels before therapy which normalised after therapy. In cervical cancer patients 14.2 % showed increased FDP and all cases normalised after therapy. Plasma FDP level was normal in all gastric cancer patients. All ovarian cancer patients had increased pre therapy FDP and all cases normalized with therapy. 62.5 % breast cancer patient had increased FDP which came down to 12.5 % after therapy. Conclusions: Thus prolongation of PT and APTT was seen in many untreated solid tumor patients, after therapy few normalized, some showed shortening while others remain prolonged. Pre therapy there was a 34 % increase in FDP which came down to 2.4 % after therapy.

Abstract P 209 Indian MDS: Do Molecular Abnormalities Alter its Biology: AIIMS Experience Rekha Chaubey, Sudha Sazawal, Manoranjan Mahapatra, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi-110029 Background: MDS in the Western countries are mainly found in the elderly population but is being increasingly seen in young adults in India. Also, the Indian patients are more severe at presentation and the response to treatment in these patients is poorer than West. There may be some molecular factors that may be responsible for the poor prognosis of these patients. The present study is an attempt to look for its effect on biology of Indian patients. Aim: To study the effect of molecular factors on the biology of Indian MDS patients. Material and Methods: Cytogentics, molecular mutations of RAS and FLT3 gene, hTERT expression, telomerase activity and methylation of TSGs were analyzed in 100 MDS patients and correlated with disease severity, progression and survival. Results: There were 67 % male

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Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 and 33 % female patients with Mean age at presentation 46 years and median 48 years. The frequency of patients with age \60 years was high as compared to the patients with age C60 years (75 vs. 25 %). 26/51 (51 %) patients had normal cytogenetics and 25/51 (49 %) patients had chromosomal abnormalities. The frequency of N-RAS mutation was 3 % and K-RAS mutation was 9 %. FLT3-LM and FLT3-TKD-mutations were not observed at all. The increased telomerase activity (TA) was found in 17/100 (17 %) cases. hTERT expression was present in 17/100 (17 %) cases. Forty patients (40 %) had p15 INK4b gene methylation. Fifty three patients (53 %) had SOCS-1 gene methylated. Forty three patients (43 %) had FHIT gene methylated. Fifty eight patients (58 %) showed calcitonin gene methylation. In multivariate analysis, of all the molecular factors studied, only p15INK4b gene methylation was found as an important predictor for progression of disease in MDS patients. Conclusion: The Indian patients showed some difference from the patients from West in terms of molecular factors like rare chromosomal aberrations, absence of FLT3 gene mutations, increased telomerase activity correlated with increased hTERT expression, and in methylation pattern.

Abstract P 210: Oral Presentation Immunohistochemical Profile and Bone Marrow Angiogenesis in Multiple Myeloma with Reference to its Clinical Significance Sarah Grace Priyadarshini, Debdatta Basu, Rakhee Kar, TK Dutta1 Departments of Pathology and Medicine1, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry Aims of the Study: To study the histomorphological features of bone marrow in multiple myeloma and to evaluate the immunohistochemical profile and angiogenesis using a panel of markers including CD38, kappa and lambda light chain, CD56, Cyclin D1, Ki67 and CD 34 and to correlate immuno-hematological profile with various clinical parameters. Materials and Methods: The study includes 48 cases of myeloma diagnosed over the period of 5 years. The histomorphological features like plasma cell morphology, percentage of plasma cells and pattern of infiltration were studied. Angiogenesis was assessed by calculating the microvessel density (MVD) using immunohistochemistry for CD34. Proliferation was assessed using both Ki67 and CD38 highlighted cells. Immunohistochemistry for CD56 and Cyclin D1 was also done. Results: A significant association was seen between the plasma cell morphology and the pattern of infiltration (p = 0.0067). The percentage of plasma cells showed a significant association with the clinical staging. The MVD was significantly associated with plasma cell morphology (p = 0.04) as well as pattern of infiltration (p \ 0.001). The proliferation index was also significantly associated with the plasma cell morphology (p = 0.003). Both the Ki67 and MVD showed an increasing trend with clinical staging and MVD was significantly associated with serum albumin (p = 0.02). CD56 negativity was associated with circulating plasma cells and also with higher clinical stage. Cyclin D1 positivity was not seen in poorly differentiated morphology and the mean Ki67 was lower in the cyclin D1 positive group. It was also found that the cases with kappa light chain restriction had significantly less of poorly differentiated morphology, diffuse pattern of infiltration, lower MVD and Ki67. Conclusion: The study of bone marrow morphology and angiogenesis with the aid of immunohistochemistry is useful for prognosticating patients with multiple myeloma.