Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 DOI 10.1007/s12288-014-0467-0

ABSTRACT

Abstracts of the Oral and Poster Presentations During Haematocon 2014 (55th Annual Conference of Indian Society of Haematology & Blood Transfusion)

Ó Indian Society of Haematology & Transfusion Medicine 2014

Topic: Hematology

Topic: Symposia on: Thalassemia—Indian Perspective

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Hematological Manifestations in Thalassemia: A Four Year Experience in a Tertiary Care Hospital

Distribution of Beta-Thalassemia Mutations and its Variance in Ethnic Groups of Saurashtra Region, Gujarat

Dr. Garima Rakheja, Dr. Richa Gupta, Dr. Tejinder Singh

JignasaGami, HeenaKasyani, Dr. KamleshDharajiya

Department of Pathology, Maulana Azad Medical College

Indian Medical Scientific Research Foundation

Summary: High performance liquid chromatography (HPLC) is a technique for detection, identification and quantification of normal and abnormal hemoglobins widely found. We have applied this technique, for cases suspected of thalassemia/hemoglobinopathies during year 2011–2014 in 1,480 patients. Introduction: Hemoglobinopathies are the most common inherited red cell disorders worldwide. Identification of these disorders is immensely important epidemiologically and for improved management protocols. HPLC offers a accurate and rapid method for quantitative estimation of abnormal hemoglobins and the diagnosis of various syndromes. However the technique is still limited by availability and cost constraints. The aim of the study was retrospective analysis of all cases with abnormal hemoglobins and their correlation with other hematological parameters which may prove to be useful. Materials and Methods: Red cell haemolysates of 1,480 patients suspected of thalassemia/hemoglobinopathy were subjected to HPLC analysis with a bio-rad-variantÒ Analyzer. A simultaneous assessment of the RBC Indices and Peripheral Smear evaluation was done. Results: Thalassaemia was diagnosed in 12 % of total cases, most common being the beta thalassemia trait. Other hemoglobinopathies which were identified included sickle cell syndrome, Hb D and HbE syndromes alone or in combination with Beta thalassemia. Conclusion: It is important to keep Thalassemia syndromes in differential diagnosis of microcytic hypochromic anemia. Careful evaluation of other hematological parameters further aids in the diagnosis. This becomes even more relevant in our set up with migrating population adding daily. Keywords HPLC, Thalassemia, Hemoglobinopathy

Introduction: b-thalassemia is one of the commonest health problems in India. More than 200 mutations described worldwide for b-thalassemia but frequency very in different geographical and ethnic groups. In west India, occurrence of this monogenic disorder is extreme in few ethnic groups of Gujarat, especially in Saurashtra. Aim: To study spectrum of mutations causing b-thalassemia and to understand diversity of mutations among different communities of Saurashtra. Materials and Methods: In present study, b-thalassemia mutations characterized in 594 thalassemia cases from January-2002 to July-2014. Known Hb mutations analyzed by reverse Dot-blot hybridization, amplification refractory mutation system (ARMS) and PCR-Restriction enzyme analysis, where as direct sequencing of amplified DNA, Southern-blotting for deletions using specified gene probe used for unknown mutations. The mutation data analyzed statistically. Results: Hb variances, IVS1-5(G[C)(44.98 %), 619 bp deletion(11.51 %), FS codon 41/42(-CTTT)(7.95 %), IVS11(G[T)(6.90 %) and FS codon8/9(+G)(6.49 %) were significant and accounted 75.73 % of studied mutant alleles. Other 14 mutations founded. Mutation at codon 1–5(G[A) and codon5(-CT) also notable, 4.88 % each. The most affected community was Patel (21.16 %); interestingly there was no case of 619 bp deletion, 41/42(-CTTT) and codon 8/9(+G). The second most affected community was Lohana(18.34 %) were all common mutations were present. However IVS1-5(G[C) was present in almost all communities. Conclusion: Appropriate knowledge of spectrum of b-thalassemia mutations is the pre-requisite for successful prenatal diagnosis. Information on prevalent mutations in ethnic groups will help in prenatal molecular diagnosis and genetic counseling specifically to couples at high risk of b-thalassemia in Saurashtra region. Keywords Spectrum of mutation, Prenatal diagnosis, Beta-thalassemia

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Topic: Proferred OR 3

S449 hypochromic anaemia with or without splenomegaly to avoid unnecessary prescription of irons. Genetic counselling should be done to decrease the prevalence of this disease. Keywords Hemoglobinopathies, HbE thalassemia, HPLC.

Presence of Alpha Mutation does not Modify HbE Levels in Heterozygous HbE Soumita Choudhuri, Aditi Sen, Maitryee Bhattacheryya Nil Ratan SIrcar Medical College & Hospital, Kolkata-700014 Introduction: Hemoglobin E is the most common abnormal haemoglobin prevalent in eastern India. However, a significant population co-inherits an alpha thalassaemia carrier gene mutation. Co-inheritance of a thalassaemia mutation has been found to modify phenotype of various hemoglobinopathies which often leads to diagnostic challenge. This study is conducted to detect the effect of co-inheritance of a thalassaemia on HbE level. Materials and Methods: The study population consisted of 1754 individuals with HbE trait detected by population screening. HbE carrier state was detected by HPLC and confirmed by ARMS–PCR. All subjects were divided into three groups depending on their HbE level: Group A with HbE levels ranging from 10 to 20 %, group B 20 to 30 % and Group C 30 to 40 %. Co-inheritance of a-thalassaemia was assessed by GAP-PCR. Results: Co-inheritance of a thalassaemia was detected in 4.13 % of subjects in group A, 16.63 % in group B and 5.47 % in group C. Conclusion: Co-inheritance of a-thalassaemia does not modify the HbE levels. Keywords HbE levels, Alpha mutation, Heterozygous HbE disease

Topic: Hematology OR 4 HbE Disease: An Uncommon Form of Hemoglobinopathy in Southern Districts of Odisha: A Report of 2 Cases Dr. Meenakshi Mohapatro, Dr. M. K. Patro, Dr. Sujata, Dr. P. K. Das, Dr. J. Nayak, Dr. A. K. Bal, Dr. A. Choudhury Pathology Department, M.K.C.G. Medical college Objective: Haemoglobin abnormalities are the most frequent genetic disease in the world. Hemoglobinopathies are a group of genetic disorders of hemoglobin, can be of 2 types: Structurally abnormal hemoglobin variant or synthesized at reduced rate, known as Thalassemia. Some show both hence referred to as thalassemic hemoglobinopathies. Hemoglobin E is a common b-chain hemoglobin variant in the world with a high prevalence in Southeast Asia. In India North-Eastern states show higher prevalence. HbE is a thalassemic hemoglobinopathy results from a single nucleotide substitution leading to a structurally abnormal variant hemoglobin causing a b+thalassemia phenotype. I am presenting 2 cases of Hb E disease to create awareness. Materials and Methods: 2 cases a 6 year female child and a 25 year female presented with anemia for evaluation. Each case was evaluated by performing CBC, sickling test, Hb Electrophoresis, & HPLC. Result: The cases show microcytic hypochromic and normocytic normochromic anemia respectively with high RDW, raised polychromasia and normoblasts along with good number of target cells in the peripheral smear suggesting a haemolytic etiology. Sickling test was negative in them. Agarose gel electrophoresis at alkaline pH and HPLC showed in the case 1 2 bands at F & A2/E regions and in the second case showed 2 bands at A2/E & A regions. Impression: Eb thalassaemia/homozygous E thalassaemia HbE heterozygous. Conclusion: All health care providers should consider it in the differential diagnosis of microcytic

Topic: Hemoglobinopathy OR 5 Antenatal Screening for Hemoglobinopathies with HPLC N. Shah, Y. Khonglah, V. Raphael, B. Swer, S. Singh* *Departments of Pathology and Obstetrics & Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences, Shillong Summary: Hemoglobinopathies are a group of genetic disorders of hemoglobin with 1.5 % of the world’s population being carriers of beta thalassemia. This hospital based study in antenatal mothers is done to ascertain the prevalence of hemoglobinopathies and thalassemia carriers. The prevalence is 14.99 % with HbE heterozygous being the commonest (10.06 %). Introduction: Hemoglobinopathies are the most commonly encountered monogenic disorders of blood in Southeast Asia and Indian subcontinent. Screening of individuals at increased risk of being carriers for thalassemia and hemoglobinopathies, can identify couples with a 25 % risk of having a pregnancy with a significant genetic disorder, for which prenatal diagnosis is possible. This study is done to know the prevalence of Hemoglobinopathies and variant of hemoglobin using cation exchange high performance liquid chromatography (CEHPL) Materials and Methods: 2 ml of venous blood was collected in EDTA vials from the pregnant mothers after informed consent. The blood was subjected to complete hemogram, peripheral blood smear and HPLC using Variant Hemoglobin Testing System (BioRad Laboratories). Beta thalassemia short programme was used. Results: 467 blood samples from various ethnic groups were evaluated .70 (14.99 %) samples showed features of hemoglobinopathies by HPLC. There were 47(10.06 %) cases of HbE heterozygous, 12(2.57 %) cases of HbE homozygous, 9(1.92 %) of Beta Thal trait, & 2(0.43 %) of double heterozygous. Conclusion: This study showed a high prevalence of hemoglobinopathies in antenatal mothers necessitating an appropriate screening strategy for antenatal mothers. We also conclude that HPLC is a sensitive technique for studying hemoglobinopathies during pregnancy and maybe utilized for screening. Acknowledgment This study was funded by Department of Biotechnology, GOI Keywords Shemoglobinopathy, Antenatal mothers, HPLC

Topic: Laboratory Haematology OR 6 Profile of Haemoglobinopathies in Odisha: A 5-year Institutional Study Siva Saumendra Sahoo, Rabindra kumar Jena, Pranati Mohanty, Bidyut Prava Das Department of Pathology, SCB Medical college, Cuttack Introduction: Thalassemias and other haemoglobinopathies are highly prevalent in Odisha & other states of eastern India. Accurate and precise separation of haemoglobin types, together with reliable quantitation, are essential for differential diagnosis and effective

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S450 management of these diseases. Objectives: To study the pattern of Hemoglobinopathy in Odisha-a tertiary centre study of 5 years duration. Materials and Methods: Patients showing features of Haemolytic anaemia in Peripheral Smear (both children and adults) were evaluated with automated capillary zone electrophoresis (Minicap Sebia): 2 ml of EDTA blood after centrifugation & discarding the supernatant was used; Effective quality control was maintained. Results: Total number of cases—5,671. Number of cases with normal study—2,458. A normal haemoglobin cases—3,213. Beta-thal(trait)— 628(19.54 %); beta-thal(homozygous)—473(14.72 %); SCD(trait)— 762(23.72 %); SCD(homozygous)—462(14.38 %). Sickle beta-thalassemia—533(16.59 %); HbE beta-thalassemia—57(1.77 %); HbE (disease)—175 (5.45 %); HbE(trait)—98(3.05 %) alpha-thalassemia— 25 (0.78 %). Conclusion: Highest no. of cases of Sickle cell trait followed by beta-thalassemia trait. Lowest no. of cases of alpha-thalassemia followed by HbE thalassemia. High prevalence of cases of sickle cell with co-existent beta-thalassemia. Traits more common than homozygous variants except in cases of HbE disease. Abnormal Haemoglobin patterns—SCD(trait) [ beta-thalassemia(trait) [ Sickle beta-thalassemia [ beta-thalassemia(homozygous) [ SCD(homozygous) [ HbE(disease) [ HbE(trait) [ HbE thalassemia [ alpha-thalassemia. Keywords Automated capillary zone electrophoresis, Thalassemia, Hemoglobinopathy

OR 7 Correlation of Serum Ferritin and Liver Enzymes Derangement in Thalassemia Dr. Reeta Meena, Prof. Dr. R. L. Suman, Dr. Jaskaran, Dr. Shivlal R.N.T Medical College, Udaipur, Rajasthan Summary: Serum ferritin increases with repeated blood transfusion deposited in liver, responsible for liver dysfunction which manifests in form of deranged liver enzymes. We studied correlation of serum ferritin with onset of liver enzyme derangement in thalassemia major patients. Out of 55 studied patients, once the serum ferritin reaches 1,000 ng/ml and number of blood transfusion cross 30, liver enzyme derangement starts beyond normal limit. So all thalassemic children should be screened and correlated with increasing serum ferritin to identify early liver dysfunction. Introduction: Thalassemia is a secondary iron loaded state. Iron overload is due to both increased absorbtion of iron from gut and from transfusion of blood. Liver is earliest site of iron deposition in regularly transfused patients and common cause of morbidity. Liver enzymes like SGOT and SGPT are raised in transfusion dependent thalassemia major patients. Iron induced liver injury is often characterized by the development of portal fibrosis and eventually, cirrhosis. Exact level of serum ferritin at which liver enzymes derangement starts is not well documented yet. Materials and Methods: The study was conducted on 55 thalassemic children registered at Balchakitsalya, Udaipur from January 2012 to November 2012. The tested group aged from 4 to 20 years. All patients were transfusion dependent at a rate one to two times monthly and all patients were HIV, HCV, & HBsAg free. Their serum ferritin was estimated and liver functions were studied by measuring liver enzymes (SGOT, SGPT). Results: In this study, every child on an average had taken 84.65 number of blood transfusion. Serum Ferritin concentration is increased in thalassemic children with average of 2130.33 ± 859.85 ng/dl against the normal value of 70–140 ng/dl. So a positive correlation exists between no. of blood transfusion & serum ferritin level with correlation(r) of +0.33. In present study SGOT & SGPT also raised with average of 71.37 ± 24.16 and 62.35 ± 23.75 IU/L. These changes crosses

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 norms of 50 and 40 IU/L as the serum ferritin reaches around 1000 ng/dl. In present study there is positive correlation between serum ferritin & liver enzymes (Pearson’s bivariate correlation coefficient r = +0.87 % = 0.84). Conclusion: We recommend that every thalassemic children should be screened of liver dysfunction as early as no. of blood transfusions are reaching around 30 or serum ferritin level exceeds [1,000 ng/dl.

Topic: Haematology OR 8 Acute Leukemias of Ambiguous Lineage: Case Series from a Tertiary Care Centre in Western India Anupama G. Patil, Tanvi Gupta, Jyoti Sawhney, Biren Parikh, Priti Trivedi, Manoj Shah Department of pathology, Gujarat cancer and research institute, Ahmedabad, India Introduction: A minority of acute leukemias (\4 %) have features that show no clear differentiation along a single lineage and hence designated as acute leukemias of ambiguous lineage (ALAL). These include leukemias with no lineage-specific antigens, i.e. acute undifferentiated leukemias (AUL) and those which express antigens of more than one lineage, i.e. mixed phenotype acute leukemias (MPAL). The diagnosis of these rare entities rests primarily on immunophenotyping and hence a constellation of markers need to be demonstrated for their definitive diagnosis. In the present study, we have reviewed the clinical data of 11 patients diagnosed at our institute, according to the 2008 WHO criteria for ALAL. Materials and Methods: Diagnosis was based on WHO classification, clinical details, morphology, cytochemistry, immunophenotyping and molecular genetics. Results: Among the 11 cases studied, 02 cases were MPAL with t(9;22) (q34; q11.2); BCR-ABL1, 03 cases were MPAL:B/myeloid, NOS, 04 cases were MPAL:T/myeloid, NOS and 02 cases were reported as AUL. Conclusion: ALAL are a rare group of disorders which arise from a multipotent progenitor cell and they carry an extremely poor prognosis. Morphologically, ALAL has a heterogeneous presentation as either lymphoblastic or myeloid leukemia. To conclude, an expertise on the part of the morphologists is required along with a comprehensive panel of immunophenotypic markers for the diagnosis of ALAL. Besides immunophenotyping, cytogenetics is also recommended in all such cases in view of its prognostic relevance. Keywords Ambiguous lineage leukemias, Immunophenotyping, Undifferentiated

OR 9 Immunological Dynamics During Intensive Acute Lymphoblastic Leukaemia Chemotherapy Alka Khadwal1, Vikas Sharma1, Amit Rawat2, Sunil Arora3, Subhash Varma1 Department of Internal Medicine, 2Department of Pediatrics, Department of Immunopathology, PGIMER Chandigarh

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Summary: In the prospective study of 34 non pediatric acute lymphoblastic leukemia (ALL) cases, both cellular and humoral immunity was found to be negatively affected during intensive chemotherapy for ALL. Background: Patients of ALL are immune-suppressed as a result of disease and/or chemotherapeutic agents. Material and Methods:

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Serum levels of IgG, IgA and IgM and lymphocyte subsets (T-, B- and NK- cell counts were measured prospectively in 34 ALL patients (age [13 years) before induction, post induction, post consolidation and at onset of maintenance chemotherapy. Immunoglobulin levels were estimated by nephlometry and lymphocyte subsets were enumerated by flow-cytometry. All values were compared with Indian reference value available. Results: Serum IgA, IgM and IgG values were lower than reference range (p = 0.00) before start of induction (1.38 ± 0.14, 1.13 ± 0.14 and 12.19 ± 0.86, g/L respectively) and continued to decrease even at the onset of maintenance (0.02 ± 0.11, 0.38 ± 0.06 and 6.04 ± 0.26 respectively).B cell counts were elevated at diagnosis(1.15 ± 0.08 9 109/L) decreased post induction(0.08 ± 0.042 9 109/L; p = 0.00), post consolidation (0.01 ± 0.00; p = 0.00) and at start of maintenance (0.01 ± 0.00; p = 0.00)T cell counts were within normal range at diagnosis (1.76 ± 0.36 9 109/L), decreased post induction (1.16 ± 0.20 9 109/L; p = 0.99), reached normal post consolidation (1.58 ± 0.36 9 109/L) and decreased again at start of maintenance (1.25 ± 0.18 9 109/L; p = 1.00).NK cell count at diagnosis was normal (0.53 ± 0.26 9 109/L), decreased post induction (0.14 ± 0.02 9 109/L; p = 0.00), recovered post consolidation (0.3 ± 0.09 9 109/L; p = 0.007) and dropped at the onset of maintenance (0.13 ± 0.02 9 109/L; p = 0.00). Only 17/34 reached maintenance (8 = induction deaths, 4 = abandoned therapy, 3 = withdrew and 3 = relapsed. Conclusion: Humoral immunity of ALL patients undergoing intensive chemotherapy was more severely affected than cellular immunity.

Topic: Haematological Malignancy OR 10 Outcome of BFM-90 Protocol in Childhood all: Experience from a Tertiary Care Centre of Eastern India Dr. Ashutosh Panigrahi, Dr. M. Kumar, Dr. T. K. Dolai, Dr. R. De, Dr. S. Dutta, Dr. P. K. Mandal, Dr. M. Bhattacharyya, Dr. P. Chakrabarti Department of Hematology, N.R.S. Medical College and Hospital, Kolkata-14 Introduction: ALL-BFM 90 is one of the established treatment protocol for childhood acute lymphoblastic leukemia. We tried to establish the efficacy and safety of the same in our pediatric patients. Materials and Methods: Prospective and retrospective analysis of records of childhood ALL patients from June 2009 to June 2014 was done with standard Statistical analysis. Results: 54 patients were analyzed. The mean age was 9.4 years (range 1.5–18 years) with M:F = 1.7:1. SRG and MRG were 78 % (n = 42) and 21 % (n = 11) respectively. The only HRG patient discontinued therapy. No Ph +ve ALL was documented. Two were acute leukemia of ambiguous lineage. Median observation time was 2.8 years. CR rate was 98.6 %. Treatment related mortality was 5(9.3 %). One-year event-free survival was 84.2 % (SRG 87 %, MRG 81 %) and OS was 91 %. All 4(6.5 %) relapses were SRG and were late relapses. Median duration of relapse was 13.4 months. Predominant adverse events were grade 2–3 cytopenia (100 %), infections (72.8 %), transaminasemia (67 %), neurological (21 %). Mean duration of interruption of therapy was 70 days without any affect on outcome. Conclusion: BFM-90 is an effective protocol with acceptable toxicity even for Indian patients. The results are comparable to previous studies. Interruption of therapy had no influence on EFS. Long term follow up on a large cohort is required. Keywords Childhood ALL, BFM-90, Indian patients

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Topic: Paediatric Haematology Oncology OR 11 Early T Precursor Acute Lymphoblastic Leukemia: Experience from a Tertiary Care Centre in India Dhwanee Thakkar1, Jasmita Dass2, Nita Radhakrishnan1, Veronique Dinand1, Sabina Langer Kumar2, Amrita Saraf2, Manorama Bhargava2, Anupam Sachdeva1, Tanvi Khanna1 1 Paediatric Haematology Oncology and Bone Marrow Transplant Unit, Sir Ganga Ram Hospital, New Delhi; 2Deparment of Haematology, Sir Ganga Ram Hospital, New Delhi

Introduction: The immunologic subtypes of T-ALL corresponding to different stages of T-cell ontogeny are thymic/cortical T-ALL, mature/ medullary T-ALL and early T-cell precursor (ETP) ALL. ETP ALL is characterised by CD1a-, CD8-, CD5weak with stem-cell/myeloid markers. ETP ALL represents about 15 % of T-ALL and carries a different response to treatment as compared to cortical T-cell ALL. Aims/Objectives: To reanalyse the immunophenotyping of all previously diagnosed T-ALL cases and compare the demographic profile and outcome of ETP ALL with other T ALL cases. Materials and Methods: We conducted a retrospective analysis of patients who presented to our center between 2009 to 2014 and were diagnosed as T-ALL based on bone marrow aspiration biopsy and immunophenotyping. Clinical/laboratory records and treatment outcome of all patients were analysed. Results: 30 patients were diagnosed as T-ALL from 2009 to 2014. Immunophenotyping identified 3 out of these 30 patients (10 %) as ETP-ALL. Of the 27 patients of T-cell ALL, M: F ratio was 3.5:1 and the mean age at presentation was 7.35 years (range 1.6–16 years). The mean TLC at presentation was 107,585 ll (range 1,700–443,000 ll) and 13 patients (48.14 %) had hyperleukocytosis (TLC [100,000 ll). Of the 27 patients, 7 died, 6 were LFU and 1 patient left treatment. 13 patients are in remission of whom 5 have completed treatment (median follow-up 3.9 years) and 8 are still on treatment. The patients with ETP-ALL had a M: F ratio of 2:1, mean age at presentation 7 years (4–10 years). The mean TLC at presentation was 83,233 ll (range 6,700–199,000 ll), 1 patient (33.33 %) had hyperleukocytosis. Of the 3 patients, 1 patient did not attain remission and discontinued treatment, whereas the other 2 are in CR1 in maintenance. One-year overall survival was 65.6 ± 11.1 % for the whole cohort. Event-free survival (EFS) was not significantly different in ETP-ALL and T-ALL (66.7 ± 27.2 % vs. 63.2 ± 10.7 %). Conclusions: With short duration follow-up, ETP-ALL didn’t show a significant difference in outcome in our cohort. A longer duration of follow-up with a larger cohort is required to establish the differences in the profile and outcomes of T-ALL and ETP-ALL. Keywords ETP-ALL, T-ALL

OR 12 Impact of State Sponsored Health Insurance Scheme in Adolescent and Adults with ALL in a Developing Country Ravisankar Arigela, Rangaraman Ganta, Srividya Nasaka, Ramu Kandula, Stalin Bala, Ashok Kumar Pillai, Vijay Gandhi Linga, Sadashivudu Gundeti, Lakshmi Srinivas Maddali, Raghunadharao Digumarti Department of Medical Oncology, Nizams Institute of Medical Sciences, Hyderabad-500082, Telangana

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S452 Introduction: Lack of treatment compliance and high treatment cost has been one of the important factors for poor outcome of ALL in the developing world. Adolescent and adult patients with ALL have poorer prognosis compared to children. These groups form a sizeable proportion of patients with ALL and are under represented. In this study, the impact of the introduction of state health insurance (SHI) on treatment compliance and overall survival in adolescents and adults with ALL have been analysed. Materials and Methods: Adolescents and adults with ALL treated during the period 2003–2011 have been studied retrospectively using medical records and tumor registry data. They were categorised into two groups, group A consisting of patients entered into the study from 2003 to 2006 (prior to introduction of SHI) and group B with patients treated from 2008 to 2011(after introduction of SHI in 2007). Compliance and overall survival (OS) have been studied. Relapse, lost to follow up and death were considered as adverse events. Overall survival of the groups was compared using Kaplan– Meier survival analysis. Results: A total of 135 patients were included in the study of which 65 were in group A and 70 in group B. The median age in years (IQR) was 18 (4–24.5) and 19 (15–26) respectively in groups A and B. Compliance rate improved significantly (p = 0.038) with defaulters being 25 (38 %) and 9 (13 %) in the groups A and B respectively. Median OS (IQR) was 9 (4–27) and 12 (5–26) months in the groups A and B respectively (p = 0.476). Conclusion: Introduction of SHI has significantly improved treatment compliance. Although statistically not significant, OS improved by 3 months.

Topic: Clinical/Adult Haematology OR 13 Prognostic Significance of CD34+CD38low/2CD123+ Leukemic Stem Cells in Acute Leukemia by Seeing the Response to the Standard Induction Chemotherapy Singh Pawan Kumar*, Maiwall Rakhi#, Sharma Rahul*, Misra Pravas C*, Seth Tulika*, Mahapatra M*, Tyagi Seema*, Pati HP*, Saxena Renu* *Department of Hematology, AIIMS, New Delhi, #Department of Hepatology, ILBS, New Delhi Introduction: It is believed that acute leukemia arise from stem cells called leukemic stem cells (LSC) and they are usually resistant to chemotherapy. These are implicated in relapse of the disease. They can easily be quantified by flowcytometry by the presence of CD34+CD38low/-CD123+ clone. We aimed to study their prognostic significance in terms of response to induction therapy and survival at induction. Materials and Methods: Prospective single center cohort study which included 80 patients of acute leukemia. Bone marrow samples at diagnosis were analysed by flowcytometry to quantify the LSC clone. Patients of both ALL and AML were given the usual induction chemotherapy. Response was assessed by peripheral blood blast clearance, interim bone marrow blast assessment, CR status and survival at induction. Results: Both ALL and AML had presence of LSC clone. T-ALL patients did not have presence of any significant LSC clone. In ALL low LSC clone size was associated with early clearance of blasts in peripheral blood (p = 0.022) and day 7 bone marrow (p = 0.013). In AML LSC clone size was associated with high risk group (p = 0.021). Low LSC clone size was associated with early blast clearance in peripheral blood in AML (p = 0.032) and good survival (0.002). Conclusions: Presence of high LSC clone is associated with poor blast clearance in peripheral blood and bone marrow and poor survival in AML patients. Keywords Leukemic stem cells, Acute leukemia, Survival, Flow cytometry

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 OR 14 Clinical Profile and Outcome of Paediatric ALL with CNS Involvement at Diagnosis D. Tarangini, Nita Radhakrishnan, Veronique Dinand, Anupam Sachdeva Paediatric Hematology Oncology and Bone Marrow Transplant Unit, Institute of Child Health, Sir Ganga Ram Hospital, New Delhi Background: CNS involvement in ALL at presentation is uncommon accounting for less than 5 % of children with ALL. Presence of blasts in CSF at diagnosis is associated with increased risk of CNS relapse. Aims/Objectives: To study the clinical profile and outcome of Acute lymphoblastic leukemia with CNS involvement at diagnosis. Materials and Methods: We conducted a retrospective analysis of ALL patients with CNS involvement at diagnosis between 2005 and 2014. Case records of all patients were analysed for clinical presentation, treatment and outcome. CNS directed therapy consisted of weekly intrathecal methotrexate during induction (2 extra LPs), high-dose methotrexate (3 cycles of 6 g/m2 in UKALL-XI; 4 cycles of 5 g/m2 in BFM-95); high-risk (HR) patients received 1 extra TIT during HR blocks 2 and 5 (BFM-HR arm). Cranial radiotherapy (CRT) was given to HR and T-cell. Results: Of 332 ALL patients, 20 (6 %) patients had CNS involvement. M:F ratio was 4:1, mean age was 6.8 years (0.5–19). 6(30 %) patients had CNS symptoms at presentation. Median WBC count was 18,250 cu mm (2,700–400,000); hyperleukocytosis was observed in 4 (20 %). 12 patients (60 %) had CNS-2, 5(25 %) CNS-3 and 3-TLP+. 14(70 %) were Pre-B CALLA +ve, 4(20 %) T-cell and 2 pro B-ALL on immunophenotyping. Cytogenetics was normal in 8(40 %), hyperdiploidy in 4(20 %). BCR-ABL and MLL rearrangement were positive in one patient each. 6 patients had HR features. 13(65 %) patients received treatment as per BFM-95 protocol, 6(30 %) UKALL-XI and 1(5 %) Interfant-06. 1 HR patient and 1 T-cell received cranial RT. 7(35 %) completed treatment, 5 (25 %) are on therapy, 5 (25 %) patients died (sepsis-2, ICH-2, relapse and progression-1(early CNS + BM)), and 3 (15 %) were LFU. During induction 2 patients died, 2 abandoned treatment at our centre and 2 didn’t achieve remission. 4 years OS as well as EFS is 70.9 ± 11.3 %. 3 year OS for Pre-B, T-cell and Pro-B are 82.5, 50.0 and 0 % respectively. Conclusions: CNS involvement in ALL is rare. Effective systemic chemotherapy with intrathecal therapy can improve outcome of ALL patients with CNS involvement.

Topic: Minimal Residual Disease in ALL OR 15 Evaluation of Minimal Residual Disease by Real-Time Quantitative Polymerase Chain Reaction (RQ-PCR) for Wilms’ Tumor 1 Gene Expression and Multicolour Flowcytometry, in Pediatric B Lineage Acute Lymphoblastic Leukemia Sandeep1, Neelam Varma1, Shano Naseem1, Man Updesh Singh Sachdeva1, Deepak Bansal2 Department of hematology and 2Department of pediatrics, PGIMER, Chandigarh

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Summary: MRD testing provides strong prognostic information in childhood ALL. WT1 gene expression has been evaluated as a candidate pan leukemic marker to simplify MRD detection. Our study demonstrates flowcytometry to be more sensitive compared to WT1 gene expression for MRD analysis and high WT1 expression at diagnosis correlates significantly with day 15 MRD levels by FCM.

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Introduction: MRD determination involves the measurement of very low levels of leukemia using sensitive techniques which at present are complex, time consuming and require expertise for performance and interpretation. A ‘‘panleukemic’’ marker such as WT1 which is frequently over expressed in acute leukemia could simplify MRD detection and serve as a useful prognostic marker. Materials and Methods: Flowcytometric (FCM) immunophenotyping and RQ-PCR for WT1 gene expression were performed using bone marrow of 23 pediatric CALLA positive B ALL cases. Day 15 MRD analysis by both these methods was performed on 11 BM samples of patients who showed WT1 over expression at day 0. Results: WT1 over expression at diagnosis was found in 69.5 % of cases (16/23). MRD was detectable in 54.5 % of cases by WT1 RQ-PCR and by FCM in 72 % of cases. A statistically significant correlation was seen between WT1 NCN at diagnosis with MRD levels detected by FCM. Conclusion: Flow cytometric immunophenotyping is a more sensitive modality for detection of mid induction MRD in ALL compared to RQ PCR for WT1 gene expression. However, WT1 levels at diagnosis correlated well with day 15 MRD by FCM. Keywords Minimal residual disease, Wilms’ Tumor 1 gene, CALLA positive B ALL

OR 16 Correlation of Bone Marrow Morphology, Flowcytometry and Immunohistochemistry in Chronic Lymphoproliferaltive Disorders Dr. Arisetty Himabindu, Dr. Sushma Belurkar Department of pathology, Kasturba medical college and hospital, Manipal, Upupi Dist., Karnataka 576104 Objective: To correlate the diagnosis of chronic lymphoproliferaltive disorders on bone marrow with flowcytometry, lymphnode biopsy & immunohistochemistry. Introduction: Though bone marrow aspiration & biopsy have been the gold standard in diagnosis of CLPD for many years, the advent of flowcytometry and immunohistochemistry have facilitated more specific diagnosis, better treatment monitoring of these cases. Materials and Methods: Retrospective study conducted in the Clinical laboratory of Kasturba Hospital, Manipal. 42 cases which were diagnosed as CLPD/CLL on bone marrow aspiration and biopsy, during the period January 2012 to August 2014 were retrieved and their diagnoses were compared with the flowcytometry report or lymph node biopsy with immunohistochemistry report wherever available. Results: Out of the 42 cases, 15 cases had only flowcytometry report, 10 cases had lymphnode biopsy, 6 cases had both flowcytometry and lymphnode biopsy, 11 cases which had neither were excluded from the study. The results are tabulated as below:

Bone marrow CLPD/CLL Flowcytometry (15) 10

CD5, 19, 23+, CD25-

Chronic lymphocytic leukaemia

3

CD5, 10-, CD103+

Hairy cell leukaemia

1

CD5, 10-, CD38, 45+

Atypical CLL

1

CD2, 3, 5+; CD19-

T-cell lymphoma

Histopathology (10) 5

CD20, 23, 5+; CYCLIND1- CLL

2

CD20, 5, CYCLIND1+

Mantle cell lymphoma

S453

Bone marrow

CLPD/CLL

2

IHC not done

NHL not subclassified

1

CD5, 20+; CD3-; CYCLIND1 focally positive

Diffuse large B cell lymphoma

Bone Flowcytometry marrow

Histopathology

CLPD 6 3

CLL

CD20, 23, 5+

1

Follicular lymphoma

CD20, CD10, BCL2+; CD3, 5-

1

Marginal zone lymphoma CD19, 20+; CD5, 10-

CLL CD20, CD5, CD23+; CYCLIND1 focally positive

1

CLL

Reactive follicular hyperplasia

18 out of the 31 (54 %) cases showed concordance between morphology and flowcytometry/immunohistochemistry diagnoses. Impression: Bone marrow aspirate and biopsy play an important role in the diagnosis of CLPD however flowcytometry and histopathology with IHC increases the sensitivity and helps in further sub classification of these cases Keywords Chronic lymphoproliferaltive disorders, Flowcytometry, Bone marrow

OR 17 An Interesting Mix of Seen and Unseen Cases of Plasmablastic Lymphoma: A Review of 11 Cases Manasi Mundada, Faiq Ahmed, Sudha Murthy, K. Suseela, M. Bharati, F. Daphne, K. Rachna, G. Sandhyadevi, Radha Rani, Senthil Rajappa*, Krishna Mohan MVT*, Rao TS* Departments of Lab Medicine, Medical/Surgical Oncology, Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad Summary: Plasmablastic lymphomas (PBL) are rare aggressive B cell neoplasms occurring predominantly in HIV setting and also increasingly been reported in immunocompetent individuals. Oral cavity is the most commonly occuring site in immunocompromised host, while different sites like lung, stomach, cervical lymph nodes, nasal cavity, small intestine, larynx has been described in HIV negative patients. We encountered 11 cases of PBL in the time period of 6 years, showing a varied clinical presentation and unique pathological features. Introduction: PBL are type of Non Hodgkin’s lymphoma in HIV scenario and constitute 2.6 % of lymphomas in this settings.These are characterized by a terminally differentiated B-cell immunophenotype with minimal or absent expression of leukocyte common antigen (CD45), epithelial markers and B-cell antigens (CD20 and CD79a) but is invariably immunoreactive for plasma cell markers such as CD138, CD38, MUM1/IRF4. Materials and Methods: Cases retrieved from the archives of BIACH& RI. Total of eleven cases diagnosed between Jan 2009 and Aug 2014, Histopathological examination along with Immunohistochemistry was carried using panel of antibodies CD45, CD20, CD10, CD5, CD138, CD30, MUM1, PAX5, Alk1 and Ki67.EBV encoded RNA In Situ Hybridisation (EBER ISH) was done in few cases. Results: The mean age of

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S454 presentation for these 11 cases PBL was 46 years. M:F ratio was 10:1. Extranodal involvement was most frequent and the various sites of involvement were oral cavity(3 cases), thyroid, jejunum, rectum, liver and nasal cavity each case each. Three cases showed lymph nodal disease. Most consistent marker which was positive was CD138 and MUM1, while CD45 and CD20 positivity was noted in 10 and 02 cases.EBV testing rapid in situ hybridisation was done in two cases. Conclusions: PBL are rare neoplasms, a structured approach is required for its varied presentation. Morphology, IHC and molecular tools should be used in congruity for definite diagnosis.

Topic: Laboratory Hematology OR 18 Genetic Basis of Hairy Cell Leukemia Reveals High Frequencies of Mutations in BRAF V600E, U2AF1 and MAP2K1 Genes Nikhil Rabade1, Asma Bibi1, Shruti Chaudhary1, Asmi Chakraborty1, Ayushi Goyal1, Shrutika Munot1, Prashant Tembhare1, P.G. Subramanian1, S.K. Hasan1, Bhausaheb Bagal2, Uma Dangi2, Hasmukh Jain2, Manju Sengar2, Navin Khattry2, Hari Menon2, Sumeet Gujral1, Nikhil Patkar1* 1

Hematopathology Laboratory, Tata Memorial Centre, 2Adult Hemato-lymphoid Disease Management Group, Department of Medical Oncology, Tata Memorial Centre

Objectives: The somatically acquired V600E mutation of the BRAF gene has been recently described as a molecular marker of hairy cell leukemia (HCL). In those cases which are BRAF negative, mutations in MAP2K1 and RNA spliceosome encoding gene U2AF1 have recently been described. In this large study we sought to evaluate the baseline frequencies of these gene mutations in cases of HCL. Methods: Genomic DNA was extracted from stained bone marrow aspirate smears of HCL patients. DNA was subjected to a modified ARMS PCR technique followed by capillary electrophoresis on an ABI3500 genetic analyzer. The PCR was designed to amplify a 200 bp control product, a 144 bp BRAF V600E specific amplicon and a 97 bp wild type specific amplicon. Exons 2 & 3 of MAP2K1 and U2AF1 genes were sequenced in BRAF negative cases by Sanger sequencing. Results: Majority of patients 88.6 % (39/ 44) with a morphological and immunophenotypic diagnosis of HCL were positive for BRAF V600E mutation. Of the five patients that were BRAFnegative a single patient harbored p. P124L mutation in exon2 of MAP2K1 gene. This mutation has been described in papillary carcinoma of the thyroid but not in patients of HCL. Conclusion: Our findings point that analysis of BRAF mutations is a potential diagnostic tool to distinguish HCL from other B-cell lymphomas with similar features such as the HCL variant and splenic marginal-zone lymphoma. This distinction is clinically relevant, since HCL, but not HCL-like disorders, responds to therapy with interferon or purine analogues. Keywords Hairy cell leukemia, BRAFV600E

Topic: Proferred OR 19 Immunophenotyping Features (FCM) and Histomorphology in Diagnosis of Chronic Lymphocytic Leukemia Nishant Sagar, N. Gupta*, N. Khurana, T. Singh Departments of Pathology and Medicine, Maulana Azad Medical College, New Delhi-110002

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Summary: 21 cases of CLL presenting to Department of Pathology, MAMC, were evaluated using a combined approach of morphology, immunohistochemistry, histomorphology (bone marrow biopsy) and flowcytometry (FCM). FCM was found to be a useful in lineage assignment, distinguishing CLL from other lymphoproliferaltive disorders and evaluation of prognostic markers. Introduction: Role of immunophenotyping in CLL, especially in under resourced laboratories is not clearly defined. The present study was conducted to evaluate efficacy of morphology of blood/BM and histomorphology with IHC of BM biopsy and immunophenotyping for diagnosis of CLL. Materials and Methods: 21 cases of CLL were included. The diagnosis was made taking into consideration the morphology, IHC and FCM on peripheral blood and/or BM using CD45, CD10, CD5, CD19, CD20, CD23, FMC7, CD79b, CD3, CD22, CD25 and CD103, CD 38, kappa and lambda. Coombs test was performed in all the cases. Results: All 21 cases were diagnosed by correlating clinical picture, peripheral blood and/or BM aspirate. BM biopsy was available for 12 cases which confirmed the diagnosis with or without using IHC. Sensitivity of clinical picture and BM findings in diagnosing CLL was 95 %. However, when these parameters are combined with FCM, the sensitivity was 100 %. FCM helped us in evaluating the monoclonality of the patient. Using FCM, 2 cases of MBL were diagnosed. CD38 was evaluated in 15 cases for prognosis and 3 cases were positive. BM biopsy was helpful in knowing the pattern of marrow involvement and thus the tumor load. Conclusion: FCM is a useful adjunct to morphology and IHC for diagnosis of CLL. It plays a vital role in defining the prognosis of CLL and to differentiate it from other CLPDs. Keywords Flowcytometry, Chronic lymphocytic leukemia, Bone marrow biopsy

Topic: Oral Paper Presentations OR 20 Chronic Lymphoproliferaltive Disorders (CLPD): Morphologic and Immunophenotypic Study on Blood and Bone Marrow C. Pooja1, T. Roshni Paul2, Shantveer G. Uppin3, Megha S. Uppin4, G. Sadashivudu5 MD student, Department of Pathology, NIMS; 2Additional Professor, Department of Pathology, NIMS; 3Associate Professor, Department of Pathology, NIMS; 4Assistant Professor, Department of Pathology, NIMS; 5Associate Professor, Department of Medical Oncology, NIMS 1

Objectives: To study the morphology along with clinical and immunophenotypic profile of CLPD and to use the scoring system introduced by Matutes. et al. to differentiate chronic lymphocytic leukemia (CLL) from other CLPD wherever possible. Materials and Methods: Cases diagnosed as CLPD in our institute between January 2010 and December 2013 on peripheral blood and bone marrow or flow cytometry were studied. Clinical parameters, morphology, marrow infiltration pattern, immunohistochemistry and cytogenetics were analysed Results: Most common CLPD was CLL seen in 36 of the total 53 cases studied (67.9 %) followed by follicular lymphoma (FL) (four cases—7.5 %). Others included Hairy cell leukemia (HCL), Marginal zone lymphoma (MZL), Prolymphocytic leukemia (PLL) etc. Of the 35 cases with flowcytometry, typical immunophenotype was noted in 25 CLL cases. Matutes et al. scoring system with a panel of four markers (CD5, CD23, FMC7, CD22/CD 79b) was helpful. Marker expression intensity differentiated PLL from CLL. CD25, CD103, CD200 were other useful markers. Conclusion: CLL is the commonest CLPD in our setting. Morphology plays a key role

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 in judicious selection of optimal flow panel in resource limited settings. Both complement each other in arriving at a proper diagnosis Keywords Lymphoproliferaltive disease, Immunophenotype, Matutes score

OR 21 Role of Flow-Cytometry in Diagnosis of Splenic Lymphoma Pragya Joshi Vakani, Amina Shaik, S. Vanajakshi, S. V. N. Anuradha, Tejal Modi, Meenakshi Swain, G. Swarnalata Department of Pathology, Apollo Health City, Hyderabad Introduction: Splenic lymphoma (SL) is rare, accounting \2 % of all lymphomas and often show leukemic manifestations. Its differentiation from other low grade B cell lymphoma is challenging. Flow-cytometric immunophenotyping on peripheral blood (PB) and bone-marrow aspirate (BMA), with immunohistochemistry (IHC) can diagnose SL accurately. Aim: To study the utility of flow-cytometry (FC) in the diagnosis of splenic lymphomas. Materials and Methods: We studied 20 cases presented with isolated splenomegaly, suspicious of lymphoma without peripheral lymphadenopathy. Immunophenotypic analysis was carried out by FC on PB (14/20) and BMA (6/20) using the Beckman-Coulter FC500 flow cytometer with a chronic lymphoproliferaltive disorders (CLPD) panel(CD45, CD34, CD10, CD19, CD22, CD20, CD23, FMC7, Kappa, Lambda, CD103, CD11c, CD38, CD5, CD56, CD16, CD3, CD4, CD8). IHC on bone-marrow biopsy(2/20) and on splenectomy(3/20) was also done. Results: Analysis revealed that SL is more common in elderly (40–60 years) with male to female ratio 3:2. The different subtypes identified were: Splenic marginal zone lymphoma-9, Hairy cell leukemia-3, Mantle cell lymphoma-2, T-cell prolymphocytic lymphoma-1, Mature T-cell lymphoma-1, diffuse large Bcell lymphoma-1 and 3 were unclassifiable. Conclusion: We conclude that the FC immunophenotype analysis is a helpful minimal invasive approach in diagnosis of SL. It serves as a guide for further subtyping by other ancillary techniques. Keywords Flow-cytometry, Splenic lymphoma, Splenomegaly, Chronic lymphoproliferaltive

Topic: Laboratory Haematology OR 22 Spectrum of Mature Lymphoid Neoplasms in Patients with ‘‘Prominent Splenomegaly Without Significant Lymphadenopathy’’ Sreejesh Sreedharanunnia, Man Updesh Singh Sachdevaa, Pankaj Malhotrab, Jasmina Ahluwaliaa, Shano Naseema, Gaurav Prakashb, Alka Khadwalb, Prashant Sharmaa, Narender Kumara, Neelam Varmaa, Subhash Varmab, Reena Dasa a Departments of Hematology and bInternal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India, 160012

Objective: Mature lymphoid neoplasms presenting with ‘‘prominent splenomegaly without significant lymphadenopathy’’ are uncommon compared to those associated with lymphadenopathy. Their description is largely limited to few case series in the literature. The aim of our study was to analyze the spectrum of these lymphomas diagnosed by peripheral blood (PB) and/or bone marrow (BM) examination. Materials and Methods: A retrospective analysis was performed over a period of 6 years; between 2008 and 2013 using hospital information system followed by review of morphological and immunophenotypic findings.

S455 Results: Seventy-five patients presented with predominant splenomegaly during this period and were diagnosed of a lymphoma from PB/BM. The commonest lymphomas were hairy cell leukemia (HCL) (32 %) and splenic marginal zone lymphoma (SMZL) (24 %). Diffuse large B cell lymphoma (8 %), Chronic lymphocytic leukemia/small lymphocytic lymphoma (8 %), mantle cell lymphoma (2.7 %) and follicular lymphoma (1.3 %), which usually presents with lymphadenopathy were also encountered. SMZL was the commonest lymphoma amongst females and those with massive splenomegaly and lymphocytosis; while HCL was commonest in patients with pancytopenia. The nature of the lymphoid cells should be analyzed by flowcytometry and/or Immunohistochemistry (IHC) on PB, BM aspirate, or BM trephine biopsy samples even if the lymphoid infiltrate is subtle and not well appreciated on routine BM examination. IHC on trephine biopsy is especially useful to identify unusual patterns or sparse infiltration of malignant lymphoid cells. Conclusion: The careful use of ancillary techniques in BM and/or PB allows accurate sub classification making it a diagnostic investigation of choice in patients suspected of splenic lymphomas. Keywords Splenomegaly, Splenic lymphoma, Mature lymphoid neoplasms

OR 23 Evaluation of Diagnostic Usefulness of CD200 Expression in B-cell Chronic Lymphoproliferaltive Disorders Abhishek Purohit, Manoranjan Mahapatra, Seema Tyagi, Renu Saxena, Hara P. Pati Senior Resident, Department of hematology, All India Institute of Medical Sciences – New Delhi, New Delhi Aims and Objectives: The aim of the present study was to evaluate the expression of CD200 in various B cell chronic lymphoproliferaltive disorders and to assess its diagnostic usefulness. Materials and Methods: We investigated expression of CD200 by multiparametric flowcytometric immunophenotyping in 130 consecutive cases of mature B cell neoplasms. Results: Of 130 cases, 86 cases were of typical CLL and 6 cases of atypical CLL, both of these groups revealed strong and homogenous expression of CD200 on neoplastic B cells whereas 11 cases of MCL included in the study showed uniform negativity for CD200 on neoplastic B cells. The difference between percent positive cells and median fluorescent intensity (MFI) between CLL (including typical and atypical) and MCL groups was statistically significant (p \ 0.0001). Furthermore, CD200 expression in hairy cell leukemia was strong and in cases of splenic marginal zone lymphoma the expression was dim and difference of MFI in cases of HCL and SMZL was statistically significant (p = 0.009). Conclusion: In conclusion, present study expands the understanding of the CD200 expression in MBNs supports inclusion of CD200 in the routine flow cytometric panels for the differential diagnosis of MBNs.

Topic: Hematopathology OR 24 High Frequency of Beta Thalassemia Trait and G6PD Deficiency in Patients Referred as gilbert’s Syndrome for Persistent Unconjugated Hyperbilirubinemia Ganesh Kumar V, Manu Jamwal, Sanjeev Chhabra, Jasbir Kaur, Pankaj Malhotra*, Ajay Duseja#, Reena Das Departments of Hematology, *Internal Medicine and #Hepatology, PGIMER, Chandigarh

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S456 Summary: A high frequency of beta thalassemia trait and G6PD Mediterranean was found in adults referred for genetic workup for diagnosing the cause of unconjugated hyperbilirubinemia. In addition a high frequency of A(TA)7TAA genotype in UGT1A1 promoter was also found. Introduction: Causes of unconjugated hyperbilirubinemia include hemolytic anemia arising from different causes, Gilberts’ syndrome, Crigler–Najjar syndrome and drugs. We studied the prevalence of b-thalassemia trait (bTT), Gilberts’ syndrome (GS) and glucose-6-phosphate dehydrogenase (G6PD) deficiency among patients with a clinical suspicion of GS. Materials and Methods: Ninety three patients with jaundice referred for mutational study of Gilbert syndrome were analyzed. Complete blood counts (Beckman CoulterÒ LH750) and HPLC (b-Thal Short programÒ, Bio-Rad Variant II) were done. Genomic DNA samples from leucocytes were amplified using specific primers for UGT1A1 promoter and products were sequenced on ABI 3130 Genetic Analyzer, Applied Biosystems. Analysis of results was performed using the Geospiza software. PCR– RFLP for G6PD Mediterranean (563 C ? T) and G6PD Orissa (131 C ? G) were done using restriction enzymes MboII and HaeIII respectively. Beta mutations were done by ARMS PCR in cases found to be bTT. Results: Among 93 patients, we found a very high prevalence of homozygosity for GS genotype A(TA)7TAA (73.2 %); heterozygosity in 19.5 % and wild type genotype i.e. homozygosity for A(TA)6TAA in 7.3 % individuals. We found high prevalence of bTT 10.8 % in this group in comparison to the prevalence of bTT in the general population of Punjab which is 3.5 %. Seven patients (8.2 %), all males, were hemizygous for G6PD Mediterranean; G6PD Orissa was not found in any sample. Conclusion: Our study shows a high prevalence of bTT, GS and G6PD deficiency in jaundiced patients referred to genetic workup of unconjugated hyperbilirubinemia. Keywords Gilbert’s syndrome, Beta thalassemia trait, G6PD deficiency

Topic: Hemolytic anemia—AIHA OR 25 A Single Centre Study of Treatment Outcomes of Consecutive 32 Cases of AIHA in a Tertiary Care Centre B. Prashanthaa, Suchitra Shenoyb, R. Aseemb, M. Chakrapanic, Shrijeet Chakrabortid, Jyothi Kinid Departments of aHematology, bMicrobiology, cMedicine, dPathology, Kasturba Medical College, Manipal University, Mangalore Summary: Consecutive 32 patients with AIHA were analyzed in terms of ANA positivity, treatment outcomes and number of drugs required to attain remission. Secondary AIHA patients had higher mortality, morbidity and needed multiagent therapy as opposed to primary AIHA. Introduction: Generally primary AIHA remit after a course of therapy where as the secondary AIHA usually runs a prolonged and unpredictable disease course. We retrospectively analyzed 32 consecutive AIHA patients managed in the department of Hematology from September 2012 to June 2014. Materials and Methods: Retrospective cohort study from Sep 2012 to June 2014 involving 32 consecutive AIHA patients were followed up and their clinical and serological associations and the outcomes were reviewed. Results: Out of 32 patients diagnosed as AIHA, 6 patients had Evan’s syndrome. 12 patients were primary AIHA and 20 were secondary. Of the 20 patients with secondary AIHA, 15 had ANA positivity and amongst them 9 had definite SLE. There was female preponderance in secondary AIHA (17/25). 10 in the cohort achieved CR with initial steroids alone. 11 patients required additional 2nd line therapy to reach

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 remission or stable disease. Rest of the 11 patients required multiagent treatment including chemotherapy. Out of the 12 primary AIHA, 5 achieved CR and 7 patients attained a stable disease. In secondary AIHA, 12 are in partial remission, 5 patients had complications and 3 patients died. Conclusion: Most patients with ANA positive autoimmune haemolytic anaemia were found to have lower remission rates and higher chances of turning refractory to treatment. AIHA associated with malignancy had poor outcomes. Keywords AIHA, ANA, Evan’s syndrome

Topic: Haematology OR 26 Clinicohaematological Profile of Hereditary Haemolytic Anemias Dr. Rachana Lakhe1, Dr. Rajashree Ingin1, Dr. Sainath K. Andola1 Department of 1Pathology, Mahadevappa Ramapure Medical College, Gulbarga, Karnataka, India Summary: A 2 year study was taken up to emphasise the utility of routine haematological investigations in the diagnosis of hereditary haemolytic anaemias and special tests to confirm the diagnosis. A total of 51 cases were studied and majority of them were beta thalassemia major. Introduction: Hereditary haemolytic anemias are common inherited disorders causing varying degree of morbidity and mortality. These include disorders due haemoglobin defect, membrane defect, and enzyme defect. Among these, the incidence of hemoglobinopathies worldwide is 5 %. The clinical findings and preliminary haematological studies suggest the diagnosis and haemoglobin electrophoresis demonstrates the presence of an abnormal haemoglobin. Materials and Methods: Blood samples from suspected cases received in the laboratories attached to Mahadevappa Ramapure Medical College, hospitals and laboratories in and around Gulbarga were examined. Basic haematological investigations, special tests including haemoglobin electrophoresis were done. Results: Among the 51 cases were investigated for hereditary haemolytic anaemia, majority were in the age group of 1–5 years with a slight female preponderance (M:F: = 1:1.27). Most of the patients presented with severe anaemia, splenomegaly and growth retardation.

Hereditary haemolytic anemias

Number

Beta thalassaemia major

41

80.3

Beta thalassemia trait

4

7.8

Sickle Cell Anaemia

2

3.9

Sickle cell trait

2

3.9

Hb-E Thalassaemia

1

1.9

1 51

1.9

Hereditary spherocytosis Total

%

Conclusion: In the present study, the paediatric cases of hereditary haemolytic anaemias were identified based on clinical data, haematological parameters and haemoglobin electrophoresis. The definitive identification of hereditary haemolytic anaemias usually requires an array of sophisticated biochemical tests, but initial investigations and few special tests suffice to come to a preliminary diagnosis. Keywords Hereditary Haemolytic Anaemia, Thalassemia, Sickle cell

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Topic: Chronic Myeloid Leukemia

S457

Response ;

EUTOS low risk n = 38

EUTOS high risk n = 19

P value (fishers test)

CHR at 3 months

38/38 (100 %)

18/19 (94 %)

0.33

OR 27 Study of Imatinib Mesylate and Low Dose Cytarabine Versus Imatinib Mesylate Monotherapy in Patients with Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia in Tertiary Care Centre

CCyR at 12 months

30/38 (79 %)

13/19 (69 %)

0.51

MMR at 18 months

32/38 (95 %)

15/19 (79 %)

0.71

FS at 24 months

34/38 (90 %)

17/19 (90 %)

1

Priyanka Samal, Dr. U. K. Nath, Dr. S. S. Roy, Prof. M. Bhattacharya, Prof. P. Chakraborty Institute of Haematology and Transfusion Medicine Objective: to assess & compare the outcomes in patients receiving the combination of imatinib and low dose cytarabine with that of imatinib as a single agent in newly diagnosed CML-CP patients presenting in a tertiary care centre. Materials and Methods: A prospective study of 30 denovo CML-CP cases were randomized into 2 arms & compared for their outcomes, arm A—imatinib only arm and arm B—imatinib + low dose ara-c arm during the period from Jan 2013 till Aug 2014. Results: Of the 16 patients randomized to the imatinib only arm, response was assessed at 3 months by RQ-PCR for BCR-ABL1 transcript levels. BCR-ABL1 transcript levels \10 % was seen in 75 % while [10 % in 25 %. At 6 months, FISH showed CCyR in 25 %, 1–35 % ph + metaphases in 56.25 % and [35 % ph + metaphases in 18.75 %. At 12 months, conventional karyotyping showed absence of t(9;22) in 56.25 % while presence in 43.75 %. Among the 13 patients receiving imatinib +low dose ara-c, RQ-PCR for BCR-ABL1 transcripts at 3 months revealed\10 % in 85.71 % & [10 % in 14.28 %. At 6 months, FISH showed CCyR in 35.71, 1–35 % ph + metaphases in 64.28 % and [35 % ph + metaphases in 0 % cases. At 12 months, conventional karyotyping showed absence of t(9;22) in 85.71 % and presence in 14.28 %. Conclusion: The p value for the response to therapy at 3, 6 and at 12 months of starting therapy was not significant ([0.05) in the 2 arms though toxicity and days of interruption of therapy was more in imatinib + low dose ara-c arm as compared to the imatinib only arm. Keywords CML-CP, Imatinib, Imatinib + ara-c

OR 28 EUTOS Score Prediction in Pediatric CML CP Response and Outcome; Single Institute Retrospective Study G. Ranga Raman, N. Srividya, A. Ravi Sankar, G. Sada Shivudu, M. Laxmi Srinivas Nizams Institute of Medical Sciences, Hyderabad Summary: EUTOS low risk pediatric CML patients on Imatinib responded better than high risk but not statistically significant. Both groups had similar outcome at the end of 2 years. Introduction: European Treatment and Outcome Study (EUTOS) scoring system is validated recently in predicting the response and outcome in CML patients on Imatinib treatment. Data on risk stratification in pediatric CML was lacking due to its rarity [\3 %]. We attempted to the correlate response and outcome among EUTOS risk groups in pediatric CML on Imatinib treatment. Materials and Methods: Hospital records of CML CP patients aged B18 years on Imatinib from year 2010 to 2012 were analyzed for EUTOS risk score, response and outcome at the end of 2 years. Results: Data of 57 children was analyzed, with median age of 14.5 years [range 5–18 years], male preponderance [M:F = 1.14:1] and majority in low risk of EUTOS score.

At the end of 2 years, 1 had loss of CHR, 3 progressed to AP/BC in low risk and 1 had loss of CHR and 1 progressed to AP/BC in high risk group. Conclusion: EUTOS score did not predict the response and outcome in pediatric CML. Limitations: Retrospective study, small sample size, short follow up duration

Topic: Free Paper Session OR 29 Characteristics and Outcome of Patients Receiving Second Generation Tyrosine Kinase Inhibitors (TKI) as Switch Therapy for Chronic Myeloid Leukemia (CML) Uday Kulkarni, Abhijeet Ganapule, Fouzia NA, Anu Korula, Aby Abraham, Auro Viswabandya, Biju George, Alok Srivastava, Poonkuzhali Balasubramanian, Vikram Mathews Department of Clinical Haematology, Christian Medical College and Hospital, Vellore, Tamil Nadu, India Introduction: For patients with CML treated upfront with imatinib, switch over to second generation TKI is considered for either intolerance or resistance. There is limited data from India on the clinical outcomes and the variables that affect the outcome following such switch therapy. Materials and Methods: We undertook a retrospective analysis of patients treated with second generation TKI for imatinib intolerance or resistance at our centre. Results: From January 2008 to April 2014, 35 patients were initiated on second generation TKI for CML. Mean age at diagnosis was 40.7 ± 12.93 years. Twenty-six were males. Median Sokal score was 1.13 (range 0.49–145.55). Median Hasford score was 1171.08 (range 0–3,827.3). Sixteen patients had tyrosine kinase mutations and all these mutations were reported to be sensitive to second generation TKI. The median time from diagnosis to switch-over to second generation TKI was 30 months (range 3–188 months). The indications for switching therapy were intolerance alone in 2 patients, failure alone (according to ELN guidelines) in 24, failure with intolerance in 4 and failure with disease progression in 5. Nine patients were initiated on dasatinib, 1 on bosutinib and 25 on nilotinib. The median duration of follow-up was 10 months (range 1–76 months). Following the switch over, 2 patients developed intolerance, 14 patients had optimal response, 2 had a sub optimal response, 2 had intolerance after a suboptimal response, 7 had failure and response was not assessed in 8. Amongst these patients, 5 underwent additional switch to third line therapy (nilotinib—4; dasatinib—1). The median duration between imatinib failure and initiation of second generation TKI was 8 months (range 0–104 months). Conclusion: Patients treated with second generation TKI for imatinib resistance or intolerance have clinically relevant response rates. Keywords Second generation tyrosine kinase inhibitors, Chronic myeloid leukemia

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OR 30 Role of NOA Program Impacting Survival in CML: Study from North India Uday Yanamandra, Kamal Kant Sahu, Kanagaraja Karunakaran Parathan, Nidhi Jain, Shanoo Naseem, Alka Khadwal, Gaurav Prakash, Neelam Varma, Subhash Varma, Pankaj Malhotra* Post Graduate Institute of Medical Education and Research Summary: The Novartis Oncology Access (NOA) or Glivec International Patient Assistance Program (GIPAP) is a patient-access program sponsored by Novartis Oncology and administered by The Max Foundation (MAX) that provides imatinib free of charge to patients in resource-restricted countries who are not able to afford this treatment. There are no studies to-date from India on the efficacy of this program. Introduction: Socioeconomic status of the patient is an important component in deciding the outcomes of cancer treatment in the resource constraint settings. We studied the influence of NOA program versus patients on self-purchased Imatinib and compared the compliance/survival of CML patients at tertiary care center. Materials and Methods: It is a retrospective observational study wherein survival of 225 adolescents and young adults (AYA) and 110 elderly CML patients managed at tertiary care center in North India over last 14 years were analyzed. Results: The mean age of AYA-CML patients was 23.08 years (12–29) and elderly-CML was 65.35 years (60–80). Compliance was better in the self-purchased group when compared to the patients under NOA, this could be explained either because of the complacency on part of the patients due to free drug supply or partly also attributable to intermittent break in supplies of the drugs. Frequency of change in dose/type TKI was significantly lower amongst patients under NOA (p \ 0.001). Cumulative Survival was much better in patients under NOA program than self-purchased group, with the difference being statistically significant (p \ 0.001) in both AYA-CML and in Elderly CML (Figs. 1, 2). The difference was much more pronounced in AYA group when compared to the Elderly group. In the elderly CML we have certain patients who were never on TKI and were found to have the worst survival. Conclusion/ Diagnosis/Impression: Patient care program which is carried out in a systematic manner would help in improving the outcomes of CML patients.

Fig. 1 .

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Fig. 2 .

Topic: Hemostasis and Thrombosis OR 31 Platelet Function Disorders in patients with Mild Bleeding Symptoms Dr. S. Karthik, Dr. Shyamali Dutta, Dr. T.K. Dolai, Dr. Rajib De, Dr. Prakas K. Mondal, Dr. Prantar Chakrabarti NRS Medical College and Hospital, Kolkata-14 Summary: Light transmission aggregometry was performed to investigate the cause of mild bleeding symptoms in 51 patients. Abnormal patterns of platelet aggregation suggestive of an inherited platelet function defect was present in 25.4 %. The commonest abnormality was Aspirin like Defect (13.7 % of patients with mild bleeding). Introduction: We present results of light transmission aggregometry (LTA) done in our institution on patients with mild bleeding symptoms, in whom routine coagulation work up and platelet counts were normal. Materials and Methods: 80 patients who had undergone LTA over last 1 year were reviewed for history of mild bleeding with objective scoring system. Patients were considered to be mild bleeders if the bleeding score was less than 5. Patterns of maximal platelet aggregation with various agonists were analysed for evidence of an inherited platelet function defect (IFD). Results: 51/80(63.75 %) cases had bleeding scores\5 and were mild bleeders. 30/51(58.8 %) of mild bleeders had normal platelet aggregation. Abnormal platelet aggregation was present in 17/51(33.3 %) patients and in 4/51 (7.8 %) cases were suspected von Willebrand disease. The patterns of abnormal platelet aggregation obtained with various agonists were suggestive of an IFD in 13/51 (25.5 %) cases. The commonest IFD found in our cases, was aspirin like defect (7/51, 13.7 %). Less common were Gi-like defect (2/51, 3.9 %), thromboxane A2 receptor/pathway defect (2/51, 3.9 %), adrenoreceptor defect(1/51, 1.9 %), Glanzmanns thrombasthenia (1/51, 1.9 %). Conclusion: Mild bleeding symptoms can be due to an inherited platelet function defect, and LTA should be advised if platelet count and morphology and routine coagulation tests are normal. Keywords Light transmission aggregometry, Inherited platelet function defects

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OR 32

OR 34

A Review of 20 Hellp Syndrome Cases in Thrombocytopenic Pateints-One Year Study

Evaluation of Risk Factors for Development of PTS in Patients of DVT and Co-relation to D-Dimer, Role of ECS in PTS Prevention

Dr. Seema Afroze, Dr. Sharada, Dr. M. Padmavathi, Dr. N. Ezhilarasi Department of pathology, Government maternity hospital, Petlaburz, Hyderabad Summary: HELLP syndrome is a life threatening condition associated with pregnancy and pre-eclampsia early diagnosis helps in good prognosis. Introduction: Pre-eclampsia is a well known obstetric condition characterised by hypertension with proteinuria. HELLP syndrome unlike pre-eclampsia is a multisystemic condition usually seen in 3rd trimester and in postpartum period, usually in 48–72 h following delivery. This condition was first described by Pritchard JA in 1954 and later by Dr Louis Weinstein in 1982 as a distinct clinical entity as opposed to severe pre-eclampsia. Materials and Methods: 20 patients diagnosed with HELLP syndrome between may 2013 to June 2014 were retrospectively evaluated with regard to symptoms, platelet count and elevated liver enzymes and pre-eclampsia during their pregnancy. Results: Out of 200 patients of thrombocytopenia there were 20 patients showing elevated liver enzymes, clinical features correlating with HELLP SYNDROME. Conclusion/Diagnosis/ Impression: Earliest sign of HELLP SYNDROME in present study was a low platelet count. Clinical and diagnostic evaluation helps in early identification of HELLP syndrome for further management.

Topic: Haematology Free Paper OR 33 Clot Signature Curve as an Adjuvant in the Investigation of Disorders of Haemostasis in the Paediatric Age Group: A Tertiary Care Hospital Experience N. Smitha1, S. H. Rajeshwari1 1

Department of pathology, KLE’s Dr. Prabhakar Kore Hospital and MRC, Belgaum, India Objectives: (1) To study the clot signature curve in paediatric samples. (2) To evaluate the utility of an abnormal clot signature curve for the investigation of coagulation disorders. Materials and Methods: Age group: 0–12 years. Attending a tertiary care hospital. Type of sample: 3.2 % trisodium citrate (109 mM). The ratio of blood to anticoagulant 9:1 was ensured. These sample were analysed with 4 h of collection on ACL Elite Pro coagulation analyser and the clot signature curve obtained were interpreted. A 2 year cross sectional study. Results: The data collected over a period of 2 years was analysed and correlated with the clinical history. Significant correlation was found between the interpreted clot waveform and clinical history, thus proving useful in clinical diagnosis. Conclusion: Automated coagulation analyzers are being relied upon increasingly in response to the heightened demand for blood clotting investigations and the need for rapid results. The automated systems can provide a wealth of information which are often useful clinically. A clot signature curve is a visual representation of the clot formation during a coagulometric test using an optical end point analyser. The APTT clot waveform has been used to evaluate cases of disseminated intravascular coagulation, septicaemia and Haemphilia. These values have shown significant clinical correlation. However, the conventional ‘Manual’ clotting method remains the gold standard. Keywords Clot waveform, Coagulation, aPTT

Sudhir K. Atri, M. Mahapatra, R. Saxena, S. Sharma PGIMS Rohtak/AIIMS New Delhi Summary/Introduction: PTS is chronic complication of DVT and it is associated with significant morbidity and loss of productive life. Hence identification of its risk factor, their prevention and treatment plays an important part in management. Materials and Methods: All patients of deep vein thrombosis at least 6 months after the diagnosis and or treatment attending the hematology OPD and Hemostasis and Thrombosis clinic from April 2012 to December 2013 were analyzed and 102 patients were finally recruited and completed the study. They were screened for the symptoms and signs of PTS and severity was graded according to Villalta Scale. Results: In univariate analysis development of PTS was not related to the Age of the patients (p = 0.70), Body Mass Index (p = 0.76), Number of vessels involved in DVT (p = 0.76), and Laterality of DVT (right versus left) (p = 0.101) but was strongly associated with Male sex (p = 0.002), Proximal DVT (p = 0.001), Lesser achievement of therapeutic INR(less than 50 % of total visits) (p = 0.048), Prolonged symptoms duration of DVT at the time of diagnosis (p = 0.001) and Prolonged symptoms resolution of DVT (p = 0.001). In multivariate analysis also development of PTS was strongly associated with Male sex (odds ratio 0.067, 95 % CI 0.004–1.286, p = 0.001), Proximal DVT(odds ratio 1.234, 95 % CI 1.026–2.625, p = 0.03), prolonged symptoms duration of DVT at the time of diagnosis (odds ratio 1.029, 95 % CI 1.009–1.033, p = 0.004) and prolonged symptoms resolution of DVT (odds ratio 1.289, 95 % CI 1.150–1.427, p = 0.001) but therapeutic INR has lost its significant correlation(odds ratio 0.081, 95 % CI 0.001–4.601, p = 0.223). D-Dimer remained elevated in all patients having chronic DVT and moderate and severe variety of PTS but only in few cases of mild PTS and had high statistical significance (p = 0.001) as its elevation indicated persistent of significant DVT/PTS. D-Dimer became negative at the end of the study in patients who had complete resolution of DVT and having no significant PTS (in 5 cases of mild PTS in our study and in 23 patients of DVT who had complete resolution of DVT). Regular use of elastic compression stockings (ECS) was observed in 72 patients (42 patients of DVT only and 30 patients of DVT/PTS) and irregular use in 30 patients (4 patients of DVT and 26 patients of DVT/PTS) in the study. Its regular use made no statistical significant difference regarding resolution of DVT (p = 0.109) and improvement in PTS (p = 0.999) versus irregular use. Conclusion: Development of PTS was strongly associated with Male sex (odds ratio 0.067, 95 % CI 0.004––1.286, p = 0.001), Proximal DVT (odds ratio 1.234, 95 % CI 1.026–2.625, p = 0.03), Prolonged symptoms duration of DVT at the time of diagnosis (odds ratio 1.029, 95 % CI 1.009–1.033, p = 0.004) and Prolonged symptoms resolution of DVT (odds ratio 1.289, 95 % CI 1.150–1.427, p = 0.001). D-dimer had strong correlation with DVT and PTS while use of elastic compression stockings made no statistically significant difference regarding resolution of DVT & PTS.

OR 35 Molecular Basis of Von Willebrand Disease in 67 Patients from India E. Sumitha, A. Viswabandya, A. Abraham, B. George, V. Mathews, A. Srivastava Department of Haematology, Christian Medical College, Vellore-632004, Tamil Nadu, India

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S460 Introduction: Type 3 Von Willebrand disease (VWD) is an autosomal recessive bleeding disorder causing mutations in the VWF gene. Identification of mutations is important for understanding the biology of von willebrand factor as well as offering genetic testing to families affected by disorder. Materials and Methods: Sixty seven patients evaluated between 2012 and 2014 were diagnosed to have type 3 VWD. All these patients presented with history of variable skin and mucosal bleeding. Genomic DNA was screened for mutations in the VWF gene by PCR and CSGE. For ascribing causality to novel mutations identified, we performed in silico analysis. Results: Mutations (n = 27) were identified in 52 patients. These included frame shifts (44.4 %) missense (29.6 %), non sense (14.8 %) and splice site mutations (11.1 %) mutations, of which 19 were novel. The molecular pathology of the twelve novel frame-shifts is self-evident as they resulted in truncated von willebrand factor proteins. One novel splice site mutation (c.3675 + 1G[C) abolished the donor splice site possibly leading to exon skipping or intron retention. Among the eight missense mutations identified five were novel (p.Gln2266His, p.Gly74 Arg, p.Gln1188Lys, p.Cys2184Tyr, p.Arg1136Trp). p.Gln2266His is highly conserved across species. Mutations in this region are known to impair the polymerization of the multimers. The p.Gln1188 residue lies in the FVIII binding site which possibly affects the extracellular secretion of VWF protein. The residue p.Asp47Val on SIFT prediction analysis had a damaging impact on the proteins which may impair the process of multimerization of the proteins. The residue p.Arg1136Trp and p.Cys2184Tyr lies at the D3 domain of the polypeptide. This mutation possibly affects the extracellular secretion of VWF proteins. Three common mutations (p.Trp2107*; n = 5: p.Arg373*; n = 7: c.2443-1G[C; n = 9) affecting 21 patients were identified in this series. Conclusion: The mutations identified in patients with VWD are as heterogeneous as reported in other populations. Three common mutations account for 40 % patients with VWD in India and should be screened first for the genetic diagnosis of this condition. Keywords Von Willebrand Disease, Mutations, Heterogenous

Topic: Hematopathology OR 36 Megakaryocyte Morphology: Possible Diagnostic Contribution in Myeloproliferative Neoplasms and Myelodysplastic Syndromes: A Case Series A. Arun Kumar1, Ashutosh Kumar2, Rashmi Kushwaha3, Mili Jain4, U. S. Singh1, A. K. Tripathi5 1

Junior Resident, Department of Pathology, KGMC, Lucknow; Professor, Department of Pathology, KGMC, Lucknow; 3Assistant Professor, Department of Pathology, KGMC, Lucknow; 4Lecturer, Department of Pathology, KGMC, Lucknow; 5Professor & Head, Department of Clinical Hematology, KGMC, Lucknow

2

Introduction: The myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterised by proliferation of cells of the myeloid lineage. Myelodysplastic syndrome (MDS) represents a spectrum of stem cell malignancies that manifest ineffective as well as dysplastic blood cells production. Changes in the megakaryocytic lineage forms an important hallmark of both these disorders. Materials and Methods: A total of 41 diagnosed cases of MPN and MDS over a period of one year were included in the study. An evaluation of clinical profile, complete blood count and bone marrow examination were done in these cases. Silver stain was also done for

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 reticulin fibrosis which was graded as per WHO guidelines. The megakaryocyte parameters analysed in our study include assessment of distribution, count, number of nuclear lobes, cytoplasmic granulations, clusters, emperipolesis, nuclear fragmentation and micromegakaryocytes. Results: A majority of these morphological parameters of megakaryocytes have a statistical significance (p \ 0.05) in diagnosing MPN and MDS. Also specific disease entities were characterised by specific megakaryocytic features like, micromegakaryocytes with hypolobation were prominent in CML, hypolobation and dysplastic features like nuclear fragmentation were characteristic of PMF, hyperlobation, prominent clusters and emperipolesis without nuclear fragmentation was seen in ET and micromegakaryocytes, hyperlobation with prominent nuclear fragmentation, hypogranularity and paratrabecular distribution were features of MDS. Also a significant correlation was seen between the marrow fibrosis, megakaryocyte count and splenomegaly. Conclusion: We conclude that the assessment of megakaryocytic parameters provides a useful diagnostic clue in cases of MPN and MDS even in the era of molecular genetics. Keywords Myeloproliferative neoplasms, Myelodysplastic syndromes, Megakaryocytes

OR 37 Calreticulin (CALR) Mutations in Philadelphia Negative-Classic Myeloproliferative Neoplasms: A Single Center Experience Dr. Arpan Mehta, Dr. Deepak Mishra, Dr. Mayur Parihar, Dr. Saurabh Bhave, Sourabh Sharma, Dr. Anupam Chakrapani, Dr. Reena Nair, Dr. Mammen Chandy, Dr. Neeraj Arora Tata Medical Center, Kolkata Summary: Calreticulin mutations(CALR) in Philadelphia negativeclassic myeloproliferative neoplasms(MPNs). Introduction: Philadelphia negative-classic myeloproliferative neoplasms(MPN) encompass polycythemia rubra vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), wherein, the most common mutations described are JAK2-V617F, JAK2-exon12 and MPLW515 K/L. Recently, CALR mutations have been described in a high percentage of these cases. CALR is a multifunctional chaperone protein involved in Ca++ homeostasis. We describe our experience of CALR mutations in these MPNs. Materials and Methods: A total of 30 cases of BCR-ABL negative suspected MPN were analysed for JAK2-V617F, MPL-W515 K/L, JAK2-exon12 and CALR-exon9 mutations. These cases were diagnosed based on combination of clinical findings and BM findings as per WHO-2008 guidelines. JAK2-V617F mutation analysis was done by already described allelespecific PCR. CALR-exon9, MPL-W515 K/L and JAK2-exon12 mutations were done by already described protocol of PCR amplification followed by gene scan analysis. Results: These included 6 cases of PV, 8 cases of PMF, 4 cases of ET, 1 case of MDS, 1 case of CMML and remaining 10 cases were of MPN, which could not be categorized into particular phenotype. JAK2-V617F mutation was found in 12/28 cases(42.85 %). CALR-exon9 mutation was found in 4/28(14.28 %) cases of Philadelphia negative-classic MPNs, all of which were diagnosed as PMF. Of these four cases, three cases were of Type-1(52-bp deletion) and one case of type-2(5-bp insertion) type of CALR mutation. Conclusion: We describe here for the first time a comprehensive mutation analysis in BCR-ABL negative classic MPNs. Keywords Calreticulin, JAK2 V617F, BCR-ABL, MPL W515 K/L

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Topic: Haematopathology OR 38 Clinicohematological Profile and Bone Marrow Histomorphology in Imatinib Treated Chronic Myeloid Leukemia Dr. Debdatta Basu1, Dr. Rakhee Kar1, Dr. Biswajit Dubashi2, Dr. T. K. Dutta3 Departments of 1Pathology, 2Medical Oncology & 3Medicine, JIPMER, Puducherry Summary: Bone marrow (BM) response criteria in Imatinib treated CML patients are less well defined. In this study, the morphological changes in the BM three months post Imatinib were correlated with hematological and cytogenetic responses (HR and CR). Introduction: Post Imatinib treatment BM changes in CML are less well defined than HR and CR. Materials and Methods: Forty six newly diagnosed patients of CML (BCR-ABL positive), irrespective of phase, were included. Peripheral blood and BM were compared at diagnosis and at 3 months of Imatinib therapy. Marrow responses were classified as normalized marrow, persistence of disease, progression of disease and hypocellular marrow and correlated with HR and CR. Results: Twenty nine (64.5 %) of the post treatment marrow aspirates were diluted and imprints/biopsy yielded information. Twenty eight (63 %) patients showed marrow normalization, eleven (23 %) persistence of disease, four (8 %) progression of disease to blast crisis (BC) and three (6.5 %) hypocellular marrow. Forty two (91 %) patients had achieved complete HR including three patients who showed excess blasts in post treatment biopsies. Cytogenetic analysis was done in thirty patients of whom 70 % achieved complete CR. On follow up of 4 to 24 months, 40 patients had an uneventful course and 6 patients had BC; 3 of whom succumbed. Conclusion/ Diagnosis/Impression: BM response lags behind HR since 92 % of the patients had an uneventful course irrespective of the post therapy biopsy findings. Post therapy BM in CML, though not mandatory, when undertaken should have biopsy rather than an aspirate alone, as BM aspirates are often diluted and biopsy may yield information like impending blast crisis. Keywords CML, Imatinib, Trephine biopsy

OR 39 Clinio-Haematological Profile of JAK2 Positive Myeloproliferative Neoplasms in a Tertiary Care Center Rohit Tapadia, Jyoti R. Kini, Urmila N. Khadilkar, Prashanth Bhat, K. S. Pooja, Krishna Prasad Kasturba Medical College, Manipal University, Mangalore Introduction: Chronic myeloproliferative neoplasms (MPN) include chronic myelogenous leukemia (CML) which is BCR/ABL positive and polycythemia rubra vera (PRV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) which are BCR/ABL negative. These BCR/ABL negative chronic myeloproliferative disorders show a high incidence of acquired point mutation (V617F) in the JAK2 kinase. In the present study the incidence of JAK 2 mutation in BCR/ ABL negative MPN and their clinico-haematological profile has been evaluated. Materials and Methods: Cases where V617F mutation in JAK 2 kinase was detected on genetic analysis between January 2012 and August 2014 were included. Clinical features, peripheral smear, bone marrow findings and molecular analysis of the cases were

S461 reviewed. Results: Twenty five cases which were positive for V617F mutation were analysed. The age group ranged from 40 to 75 years with males accounting for 88 % of the cases. Peripheral smear and bone marrow evaluation showed that 19 cases were PRV (76 %) and 6 cases were PMF (24 %). Of the 19 PRV cases, it was found that one case showed secondary Myelofibrosis and another case showed PRV transformation to Acute Leukemia and associated Myelofibrosis. In addition to the 25 cases, 7 cases which were diagnosed as PRV based on low Erythropoietin level and concurrent bone marrow panmyelosis were included. Of the 7 cases, one was JAK2 negative and the mutation status of the remaining 6 cases was not known due to financial constraints. Conclusion/Diagnosis/Impression: The study showed increased sensitivity of JAK2 mutation for PRV compared to other MPN. Therefore JAK2 mutation analysis is an important tool in diagnosing PRV in cases presenting with primary erythrocytosis. Keywords JAK2 positive, Myeloproliferative neoplasm, BCR/ABL negative

Topic: Free Oral OR 40 Immunohistochemical Analysis of Bone Marrow Angiogenesis in Primary and Secondary Myelofibosis S. P. Verma1, Debdatta Basu2, Rakhee Kar2, T. K. Dutta1, K. V. Vinod1 1

Department of clinical Haematology, 2Department of Pathology, JIPMER Puducherry

Summary: CD34 staining appears better compared to CD105 for studying microvessel density at least in primary myelofibrosis. PMF patients have significantly higher angiogenesis in marrow compared to secondary myelofibrosis. Higher megakaryocytic expression of VEGF in PMF compared to secondary myelofibrosis may be an important differentiating feature. Introduction: Increased angiogenesis in marrow is a common finding in hematological malignancies. Primary and secondary myelofibrosis is associated with increased angiogenesis. Various markers including CD34, CD105 and tissue expression of VEGF have been used to study angiogenesis. We analyzed comparative utility of these markers in studying angiogenesis in cases of myelofibrosis. Materials and Methods: Bone marrow biopsies of patients with PMF (Group-1, n = 15), MPNs other than PMF (Group-2, n = 15), secondary myelofibrosis excluding MPNs (Group-3, n = 17), with WHO grade 2 and grade 3 myelofibrosis were included in this study along with 16 patients (Group-4) with normal marrows as controls. Visual microvessel grade (MVG) & microvessel density (MVD) was studied with CD34 and CD105 by standard IHC methods. Marrow expression of VEGF was also studied. Results: Median CD34 MVG was significantly higher (grade 4) in PMF group compared to all other groups (grade 2, 1.5 and 1 in group 2, 3 and 4 respectively). Mean MVD by anti CD34 was significantly high (26.93) in PMF compared to mean MVD of 18.67, 14.06 and 10.50 in group 2, 3 and 4 respectively. Mean MVD assessed by anti CD105 was not significantly different between the groups. Comparative assessment of MVD by CD34 and CD105 in all groups using paired t test showed significantly higher CD34 MVD in group 1. Cellular expression and megakaryocytic expression of VEGF evaluated by Immunoreactive scale was higher in group 1 compared to other groups. Conclusion: Marrow angiogenesis is significantly more in PMF compared to secondary myelofibrosis. CD34 appears to be a better angiogenic marker compared to CD105. Megakaryocytic expression of VEGF is significantly higher in PMF. Keywords Primary myelofibrosis, Angiogenesis

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S462 OR 41 Clinical Profile and Response Rates in CLL: An Institutional Experience Dr. Bala Joseph Stalin, K. Ashok, K. Ramu, K. Krishnamani, G. Sadashivudu, M. Lakshmi Srinivas Nizam’s Institute of Medical Sciences Summary: More than half of the patients had advanced stage at diagnosis in CLL and needed treatment. Bendamustine based regimes when used resulted in early response and improved outcomes with acceptable side effect profile compared to chlorambucil based regimens. Introduction: Chronic lymphocytic leukemia (CLL) is one of the chronic lymphoproliferaltive disorders. Newer regimens in the management of CLL had improved outcomes with early and sustained response rates. Materials and Methods: All patients with CLL diagnosed at our institute from June 2010 to April 2014 were retrospectively analyzed with respect to demographic characteristics, treatment, responses, toxicity and outcomes. Results: Fifty two patients with diagnosis of CLL were analysed. The median age at presentation was 63 years (range 35–84) with male preponderance (3.7:1). The most common presenting symptom was swellings over neck(57 %) followed by weight loss(46 %),fever(44 %),anorexia(44 %).The most common sign at presentation was Lymphadenopathy (80 %). Splenomegaly and hepatomegaly were seen in 46 and 15 % of patients. None had Rai Stage 0 and 35, 27, 10 and 28 % had Stage I, II, III, IV respectively. ZAP 70 expression was seen in 5 of 18 patients. The treatments received were bendamustine based (40 %) followed by chlorambucil based (27 %), observation only (23 %). Among 23 % patients planned observation, median follow up was 10.3 months and all had lymphocyte doubling time[6 months. Median number of cycles of Bendamustine regimen taken was 6 cycles and chlorambucil was 4.5 cycles. 98 % patients achieved complete hematological response (CHR) at the end of treatment. The median number of cycles among bendamustine and chlorambucil to achieve CHR was 1 and 2.5 cycles respectively. The most common toxicity seen in Bendamustine regimen was skin toxicity. (42 %). Only 1 patient had discontinued Bendamustine due to toxicity. Conclusion/Diagnosis/Impression: Although all the patients at diagnosis may not need treatment in CLL, Bendamustine based regimens used in patients who need treatment had early and improved response rates with tolerable toxicity.

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 affecting PFS and OS. Results: Out of 141 NHL patients, 67 patients were identified subjected to the inclusion criteria. The median age was 68 years (60–92). Majority were B cell NHL (86.6 %). The commonest subtype in B cell was DLBCL (55.2 %). 54 patients took treatment. The treatment intent was curative in 41 patients (61.2 %). Among the patients receiving curative treatment, 16 patients couldn’t receive treatment in accordance with NCCN guidelines due to financial issues. Two year PFS was 55 %. Two year PFS for B cell NHL and T cell NHL were 55 and 50 % respectively (p = 0.982). Two year PFS for standard Rx and non standard Rx were 62 and 50 % respectively but it didn’t reach statistical significance (p = 0.537). Two year OS for entire cohort was 84 %. Conclusion/Diagnosis/Impression: Standard treatment in accordance with guidelines can be delivered in elderly patients irrespective of age. There is need for creating financial assistance for patients so that potentially curative treatments are not denied.

OR 43 Primary Extranodal Non-Hodgkin’s Lymphoma: Clinicopathological features, prognostic factors and treatment outcome N. Srividya, G.R. Raman, A. Ravi, G. Sadashivudu, M. Lakshmi Srinivas Nizam’s Institute of Medical Sciences, Hyderabad Introduction: Non-hodgkins lymphoma (NHL) arising from extranodal site is uncommon. Data on primary extranodal NHL(pENL) is scarce. Materials and Methods: We analyzed hospital records of pENL from year 2005 to 2012, for their site, histology, prognostic factors, treatment response and outcome. Results: pENL constituted 23 % (120/521) of NHL, with median age of 45 years (3–79 years) and male predilection (M:F = 2:1). The sites of involvement were gastrointestinal tract (GIT) (34 %), bone (15 %), testis (8 %) and brain (6 %). DLBCL was the most common histology (85 %). CHOP was administered in 83 % and Rituximab was given in 30 %. Complete remission was achieved in 64 %, partial remission in 16 %; stable disease in 10 % and progressive disease in 10 %. The median PFS and OS were 65 and 71 months respectively. The 2-year OS was 70 %. Bulky disease (p = 0.003), B symptoms (p = 0.004), poor PS (p = 0.000), high LDH (p = 0.04) was associated with poor OS. Conclusion: Around one fourth of NHL were pENL. GIT is the most common site and DLBCL is the most common histology. Bulky disease, B symptoms, poor PS and high LDH predicted poor survival.

OR 42 Retrospective Audit of Clinico-Pathologic Features and Treatment Outcomes in a Cohort of Elderly Non Hodgkin’s Lymphoma Patients in a Tertiary Cancer Centre

Topic: Proferred

Dr. Chandran K. NaIr, Dr. Vijay M. Patil, Dr. Vineetha Raghavan, Dr. Satheesh Babu, Dr. Sangeetha Nayanar

Use of VTD in Newly Diagnosed Myeloma: An Institutional Experience

Malabar Cancer Centre, Kerala

Meet Kumar, Tuphan Kanti Dolai, Asutosh Panigrahi, Rajib De, Prakas Kr Mandal, Prantar Chakrabarti, Maitryee Bhattyacharyya

Summary: A retrospective audit of elderly NHL patients revealed that potentially curative treatments can be delivered in this age group as per standard guidelines with reasonable good results. Introduction: There is limited data from India regarding elderly NHL patients. Hence this audit was planned to study the clinic-pathological features and treatment outcomes in elderly NHL patients. Materials and Methods: Retrospective analysis of all NHL patients above age of 59 years treated at the author’s institute, between December 2010 and December 2013 was done. Case records were reviewed for baseline details, staging details, prognostic factors, treatment delivered, response, toxicity and efficacy. SPSS version 16 was used for analysis. Descriptive statistics was performed. Kaplan–Meir survival analysis was done for estimation of PFS and OS. Univariate analysis was done for indentifying factors

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NRS Medical College & Hospital, Kolkata Introduction: VTD is important chemotherapy for newly diagnosed myeloma. We evaluated outcome of patients treated with upfront VTD at a single centre in eastern India. Materials and Methods: All newly diagnosed patients of multiple myeloma (diagnosed June 2011 to June 2013) who received VTD as induction therapy were eligible for the study. Primary end point was response assessment (as per IWG criteria). Secondary endpoints were safety and overall survival. Results: A total of 37 patients were enrolled for the study. Of these, 35 had myeloma and 2 had plasma cell leukemia. Two patients died during induction, remaining 35 patients completed 6 cycles of

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 therapy. After 6 cycles, 14 (40 %) achieved CR, 10 (25.5 %) VGPR, 8(22.8 %) PR and 3(8.5 %) had stable disease. After induction, 5 patients underwent autologous SCT and 18 (51.4 %) patients received thalidomide as post-remission therapy. Ten patients did not receive any post-remission therapy. At average follow-up of 18.2 months (8–43 months), 5 patients relapsed, 3 more patients died due to disease progression and 3 patients were lost to follow-up. Probability of survival at 30 months was 85.71 %, and relapse-free survival at 26 months was 83.3 %. Major toxicities were nonhematological, with neuropathy and constipation being most common, followed by infections and thrombosis. Conclusion: VTD is effective as an upfront therapy for newly diagnosed myeloma patients. Keywords Myeloma, VTD, Outcome

OR 45 Bortezomib is a Safe and Effective First Line Treatment in Multiple Myeloma Sriram Ravichandran1, Saurabh Bhave Jayant1, Anupam Chakrapani, Rohit G. Chitrapur1, Vishvdeep Khushoo1, Neeraj Arora2, Mayur Parihar3, Deepak Mishra2, Reena Nair1, Mammen Chandy1

S463 Objective: Multiple myeloma (MM) is a paraproteinemia characterized by clonal proliferation of plasma cells in the marrow. Morphology of the plasma cells, beta-2 microglobulin levels, end organ damage and the stage of the disease are the prognostic parameters. It is also associated with complex cytogenetic and molecular abnormalities which can be of prognostic significance. Materials and Methods: All the case of MM diagnosed between January 2012 to July 2014 were include in the study. The clinical details including presentation, electrophoresis, renal parameters were taken from clinical records. The bone marrow slides were retrieved and reviewed. Conventional cytogenetics and FISH analysis was performed in 14 and 8 patients respectively. Results: The study includes 45 patients in the age range of 30–82 years with male predominance (M:F = 1.5:1). Most of the patients (21) presented with complaints of back pain and weakness. Renal dysfunction was seen in 29 patients with mean creatinine of 2.5 mg/dl. Immunoelectrophoresis revealed M-band in 19 cases. The bone marrow plasma cell ranged from 15 to 90 %.The commonest morphology was that of Marshalko type where as plasmablastic type was rare. The cytogenetic abnormalities detected were aneuploidy, trisomy 1 and 9, 13q deletion. Conclusion: This study reflects our experience of 45 patients of MM with respect to clinical features, bone marrow morphology and cytogenetic analysis. Keywords Multiple Myeloma, Bone marrow, Cytogenetics

1

Department of Clinical Haematology, Tata Medical Center, Kolkata, Department of Haematopathology, Tata Medical Center, Kolkata, 3 Department of Cytogenetics, Tata Medical Center, Kolkata 2

Introduction: Multiple myeloma is a clonal malignancy of the mature plasma cells. The introduction of bortezomib, a proteosome inhibitor has improved the outlook for what was otherwise, an exigent disease to treat. Here, we report our experience of using bortezomib, upfront in multiple myeloma. Materials and Methods: This is a retrospective analysis of all patients with multiple myeloma, seen in our center between May 2011 and April 2014. All patients treated upfront with bortezomib have been included in this analysis. The diagnosis and responses have been characterised as per IMWG criteria. Results: A total of 393 patients were identified for this analysis. 103 patients were treated with bortezomib upfront. The median age of this cohort was 60 yrs and a quarter of this cohort was above 65 yrs of age (26/103). 30 % of patients presented in advanced stage(ISS III—37/82). Cytogenetics was available for 26/91 patients. Cytogenetic abnormalities were found in 12/26 patients. VTD (Bortezomib + Thalidomide + Dexamethaosne) was the most commonly used regimen (76/91). Median number of cycles was 6(range 1–8). The overall response rate(ORR) was 90 % (sCR + CR + VGPR + PR). The median OS was 13 months and the median PFS was 11 months. There were 10 deaths in this cohort, all due to disease progression. Peripheral neuropathy was the most common complication occurring in 25 % of patients (21/91). Lung toxicity was seen in 2 of the patients in this cohort. Bortezomib was discontinued in only 5 patients due to toxicity. Conclusion: Upfront Bortezomib is a safe and effective treatment for Multiple Myeloma with tolerable side effect profile. Keywords Multiple myeloma, Bortezomib

Topic: Multiple Myeloma OR 46 Multiple Myeloma: A Clinicopathologic and Cytogenetic Analysis of 45 Patients in a Tertiary Care Centre P. Madhavi, Ashwani Tandon, Megha Uppin, Shantveer Uppin, G. Sadashividu, AMVR Narendra Department of Pathology, Medical oncology and General Medicine, Nizam’s Institute of Medical Sciences, Hyderabad

Topic: Haematology OR 47 Bone Marrow Immunohistochemical Expression of Vascular Endothelial Growth Factor in Patients of Multiple Myeloma and its Correlation with Other Prognostic Factors Ritika Singh, Meera Sikka, Usha Rusia Department of Pathology, University College of Medical College, Delhi Introduction: In multiple myeloma (MM), angiogenesis is correlated with an adverse prognosis. With the availability of antiangiogenic drugs, evaluation of angiogenesis assumes importance. It can be measured by assessing Microvessel density (MVD) or the expression of proangiogenic molecules (VEGF). This study aimed to ascertain bone marrow (BM) expression of VEGF in MM and correlate it with MVD and other prognostic factors. Materials and Methods: VEGF immunoexpression and MVD were evaluated in BM of 25 MM patients (monoclonal VEGF antibody and CD34 antibody) and 25 age matched controls. Results: VEGF expression was positive (staining in [10 % plasma cells) in 20 (80 %) patients. MVD/hpf was significantly (p \ 0.0001) higher in patients as compared to controls. MVD/ hpf was significantly (p \ 0.001) higher in VEGF positive cases than VEGF negative cases. Plasma cell number was significantly (p = 0.024) higher in VEGF positive cases as compared to VEGF negative cases. VEGF positivity was not significantly associated with haematological and biochemical parameters, pattern of plasma cell infiltration and stage of disease. MVD correlated significantly with poor risk factors. Conclusions: This study observed a frequent expression of VEGF in MM patients. The increased MVD and its association with poor risk factors suggested that angiogenesis is of significance in MM and is a useful prognostic marker. A significant correlation was seen between VEGF and MVD indicating the contribution of VEGF to angiogenic pathway in MM. Thus measurement of angiogenesis at diagnosis will help in identifying high risk patients who may benefit with new aggressive treatment strategies and also provide a rationale for use of anti angiogenic therapy. Keywords Multiple Myeloma, VEGF, CD34, MVD

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Topic: Acute leukemia OR 48 Immunophenotypic Profile of Acute Promyelocytic Leukemia Patients A. Saksena, V. Chauhan, N. Gupta#, T. Singh #

Department of Pathology and Medicine, Maulana Azad Medical College, New Delhi

Summary: Flowcytometry was evaluated to be useful in early diagnosis of APML Introduction: Acute Promyelocytic leukemia (APML) needs to be suspected early so that ATRA therapy can be started early. RT PCR for PML RARA detection is time consuming and may delay treatment Materials and Methods: In the present study, role of flowcytometry was evaluated for early diagnosis of APML. 13 cases of APML were diagnosed in MAMC from 2010 to 2014. These comprised of 6 males and 7 females and their ages ranged between 2 and 36 years. Cytochemistry and flowcytometry were performed in all cases. Results: All cases showed 3+ cytochemical MPO positivity, diffuse flush PAS positivity and NSE positivity. Side scatter was moderate in 4 cases and high in 9 cases. The former were AML M3v whereas the latter were classic APML. CD13 was a heterogenous dim to moderate positive in 12/13 cases. CD 33 was homogenous positive in all 13 cases; dim in 3 and moderate in 10. CD 34 was moderate positive in 1/13 case and HLADR was positive in 2/13 cases. 11/13 cases were dual negative for CD 34 and HLADR. Aberrant marker CD2 was positive in 1/13 case. Thus, despite being NSE positive, the APML cases could be distinguished from monocytic cells by typical flow findings. Conclusion/Diagnosis/Impression: It is concluded that flow cytometric pattern of APML is characteristic with moderate to high side scatter. Dual negativity for CD 34 and HLA DR is highly suggestive of APML however occasionally it may be positive in APML also. Thus flow cytometry aids in early diagnosis of APML. Keywords APML, Flowcytometry, Early diagnosis

Topic: proffered OR 49 Flowcytometry in the Diagnosis of Acute Lymphoblastic Leukemia Parul Gautam, Vijaya Vaishnav, Tejinder Singh, A. P. Dubey*, N. Gupta# Department of Pathology, *Pediatrics and #Medicine, Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi Summary: Immunophenotyping is the key factor in the diagnosis and prognosis of acute lymphoblastic leukemia (ALL), turning the morphological analysis method complete. Materials and Methods: Morphology of blasts was assessed in all the cases on giemsa stained peripheral blood and bone marrow aspirate smears and cytochemical tests were carried out. Flow cytometric analysis was carried out on blood and/or bone marrows of 112 cases of ALL. Results: The study included a total of 112 cases of ALL. M:F ratio was approximately 2:1. The patients’ age ranged from 3 months to 62 years. Out of 112 cases, 80 were pediatric cases (age B14 years). TLC ranged from 840 to 418,340 cu mm. In 6 of the cases, peripheral smear did not show any atypical cells. On peripheral smear/bone marrow evaluation, 79 cases had L1 morphology and 33 had L2 morphology. Cytochemistry showed MPO negativity in all the cases. Block positivity with PAS

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 was seen in 26 % of B-ALL. In our study focal acid phosphatise positivity was 100 % specific for T-ALL, seen in 9 out of 17 cases (53 %). NSE stain was positive in 2 cases of T-ALL. Out of 112 cases, 95 were diagnosed as B-ALL and 17 as T-ALL on Immunophenotyping by flowcytometry. 8 cases of T-ALL could be diagnosed only on flowcytometry. Aberrant expression of myeloid markers was seen in 24 % cases of B-ALL and 17.6 % cases of T-ALL. Conclusion: Imunophenotyping by flowcytometry is mandatory in all the cases of ALL for characterization of leukemic cell population and for lineage assignment in each individual patient. Keywords ALL, Immunophenotyping, Flowcytometry

Topic: Laboratory Hematology OR 50 A retrospective Evaluation of Diagnostic Utility of Five Color Primary Screening Antibodies Panel Used in Flowcytometric Immunophenotyping of Acute Leukemia Dr. Pranay Tanwar, Dr. Amar Ranjan Singh, Dr. Ritu Gupta Laboratory Oncology Unit, Dr. B.R.A.I.R.C.H., AIIMS, New Delhi Summary: The diagnosis of acute leukemia is based on morphology, cytochemistry, cytogenetics and immunophenotyping. Primary screening antibodies panel is used with the objective of stratifying the lineage followed by extensive secondary antibodies panel to reach a conclusive diagnosis. In this study the diagnostic utility of primary screening antibodies panel has been evaluated retrospectively based on the final immunophenotype. Aims and Objective: To evaluate the diagnostic utility of five-color primary screening antibodies panel used in flowcytometric immunophenotyping of Acute Leukemia. Materials and Methods: A total of 292 diagnosed cases of Acute Leukemia from Jan 2013 to Aug 2014 were included in this study. All these cases were stratified to lineages with primary screening antibodies panel comprising of cytoplasmic myeloperoxidase (MPO), cytoplasmic 79a (c79a), CD34, cytoplasmic CD3 (c3) and CD45. Once the lineage has been stratified, then extensive secondary panel with more specific marker of myeloid, B-ALL and T-ALL were used to reach the final diagnosis. Results: Of all the cases (292) evaluated 235(81 %) were successfully identified on primary screening panel only, 57(19 %) cases failed to show any specific lineage in primary screening panel. These 57 cases included 31/125(25 %) AML, 22/131(17 %) B-ALL and 4/36(11 %) cases of T-ALL. Conclusion: The five-color primary screening antibodies panel is proved useful in identify the correct lineage in 81 % of cases. The absence of lineage specific marker was the most common reason for failure of the predictive power of the primary panel. Keywords Acute leukemia, Immunophenotyping, Flowcytometry

OR 51 A State of the Art Custom ‘‘Duplexed’’ Real Time PCR Assay for Minimal Residual Disease Monitoring of Chronic Myeloid Leukemia Using Locked Nucleic Acid Probes Heena Doshi, Swapnali Joshi, Shruti Chaudhary, Ridhima Bane, Shashikant Mahadik, Sheetal Gaware, Devanand Vishwakarma, Syed Hasan Khizer, PG Subramanian, Prashant Tembhare, Sumeet Gujral, Nikhil Patkar* Hematopathology Laboratory, Tata Memorial Centre

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Objectives: We have high workload of samples for chronic myeloid leukemia (CML) minimal residual disease (MRD) necessitating automation and innovative strategies. It is currently recommended to test for BCR-ABL real time PCR (qPCR) in triplicates, however this places economic strain. We cloned a duplex qPCR compatible plasmid that enables multiplexing BCR-ABL and ABL reaction in a single well using FAMblack hole quencher1 (BHQ1) and HEX–BHQ1 probes modified with locked nucleic acids (LNA). This is first paper to use LNA probes for CML MRD monitoring. Methods: We customized blood processing/RNA extraction protocol for two QIAcube instruments. A 1600 bp-amplicon was cloned from cDNA of a CML patient into a pJET1.2/blunt cloning vector. Modified EAC BCR-ABL probe was truncated to 16 nucleic acids labeled with FAM and BHQ1 with three LNA modifications whereas the ABL probe was 19 nucleic acids (HEX and BHQ1) with 4 LNA modifications. Calibration to the WHO calibrator was done using a secondary standard. Results: Median ABL copy numbers on 700 consecutive samples was 33855. In all runs, slope was between -3.2 and -3.6, R2 [ 0.98. The international scale conversion factor was 0.79. For 235 samples the median BCR-ABL and ABL copy numbers were 132.95 and 38925. The BCR-ABL assay has a CV of 16.47 % for the highest (1E1) dilution of the plasmid. Conclusion: Due to multiplexing, 24 samples in triplicates could be processed in a single run (including two controls and 6 plasmid dilutions) reducing the cost as compared to the legacy assay. Keywords Chronic myeloid leukemia, Real time PCR, Locked nucleic acid, Minimal residual disease

Topic: Proffered OR 52 JAK2V617F Mutation in Primary Myelofibrosis in India: Its Correlation with Clinico-Hematologic Characteristics and IPSS Scoring Neha Singh, Sudha Sazawal, Sunita Chhikara, Manoranjan Mahapatra, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi, India Summary: A retrospective study of sixty eight patients with primary myelofibrosis (PIMF) was done to correlate the JAK2V617F mutation status with the clinico-hematologic characteristics as well as the IPSS scoring. Homozygous patients were significantly associated with thrombocytosis and JAK negative had significantly higher transfusion requirement than the other groups. No significant correlation was detected between JAK2V617F mutational status and clinical features, haematologic profile or prognostic risk stratification of the PIMF patients. Introduction: Somatic mutation in the exon 14 of Janus Kinase 2 gene is an established diagnostic marker in bcr-abl negative myeloproliferative neoplasms, especially primary myelofibrosis. Materials and Methods: Clinical and hematologic features were reviewed for 68 patients with PIMF. The status of JAK2V617F mutation was analyzed by DNA tetraprimer amplification refractory mutation system and the results correlated. The patients were further stratified into low, intermediate 1, intermediate 2 and high risk groups on the basis of the IPSS scoring. Results: The JAK2V617F mutation was detected in 40/68 (58.8 %) patients. Univariate analysis of variables at presentation identified that JAK2 negative patients were significantly associated with higher transfusion requirement (p = 0.017). Patients homozygous for JAK2 mutation were associated with thrombocytosis (p = 0.014) and also had higher TLC than the other groups. No significant correlation was detected between the JAK2V617F mutational status and the patient age, presence of constitutional symptoms, TLC, hemoglobin, spleen size, grade of fibrosis or prognostic risk stratification of the PIMF patients. Conclusion: Use of JAK2 quantification

S465 methods and other newer molecular markers is required to develop deeper insight into various molecular alterations involving PIMF patients in India as well as worldwide. The variations in the prognostic implication of PIMF patients with mutation status as stated by various publications worldwide, calls for the need for larger prospective studies. Keywords PIMF, JAK2V617F mutation, ARMS-PCR, IPSS Scoring

OR 53 Genetic Profile of the Transformed MDS Patients: AIIMS Experience Dr. Rekha Chaubey, S. Sazawal, M. Mahapatra, S. Chhikara, R. Saxena Department of Hematology, All India institute of Medical Sciences, New Delhi-110029 Summary: Molecular basis of MDS for its progression to AML remain largely undefined. We studied several molecular factors; cytogenetics, RAS gene mutations, Telomerase activity, hTERT expression and methylation status of TSGs; p15, SOCS-1, FHIT and Calcitonin. Of all these, only p15 gene methylation was found to be an independent predictor of progression MDS patients in India. Introduction: Myelodysplastic syndromes are characterized by bone marrow failure and risk of progression to acute leukemia. So far, blast percentage and cytogenetic abnormalities are known as major determinants of the risk for transformation. Molecular changes in MDS with leukemic transformation have not been studied in Indian settings. Hence, this study aims towards finding out the major molecular alterations in MDS transformed patients. Materials and Methods: DNA/RNA was extracted from peripheral blood/BM. Conventional cytogenetics was used for karyotyping, Nested PCR and RT-PCR was used to detect RAS mutations and hTERT expression respectively. Telomerase activity was measured by PCR-ELISA—TRAP Assay. Methylation specific PCR was used to detect the methylation status of tumor suppressor genes (TSGs) (p15, SOCS-1, FHIT and Calcitonin). Results: Of the twenty-one patients (median age 48.0 years, range 9–77 years, M:F: 2.5:1) who progressed from MDS to acute leukemias, the transformation was seen in two RA, ten RAEB-II, and five RAEB-I patients. One patient of RAEB-II progressed to ALL while another with 5q deletion transformed to myelofibrosis, (median time of progression 15 months,). Out of 9 patients in which cytogenetics was available 3 belonged to good risk and 4 had poor cytogenetics. (4/21) patients with K-RAS mutation, (2/21) with N-RAS mutation and (5/21) with hTERT expression progressed to acute leukemia. Hypermethylation of TSGs was as follows: p15INK4B gene in 13 (32.5 %) patients (p \ 0.02), SOCS-1 gene in 17(32.1 %) (p \ 0.006), Calcitonin gene in 15 (25.9 %) (p \ 0.08) and FHIT gene in 16 (37.2 %) (p \ 0.001). Multivariate analysis showed only p15INK4b gene methylation as an independent predictor for progression of disease (HR 5.15, 95 % CI 1.64–16.1, p = 0.005). Conclusion/Diagnosis/ Impression: Aberrant methylation of p15INK4b gene is an independent predictor for progression of disease in Indian patients with MDS.

Topic: Acute leukemia (Acute Lymphoblastic Leukemia) OR 54 Prevalence of IKZF1 (Ikaros) Deletions in B-ALL at a Tertiary Care Hospital in North India Dr. Sanjeev Kumar Gupta1,*, Rajive Kumar1, Sameer Bhakhshi2 1 Laboratory Oncology Unit, and 2Department of Medical Oncology, IRCH, All India Institute of Medical Sciences (AIIMS), New Delhi

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S466 Summary: This study attempted to find out the prevalence of IKZF1 (Ikaros) deletions in B-ALL patients using multiplex ligationdependent probe amplification (MLPA). Introduction: IKZF1 deletions have been reported in 15–20 % of pediatric B-ALL and [75 % of BCR-ABL-positive B-ALL patients and are associated with poor outcome, both in BCR-ABL-positive as well as BCR-ABL-negative ALL. We undertook this study to check if Indian B-ALL patients have a higher prevalence of IKZF1 deletions compared to the western patients. Methods and Materials: 50 cases of newly-diagnosed, untreated B-ALL were prospectively analyzed for IKZF1 deletions in last 6 months. The cases were diagnosed using morphology, cytochemistry and flowcytometry immunophenotyping (FC-500, Beckman Coulter). RT-PCR was done to detect their BCR-ABL status. MLPA was done using SALSA MLPA kits P335-B1 ALLIKZF1 and P202-B1 IKZF1 (MRC-Holland) for IKZF1 deletions. Ten healthy controls were also included. Results: The median age of 50 cases was 10.5 years (1–60 years) and included 34 males and 16 females. Ten out of 50 (20 %) cases were positive for BCR-ABL. Overall, the IKZF1 deletions were detected in 17/50 (34 %) cases. The IKZF1 deletions were detected in 7/10 (70 %) of BCR-ABL positive cases and 10/40 (25 %) of BCR-ABL negative cases and included both complete and partial IKZF1 deletions in both the groups. Conclusion: The IKZF1 deletions are more common in BCRABL positive B-ALL compared to the BCR-ABL negative cases. The prevalence of IKZF1 deletions in the current study is similar to the reported literature. However, larger studies using more sensitive techniques are desirable to ascertain the same. Keywords Ikaros deletion, BCR-ABL like ALL, B-ALL prognosis, MLPA in B-ALL

OR 55 Gene Mutations in FLT3-ITD, NPM1, CEBPA Genes in Paediatric Acute Myeloid Leukemia Sheetal Gaware, Swapnali Joshi, Shruti Chaudhary, Heena Doshi, PG Subramanian, Prashant Tembhare, Sumeet Gujral, Nikhil Patkar* Hematopathology Laboratory, Tata Memorial Centre Objectives: Mutations in FLT3 (esp FLT3-internal tandem duplications; FLT3-ITD), NPM1 and CEBPA genes have profound prognostic significance in AML. Pediatric acute myeloid leukemia (AML) is a relatively rare hematolymphoid malignancy. We evaluated the baseline frequencies of the mutations in FMS-like tyrosine kinase 3 (FLT3), nucleophosmin1 (NPM1), and CCAAT/enhancerbinding protein alpha (CEBPA) genes in the pediatric AML patients and found that 41.50 % of AML patients harbor gene mutations. Materials and Methods: A total of 53 patients of AML [other than AML with t(15;17)] were received from January 2014 to August 2014 (age range: 1–16 years, 62.26 % male). Genomic DNA was extracted and subjected to multiplex PCR that amplified FLT3, NPM1 genes TAD1, TAD2 and b-ZIP domains of the CEBPA gene using fluorescently labeled primers followed by capillary electrophoresis and analyzed for any duplication, insertion and/or deletion. Results: FAB subtypes: AML-M0 (35.84 %), AML-M2 (28.30 %), AML-M4 (9.43 %), AML-M5 (5.66 %), & AML-M7(3.77 %) & t (8;21), t(11q23), & inv16 were seen in 15.09, 9.43 and 5.66 % patients respectively. Allelic ratio for FLT3-ITD ranged from 0.04 to 0.6. The subcategories of mutations were as follows: [FLT3+/NPM1/CEBPA(9.40 %), FLT3+/NPM1+/CEBPA(1.88 %), FLT3+/NPM1/CEB PA+(1.8 %)], FLT3-/NPM1 +/CEBPA-(7.54 %) and CEBPA mutations (FLT3-/NPM1-/CEBPA+) accounted for 11.32 %. No biallelic mutations in CEBPA genes were found. No mutation could be detected in 56.60 % of patients. Conclusion: The requencies of these

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 mutations are comparable with published data. As majority of these mutations are of in-del type, fragment length analysis can be used to detect these mutations. Keywords Pediatric acute myeloid leukemia, Gene mutations, India

Topic: Oral/Proffered/Platform OR 56 Clinicohematological Profile of Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia S. Venkatesan, A. Purohit, M. Aggarwal, P. Mishra, T. Seth, M. Mahapatra, S. Tyagi, H. P. Pati, R. Saxena Department of Haematology, All India Institute of Medical Sciences, New Delhi Objectives: The aim of this study was to investigate the occurrence of Philadelphia chromosome in Acute lymphoblastic leukemia (ALL) and their clinicopathological profile. Materials and Methods: In this study, we analysed occurrence of Philadelphia chromosome by conventional cytogenetics (CTG), FISH and RT PCR for BCR-ABL-1 from July 2007 to August 2014. Cases who received chemotherapy were included and cases of CML with lymphoid blast crisis were excluded. Clinical details and response to induction chemotherapy was noted and cases were followed-up. Results: Of 37 cases, Ph chromosome was detected by CTG and FISH in 18 cases where as BCR-ABL1 fusion transcripts were detected by RT-PCR in 19 cases. Of later 19 cases, 08 and 11 cases were positive for p190 and p210 fusion transcript respectively. Male to female ratio was 3.1 and median age at the time of presentation was 30 years (range 9–56 years). Median presenting total leucocyte count (TLC) was 48,000 cu mm. CNS status at the time of induction were CNS-1, CNS-2 and CNS-3 in 30, 05, 02 cases respectively. 30 cases achieved remission after induction chemotherapy, four died during induction, one case did not achieve remission, however two cases received only Imatinib due to associated comorbidities. On follow up 20 cases relapsed with 05 cases had isolated CNS relapse and 15 cases had systemic relapse. On subgroup analysis there was no difference in clinical or laboratory features between cases with p190 and p210 fusion transcripts. Conclusion: To conclude Philadelphia chromosome positive ALL presents with high TLC, CNS disease and male predominance. Even though these cases achieve remission, are associated with high incidence of CNS and systemic relapse. Keywords ALL, Ph chromosome, BCR-ABL1

OR 57 VCS Parameters: A Comparative Study of Dengue Patients Dr. Piyush Deshpande, Dr. Anjali Kelkar Bharati Vidyapeeth Medical College, Pune Summary: Dengue is a vector borne disease caused by mosquito A. aegypti. It presents with fever, joint pain as well as low platelet count causing bleeding. Treatment is largely supportive. Introduction: Febrile illnesses like dengue are increasingly becoming difficult to diagnose clinically. In such a scenario, automated analyser can help bridge the gap between the disease and its diagnosis. Materials and Methods: VCS parameters from 100 dengue patients, and 100 controls without dengue were analyzed. Statistical discriminant functions were generated and used as parameters to compare the two. Results: Statistical functions were generated: Controls-vs. dengue factor were

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 obtained by incorporating platelet count, lymphocyte percentage and standard deviation of lymphocyte volume that distinguished dengue from other febrile illnesses are to be analyzed. We compared the factor given in the literature and found it useful in diagnosing dengue patients. Conclusion: Dengue patients were successfully segregated using the dengue factor. These factors can be generated effectively by the analyser and can be of great gain in diagnosis in the future.

OR 58 Exploring the Role of Cell Population Data as a Novel aid in the Diagnosis of Dengue Fever: The Manipal Experience! Dr. Pratyusha Gudapati, Dr. Deepak Nayak M. Assistant Professor Kasturba Medical College, Manipal; Manipal University Introduction: Dengue fever is a rapidly emerging vector-borne viral disease. Clinically, it still remains a diagnostic challenge as there are no specific symptoms to distinguish it from other common febrile viral illnesses. The VCS (volume, conductivity and scatter) technology available in automated hematology analyzer and cell population data are capable of quantifying leukocyte morphologic characteristics and also known to reflect subtle yet decisive changes in this regard. Thus, we decided to explore the role of the same in making a diagnosis of dengue fever. Aims and Objectives: To analyse diagnostic performance of VCS parameters and cell population data in dengue fever. Materials and Methods: Type of Study Cross-sectional study, Period of study June 2014 to September 2014. Cases Dengue IgM positive cases (n = 100). Controls acute febrile illness with or without thrombocytopenia with dengue IgM negative status and negative for malaria (n = 100 subjects) Volume & scatter parameters of neutrophils, lymphocytes and monocytes in both cases and controls were retrieved from Beckman Coulter LH 780; with ensuing appropriate statistical analysis. Results: By using Mann–Whitney Test, neutrophil volume—Standard deviation(SD), neutrophil scatter—SD, lymphocyte volume—SD, mean lymphocyte scatter, lymphocyte scatter—SD, monocyte volume—SD & monocyte scatter—SD showed statistically significant difference among cases and controls. Conclusion: Research population data of WBCs were found to be effective in differentiating dengue from other acute febrile viral illnesses. Further studies in this vein and development of discriminant factors using these parameters could be the way ahead in the diagnosis of dengue. Keywords Dengue, Automated hematology analyzer, VCS parameters

Topic: Oral Paper Presentations OR 59 Macrothrombocytopenia in North India: Role of Automated Platelet Data in the Detection of an Underdiagnosed Entity Priyageet Kaur Kalsi1, Naveen Kakkar1, M. Joseph John1, Amrith Mathew2 1

Departments of Pathology and 2Clinical Haematology, 2HaematoOncology and 2Bone Marrow (stem cell) Transplant Unit, Christian Medical College & Hospital, Ludhiana, Punjab, India Objectives: Congenital macrothrombocytopenia is being increasingly recognised because of the increasing availability of automated platelet counts during routine complete blood count (CBC). If not recognised, these patients may be unnecessarily investigated or treated. This study

S467 reports the occurrence of macrothrombocytopenia in a North Indian population. Materials and Methods: This prospective study was done on patients whose blood samples were sent for CBC to the hematology laboratory of a tertiary care hospital. Samples were run on Advia-120, a 5-part differential automated analyzer. Routine blood parameters including platelet count, mean platelet volume (MPV) and platelet cytogram pattern and platelet flagging was studied along with peripheral blood smear examination. ANOVA was used to compare difference in mean MPV in patients with macrothrombocytopenia, and those with secondary thrombocytopenia and ITP. Results: Seventy five (0.6 %) patients with CBC evaluation were detected to have macrothrombocytopenia, majority (96 %) of North Indian origin. The MPV (fl) in the 75 patients ranged from 10.9 to 23.3 (Mean 15.1 ± 3.1 fl) with a dispersed cytogram pattern distinct from that seen in patients with normal platelet count, raised platelet count or low platelets due to secondary thrombocytopenia (MPV 10.9 ± 2.6) or ITP (10.8 ± 3.5). The difference in mean MPV in these patients was statistically significant (p \ 0.00001). Conclusion: Macrothombocytopenia is an under diagnosed condition and may be initially suspected on automated blood counts. Along with a blood smear examination, automated data (MPV and platelet cytogram pattern) aids the diagnosis and can avoid unnecessary investigations and interventions for these patients. Keywords Macrothrombocytopenia, Automated data, Mean platelet volume, Cytogram

Topic: Morning OR 60 Role of Immature Reticulocyte Fraction in Evaluation of Aplastic Anemia in Cases of Pancytopenia Dr. R. Sindhu, S. K. Behera, D. P. Mishra Department of Pathology, M.K.C.G Medical College, Berhampur, Odisha Summary: The immature reticulocyte fractions (IRF) which are derived from automated reticulocyte counts are based on flow-cytometric determination of erythrocyte RNA content. Most of the physicians use only reticulocyte percentage and absolute reticulocyte count in evaluating anemia’s. We attempted to emphasize the importance of IRF in evaluating pancytopenia cases before going for a bone marrow. Introduction: Reticulocyte indices like MFR, HFR, LFR plays an important role in evaluation of hypoproliferative anemia’s and segregating aplastic anemia from other causes which is not possible by a reticulocyte count since it is more or less equal in all cases. Materials and Methods: A prospective study was done in M.K.C.G Medical College in Department of Pathology from November 2013 to August 2014. Patients who satisfied the criteria for pancytopenia were evaluated with clinical details. CBC, peripheral smear and bone marrow were done. Result: 86 pancytopenia cases were studied. CBC done with Sysmex XT-2000i automated cell counter showed 12 cases with IRF (MFR + HFR) 0 % and LFR 100 %. Simultaneously bone marrow was done in all, out of which 10 had aplastic marrow and 2 had pure red cell aplasia. Remaining cases showed most commonly megaloblastic bone marrow, hypoplastic marrow and less commonly acute leukemia, myelodysplastic syndrome, marrow of infection and storage disorders. Reticulocyte count and IRF was found to be variable in these cases. Conclusion: Hence, IRF helps in differentiating aplastic anemia from common nutritional deficiency anemia’s and avoids unnecessary blood transfusion in all cases. It also helps in monitoring the response during and after therapy. Keywords Immature reticulocyte fraction (IRF), Aplastic anemia, Pancytopenia.

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Topic: Laboratory Haematology OR 61 Microcytic Hypochromic Anaemia in Antenatal Women in a Tertiary Care Centre in Southern India Dr. Anita Ramdas, Dr. Priyanka Joseph Koshy, *Ms Meenakshi Vedavyasan, **Dr. Padma Alaganandan, Dr. Renu G’Boy Varghese

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 January 2012 to August 2014. Among these, 43,771(93.82 %) were Rh-D positive and remaining 2,883(06.18 %) were Rh-D negative. A total of 30 individuals (16 donors and 14 patients) were weak D positive constituting 1.04 % of Rh D negatives and 0.06 % of total individuals screened. Conclusion: This study shows the prevalence of weak D antigen in our population who are representative of Rayalaseema region of Andhra Pradesh. It also stresses the need to identify individuals with weak D to prevent sensitization/alloimmunisation and haemolytic transfusion reactions. Keywords Weak-D, Rh-D negative, IgG anti-D.

Departments of Pathology,*Biochemistry and **Obstetrics and Gynaecology, Pondicherry Institute of Medical Sciences Summary: Iron deficiency anaemia remains a problem in antenatal women. We found a 13 % prevalence of microcytic hypochromic anaemia and tried to determine utility of RBC discriminative indices to differentiate iron deficiency and thalassemia trait. Introduction: We aimed to screen all the antenatal women on their first visit for microcytic hypochromic anaemia with the objective to determine the prevalence and identify any b thalassemia traits based on the Shine and Lal Index and Red Cell Distribution Width Index. Materials and Methods: A total of 433 antenatal women were included. Haemoglobin estimation was done using AutoSysmex autoanalyser. Anaemia was graded according to the WHO criteria. Shine & Lal Index, RDW Index and RBC parameters were used to differentiate between iron deficiency anaemia and thalassemia trait. Results: 36 % of antenatal women had anaemia with 13 % having microcytic hypochromic type. Based on discriminative indices no conclusive evidence of thalassemia trait was identified. However, based on the RBC parameters, five women were selected for haemoglobin electrophoresis and all had Hb A2 levels[3.5 %. Conclusion: Significant number of microcytic hypochromic anaemias were detected. Studies on methods and compliance of iron supplementation to antenatal women need to be done. Keywords Antenatal, Microcytic, Anaemia, Screening, Indices

OR 62 Prevalence of Weak D Among Blood Donors and Patients at a Tertiary Care Teaching Hospital in Southern India i.e. SVIMS, Tirupati, AP Dr. M. Anitha, Dr. G. Deepthi Krishna, Dr. B. Suresh Babu, Dr. R. Arun, Dr. K. V. Sreedhar Babu, Dr. D. S. Jothi Bai Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh Summary: To study the prevalence of weak D antigen in the blood donors and patients of a tertiary care teaching hospital in Tirupati i.e. SVIMS, Tirupati, AP. Introduction: Rh-D antigen is the next most important after ABO system and it is one of the clinically significant antigens in terms of transfusion and pregnancy. Weak D refers to reduced expression of D antigen on the red blood cell that requires an extended testing with indirect antiglobulin test. Clinical importance arises when labeling the donor and patient, as the donor is labeled as D positive, patient as D negative. Materials and Methods: This is a retrospective and prospective, cross-sectional descriptive and analytical study carried out in our department. All blood donor and patient samples were tested for ABO and Rh (D) using two anti-D reagents (IgM monoclonal/IgM and IgG blend). The blood samples which were negative were further tested for weak-D using IgG anti-D. Results: A total of 46,654 blood samples were tested (22,326 donors and 24,328 patients) from

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Topic: Free Paper OR 63 Hemolytic Disease of the Newborn Secondary to Rh and other Minor Blood Group Antibodies Dr. B. Shanthi1, Dr. S Vijalakshmi2, Dr. C. Vishala Sarma3 1

Associate Professor in Department of Transfusion Medicine, Lecturers in Department of transfusion Medicine Department of Transfusion Medicine, Nizam’s Institute of Medical Sciences, Punjagutta 1,3

Introduction: Hemolytic disease of the fetus and newborn (HDFN) is a condition in which transplacental passage of maternal antibodies results in immune hemolysis of fetal/neonatal red cells. The implicated antibodies could be naturally occurring (anti A, anti B) or immune antibodies which develop following a sensitizing event like transfusion or pregnancy. The hemolytic process may result in anemia or hyperbilirubinemia or both; thereby affecting fetal/neonatal morbidity and mortality. Alloantibodies other than anti D have emerged as an important cause of HDFN and are now responsible for greater proportion of these cases. Timely detection and close follow up of this condition is necessary to reduce harmful effects on the newborn. Transfusion services play a vital role in the antenatal detection, monitoring and providing transfusion support to such cases. The objectives of the study 1. To determine the incidence and Associated risk factors for neonatal hyperbilirubinemia resulting from ABO, RH incompatibility and other minor blood group antibodies in a tertiary care hospital. Materials and Methods: A retrospective study was done for a period from January 2013 to August 2014. Ref Data on mothers and babies blood group, Rh and kell extended phenotyping, direct Coombs test, indirect Coombs test, Auto control, Antibody screening by 3 cell and 11 cell panels. Correlated with hyperbilirubin levels, haemoglobin and other laboratory values. Previous obstetric history, transfusions were also analyzed. Results: Reviewed 45 cases all presented with neonatal jaundice, with in 24 h to 2 months age of the baby. 90 % male babies, 75 % of the cases showed ABO incompatibility between mother and baby. 20 % showed Rh incompatibility. Direct Coombs test was positive in 25 % of the cases. 20 % of the cases showed Indirect Coombs positive in mother. Auto control was positive in one case. Anti-D, c, E, C Rh system antibodies in 90 % of the cases followed by Duffy and Anti-M antibodies. Conclusion: Thus, it highlights the importance of thorough antenatal ABO, RhD blood grouping and antibody screening, and if necessary, antibody identification and regular monitoring of antibody screening and antibody levels for prevention or early detection of hemolytic disease of the fetus and newborn, especially in cases of mothers with clinically significant red cell alloantibody. Keywords HDFN, Minor blood group antibodies, Rh syetem

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Topic: Laboratory Hematology—Reference Range OR 64 Reference Range for Haematological Profile of Umbilical Cord Blood: A Study Done at a Tertiary Care Centre in South India Dr. Febe R. Suman, Dr. Priyathersini, Mr. Reddy S. Sudheer Raj, Dr. Rithika Rajendran, Mrs. Umalakshmi Department of Pathology, Sri Ramachandra Medical College and Research Institute, Chennai, India Objectives: Umbilical cord blood (UCB) a source of hematopoietic stem cells, is also an acceptable sample to assess neonatal sepsis. Though reports are available for stem cell counts very minimal literature is available regarding hematologic parameters, which may vary on ethnicity. This study is intended to establish reference ranges for haematological parameters of umbilical cord blood to guide neonatologists, hematopoietic stem cell transplant specialists and future analysis. Materials and Methods: Prospective longitudinal study was done from January 2014 to April 2014 after ethics committee approval. UCB from 80 full term new born of normal birth weight born out of uneventful pregnancy to mothers aged between 21 and 45 years with hemoglobin above 10 g/dl were processed in Beckman Coulter LH780 analyzer for complete blood count and counter checked by peripheral smear. Results tabulated in Microsoft excel are analyzed using IBM SPSS statistics 19 software. Results: Male to female ratio is 1:1.05. Reference ranges are Hb 14.9 ± 1.7 g/ dl, RBC 4.1 ± 0.4 9 106/ll, HCT 44.6 ± 5.3 %, MCV 108.1 ± 4.8 fl, MCH 36 ± 1.7 pg, MCHC 33.3 ± 0.8 g/dl, RDW 13.7 ± 3.5, NRBC 3.7 ± 3.5/100WBC, WBC 15.9 ± 5.1 9 103 ll, Polymorph 8 ± 1.2 9 103 ll (50.3 ± 12.2 %), Lymphocyte 5.7 ± 0.9 9 103 ll (35.9 ± 12.2 %), Monocyte 1.4 ± 0.4 9 103 ll (8.9 ± 3.1 %), Eosinophil 0.46 ± .06 9 103 ll (2.9 ± 2 %), Basophil 0.2 ± 0.05 9 103 ll (1.5 ± 1.2 %), Platelets 215 ± 67 9 103 ll, MPV 8.4 ± 0.9 fl. Gender difference nil. Conclusion: Reference ranges are comparable with existing ranges for UCB and at birth. This study can be a useful guide to neonatologists, hematopoietic stem cell transplant haematologists and future analysis. Keywords Umbilical cord blood, Reference range

Topic: Free Oral Paper

S469 red cell concentrate units were wasted for the reason of time expiry due to open system. 3.21 % of fresh frozen plasma units had to be discarded due to breakage during storage and transfer. Conclusion: The wastage of components can be utilized as a quality indicator for continuous assessment of the quality improvement plan of the blood bank further helping in refining the blood transfusion system. Keywords Blood Bank, Blood, Blood Component, Wastage, Transfusion

OR 66 A Descriptive Study on Clinical Efficacy and Adverse Effects of Apheresis Platelets Nittin Henry, Vinod Kumar Panicker, R. Krishnamoorthy Sri Ramachandra Medical College and Research Institute Summary: A descriptive study to enumerate the indications, analyze the efficacy and to evaluate the risk to single donor platelet transfusions. Introduction: This study was a prospective cum retrospective descriptive study conducted in a tertiary health center to identify the various indications, diagnostic groups, efficacy and incidence of adverse effects with respect to SDP transfusions. Materials and Methods: The study was done in 50 patients who underwent SDP transfusions between 2010 and 2012. All the platelets were prepared by Fresenius Kabi ‘COM TECH’ cell separator. The study group included both the gender, age groups from 10 to 80 years and wide variation in ethnicity. The study group belonged to 19 different diagnostic groups which was further classified into thrombocytopenia due to increased platelet destruction and decreased platelet production. 24 % were Dengue patients and 22 % were acute myeloid leukemia patients among the study population. Descriptive statistics was used. Results: In the present study 40.57 % of SDP transfusions were therapeutic 59.4 % were prophylactic platelet transfusions. Therapeutic response was observed in 48.8 % as shown by the corrected count increment (CCI) and percentage platelet recovery (PPR). The incidence of transfusion reaction was 0.94 % which manifested as allergic. Conclusion: Considering the efficacy and the lesser incidence of adverse effects, single donor platelet transfusions hold great promise for patients needing platelet transfusions in view of their clinical condition, more so for hematology patients on chemo/ radiotherapy.

OR 65 Wastage of Blood Components in a NABH Accredited Blood Bank Dr. Kashish R. Panchal, Dr. Shayaz Ali Siddique, Dr. Mustafa F. Ranapurwala, Dr. Monica Gupta Pramukh Swami Medical College, Karamsad, Anand, Gujarat Summary: The purpose of this study was to determine the rate and causes of wastage of blood components in a NABH accredited blood bank and a few steps that can be taken to reduce the wastage. Introduction: Blood transfusion is an essential part of patient care. While the blood supply is seriously limited, the demand for blood is increasing every year. In spite of various measures taken to reduce the wastage of components, we still encounter wastage of components each year. Materials and Methods: The study was conducted in A.D. Gorwala blood bank. This descriptive study was based on available data from blood bank records. The collected data were analyzed using descriptive statistic methods. Results: In 2012 and 2013, 3.96 % of whole blood units and 2.57 % components were discarded. 0.40 % of

Topic: Hematology OR 67 Incidence of Post Transfusion Reactions in the Thalassemics using RBCS and Leucodepleted RBCS Dr. Sarita Nair, Dr. Surekha Patil, Dr. Ashok Patil, Dr. Nadia Shafi Al Ameen medical college Bijapur, Karnataka Objective: Any untoward event occurring in a patient following transfusion of whole blood or components is called transfusion reaction. Multiple transfused patients like thalassemics are known to develop agglutin. Leukocyte removal from blood components before transfusion prevent or reduce the incidence of leukocyte mediated adverse reactions. Our aim in this study is to evaluate post transfusion reaction of blood in thalassaemics with RBCs. Leucodepleted RBCS prepared using top and bottom quadruple blood bags by semi automated plasma expressor Materials and Methods: 2 different types of

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S470 red blood concentrate were used in this study. 100 thalassaemics who received transfusion in the blood bank outside Bijapur are included in this study. They received transfusion on an average once 3 weeks. Age between 5 and 14 years was included. Red blood concentrate prepared from whole blood using double bag and Leukodepleted RBC prepared by semi automatic method using Top and bottom Quadruple blood bags were used. Results: Post transfusion reactions with RBC transfusion are 3.5 % and with leucodepleted RBC prepared from Quadruple blood bags is 1 % Conclusion/Diagnosis/Impression: Use of leucodepleted RBC reduces the incidence and severity of number of adverse transfusion reactions. Post transfusion reactions in thalassaemics are found minimum in our study using quadruple top and bottom blood bags. Keywords Thalassemia, Leucodepletion

Topic: Free paper OR 68 Therapeutic Phlebotomy: An Approach to Medical EvidenceBased Phlebotomy from Thousands of Years Old Blood Letting Practice, Review of Current Practice in a Tertiary Care Center Dr. V. Sudhir, Dr. B. Shanthi2, Dr. S. VijayaLaxmi1, Dr. C. Vishala Sharma3 Lecturers in Department of transfusion Medicine, 2Associate Professor in Department Of Transfusion Medicine Department of Transfusion Medicine, Nizam’s Institute of Medical Sciences, Punjagutta

1,3

Background: Originally, several 1,000 years ago, phlebotomy was used for the treatment of various disorders, but in addition to its therapeutic benefits, phlebotomy also had a preventive role. In present day medicine, phlebotomy can be performed in physicians’ offices, at a blood bank or in hospital under the supervision of a doctor after obtaining a medical prescription stating the indication. Currently, therapeutic phlebotomy is approved for three main indications: haemochromatosis, polycythaemia vera and porphyria cutanea tarda. In our Institute this review focuses not only on the three main indications but also discusses some of the other possible indications. Aim of the study: To review the indications and current practice of therapeutic phlebotomy in our department of transfusion medicine. Materials and Methods: Reviewed all the procedures of therapeutic phlebotomy performed during the period August 2008 to August 2014. Indications, weight, haemoglobin, haematocrit, volume collected, peripheral vascular access, post phlebotomy were reviewed. Results: Total 95 patients who under went multiple sessions of therapeutic phlebotomy were reviewed. Age of the patients ranged from \10 to 85 years peak being second, third and fourth decades. Based on the indications Haemoglobin ranged from 10 to 25.8 g/dl, highest being 18–20 g/dl. Packed cell volume ranged from 35 % to 73 % maximum no. of cases 51–60 %. 50 % of the patients had single session of therapeutic phlebotomy. Commonest indication being Polycyathemia vera(25 %),Congenital heart disease(15 %), secondary polycythemia vera, post renal transplant, COPD, Myelofibrosis, Obesity induced Breathlessness, heamochromatosis.etc. Volume collected during the procedure ranged from 150 to 450 ml maximum no. of procedures 301–400 ml. Complications were seen in 2 % of the cases Breathlessness and cyanosis. Conclusion: In summary, therapeutic phlebotomy is an essential part of the treatment of various diseases especially those associated with iron overload. In our opinion, it is a safe and costeffective treatment that should also be considered for use as adjunctive therapy in the treatment of the other disorders discussed in our review. Keywords Therapeutic phlebotomy, Polycyathemia vera, Chd.copd

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Topic: Free paper OR 69 A Study of Chronic Red Cell Transfusions in Thalassemia and Sickle Cell Disease Patients with Emphasis on Auto/ Alloimmunization and Selection of Blood Units, Review of 230 Transfusion Episodes in 33 Patients Dr. S. VijayaLaxmi1 Dr. B. Shanthi2, Dr. C. Vishala Sharma3, Dr. Suman Jain4 1,3

Lecturers in Department of transfusion Medicine, 2Associate Professor in Department Of Transfusion Medicine Department of Transfusion Medicine, Nizam’s Institute of Medical Sciences, Punjagutta, 4Thalassemia and Sickle Cell Society Hyderabad

Background: Transfusions are the primary therapy for thalassemia and sickle cell disease but have significant complications and expose the patients to a variety of risks. Continuous blood transfusion can causes alloimmunization against RBC antigens and complicate further treatment in these patients, Alloimmunization to red cell antigens is one of the most important immunological transfusion reaction and results in ineffective transfusion. However, few data are available on the frequency of RBC alloimmunisation in the Indian population with history of chronic transfusions. Extended phenotype matched blood may help in decreasing the rate of alloimmunization and effectiveness of transfusion. Aim of the Study: The purpose of this study is to evaluate the frequency of RBC allo and auto antibodies and the role of extended Rh and Kell phenotype matched blood to prevent alloimmunization. Materials and Methods: Reviewed all the red cell transfusions given to clinically diagnosed patients of thalassemia major, intermedia and sickle cell anemia/disease patients between Aug 2011 and Aug 2014 Total 33 patients and 230 blood transfusion work up were reviewed. ABO Rh and Kell grouping was done, direct and indirect Coomb’s test, Antibody screening for auto and allo antibodies, Anti body identification and Coomb’s cross match were performed by column agglutination method. Results: Allo antibodies like anti-c, E, e, C, D(Rh system), Kell, Xga, Duffy were identified in the patients. 9 patients presented with both auto and allo antibodies. Rh extended and Kell along with ABO typing done for the donors for each transfusion. Conclusion: Rh and Kell extended phenotype matched blood helped in preventing delayed haemolytic reactions.RBC allo antibodies in our patients with b-thalassemia were mostly Rh system antibodies. The development of RBC allo and auto antibodies was associated with previous transfusion reactions. Rh and Kell extended Phenotyping also helped in preventing further alloimmunization. Keywords Alloimmunisation, Thalassaemia, Sickle cell disease, Transfusion, Rh an

OR 70 Quality Control of Blood Components: A step Towards Efficient Supply of Blood Products Dr. Swapnil N. Patel, Dr. Vishva C. Patel Department of Pathology, Pramukh Swami Medical College & Shree Krishna Hospital, Karamsad, Gujarat Introduction: Quality Control describes steps taken by Blood Bank to ensure that tests are performed correctly. Primary goal of Quality Control is Transfusion of safe quality of blood. It is to ensure availability of efficient supply of blood, blood components. Materials and Methods: All donor blood bags at A.D. Gorwala Blood Bank in Pramukh Swami Medical College & Shree Krishna Hospital,

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Karamsad, collected in between year 2012–2014 were included in our study. Monthly QC of this blood bags were done. Selection criteria was 1 % of total collection or minimum 4 bags per month. Results: RCC—91.30 %, FFP—87.89 %, PC—97.98 %, CP—84.44 % of QC were found within range in our study. While RCC 8.70 %, FFP— 12.10 %, PC—2.04 %, CP—15.55 % were out of range. Conclusion/ Diagnosis/Impression: At present era Quality Control is very important step in maintaining quality of blood bank. So that we ensure most efficient blood transfusion to patients. Keywords Quality Control, RCC, FFP, CP, PC

Topic: Proffered OR 71 Prevalence of Transfusion Transmitted Infections in Thalassemia Major Patients: Data from a Tertiary Care Centre in North India Dr. Alka yadav, Dr. Jasbeer, Dr. Sanjeev Nanda, Dr. Geeta Gathwala, Dr. Nishu Pt. B.D.Sharma PGIMS, Rohtak. Haryana Introduction: Transmission of various infectious agents remains the most significant concern for multitransfused individuals such as thalassemics and hemophillics. Even after mandatory screening of blood products for HIV, HBV, HCV, syphilis and malaria, blood safety remains questionable in developing countries like India. Materials and Methods: All the enrolled patients who were on regular transfusions for at least 1 year at the institute were enrolled. Data regarding transfusions received and the viral markers for HIV, HBV and HCV were collected at enrolment, 6 months and then at 1 year of study. Results: Out of 285 patients enrolled at the institute, 276 fulfil the inclusion criteria. Out of 276, 171 were males and 105 were female patients. The mean age at first transfusion was 6.4 ± 3.8 months. The average number of transfusions and the amount of blood received per year were 18 ± 3 and 112 ± 15 ml/kg respectively. Total 80 patients (54 males and 26 females) screen positive for at least one infection at the time of enrolment in the study. Amongst them one male was HIV ELISA, 5 were HBsAg (3 males), and 74 (50 males, 24 females) were HCV positive. One patient died during the study during study due to cardiac complications. At one year 3 new patients (2 females and one male) got anti-HCV antibodies positive. Conclusion: Blood safety is a major concern for thalassemia patients in India. Keywords Multitransfusion, Transfusion transmitted infections

Topic: Free Paper Session OR 72 Towards Fertility Preservation in Children Undergoing Low Risk Bone Marrow Transplantation for Severe Thalassemia: Can ATG Substitute Thiotepa? Priya Marwah1, Rajpreet Soni1, Sadaf Khalid2, Naila yaqub3, Fatima Itrat3, Sarah Khan Gilani3, Mohammed H. Hussain3, Tatheer Zahra3, Lallindra Gooneratne4, Ruwangi Dissanayake4, Rachna Narain5, Jagdish Singh5, Mohammed El Missiry2, Cornelio Uderzo2, and Lawrence Faulkner2 1 South East Asia Institute for Thalassemia, Jaipur, India, 2Cure2 Children Foundation, Florence, Italy, 3Pakistan Institute of Medical Sciences, Islamabad, Pakistan, 4Central Asiri Hospital, Colombo, Sri Lanka, 5Sawai Man Singh Medical College and Hospital, Jaipur, india South East Asia Institute for Thalassemia, Jaipur, India

S471 Summary: Bone marrow transplantation (BMT) offers a definitive cure with improved health related quality of life (HRQoL) in over 90 % of low-risk children with a matched related donor. This study retrospectively compares BMT outcomes in two groups of low risk patients with severe thalassemia (ST) receiving thiotepa–busulfan– cyclophosphamide (TTBu–Cy) or ATG–Bu–Cy as preparative regime. No difference in BMT outcomes was detected while higher fertility rates are expected for patients on ATG–Bu–Cy regime. Introduction: Many centres currently incorporate thiotepa in busulfan or treosulfan based BMT regimens for thalassemia. This combination however may permanently impair fertility in most patients. In the era of increasingly effective supportive care in which many thalassemia patients may have children, this is increasingly concerning. Very long term follow up studies have shown how the standard Bu–Cy regime may be associated with birth rates comparable to the control population (La Nasa et al., Blood, 2013). Materials and Methods: This is a retrospective multicentre comparative study of the safety and efficacy of substituting TT with ATG in low risk ST patients undergoing matched related BMT. Between January 2009 and July 2013, a group of 35 patients were transplanted after conditioning with TT-Bu-Cy, while between August 2013 and July 2014, 14 patients were conditioned with ATG–Bu–Cy Results: The actuarial overall survival in the TT–Bu–Cy and ATG–Bu–Cy group is 94.2 and 92.8 % and thalassemia- free survival is 91.3 and 92.8 % respectively, with no statistically significant difference by logrank test. Conclusion/Diagnosis/Impression: Substituting thiotepa with ATG in the standard Bu–Cy context seems safe and effective and may substantially improve fertility rates and thus long term HRQoL in children undergoing BMT for severe thalassemia. Keywords BMT, Fertility, Thiotepa, ATG

OR 73 Challenges of Managing a New Thalassemia Clinic in a Municipal Hospital in Delhi Priyanka Verma, Payal Malhotra, Sandeep Jain, Gauri Kapoor, Sanjay Chaudhary Rajiv Gandhi Cancer Institute and Research centre New Delhi, Dr Baba Saheb Ambedkar Hospital New Delhi Summary: Holistic approach to education is required to optimize care of children with thalassemia major. Facilities for free transfusion and chelation are clearly not enough and require supplementation with structured counselling. Introduction: Managing children with thalassemia major in a resource limited setting remains a challenge. The objective of this study was to analyze the clinical profile, transfusion and chelation status and scope of improvement in their management. Materials and Methods: Between July 2012 and 2014, 39 children of b thalassemia major were registered in a free hematology clinic of a municipal hospital. Demographic profile, transfusion and chelation data were retrieved from the medical records. Results: Median age at diagnosis was 6 months (3–18 months), 69 % (22/39) were males and 85 % (33/39) belonged to lower socioeconomic status. The median pre-transfusion hemoglobin was 8 gm/dl (5–9.6 gm/dl), transfusion frequency of 30 days (21–45 days), despite non-replacement policy. Chelation with defrasirox (free supply) was started at median age of 3.5 yrs (1.5–5 years) after median transfusion volume of 175 ml/kg. First serum ferritin level was 650 ng/ml (166–1302 ng/ml) at a median age of 3 years (1–6 years), the highest ferritin was 1,580 ng/ml (400–4,283 ng/ ml).Overall 82 % (32/39) patients were on defrasirox @30 mg/kg (15–40 mg/kg). Undernutrition and short stature occurred in 23 % (9/ 39) and 56 % (22/39) of children and 51 % (16/39) were incompletely immunize. Conclusion/Diagnosis/Impression: Despite availability

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S472 of free transfusion and chelation this study identifies nutrition, immunization and regular transfusion as basic thrust areas for parental counseling. Holistic approach to education is required to optimize care of children with thalassemia major.

OR 74 How Structured data Management using BMTPlus is Enabling Favourable Bone Marrow Transplantations Outcomes in Developing Countries Rajat K. Agarwal1, Amit Sedai1, Sunil Dhimal1, Mohamed El Missiry2, Ankita Agarwal1, Sulman Siddique2, Luigi Clemente2, Sadaf Khalid2, Naila Yaqub3, Priya Marwah4, Rajpreet Soni4, Fatima Itrat3, Sarah Khan Gilani3, Mohamed H. Hussain2, Tatheer Zahra3, Lallindra Gooneratne5, Ruwangi Dissanayake5, Corrado Zuanelli2, Nunzia Manna2, Cornelio Uderzo2 and Lawrence Faulkner2 Jagriti Innovations, Bangalore, India, 2Cure2Children Foundation, Florence, Italy, 3Hematology-Oncology, Children’s Hospital Pakistan Institute of Medical Sciences, Islamabad, Pakistan, 4South East Asia Institute for Thalassemia, Jaipur, India, 5Central Asiri Hospital, Colombo, Sri Lanka 1

Introduction: There is a urgent need to setup more BMT centers where reliable and cost-effective transplants can be offered. Jagriti Innovations developed a data management tool in partnership with the Cure2Children Foundation, Italy that has been used by health professionals in India, Pakistan and Italy and Sri Lanka since August 2008. Materials and Methods: BMTPlus was built using free and open source tools including LAMP (Linux, Apache, MYSQL, and PHP) software stack and using Drupal CMS (http://drupal.org) as the application development framework. This online database covers data recording, analyzing, and reporting besides enabling knowledge exchange, telemedicine, capacity building, and quality assurance. Results: As of July 2014, over 2,600 patients have been registered and 140 HLA-compatible related bone marrow transplantations (BMT) primarily for patients with TM. Actuarial thalassemia-free survival is 87 % in the whole group of 60 TM patients (median follow-up of 13 months) and 92 % in the 48 low-risk cases (no hepatomegaly and age \7 years) (median follow-up of 15 months) treated homogeneously in institutions consistently relying on BMTPlus. These outcomes compare favorably with international standards and where obtained at a fraction of the cost of BMTs in affluent countries. Conclusion/Diagnosis/Impression: BMTPlus seems to have streamlined and simplified key processes providing stakeholder with real-time information and alerts as well as facilitated structured quality assurance and telemedicine collaboration. This cost-conscious, simple and yet robust approach was associated with favorable outcomes in a well-defined group of patients undergoing BMT in start-up centers.

OR 75 Evaluation of Endocrine Dysfunctions in Children with Beta Thalassemia Major on Long Term Transfusions Dr. K. Sravanthi, Dr. Shrikiran Hebbar, Dr. Suneel Mundkur Department of Paediatrics, Kasturba Hospital, Manipal Introduction: Blood transfusion, which is critical for survival of thalassemia patients is double edged sword, prolonging life while eventually leading to iron overload resulting in various endocrine

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 dysfunctions. Materials and Methods: A cross sectional study was carried out in Kasturba Hospital, over a period of 2 years, on all children with b-thalassemia major more than 5 years of age receiving regular blood transfusions. Patients with transfusion dependent anemia other than b-thalassemia major were excluded. Growth assessment was measured by Height SDS, height and weight according to age. For all patients serum ferritin concentration, fasting blood sugar and postprandial blood sugar, thyroid stimulating hormone were measured. Results: The total number of children included in the present study was 33. Median age of children at the time of enrollment in the study was 9 years. Mean Pretransfusion Hb was 6.4 ± 0.94 g/dl and the median serum ferritin level was 2,926 ng/ml (2,406, 4737 ng/ml). The most common endocrine abnormality was delayed puberty (64 %). Short stature was the second most frequent endocrine complication with prevalence of 60.6 %. Impaired glucose tolerance (IGT) and diabetes mellitus were seen in 21 and 3 % patients respectively. Hypothyroidism was not seen in any child in the study group. Mean age of patients with Short stature was 11 ± 1.63 years, delayed puberty was 14.33 ± 1.8 years and IGT and DM was 14 ± 1.63 years, implying that endocrine abnormality usually occurs in the second decade of life. In the present study, 63 % of patients with thalassemia major suffered from at least 1 endocrine abnormality mostly in the second decade of life. 36 % of patients had no endocrine abnormalities. Conclusions: In this study, delayed puberty and short stature were the most common abnormalities observed. Most of the children had abnormalities during the second decade of life. No relationship between serum ferritin and development of endocrinopathy was noted. Keywords Beta thalassemia major, Growth, Puberty, Glucose intolerance

Topic: Hematology OR 76 Clinico-Hematological Profile of HbE Hemoglobinopathy Diagnosed in a Tertiary Care Centre in Delhi Dr. Pragya Jain, Dr. Neha Garg, Dr. Meera Sikka, Dr. Vandana Puri UCMS and GTBH, New Delhi Objectives: Hemoglobinopathies are the most common inherited red cell disorder seen worldwide. HbE is a structural variant with a high prevalence in north eastern India (allele frequency 10.2 %).This study aims to evaluate the clinic-hematological profile of patients with HbE diagnosed over a 5 year period at a tertiary care centre. Materials and Methods: Data of 272 patients diagnosed with hemoglobinopathy was analysed. Of these HbE was diagnosed in 34 (12.5 %) patients. Complete blood count (automated hematology analyser LH 500), Hb electrophoresis(cellulose acetate using TEB buffer ph 8.7) were done in all the patients. Iron profile was done whenever necessary. Hemoglobinopathy was diagnosed using standard criteria. Cases with Hb E structural variant were further categorized into HbE heterozygotes, HbE homozygotes and compound heterozygotes. Results: Of the patients diagnosed with Hb E there were 21 (61.7 %) patients of HbE trait, 2 (5.8 %) with HbE disease and 11 (32.3 %) were compound heterozygotes. There were 16 (47 %) females and 18 (53 %) males. The age of the patients ranged from 2 to 55 years. Mean ± SD hemoglobin was 9.01 ± 1.06 g/dl. Anemia was detected in 94 % patients. Concomitant iron deficiency was detected in 5 (15 %) patients. HbE ranged from 20 to 88.6 % in these patients. Conclusion: This study highlights the migration related fluctuation in prevalence of HbE hemoglobinopathy in Northern India. It’s

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 interaction with other hemoglobinopathies especially beta thalassemia trait leads to a more severe clinical form akin to b thalassemia major Keywords Hemoglobinopathy, HbE trait, HbE disease

OR 77 Comparison of Hematological and Molecular Data Between Transfusion Dependent Thalassemia and NTDT Soumi Chaudhuri, Sujana Biswas, Meet Kumar, Aditi Sen Nil Ratan Sircar Medical College & Hospital, Kolkata-700014 Summary: Varying clinical severity in Hb E-beta thalassemia is perplexing. We found that high HbF levels and presence of xmn-I polymorphism are associated with milder phenotype. Introduction: Clinical severity of E-beta thalassemia varies from transfusion dependent to mildly symptomatic patients who sparsely require transfusion. We evaluated for hematological and molecular differences between two groups. Materials and Methods: We retrospectively evaluated hematological and molecular differences between transfusion dependent and independent patients with Hb E-beta thalassemia. Results: We found high HbF levels in NTDT patients, and also high frequency of xmn-1 polymorphisms in these patients (as shown in Table 1 below).

levels correlates positively with the severity of the disease. This suggests the role of immune attack on hematopoietic stem cells in these cases and such patients may benefit from immunosuppressive therapy. Materials and Methods: Thirty children with AA between age of 6 months–18 years were included along with age & sex matched controls. CBC and bone marrow examination was done. They were grouped as very severe aplastic anemia (VSAA), Severe aplastic anemia (SAA) and Non severe aplastic anemia (NSAA) as per standard guidelines. The serum levels and intracytoplasmic expression of Type 1 cytokine (IFNc) and Type 2 cytokine (IL4) was done by (ELISA) and Flowcytometry (FCM) respectively. CD8 and CD4 cell counts were also determined. Results: The CD8 cell count was higher than the CD4 counts with reversal of CD4:CD8 ratio in all patients and had significant correlation with clinical severity. The mean serum IFNc (ELISA) and intracytoplasmic IFNc expression (FCM) was higher in cases than in controls (p \ 0.0001) and both showed graded positive correlation with the severity of the disease (p \ 0.0001). Type 2 cytokine (IL4) did not show any such correlation. The percentage of CD8 cells expressing IFNc was higher than the percentage of CD4 cells expressing the same(FCM). Conclusion: IFNc is the predominant effector cytokine. The CD8 cells are main source of IFNc. The reversal of CD4:CD8 ratio and IFNc levels are predictors of clinical severity of Aplastic Anemia. Keywords Aplastic anemia, Cytokine, Flow cytometry, IFN gamma

Topic: Clinical Hematology

Table 1 . Parameter

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TDT (N = 23)

NTDT (N = 24)

Age at diagnosis (years) 3.4

7.8

Baseline Hb (g/dl)

6.7

7.9

S. ferritin

1,160.3

972.2

HbF

14.4

24.2

HbA2+E

47.4

57.7

Yearly transfusion

14.2

Occ

Beta mutation

IVS 1–5 (20), Cd15 (3)

Cd15 (4), Cd41/42 (2), IVS1–5 (18)

Xmn-1 polymorphism

Hetero (2), Hetero (6), Homo (3), N (18) Homo (13), N (5)

Conclusion: We conclude that presence of high HbF levels and presence of xmn-1 polymorphism portend milder phenotype in patients with Hb E-beta thalassemia.

Topic: Award OR 78 Study of Cytokine Profile of Type 1 and Type 2 T Cells in Children with Aplastic Anemia B. Aasha, A. Nangia, S. Sharma and J. Chandra* *Department of Pathology and Paediatrics, Lady Hardinge Medical College and Associated Kalawati Saran Children’s hospital New Delhi 110001 Summary: The intracytoplasmic and serum levels of IFNc and IL4 were studied in aplastic anemia patients and we found that IFNc

OR 79 Single Centre Experience with Indigenous Equine Anti-thymocyte Globulin in Aplastic Anaemia Dr. Chandrakala Shanmukhaiah1, Dr. Farah Jijina2, Dr Shailesh Bombarde3, Dr Rajesh Patil3 1 Associate Professor, Department of Haematology, Seth GS Medical College & KEM Hospital, Mumbai, 2Consultant, P.D. Hinduja National Hospital And Medical Research Centre, Veer Savarkar Marg, Mumbai, 3DM Registrar, Seth GS Medical College & KEM Hospital, Mumbai

Introduction: This retrospective analysis aimed to evaluate the benefit of indigenous equine anti-thymocyte globulin (eATG) in patients with aplastic anemia (AA). Materials and Methods: Patients with AA admitted to KEM Hospital, Mumbai from 2008 till February 2014 were allocated to 2 groups—indigenous eATG (Thymogam) and non-eATG. Dose of eATG, was 40 mg/kg/day for 4 days. All patients received cyclosporine and appropriate anti-microbials. Severity and response to treatment were classified as per standard guidelines. Response (complete-CR, partial-PR) was calculated at 6 (±1) months and 1 year (±2 months). Results: Eighty five patients (38 in Thymogam and 47 in non-eATG groups) were admitted. Median age was 24 years with male:female ratio of 1.6:1. Seventy-five of these were analyzable at 6 months. Sixty-four percent (11 CR, 10 PR) responded in Thymogam and 33 % (2 CR, 12 PR) in non-eATG group. Fifty-six patients (19 in Thymogam and 37 in non-eATG groups) were analyzable at 1 year’s follow-up. Eighty-four percent (10 CR, 6 PR) responded in Thymogam and 49 % (3 CR, 15 PR) in non-eATG group. Fourteen patients died (5 patients in each group died within 4 weeks were non analyzable); 6 in the Thymogam and 8 in noneATG group. Sixty-one adverse drug events were noted (rash and fever being the most common), distributed evenly across both groups. Conclusion: Thymogam is a safe and cost-effective indigenous eATG for immunosuppressive treatment of AA. Keywords Aplastic anemia, Antithymocyte globulin, Cyclosporine

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Topic: Hematology OR 80 A Clinicopathological Study of Bone Marrow Failure Syndrome with Reference to Treatment and Outcome in Paediatric Age Group Dr. Sadasiba Padhy, Dr. Pravakar Sethy, Dr. N. N. Soren M.K.C.G. Medical college, Berhampur, Orissa Objectives/Introduction: Bone marrow failure syndrome (BMFS) is a disorder of haematopoiesis which includes peripheral blood single cytopenias as well as pancytopenias due to inability of the marrow to effectively produce blood cells. AIM: To study the spectrum of clinical presentations of BMFS, to find out the etiological factors of BMFS, to compare the hematological profile and bone marrow aspiration cytological picture between pre-treatment and post-treatment, to assess the effectiveness of response to drug therapy. Materials and Methods: cases presented with symptoms and sign of BMFS with peripheral smear showing features of bicytopenia or pancytopenia, in the paediatric age group were included in my study. Bone marrow aspiration and haematological test were carried out. Aplastic anaemia cases were treated with Cyclosporine (5 mg/kg/day q12hrlyx 6 months).Other cases were treated with appropriate therapy. Stastistical analysis: by ANOVA followed by Bonferroni’s Multiple Comparison Test. Results: Out of 40 children, 21(55 %) were diagnosed as aplastic anemia. Pallor was present in all followed by fever (97.5 %). Out of all cases, 31(77.5 %) were treated, 3(7.5 %) were died before therapy and 6(15 %) were referred to highercentre. 21 cases of aplastic anemia were treated with cyclosporine, 6(i.e. 28.57 %) were completely responded, 7(i.e. 33.33 %)were partial responders and 8(38.10 %) were non responders to therapy. Others (malaria, megaloblastic anaemia, sickle cell anaemia) were cured (i.e. 100 %) after the appropriate treatment. Conclusion: Pallor was the most prominent sign of BMFS irrespective of causes. Aplastic anaemia was responsible for most ([50 %)cases of BMFS. Majority had acquired aplastic anaemia. Cyclosporine monotherapy on aplastic anemia had 28.57 % of response. The mortality rate of BMFS found to be 7.5 %. Keywords Bone marrow failure syndrome, Aplastic anaemia, Pallor, Cyclosporine A

Topic: Bone Marrow Failure OR 81 Outcome of Cyclosporine in Patients of Aplastic Anemia in Tertiary Care Hospital

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 16(28 %) non severe AA and 6(10 %) were very severe AA. At 3 months overall response rate (OR) was 7 (12 %) with 1(14.2 %) had complete response (CR), 6 (85.8 %) partial response (PR) and 50(88 %) had no response (NR). At 6 months the overall response (OR) rate of 19.6 % was achieved with 3 (27 %) had CR, 8 (73 %) PR and 46 (80.4 %) had no response (NR). Transient raised creatinine and increased bilirubin, SGOT and SGPT was the main side effects observed in this cohort. Conclusion: Oral cyclosporine at dose of 5 mg/kg/day is one of the treatment options for Indian patients who could not afford the cost of BMT or ATG therapy. Keywords Cyclosporine, Aplastic anemia

Topic: Free Paper OR 82 Response to Immunosuppressive Therapy in a Large Cohort of Adult Patients with Aplastic Anemia Yasir Jeelani Samoon, Auro Viswabandya, Aby Abraham, Abhijeet Ganapule, N. Fouzia, Anu Korula, Kavitha N. Lakshmi, Alok Srivastava, Vikram Mathews, Biju George Department of Clinical haematology, Christian Medical College, Vellore Summary: Immunosuppressive therapy (IST) with ATG and cyclosporine results in good responses in a large cohort of adult patients with aplastic anaemia (AA) from a single centre. Objectives: IST is the treatment of choice for patients with AA who lack a HLA matched donor. Limited data available on outcome in Indian patients. Materials and Methods: Adult patients who received ATG for AA between 1986 and 2013 were studied. IST included Pasteur Merieux ALG for 5 days or Pharmacia UpJohn ATGAM for 4 days followed by prednisolone prophylaxis and low dose cyclosporine for 6 months. Response was assessed at 6 months. Results: 416 patients (276 males; 140 females) with a median age of 40 years (range: 16–73) received ATG. 93 had NSAA, 254 had SAA and 69 had VSAA. 6 month response rates (RR) were 64.6 % with CR in 23.7 % and PR in 40.9 %. RR with NSAA was 69.8, 64.5 % with SAA and 57.9 % with VSAA. RR was 66.8 % between ages 16–30 years, 67.3 % between 31 and 50 years and 60.6 % for [50 years. At a median follow up of 38 months (range: 1–264), loss of response/relapse was seen in 27, clonal evolution to PNH in 12 and transformation to MDS/AML in 5. Repeat ATG given in 14 and BMT performed in 7. The 5 year KM estimates for OS for the entire group is 68.2 ± 2.2 %. Conclusion: IST results in reasonable responses in adult patients with AA and is a good alternative for patients who do not have a HLA matched sibling donor. Keywords IST Immunosuppressive therapy, AA Aplastic anemia

Dr. Shuvra Neel Baul, Dr. Tuphan K. Dolai, Dr. Pooja Prasad, Dr. Rajib De, Dr. Shyamali Dutta, Dr. P. K. Mandal, Prof. Prantar Chakrabarti

Topic: Free Paper

Department of Hematology, NRS Medical College, Kolkata

OR 83

Introduction: Most of the patients of aplastic anaemia (AA) attending in OPD could not afford the cost of allogeneic bone marrow transplant or ATG. The aim of the study was to show the efficacy and toxicity with oral cyclosporine at dosage of 5 mg/kg/day in aplastic anaemia Materials and Methods: This prospective study was conducted over a period of 2 years. The diagnosis and response to treatment of AA was established as per published criteria. Follow- up was done at 3 and 6 months in order to assess the response. Results: 57 patients of acquired aplastic anaemia with median age of 37 years (6–81 years) were included in the study. Male and female were 35(61 %) and 21(39 %) respectively. 35(62 %) with severe AA,

Phenotypic Profile of Fanconi Anemia Patients in a Tertiary Centre in Southern India

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Abhijeet Ganapule, Vikram Mathews, Auro Viswabandya, Aby Abraham, Kavitha N. Lakshmi, N. Fouzia, Anu Korula, Nancy Gabriel, Vivi Srivastava, Alok Srivastava, Biju George

Summary: This analysis attempted to describe the mode of presentation, phenotypic features and response to androgens in patients with

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Fanconi anemia (FA). Introduction: Fanconi anemia is a rare bone marrow failure syndrome characterized by bone marrow failure and skeletal abnormalities. Bone marrow transplant and androgens are the mainstay of treatment. Materials and Methods: 160 patients with a clinical phenotype of FA seen in CMC Hospital between September 1994 and October 2013 were analyzed. Results: There were 86 males and 74 females with a median age of 11 years (range 3–36). At presentation, 157(98 %) had anemia with 117(73 %) having received transfusions. Bleeding symptoms occurred in 50 (31 %) with documented infection in 4 (2.5 %). Skeletal abnormalities were seen in 113 (70 %), facial dysmorphism in 85(53 %), skin changes in 136(85 %) and genital abnormalities in 1(0.6 %). Renal and cardiac abnormalities were seen in 24 % and 2.4 % of patients tested. Bone marrow findings in 155 patients showed aplastic anemia in 129 (81.2 %), Myelodysplastic syndrome in 25(15.6 %) and Acute myeloid leukemia in 1(0.63 %). Stress cytogenetics data in 158 patients was positive (score [40) in 84(53.1 %) and negative in 74(46.9 %). Androgens (stanazolol or danazolol) were used in 108(67.5 %) patients while 35(21.8 %) underwent an allogeneic transplant. Conclusion: Skin changes and skeletal abnormalities were common in FA patients and commonest marrow finding was aplastic anemia. Androgens are a common modality of treatment with a few proceeding for a stem cell transplant.

Topic: Free Paper OR 84 Decitabine in Elderly AML: The Road Less Travelled K. Ashok kumar, Stalin, Ramu, Krishnamani, G. Sadashivudu, M. Lakshmi Srinivas Nizams Institute of Medical Sciences Summary: AML in elderly is difficult to treat with conventional chemotherapy due to poor PS and Organ reserve. So hypomethylating agents can be tried in elderly AML patients with poor PS which showed good tolerance, effective outcomes and less toxicity. Introduction: Acute myeloid leukemia (AML) is usually a disease of the elderly. There is a growing interest in the use of hypomethylating agents as frontline therapy in elderly AML patients as they have difficulty in tolerating chemotherapy due to comorbidities, poor performance status and poor organ reserve. Data from India is scarce on this subject. Materials and Methods: To study the clinical benefit of Decitabine in elderly AML patients with PS C 2 at our center. Elderly AML patients diagnosed at our institute from 2012 to 2014 were analyzed with respect to demographic characteristics, treatment offered, responses, toxicity and outcomes. Results: The study included 11 patients (5 males, 6 females) with a median age of 66 years. Three patients had a history of MDS with the median duration prior to AML being 14 months. All patients received Decitabine (20 mg/m2, day 1–5 q 4 weekly). The median number of days to achieve complete hematological response was 28 days and the median number of cycles taken was six. The median nadir leucocyte count, median nadir ANC and platelet count were 1,800,585 and 20,000 cu mm respectively. Time taken for the leucocyte recovery was 12 days. The median PFS and OS were 3.2 and 4.4 months respectively Conclusion/Diagnosis/Impression: The median PFS and OS were comparable to reported studies. Small sample size is a limiting factor. Larger numbers are needed to draw definite conclusions

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Topic: Free Paper OR 85 Use of Hypomethylating Agents in Patients with Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) Mobin Paul, Vikram Mathews, Auro Viswabandya, Aby Abraham, Abhijeet Ganapule, Alok Srivastava, Biju George Department of Hematology, Christian Medical College, Vellore Summary: Retrospective analysis of consecutive patients with MDS and AML who received hypomethylating agents based therapy in the Department of Haematology, CMC Vellore between January 2012 and June 2014. Introduction: Hypomethylating agents are used in the treatment of high risk MDS and in elderly AML patients but there is limited data from India. We studied the response to therapy, side effects and outcome in patients treated with hypomethylating agents. Materials and Methods: Retrospective analysis of consecutive patients who received hypomethylating agent based therapy for various hematologic malignancies in the Department of Haematology, CMC Vellore between January 2012 and June 2014. Patient demographics along with the clinical & laboratory parameters were recorded. Patients were monitored for progression of the disease, neutropenic sepsis and other major life threatening complications. Results: There were 63 patients with a median age of 47 years (range 11–80). Twenty seven (43 %) had MDS, 36(57 %) had AML (primary and secondary). Forty-eight patients received decitabine while 15 received azacytidine. Of the MDS patients, 7 (27 %) were intermediate I by IPSS, 13 (50 %) were intermediate II & 6 (23 %) were high risk. The median number of cycles given were 3(1–25). Response to therapy was assessed for 28 patients who received at least 3 cycles and response was seen in 36 %. Majority (76 %) had neutropenic sepsis necessitating inpatient therapy & 27 % developed fungal pneumonia. Conclusions: Hypomethylating agents are a useful mode of therapy in patients with MDS and AML and responses lead to improved quality of the life. Keywords Hypomethylating agents, AML, MDS

OR 86 Myeloablative Haploidentical Stem Cell Transplantation (PTCy) in Relapse/Refractory Acute Myeloid leukemia: A Single Center Experience Rohith G. Chitrapur, Mammen Chandy, Sriam Ravichandran, Damodar Das, Roshan Dixit, Neha Ganju, Viswadeep Khusoo, Jeevan Garg, Saurabh Bhave, Mayur parihar, Neeraj Arora, D. K. Mishra,. Reena Nair, Anupam Chakrapani Tata Medical Center, Kolkata, Department of Clinical Hematology and Transplant Summary: Myeloablative Haplo-identical transplant (PTCy) is effective option in Relapse/Refractory Acute Myeloid Leukemia. At our center this novel protocol used in 8 relapsed/refractory AML showed very good outcome and the longest survivor of this cohort is 730 days. Materials and Methods: Total of 8 AML patients not in remission were conditioned with IV Flu 30 mg/m2/day for 4 days and IV Busulfan—3.2 mg/kg/day for 4 days. For GVHD prophylaxis PTcy 50 mg/kg 9 2 days (D+3, +4) and Tac/MMF with GCSF from Day +5.

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S476

No. Age/sex

1

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Cytokine Engraftment Chimerism Present status Cell dose: reaction NEU-1 PLT-2 D+28 CD34 (10 9 6 kg)

28 years/M 4.6

Yes

NO

D:0 %

Death D + 43 days

R:100 % 2 3 4 5 6 7

32 year/M 29 year/M 27 year/M 21 year/M 43 year/F 28 year/M

9.0 5.88 4.4 21.10 11.5 11.8

Yes Yes Yes Yes Yes Yes

1-DAY + 18

D:100 %

2-DAY +19

R:0 %

1-DAY +14

D:100 %

2-DAY +19

R:0 %

1-DAY + 15

D:100 %

2-DAY + 16

R:0 %

1-DAY +15

D:100 %

2-DAY + 18

R:0 %

1-DAY + 21

D:100 %

2-DAY + 21

R:0 %

1-DAY +19

D:100 % R:0 %

8

58 year/M

5.38

Yes

1-Day + 17

D:100 %

2-DAY +15

R:0 %

Alive 60 days in CR Alive 730 days in CR Alive 270 days in CR Alive 120 days in CR Alive 69 days in CR Death D +30 days in CR Alive D + 210 days in CR

Results: The median age of this cohort was 33 years[range 21–58 years] and M/F:7:1. The median PBSCT cell dose infused was 9.2 9 106/kg CD34 [range 4.4–21.10 9 106/kg CD34]. All patients had cytokine reaction (fever, chills and rash) which resolved after PTCy and three patients treated with short course IV Hydrocortisone 7/8[87.5 %] patients had 100 % donor chimerism at day + 28. One patient died of acute hepatitic variant of GHVD and one died due to non engraftment and sepsis. 6/8[75 %] surviving in CR with longest follow up 730 days [range 60–730 days]. Conclusion: Myeloablative Haplo-identical (PTCy) with IV Busulfan is feasible in Relapse/ Refractory AML with good engraftment and less TRM/relapse rate.

bone marrow aspiration & biopsy, flowcytometry for acute leukemia panel & FLT3 mutation status. Hemogram, clinical chemistry & coagulation profile were intensively monitored. Other relevant investigations were done as required. Results: A total 25 (M: F:: 19:6) patients of APL presented to Dept. of hematology, SGPGI during July 2012 to June 2014.3 patients were FLT 3-ITD mutation positive. Single patient was having micro-granular variant. Sanz scoring risk stratified them into 8-low, 9- intermediate & 8 high risk patients. Patients were treated as par PATHEMA protocol. 23/25 patients opted for treatment, 3 patients died in pre induction phase, chemotherapy (ATRA + IDA) was given to 20 patients. 17/20 patient achieved complete remission, with 3 induction deaths. 3/17 patients relapsed during follow up, 2/3 relapsed patients presented with IC bleed & expired. 3rd relapsed patient was treated with ATRA + ATO +chemo & achieved remission. The same patient presented few months back with isolated CNS relapse, for which he was treated with cranial irradiation & planned for ASCT.15/20 patients are presently in molecular remission & follow up. Conclusion/Diagnosis/Impression: APL has overall a good prognosis among all AMLs, with a low relapse rate; hence it is a rewarding disease to treat. Early death are important to deal with, it needs prompt diagnosis & proper management. Keywords APL, ATRA, PML-RARA

Topic: Acute Leukemia OR 88 Analysis of FLT3-ITD and FLT3-ASP835 Mutations in De Novo Acute Myeloid Leukemia: Evaluation of Prevalence, Distribution Pattern and Correlation with FAB Subtype, and Cytogenetics from a Tertiary Referral Hospital Dr. S. Kishore Kumar, Uttam K. R. Nath, S. S. Roy, Prantar Chakrabarti, Utpal Choudhuri

Topic: Acute Myeloid Leukemia OR 87 Acute Promyelocytic Leukemia Diagnosis and Management: SGPGI Experience Dr Sanjeev, Dr. Akansha Garg, Dr. Sachin Bansal, Dr. Ruchi Gupta, Prof. (Dr.) S. Nityanand* Department of Hematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh Summary: APL is a hematological emergency. It has a very good prognosis if diagnosed early and properly treated. There is a high early deaths due to catastrophic bleeding at presentation & ATRA syndrome during induction. Very few centers in northern India treat APL. We present our institutional experience in last 2 years between July 2012 to June 2014. Introduction: APL is a hematological malignancy, characterized by PML-PARA protein due to t(15;17) which leads to maturation arrest of myeloid precursors at promyelocytes stage. It usually present with DIC & catastrophic bleeding, needs aggressive and prompt treatment, especially adequate transfusion support (FFP & Cryo-ppt). All Trans Retinoic Acid (ATRA) is the key chemotherapeutic agent as it causes differentiation in the abnormal promyelocytes. With proper monitoring, there is very good prognosis With ATRA ([90 % CR rate). Materials and Methods: All clinically suspected cases of APL were confirmed & risk stratified by the panel of investigations which include PML-RARA RQ PCR,

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Miot International, Chennai & IHTM, Medical College, Kolkata Summary: Many submicroscopic lesions such as FLT3 mutations which usually remain undetectable at chromosomal levels have been described in AML. FLT3 and NPM1 mutations have been shown to be the most prevalent somatic alterations in AML, particularly in cytogenetically normal cases. These mutations play significant role in the diagnosis, risk assessment, and guidance of therapy. The current study, done to assess the incidence of FLT3 mutations in de novo AML. Introduction: While several reports on FLT3 mutations in AML are available globally, no precise published data are available from India with respect to the incidence and distribution pattern of FLT3 mutations. Materials and Methods: A prospective observational study of 51 de novo AML cases referred to IHTM, Medical College, Kolkata were included in this study. FLT3-ITD mutation and FLT3-Asp835 Point mutation were detected by DNA Polymerase Chain Reaction. Results: (1) Nearly 67 % of our patients belong to Normal Karyotype followed by inv (16) and t (8:21) in around 10 % of cases each. (2) FLT3-ITD was present in 24 % of cases and FLT3Asp835 mutations were present in 6 % of cases. (3) At presentation FLT3-ITD positive patients reveal significantly high total count, low hemoglobin, low platelets and high Serum LDH values. (4) FLT3ITD positivity correlated with dim or no expression of immaturity markers namely CD34 and HLA-DR in Immunophenotyping. (5) Induction failure significantly correlated with FLT-ITD positivity but not induction death. Conclusion: In conclusion the prevalence FLT3 mutations in our population is similar to reported literature. Keywords AML, FLT-mutation

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Topic: Acute Myeloid Leukemia OR 89 AML with Abnormal Marrow Eosinophils (AML M4Eo): A Case Series from a Tertiary Care Hospital T Roshni Paul, Shantveer G. Uppin, Megha S Uppin, Ashwani Tandon, G Sadashivudu*, AMVR Narendra** Departments of Pathology, Medical Oncology* & General Medicine**, Nizam’s Institute of Medical Sciences, Hyderabad Objectives: AML M4Eo is an uncommon variant of AML with monocytic and granulocytic differentiation with abnormal eosinophils and characteristic inv(16)(p13;q22)/t(16;16)(p13;q22). This work was done to study the clinico-pathologic and cytogenetic profile of all cases diagnosed at our Institute. Materials and Methods: The cases were retrieved from the hematopathology records (2002–2014). The clinical & followup details were obtained from the patient’s records. The morphology stained with Giemsa and cytochemistry, was reviewed. The immunophenotypic, cytogenetic &/or molecular genetic reports, were retrieved, wherever available. Results: During the 12 year study period, 8 cases of AML M4Eo were diagnosed. The patients ranged from 18 to 64 years of age (Median age -42 years) with a M: F ratio of 5:3. Patients presented with fever, symptoms of anemia. Two had palpable lymphadenopathy, one each with subcutaneous nodules and gum hypertrophy, suggestive of extramedullary infiltration. All patients had anemia, thrombocytopenia and 7/8 patients had leucocytosis. The diagnosis was made on classical morphology—(greater than 20 % blasts with granulocytic and monocytic differentiation and C5 % abnormal eosinophils, SBB & NSE positivity). FISH technique revealed inv 16 in 2 patients, 3 had apparently normal karyotype and 3 were not tested. Discussion: AML M4Eo is an uncommon sub-type of AML with characteristic morphology, cytogenetics and propensity for extramedullary infiltration. Conventional cytogenetics may not be sufficient to make a diagnosis. Conclusion: This was presented because of its rarity, and to emphasize the need for correlation of morphology, clinical features, immunophenotyping with cytogentics, FISH & PCR techniques to make the final diagnosis. Keywords Abnormal eosinophils, Inversion 16, FISH

Topic: Clinical/Adult Hematology OR 90 Profile of Myelodysplastic Syndrome: A Study Done at a Tertiary Care Centre from South India Dr. Rithika Rajendran1, Dr. Febe Renjitha Suman1, Dr. Krishnarathnam K2, Ms. Teena Koshy3 1

Department of Pathology, 2Department of Hematology, Department of Human Genetics Sri Ramachandra Medical College and Research Institute, Chennai, South India

S477 significantly longest in RAEB-1 and del(5q). Survival decreased as the IPSS and IPSS-R score increased; it is about 50 % of that established in literature for each group. AML transformation and mortality were high with RAEB, RAEB-T, RAEB-2, IPSS[2.5 and IPSS-R [ 6. Comparable with other Indian studies, 53.8 % had cytogenetic aberrations of which 21.4 % had complex abnormalities. 65.4 % were of good prognostic subgroup. Mortality was high in the very poor cytogenetic group. Conclusion: Though survival, AML transformation and mortality were comparable with existing reports, the duration of survival is only 50 % in each group. This may be due to socioeconomic and ethnic reasons. The rare cytogenetic abnormalities of t(15;17) and t(8;16) seen can be a predictor of leukemia. This study will help in adding to the database of MDS as minimal data is available for Indian population. Keywords Myelodysplastic syndrome, Cytogenetics, MDS

OR 91 Hemophagocytic Lymphohistiocytosis Dr. Neha Dhavalpure, Dr. Jyoti R Kini, Dr. Pooja K Suresh, Dr. Urmila N. Khadilkar, Dr. Shrijeet Chakraborthy, Dr. Deepa Adiga, Dr. Prashanth Bhat Kasturba Medical College, Mangalore Manipal University Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a histiocytic disorder characterized by a highly stimulated, but ineffective, immune response to antigens, which results in life threatening cytokine storm and inflammatory reaction. HLH is not a single entity, but a clinical syndrome which occurs due to underlying genetic defects as well as ‘‘trigger’’ factors such as infections, malignancies and autoimmune disorders. Materials and Methods: We retrospectively reviewed all the cases which showed hemophagocytosis in the bone marrow aspirates in our center at Kasturba Medical College and Hospital, Mangalore between January 2012 to August 2014. The final diagnosis of HLH was based on fulfilling the clinical and laboratory criteria. Results: During the study period, 23 cases showed hemophagocytosis in bone marrow aspirate, out of which 6 were reported as HLH. Among these cases, there was one paediatric case of a 7 months old female. Remaining were adults with age ranging from 20 to 67 years. Male:Female ratio was 1:1. Patients initially presented with unremitting prolonged fever (n = 4), splenomegaly (n = 2), lymphadenopathy (n = 3), and bi- or trilineage cytopenias (n = 5). Blood investigations showed elevated ferritin (n = 4), elevated liver enzymes (n = 3), increased triglycerides (n = 2) and high LDH (n = 2). The associated infections were dengue, falciparum malaria, tuberculosis and candidiasis. One patient succumbed to multiorgan failure and DIC. Others were stable at discharge. Conclusion: HLH constitutes a medical emergency at any age. A persistent and prolonged fever with splenomegaly and cytopenias should raise the suspicion of HLH. Early diagnosis and prompt aggressive treatment are vital for patient survival and favourable outcome.

3

Objectives: Prognosis of myelodysplastic syndrome (MDS) depends on the cytogenetic profile, blast percentage and cytopenias. This study intends to categorize MDS patients according to established classifications and scoring systems and to assess mortality, acute myeloid leukemia (AML) transformation and survival. Materials and Methods: This is a retrospective study done on 26 completely evaluated and followed up adult MDS patients from July 2009 to June 2014. The demographic, laboratory (FAB, WHO, International Prognostic Scoring System (IPSS) and IPSS-R (Revised)) and patient details, retrieved from records after ethics committee approval were tabulated in Microsoft Excel and statistically analyzed. Results: Male/female ratio is 1.2:1. Females were younger. Survival was up to 56 months with a mean of 20.6 ± 15.7,

Topic: Clinical Haematology OR 92 A Study of 92 Patients of Paroxysmal Nocturnal Hemoglobinuria from a Tertiary Center in Western India Nilesh Wasekar*, S. Chandrakala*, Toshniwal Manoj*, Farah Jijina*, Sonali Sadawarte*, K. Ghosh**, Maya Gupta**, Manisha Madkaikar** *Department of Hematology, Seth GS MC & KEM Hospital, Mumbai, **National Institute of Immunohematology (ICMR), KEM Hospital Campus, Mumbai

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S478 Introduction: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal disorder of haematopoietic stem cells. The molecular defect in PNH is somatic mutation in the PIG-A gene causing defect in glucosylphosphatidylinositol (GPI) anchored proteins. The clinical course of PNH is highly variable. There are very few studies of PNH from India. Hence there is a need to sub-classify PNH patients and study the natural history and the treatment outcomes on the basis of flowcytometric clone size in Indian population. Materials and Methods: Ninety two patients with clinico-hematological features were diagnosed as PNH on flowcytometry and/or FLAER of granulocytes using either anti-CD55 or anti-CD59 (clone size[3 %) included in this study from 2003 to 2013. They were classified into hypoplastic PNH, PNH-MDS and classical PNH. Patients were treated with prednisolone, danazol and immunosuppressive therapy. Results: Of 92 patients, 48, 17 and 27 had hypoplastic PNH, PNH-MDS and classical PNH respectively. Pancytopenia was present in 36(75 %), 10(59 %) and 8(30 %) in hypoplastic PNH, PNH-MDS and classical PNH respectively. There were 14 (17 %) episodes of thrombosis. Thrombosis, hemoglobinuria and death were almost equally distributed in all the three groups. Twenty seven (30 %) patients had response to therapy in the form of freedom from transfusion. Ten patients died during the follow up. Conclusion: Thrombosis, hemoglobinuria and death were more common in patients with PNH clone [50 %. Thirty percent patients had response to cheap and easily available therapy. Median survival was 80, 67, 130 months from the diagnosis in hypoplastic PNH, PNH-MDS, classical PNH respectively. Keywords PNH, Thrombosis, Immunosuppression

Topic: Hemostasis OR 93 Thrombophilia Profile in Extrahepatic Portal Vein Obstruction in Children

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Topic: Supportive Care OR 94 Outbreak of Burkholderia Cepacia Infection in an Hematology Unit of a Tertiary Care Teaching Hospital Dr. Shuvra Neel Baul, Dr. Rajib De, Dr. Tuphan.K.Dolai, Dr. Pooja Prasad, Dr. Shyamali Dutta, Dr. P.K. Mandal, Dr. Swagnik Roy, Prof. Prantar Chakrabarti Department of Hematology, NRS Medical College Introduction: Burkholderia cepacia outbreak is uncommon in hematological patients. This may occur in patients with febrile neutropenia as an opportunistic infection. We assessed the outcome of this outbreak, traced the source & took necessary action to stop this outbreak. Materials and Methods: Febrile neutropenic episodes were detected by IDSA guideline & blood cultures were sent in all the events, in FA plus aerobic medium. Culture was done by automated method using Bactalert 3D biomeriux & sensitivity pattern by Microscan Siemens method. Results: During 4 months (September 2013 to December 2013) 498 no of blood cultures send during febrile neutropenic episodes. Out of which 60 (12 %) came out to be positive. Burkholderia was detected in 29(6 %) of total cultures. Out of positive blood culture, Burkholderia was detected in 29 (48 %) of patients, which reduced drastically following change of antibiotic preparation practice. All isolates showed sensitivity to pipercillin + tazobactum, cefoperazone + sulbactum, fluoroquinolones, cotrimoxazole & carbepenems with all showing resistance to polymyxin B & colistin. Conclusion/Diagnosis/Impression: With appropriate & timely intra venous antibiotics as per culture sensitivity result mortality was low 1(4 %) out of 29 patients. With change of antibiotic preparation practice this outbreak was reduced drastically. Keywords Burkholderia, Bactalert, Blood culture

Pooja Prasad, T. K. Dolai, Shuvraneel Baul, P. K. Mondal, S. Dutta, Rajib De, P. Chakrabarti

Topic: Haematology

Department of Hematology, NRS Medical College, Kolkata

OR 95

Summary: Extrahepatic portal vein obstruction (EHPVO) is a common cause of portal hypertension in the developing countries, accounting for 70 % of pediatric cases. The etiology of EHPVO in children has not been well documented. Hereditary deficiencies of natural anticoagulant proteins have been implicated in its pathogenesis. Objectives: To characterize the coagulation profiles in children less than 12 years age having EHPVO and to investigate the possible role of deficiency of anticoagulant proteins. Materials and Methods: Fifty cases of EHPVO of less than 12 years age (mean age 8 years, M = F), were included. Detailed clinical history and routine investigations including CBC, LFT and viral serology, USG abdomen, Doppler and UGI Endoscopy were performed. Tests for thrombophilia comprising of Protein C & S, antithrombin-III and lupus anticoagulant along with Factor V Leiden mutation were carried out. Results: In thrombophilia work-up, antithrombin-III deficiency was found in 29 % cases. Protein C&S deficiency was found in 60 and 57 % cases respectively. Lupus anticoagulant was detected in 30 % children. Factor V Leiden mutation was seen in 3.3 % cases. Combined deficiency of Protein C, S and antithrombin-III was detected in 12.9 % cases; Protein C&S in 44.4 % cases; Protein C & antithrombin-III in 23.3 % and Protein S and antithrombin-III in 17.9 % cases. In 28 % cases, no deficiency of these proteins was found. Conclusions: Deficiency of natural anticoagulant proteins in common in EHPVO in children and the risk of EHPVO is increased in the presence of thrombophilia. Keywords EHPVO, Thrombophilia

They are Anemic, but due to Xenobiotic Exposure!!

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Rajlaxmi Basu, Siddhartha Sankar Ray, Sila Chakrabarti, Sambit Samanta, Arnab Chatterjee, Uttam Kumar Nath, Maitreyee Bhattacharyya Institute of Hematology and Transfusion Medicine, Medical College, Kolkata Objectives: Sewage workers are exposed to pollutants including toxic heavy metals. Exposure to such pollutants results in health hazards. Heavy metal input in blood stream in different mode is one of the main reasons for imbalance in essential elemental profile especially depletion in Iron concentration and make the workers anemic. Our objective was to find out the incidence of Iron deficiency and analyse the cause among these workers Materials and Methods: Lead and Cadmium concentration in blood was estimated (AAS) and whole blood Iron, Zinc concentration was estimated (ED-XRF). Results: workers have significantly high (p \ 0.05) lead, significantly high (p \ 0.001) Cadmium concentration than control population. Here, workers are rich in heavy metals and deficits in blood iron which is significantly lower (p \ 0.001) than the control population, also significantly (p \ 0.001) low Zinc concentration Conclusion: Heavy metals responsible for anemic tendency in drainage workers and responsible for other complication and other trace elemental imbalance and turn in vulnerable condition. Keywords Iron deficiency, Lead, Cadmium, Sewage workers

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Topic: Hemostasis and Thrombosis OR 96 Effect of Addition of Dapsone on Platelet Count Following Steroid Withdrawal: A Prospective Study in Steroid Responsive Chronic and Persistent ITP Patients Dr. S. Karthik, Dr. Shyamali Dutta, Dr. T. K. Dolai, Dr. Rajib De, Dr. Prakash K. Mondal, Dr. Prantar Chakrabarti

S479 were peripheral blood cultures, 13 PICC samples and 44 were from Hickman catheter lumens. Among Gram negative bacilli Klebsiella pneumonia was the most predominant organism causing sepsis followed by coagulase negative staphylococcus among the Gram positives. Three instances of candidemia were also noted. Conclusion: Blood cultures from PICC line showed higher positivity than peripheral blood cultures & Hickman catheters. Most predominant organisms causing bacteraemia were Gram negative bacilli of which Klebsiella pneumonia was the most predominant one.

NRS Medical College and Hospital, Kolkata-14 Summary: Dapsone was given to a group of patients with persistent and chronic ITP responding to steroids, during the phase of steroid withdrawal. Platelet count response was assessed at 6 months following steroid withdrawal and compared to a similar group of patients who did not receive dapsone during steroid withdrawal. The addition of dapsone had a significant effect on the duration of sustained platelet count responses after steroid withdrawal. Introduction: Steroids are the most commonly used first line therapy in symptomatic persistent and chronic ITP. However steroid withdrawal is followed by relapse in majority of patients. We investigated whether addition of dapsone during steroid taper had an effect on duration of platelet count response after steroid withdrawal in steroid responsive ITP. Materials and Methods: 28 patients with persistent and chronic ITP were given dapsone (50–100 mg/day) during steroid taper. In a comparable group of 28 patients, steroid withdrawal was done without dapsone. Platelet count response at 6 months following steroid withdrawal was assessed. Results: In the dapsone group, 46.4 % were in CR, 25 % in PR at 6 months, while 28 % had lost response at 6 months. In comparison, 18 % in steroid only group maintained CR at 6 months, none of these patients were in PR and 82 % had lost response at 6 months. The results were statistically significant (p \ 0.001). Conclusion/Diagnosis/Impression: Addition of dapsone during steroid taper is effective in maintaining longer duration of platelet count response in patients with persistent and chronic ITP. Keywords Dapsone, ITP (chronic and persistent), Platelet count

OR 97 Comparative Evaluation of Blood Cultures from PICC, Peripheral Line and Hickman Catheter in Hematolymphoid Cancer and Bone Marrow Transplant Patients (TMP 048) Nayana P. Barskar, Dr. Viek Bhat, Dr. Preeti Chavan, Rajni Mohite, Reshma Kamble, Umakant Gavhane, Ashwadeep Karmore Summary: A comparative evaluation of peripheral line blood cultures and Hickman catheter blood cultures from oncology patients was performed. Out of 2,092 blood cultures processed, 676 were peripheral blood cultures, 219 PICC blood and 1,200 from Hickman catheter lumens (red, white, grey and blue). PICC line blood culture found to be more promising; Gram negative bacilli were the more predominant organism causing sepsis. Introduction: Blood culture remains a valuable microbiological procedure for diagnosis of bacteraemia and fungemia. Many patients undergo intravascular catheter placement at some time during their hospitalization Ensuing infection is the most common complication associated with intravascular devices and subsequent bacteremia may be associated with morbidity and mortality. Materials and Methods: We processed 2092 blood culture samples in Bactec 9050 automated blood culture system during the period of June 2013- May 2014 from oncology units. The positive cultures were identified as per standard laboratory protocol. Result: Out of 2092 blood cultures 89 were positive, among them 32

OR 98 Hemophagocytic Lymphohistiocytosis in Children: A Tertiary Care Center Experience from India Shirali Agarwal, Nita Radhakrishnan, Veronique Dinand, Vasant Chinnabhandar, Anupam Sachdeva Pediatric Hematology Oncology Unit, Institute of Child Health, Sir Ganga Ram Hospital, New Delhi Introduction: Hemophagocytic lymphohistiocytosis (HLH) although rare, is being increasingly diagnosed in pediatric centers across the world. Literature from developing world is mostly related to case reports. Here we discuss the clinical profile and treatment of patients who were diagnosed with HLH at our center in the last 48 months. Materials and Methods: We retrospectively analyzed patients diagnosed with HLH between January 2010–July 2014. They were diagnosed based on HLH 2004 criteria. Search for secondary causes of HLH was performed in all patients. Patients \5 years at diagnosis and with positive family history were evaluated for familial HLH. Results: A total of 35 cases were diagnosed during this period. Male: Female ratio was 3:1. Median age of this group was 5 years (range 8 weeks–16 years). 6 children had family history of consanguinity and 5 had history of sibling death. Presenting features included fever and hepatosplenomegaly (34), bleeding manifestations (7), lymphadenopathy (8), skin rash (5), shock (6), jaundice (13), CNS manifestations (8), renal failure (6), liver failure (2) and arthritis (1). Cytopenias affecting two or three lineages was present in all while hemophagocytosis in bone marrow was documented in 27 patients. Other important laboratory parameters were raised ferritin (31), hypertriglyceridemia (30) and hypofibrinogenemia (30). Secondary cause of HLH was identified in 12 patients. 2 patients had EBV Infection, 3 had Enteric fever, and 1 each had malaria, tuberculosis, juvenile rheumatoid arthritis, bacterial (pseudomonas sepsis), fungal infection and CMV infection. Malignancy associated HLH was seen in 1 patient each with Hodgkin lymphoma and anaplastic large cell lymphoma. Genetic workup was sent in 14 children with suspected familial HLH. Mutation analysis was positive in 6 patients. Out of this 2 patients had FLH-3 (MUNK 13-4), 1 had FLH-4 (STX11), 1 patient had FLH-2 (Perforin) and 1 patient has Griscelli syndrome. 1 child who presented at 8 weeks of life with HLH was heterozygous for MUNK 13-4; however sequencing for another mutation to explain the clinical presentation could not be done. Treatment given was according to HLH 2004 protocol. Steroids were given to all 33 patients and combination of steroids with cyclosporine was given to 19 patients. Stem Cell transplantation was done in 3 patients (2 umbilical cord blood, 1 matched sibling bone marrow). 1 of the patient is still alive but the other 2 patients died later on due to secondary complications. Overall 19 patients are dead (54 %), 10 patients are alive, 4 were lost to follow up and the rest are presently being treated. Conclusions: Diagnosis of HLH should be considered in patients presenting with fever, hepatosplenomegaly and pancytopenia. Despite early and aggressive treatment we have encountered mortality in more than 50 % of our patients.

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Topic: Clinical Haematology

Topic: Haemophilia

OR 99

OR 100

Surgeries in Patients with Hemophilia: Experience from a Comprehensive Hemophilia Care Center of Western India

A Study of Clinico-Haematological Profile of Patients with Haemophilia

Dr. Manoj Toshniwal, Dr. S. Chandrakala, Dr. Nilesh Wasekar, Dr. Farah Jijina*, Dr. Ghosh K# *Department of Hematology, KEM Hospital, #National Institute of Immunohematology (NIIH), KEM Hospital, Mumbai, India Introduction: Patients with hemophilia (PWH) frequently suffer from disabling chronic arthropathy, osteoporosis and need orthopedic surgeries for various complications. Other surgical problems also remain same as in general population in these patients. Although most surgical and invasive procedures can be performed safely in patients with hemophilia, the optimal level and duration of replacement therapy required to prevent bleeding complications have not been established conclusively. Objective: A retrospective analysis of surgical procedures carried out, amount of factor used and complications at a comprehensive hemophilia care center in western India. Materials and Methods: This is a retrospective study of 130 different surgeries done in 116 hemophilic patients at our comprehensive hemophilia care center in western India from the period of 2004–2014, in age range of \1–62 years. Of these 116 patients, 89 had hemophilia A (mild 01, moderate 9, severe 80), three patients had hemophilia A with Inhibitors and 24 patients had hemophilia B. 98 surgeries were major and 32 were minor. Out of 98 major surgeries, 69 were orthopedic surgeries like pseudotumors (13), amputation (08), joint replacement surgery (13), and internal fixation (22). Twenty General surgeries of which 11 cases were of circumcision, 07 of hernia repair and 02 appendicectomy. Neurosurgeries were performed in 12 cases. Urological intervention was required in 6 patients. Other surgeries like tracheostomy or ophthalmic procedures were done for 8 patients. Circumcision was done in 09 cases. The Factor concentrate used was optimized from our previous experience, along with generous use of antifibrinolytic drugs, oral, parenteral and local. Results: The factor concentrate used in 130 surgeries ranged from 300 to 62200 IU (mean 15605.02 IU), with 293.79 IU/kg/surgery. Three patients with inhibitors received modest dose of FEIBA+ factor VIIa. Most of them received parenteral antifibrinolytic drugs. Factor support was given for only 7–10 days in majority of the patients. Six patients (7.62 %) developed bleeding complications post operatively that were managed with factor correction. Eight patients (10.16 %) developed inhibitors post operatively. There was no death due to bleeding. Most of them were discharged with good functional activity except one elderly patient who developed massive pulmonary embolism on 2nd post operative day and succumbed to the complication. Conclusion: The present study clearly shows that major surgeries in patients with hemophilia can be accomplished with only 30–40 % of total factor concentrate as compared to western studies for factor concentrate prophylaxis in PWH undergoing surgery. As there are no guidelines to define the safe lower limit of factor concentrate prophylaxis for surgery, the need to define these lower limits is essential in the developing world so that the limited resources may be used optimally for this cohort of PWH. Keywords Surgery, Haemophilia, Factor concentrate, FEIBA

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Dr. Parismrita Borah, Dr. Hanagavadi Suresha, Dr. S. S. Hiremath Department of Pathology, JJM medical college, Davangere Summary: Clinico-haematological profile of 148 cases of haemophilia were studied. Basic coagulation work up including inhibitor screening and quantification was done and correlated with clinical manifestations. The available literature was reviewed to understand the coagulation disorder. Introduction: Haemophilia, the most common inherited X linked recessive bleeding disorder is caused by deficiency of factor VIII/factor IX. The manifestation includes prolonged bleeding on trivial trauma and if not treated early may lead to disabling arthropathy. Knowledge of spectrum of its presentation helps in early diagnosis and management of these patients, thereby preventing them from complications. Objectives: (1) To study the pattern of clinical presentation in Haemophilia and its correlation with factor level. (2) To document the incidence of inhibitors. Materials and Methods: Prospective study involving detailed clinical history and coagulation profile of 148 cases of Haemophilia, referred to the Department of Pathology, JJM Medical College, Davangere during July 2013 to August 2014 is done. Results: In total of 148 cases (147 males and 1 female) 81.75 % cases had deficiency of factor VIII while 18.24 % were factor IX deficient. 62.16 % had severe, while 26.35 % had moderate and 11.48 % had mild form of the disorder. 92 % cases had initial bleeding episode before 10 years of age. Haemarthrosis was the most common clinical manifestation (81.75 % cases) with knee joint being most commonly involved (70.2 %). 9 cases (4 low titre and 5 high titre) were positive for inhibitors. Conclusion: Classical haemophilia caused by factor VIII deficiency is the most common type. The clinical manifestation depends upon the level of factor deficiency, predominant being musculoskeletal bleeding episodes. Development of inhibitors is a major challenge in the management of these cases. Keywords Haemophilia A, Haemophilia B, Haemarthrosis, Inhibitor

Topic: Haemostasis/Haemophilia OR 101 A Comprehensive Analysis of Risk Factors for FVIII Inhibitor Development in Indian Haemophilia A Patients Patricia Pinto, Kanjaksha Ghosh, Shrimati Shetty National Institute of Immunohematology (ICMR), Mumbai Objectives: FVIII Inhibitor development is a serious complication in congenital HA patients; and a complex, multi-factorial process. The study was planned to comprehensively analyse risk factors (genetic and non-genetic) for Factor VIII (FVIII) Inhibitor development in Indian Haemophilia A (HA) patients. Materials and Methods: 170 severe HA patients, 82 inhibitor positive & 16 severe HA family members, and 72 control inhibitor negative patients, were included in the study after informed consent & ethics approval. A clinical proforma was designed to include patient information. F8 mutations and

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 polymorphism haplotypes, HLA-DRB1 & DQB1 alleles, fourteen immune response gene (IL10, TNFA, IL1b, IL4, CTLA4) polymorphisms were analysed by CSGE, DNA Sequencing, MLPA, RFLPPAGE, SSP typing etc. respectively. The results were analysed for statistical significance. Results: The type of treatment products and exposure before the first year of life were not significantly different. Intron 22 Inversions (P = 0.0115; OR 2.600), large F8 deletions (P = 0.0280, OR 13.452), the IL10 promoter GCC/ATA haplotype (P = 0.0273, OR 2.732), and TNFA rs1799724 C/T genotype (P = 0.0401, OR 2.542) were significantly higher in the inhibitor positive patients, and are thus ‘risk’ associations for FVIII inhibitor development. F8 missense mutations (P \ 0.0001, OR 0.04335), the IL10 promoter ATA/ATA haplotype (P 0.0200, OR 0.3421) and DRB1*03 allele (P = 0.0365, OR 0.3636) were significantly higher in the inhibitor negative patients, and are thus ‘protective’ associations for FVIII inhibitor development. Conclusion: F8 Intron 22 inversions, large deletions and IL10 promoter haplotypes seem to be the strongest risk factors (genetic) for inhibitor development, in this comprehensive study in Indian severe HA patients. Keywords Haemophilia A, FVIII, Inhibitors, Risk factors

Topic: Clinical Haematology OR 102 Application of BCL 1–RFLP for Carrier Detection in Haemophilia A Dr. Riya Ballikar, Dr. Sheela Chakrabarti, Dr. Maitreyee Bhattacharya Institute of Haematology and Transfusion Medicine, Kolkata Summary: The incidence of BCL 1 heterozygosity in mothers of patients with Hemophilia A has been studied. An incidence of 35 % has been demonstrated in our study. BCL 1 and other related RFLP markers may be used to screen the general population and for antenatal screening in affected families. Introduction: Haemophilias are the most common severe inherited bleeding disorders recognized with prevalence of 1 in 10,000. The factor VIII gene is very large and complex, making its detection very difficult and cumbersome. This can be overcome using polymorphic DNA markers in the factor VIII gene. Polymorphic markers are slight sequence variations usually present in the non-coding regions of a gene. Some of these markers can be identified by restriction enzymes using RFLP. The more commonly used ones in haemophilia A are BCL 1, Hind III and Xba1. Materials and Methods: This is an ongoing study being conducted in the Department of Haematology IHTM with help and guidance from Dr K Ghosh and team at NIIH. The heterozygosity rate for BCL 1 was first determined in the general population. It was found to be 38 %. Later testing for BCL 1 heterozygosity in the study population was done. Till date 17 patients with Haemophilia A and their mothers have been included. The mothers were tested for presence of heterozygous state of BCL 1. The method used for BCL 1 detection was PCR RFLP. Results: Of the 17 patients tested, 6 mothers had BCL 1 heterozygosity. Thus the incidence of BCL 1 heterozygosity was 35 % in the carriers of the factor 8 gene. Conclusion: BCL 1 heterozygosity may be used for detection of carrier state of Haemophilia A in the general population and especially for antenatal screening. This will overcome the shortcomings associated with detection of the Factor 8 gene. Especially in a resource poor country like India with such a high burden of the disease, this can go a long way in preventing Haemophilia and the morbidity associated with this disease. Keywords Haemophilia, BCL 1, RFLP

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Topic: Proffered OR 103 Hemophagocytic Syndrome: Report of 40 Cases Poonam Rani, Udaya Kumar, Amita Jain, Vijaya Vaishnav, Tejinder Singh Department of Pathology, Maulana Azad Medical College and associated Lok Nayak Hospital Summary: We studied 40 cases of reactive hemophagocytic syndrome. We encountered multiple diseases associated with RHS. Among our study population, HIV was the most common followed by tuberculosis and enteric fever. Introduction: Hemophagocytic lymphohistiocytosis (HLH) is an uncommon life threatening hematologic disorder due to severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and macrophages that secrete high amounts of inflammatory cytokines. Two forms of the syndrome have been described-familial hemophagocytic lymphohistiocytosis (FHL) of infants and reactive hemophagocytosis syndrome (RHS) encountered at any age. Materials and Methods: 40 cases of hemophagocytosis were studied retrospectively over a period of 7 years. Complete clinical and laboratory details were retrieved from the medical record. Results: All cases presented with fever of more than 5 days duration. Mean age of presentation is 27 years ranging from 2 to 70 years. Males were affected more than females. The peripheral blood demonstrated cytopenias. Bone marrow revealed hemophagocytosis in all the cases. Reactive hemophagocytosis syndrome was associated with dengue fever (2 cases), HIV infection(8 cases), enteric fever(5 cases), tuberculosis(7 cases), Kala-Azar (1 case), falciparum malaria (1 case), associated NHL(2 cases), staphylococcal infection(1 case), both HIV and tuberculosis (4 cases) and ? viral etiology (9 cases). 5 patients expired. Conclusion: The underlying diseases of RHS are heterogenous which include infections- bacterial, viral, fungal and parasitic. Mechanism of RHS remains unclear, but cytokines may play a role. In RHS, though characterized by certain laboratory features, demonstration of hemophagocytosis is essential for diagnosis; hence the need for bone marrow evaluation in cases of unresponsive fever of [1 week duration. Keywords Hemophagocytosis, HIV, Reactive hemophagocytic syndrome

Topic: 7th November OR 104 Case Series of Haemophagocytic Lymphohistiocytosis Savita Kumari, Vijay Kumar, Sadhna Marwah, Gurdeep Buxi Department of Pathology, Dr. Ram Manohar Lohia Hospital, PGIMER, New Delhi Objectives: Haemophagocytic Lymphohistiocytosis is a disorder of the mononuclear phagocyte system. It is a life-threatening hyperinflammatory syndrome, caused by severe hypercytokinemia associated with ineffective immune response. It is a rare fatal disease, seen in all ages, having no age and sex predilection. It is important to categorise HLH patients in familial(primary) and secondary(sporadic) for prognostic and therapeutic importance. Untreated HLH has very high mortality rate. Early diagnosis of HLH is important so that appropriate treatment can be initiated. Materials and Methods: The bone marrow examination were done between 2012 and 2014 July in the department of Pathology, Dr. RML Hospital, New Delhi. The diagnosis of HLH was suggested in seven patients who fulfill the 2004 diagnostic criteria of HLH. Results: Age of

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S482 the patients ranges from 3 months to 32 years, male:female = 2.5:1. All patients presented with fever and cytopenia. Bone marrow smears showed reactive feature along with increased number of histiocytes, showing haemophagocytosis. Two patients showed positive marker for hepatitis B(HBs) and Cytomegalovirus(CMV),one was of disseminated tuberculosis (TB),one was diagnosed case of B cell ALL on chemotherapy and one had multiple joint pain(soJIA).On follow-up two patients died and rest survive with appropriate treatment. Conclusion: It is important to make early diagnosis for proper treatment and survival of the patients. Since viral trigger is most important to develop HLH both in familial and acquired. So it is very important to correlate the cause, clinical and laboratory findings to make early diagnosis of HLH. Immunochemotherapy followed by allogenic BMT hope for long term survival in these patients. Keywords HLH—Haemophagocytic lymphohistiocytosis, Cytomegalovirus, Hepatitis B

OR 105 Comparison of Blood Cyclosporine Level Estimation in Bone Marrow Transplant Patients on Two Analyzers with Different Principles Ulka Gosavi, U. Gavhane, P. Poladia, B. Pillai, M. Tiwari, S. Pal, A. Karmore, H. Dethe, P. Chavan, V. Bhat TATA Memorial Centre, ACTREC, Kharghar, Navi Mumbai Summary: This comparative study was performed on 211 whole blood samples obtained from allogenic bone marrow transplant patients to detect the cyclosporine levels. Samples were analysed simultaneously on ARCHITECT i1000 by Abbott Diagnostics and RXL-MAX by Siemens Healthcare Statistical. Statistical analysis was performed on the values obtained for correlation. Both methods showed good correlation and may be used for detection of cyclosporine levels interchangeably. Introduction: Cyclosporine is an immunosuppressant drug used in patients of allogenic bone marrow transplant. It has severe toxic effects and the drug levels need careful monitoring at designated intervals. This study was done to explore the possibility of using two methods of cyclosporine estimation with different technology interchangeably. Materials and Methods: 211 whole blood samples were obtained from post-transplant patients to detect the cyclosporine level in the blood and were analysed simultaneously on ARCHITECT i1000 by Abbott Diagnostics with chemiluminescent microparticle immunoassay (CMIA) principle and RXL-MAX by Siemens Healthcare with hetero-immunoassay technique. Bland–Altman and Passing-Bablock regression analyses were done. Result: Linear regression analysis showed R2 value of 0.9181. Bland–Altman showed a mean difference of 2.073. Conclusion: The results obtained by both methods showed good correlation with R2 value 0.9181 indicating low level of variation, hence either of the methods (ARCHITECT i1000 & RXL-MAX) may be used for detection of cyclosporine levels in oncology patients interchangeably. Keywords Cyclosporine, Allogenic

Topic: Hematological Malignancies OR 106 Pure Red Cell Aplasia in Haematological Malignancies: A Mimicker of Disease Relapse Nabhajit Mallik, Sanjeev Kumar Gupta Department of Laboratory Oncology, IRCH, All India Institute of Medical Sciences (AIIMS), New Delhi

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Introduction: Acquired pure red cell aplasia (PRCA) may be primary or secondary to a variety of causes including infections, solid tumours, autoimmune haemolytic anaemia, collagen vascular disorders, drugs, and chemical exposure. Uncommonly, it may complicate the course of a haematological malignancy as well, where the unexplained anaemia may be confused with a disease relapse. We present four such cases of haematological malignancies, in different age groups, but similarly complicated by acquired PRCA. Case reports: Case 1: A 6 year old boy, treated for AML, presented with progressive pallor after completion of consolidation chemotherapy. Case 2: A 7 year old girl with ALL on maintenance chemotherapy, presented with progressive pallor and CNS relapse. Case 3: A 12 year female, earlier treated for NHL, presented with anaemia requiring blood transfusion for the last 6 weeks. Case 4: A 75 year old male being treated for multiple myeloma presented with anaemia which recurred after an initial rise in haemoglobin. Bone marrow examination was performed for all the above mentioned patients in view of suspected disease relapse. All these cases showed a cellular bone marrow with paucity of erythroid precursors and few giant proerythroblasts. Myeloid and megakaryocytic lineages were unremarkable. There was no evidence of disease relapse in any of these cases. Conclusions: There should be a high index of suspicion of PRCA as one of the causes of anaemia, besides disease relapse, in the setting of hematological malignancies. A correct recognition of this entity can avoid unnecessary investigations, and help in timely diagnosis. Keywords PRCA, Parvovirus, Hematolymphoid malignancies

Topic: Laboratory Haematology OR 107 Telepathology as a Tool for Online Consultation and Continuing Education in Hematopathology 1

Dr. Prasanna N. Kumar, 2Dr. Nina Hurwitz, 3Dr. Andre Tichelli

1

Department of Pathology, PSG Institute of Medical Sciences & Research, Coimbatore, 2Insititute of Pathology University of Basel, Switzerland, 3Department of Haematology, University Hospitals, Basel, Switzerland Summary: Online consultations through IPath programme are aimed at improving standards of diagnostic pathology, particularly of cancer in resource restricted countries, by utilizing expertise available elsewhere. This programme has succeeded in developing international networking of pathologists to optimize technology transfer and Pathology based collaborative projects. Introduction: ‘IPath’ programme is an open sourced software, which is used for online diagnosis in pathology. This form of telepathology which encompasses the domains of consultation, teaching and research is an efficient tool, especially for low resources countries. Materials and Methods: Cases, submitted digitally for diagnosis are examined by an expert pathologist at a distant location. Subsequent consultations or interactive case discussions take place through Skype. The technologists’ forum of IPath contributes by providing online support for training in new methodologies. This programme, in its present form can also be used for training residents and conducting online CMEs and case discussions. These online teaching sessions are complemented by periodic visits to respective institutions, helping in solving problems which telepath alone cannot handle. Results: IPath has helped to considerably reduce the turnaround time (TAT) for Pathology diagnosis in low resource countries. It has helped in improving the reliability of diagnoses and diagnostic accuracy is adapted to locally available therapeutic options. It has also achieved the goal of increasing the credibility of local pathologists.

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Conclusion/Diagnosis/Impression: The combination of online teaching and onsite visits is helping to overcome limitations in pathology in resource limited countries. Keywords IPath, Telepathology, Resource limited countries

S483 PO 3 Mixed Phenotype Acute Leukemia (B/T), with t(9;22) (q34,q11.2) Translocation: A Rare Entity Pankaj Punetha, Faiq Ahmed, Sandhya Devi, Daphne Fonseca, Sudha S Murthy, M. V. T. Krishna Mohan

PO 1 Acute Megakaryoblastic Leukemia: A Case Report

Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad

Introduction: Acute Megakaryoblastic Leukemia (AMKL) is a rare entity with poor prognosis and accounts for 3–5 % of all Acute Myeloid Leukemias. The clinical profile and morphological criteria for its diagnosis remain ill-defined and can be confused with Acute lymphoblastic Leukemia (L1) or Acute Myeloid Leukemia (M0). AMKL has a bimodal peak of distribution, in adults and in children (1–2 years of age). It can present in a variety of ways. It may arise denovo or may be secondary to chemotherapy, progress from myelodysplastic syndrome and/or myeloproliferative neoplasms. The course of illness is one of rapid deterioration and death from hemorrhage and infections occur despite aggressive therapeutic measures. Method and Material: A 26 year old female was referred to our hospital for evaluation of pancytopenia. The patient presented with fever and rashes all over the body for 1 week duration. On examination, multiple petechial rashes were noted all over the body along with pallor. In view of the above findings, bone marrow aspirate and biopsy were performed along with flow cytometry. Results: Bone marrow studies had given a diagnosis of Acute Myeloid Leukemia (AML) which was further confirmed by flow cytometry studies to be AML-M7/AMKL. Conclusion/Diagnosis/Impression: AMKL is a rare leukemia. It is important to correctly diagnose this variant in view of its prognostic implications. Keyword Acute Megakaryoblastic leukemia

Summary: Mixed phenotypic acute leukemia (MPAL) with t(9;22) or BCR-ABL rearrangement is most commonest recurrent genetic abnormality seen in the MPAL. Immunophenotypic profile of B/T lineage assignment is a rarely seen in MPAL associated with BCRABL gene rearrangement. We present a case of 54 year old male whose immunophenotype and Fluorescence in situ hybridization (FISH) rendered a diagnosis of MPAL (B/T) with BCR-ABL gene rearrangement. Introduction: Blasts which are evidenced with expression of both lymphoid and myeloid lineage—specific antigen are referred as MPAL. Common subtypes encountered are B/myeloid, T/myeloid, while B/T MPAL being the exception. B/T MPAL with BCR-ABL gene rearrangement constitute 1 % of all acute leukemias. Materials and Methods: Peripheral blood sample was subjected to flowcytometry evaluation in view of circulating blast. Immunophenotyping was carried on 4 coloured BD FACS Calibur using standard protocol. Simultaneously the patient sample was subjected for testing of MLL and BCR-ABL gene rearrangement using break apart probe for MLL and dual color fusion probe for BCR-ABL gene. Results: The gated population of cells show bright expression for HLA DR, CD19, moderate expression for CD10, CytCD3, CD13 and dim expression for Tdt, CD22, Cyt CD22, Tdt, sCD3, CD5, CD7, CD56 and CD33. FISH was positive for BCR-ABL fusion gene rearrangement. Conclusion: Mixed phenotypic acute leukemias with B/T phenotypic profile and BCR-ABL translocation are uncommon, which are usually associated with poor outcome. The diagnosis should not be made in patients known to have had chronic myeloid leukemia.

Topic: Pathology

PO 4

Dr. Datla Swathi Priya, Dr. Jyoti Kini, Dr. Krishna Prasad, Dr. B. Prashanth Kasturba Medical College, Manipal University, Mangalore

PO 2 Large Granular Lymphocytic Leukemia Dr. Rohita Kumar, Dr. Prajakta Gupte, Dr. Daksha Prabhat Department Of Pathology, Seth G S Medical College & KEM Hospital, Mumbai Objective: Large granular lymphocytic leukemia constitutes 2–3 % cases of lymph proliferative disorders. These can arise from a CD3+ T-cell lineage or from a CD3- NK-cell lineage. This presentation represents clinical course & diagnostic approach for this rare entity. Methods: Clinical presentation along with peripheral smear findings, bone marrow study and immunophenotyping will be used for the diagnosis. Results: Clinically, patient presents with fever, recurrent infections, loss of weight, lymphadenopathy and mild to moderate Hepatosplenomegaly. Peripheral smear shows lymphocytosis predominantly large granular atypical lymphocytes along with majority of cases showing chronic neutropenia and low hemoglobin. Bone marrow study also shows presence of similar atypical lymphocytes. Immunophenotyping determines whether the origin is of T-Cell or natural killer (NK) type. Conclusion: Clinical presentation, peripheral smears findings & bone marrow studies concludes that it is a case of large granular lymphocytic leukemia and immunophenotyping shows it’s a case of natural killer (NK) cell variant. Keywords Large granular lymphocytic leukemia, T-Cell or NK cell

Metronomic Therapy with Oral 6-Mercaptopurine and Prednisolone in Relapsed Acute Lymphoblastic Leukemia in Elderly Patients: A Prospective Study Dr. Akhil Kapoor, Ashok Kalwar, Surender Beniwal, Harvindra S. Kumar Acharya Tulsi Regional Cancer Treatment & Research Institute, Bikaner, Rajasthan Summary: We evaluated the role of Metronomic chemotherapy with oral 6-Mercaptopurine and Prednisolone as a treatment option with minimal toxicities in elderly ALL patients not amenable to curative therapy. Introduction: Acute Lymphoblastic Leukemia (ALL) in elderly patients differs biologically from that in younger patients with unfavorable chromosomal rearrangements. These patients are more likely to relapse and in situations where Bone Marrow Transplantation is not feasible, the therapeutic options are limited. In such circumstances, instead of giving full blown chemotherapy, palliative treatment in the form of Metronomic chemotherapy (MC) may be a viable option. Method and Material: We performed retrospective analysis of prospectively collected data of elderly ALL patients (age [ 60 years) not amenable to curative treatment. Between July 2013 to June 2014, 14 patients were enrolled into the study and were treated with daily oral 6-Mercaptopurine 75 mg/m2 and Prednisolone 40 mg/m2 till progression or death of the patient. Results: The median age of the patients was 64 years (Standard deviation: ±6.43)

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S484 with male: female ratio of 2.5:1. 64.3 % patients had Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 2 while rest had status of 3. The median OS was 7 months [95 % confidence interval (CI) 4.44–9.56]. The patients of age group 61–70 years had highest median OS of 9 months (95 % CI 7.93–10.06) while both 71–80 and 81–90 years had median OS of 4 months (p = 0.014). The MC was well tolerated with no grade 4 toxicities and no episode of febrile neutropenia. Conclusion/Diagnosis/Impression: Metronomic chemotherapy with oral 6-Mercaptopurine and Prednisolone is an attractive treatment option with minimal toxicities in elderly ALL patients not amenable to curative therapy.

PO 5 Methotrexate Induced Chemical Meningitis in Patients with Acute Lymphoblastic Leukemia/Lymphoma Dr. S. Aparna, Dr. Linu A. Jacob, Dr. K. C. Lakshmaiah, Dr. D. Lokanatha, Dr. T. M. Suresh, Dr. Govind Babu, Dr. Suresh Babu Department of Medical Oncology, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India Introduction: Intrathecal Methotrexate (ITMTX) is an important component in the treatment as well as prophylaxis of Leukemia/lymphoma. ITMTX can cause chemical meningitis characterized by vomiting, headache and fever lasting 2–5 days with spontaneous resolution of symptoms which differentiates this syndrome from bacterial meningitis. Objective: This prospective observational study was carried out to determine incidence of Post ITMTX syndrome in patients receiving prophylactic ITMTX as part of BFM protocol. Materials and Methods: Patients aged 15–50 years receiving BFM 90 or BFM 95 protocol for Acute Lymphoblastic Leukemia or Lymphoblastic Lymphoma were followed up for Post ITMTX syndrome, defined as vomiting, headache and fever following ITMTX. Results: 33 patients received a total of 297 courses of ITMTX. Of the 297 doses of ITMTX 20 episodes (6.7 %) of post ITMTX syndrome were observed. The incidence of post ITMTX syndrome was highest after the second dose of ITMTX (24 %).The most common symptom of post ITMTX syndrome was headache which was seen in 17 (85 %) patients. 17 (85 %) patients had vomiting, 10 (50 %) patients had fever and 4 (20 %) patients had backache. Meningeal signs were present in 2 (10 %) patients. Conclusions: Post ITMTX syndrome is not uncommon in adult patients receiving prophylactic ITMTX for treatment of acute lymphoblastic leukemia and lymphoblastic lymphoma. Patients develop a toxic syndrome closely mimicking acute bacterial meningitis but spontaneous recovery is seen without any neurological sequelae. Keywords ITMTX intrathecal methotreaxate, ALL acute lymphoblastic leukemia, BFM

Topic: Miscellaneous PO 6

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 our knowledge, less than five cases of osteogenesis imperfect with acute leukemia have been reported. Its association with hemophilia has not yet been documented. Here we present two cases of OI, one with Acute Lymphoblastic Leukemia (ALL) and another with severe Hemophila A. Case 1: RD, a 17 year old boy presented with history of fever and gum bleeding for 1 month. He had past history of multiple fractures of long bones since childhood. There was history of generalized seizures at the age of 14 years for which he was put on Clobazam and valproic acid. The general survey revealed pallor, petechiae, dolicocephalic skull with reduced head circumference (\3rd percentile), blue sclera and stunted growth. Abdominal examination revealed hepatosplenomegaly. Immunophenotyping confirmed a diagnosis of CALLA positive B-ALL. MRI brain revealed gliotic lesions in both occipital lobes but a normal EEG at rest. He was treated with BFM 2002 ALL protocol and is now on maintenance. Case 2: SD, a 52 year old man presented with history of prolonged bleeding following minor trauma & recurrent episodes of right knee hemarthrosis since childhood. He also had multiple fractures of long bones since his childhood. He had no family history of similar illness. On examination he had blue sclera and fixed flexion deformity of right knee joint and left elbow. His factor VIII level was\1 % with no inhibitor. He was treated with on demand factor VIII therapy and is on regular follow up at present. Keywords Osteogenesis Imperfecta, Acute Lymphoblastic Leukemia, Hemophilia A

Topic: Haematology PO 7 Adult T Cell Lymphoma/Leukemia: An Unusual Presentation Dr. Merin Jose, Dr. K. K. Prabhalakshmy, Dr. Sunitha Balakrishnan, Dr. Joy Augustine Pathology, Government Medical College, Thrissur Summary: A case report of Adult T Cell Lymphoma/Leukemia presented with SDH. Introduction: ATLL is a clinicopathologic syndrome characterized by presence of flower cells with lobulated nuclei in the peripheral blood. HTLV is causally linked to ATLL. It has a long latency. The affected individuals are exposed to virus very early in life. Method and Materials: Clinicopathological data, biochemical investigations, and HTLV serology. Result: 55 years old female presented with left sided hemiparesis. CT head showed right temporal SDH with subarachnoid hemorrhage. On clinical examination patient had hepatosplenomegaly and cervical lymphadenopathy. Patient had history of skin rashes 3 months before this episode. Blood Investigations: Hb—10.8 g/ dl, total count—1.3 lakh/mm3, platelet count—61,000/mm3, serum LDH level—2,530 IU/L, serum calcium—11 mg/dl. Peripheral smear—lymphoid cells with flower shaped nuclei, CD3+ BM showed infiltration by lymphoma cells. HTLV I and II antibody assay (EIA) showed positivity. Patient was started on CHOP regimen and Zidovudine. Expired 2 months after treatment. Conclusion: Adult T Cell Lymphoma/Leukemia can be diagnosed with characteristic morphology in peripheral smear and supported by serological assay. Keywords ATLL, HTLV, Flower cells

Rare Associations of Osteogenesis Imperfecta with Acute Lymphoblastic Leukemia and Severe Hemophilia A D. Bhattacharyya, D. Halder, U. R. Ballikar, S. Bhattacharya, U. K. Nath, M. Bhattacharyya Insititue of Haematology and Transfusion Medicine, Medical College, Kolkata Osteogenesis imperfecta (OI) is an uncommon disease in general population. Its coexistence with hematological diseases is even rarer. To the best of

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PO 8 Invasive Aspergillosis in Childhood Acute Leukemia: Is There a Need for Prophylaxis? Payal Malhotra, Sandeep Jain, Gauri Kapoor Rajiv Gandhi Cancer Hospital and Research Centre

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Summary: Invasive aspergillosis (IA) jeopardizes the anti-leukemia therapy and contributes to significant morbidity and mortality in acute leukemia patients. Incidence of IA in acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) was 9 and 13 %, respectively. Risk factors associated with high incidence of IA included older age, ALL induction, high risk disease, hyperglycemia and latter half of study period. We recommend anti-aspergillus prophylaxis in this high risk subset. Introduction: IA is one of the dreaded complications of acute leukemia. Use of anti-aspergillus prophylaxis remains controversial and a challenging decision for the physician. Materials and Methods: Among 356 children with acute leukemia, 34 cases of IA were identified between 1996 and 2011 and their demographic, disease and IA related data was retrieved from medical records. Results: Relative incidence of IA (proven 5 and probable 29) among patients of ALL and AML was 9 % (28/310) and 13 % (6/46), respectively. Incidence of IA was significantly higher after 2004 especially among ALL patients. While all children had neutropenia and pulmonary involvement, those [10 years, with high risk disease, on ALLinduction and with hyperglycemia were statistically more predisposed to develop IA (p \ 0.01). IA led to median 17 (2–44) days additional hospital stay and contributed to delay or reduction in planned chemotherapy in 30/34 (88.2 %) patients. The 120-day aspergillusattributable mortality rate was 14.7 %. Conclusion/Diagnosis/ Impression: High incidence of IA in recent years warrants identification of patient subsets likely to benefit from anti-aspergillus prophylaxis.

PO 9 Fluorescent In Situ Hybridization using ETV6/RUNX1 Probe Identifies Multiple Prognostic Markers in Paediatric ALL Arun SR1, Anil Yadav1, Umang Patel1, Akshay Gore1, Neeraj Arora2, Deepak Mishra2, Saurabh Bhave3, Anupam Chakrapani3, Reena Nair3, Arpita Bhattacharya4, Shekhar Krishnan4, Vaskar Saha4, Mammen Chandy3, Mayur Parihar1, Mayur Parihar 1

Dept of Cytogenetics, Tata Medical Center, Kolkata, 2Department of Lab Hematology, Tata Medical Center, Kolkata, 3Department of Clinical Hematology, Tata Medical Center, Kolkata, 4Department of Paediatric Oncology, Tata Medical Center, Kolkata Tata Medical Center, Kolkata Summary: We describe FISH patterns in ALL using the ETV6/ RUNX1 probe highlighting the identification of t(12;21) and additional abnormalities like hyperdiploidy, ETV6 deletions and intrachromosomal amplification of chromosome 21(iAMP21). Introduction: The t(12;21) resulting in ETV/RUNX1 fusion is associated with a good risk in paediatric ALL. The cytogenetic abnormality being cryptic on karyotype is best detected by FISH. The ETV6/RUNX1 probe provides additional information about the presence of hyperdiploidy, iAMP21 and deletion of the ETV6 gene. Materials and Methods: FISH for t(12;21) using ETV6/RUNX1 extra signal dual colour probe was performed on 100 patients with pediatric ALLs (B18 years) using standard protocols. The FISH and karyotype results were correlated. Results: Thirteen patients were found to harbor t(12;21) of which 8 had concurrent ETV6 deletion. Extra RUNX1 signals were observed in 46 patients suggesting hyperdiploidy of which 25 had hyperdiploidy on karyotype as well. Of the remaining 21 patients 8 had a normal karyotype and 13 didn’t have optimum karyotyping results. Five patients showed deletion of the ETV6 gene only. Metaphase FISH showed iAMP21 in two patients of which one had concomitant t(12;21) and ETV6 gene deletion. Conclusion: The incidence of t(12;21) in our study is less compared to the Western literature and similar to previous reports from India. FISH using the ETV6/RUNX1 probe can detect hidden

S485 hyperdiploidy, iAMP21 and other cryptic abnormalities that can be missed by conventional karyotyping. The detection of chromosome abnormalities by conventional cytogenetics, combined with FISH analyses, is an important component in risk stratifying ALLs. Keywords ETV6/RUNX1, Hyperdiploidy, iAMP21, FISH, Paediatric ALL

PO 10 Copy Number Alterations (CNA) in Ikaros, CDK2NA/B and Retinoblastoma Genes in a Case of Ph Positive Acute Lymphoblastic Leukemia (ALL) Shruti Chaudhary, Satish Mirgal, Swapnali Joshi, P. G. Subramanian, Prashant Tembhare, Sumeet Gujral, Nikhil Patkar* Hematopathology Laboratory, Tata Memorial Centre Objectives: Genome wide analysis of Ph+ALL has revealed presence of recurrent CNA. Most notable is presence of deletions in the transcription factor Ikaros (IKZF1), which occur in more than 80 % of Ph+ALL. As these patients are associated with a poor outcome it is important to identify them. Multiplex ligation dependent probe amplification (MLPA) is a relatively low cost alternative to SNP/CGH arrays for identifying CNA. In this case report we describe CNA occurring in IKZF1, CDK2NA/B and Retinoblastoma (RB) genes in a case of Ph positive ALL (Ph+ALL) using MLPA. Methods: Genomic DNA was obtained from three healthy controls as well as a 36 yearold patient of Ph+ALL at diagnosis. DNA was examined using the SALSA MLPA kit P335 which includes 57 probes including IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1 and the PAR1 region (CRLF2, CSF2RA, IL3RA). Approximately 150 ng of DNA was hybridized to the probe mix and subjected to amplification as per manufacturers recommendations. PCR amplicons were subjected to capillary electrophoresis on an ABI3500 genetic analyzer. Analysis was done on Coffalyzer software (v.140). Results: In this patient as compared to the normal the probes showed abnormal ratio for IKZF1 from regions 4–7 indicating a deletion IKZF4-7. Similarly abnormalities were also observed for RB1-19, RB1-24 CDK2NA-5,2a and 2, indicating presence of deletions at these loci. Conclusion: In this patient we identified deletions in IKZF1 and CDK2NA/B, which are known to occur synchronously. Keywords Acute lymphoblastic leukemia, Copy number alterations, Ikaros gene

Topic: AML, MDS/MPN PO 11 A Case of Juvenile Myelomonocytic Leukemia Progressing to Myeloid Sarcoma A. K. Jitani, Y. Khonglah, J. Mishra, V. Raphael, T. Natung* Department of Pathology and Ophthalmology*, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences, Shillong Summary: Juvenile myelomonocytic leukemia (JMML) is a rare childhood neoplasm and its progression to acute leukemia is a rare entity. We present a case of JMML, which subsequently progressed to myeloid sarcoma. Introduction: JMML is a paediatric leukemia with features characteristic of both myelodysplastic and myeloproliferative disorders. It is a lethal disease with rapid progression and early demise. Materials and Methods: A three and half year boy presented with fever,

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S486 splenomegaly, pre-auricular lymphadenopathy, skin rash and pallor. Results: Complete blood count showed haemoglobin of 98 gm/dl, total count of 22 9 109/L and platelet 160 9 109/L. PBS showed 9 % myeloid blast and promonocyte, 19 % myeloid precursors and 11 % monocytes. Subsequently, bone marrow aspirate was performed and myelogram showed 15 % blast, 15 % cells were of monocytic lineage. There was evidence of marked dyspoiesis in myeloid lineage. HbF was raised and bcr-abl was negative. The child was diagnosed as JMML. Subsequently, 2 months later, the child presented with swelling of the left eye, with dimness of vision, proptosis and total corneal opacity. Debulking of the stony hard ocular mass was done. Histopathology revealed effaced tissue architecture. The tumour mass was composed of myeloblasts, which were MPO positive. Also seen were other myeloid and monocytes precursors. Based on these features, the case was diagnosed as myeloid sarcoma. Conclusion: JMML is rare and constitute 2–3 % of paediatric leukemia. It is a rapidly progressive fatal disease. However, transformation to acute leukemia, especially myeloid sarcoma is quite rare. Keywords JMML, MDS/MPN, Myeloid Sarcoma

PO 12 Apml the Age Old Harlequin: Case Report and Review of Literature

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 about 5–10 %. This leukemia has three morphologic variants; the typical hypergranular, microgranular and basophilic type. The incidence of basophilic variant is rare. Diagnosis of basophilic variant is made by predominance of hyperbasophilic cells in the Peripheral smear and Bone marrow. Morphologically AML-M2, M4 and M7 can come as differential diagnosis of this variant. Materials and Methods: Haematological investigations, Bone marrow study, Cytogenetic study was done. Results: Hb: 7.8 %, TLC-68 9 109/L and TPC 64 9 109/L. PS showed normocytic normochromic RBCs with moderate hypochromasia and occasional normoblasts. DC showed neutrophils 21 %, Basophil 1 %, Monocyte 2 %, Lymphocytes 8 % with 68 % promyelocytes. Good number of promyelocytes showed bundles of Auer rods(faggots) in cytoplasm. Indented and bilobed nuclei were seen. Bone marrow was hypercellular with suppressed erythropoiesis and megakaryopoiesis. Granulopoiesis was accelerated with more than 70 % promyelocytes having similar morphology. M:E ratio was 58:1. Promyelocytes were strongly MPO and SBB positive. Cytogenetic study showed 46XX, t(15;17) & patient was advised ATRA therapy. Conclusion: Though unequivocal diagnosis of APML can only be established by the above tests, they take several days. So, a diagnosis based on morphology supported by immunocytochemistry can give a rapid diagnosis for early institution of therapy. Keywords Acute leukaemia, Promyelocytes, Basophilic, Auer rods

Aroonima Misra, Sushant Soni, Anjali Dutta, Anita Chopra, Sameer Bakshi, Rajive Kumar

Topic: Hemato-Pathology

Laboratory Oncology, AIIMS, New Delhi

PO 14

Summary: APL can have varied molecular signature, apart from the conventional reciprocal translocation t(15;17)(q22;q21) disrupting the PML and RARa genes, other chromosomal abnormalities. All of these are characterized by PML RARa fusion transcripts. We report a rare case of APL with iso(17) and absence of RARa managed by ATRA and daunorubicin. Introduction: Here we describe a case of APL, with classical morphological and immunophenotypic features, which was of interest because of presence of isochromosome (17) along with the absence of rearrangement of RARa. Materials and Methods: An 11-year-old male presented with fever and cervical lymphadenopathy for 2 months. Laboratory investigations showed TLC of 14,800/lL with 85 % abnormal promyelocytes with hemoglobin of 7.7g% and platelet count of 20,000/lL. On IPT abnormal promyelocytes were positive for cMPO, CD117, CD13, CD33, CD9, CD64 and CD65. Results: A diagnosis of APL was made and genetic studies performed. RT-PCR was negative for PML-RARa fusion transcript. Metaphase karyotyping revealed 45–48,XY,i(17)(q10),6,+8,-14,-21,+21[cp10]. ATRA with Daunomycin was given. Patient was in morphological remission at the end of first consolidation cycle. Conclusion/Impression: Absence of rearrangement of RARa indicates alternative mechanisms could mediate the differentiation block that typifies this disease.

Granulocytic Sarcoma: A Rare Case Report

PO 13 Basophilic Variant of Acute Promyelocytic Leukaemia: A Case Report D. Das Mohapatra, K. L.Tripathy, P. Mohanty, M. Nayak, B. P. Das, R. Panda Department of Pathology, S.C.B. Medical College, Cuttack Summary: A 37-year female presented with fever for 1 month and diagnosed as acute leukemia. Introduction: Among acute myeloid leukemia with maturation, acute promyelocytic leukemia accounts for

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Dr. Gurleen Oberoi, 2Dr. Ashutosh Kumar, Dr. Rashmi Kushwaha, 4Dr. Mili Jain, 5Dr. U. S. Singh, 6 Dr. Archana Kumar 3

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Junior Resident, Department of Pathology, KGMC, Lucknow. Professor, Department of Pathology, KGMC, Lucknow. 3 Assistant Professor, Department of Pathology, KGMC, Lucknow. 4 Lecturer, Department of Pathology, KGMC, Lucknow. 5 Professor, Department of Pathology, KGMC, Lucknow. 6 Professor Department of Pediatrics, KGMC, Lucknow 2

Introduction: Granulocytic sarcoma (chloroma) is a rare entity. It is defined as a tumor mass consisting of myeloid blasts with or without maturation effacing tissue structure occurring at an anatomical site other than bone marrow. Involvement of multiple anatomic sites is even rarely reported. Materials and Methods: We report a case of 7 year old male who presented with complaints of proptosis in left eye for 3 months. The patient had been operated for the same but no histo-pathological examination had been done. Following this he developed proptosis in other eye. On examination, no white eye reflex was seen. Submandibular lymph nodes were enlarged. CT scan of the skull revealed a soft tissue swelling in superolateral part of left orbit without central nervous extension. Also a soft tissue density lesion was seen in D7–D8 pre and paravertebral axial region. A biopsy from the orbital region showed diffusely proliferated medium to large sized tumor cells with round-oval nuclei, fine chromatin, conspicuous nucleoli and moderate cytoplasm. His peripheral blood smear examination revealed pleomorphic blast cell. Subsequent bone marrow aspiration had similar blasts, few showing maturation towards granulocytic lineage. Results: Final diagnosis of myeloid sarcoma was concluded. Conclusion: Granulocytic sarcoma although a rare entity in paediatric age group should be considered in the differential diagnosis of round cell tumours. Multiple anatomical sites may be involved rarely. Keywords Myeloid Sarcoma, Acute Myeloid Leukemia

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Topic: Hematology PO 15 Therapy Related Acute Myeloid Leukemia: A Case Series Dr. S. Neethu, Dr. K. K. Prabhalakshmy, Dr. Sunitha Balakrishnan, Dr. Joy Augustine

S487 (1 h) than DES (1.5–2 h) and involves fewer technical steps (one step) than DSE (two steps). Conclusion: 1—NSE and DES cytochemical stains are better than immunophenotypic markers for subclassification AML with monocytic differentiation. 2—NSE is easy and less time consuming stain than DES. Keywords NSE and DES in comparison with immunophenotypic markers

Department of Pathology, Govt Medical College, Thrissur Objectives: Four cases of therapy related acute myeloid leukemia observed in Govt Medical College, Thrissur over a period of 1 year (2013–2014) is presented. Introduction: Therapy related AML (tAML) accounts for 10–20 % of all cases of AML. The common cytotoxic agents implicated are alkylating agents, topoisomerase II inhibitors and radiation. The oucome is poor when compared to denovo AML. Materials and Methods: We had studied all the AML s attending Govt Medical College, Thrissur from Aug 2013–Aug2014. Results: 40 A/c Leukaemia cases were diagnosed. There were 4 cases of t-AML and are reviewed. Three of the 4 cases were following treatment for carcinoma breast with anthracyclines, and one case following treatment for carcinoma of ovary with etoposide and paclitaxel. One of the case was AML-M 3 with t(15,17) translocation. 3 remaining cases were AML with monocytic differentiation (AML-M 4, AML-M 5). Conclusion: Therapy related acute myeloid leukemia is not a rare entity. The unusual feature of the present series was that all the cases were diagnosed within a short time interval of 6–12 months. Monocytic differentiation appears to be a feature in the present series. The significance of this frequency is rather intriguing and is discussed Keywords t-AML, AML M3, AML M4, AMLM5

PO 16 Study of NSE and DES Cytochemical Staining in Comparison with Immunophenotypic Markers for Sub-classification of AML with Monocytic Differentiation 1

Sona Duseja, 1Sarika Narkhede, 2Kiran Ghodke, Shashikant Mahadik, 2Asma Bibi, 3Shilpa Kushte, 3 Y. Badrinath, 3Ashok Kumar, 3Sitaram Ghogle, 3 Rashida Ansari, 2Nikhil Patkar, 2Sumeet Gujral, 2 P. G. Subramanian, 1Prashant Tembhare 3

Haematopathology Laboratory, Department of Pathology, Tata Memorial Centre, Mumbai 400012 Introduction: WHO (2008) has defined acute monocytic leukemias based on cytochemistry (non specific esterase, NSE) and immunophenotypic markers (any two of CD11c, CD14, CD64 and lysozyme). NSE and double esterase stains (DES) are used to differentiate monocytic blasts from myeloid blasts. In this study, we evaluated the use of NSE and DES against immunophenotypic profile of acute monocytic leukemia. Materials and Methods: We studied 36 cases of myeloid leukemia, which include 18 acute myeloid leukemia (AML) with monocytic differentiation, 13 AML (M1, M2 and M3), and 5 CML. NSE and DES staining was performed on peripheral and bone marrow smears. Immunophenotyping was performed using 6–8 color antibody panel using Navios (BC). Results: Of 18 with AML with monocytic differentiation, 14 cases (78 %) were strongly positive for NSE, one case was weak positive (5 %) and 3 (17 %) were negative. DES showed positivity for monocytic cells in 9 cases (50 %), weak positive in 5 (28 %) and 4 (22 %) were negative. On immunophenotyping, only 22 % cases showed [2 monocytic lineage sensitive markers (CD14, CD64 and CD11b). In AML (M1, M2 and M3) and CML cases DES show positivity for myeloid cells only. On inter-comparison between NSE and DES, NSE less time consuming

Topic: Proffered PO 17 FLT3 and NPM1 Mutations in AML Patients with Normal Karyotype: Their Correlation with Prognosis Sudha Sazawal, Neha Singh, Sonal Jain, Sunita Chhikara, Rahul Sharma, Manoranjan Mahapatra, P. Mishra, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi, India Summary: The clinical significance and prognostic outcome of the FLT3-ITD and NPM1 mutation has been extensively studied in recent years. We found no statistically significant difference in the CR rates as well as disease-free survival of the four subgroups including only NPM+, only FLT3-ITD+, both FLT3-ITD and NPM+ and both FLT3ITD and NPM-groups in AML patients with normal karyotype. Introduction: Mutations in fms-like tyrosine kinase 3 (FLT3) gene including internal tandem duplication (ITD) as well as in nucleoplasmin (NPM1) gene are considered to be major determinants of the patient response to therapy and outcome, besides their role in the pathogenesis of the disease. Materials and Methods: All the AML patients underwent complete diagnostic work-up including immunophenotyping, conventional cytogenetics along with molecular analysis for FLT3-ITD and NPM1 mutations by RT-PCR at presentation. Response to standard AML treatment protocol was analyzed in terms of CR rates and disease-free survival. Results: The prevalence of FLT3-ITD and NPM1 mutations was 10.7 and 17.8 %, respectively. The mean TLC was significantly higher in patients with NPM1+/FLT3-ITD- (p = 0.0019). CR was better in NPM1+/FLT3ITD- patients than the other groups but long term survival did not differ between mutated and unmutated NPM patients. Unlike most other studies CR rates as well as disease-free survival was better in the NPM-/FLT3-ITD- group than the FLT3-ITD+ groups, although not reaching statistically significant levels. No statistically significant correlation could be established for CD7 expression with TLC, FLT3 mutation or prognosis of the patients. Conclusions: Differences in the clinical outcome of patients from the West in the presence of NPM1 and FLT3-ITD suggests that comprehensive studies are required to confirm the definitive role of these mutations among AML patients, especially with normal karyotype. Keywords AML, Complete Remission, FLT3-ITD mutation, Nucleophosmin1, RT-PCR

PO 18 Acute Myeloid Leukemia with a Rare t(4;12)(q12;p13) 1

S.. Suganya, 3M. Angi, 2U. Sitaram, 1N. A. Fouzia, A. Ganapule, 1A. Abraham, 1A. Viswabandya, 1B. George, 1 V. Mathews, 1A. Srivastava, 3V.M. Srivastava 1

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Departments of Haematology, 2Transfusion Medicine and Immunohaematology and 3Cytogenetics Unit, Christian Medical College, Vellore

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Introduction: The t(4;12)(q12;p13) is a rare translocation in AML, with only about 20 cases reported till date. It involves the CHIC2 gene at 4q12 and ETV6 gene at 12p13 and is associated with ectopic expression of the homeobox gene, GSX2 on chromosome 4 with or without the formation of a CHIC2/ETV6 fusion gene. The 4q12 breakpoints are heterogeneous. The marrow in t(4;12) AML has been reported to show dysplastic changes, basophilia and blasts which may resemble lymphoid cells with low/absent myeloperoxidase activity. However, the blasts express myeloid antigens as well as CD7. It is associated with a poor prognosis. We describe the cytogenetic and clinicopathological features of three patients with AML and the t(4;12)(q12;p13). Patients and Methods: All patients with AML and the t(4;12) seen in the Department of Haematology, Christian Medical College, Vellore, between January 2003 and December 2013 were included. Karyotypes were correlated with blood and bone marrow findings. For each patient, 20 G-banded metaphases were studied from unstimulated overnight cultures of bone marrow. Results: The t(4;12) was seen in 0.15 % (3/1975) of all AML. The patients were 14, 24, and 52 years of age. Two were females. The haemoglobin was 6.0–8.8 g/dl, WBC count: 16.3–21.3 9 109/L, platelet count: 15–69 9 109/L, blast percentage: 21–54 %. The blasts were myeloperoxidase positive (all three) and showed aberrant expression of CD7 (one) and CD56 (two). One patient also had trilineage dysplasia and basophilia. Two of the three patients had a complex karyotype ([2 abnormalities), with abnormalities of 17p in two, and the t(8;21)(q22;q22) in one. Follow up details are not available. Conclusion: The t(4;12)(q12;p13) was seen in 0.15 % of AML. All three patients were below 60 years of age. An unusual finding is its association with the t(8;21) in one patient. These three cases will add to the reports of this rare entity in AML. Keyword AML,t(4;12)

Introduction: Hematological malignancies are primary cancers of the bone marrow and lymphoid tissues. They are often associated with chromosomal abnormalities. Their clinical presentations are variable and depend largely on the nature of the disease and its extent. Materials and Methods: All patients with bone marrow involvement by lymphomas and leukaemias in aspiration and trephine biopsy. Other relevant data of the patients was retrieved from Medical Records Department (study period March 2013–August 2014). Results: Total number of lymphoma and leukemia cases with bonemarrow involvement-62. Out of them, Hodgkin lymphomas-2, NonHodgkin lymphomas-12, Acute Lymphoblastic Leukemia/lymphoma(ALL)-5, chronic granulocytic leukemias (CGL)-25, Acute myeloid leukemias (AML)-15, Chronic lymphocytic leukemia(CLL)3. The data was analyzed according to the type, age, sex distribution and other important clinical findings. Conclusion: In CGL, peripheral blood features are more important than bone marrow findings. Bone marrow findings depends on phase of CGL. In AML, blood findings and bone marrow aspirate features are more important than trephine biopsies. Cytochemical stains like myeloperoxidase (MPO) can be used to detect myeloid origin of neoplastic cells. In lymphomas, pattern of infiltration can be fully assessed on trephine biopsies. Overall infiltration is more common in B-cell lymphomas than T-cell lymphomas. Immunohistochemistry can be done to confirm the nature of infiltration. Keywords Lymphoma, Leukemia, Bonemarrow

References

Significance of Bone Marrow Studies in Underequipped Laboratories: A Case of Gaucher’s Disease

(1)

(2)

(3)

Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (2014). Mitelman F, Johansson B and Mertens F (Eds.). http://cgap.nci.nih.gov/Chromosomes/Mite lman Cools J, Mentens N, Odero MD, Peeters P, Wlodarska I, Delforge M, et al. Evidence for position effects as a variant ETV6-mediated leukemogenic mechanism in myeloid leukemias with at (4; 12)(q11-q12; p13) or t (5; 12)(q31; p13). Blood. 2002; 99(5):1776–84. Harada H, Harada Y, Eguchi M, Dohy H, Kamada N. Characterization of acute leukemia with t(4;12). Leuk Lymphoma. 1997 Mar; 25(1–2):47–53.

Topic: Haematology PO 19 Study of Bone Marrow Infiltration in Leukemias and Lymphomas: Experience at a Tertiary Care Centre in South India 1

M. Alekhya, 1Y. C. Spoorthy Rekha, 1T. Asha, Manicka Vasagam

2 1

Departments of Pathology, 2Medical Oncology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh Summary: Bone marrow infiltration of lymphomas and leukemias vary according to their subtype. Their study is important for definitive diagnosis, staging, treatment and prognosis of the patient.

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Topic: Clinical Hematology PO 20

Dr. Aneet Singh, Dr. Ramesh Chavan Jawaharlal Nehru Medical College, KLE University, Belgaum Objectives: Hematologists are often referred pediatric cases with global developmental delay. One of the non-infectious differential diagnosis is that of storage disorders. Early diagnosis and treatment can be life saving and life prolonging for the patient. Gaucher’s disease is a rare autosomal recessive lysosomal storage disease due to deficiency of enzyme glucocerebrosidase, incidence being 1/50,000 to 1/100,000. This case is presented to highlight the significance of bone marrow studies in underequipped laboratories where advanced enzyme assays cannot be undertaken. Methods: Case: A 3 and 1/2 year old child presented with growth retardation, progressive abdominal distention and indigestion since birth. On examination the patient had pallor, frontal bossing and gross hepatosplenomegaly. The child was a product of consanguineous marriage. Results: Investigations—RBC count was 3.9 million/mm3, hemoglobin was 9 g%, total WBC count was 8,100/mm3, platelet count was 1,32,000/mm3. RDW was 19, the retic count was 3.6 %. PCV was 31.5. The peripheral smear showed normocytic hypochromic anemia with mild thrombocytopenia. Liver function tests were normal. The bone marrow report showed suppressed erythroid, myeloid and megakaryocytic series. Large cells with eccentrically placed nucleus, fibrillary, crumpled tissue paper like cytoplasm (Gauchers cells) suggestive of storage disorders most likely Gaucher’s disease was diagnosed. Liver biopsy and b glucosidase test was suggested. b glucosidase level was 1.00 nmol/h/mg which confirmed our diagnosis. Conclusion: Final diagnosis: Gaucher’s disease Type 1. Keywords Gaucher’s disease, Gaucher cells, Storage disorder, Bone marrow studies

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Topic: Hematology PO 21 Role of Bone Marrow Biopsy in Detection of Metastasis Dr. Ashna Mittal, Dr. T. Singh, Dr. Neha Singh Department of Pathology, Maulana Azad Medical College Summary: Spectrum of bone marrow involvement by solid tumor metastasis was studied for 66 cases. Special stains and immunohistochemistry were used to classify these tumors. Involvement of marrow suggested higher stage and poor prognosis for these patients. Aim: To study the spectrum of metastasis and the associated morphological changes in bone marrow. Introduction: Bone marrow involvement by tumor upgrades the tumor to stage 4 and is a harbinger of poor prognosis. It can be easily picked up in the bone marrow aspirate/biopsy which is a relatively inexpensive and reliable procedure to diagnose tumor metastasis. Materials and Methods: Case records, bone marrow aspirates and trephine biopsies for the cases of bone marrow metastasis were retrospectively analysed for the period January 2007 to September 2014. Special stains and immunohistochemistry were used to classify the tumor. Results: A total of 66 cases of bone marrow metastasis were found, 22 belonged to the paediatric age group and rest 44 to the adult age group. Bone marrow aspirate only was available for 16 patients, bone marrow biopsy only for 27 patients and both for 23 patients. Malignancies detected in the marrow included neuroblastoma (18), prostate (5), adenocarcinoma (13), undifferentiated carcinoma (18), small round cell tumor (3), breast (3), melanoma (3), Ewings (1), thyroid (1) and renal cell carcinoma (1). The tumor cells were present as sheets, clusters and infiltrates into the marrow spaces. Bone marrow was completely or nearly completely replaced in 16 cases. The marrow response to metastasis included fibrosis (28), necrosis (12), gelatinous marrow transformation (4), plasmacytosis (4), eosinophilia (6) and new bone formation (5). Conclusion: Bone marrow is an important site of solid tumor metastasis. It can also serve useful to determine the primary site of occult malignancy by morphology, special stains and immunohistochemistry. Marrow positivity for metastatic deposit upstages the case to stage IV and hence the need to carry out a bone marrow biopsy in solid malignancies. Keywords Bone marrow, Solid tumor, Metastasis

Topic: Haematology PO 22 Comparison of Touch Imprints with Aspirate Smears for Evaluating Bone Marrow Specimens

S489 than in bone marrow aspirate smears. (3) No statistical difference was found between touch imprints and bone marrow aspirate smears in the final diagnosis. (4) Touch imprints were reliable and helpful in cases where smears failed to demonstrate an abnormal population. (5) In clinically suspected cases of malignancy, touch imprints showed high cellularity due to less contamination of peripheral blood. Conclusion: Touch imprints are extremely helpful in cases where bone marrow aspirate smears fail to demonstrate an abnormal cell population. Touch imprints should be routinely done on all bone marrow biopsy specimen. Hence touch imprints can serve as better diagnostic modality than bone marrow aspirate smear and thus can be an independent and reliable technique. Keywords Touch imprints, Bone marrow, Leukaemia

PO 23 A Case of Gaucher Disease Diagnosed on Bone Marrow Aspiration and Biopsy Himanshu Srivastava, Amit Nisal, Anjali Kelkar Bharati Vidyapeeth Hospital & Research Center, Dhankawadi, Katraj, Pune 411043 Summary: A 40 year old man presented with history of abdominal discomfort, lump in abdomen and anorexia of almost 8 years duration. He was born of second degree consanguineous marriage. He did not have fever, jaundice or bleeding from gastrointestinal tract at the time of presentation. There was no past history of tuberculosis or blood transfusion. On clinical examination, he had pallor, massive splenomegaly and hepatomegaly with no other physical findings. Based on these findings differential diagnoses of tropical splenomegaly, storage disorder or hematological malignancy were considered. Investigations revealed Haemoglobin of 6.8 g/dl, WBC count of 1,500/cubic mm and platelet count of 20,000/cubic mm, with reticulocyte count of 1 %. His renal function tests and liver function tests were within normal limits. Ultrasonography of abdomen revealed hepatomegaly with coarse echo-texture and massive splenomegaly. CT scan of abdomen showed hepatosplenomegaly. Bone marrow aspiration & biopsy showed many Gaucher cells on smear. Introduction: Gaucher disease is classified in three types, type I or chronic non-neuronopathic, type II or acute non-neuronopathic and type III or Subacute neuronopathic form. Type I Gaucher disease is most common of all the three types, in which there is reduced but detectable activity of b-glucocerebrosidase, and glucocerebroside storage is limited to mononuclear phagocytes. Materials and Methods: Peripheral blood smear showed pancytopenia. Ultrasonography of abdomen revealed hepatosplenomegaly. Bone marrow aspiration and biopsy revealed many Gaucher cells. Results: On investigating, serum b-glucocerebrosidase was found to be reduced, which confirmed the diagnosis of Gaucher disease. Diagnosis: Type I Gaucher disease

Goutami Dasnayak, S. Palei, A. Mishra, K. L. Tripathy, B. P. Das Department of Pathology, SCB Medical College, Cuttack, Orissa Objectives: (1) To assess whether differential count from touch imprints can be a reliable and independent test for routine examination of bone marrow aspirate. (2) To assess whether touch imprints can be a better technique for morphological classification and detection of haematological abnormalities or malignancies. Materials and Methods: 100 cases belonging to all age groups and both sexes diagnosed and confirmed by investigations as abnormalities or malignancies. Comparative evaluation of touch imprints and bone marrow aspirates was done. Results: (1) Acute leukaemia constituted 80 % of the total cases wih a male predominance. (2) Higher percentage of blasts and abnormal cells were observed in touch imprints

Topic: Hematology PO 24 Spectrum of Pancytopenia at a Tertiary Care Center Monika Gupta, Sant Prakash Kataria, Gajender Singh, Sanjay Kumar, Rajeev Sen Department of Pathology, Pt BDS, PGIMS, UHS, Rohtak Introduction: Pancytopenia is an important clinicohaematological entity encountered in our day today clinical practice. A multitude of disorders primarily or secondarily affecting the bone marrow

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S490 manifests with various haematological derangements which is commonly presented as pancytopenia. Diagnosis of pancytopenia requires microscopic examination of bone marrow aspirates to assess the overall cellularity and morphology. Materials and Methods: In this study, 169 consecutive patients with evidence of pancytopenia on peripheral blood from both the sexes and all age groups were included. The inclusion criteria for pancytopenia were haemoglobin \10 g/dl, total leukocyte count\4.0 9 109/ll and platelets\100 9 109/ll5. A detailed relevant clinical history and physical examination was done. Observations: Bone marrow examination was helpful in identifying the aetiology of pancytopenia. There were 94(55.6 %) males and 75(44.4 %) females with slight male predominance (1.2:1) and age ranged from 1 to 75 years. Among non-malignant causes megaloblastic anemia was the predominant cause 64(37.87 %) followed by mixed nutritional deficiency anemia 27(15.98 %) and aplastic anemia 19(11.24 %). Other non-malignant causes were normoblastic erythroid hyperplasia, infections and drug induced bone marrow suppression. Among malignant causes acute leukemia forms the major part 21(12.43 %) followed by non Hodgkins lymphoma and myelodysplastic syndrome. Conclusions: The main cause of pancytopenia in our study was megaloblastic anemia followed by mixed nutritional deficiency anemia. This study emphasize that in developing countries like India majority of the patients had reversible aetiology. In pancytopenia cases, attempt should be made for an early recognition of underlying aetiology so that treatable causes are identified without delay and prognosis can be improved. Keywords Pancytopenia, Bone marrow aspirate, Megaloblastic anaemia

Topic: Haematopathology PO 25 Bone Marrow: Primary Diagnosis of non Hodgkins Lymphoma on Bone Marrow Examination Dr. Richa Pawarco, Monika Gupta, Swagatika Samal, Sonia Chhabra, Gajender Singh, Sanjay Kumar, Rajeev Sen

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 although bone marrow aspiration and bone marrow biopsy are not entirely complimentary. Keywords Bone marrow, Non-Hodgkins lymphoma, Lymph node biopsy

Topic: Hematology PO 26 A Case of Mono-ostotic Paget’s Disease 1

S. V. R. Raja Sekhar, 1Rashmi Patnayak, 2Manickavasagam, V. Siva Kumar

3 1

Departments of Pathology, 2Medical Oncology, 3Nephrology

Summary: 60 year old diabetic male came with complaints of haematuria and right flank pain associated with fever and chills since 2 months. Thorough clinical examination did not reveal any abnormality.On investigating with imageology, Dimercaptosuccinic acid scan (DMSA) cortical renography showed bilateral chronic pyelonephritic related changes, spiral computed tomography (CT) abdomen and pelvis showed sclerotic and lytic foci in right hip bone ? pagets/? metastasis. BONE SCAN showed intense radio-tracer uptake involving right hip bone suggestive of mono-ostotic pagets disease which was later confirmed by trephine biopsy. Introduction: Paget’s disease of bone is a chronic disorder, characterised by focal areas of excessive osteoclastic bone resorption accompanied by a secondary increase in osteoblastic bone formation. The disease results in bone expansion and structural weakness, which can cause pain, deformity, and a range of complications. The disease is often asymptomatic and commonly seen in an aging population. The diagnosis of the disease is mostly based on radiological examination and on biochemical markers of bone turnover. It is being successfully treated by biphosphonates, a group of anti-resorptive drugs, thereby decreasing the morbidity and mortality associated with the disease. Conclusion: Paget disease of bone may be asymptomatic initially and it can be diagnosed by its classic radiologic features. An early diagnosis can help to prevent potential complications. Keywords Pagets disease, Sclerotic and lytic foci, Trephine biopsy

Pt. B.D. Sharma, PGIMS, Rohtak Objective: Bone marrow examination is an essential investigation in the evaluation and staging of NonHodgkin’s Lymphoma (NHL). Correlation with bone marrow biopsy, flow cytometry and lymph node biopsy was done wherever possible. Introduction: Bone marrow aspiration (BMA) is a simple and minimally invasive technique that allows cytological assessment of bone marrow cells. It is a retrospective study to evaluate the sensitivity of BMA in detecting involvement of bone marrow by NHL. Materials and Methods: A total of 22 consecutive BMAs with lymphoid infiltrates were seen in the Department of Pathology, Pt. BDS PGIMS, Rohtak during the period of March 2012 to August 2012. Correlation with bone marrow biopsy, flow cytometry and lymph node biopsy was done wherever possible. Results: The incidence of bone marrow involvement by lymphoid infiltrate was 8.3 %. The cases comprised of 11 males and 11 females. The age ranged from 18 to 85 years. The criteria for positive involvement of bone marrow was [30 % of lymphocytes or presence of atypical lymphocytes and blastoid cells even if the proportion was \30 %. Out of 22 cases, two were fully diagnosed Chronic Lymphocytic Lymphoma, three case of plasma cell dyscrasias and three cases of NHL (confirmed on biopsy). In one case Immunohistochemistry confirmed Peripheral T cell Lymphoma on sections from cell blocks of BMA. Conclusion/Diagnosis/Impression: BMA is an easily available technique and provides large amount of marrow that can give us the clue to early diagnosis of lymphoma,

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Topic: Haematology PO 27 Bone Marrow Infections in Patients with HIV/AIDS, A Study of 40 Patients 1

Vikram Raj Gopinathan, 1Tejinder Singh, 1Nita Khurana, Richa Dewan, 3Preena Bhalla

2

1 Departments of Pathology, 2Medicine & 3Microbiology, Maulana Azad Medical College, New Delhi, India

Summary: Bone marrow of 40 patients with HIV infection were examined. 14 patients had Tuberculosis (TB), 1 patient had Mycobacterial avium-intracellulare complex (MAC), 1 patient had Histoplasmosis and 1 patient had Pure red cell aplasia due to Parvovirus B19 infection. Introduction: Human immunodeficiency virus (HIV) primarily infects CD4+ T cells and the CD4+ cell count decreases, predisposing the patient to develop opportunistic infections. These pathogens disrupt the marrow microenvironment and present with peripheral blood abnormalities. Bone marrow examination is an essential investigation in HIV/AIDS patients presenting with pyrexia of unknown origin (PUO) and cytopenias.

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Materials and Methods: 40 adult HIV infected patients were enrolled in the study. The patients had cytopenias, pyrexia of unknown origin and suspicion of hematological malignancy as indications for bone marrow examination. A thorough clinical history and examination, complete blood count and peripheral smear examination, and CD4+ cell counts were obtained for all the patients. Results: 15 (37.5 %) patients demonstrated granulomatous inflammation in the bone marrow. 2 of these 15 patients showed AFB positivity. Of the 2 patients, a diagnosis of disseminated MAC infection was made in one patient by demonstrating AFB positive granulomas in liver biopsy and blood culture positivity. The remaining patients were diagnosed as Tubercular infection. In addition, 1 (2.5 %) patient showed both extracellular and intracellular yeasts of Histoplasma capsulatum in the bone marrow. Another (2.5 %) patient showed pure red cell aplasia and giant pro erythroblasts in the marrow suggestive of Parvovirus B19 infection. Majority (65 %) of patients included in the study had CD4+ cell count \200/lL. Conclusion: Bone marrow biopsy offers the possibility of providing relatively rapid information regarding the presence of opportunistic infections in HIV infected patients presenting with PUO and cytopenias. Keywords Marrow infections HIV, Marrow Histoplasma, Marrow Granuloma

Topic: Haematology PO 28 Role of Bone Marrow Histopathology as Compared to Aspiration in the Diagnosis of Tuberculosis 1

Dr. Sameera Karnam, 1Dr. Amit Kumar Chowhan, Dr. K. Radhika, 2Dr. Alladi Mohan, 3Dr. V. Siva kumar

1 1

Departments of Pathology, 2General Medicine and 3Nephrology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh

Summary: Bone marrow biopsy is superior to bone marrow aspiration in diagnosing granulomas. Introduction: Tuberculosis is a major health problem world wide, particularly in developing countries. One of the health targets set in 2010 Millenium development goals is to halt and reverse the incidence of tuberculosis by 2015. Tuberculosis is caused by Mycobacterium tuberculosis. Tuberculosis infection is classified into pulmonary and extra pulmonary. Common extra pulmonary sites involved are lymphnode, pleura, genitourinary tract, bones, joints, meninges, peritoneum and pericardium in order of decreasing frequency. Bone marrow involvement occurs in miliary or disseminated tuberculosis when bacteria disseminates through systemic arterial system. Materials and Methods: Here we are presenting bone marrow examination of two patients who had high index of suspicion to tuberculosis. One male patient of age 24 years, presented with pyrexia of unknown origin since 1 month. On further evaluation patient suspected to have tuberculosis and showed clinical response to empirical antitubercular therapy. Another male patient of age 32 years was a known case of post renal transplant with graft failure and on immunosuppressive agents since 6 years Leishman, Giemsa stained smears of bone marrow aspiration, Haematoxylin, eosin and Ziehl–Neelsen stained sections of trephine biopsy were studied. Results: Bone marrow aspiration was reported as megaloblastic marrow with plasmacytosis and mild erythroid hyperplasia with megaloblastic change, respectively. But trephine biopsy showed granulomas of variable size with epithelioid cellular clusters, langhan giant cells and caseous necrosis. Conclusion: It is not easy to demonstrate granulomas in Bone Marrow Aspiration but biopsy is always better for demonstration Keywords Bone marrow biopsy, Bone marrow aspiration, Tuberculosis

S491

Topic: Haematology PO 29 Experience of Autologus Hematopoietic Stem Cell Transplantation (HSCT) in the Patients Infected with Either Hepatitis B or Hepatitis C Virus: Case Series from the Western India Dr. Gaurang Modi Gujarat Cancer Research Institute, Ahmedabad Summary: We are showing here the result of autologus HSCT of 11 patients infected with hepatitis B or C during the year 1999–2010. Introduction: Autologus HSCT is challenging in HBV or HCV positive patients due to fear of hepatitis and TRM (transplant related mortality). Very few data are published internationally till date. Materials and Methods: It’s a retrospective analysis. All the positive patients (HBV/HCV) underwent auto HSCT for malignant causes during the year 1999–2010 were selected. 11 patients underwent autologus HSCT. All the included patients had performance score 1, normal liver function test, non-infectious state of HBV before auto HSCT. Antiviral treatment continued according to gastrologist opinion. Results: The median age of the seropositive patients was 29 year (range 15–54); 10 and 1 patients were HBV and HCV positive, respectively. Most common indication of HSCT were lymphoma (n = 10), in which 7 patients were of Hodgkin disease (HD) and 3 patients were of non Hodgkin disease (NHL) and 1 of multiple myeloma (MM).9 patients were in 3rd CR; 2 patients were in 2nd CR. We had used BEAM in lymphoma and melphalan in MM as conditioning regimen. Death due to disease and TRM is 0 %. None of the patient developed hepatitis post transplant. Average cost of auto HSCT was 8100 USD. Conclusion: We have described our experience of HSCT in heavily pre-treated and high risk HBV/HCV infected patients. Being the developing country we had completed this challenge by confronting with limited resources, significant cost issue, inbuilt administrative hurdles of government set up.

Topic: Platelet Related Disorders PO 30 Role of Mean Platelet Volume in Patients with Autoimmune Disease Garima Goel, Kaushik Majumdar, Deepti Joshi, Rajneesh Joshi*, Neelkamal Kapoor Department of Pathology & Lab Medicine, Department of Medicine*, All India Institute of Medical Sciences, Bhopal Summary: Mean Platelet Volume (MPV) is a simple platelet parameter which increases during platelet activation and can be taken as a risk marker for atherothrombotic complications in autoimmune diseases. Introduction: MPV is a reliable index of platelet size that correlates with the functional status of platelets. The patients with autoimmune diseases are at increased risk of cardiovascular complications and MPV levels are usually increased in autoimmune diseases. With the present study we aimed to investigate the levels of MPV in patients with autoimmune diseases as compared to normal healthy controls. Materials and Methods: A prospective study carried out in Department of Pathology & Department of Medicine including 30 patients with proven autoimmune disease and 30 healthy controls matched for age and gender. The diagnosis of autoimmune diseases was given based on the clinical presentation and presence of

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S492 autoantibodies. MPV was measured in a blood sample collected in EDTA. Results: Out of the 30 patients with proven autoimmune diseases 15 were of Hashimoto thyroiditis, 8 of Rheumatoid arthritis, 5 of Graves disease and 2 cases of mixed connective tissue disease. The mean MPV level of patients with autoimmune diseases was higher than that of control group. Conclusion: To conclude the present study demonstrated that patients with autoimmune diseases tend to have increased platelet activation and in turn are at an increased risk of atherothrombotic complications. Keywords Mean Platelet Volume, Autoimmune, Atherothrombosis

Topic: Oral Paper Presentations PO 31 Mean Platelet Volume in Chronic Obstructive Pulmonary Disease Gurdeep Buxi, *A. K. Malhotra, Vijay Kumar, Sadhna Marwah, A. S. Nigam Department of Pathology, *Medicine, PGIMER, Dr. Ram Manohar Lohia Hospital, New Delhi Introduction: Mean Platelet Volume (MPV) is one of the most widely used surrogate markers of the platelet function and has been shown to reflect inflammatory burden in different chronic diseases. Aim-To study Mean Platelet volume in patients with acute exacerbation of Chronic Obstructive Pulmonary Disease (COPD) and stable phase and to compare these results of MPV with normal healthy control. Materials and Methods: 65 diagnosed cases of COPD in acute exacerbation were taken and reviewed again after a period of 3 months in their stable phase and compared to age and sex matched controls. Routine haemogram (complete blood count, platelet count and MPV) and Spirometry (Actual and percentage predicted values of FEV1, FVC, FEV1/FVC, PEF, FEF 25–75 %) were done. Statistical analysis was done with p value of 0.05 or less taken as statistically significant. Results: The values of MPV was found to be lower in patients during acute exacerbation of COPD and higher in stable phase of COPD as compared to controls and this difference was statistically significant. (p = 0.00). Conclusion/ Summary: MPV is significantly lower during acute exacerbation and higher in stable phase of COPD. Therefore, MPV could be used as an acute-phase reactant in acute COPD exacerbation. Measurement of changes in MPV values during follow-up may be considered as a quick and reliable tool in the assessment of inflammatory response. Keywords Mean Platelet Volume, Spirometry, COPD

Topic: Hemato-Pathology PO 32 Variation in the Platelet Parameters in Myeloproliferative Neoplasms and Myelodysplastic Syndromes: A Case Series 1

A. Arun Kumar, 1Ponwang Konyak, 2Ashutosh Kumar, Rashmi Kushwaha, 4Mili Jain, 1U. S. Singh, 5A. K. Tripathi

3 1

Department of Pathology, KGMC, Lucknow, Department of Pathology, KGMC, Lucknow, 3Department of Pathology, KGMC, Lucknow, 4Department of Pathology, KGMC, Lucknow, 5Department of Clinical Hematology, KGMC, Lucknow

2

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Introduction: The myeloproliferative neoplasms (MPNs) are characterised by proliferation of one or more of the hematopoietic lineages. In CML, it is predominantly granulocytes, in Polycythemia Vera it is predominantly cells of erythroid series while it is the megakaryocytes and platelets in Essential Thrombocytosis. On the other hand, Myelodysplastic Syndrome (MDS) is characterised by dysplasia in one or more cell lines. Both these disorders have significant variations in the platelet parameters. Materials and Methods: The study included a total of 41 diagnosed cases of MPN and MDS. An evaluation of clinical profile and complete automated blood count using ADVIA 2120, SIEMENS automated 5 part cell counter were done in all these cases. Platelet count, Mean Platelet Volume (MPV), Platelet Distribution Width PDW), Plateletcrit (PCT), Mean Platelet Concentration (MPC), Large Platelets (LP) and a calculated new parameter namely ratio of Large Platelets and Mean Platelet Volume (LP/MPV) were assessed. Results: A majority of these automated platelet parameters were seen to have a statistical significance (p \ 0.05) in diagnosing MPN and MDS. Within MPN, it was seen that there was a wide variation in parameters like Platelet count, Plateletcrit, MPV and large platelets among CML and other Ph(-ve) MPNs. Even within CML there was a significant difference between chronic phase and blast crisis patients. Conclusion: The platelet parameters can serve as a useful diagnostic aid to the molecular analysis and bone marrow morphology in cases of MPN and MDS. Also, the LP/MPV parameter used in our study can serve as a significant diagnostic parameter Keywords MPN, MDS, Platelet parameters.

PO 33 Evaluation of ADVIA2120i for Body Fluid Analysis and Its Correlation with Microscopic Examination Rashida Ansari, Kanchan Kulkarni, Sona Disuja, Mansi Joshi, Shashikant Mahadik, Nikhil Patkar, P. G. Subramanian, Sumeet Gujral, Prashant Tembhare Hematopathology Laboratory, Pathology Department, Tata Memorial Centre, Mumbai 400012 Introduction: Body fluids (BF) analysis provides valuable diagnostic information to the clinicians inseveral serious medical conditions. The microscopic counting and the differentiation of WBC in a BF smear are being used as a reference; however it has high interpersonal variability. Automated methods of analysis by hematology analyzers have reduced interpersonal variability up to a great extent in the peripheral blood counts and improved turnaround time and precision. In this study we evaluated the accuracy of BF analysis by ADVIA2120i and correlated with microscopic findings. Materials and Methods: We prospectively studied 50 BF specimens (Plural fluids 21, Ascitic fluid 14, and CSF 15) from different cancer patients on ADVIA2120i and correlated with microscopic counts and differentials. Microscopic counts were done using neubauer chamber and cytomorphology using Wright’s staining. Results: Range, Mean, Median and coefficient of correlation of counts of different BFs by ADVIA2120i and microscopic examination is shown in Table 1. However, CBC parameter ‘‘LUC’’ that indicates atypical Lymphoid cells or blasts does not match with morphological findings as large cells like mesothelial cells are also counted as LUC. Conclusion: Automated evaluation of body fluids using ADVIA2120i can provide easiest, simple and useful method for counting white blood cells in effusions. Keyword Automated Body fluids analysis

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Table 1 . S. no.

Body fluids

Conuts on ADVIA2120i Range (cells/ll)

Mean (cells/ll)

Microscopic counting Median (cells/ll)

Range (cells/ll)

Mean (cells/ll)

Correlation coefficient Median (cells/ll)

1

Plural fluid

40–17,700

177

660

2–23,464

189

325

0.92

2

Ascitic fluid

50–81,000

6,533

555

2–78,600

6,107

174

0.99

3

Cerebrospinal fluid

21

1

20.4

2

0.99

0–270

0–280

Topic: Hematopathology PO 34 WBC Histogram, Graying of Population, A Diagnostic Kaleidoscope Sarika Singh, Sunita Sharma, Anita Nangia, Deepshikha Dhiman Lady Hardinge Medical College, New Delhi Summary: WBC/Diff graph gives enormous information in automated 5 part differential counters if looked upon well. Information which can be obtained is graying, merging, shifting and multiplicity of populations. Based on these, various diagnostic interpretations can be made, which are atypical cells, abnormal/immature cells, blasts, malarial infection and nucleated red blood cells. Introduction: Automation in hematology in past 3 decades has progressed from 3 part to 5 part and now to 6 part. Progressing from basic impedence based technique to impedance/flow cytometry to volume, conductivity and scatter (VCS) i.e. combination of flow cytometry and impedance technique. This advancement gives us great insight into pathology and help us make correct and quick diagnosis. Present study is intended to highlight the importance of 5 part differential counter and its role in picking up diagnosis. Materials and Methods: This study was conducted over a month and 600 samples collected in K2 EDTA BD, Vacutainer (3.0 ml blood) were run on Sysmex XP 2000i and analysed in research mode graph and Q flag. The findings of CBC, scatter plot and peripheral blood smear findings were correlated. Results: 50/600 samples screened showed graying of population. Patient’s age ranged from 05 days to 71 years. M:F 30:20. TLC 0.37–173.1 9 109/L, Hb 3.5–22.5 g/dl, Plt 20–658 9 109/L. 30/50 patients, showed complete and 20/50 showed partial graying. Division of cases as per the diagnosis was 10/50 thalassaemia, 6/50 septicemia,06/50 malaria,26/50 leukemia,02/50 cases no conclusive diagnosis. 38/50 cases showed correlation with clinical diagnosis, while in 12/50 suggestion to diagnosis was made. Conclusion/ Diagnosis/Impression: WBC/Diff, WBC/Baso scatter plot, showing graying of population are very sensitive and reliable for screening samples in a busy and specialized laboratory, if carefully looked upon. Keyword WBC Histogram

Topic: Hematology PO 35 Significance of Red Cell Distribution with as a Severity Marker in Pre-eclampsia 1

Dr. G. Shilpa, 1Dr. M. L. Harendra Kumar, 1Dr. Geethanjali, Dr. Puspha P. Kottur

2 1

Department of Pathology, 2Department of Obstetrics and Gynecology, Sri Devraj Urs Medical College, Kolar Objective: To evaluate the use of RDW as a severity marker in preeclampsia. Methods: This prospective study was done on 87 cases of

S493 severe pre-eclampsia, 96 patients with of mild pre-eclampsia and 100 cases of normal pregnant females taken as controls, who were admitted in RL Jalappa Hospital during the study period of June 2013 to June 2014. Women with anemia (Hb \ 11 g%), iron deficiency status, hypertension and hemolysis were excluded from the study. The RDW obtained by analyzing anticoagulated blood at the time of admission was recorded and compared among three groups by ANOVA and t-test. Correlation coefficient between RDW and mean arterial blood pressure (MAP) was analyzed in mild and severe pre-eclampsia groups. Receiver operating characteristic curve (ROC) was calculated to estimate the cut off value for predicting disease progression of pre-eclampsia. Results: Among the three groups RDW was significantly higher in the severe pre-eclampsia cases (p \ 0.001). In mild and severe pre-eclampsia group, RDW was statistically correlating with MAP (r = 0.34, p \ 0.001). The area under curve of RDW to predict the severe pre-eclampsia was 0.76 in ROC curve. RDW at a cut off value of[15.85 % was found to have significant predictive value for severity in pre-eclampsia cases, which had a sensitivity and specificity of 71 and 65 %, respectively. Conclusion: RDW values significantly increased in severe pre-eclampsia group and were positively correlating with MAP. Therefore, RDW can be a candidate marker for predicting the severity of pre-eclampsia. Keywords Red cell Distribution Width, Pre-eclampsia, Mean arterial Pressure

Topic: Laboratory Haematology PO 36 P6/D1-PANDA-Pattern on ADVIA2120i is Highly Sensitive and Very Specific Tool in the Identification of Acute Promyelocytic Leukemia Shilpa Kushte, Sampat Godage, Nikhil Rabade, Kanchan Kulkarni, Vrushali Mohite, Shashikant Mahadik, Nikhil Patkar, P. G. Subramanian, Pratibha Amare, Sumeet Gujral, Prashant Tembhare Haematopathology Laboratory, TATA Memorial Hospital, Mumbai Introduction: Acute promyelocytic leukemia (APML) is a highly curable acute leukemia with disseminated intravascular coagulation (DIC) as a major cause of early death. Therefore the intial diagnosis of APML needs to be made on an urgent basis. A suspicion of APML if made on a blood counter is of paramount importance. We evaluated CBC on ADVIA 2120i to confirm these findings based on its properties to derive cellular type information based on peroxidase activity (PA) and nuclear density (ND)—PANDA profile. Methods and Materials: We studied CBCs in 107 patients (APML 42, NonAPML-AMLs 50 and CML 15) for PANDA profile on ADVIA2120. Results: Of 42 APML cases, 33 revealed a specific PANDA profile (P6/D1 pattern) however remaining cases didn’t show P6/D1 pattern. Of remaining 9 cases, 7 had WBC count \3,000/ll, 2 had [3,000/ll. This indicates that P6/D1 pattern cannot be seen in APML with low counts. Non-M3-AML and CML cases didn’t show P6/D1 pattern. Hence APML is the only AML subtype allocated to P6/D1 PANDA profile and crucially none of the remaining 65 cases of Non-APML displayed this characteristics profile. Positive predictive value (PPV) and negative predictive value (NPV) of P6/D1 PANDA-pattern on ADVIA-2120 in identification of APML was 100 and 88 %, respectively. NPV is further increased to 97 % if total count is [3,000/ll. Conclusion/Diagnosis/Impression: P6/D1-PANDA-pattern is highly specific and very sensitive tool in the identification of APML. It is simple and cost effective tool as a first indicator of APML diagnosis in routine hematology laboratory for early detection. Keywords APML, Panda System

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Topic: Haematology PO 37 Evaluation of Haematological Parameters among Alcoholics Dr. S. Smitha, Dr. Taranath Sitimani Department of Medicine, Al-Ameen Medical College Bijapur, Karnataka Introduction: Alcoholism represents one of the most serious worldwide socio-economic health problem and one of the leading cause of preventable mortality. Alcohol affects multiple systems like Hepatobiliary system, Cardiovascular system, Haematopoietic system. Alcohol has direct and indirect effect on haematopoietic system. Many a times haematological changes are left undetected and untreated which could lead to various complications. Ojective/Aim: Study conducted to evaluate the haematological parameters in Alcoholics. Materials and Methods: This Case–Control study was conducted among 30 adult male alcoholic patient taken as case and 30 adult male non alcoholic as control group who were admitted in medicine department in Al-Ameen Medical College. Complete Blood count and peripheral blood smear study were analysed and statistically assessed. Results: The mean Haemoglobin were significantly reduced among alcoholics [p = 0.005]. The mean WBC count and platelet count were significantly reduced in alcoholics. MCV in alcoholic were significantly increased when compared to non alcoholic. Peripheral blood smear showed macrocytic anemia in significant number of alcoholic patients. Conclusion: In the above study conducted we observed that Anemia, Thrombocytopenia, Granulocytopenia are seen among alcoholics when compared to non alcoholics. So by help of this study we can alert physician that ethanol consumption history should be systematically included in the clinical assessment of patient presenting with deranged haematological indices along with other causes as there was significant correlation between anemia, thrombocytopenia and alcohol consumption. Keywords Anemia, Thrombocytopenia, Megaloblastic anemia

Topic: Free

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 within 1 h of collection by automatic blood counter. Statistical evaluation was performed by descriptive studies and student t test. Results: All the platelet indices i.e. MPV, PDW, PLCR were higher in patients with poor glycemic control than in patients with normal glycemic control. Conclusion: Platelet Indices can be considered as easy, rapid, economical, cost effective tool to assess thromboembolic events in diabetes mellitus, which could reduces the morbidity & health care costs. Keywords Platelet indices, Glycemic control, Diabetes Mellitus

PO 39 Evaluation of Novel WBC VCS Parameters Obtained on LH750 for Early Diagnosis of Sepsis and Differentiation from SIRS Dr. Shanaz Khodaiji, Raajeev Hingorani, Kunal Sehgal, Reeta Dalal, P. D. Hinduja Hospital & MRC, Mumbai Summary: Conventional methods for diagnosing sepsis are expensive and time consuming. The aim of our study was to evaluate cell population data from Beckman Coulter LH750, for early and accurate detection of sepsis. We concluded that CPD like cell volume, conductivity, scatter and their standard deviations are capable of early prediction of sepsis and can differentiate sepsis from SIRS at no extra cost. A formula can be incorporated into the LIS to generate flags for sepsis and/or SIRS. Introduction: Sepsis is the body’s systemic response to severe infection. Systemic inflammatory response syndrome (SIRS) is associated with infections and noninfectious insults. Early differentiation of sepsis from SIRS is important to prevent severe sepsis and multi organ failure. Materials and Methods: Our study consisted of 3 groups. Group1–43 sepsis patients (blood culture +ve and/or PCT [ 2), Group2–29 SIRS patients and Group3–1528 controls (CBC only). CBC with CPD, blood culture and PCT were performed on group 1 and 2.

ROC analysis for single parameters to-differentiate Sepsis versus Normal and Sepsis versus SIRS AUC

Cut-off

Sensitivity

Specificity

PO 38 Study of Relationship Between Platelet Volume Indices and Glycemic Control Among Patients with Diabetes Mellitus Attending Tertiary Care Hospital: A One Year Cross Sectional Study Dr. Tanima Dwivedi, Dr. D. Reshma, Dr. G. S. Pilli Dept of Pathology, JNMC Belgaum, KLES Dr. Prabhakar Kore Charitable Hospital & MRC Introduction: Diabetes mellitus is a major global health problem. Diabetes mellitus is considered as prothrombotic state with enhanced platelet activity. The platelet activation may alter platelet morphology and function which may result in development of microvascular and macrovascular complications of this metabolic disorder. Platelet Indices i.e. MPV, PDW and PLCR are routinely available in almost all the laboratories but are underutilized. Objective: To study the relationship between platelet volume indices and glycemic control among patients with diabetes mellitus. Materials and Methods: A Cross sectional study was carried out in 210 Diabetic patients. Patients suffering from malignancy, anemia, platelet disorders and those who developed hypertension before diabetes were excluded. 5 ml of venous blood was collected under strict aseptic precautions in 2 ml of EDTA tubes. Sample was tested

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Sepsis versus normals NDW

0.975

[20.91

95.35

90.04

MNV

0.849

[149.25

74.42

88.1

Sepsis versus SIRS NDW

0.786

[25.517

58.14

96.55

MNV

0.734

[154.144

58.14

82.76

Results: Sensitivity and specificity of Mean Neutrophil Volume (MNV) of [149.25 and Neutrophil Distribution Width (NDW) of [20.91 in early diagnosis of sepsis are shown in table. An NDW [ 25.52 and MNV [ 154.14 can readily distinguish sepsis from SIRS as seen in the table Conclusion/Diagnosis/Impression: Multivariate analysis was used which defined a model with 9 parameters—TLC, Neutrophil%, ANC, Eosinophil%, AEC, NDW, Mean Monocyte Volume (MMV), Monocyte Distribution Width (MDW) and Mean Neutrophil Scatter (MNS). Using a cutoff of 0.4109, sepsis can be differentiated from SIRS with sensitivity of 90.48 %, specificity of 75.86 %, PPV of 84.4 % and NPV of 84.6 %.

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Topic: Poster Presentations PO 40 Comparative Analysis of Molecular Genetic and Conventional Cytogenetic Detection of Diagnostically Important Translocations Dr. Deepak Mishra, Dr. Mayur Parihar, Poonam Santra, Sourav Sarma Chowdhury, Dr. Saurabh Bhave, Dr. Anupam Chakrapani, Dr. Arpita Bhattacharya, Dr. Shekhar krishnan, Dr. Reena Nair, Dr. Vaskar Saha, Dr. Mammen Chandy, Dr. Neeraj Arora Department of Molecular Genetics & Cytogenetics, Tata Medical Center, Kolkata Summary: We compare the results of cytogenetics and molecular genetics based detection of chromosomal translocations in leukaemias. Introduction: The contemporary diagnosis of leukaemia relies on a multifaceted approach. Techniques like cytogenetics, RT-PCR and FISH are the commonly used methods in the diagnostic laboratory. The aim of the study was to test the usefulness of these three techniques for detection of common recurrent chromosomal translocations in leukemia so that they can be used in a cost effective manner. Materials and Methods: This is a retrospective study carried out in the Department of Molecular Genetics and Cytogenetics, Tata Medical Center, Kolkata. All newly diagnosed cases of acute leukemia from August 2012 to August 2014 referred for RT-PCR analysis were included. Patients were diagnosed and sub classified according to the WHO 2008 classification. All RT-PCR positive cases were compared to the karyotyping, and/or FISH wherever available. Results: 486 specimens from 239 unique patients were tested by RT-PCR for seven common translocations: t(8;21), t(15;17), inv(16), t(9;22), t(12;21), t(4;11), and t(1;19), with a detection rate of 18.9 % (92/486) in 2 years. Of 92 positive cases, 30 having previous treatment history were excluded. Of remaining 62, FISH analysis was performed on 44 (71 %) and karyotyping in 30 (48.4 %) cases only. Discordance rate of RT-PCR/ FISH and RT-PCR/karyotyping was 4.5 % (2/44) and 6.6 % (2/30). Both these cases were diagnosed as APML. Conclusion: Although there is sporadic discordance between these two techniques and each method has its own limitations, the judicious use of these techniques will deliver the best cost effective treatment in leukaemias. Keywords Leukemia, RT-PCR/FISH comparison, RT-PCR/cytogenetics comparison PO 41 HbTianshui (b39; CAG[CGG, Gln[Arg): First Report of a Rare Hemoglobin Variant from India Satyabrata Meher, Dilip Kumar Patel, Siris Patel, Kishalaya Das, Snehadhini Dehury, Prasanta Purohit, 1Arpita Jana, 1 Subhra Bhattacharya, Pradeep Kumar Mohanty, 1 *Biswanath Sarkar Sickle Cell Clinic & Molecular Biology Laboratory, VSS Medical College & Hospital, Burla, Odisha, 1Anthropological Survey of India, 17 J N Road, Kolkata, West Bengal Summary: HbTianshui is rare Chinese Hb variant mimicking HbDIran in HPLC and alkaline electrophoresis. We report the first case of HbTianshui from South-East-Asia co-inheriting with HbS. The doubleheterozygote of HbS/HbTianshui does not present typical SCD phenotype and is clinically benign. Introduction: This is the first case of HbTianshui reported in an individual from Deogarh district of Odisha, India, with co-inherited HbS. We detail here the investigation of CEHPLC, CBC, biochemical parameters, genealogical study of the family and g-DNA sequencing of this rare variant. Materials and Methods: Sickling test, CBC, alkaline electrophoresis, Biochemical

S495 investigation, CE-HPLC were carried out. HbS mutation was confirmed by ARMS-PCR. g-DNA sequencing of b-globin gene was done with Big Dye terminator protocol using ABI-Prism 3730 and SeqScape software. a-3.7 and a-4.2 kb deletions were detected by gap-PCR. Results: The case revealed prominent band in HbS/HbD zone slower to HbA on electrophoresis. HPLC revealed HbTianshui peak of 47.15 ± 0.21 % at HbA2 window (RT 3.75 ± 0.01) and HbS peak (40.1 % ± 1.56, RT 4.44 ± 0.01). Interestingly HbTianshui had two visible characteristic structures evident in CE-HPLC chromatograph. A peak of 2.0 % ± 0.28 (RT 2.025 ± 0.02) was found with a small hump at the base of HbA2 peak. CBC and Biochemical findings were normal. Deletional athalassaemias were absent. DNA sequencing confirmed HbS mutation at codon-6(GAG[GTG) and a substitution (CAG[CGG) at codon-39 confirming HbTianshui (HBB: c.119A[G, b-39(C5), Gln[Arg). Conclusion: The HbS/HbTianshui presents with normal hematological & biochemical findings, clinically benign and has no visible symptoms of SCD. Possibility of a false characterization of HbTianshui as HbDIran in CE-HPLC and electrophoresis necessitates g-DNA sequencing for confirmation. Keywords HbTianshui, Sickle Cell Disease, HPLC

Topic: Poster Presentations PO 42 Molecular Diagnosis of HPFH and (db)0-Thalassemia Deletions in Indian Population G. Senthil Kumar, Prashant Deshpande, Thiyagaraj Mayuranathan, Eunice S. Edison, Shaji R. Velayudhan Department of Haematology, Christian Medical College, Vellore, India Introduction: Hereditary persistence of foetal haemoglobin (HPFH) and (db)0 thalassemia are heterogeneous disorders caused by large deletions involving d and b globin genes with or without Ag globin gene and they are characterized by elevated Hb F in adult life; *100 % in homozygous and 5–30 % in heterozygous states. Two deletions, Gc(Acdb)O deletion/inversion and HPFH-3, are the commonest mutations in Indian patients with these disorders. A recent study from our group has shown high prevalence of other deletions and we describe PCR based methods for screening five common deletions for cost effective and rapid molecular diagnosis of (db)0 thalassaemia and HPFH in Indian population. Materials and Methods: For the detection of HPFH-3, Asian Indian deletion/inversion Gc(Acdb)O and Hb Lepore the primers described by Craig et al. were used. We designed primers across the breakpoints of 49.3 kb Gc(Acdb)O and 32.6 kb GcAc (db)O deletions. For identification of the heterozygous state, we included primers that amplify a region in non-deleted d globin gene. PCR products were analysed by agarose gel electrophoresis. Results and Discussion: Gap PCR for HPFH-3, deletion/inversion Gc(Acdb)O and Hb Lepore resulted in PCR products of 607, 371, 777 bp, respectively while 49.3 kb Gc(Acdb)O Asian deletion and 32.6 kb GcAc (db)O Indian deletion formed amplicons of 1526 and 307 bp, respectively. In all the heterozygotes normal allele was amplified as 915 bp product from d globin gene while this product was absent in homozygotes. In 51 families screened, the most frequent mutation was del/inv Gc(Acdb)O (49.01 %) followed by HPFH-3 (27.45 %), 49.3 kb Gc(Acdb)O deletion (13.74 %) and 32.6 kb GcAc (db)O deletion (5.88 %) while no mutations were identified in 2 families (3.92 %). Conclusion: Using the gap PCR based approach described here the causative mutations can be identified in *96 % of patients with HPFH and (db)0 thalassemia in Indian population. This method is suitable for high throughput analysis of these mutations in this population.

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S496 Keywords HPFH, Delta–beta thalassemia, Indian population

PO 43 A Rare Dominant Beta Thalassaemia Mutation in Association with HbS: First Report of HbWestdale (cd.126_131:17 bp deletion) from Odisha, India Snehadhini Dehury, 1Biswanath Sarkar, Dilip Kumar Patel, Satyabrata Meher, Prasanta Purohit, 1Arpita Jana, 1 Subhra Bhattacharya, Kishalaya Das, Pradeep Kumar Mohanty, *Siris Patel Sickle Cell Clinic and Molecular Biology Laboratory, V.S.S. Medical College, Burla, Odisha. 1Anthropological Survey of India, 17 J N Road, Kolkata, West Bengal Summary: FS-17bp-del (HbWestdale) in b-Globin-gene produces severe b0-thalassaemia. We carried out clinico-haematological comparison of four cases of HbS/HbWestdale double heterozygote with matched HbS-b-Thalassaemia with IVS-I-5-G[C mutation and HbSS patients from Odisha. The findings agree to earlier inferences on HbWestdale from Trinidad and Pakistan. Introduction: We report here the first series of severe SCD with 17 bp deletion b0-thalassaemia (Hb-Westdale, Cd. 126–131, -17bp-del), hitherto unreported from Odisha. Detailed study of clinico-haematological characterisation of 4 HbS/HbWestdale agreed to earlier reports of b-thalassaemia with this mutation. The finding has implications in PND of SCD in India. Materials and Methods: Screening with haematological, biochemical-investigations and parent-screening revealed large cohort of HbS-b-thalassaemia during 2011–2014 under Odisha Sickle Cell Project (NHM). Mutation confirmation of HbS and b-thalassaemia were done by ARMS-PCR and DNA Sequencing. Clinico-haematological data of matched HbSS and HbS/b-Thalassaemia (IVS-I-5G[C mutation) patients has been done. Results: Four of seven HbWestdale cases had co-inherited HbS. HbA0, MCV, MCH and TPC were significantly lower in HbS/HbWestdale cases compared to both HbS/b-thalassaemia [IVS1-5(G ? C) mutation] and HbSS. HbS/HbWestdale patients had more painful episodes than that in both HbS/b-thalassaemia [IVS1-5(G ? C) mutation] and HbSS patients, where as blood transfusion among them was similar with HbSS patients. Hepato-splenomegaly was common clinical presentation. Higher LDH in HbS/HbWestdale imply the possible increased haemolysis consequent to the instability of the HbWestdale tetramer and corroborates to the earlier reports. Conclusion/Diagnosis/ Impression: This is the first report describing four patients with HbS/ HbWestdale in literature. We found that SCD with HbWestdale present severe clinical manifestation compared to patients with other HbS/b-thalassaemia types. Keywords HbS/b-thalassaemia, HbWestdale, Sickle Cell Disease

PO 44 Gene mutations in IDH1, IDH2, DNMT3A, c-KIT, ASXL1 in adult acute myeloid leukemia (AML) Swapnali Joshi, Shruti Chaudhary, P. Amare, Syed Hasan Khizer, P.G. Subramanian, Hari Menon, Navin Khattry, Bhausaheb Bagal, Manju Sengar, Uma Dangi, Hasmukh Jain, Prashant Tembhare, Sumeet Gujral, *Nikhil Patkar 1

Hematopathology Laboratory, Tata Memorial Centre, 2Adult Hemato-lymphoid Disease Management Group, Department of Medical Oncology, Tata Memorial Centre

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Objectives: There has been a deluge of data which has identified gene mutations prevalent in AML affecting a diverse range of biological processes. Some of these are prognostically relevant and in the future may offer newer avenues for risk stratification of AML-NOS in CR1. We evaluated the baseline frequencies of gene mutations in isocitrate dehydrogenase 1 and 2 (IDH1, IDH2), additional sex comb-like 1 (ASXL1), cKIT-D816V and DNA methyl transferase 3A (DNMT3A) genes in 91 patients of adult acute myeloid leukemia (AML). Methods: A total of 91 patients of AML [other than AML with t(15;17)] were accrued from July 2012 to January 2014 (median age 26, range; 15–55 years, 65.9 % males). FLT3-ITD, NPM1 and CEBPA mutations were detected using fluorescently labelled primers followed by capillary electrophoresis. cKIT D816V: Detected by a two tube PCR format that amplified c-kit exon 17 as a control and an allele specific reaction to selectively amplify the D816V mutant allele. ASXL1,DNMT3A, IDH1 and IDH2 mutations were detected by bidirectional Sanger sequencing using in-house primers. Results: Mutations in FLT3-ITD, NPM1 and CEBPA were seen in 19.8, 18.7 and 18.7 % of patients, respectively. cKIT D816V was mutated in 11 %, IDH was mutated in 13.1 % (IDH1: 4.8 % and IDH2: 8.2 %), DNMT3A mutations in 7.6 % and ASXL1 mutations in 6.2 % of our patients. commonest IDH2 and IDH1 mutations were p.R140Q and p.R132C. Commonest ASXL1 and DNMT3A mutations were G646WfsX12 and mutations at R882, respectively. Conclusion: Amongst intermediate cytogenetic risk patients, 41.5 % patients could not be adequately prognosticated because of lack of conventional genetic biomarkers (FLT3, NPM1, CEBPA). Of these, in 6 patients, additional mutations in IDH1, IDH2, DNMT3A ASXL1 and cKIT could be detected, further justifying the need for incorporation of additional genetic biomarkers in the workup of AML Keywords Adult acute myeloid leukemia, Gene mutations

PO 45 Prevalence of Chromosomes 5, 7 and 8 Aberrations: Correlation with Sole Recurrent Cytogenetic Markers, Monosomal Karyotype and Complex Karyotype in AML 1

Hemani Jain, 1Pratibha Kadam Amare, 1Sharayu Kabre, Pranita Pawar, 1Deepa Sarang, 2Hari Menon, 2Brijesh Arora, 2 Navin Khatri, 3P.G. Subramanian, 3Sumit Gujral, 2 Sripad Banavali 1

1

Cancer Cytogenetics Laboratory, 2Hematopathology Laboratory, Department of Medical Oncology, Tata Memorial Hospital, Parel, Mumbai 400012

3

Introduction: Cytogenetic analysis at diagnosis is one of the most important prognostic factors in AML. Apart from monosomal karyotype (MK), complex karyotype (CK), and patients with recurrent cytogenetic markers, AML with 5 and 7 aberrations has been identified as an independent prognostic factor. Objectives: (1) To seek the prevalence of chromosome 5, 7 and 8 aberrations in newly diagnosed adult and pediatric AML, (2) to find association of 5, 7 and 8 aberrations with cytogenetic risk groups such as MK, CK, RCM (recurrent cytogenetic marker/s) and AKMA (abnormal karyotype with miscellaneous aberrations). Materials and Methods: FISH studies with locus specific target probes, dual fusion translocation probes, breakapart and centromeric probes in 805 newly diagnosed AML patients which included conventional karyotyping in 152 cases. Results: Overall incidence of aberrations of 5, 7 and 8 in 805 cases was 17 %. There was no difference in incidences of

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 adult and pediatric AML. The incidence of -5/del(5q) was 5 %, 7/del(7q) was 8 % and trisomy 8 was 8 %. The incidence of CK group was 11 %, RCM 13 %, MK 18 % and AKMA group was 36 %. Aberrations of 5, 7 and 8 were more prevalent in CK group (24 %). Conclusion: There was no difference in the incidences of chromosome 5, 7, 8 aberrations in adult and pediatric AML. We could identify 4 cytogenetic groups, (a) AML with recurrent cytogenetic markers group, (b) monosomal karyotype, (c) complex karyotype and (d) abnormal karyotype with miscellaneous aberrations group. The occurrence of 5, 7, 8 aberrations was more prevalent in CK group. AML with abnormal karyotype in association with 5, 7 and 8 aberrations needs more attention as a cytogenetic high risk group for better management of disease. Keywords Acute Myeloid Leukemia, FISH, Monosomal karyotype, Complex karyotype

PO 46 Unusual Morphology of Blasts in BCR–ABL Fusion Transcript Positive B-Lineage Acute Lymphoblastic Leukemia: A Study of 5 Cases from Single Tertiary Care Center Man Updesh Singh Sachdeva, Shano Naseem, Reena Das, Neelam Varma, Amita Trehan Summary: Blasts in B-lineage acute lymphoblastic leukemia (BALL) are typically small with scanty cytoplasm, condensed chromatin and usually rounded nucleus. This study retrieved five cases of B-ALL with unusual morphology. The blasts were large with irregular nuclear membrane, 1–3 prominent nucleoli, and moderate amount of basophilic cytoplasm with blebs. Fine granules were noted in some blasts. These cases were eventually diagnosed to be B-ALLs on flow cytometry. Remarkably, all cases showed presence BCR–ABL fusion transcripts. Introduction: Blasts in B-lineage acute lymphoblastic leukemia (B-ALL) are typically small with scanty cytoplasm, condensed chromatin and usually rounded nucleus. We present five cases of acute leukemia with unusual morphology of blasts (resembling monoblasts), eventually diagnosed to be B-ALLs on flow cytometry, with presence BCR–ABL fusion transcripts. Materials and Methods: Five cases of acute leukemia which on routine morphological examination of bone marrow aspirate were thought to be monoblastic in nature, but finally diagnosed as B cell ALL on immunophenotyping, were retrieved for further analysis. Results of RT-PCR for fusion transcripts were also retrieved. The demographic profile, clinical presentation, haematological investigations were analysed. Results: Five cases of B-ALL showing unusual morphology of blasts were retrieved. The mean age of patients was 15 years (range 10–36 years) with male to female ratio of 4:1. The mean haemoglobin was 10 g/dl, mean platelet count 163 9 109/L range (467 9 109 to 10 9 109). Mean leukocyte count was 40 9 109/L range (41 9 109 to 10 9 109). Morphology in all cases showed presence of large blasts with irregular nuclear membrane, 1–3 prominent nucleoli, moderate amount of basophilic cytoplasm with blebs. Fine granules were noted in some blasts. However no Auer rods were seen. Flow cytometry confirmed B-lineage of blasts with all cases. No other lineage specific markers like, MPO, markers of monocytic lineage (CD64, CD14, CD11c) and cytoplasmicCD3 were present. Remarkably, BCR– ABL fusion transcript was present in all five cases. Conclusion/ Diagnosis/Impression: B-ALL cases having BCR–ABL fusion transcript might show unusual morphology of blasts, and hence comprehensive immunophenotyping by flow cytometry is necessary for lineage determination.

S497 PO 47 Prevalence of BCR/ABL Positive Cases in Pediatric Acute Lymphoblastic Leukemia: AIIMS Experience Nivedita Pathak, Akhilesh Mishra, Sameer Bakhshi, Vinod Raina, Lalit Kumar Department of Medical Oncology, Dr BRAIRCH, All India Institute of Medical Sciences, New Delhi 110029 Summary: To investigate the frequency of BCR/ABL fusion transcript in pediatric ALL patients and correlation with clinical outcome. Introduction: Philadelphia chromosome is reported in 25–50 % of adult Acute Lymphoblastic Leukemia (ALL) cases and in about 5 % of childhood ALL cases. Translocation t(9; 22) or BCR/ABL fusion is a common structural aberration found in ALL patients confers a worse prognosis. Materials and Methods: Total RNA extracted from bone marrow/peripheral blood mononuclear cells of ALL patients, was reverse transcribed to cDNA. Nested PCR was carried out to detect BCR/ABL fusion transcript. Results: A total of 38 pediatric patients (median age 6 years, range 1–9 years; M: F 3:1; median TLC-13 9 103/mm3, range 0.8–810 9 103/mm3) were studied. BCR/ ABL fusion transcript was observed in 5/38(13 %) of our patients. In 4/5 patients the fusion transcript was e1a2 while in one patient the transcript was b2a2. This frequency of BCR/ABL transcript was found to be quite higher in pediatric patients as compare to that of west. Conclusion/Diagnosis/Impression: Prevalence of poor prognostic aberrations might be contributing to the relatively poorer outcome in our ALL patients.

Topic: Poster Presentations PO 48 Fms Like Tyrosine Kinase (FLT3) and Nucleophosmin 1 (NPM1) Mutations in Acute Myeloid Leukemia (AML): A Descriptive Analysis 1

M. Sathya, 1Ajay Abraham, 1Savitha Varatharajan, Sreeja Karathedath, 1Nancy Arthur, 1J. Ashok Kumar, 2 Vivi M. Srivastava, 1Aby Abraham, 1Auro Viswabandy, 1 Biju George, 1Alok Srivastava, 1Poonkuzhali Balasubramanian, 1 Vikram Mathews 1

1

Department of Haematology & 2Cytogenetics Unit, Christian Medical College, Vellore

AML patients with normal karyotype (NK) form the most heterogeneous group both clinically and biologically. Advances in molecular genetic technologies resulted in identifying several molecular markers to further prognosticate this subgroup. Of these markers, mutations in nucleo-phosmin (NPM1) and FMS-like tyrosine kinase 3 (FLT3) are the most common good and poor prognostic markers respectively, in NK-AML. The frequency of exon 12 NPM1 mutations (NPM1c) in adult AML ranges between 25 and 35 %, and accounts for more than 50 % in adult NK-AML. Two major classes of activating FLT3 mutations have been identified in patients with AML, which include the internal tandem duplications (ITD) and tyrosine kinase domain (TKD) point mutations. FLT3-ITD occurs in approximately 30 % of cases of NK whereas FLT3-TKD mutations are relatively rare (10 %). The present study aims to describe the spectrum of NPM1 and FLT3 mutations in a large cohort of AML patients diagnosed at the Department of Haematology, Christian Medical College-Vellore. AML patients (n = 410) diagnosed between June 2009 and August 2014 were included in this study (patient demographics: Table 1).

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

FLT3-ITD and NPM1 mutation detection were done using genomic DNA sample at diagnosis by PCR followed by GeneScan analysis. FLT3-TKD (D835) mutation were detected by PCR and restriction fragment length polymorphism (RFLP) analysis using EcoRV restriction enzyme and size fractionated through 3 % agarose gel. The type of NPM1 mutation was determined by sequencing. The frequency of NPM1 and FLT3 mutations in the total cohort as well as in NK-AML (Table 1) is similar to previous reports. NPM1 mutations were also identified in AML patients (n = 18/208; 8.6 %) with structural cytogenetic abnormalities. As reported earlier, NPM1 mutations were significantly associated with lower bone marrow blast percentages and low CD34 expression (15.5 vs 82.4 %; p \ 0.0001). The most common type of NPM1 mutation was Type-A (78.7 %) followed by Type-B; six novel NPM1 mutations were also identified in this cohort and are listed in Table 2. 63 % patients were positive for both NPM1 and FLT3 mutations. Of the 71 patients with FLT3ITD, 11 had multiple ITDs ranging from (16–208 bp) and were found associated with high CD34 [78.5 % (1–91)] expression and increased blast percentage [69 % (28–100)]. Our analysis has identified enormous heterogeneity within NPM1 and FLT3 mutations. The data highlights the importance of risk stratification by combining both NPM1 and FLT3 mutations since a good majority of FLT3-ITD cases are shown coexisted with NPM1 mutations. The study emphasizes the importance of sequencing in addition to GeneScan for identifying different NPM1 subtypes and warrants further studies for validating its prognostic relevance. Table 2 Types of NPM1 mutations

Background: Substitution of T to C at nucleotide position 16189 in the hypervariable D-loop of the control region (CR) of mtDNA has attracted research interest because of its suspected association with various multifactorial diseases. The mtDNA4977(4977 bp deletion) accounts for 30–50 % of all deletions and has been commonly used as an indicator of somatic mitochondrial injury. Objectives: To study the association of mtDNA T16189C polymorphism in patients of CKD with CAD and association of mtDNA4977 in patients of CKD. Methods: The study comprised of 50 patients of CKD and 50 healthy controls. The mtDNA T16189C polymorphism was assessed by direct PCR–RFLP and mtDNA4977 was assessed by nested PCR followed by RFLP analysis. Results: The T16189C polymorphism was present in 12 % (6/50) of CKD patients absent in healthy controls. It was present in 10.3 % (3/29) of CKD without CAD patients and 14.3 % (3/21) of CKD patients with CAD. The mtDNA4977 was present in 11 out of 50 cases (22 %) but it was absent in all the healthy controls. The mtDNA4977 was significantly associated with older age and diabetes. The prevalence of mtDNA4977 in the age groups of 31–40, 41–50 and 51–60 years was 0, 27.2 and 72.7 %, respectively. Conclusions: There was significant association of T16189C polymorphism and mtDNA4977 with CKD The association of mtDNA T16189C polymorphism in patients of CKD with and without CAD may indicate that patients of CKD with mtDNA T16189C polymorphism are at high risk of developing CAD. The mtDNA4977 was associated with older age and diabetes in patients with CKD. Keywords Mitochondrial DNA, T16189C polymorphism, mtDNA49 77

Table 1 . Patient characteristics (n = 410) Gender

Male = 248; female = 162

Age

40 years (1–78 years)

Cytogenetics risk group (*available in n = 386) Favorable

52 (13.5 %)

Topic: Hematology PO 50 Inversion(14)(q11.2q32) in T Cell Acute Lymphoblastic Leukemia

Intermediate

225 (58.3 %)

3

Adverse

109 (28.2 %)

1

NPM1 mutation

108/410 (26.3 %)

FLT3-ITD FLT3 (multiple ITD)

71/410 (17.3 %) 11/71 (15.5 %)

Both FLT3-ITD and NPM1

45/71 (63 %)

FLT3-ITD and FLT3TKD P05

2/71 (2.8 %)

FLT3-TKD (DS35)

19/410 (4.6 %)

Normal karyotype

171 /386 (44.3 %)

NPM1 mutation

88 [51.5 %)

FLT3-ITD

48 [28.1 %)

FLT3-TKD

9 (0.56 %)

Topic: Hematology PO 49 Detection of mitochondrial DNA T16189C polymorphism and 4977 bp deletion in patients of chronic kidney disease with or without coronary artery disease Vaibhav Girotra, Satendra Sharma, Om Prakash Kalra, Sonal Sharma University College of Medical Sciences and GTB Hospital, Dilshad Garden, Delhi 110095

123

M. Angi, 2U. Sitaram, 1N. A. Fouzia, 1A. Ganapule, A. Abraham, 1A. Viswabandya, 1B. George, 1V. Mathews, 1 A. Srivastava, 3V. M. Srivastava 1

Departments of Haematology, 2Transfusion Medicine and Immunohaematology and 3Cytogenetics Unit, Christian Medical College, Vellore Introduction: The inversion (14)(q11.2q32) is a distinct T-cell neoplasia-associated abnormality which is usually seen in T-prolymphocytic leukemia and ataxia telangiectasia-associated T cell leukemia. It has also been reported in about 25 patients with T-ALL and less commonly, in B-ALL and AML expressing lymphoid-associated antigens. The inversion 14 is associated with activation of the TCL1 (T cell leukemia 1) gene on 14q32 due to its juxtaposition to the TRA/D (T cell receptor alpha/delta) gene on 14q11.2. Its prognostic impact in T-ALL is not well defined. We describe the cytogenetic and clinicopathological features of four patients with T-ALL and inversion 14. Patients and Methods: Karyotypes of all patients with T-ALL and the inversion 14 seen in the Department of Haematology, Christian Medical College, Vellore, between January 2003 and February 2014 were correlated with blood and bone marrow findings. For each patient, at least 20 G-banded metaphases were analysed from unstimulated overnight cultures of bone marrow. Results: The inversion 14 accounted for 0.3 % (4/ 1,450) of all ALL and 1.4 % (4/290) of T-ALL seen during the period of study. The patients were 5, 9, 20 and 29 years of age. Three were males. The haemoglobin was 7.1–15.1 g/dl, WBC count: 45–337.2 9 109/L, platelet count: 23–93 9 109/L, blast percentage: 60–95 %. The blasts were PAS positive in only one patient but immunophenotype was consistent with T-ALL in all four. Three

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 patients had a complex karyotype with two or more additional abnormalities (deletion 12 p in two, tetraploidy in one). Two patients were in remission at 5 and 8 years of follow up. The third patient who presented in 2014 is well at 7 months of follow up. The fourth patient who had a complex karyotype relapsed within 1 year of treatment. Conclusion: The inversion (14)(q11.2q32) was found in 0.3 % of all ALL and 1.4 % of T-ALL. It is important to be aware of this rare abnormality because it can be easily overlooked, especially if the morphology is suboptimal. A larger number of cases need to be studied to understand its impact on prognosis in ALL. Keywords Inversion 14, T-ALL

Topic: Laboratory Haematology/ALL PO 51 Gene Mutations in FLT3-ITD, NPM1, CEBPA Genes in Adult Acute Myeloid Leukemia Ridhima Bane, Swapnali Joshi, Shruti Chaudhary, Heena Doshi, Devanand Vishwakarma, Syed Hasan Khizer, P.G. Subramanian, Prashant Tembhare, Sumeet Gujral, *Nikhil Patkar Hematopathology Laboratory, Tata Memorial Centre Objectives: Mutations in FLT3 (esp FLT3-internal tandem duplications; FLT3-ITD), NPM1 and CEBPA genes have profound prognostic significance in AML. We evaluated the baseline frequencies of the mutations in FMS-like tyrosine kinase 3 (FLT3), nucleophosmin1 (NPM1), and CCAAT/enhancer-binding protein alpha (CEBPA) genes in patients with adult acute myeloid leukemia (AML) and found that 46.8 % harbor gene mutations. Methods: A total of 107 patients of AML [other than AML with t(15;17)] were accrued from July 2012 to January 2014 (age range 18–55 years, 66.3 % males). Genomic DNA was extracted and subjected to multiplex PCR that amplified FLT3, NPM1, TAD1, TAD2 and b-ZIP domains of the CEBPA gene using fluorescently labeled primers followed by capillary electrophoresis and analyzed for any duplication, insertion and/or deletion. Results: FAB subtypes: AML-M0 (6.5 %), AML-M1 (20.5 %), AML-M2 (43.9 %), AML-M4 (7.4 %) and AML-M5 (12.1 %) and t(8;21), t(11q23), inv16 were seen in 18.69, 4.60 and 1.80 % patients respectively. [FLT3+/NPM1-/CEBPA-(7.4 %), FLT3+/NPM1+/CEBPA-(8.4 %), FLT3+/NPM1-/CEBPA+(1.8 %)], NPM1 (FLT3-/ NPM1+/CEBPA-(11.2 %) and CEBPA mutations (FLIT3-/NPM1-/ CEBPA+) accounted for 15.8 %. Biallelic mutations (20 % of all CEBPA mutations) occurred in the TAD1 and ZIP domain. No mutation was detected in 53.2 % of patients. Conclusion: The frequencies of these mutations are comparable with published data. As majority of these mutations are of in-del type, fragment length analysis can be used to detect these mutations. Keywords Adult acute myeloid leukemia, Gene mutations, India

S499 NRAS is a member of the RAS family of GTPases and plays a central role in the MAPK signaling pathway. Activating mutations in codons 12, 13, and 61 have been found in various cancers, including myeloid leukemias leading to constitutive activation of various signaling pathways involved in proliferation and cell survival.Previous studies have not reported any prognostic impact of Ras mutations in AML, but the presence of Ras mutations appears to sensitize AML blasts to high dose cytarabine in vivo (Motyckova and Stone 2010) and these patients respond well to high dose cytarabine (HiDAC) post remission. N-Ras mutations have been found to co-occur with other molecular markers such as NPM1, FLT3-ITD mutations or with inversion 16. The aim of the present study was to investigate the presence of N-Ras mutations in adult AML patients. 328 adult AML patients (excluding AML-M3) diagnosed between June 2009 and February 2014 were included in the present study (Patient demographics: Table 1). N-Ras exon1 and exon2 mutation detection was done using genomic DNA by PCR followed by sequencing. Other molecular markers like NPM1, FLT3-ITD, and FLT3-TKD were screened by DNA-PCR. Presence of AML1-ETO [t(8;21)] and CBFB-MYH11 [inversion 16, t(16;16)] were screened by reverse transcriptase PCR (RT-PCR). Twenty nine patients (8.8 %) harbored N-Ras mutation, predominantly in codon 12 (n = 16), codon 13 (n = 6) followed by codon 61 (n = 7). Change in amino acid residues caused by these mutations and numbers of patients with the mutations are enlisted in Table 2. Eleven (7.8 %) out of 141 normal karyotype AML had N-Ras mutations. Eleven and 6 of the 29 patients with N-Ras mutations had NPM1 mutation and core binding factor translocations, respectively. Prevalence of N-Ras mutations in our cohort of patients is marginally lesser in comparison to previous reports (Ritter, Kim, et al. 2004; Illmer, Thiede, et.al. 2005). N-Ras mutations were found to coexist with other molecular markers and translocations such as NPM1 and core binding factors. Further analysis is ongoing in the lab to evaluate the effect of these mutations on treatment outcome and response to HiDAC. Keywords AML, N-RAS, Molecular markers Table 1 Patient Demographics Patient demographics De novo AML (n =328) Age

41 years (15–75 years)

Sex

males: 198; females: 130

Cytogenetics Risk Group (available in n = 301) Favorable

178 (59.1%)

Adverse

81 (26.9%)

Molecular Markers NPM1 mutation FLT3-ITD

PO 52 N-Ras Mutations in Acute Myeloid Leukemia: A Descriptive Analysis 1

Vinodhini Kumaraswamy, 1Savitha Varatharajan, A. Senthamizhselvi, 1Sreeja Karathedath, 1Ajay Abraham, 1 Nancy Arthur, 1J. Ashok Kumar, 2Vivi M. Srivastava, 1 Aby Abraham, 1Auro Viswabandya, 1Biju George, 1 Alok Srivastava, 1Poonkuzhali Balasubramanian, 1Vikram Mathews 1

Department of Haematology & Cytogenetics Unit, 2Christian Medical College, Vellore

42 (14%)

Intermediate

95 (30.74%) 57 (18.44%)

NPM1+/FLT3-ITD+

36 (11.65%)

FLT3-TKD

13 (4.2%)

N-Ras mutations

29 (8.8%)

Other mutations and translocations with N-Ras NPM1

11

FLT3-ITD

1

FLT3-TKD

2

AML1-ETO [t(8;21)]

3

CBFB-MYHll[t(16;16), invl6]

3

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Table 2 amino acid change and distribution of mutations in codon 12,13and 61 of N-Ras Codon

Amino acid change

12

Glycineto Aspartic acid

13

61

No of patients 12

Glycine to Arginine

2

Glycineto Alanine

1

Glycineto Valine

1

Glycineto Valine

3

Glycineto Aspartic acid

3

Glycineto Alanine Glutamineto Lysine

1 4

Glutamineto Arginine

2

Glutamineto Methionine

1

PO 53 Prenatal Diagnosis of Hemophilia A in Twin Pregnancies G. Sankari Devi, E. Sumitha, Eunice S. Edison, A. Viswabandya, A. Abraham, B. George, V. Mathews, A. Srivastava Department of Haematology, Christian Medical College, Vellore 632004, TamilNadu, India Introduction: Hemophilia A is an X-linked bleeding disorder caused by mutations in F8 gene. Prenatal diagnosis in carriers with twin pregnancies is more challenging than in women with singleton pregnancies. We report here prenatal diagnosis performed to three couples (families A, B, and C) with twin pregnancies who were at risk for hemophilia A. Methods: Identification of mutations in three index patients was confirmed at molecular level. Chorionic villus samples (CVS) of twins were obtained at 10–12 weeks of gestation. PCR based screening for intron1 and 22 inversions, was followed by and point mutation screening by multiplex PCR and conformation sensitive gel electrophoresis (MPCR–CSGE) strategy. Identification of each twin, followed by elimination of maternal contamination in CVS samples were ruled out by variable number of tandem repeats (VNTR) analysis. Results: We found that in family A both fetuses were affected with Hemophillia A due to a novel frameshift p.Lys713fs*4; in family B one of the two fetuses was positive for intron 22 inversion and the other foetus was normal; in family C among the two fetuses one was carrier for intron 22 inversion while the other was normal. Conclusion: Prenatal diagnosis is the major way of prevention of the genetic disorders including hemophilia A. Collection of CVS samples and precise genetic identification of affected fetus in twins can be challenging. VNTR analysis is a useful tool for confirmation of identity and detection of maternal contamination of CVS. Keywords Hemophilia A, Prenatal diagnosis, Mutations, Twin pregnancy

PO 54 Rare and Aberrant Mixed Lineage Leukemia (MLL) Gene Rearrangements 1

Umang Patel, 1Anil Yadav, 1S.R. Arun, 1Akshay Gore, Neeraj Arora, 2Deepak Mishra, 3Saurabh Bhave,

2

123

3

Anupam Chakrapani, 3Reena Nair, 4Arpita Bhattacharya, Shekhar Krishnan, 4Vaskar Saha, 3Mammen Chandy, 1 Mayur Parihar 4

1

Dept. of Cytogenetics, Tata Medical Center, Kolkata. 2Dept. of Lab Hematology, Tata Medical Center, Kolkata. 3Dept. of Clinical Hematology, Tata Medical Center, Kolkata. 4Dept. of Paediatric Oncology, Tata Medical Center, Kolkata Summary: We describe the spectrum of MLL gene rearrangements (MLLR) in haematological malignancies highlighting rare variants like t(2;11), concomitant deletion in the MLL gene, complex MLLR and intrachromosomal amplification (iAMP) of the MLL gene. Introduction: The MLL gene (11q23) is known to participate in almost 90 different rearrangements with at least 51 different partner genes. The MLLR are best identified using a dual colour breakapart FISH approach, although the common translocations can be identified by karyotyping. Materials and Methods: G banded karyotypes and FISH analysis results of patients with MLLR between July 2011 and August 2014 were correlated with blood, and bone marrow findings. Results: There were 23 patients with MLLR with an incidence of 6.4 % (16/247) in AML and 1.8 % (6/332) in ALL. There was one patient with acute leukemia of ambiguous lineage. The commonest partner chromosomes were 9 and 19 in AML and ALL, respectively. AML M5 (9 cases) was the commonest FAB subtype followed by AML M6 (3 cases). Aberrant and uncommon rearrangements included t(2;11), translocation of the whole gene on partner chromosome followed by rearrangement, translocation and subsequent deletion of 3 prime end of the MLL gene and iAMP of MLL gene (3cases). The iAMP was seen on a derivative chromosome in one patient and as a ring chromosome in another. Conclusion: We report a lower frequency of t(4;11) in ALL. In AML the pattern is comparable to the published literature. A combination of karyotyping and FISH using dual colour breakapart probe identifies rare and aberrant rearrangements. Keywords MLL, FISH, Karyotyping, Acute Leukemia

Topic: Haematopathology PO 55 Flowcytometric Evaluation of Leucocyte Activation Marker Cd 11b in Adult Sepsis Major (Dr.) Barun Kumar Chakrabarty, Surg Capt (Dr.) V. Manu, Col (Dr.) Mahadevan Kumar, WO K.B. Santhosh Department of Pathology, Military Hospital, Namkum Summary: The present study supports leucocyte activation marker CD 11b study by flowcytometry is a fairly reliable tool to assess it as diagnostic indicator in the sepsis patients. Introduction: Sepsis is a major global healthcare burden affecting millions of individuals. Leucocytes play a key role in inflammatory response and coagulation dysfunction seen in sepsis. However scanty information is available to understand the complex pathophysiology associated with leucocyte activation in adult sepsis patients. Whole blood flowcytometry is a powerful new technique which allows rapid, specific and detailed analysis of the evaluation of WBC activation parameters in sepsis and its related complications. Materials and Methods: In the present study we used flowcytometry to examine well known leucocyte activation parameter CD 11b in adult sepsis patients. To achieve this

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 objective 40 diagnosed sepsis cases with 40 healthy controls were studied. Results:

S501

Topic: Poster PO 57

CD 11b study descriptive

Role of Multicolor Flow Cytometry in Diagnosis of Autoimmune Lymphoproliferative Syndrome (ALPS)

CD 11b expression variables Variables

CD 11b%

Sepsis patients (N = 40)

Healthy controls (N = 40)

Mean

Mean

81.8

SD 14.4

69

p value Significance

Department of Haematology, Christian Medical College, Vellore

SD 11.46 0.000

S

CD 11b MeFI 567.48 305.5 433.17 151.06 0.160

n.s

CD 11b MFI

n.s

595.68 337.7 455.52 194.38 0.260

Faranaz Khamruddin, Prashant Deshpande, Ansu Abu Alex, Harikrishnan Babu, Aby Abraham, Auro Viswabandya, Biju George, Alok Srivastava, Vikram Mathews

CD 11b% CD 11b percentage positive leucocytes; MeFI mean florescence intensity; MFI median fluorescence intensity; AI adhesion index; S significant; n.s. Not significant Conclusion/Diagnosis/Impression: In our present study we observed significantly higher CD11b expression in percentage positive leucocytes in sepsis patients when compared with healthy individuals. The CD 11b MeFI and MFI were also higher than healthy individuals. Keywords Adult sepsis, Leucocyte activation marker, CD 11b, Flowcytometry

Topic: Morning session PO 56 Absolute Lymphocyte Count: A Surrogate Marker for CD4 Cell Count in HIV-Infected Individuals Dr. C. Sujitha, Dr. Smitha, Dr. K. Durga Department of Pathology, AP Govt. TB and Chest Hospital, Osmania Medical College, Hyderabad Introduction: Depletion of CD4 cell count is a hallmark of disease progression in AIDS. CD4 cell count is essential for the physicians to decide about the timing of anti retroviral therapy (ART) and for prophylaxis of opportunistic infections. WHO has recommended that, absolute lymphocyte count of B1,200/microlitre can substitute CD4 cell count of B200/microlitre in resource-constrained countries throughout the world. Aim: The aim of this study was to correlate the absolute lymphocyte count and CD4 cell count and to calculate correlation coefficient and P-value as a marker of disease progression in HIV-infected individuals attending AP Govt. TB and Chest Hospital. Methodology: This study was undertaken at AP Govt. TB and Chest Hospital for a period of 1 year from July 2013 to June 2014. A total of 5,256 samples were analyzed for absolute lymphocyte count and CD4 cell count in HIV-infected individuals. In 720 cases where the CD4 cell counts B200/microlitre, absolute lymphocyte count was found to be B1,327/microlitre. Results: Details of the study and statistical analysis results will be presented at the time of conference. Keywords CD4 cell count, ALC, Surrogate marker, HIV-positive cases

Introduction: Autoimmune lymphoproliferative syndrome (ALPS) is characterized by abnormal lymphocyte homeostasis due to defect in the apoptosis pathway, which leads to chronic lymphoproliferation, autoimmune cytopenia, and increased risk of development of malignancies. Diagnosis of ALPS requires demonstration of chronic non-malignant lymphoproliferation like lymphadenopathy and/or splenomegaly and increased double-negative (CD4-/CD8-) ab T cells (DNTs) along with other accessory criteria. Here we are describing our experience of multicolor flow cytometry in diagnosis of ALPS. Materials and Methods: In this retrospective study, we identified 13 patients over a period of 4 years, diagnosed as ‘probable’ ALPS as per revised International ALPS workshop (2010) criteria. Relevant clinical and laboratory parameters were reviewed. Identification and quantification of TCR-ab T cell subsets (CD3/CD4/CD8) in peripheral blood was performed using multicolor flow cytometry (MFC). Results: The median age of presentation was 7 years (range 4.5 months to 17 years) with 4/13 (30.77 %) patients presented during infancy. Fever with lymphadenopathy and/or splenomegaly was the most common presentation, being seen in 10/13 (76.92 %) patients. Cytopenias at diagnosis, involving one or more hematological lineages, were present in 11/13 (84.62 %) patients. One patient had co-existent pure red cell aplasia. On flow cytometry, TCR-ab DNT cells varied from 1.9 to 61 % of all lymphocytes and 2.3–64.6 % of all CD3+ T lymphocytes. TCRab CD8+ T cells were also increased in all patients with CD4:CD8 reversal in 9/13 (69.23 %) patients. Discussion: Increased TCR-ab DNT cells is hallmark of ALPS and along with typical clinical presentation and relevant laboratory investigations, a ‘probable diagnosis’ of ALPS can be made, even when data regarding other accessory criteria (defective lymphocyte apoptosis and pathogenic mutation in FAS, FASLG, or CASP10) is not available. Multicolor FC is a sensitive and relatively simple technique for precise identification and quantification of DNT cells in peripheral blood. However, DNT% can vary in different patients and interpretation must be done in context with clinical and other laboratory parameters. Conclusion: Multicolor flow cytometric estimation of DNT cells is a sensitive tool for screening and when used in proper context, is sufficient to provide a probable diagnosis of ALPS. Keywords Autoimmune lymphoproliferative syndrome, Flow cytometry

Topic: Poster PO 58 Flow cytometric diagnosis of Leukocyte Adhesion Deficiency-I Kotteeswari Kathirvel, Prashant Deshpande, Ansu Abu Alex, Harikrishnan Babu, Aby Abraham, Auro Viswabandya, Biju George, Alok Srivastava, Vikram Mathews Department of Haematology, Christian Medical College, Vellore Introduction: LAD-I is a rare disorder characterized by defective leukocyte adhesion and chemotaxis, due to deficient common subunit

123

S502 of b2-integrin CD18. As a result, normal heterodimers of three a (CD11a, CD11b, and CD11c) and b subunits cannot form leading to defective neutrophil function. Flow cytometric analysis for expression of these proteins is an effective and relatively simple method for diagnosis of LAD-I. The aim of this study was to describe the use of a simple panel of antibodies for the diagnosis of LAD-I in our institute. Methods and Materials: We retrospectively analyzed 27 LAD-1 patients, from 2005 to 2014. Multicolour flow cytometry was done on peripheral blood to get the percentages of CD11a, CD11b, CD11c and CD18 on the granulocyte population in patients and normal controls. Results: In all patients, expression of CD18, CD11a and CD11c was seen in \20 % cells with mean positive population of 1.42, 1.49 and 0.78 %, respectively as compared to more than 90 % in normal controls. Mean CD11b expression was 9.5 % with high expression in two patients ([50 %). Among 27 patients, 24 were classified as severe LAD-I (CD18 expression \2 %) and 3 were moderate type (CD18 expression [2 %). 25 out of 27 patients (92.6 %) presented during infancy (median: 5 months) with two patients presenting at 5 and 16 years. All patients presented with recurrent bacterial infections and 80 % had history of omphalitis. All patients had leukocytosis (median 63,800/mm3; range 15,900–138,200/mm3) and neutrophilia (median ANC = 52,316/mm3; range 7,476–120,234/mm3). 66.6 % of the patients were born out of consanguineous marriage while a family history suggestive of LAD-I was present in half of patients (n = 14). Conclusion: This is a descriptive experience of a large cohort of patients with LAD-I from a single center. Multicolor flow cytometry using a limited antibody panel and normal controls is an effective tool to diagnose and classify LAD-I. Keywords LAD-I, Flow cytometry

Topic: Lab Hematology PO 59 Monoclonal B Lymphocytosis (MBL) Associated with T Cell Lymphoproliferative Disorder: Calls for Revision of Diagnostic Criteria of MBL Supriya Jain, Kiran Ghodke, Asma Bibi, Shilpa Kusthe, Ashok Kumar, Nikhil Patkar, Prashant Tembhare, Sumeet Gujral, P. G. Subramanian Hematopathology Laboratory, Department of Pathology, Tata Memorial Hospital, Parel, Mumbai 400012 Introduction: Monoclonal B-cell lymphocytosis (MBL) is defined as B-cell lymphocytosis with \5 9 109 clonal B cells/L and absence of signs/symptoms of other lymphoproliferative neoplasms. MBL is considered as a potential precursor of CLL and its co-existence with a T cell neoplasm has not been reported. We present a case of T cell lymphoproliferative disorder with CLL-type MBL. Materials and Methods: 57-year-old patient presented with dry cough for 6 months, weight loss, pruritic skin lesions, hepatomegaly, mediastinal and peripancreatic lymphadenopathy. CBC and peripheral-smear examination followed by immunophenotyping using six-color antibody panel on BD Canto-II was performed. Results: CBC analysis revealed total WBC count of 57.4 9 109 cells/L with absolute lymphocyte count 46.1 9 109 cells/L, Hb 133 g/L and platelets 255 9 109 cells/L. Peripheral smear revealed 78 % atypical lymphocytes. Immunophenotyping demonstrated abnormal T cells (60 % of viable cells) expressing bright CD5, moderate sCD3, heterogenous CD7 and bright CD4 without expression of CD8 or CD56. An additional population of abnormal B cells with CLL like immunophenotype (149 cells/llitre) expressing dim CD19, CD20 with coexpression of CD5 along with CD23,CD43, CD200 and lambda light chain restriction was detected.

123

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 These findings were consistent with the diagnosis of T cell CLPD along with co-existance of MBL. Conclusion: Criteria for MBL include the absence of lymphoadenopathy and organomegaly. Our case showed that MBL can be seen in association with other lymphoproliferative disorder as well as lymphoadenopathy and organomegaly. Hence, there is a need for revision of current diagnostic criteria of MBL. Keywords CLL-type MBL, Monoclonal B-cell lymphocytosis (MBL), T cell lymphoprol

PO 60 Characterization of Leukemic Stem Cells and Associated Cell Signaling Pathways Can Predict the Outcome in Acute Leukemia 1

Swati Garg, 2S. Chandrakala, 3Supreet Marathe, Prashant Mishra, 4Bhibhas Dasgupta, 1Kanjaksha Ghosh, 1 Manisha Madkaikar 3

1

Dept. of Pediatric Immunology and Leukocyte Biology, National Institute of Immunohaematology, Mumbai, Maharashtra, India, 2Dr. J.C. Patel Dept of Hematology, KEM Hospital, Mumbai, 3Cardio Vascular and Thoracic Center, KEM Hospital, Mumbai, 4Department of Prthopedics, KEM Hospital, Mumbai Background: Acute Myeloid Leukemia is a group of heterogeneous disorders which rise from the failure of normal hematopoiesis. Even after advancements in management strategy most patients succumb to either aggressiveness of the disease or relapse. The later in turn is suggested to be caused by a therapy resistant population of malignant cells; termed as ‘‘Leukemic stem cells’’ (LSC). In the present study, we evaluated expression of cellular antigens and signaling pathways in these cells and correlated with response to treatment in the pursuit of a prognostic clue. Materials and Methods: Bone marrow samples were collected after informed consent from healthy individuals undergoing orthopedic/cardiac surgery (n29) and from patients (n28) at diagnosis. We examined expression of 13 cellular markers, 4 signaling molecules, 8 mutations, important cytogenetics and findings were then correlated with disease outcome. Results: Markers were divided in 4 groups: (1) LSC specific antigens (CD44,CD90,CD96,WT-1), (2) Epithelial related antigens (CD18,CD24,CD32), (3) Cytokine receptors(CD25,CD123,CD116, CD135), (4) Kinase units (Hck,AK-5). Out of these; CD44,CD96,CD25 and CD123 were significantly over expressed while CD90 was down regulated on LSCs. Higher frequency of LSCs was associated with overall poor outcome and was more in AML-M0. Based on survival and treatment outcome it was observed that CD90, WT-1, CD25, CD32 and AK-5 differentially over expressed on LSCs of patients with poor prognosis and had a constitutive expression of pAkt, pErk and pStat-5. Conclusion: An initial LSC screening panel consisting of these markers at the time of diagnosis and knowledge of constitutively stimulated cell signaling pathways can act as a helpful predictive tool in acute leukemia Keywords AML, Luekemic stem cells, Signal Transduction, Prognosis

Topic: Dr. JC Patel Award PO 61 Immunophetyping in Acute Leukemias in Children Dr. Tanuja Bundela, Dr. Shailaja Shukla, Dr. Sunita Sharma, Dr. Jagdish Chandra Depatment of Pathology and Paediatrics, Lady Hardinge medical College, New Delhi

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Summary: Immunophenotyping is fundamental for classifying acute leukemias, treatment selection, optimization and monitoring response to therapy and also identifying markers with prognostic implication. Introduction: Immunophenotyping differentiates ALL from AML and also classifies ALL into subgroups with prognostic implication. In childhood ALL, patients with Common-ALL and Pre-B-ALL do best; those with ProB-ALL and T-ALL do less well and those with rare mature B-ALL tend to do very poorly. Materials and Methods: Eighty cases of Acute Leukemia in children up to 18 years were included in study. Complete blood count along with peripheral blood and bone marrow aspirate smear examination was performed in all cases. Relevant cytochemistry was also performed. Immunophenotypic analysis was performed by Beckman Coulter Flow cytometer using monoclonal antibodies against various myeloid and lymphoid cell surface/cytoplasmic antigens. Results: 68/80 cases (85 %) were of ALL including 53 cases of B-ALL, 13of T-ALL and 2 of Biphentypic acute Leukemia. Twelve cases were of AML including 7 M1, 2 M2 and 3 M4. All cases of B-ALL expressed CD79a and cCD3 was seen in all T-ALL cases. CD79a and CD19 were 100 % sensitive, and sIg, CD22, CD19 showed 96 % specificity for B lineage. cCD3 and CD7 were 100 % sensitive and CD5, CD4 and CD8 showed 100 % specificity for T Lineage. MPO and CD33 were 100 % sensitive while MPO, CD14, CD15 showed 100 % specificity for myeloid lineage. Conclusion: Immunophenotyping plays an important role in sub-classification of acute leukemia and for further risk stratification. Keywords Immunophenotyping, Acute leukemia

Topic: Morning 7th Nov PO 62 Diagnostic Utilitity of CD117 in Immunophenotyping of Acute Leukemias Vandana Puri, Neha Niihariika, Meera Sikka Department of Pathology, University College of Medical Sciences, Delhi Introduction: CD117 (c-kit) is a transmembrane tyrosine kinase receptor that is encoded by the c-kit proto-oncogene. It has been proposed that CD117 or c-kit expression should be routinely evaluated in cases of acute leukemia because of its higher myeloid lineage specificity as compared with CD13 or CD33. However CD117 is not as sensitive as CD13 and CD33 for identifying acute myeloid leukemias (AMLs), and recent studies have suggested that adding anti-CD117 to a flow cytometry panel that contains anti-CD13 and anti-CD33 is not essential for the assignment of blast lineage. Materials and Methods: We assessed the diagnostic usefulness of adding anti-CD117 to our existing flow cytometric profile in the analysis of 50 consecutive cases of acute leukemia (de novo or relapsed AML, blast crisis of chronic myelogenous leukemia [CML], acute lymphoblastic leukemia and biphenotypic leukemia) over a period of 6 months from 01/03/2014 to 01/09/14 at our institute. Results: Out of total 50 cases of acute leukemia, CD117 was positive in 27/50 cases. It was a consistent negative marker in B-ALL and showed 88.88 % positivity in acute myeloid leukemia. Although T cell leukemia number was less in our study, however CD117 showed positivity in 25 % of these cases. Conclusions: Our findings demonstrate that c-kit is a reliable and specific marker to detect leukemia cells committed to the myeloid lineage, and therefore should be included on a routine basis for the diagnosis of acute leukemias to demonstrate myeloid commitment of the blasts. Findings in T-ALL and acute unclassifiable leukemia suggest that c-kit identifies a subgroup of cases, which may correspond to leukemias either arising from early prothymocytes and/or early hematopoietic cells, both able to differentiate to the lymphoid and myeloid pathways.

S503 Keywords CD117, Immunophenotyping, Leukemia

Topic: Laboratory haematology PO 63 Role of Flow Cytometry in Diagnosis of Myelomatous Pleural Effusion: A Case Report 1

Dr. Parul Arora, 2Dr. Nabhajit Mallik, 2Dr. Reena Mittal, Dr. Sanjeev Kumar Gupta

2 1

Department of Laboratory Medicine, 2Department of Laboratory Oncology, All India Institute of Medical Sciences (AIIMS), New Delhi, India

Introduction: Plasma cell myeloma is a multifocal plasma cell neoplasm associated with increased monoclonal protein in serum and/or urine. Pleural effusions in patients with myeloma are uncommon (6 %). However, effusions due to direct infiltration of the pleura by plasma cells (myelomatous pleural effusion) are extremely rare (\1 %) and usually seen with IgA myeloma. The diagnosis of such cases requires pleural fluid cytology, electrophoresis or pleural biopsy. We present a case of myelomatous pleural effusion diagnosed using flow cytometry immunophenotyping in addition to the pleural fluid cytology. Case Report: A 45 year old female was diagnosed as plasma cell myeloma (IgG kappa) in 2007. She received multiple chemotherapeutic agents during the course of her treatment including thalidomide, dexamethasone, lenalidomide, bortezomib, and doxorubicin based regimens. However, the patient had progressive extramedullary disease and developed pleural effusion in 2014. Cytological examination of the pleural fluid showed degenerative changes. Few preserved areas showed mononuclear cells including abnormal looking plasma cells. Immunophenotyping of these cells by flow cytometry revealed a pattern indicating neoplastic plasma cells. There was expression of CD38, CD138 and CD56, with absence of CD19, CD10 and CD45. This confirmed the diagnosis of myelomatous pleural effusion. Subsequently, the patient was offered a dexamethasone, cyclophosphamide, etoposide and cisplatin based regimen but, she declined further treatment and was discharged on request. Conclusion: Myelomatous pleural effusion is a rare complication of plasma cell myeloma. Flow cytometry can be used as an adjunctive technique in its diagnosis particularly in cases with equivocal cytology and electrophoresis findings. Keywords Myelomatous, Pleural effusion, Flow cytometry

Topic: Delegates PO 64 Non-transfuion Dependent Thalassemia: A Therapeutic Challenge Ajit Kumar Singh, Pranati Mohanty, Bidyut Prava Das Department of Pathology, S.C.B. Medical College, Cuttack, Odisha Summary: A 7 year old female child presented with progressive pallor with splenomegally (10 cm) was diagnosed as sickle—Beta thalassemia. She gave h/o recurrent vaso occlusive crisis requiring BT-1–2 units at an interval of 6–8 months. Introduction: Non transfusion dependent thalasemia is a distinct entity characterized by no regular blood transfusion for survival. A variety of clinical complications, mild to moderate elevation of serum ferritin and paradoxically a high iron overload in the tissue or organ. Materials and Methods: Hb-8gm/dl, Reti count-2.4 %, LDH-380 IU/L, S.ferritin-945.7 ng/ml and Transferrtin saturation-58 %, liver biopsy

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S504 shows iorn overload detected in Prussian blue stain which was disproportionately high in compariosion to Serum ferritin level. Echocardiography appeared normal with EF-74 %. The child was treated with hydroxyurea 20 mg/kg, Deferasirox-10 mg/kg with Folvite and Sodamine. On follow up after 1 year S.ferritin was -735 ng/ml and liver biopsy showed depleted iron deposition. Result: The child was experienced improvement physically along with improvement in the haematological parameters. Conclusion: In absence of increased hemolysis and regular BT increased iron deposition in the tissue can be attributed to increased iron absorption in the G.I. bed due to decreased hepcidin. Sickle-beta cell thalassemia coulde be considered as NTDT syndrome. Monitoring and management of iron overload should be adopted occasionally. Keywords Sickle-beta Thalassemia, Hepcidin

Topic: Hemoglobinopathies PO 65 Hematological Profile and Clinical Severity Scoring in E-Beta Thalassemia 1

M. Murari, 1K.C. Pani, 1S. Sharma, 2S. Agrawal, 2S. Phadke

1

Departments of Pathology and 2Medical Genetics, SGPGIMS, Lucknow Background: Hemoglobin E, a structural hemoglobin variant is the most common hemoglobin variant in South East Asia and the second most hemoglobin variant worldwide. In addition compound heterozygous states occur such as hemoglobin E/b Thalassemia (E/b thal) and rarely sickle cell/hemoglobin E disease (SE genotype). Patients and Methods: Present study includes clinico-hematological findings in 20 cases of Hb E-beta thalassemia diagnosed within 1 year period. All patients had detailed clinical, and hematological examination and HPLC. Mutational analysis was done in 12 patients. Clinical severity was assessed according to the scoring system by Sripichai et al. Results: Sixteen male and four female patients with wide age distribution (1 year to 38 years) were diagnosed as E-b Thalassemia, on the basis of HPLC and molecular findings. Thirteen patients had severe anemia and seven had moderate anemia at presentation. Significant negative correlation was seen between Hb E and red cell indices (MCV and MCH). Significant positive correlation was also observed between Hb E and RDW. We stratified the patients into mild, moderate and severe category according to the scoring system. Patients with severe disease show earlier age of presentation, more dependency on transfusion, larger spleen size and lower hemoglobin at presentation, however there were no statistically significant differences in the hematological parameters between the three groups. Conclusions: Hemoglobin E-beta Thalassemia results in chronic hemolytic anemias with variable clinical severity, most of the cases required regular transfusions. In our study we did not find statistically significant correlation of haematological findings with clinical severity scoring. Keywords e Beta thalassemia, Clinical severity score

Topic: Clinical Haematology PO 66 Sickle Cell Anaemia Presenting as Inflammatory Polyarthritis Dr. Riya Ballikar, Dr. Nilanjan Sinha, Dr. De Dibyendu, Dr. Debmalya Bhattacharya, Dr. Subham Bhattacharya, Dr. U.K. Nath, Dr. M. Bhattacharya Institute of Haematology and Transfusion Medicine, Kolkata

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Summary: This is a case report of a young male presenting with inflammatory polyarthritis as the sole manifestation of sickle cell disease and who was misdiagnosed and treated outside as a case of rheumatoid arthritis. Introduction: Sickle cell anaemia is a hemoglobinopathy which predominantly presents in early childhood with features of hemolytic anaemia and painful crises. Case Discussion: A 27-year-old male patient presented with history of recurrent episodes of painful swelling of large and small joints of all four limbs associated with morning stiffness, without axial skeleton involvement. The patient also had chronic pain in both the hip joints and bursitis of knee joints. Patient was investigated for polyarthritis for 3 years. Rheumatoid factor and antinuclear antibody were positive and CRP was elevated. X rays of hands and feet done revealed erosive arthritis, with patchy areas of osteosclerosis and osteopenia. Patient was labeled as having rheumatoid arthritis and treated with hydroxychloroquine. When his hip pain worsened with restriction of movements, X ray of the hip joint revealed bilateral loss of femoral head, which was later by MRI confirmed to be avascular necrosis. Retrospectively, patient gave history of intermittent jaundice, but there was no history of blood transfusion. On examination there was no organomegaly. Hemogram with peripheral smear revealed sickled red cells. Hemoglobin HPLC revealed Homozygous Sickle cell disease. Both parents had HbS carrier state. Patient was started on therapy with Hydroxyurea, with symptomatic treatment of the painful crisis with analgesics and hydration. Patient is currently doing well with marked reduction of his musculoskeletal complaints. For the avascular necrosis, bilateral Total hip Replacement has been done. Conclusion: Sickle cell anaemia may present only with arthropathy or arthritis with no other evidence of hemolytic disease. This should be kept in the differential diagnosis of musculoskeletal disorders whenever applicable. Early and prompt diagnosis will result in preventing or limiting the debilities associated with this disease. Keywords Sickle cell anaemia, Inflammatory polyarthritis, Arthropathy

PO 67 Alpha-thalassemia Protects Against Severity of P. falciparum Malaria in Eastern India: A Hospital Based Study Prasanta Purohit, Dilip Kumar Patel, *Siris Patel, Pradeep Kumar Mohanty, Snehadhini Dehury, Satyabrata Meher, Kishalaya Das Sickle Cell Clinic & Molecular Biology Laboratory, V.S.S. Medical College, Burla, Odisha, India Summary: The prevalence of alpha-thalassemia in patient with P. falciparum malaria was found to be 32.3 % and protect against severity of the disease manifestation. Introduction: Malaria is an important public health problem in Odisha. Alpha-thalassemia has been hypothesized to prevent against severe malaria. The prevalence of alpha-thalassemia in this area has found to be *50 %. Materials and Methods: Ninety six adult patients with P. falciparum malaria admitted in the Department of Medicine, were analyzed for alphathalassemia at Sickle Cell Clinic and Molecular Biology Laboratory, V.S.S. Medical College, Burla, Odisha. Alpha-thalassemia was analyzed by Multiplex PCR. On the basis of alpha-thalassemia, 96 patients were divided into two groups. Group-1: Patients without alpha-thalassemia and Group-2: Patients with alpha-thalassemia (both heterozygous (-a/aa) and homozygous (-a/-a) alpha-thalassemia). Results: The prevalence of alpha-thalassemia was found to be 32.3 % both in heterozygous (19.8 %) and homozygous (12.5 %) state. Both 3.7 and 4.2 kb deletional alpha-thalassemia were found with a gene frequency of 0.203 and 0.020, respectively. Hemoglobin and WBC level were significantly high where as MCV, MCH, serum-bilirubin

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 and serum-creatinine were significantly low in Group-2 compared to Group-1. Clinical evaluation revealed that patients in Group-2 had a lower degree of having anemia, acute renal failure, jaundice and death compared to patients in Group-1. Conclusion: Presence of alphathalassemia in patients with P. falciparum malaria has a protective effect on the severity of the disease manifestation. High incidence of alpha-thalassemia in this area probably reflects a selective advantage of these people against death from P. falciparum malaria. Keywords Malaria, Alpha Thalassemia, Plasmodium falciparum

PO 68 Molecular Hematological and Clinical Characterization of Sickle Cell HbE in Eastern India: The Largest Series in World Siris Patel, Satyabrata Meher, Snehadhini Dehury, Prasanta Purohit, Kishalaya Das, Dilip Kumar Patel, Pradeep Kumar Mohanty Sickle Cell Clinic & Molecular Biology Laboratory, VSS Medical College & Hospital, Burla, Odisha Summary: This is the first report of a large cohort (14 cases) of HbSE double heterozygote state from Odisha, India. Patients with HbSE presents varied clinical manifestations. Introduction: Sickle Cell Disease (SCD) is the commonest hemoglobin disorder in Eastern India. Co-inheritance of Hb E (cd26, Glu[Lys, GAG[AAG), with HbS (HbSE) presented with varied clinical presentation ranging from asymptomatic to severe disease. Materials and Methods: Sickling test, alkaline electrophoresis, CBC, Biochemical examination, CEHPLC were carried out at Sickle Cell Clinic & Molecular Biology Laboratory, VSS Medical College & Hospital, Burla, Odisha. HbS/HbE mutation characterized by ARMS-PCR/RFLP & DNA sequencing. Xmnl polymorphism and a-thalassemia were done by PCR. HbSE cases compared with matched HbSb-thalassemia and HbSS. Results: Fourteen HbSE cases revealed HbE (31.69 ± 4 %) eluting at HbA2 window (RT 3.75 ± 0.01) and HbS 57.35 ± 3.15 (RT 4.44 ± 0.01) in CE-HPLC. DNA sequencing confirmed HbS mutation at codon-6 (GAG[GTG) and HbE at codon-26 (GAG[AAG). Rate of VOC and Blood-Transfusion were lower in HbSE, where as incidence of splenomegaly, AVN and cholelithiasis were comparable. MCV, MCH and MCHC were lower than HbSS and higher than HbSb. All biochemical parameters were lower in HbSE compared to both HbSb and HbSS patients. Six cases are on Hydroxyurea therapy (10 mg/kg/day). Six cases were co-inherited with a-thalassemia (three a-3.7 and three a-4.2) in heterozygous state. Xmnl polymorphism was found in HbSE cases. Conclusion: This is the first report on 14 cases of HbSE, the largest number of subjects studied. HbSE patients presented with microcytic-hypochromic blood picture and clinically less severe compared to both HbSb and HbSS patients. Keywords Sickle Cell Anemia, HbSE, HbSb

Topic: Thalassemia

S505 Introduction: HbE-b thalassemia is the most common form of hemoglobinopathy in Southeast Asia and eastern India. Accumulation of iron resulting from blood transfusion and increased intestinal iron absorption in thalassemia promotes the formation of reactive oxygen species (ROS), which leads to oxidative stress, organ dysfunction, and tissue damage. Of these, cardiovascular complications are the leading cause of mortality. Impaired endothelial function is a biomarker in patients with cardiovascular risks. Therefore, assessment of endothelial function is a useful prognostic tool. Materials and Methods: 60 E-b thalassemia patients were included in the study and baseline parameters and iron status were measured. 60 healthy non hypertensive, non diabetic, non smoker, age sex matched control subjects were also taken. The endothelial function measured by measuring flowmediated vasodialation (FMD) and arterial elastic parameters. The carotid intima-medial thickness (CIMT) also measured. Results: The baseline parameters of thalassemic patients and control group were matched except Hemoglobin and Ferritin level. The mean hemoglobin and ferritin of thalassemic patients were 7.43gm/dl and 1,032 mcg/dl, respectively. There was lower FMD (7.49 %) and higher CIMT (0.46 mm) in thalassemic group than control (10.52 % and 0.36 mm, respectively) (p value\0.05). Also the derived parameters of vascular functions, like arterial stiffness is elevated and arterial distensibility is lower in thalassemic patients than control. Among the thalassemic patients FMD or CIMT did not correlate with serum ferritin value or cholesterol level. Conclusion/Diagnosis/Impression: The E-b thalassemia patients have endothelial dysfunction, and also higher intima medial thickness. So they are at a higher risk of developing atherosclerosis and cardio-vascular complication than normal population. The vascular dysfunction does not correlate with serum ferritin value, so regular monitoring with Doppler study is required for early diagnosis of subclinical atherosclerosis in this group of patients. Keywords E-b thalassemia, Endothelial dysfunction, Atherosclerosis

PO 70 The Role of Hydroxyurea and Valproic Acid in the Management of Severe HbE-b Thalassaemia Sujana Biswas, Soumi Chaudhuri, Meet Kumar, Aditi Sen, Maitryee Bhattyacharyya, Kanjaksha Ghosh Nil Ratan Sircar Medical College & Hospital, Kolkata Summary: We evaluated the efficacy of HbF inducing agents (hydroxyurea and valproic acid) in E-beta thalassemia. We found Hydroxyurea is effective in significant increases in HbF levels. Introduction: Hb E-beta thalassemia is a common disorder in eastern India with varying clinical presentation. Pharmacological induction of fetal haemoglobin is shown to reduce disease severity in sickle cell disease. We evaluate the efficacy of hydroxyurea and valproic acid in Hb E-beta thalassemia. Materials and Methods: Patients with severe Hb E-beta thalassemia were randomised to receive either hydroxyurea (10 mg/kg) or valproic acid (5 mg/kg). Patients were followed up for response with frequent scheduled visits. Results: Baseline parameters for patients in both arms were matched as shown in Table 1.

PO 69 Early Vasomotor Dysfunction and Subclinical Atherosclerosis in Patients of E B Thalassemia Dr. De Dibyendu, 1Dr. Subham Bhattacharya, Dr. Uttam K. Nath, 1Dr. S.S. Ray, 1M. Bhattacharyya, 2P. Chakrabarti

Table 1 . HU arm

1

Valproate arm

1

Age at 1st transfusion (years)

1

Spleen size (cm)

14.5

12.6

Yearly transfusion

13.4

14.8

Institute of Haematology and Transfusion Medicine, Medical College, Kolkata. 2Dept. of Haematology, NRS Medical College, Kolkata

4.1

4.9

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

All patients were followed prospectively for transfusion requirement, drug effectiveness and toxicity. With median follow-up of 6 months, we found hydroxyurea to be associated with significant increases in HbF levels (Table 2).

Interesting Cases of Haemoglobinopathies on HPLC: A Series of 9 Cases Deepshikha Dhiman, Sunita Sharma, Jagdish Chandra, Nupur, Preeti Rai

Table 2 . Parameter Hydroxyurea arm (N = 36) Valproic acid arm (N = 15) Baseline

After

7.0

7.1

Ferritin

1,045.3

878.9

HbA2+E HbF

44.7 12.6

38.7 30.6

Hb

PO 72

P

Baseline

After

6.3

6.4

P

0.06

1,263

1,190

0.09

0.001

41.8 14.6

25.7 15.3

0.02

Conclusions: Hydroxyurea as compared to valproic acid results in significant increase in HbF and decrease in serum ferritin, although it does not translate into reduced transfusion requirement. Longer follow-up with more number of patients is warranted for confirmation of these findings.

PO 71 Pattern of Hemoglobinopathies and Thalassemias in a Tertiary Centre Laboratory in Eastern India, A 1 Year Study Dr. B. A. Savitha, Dr. Susruta Sen, Dr. T. K. Ghosh

Department of Pathology and Department Of Paediatrics, Lady Hardinge Medical College, New Delhi Introduction: Haemoglobinopathies are major healthcare problem and commonest inherited disorders. Haemoglobinopathies have varied clinical presentations, as doubly heterozygous can modify the clinical presentation thus, misleading the clinical diagnosis. An accurate diagnosis of haemoglobinopathies is immensely important for management and prevention of more serious haemoglobin disorders. Materials and Methods: A total of 3,700 blood samples were screened from January 2013 to August 2014 for haemoglobinopathies and structural haemoglobin variants by HPLC, b-THAL short programme, Bio-Rad variant II for evaluation of anemia and for family and antenatal screening studies. Results: Among 3700 samples tested, 9 interesting cases were found comprising of HbQ India—3 cases (2 with b thalassemia trait and 1 of transfusion associated peak), Hb Agenogi(1case), Doubly heterozygous for HbD Punjab/b+ thalassemia-1 case(unusual chromatogram pattern), HbD Iran(1 case), Compound heterozygous for HbE/b thalassemia trait with varied clinical presentation in two siblings (1 case), HbH disease(2 cases) These cases were diagnosed on the basis of abnormal peaks on HPLC and some were further confirmed by serum electrophoresis and DNA sequencing. Conclusion: HPLC is a simple, easy and accurate technique for diagnosis of hemoglobinopathies. An accurate diagnosis can be provided in majority of cases based on retention time, proportion of total haemoglobin, and peak characteristics of HPLC. Family studies, electrophoresis and DNA sequencing play valuable role in difficult cases.

The Calcutta Medical and Research Institute, Kolkata Summary: An analysis of cases presenting for hemoglobinopathy screening by HPLC in a referral laboratory in Eastern India. Introduction: Hemoglobinopathies are common disorders of haemoglobin which are caused due to globin gene disorders that cause diminished rate of synthesis of one or more globin chains. Detection and accurate diagnosis is useful in genetic counselling. Aim: HPLC is a commonly used method for the diagnosis of Hemoglobinopathies and thalassemias. A total of 599 cases, who underwent HPLC testing in a tertiary reference laboratory in Kolkata, in the past 1 year duration were analysed. Materials and Methods: This study was performed by high performance liquid chromatography (HPLC) using BIORAD D 10 system. Results: 599 cases were analysed over a period of 1 year, belonging to predominantly the Eastern Indian region. 67.9 % of cases showed a normal chromatogram pattern. Beta thalassemia Trait was the most prevalent hemoglobinopathy seen here, accounting for 11.4 % of cases. Haemoglobin E Trait was observed in 6.2 % and Homozygous Haemoglobin E in 3 % of cases. Heterozygous Haemoglobin E- beta Thalassemia was seen in 1 % of cases. Reduced haemoglobin A2 levels suspicious of Alpha thalassemia, was seen in 3.5 % cases. However the same could not be confirmed with genetic testing. Conclusion: The prevalence of Hemoglobinopathies is quite high in this region and such screening help in prevention by genetic counselling. Keywords Hemoglobinopathy, HPLC, Thalassemia, Prevalence, Eastern India

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PO 73 Antenatal Carrier Screening for Thalassemia and Related Hemoglobinopathies Kalpana Singh, Divyanshu Singh, Shailaja Shukla, Sunita Sharma, S. S. Trivedi Department of Pathology, Lady Hardinge Medical College & Associated Hospitals, New Delhi 110001, Department of Obstetrics and Gynaecology, Lady Hardinge Medical College & Associated Hospitals, New Delhi 110001 Objectives: In India there are around 45 million B-thalassemia carriers with carrier frequency of 1–17 % in different regions of the country, average being 3.3 %. Prevalence of sickle cell trait, HbE trait and HbD trait are 29.8, 0.9 and 0.2 %, respectively. This study is an attempt to recognise the carrier status of pregnant females in National Capital Region of Delhi and to prevent the homozygous/major births. Materials and Methods: 2,000 antenatal patients were subjected to complete blood count. All cases with microcytic hypochromic indices (MCV \ 77fl and MCH \ 27pg) were subjected to serum ferritin estimation, peripheral blood smear examination and reticulocyte count. Cases with reduced serum ferritin levels were treated as iron deficiency anemia while cases with normal or raised serum ferritin levels were screened for heamoglobinopathies by HPLC. Partners of

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 carrier women were also identified on HPLC. The carrier couple was then referred to tertiary care centre for prenatal counselling and diagnosis. Results: 1,100/1,163 cases with microcytic hypochromic indices were of iron deficiency anemia with reduced levels of serum ferritin. The remaining 63 cases with normal serum ferritin were carriers of beta thalassemia and other hemoglobinopathies identified on HPLC. Out of 63 hemoglobinopathic carriers, 59 were of beta thalassemia trait, one case each of HbE, HbS and HbD heterozygous and one was double heterozygous for beta thalassemia and HbE. The prevalence of beta thalassemia trait was 2.95 % and other hemoglobinopthies being 0.05 % each. Two couples at risk of beta thalassemia major births were identified after screening 59/63 partners of the carriers. Conclusion: Antenatal screening for beta thalassemia and related hemoglobinopathies should be mandatory. Complete blood count and serum ferritin estimation can exclude almost all cases of iron deficiency anemia and further need not be evaluated on HPLC for identification of hemoglobinopathic carriers.

S507 imposed by betathalassemia allele. Drug induced oxidation of HbE occurs faster than HbS,A and F.Co existing G6PD deficiency will further worsen the clinical condition. Materials and Methods: 20 year male presented with fever with chills since 5 days for which antimalarials and paracetamol were started from local hospital. Following this he developed jaundice and cola coloured urine. Clinicopathological data and biochemical investigations analysed. Results: pallor+; icterus+; splenomegaly+; Hb dropped from 8.4 to 5.9 g%. Peripheral smear showed hemolytic blood picture with bite cells. S. bilirubin total/direct = 5.7/1.0 mg/dl; S.LDH = 4578 IU/L; Heinz body demonstrated. Urine spectrometry-evidence of methemoglobinuria. HPLC: HbE-betathalassemia G6PD = 3.5 U/g Hb(6.97–20.5). Initially antimalarials was stopped but symptoms persisted. Later paracetamol was stopped following which patient improved dramatically. Conclusion: Combined hemoglobin E/betathalassemia and G6PD deficiency can cause severe clinical manifestations following oxidant stress even with paracetamol. Keywords HbE, Beta thalassemia, G6PD deficiency

PO 76

Topic: Oral Paper Presentations PO 74 Analysis of Uncommon Haemoglobinopathies from Tertiary Care Center Kaniyappan Nambiyar, Anjali Sharma, Vijay Kumar, Sadhna Marwah, A. S. Nigam, Gurdeep Buxi Department of Pathology, PGIMER, Dr. Ram Manohar Lohia Hospital, New Delhi Objectives: Haemoglobinopathies and thalassemias are the most common single gene disorders in the world. HPLC is an important investigation for accurate and speedy diagnosis of various hemoglobin disorders. This study is aimed to analyse uncommon variants of haemoglobinopathies in northern part of India and correlating with respective hemogram parameters. Methods: Retrospective analysis of hemoglobin HPLC using VARIANTTM (Bio Rad Laboratories, Hercules, CA, USA) was done from January 2006 and August 2014. Hematological parameters of these patients were also analysed. Results: Out of 6,180 cases, 267 cases were having abnormal hemoglobin fractions with 180 cases of b-thalassemias, 29 cases of Hb S (including double heterozygous), 27 cases of Hb E (including double heterozygous), 18 cases of Hb D, 7 cases of suspected a-thalassemias, 2 cases of Hb Lepore, 2 cases of Hb J Meerut, 1 case of Hb Q, and 1 case of Hb Hope. Conclusion: Timely detection of Hb variants can prevent occurrence of more serious disorders of hemoglobin. Correlating HPLC with respective hemogram parameters is also important. Keywords HPLC, Haemoglobinopathies, Thalassemias

Topic: Hematology PO 75

‘And It Made all the Difference’: Introduction of High Performance Liquid Chromatography, A Key Tool in the Diagnosis of Thalassemias and Hemoglobinopathies Dr. Mahathi Krothapalli, Dr. Jyoti R. Kini, Dr. Anupama Hegde, Dr. R. Madan Gopal, Dr. Harsha Prasad, Dr. B. Prashanth, Dr. Urmila N. Khadilkar, Dr. Debarshi Saha, Dr. M. Nirupama Kasturba Medical College, Manipal University, Mangalore Introduction: Identification of hemoglobinopathies and Thalassemia traits is a challenge based on the routine laboratory parameters. Use of high performance liquid chromatography (HPLC) in association with hematological findings has improved the recognition of these. Materials and Methods: A prospective cross-sectional study was undertaken at the Kasturba Medical College Laboratory Services, Mangalore, in the period from September 2013 to August 2014. All patients with request for hemoglobin variant analysis by HPLC were included. The hemoglobin variants were detected using ion-exchange HPLC method with Biorad D10 Hb Variant analyser which had been newly installed in the laboratory. Complete hemogram, iron profile, ferritin were collected and indices were derived at the time of analysis, as corroborative evidence for evaluating the chromatograms. Results: A total of 167 cases were analysed of which 34.1 % cases were in the pediatric age group, 65.6 % were amongst adults. The distribution of cases based on HPLC chromatogram were—Thalassemia trait (31.6 %), Thalassemia intermedia (2.6 %) Thalassemia major (4.6 %), HbS (2 %) and others (Heterozygous-8.6 %). Of these, 40 cases had relevant complete data. The cases (16 indecisive based on HPLC and 24 Thalassemia traits) were analysed with respect to three indices— Mentzer, Green & King & Ricerca. Green & King (p \ 0.005) & Ricerca (p \ 0.05) indices showed significance in detecting Thalassemia trait. Conclusion: Though differentiation of traits can be done with the help of various indices, introduction of HPLC has improved the detection of the Thalassemia traits and Haemoglobinopathies. Keywords Thalassemia, Hb variants, HPLC

An Unusual Case Report of Hemoglobin E/Beta Thalassemia and G6pd Deficiency

PO 77

Dr. Linta Thampi, Dr. K. K. Prabhalakshmy, Dr. Joy Augustine

Haemoglobin Analysis in Heterozygous States of B Thalassaemia, Bs and Be

Pathology, Government Medical College, Thrissur Summary: Case report of combined hemoglobin E/betathalassemia and G6PD deficiency. Introduction: Compound heterozygosity for HbE and betathalassemia causes moderate to severe anemia. Severity of interaction reflects instability of HbE consequent upon globin chain imbalance

K. Neelagandan, S. Eswari, S. V. Rajkumar, G. Sankari Devi, R. V. Shaji, Eunice S. Edison Department of Haematology, Christian Medical College, Vellore, India

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Introduction: Haemoglobinopathies are one of the commonest monogenic diseases affecting the world’s population. Diagnosis of haemoglobin disorders is based on clinical features, haematological parameters, peripheral smear examination and quantification of haemaoglobins F (HbF), A2 (HbA2) and haemoglobin variants. There is limited data on levels of HbF, Hb A2 and haemoglobin variants in various haemoglobin disorders in Indian population. Materials and Methods: We did a retrospective analysis of 9,000 samples for which the levels of HbF, HbA2 and variant haemoglobins were quantified between 2004 and 2013. Haematological parameters were measured by standard methods. Haemoglobin analysis was performed using Biorad variant High Performance Liquid Chromatography. Results and Discussion: Out of the 9,000 samples, 2404 had heterozygous b thalassaemia, 417 were heterozygous for HbE, 275 were heterozygous for HbS. The haematological parameters and the haemoglobin analysis are listed in Table 1. Total haemoglobin (p = 0.00) and HbA2 levels (p = 0.025) were significantly lower in women than in men. The HbA2/E levels were within a narrow range whereas the sickle levels varied widely. Conclusion: This is a retrospective analysis with a large sample size. This data gives an overview of HbF, HbA2 and variant Hb levels in heterozygous b-thalassaemia, heterozygous HbE and heterozygous sickle (HbS) in the Indian population. This study will aid in interpreting the HbF and HbA2 levels in diagnostic laboratories. Keywords HPLC, Beta thalassemia, Hemoglobinopathy Table 1

The haematological parametersand Hb-HPLC percentages in patients Hb (g/dl) Median (range)

MCV (fl) Median (range)

HbF% Median (range)

Heterozygous b Thalassaemia

10.87 (3.0–16.8)

63.78 (46.9–92.3)

0.87 (0–12.9)

Heterozygous HbS

12.32 (5.4–17.2)

80.19 (50.9–108.7)

Heterozygous HbE

11.67 (4.4–16.3)

75 (54.2–96.7)

1.01 (0–9) 1.01 (0–6.8)

HbA2/E% Median (range)

HbS% Median (range)

4.9 (3.4–7.9) 3.23 (1.8–5.30)

34.70 (22.90–44.9)

27.45 (19.7–35.9)

Topic: Hemolytic anemia/Hemoglobinopathy PO 78 Frequency of B Thalassemia Trait and Other Hemoglobinopathies in a Tertiary Care Hospital in Delhi Neha Garg, Vandana Puri, Mrinalini Kotru, Meera Sikka, Satendra Sharma Department of Pathology, University College of Medical Sciences and GTB Hospital, Delhi Introduction: Hemoglobinopathies are common, preventable hereditary disorders with some associated with major morbidity. The frequency of beta-thalassemia trait (BTT) has been reported to be 5.5–6.0 % in Delhi. The prevalence of hemoglobinopathies varies in different population groups in the country with a higher frequency of HbD in Punjab, HbE in east and HbS in tribal regions. Objective: This study aimed to assess the frequency and hematological profile of various hemoglobinopathies diagnosed in our institute over a period of 5 years. Materials and Methods: On retrospective analysis of data over a 5 years period, 272 patients in whom a hemoglobinopathy was diagnosed were studied. CBC (Automated Hematology analyser LH500) and Hb electrophoresis (cellulose acetate using TEB buffer pH 8.7) were done in all the patients. Standard criteria were used for diagnosis of hemoglobinopathy. Results: BTT was diagnosed in 63.46 % patients, beta thalassemia major (BTM) in 6.64 %, bthalassemia intermedia in 2.2 %, Hb E trait in 7.74 %, E-bthalassemia in 4.05 %, homozygous HbE in 0.73 %, HbD trait in 2.25 %, HbD-bin 0.73 %, HbS trait in 1.84 %, HbS-b in 1.47 %, HbSS in 1.1 % and

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HPFH in 0.36 %. Majority of patients with BTT were anaemic. Coexistent iron deficiency was detected in 30 % traits. Conclusion: In view of the high prevalence of hemoglobinopathies in this region, there is a need of increasing awareness about them to enable couples to opt for premarital screening thus enabling them to prevent the birth of a child with BTM. Keywords Hemoglobinopathy, Beta thalassemia trait

PO 79 Prevalence of Hemoglobinopathies in Eastern India: Results of a Population-Based Screening Programme Pooja Prasad, T.K. Dolai, Shuvraneel Baul, De Rajib, P. K. Mondal, S. Dutta, P. Chakrabarti Department of Hematology, NRS Medical College, Kolkata Introduction: Haemoglobinopathies are a leading cause of child mortality worldwide, although with a variable geographical incidence. In India, which lies in the thalassemia belt, both alpha and beta thalassemia are found in West Bengal. A reliable estimate of prevalence of the disease is necessary for reducing its burden. Materials and Methods: The present study includes 69, 803 individuals in school and colleges, newly married couples and pregnant women, who were screened for hemoglobinopathies after proper counselling from June 2010 till July 2014. Blood samples were collected in EDTA and complete blood count was done by Sysmex KX-21 and thalassemia screening was carried out by high performance liquid chromatography (HPLC) using Bio-Rad variant analyser. Results: Hemoglobinopathies were identified in 11.6 % of the screened individuals. The prevalence of Beta thal trait and Hb E trait was 6.8 and 2.8 %, respectively. Carriers for Hb S and Hb D comprised 0.5 and 0.2 %. Among the compound heterozygotes, HbE-Beta thal was seen in 0.4 % cases and HbS-Beta thal in 0.2 %. Beta thal major and the homozygous state of Hb E and HbD disease were seen in a minority (less than 1 %). Other hemoglobinopathies including HPFH and HbLepore were noted in less than 1 % cases. Conclusions: The prevalence of carrier state of Beta thal and Hb E is higher in West Bengal than in other parts of India. In view of high prevalence of hemoglobinopathies in this region, a routine premarital screening program should be continued for the identification and prevention of high-risk marriages. Keywords Hemoglobinopathies, HPLC, Screening

Topic: Poster PO 80 HPLC in Antenatal Screening of Thalassemia and Other Hemoglobinopathies: An Experience at DDU Hospital Pranshuta Sharma, Neelam Sood, Garima Goyal Department of Pathology, Deen Dayal Upadhyaya Hospital, Hari Nagar, New Delhi Summary: Antenatal screening is pivotal to check the growing disease burden of thalassemia in the community. HPLC is one of the recommended techniques for this purpose. Besides detecting thalassemia major, it can also detect other hemoglobin variants reliably. Introduction: Thalassemia is a major public health issue in the Indian subcontinent, requiring lifelong treatment. Annually, 6000–8000 children are born with thalassemia major. Antenatal screening thus becomes vital to prevent the birth of a thalassemic child. NESTROFT is often used for low-cost initial screening, with

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 subsequent confirmation by HPLC. Materials and Methods: This study was conducted in the department of Pathology of DDU Hospital from 2010 to 2014. All pregnant women attending the antenatal clinic and referred peadiatric patients were included in the study, being conducted as part of Thalassemia Control Programme. A total of 5563 patients were studied using HPLC (BioRad variant). A cut-off value of HbA2 of 3.8 % was used to define thalassemia trait. The spouses of antenatal patients testing positive on HPLC were also studied for the purpose of genetic counseling. Results: Out of 5563 patients tested, 997 (17.9 %) were found positive. 812 cases were thalassemia minor (14.5 %). Some compound heterozygotes were also observed in this cohort (such as HbD with b thal).Also some unusual cases were detected which will be presented. Conclusion: 14.7 % of patients were thalassemia carriers. HPLC is a useful test for detecting thalassemia carriers as well as unusual hemoglobin variants. Keywords HPLC, Thalassemia, Hemoglobin variants

PO 81 Diagnosis of a Novel Hemoglobinopathy of Compound Heterozygosity of Hemoglobin S/Hemoglobin Q India Dr. Suhas Sakhare, Sushama Parab, Dr. Caesar Sengupta, Dr. A. Velumani Thyrocare Technologies Limited, Turbhe, Navi Mumbai 400703 Summary: Thalassemia is a quantitative genetic disorder resulting due to mutations in the globin genes of hemoglobin. Double heterozygosity of HbS with various other hemoglobinopathy (like Hb E, HbD Punjab and Hb C) is already reported. However HbS along with HbQ India is still not reported in literature. Introduction: A novel double heterozygosity for the alpha chain variant Hb Q India and beta chain variant Hb S is described. Hb S is prevalent in central part of India while Hb Q India in its heterozygous state is found mainly in Sindhi families. Materials and Methods: Identification of both the variants, Hb S and Hb Q India was done based on chromatograms of HPLC (Tosoh G8 HPLC analyser, Japan) and capillary zone electrophoresis (CE, Capillarys 2 Flex piercing, Sebia, France). Confirmation of variants was done by PCR based amplification refractory mutation system (ARMS) technique. Results: Both HPLC and CE confirmed presence of Hb S. HPLC showed a pointed narrow peak of Hb Q India at retention time of 4.55–4.56 min while it is eluted in Hb D zone on CE. A hybrid variant of this alpha and beta globin chain was eluted in Hb C window and Hb C zone on HPLC and CE, respectively. Molecular studies using ARMS technique confirmed these findings. Both the cases showed positive sickling test and presented with mild anemia. Conclusion: We are presenting for the first time two index cases for compound heterozygosity of Hb S with Hb Q India.

Topic: Afternoon PO 82 Study of Double Heterozygous Cases by Using Cation Exchange High Performance Liquid Chromatography (H.P.L.C.) in Nanded Region of Maharashtra Dr. Sundar Shewale, Dr. M. A. Sameer, Dr. S. A. Deshpande Dr. Shankarrao Chavan Govt. Medical College, Nanded Summary: The double heterozygous hemoglobinopathy cases

S509 includes both the qualitative and/or quantitative disorders of haemoglobin inherited from both the parents. The early detection of double heterozygous cases is important so as to reduce the rise in new cases. Introduction: The study of compound heterozygous hemoglobinopathies are all clinically significant. Correct identification of the subtype would be useful in defining the clinical course of the disease. Materials and Methods: We conducted retrospective study from Jan 2013 to Dec 2013 Inclusion criteria: (1) All the patients of anemia showing sickling test, solubility test and NESTROFT positive and their family members. (2) Positive samples received from RH and PHCs on Hb Electrophoresis. Exclusion criteria: Patients WHO received blood transfusion in the last 6 months. The routine tests were performed on Mythic cell counter and HPLC was performed on BIO RAD variant HPLC machine. Positive cases were further called for family screening. Results: 1,043 cases of hemoglobinopathies were screened out of which 250 cases of sickle disorder were found. Total double heterozygous cases were 117. 111 cases of Sb thal, 2 cases of SD Punjab, 2 cases of S-Lepore, 1 case each of ES and Eb thal. Maximum cases were found in the age group 11–20 years. Average age of was 13.4 years. Consanguinity was seen in 65 cases. Maximum number of cases were found in Boudha (29) followed by Muslims (25). Conclusion: Prompt diagnosis and early institution of supportive care has reduced morbidity and mortality and ultimately has been of crucial importance in better outcome of diseased patients. Keywords Heterozygous, HPLC, Sickling, Electrophoresis

PO 83 Detection of Hemoglobinopathies in New Born Screening (NBS): An Experience at a Reference Laboratory Vishal Mehrotra, Pradnya Chaudhari, Narendra Gupta, Sunita Salvi, Manisha Ramani and Amar Das Gupta Sections of Hematology and Flow Cytometry, SRL Limited, Mumbai Introduction: Diagnosis of sickle cell disease prior 4 months of life allows early administration of penicillin prophylaxis with resultant decrease in morbidity and mortality. Hemoglobin analysis of eluates from dry blood spots (DBS) by high-performance liquid chromatography (HPLC) permits centralized testing of new-born blood samples for hemoglobinopathies. We wanted to find out the incidence and spectrum of hemoglobinopathies in neonates using HPLC of DBS samples and to identify the issues faced in this approach. Materials and Methods: Eluates from DBS were analyzed using Sickle Cell Short Program on Bio-Rad Variant Classic HPLC system for the presence of hemoglobins F, A, S, D, C and E. Results: Of the total 891 neonatal DBS (467 males and 424 females) examined, 876 showed a normal pattern (Hb F [ Hb A). Fifteen cases (1.7 %) showed presence of hemoglobinopathies, viz. Hb S trait (6 cases); Hb D trait (5 cases); Hb E trait (4 cases). No case of homozygosity for these hemoglobinopathies was identified, as evident from the presence of HbA. The percentages of abnormal hemoglobins varied from 3.8 to 5.1 % for Hb S, 2.3–11.2 % for Hb D and 1.6–4.1 % for Hb E/A2. Conclusion: There is no policy for neonatal screening for hemoglobinopathies in India. Therefore, even though the incidence of hemoglobinopathies in neonates in this study is low, the findings justify the inclusion of testing for hemoglobinopathies in the newborn screening panels. The very small amount of blood required for this purpose and the ease of collection and transportation of the samples in the form of DBS are an added advantage.

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S510 PO 84 A Simple Approach to Distinguish Between Artifactual and Inherited P3 Hemoglobin Peak in HPLC Vishal Mehrotra, Pradnya Chaudhari, Manisha Ramani, Payal Pacchigar and Amar Das Gupta Sections of Hematology and Flow Cytometry, SRL Limited, Mumbai Introduction: A ‘‘P3’’ abnormal hemoglobin (Hb) peak with retention time of 1.4–1.9 min is seen in a number of samples investigated for the presence of hemoglobinopathies using high performance liquid chromatography (HPLC). In addition to representing structurally abnormal Hb peaks (e.g. Hb J variants) ‘‘P3’’ peaks can also occur as an artifact in association with ageing of samples. It is therefore, necessary to distinguish between these two types of P3 peaks. However, this job becomes difficult in the absence of Hb studies of family members and non-availability of sophisticated technologies. We have devised a simple approach for distinguishing between acquired and inherited P3 peaks. Materials and Methods: 57 consecutive cases with ‘‘P3’’ peaks of [10 % on HPLC were selected for the study. Blood samples were incubated at 37 °C for 48 h and an aliquot was retested from each sample at an interval of 24 h by HPLC. Results: 17/57(29.8 %) cases showed presence of other abnormal Hb peaks while in 40/57(70.1 %) cases no hemoglobinopathy could be detected. In 51/57(89.4 %) cases incubation of the sample led to increase in the level of the ‘‘P3’’ peak thereby suggesting their acquired nature. Only in 8/57(14 %) cases the ‘‘P3’’ peak remained stable after incubation suggesting their inherited nature. In cases with acquired P3 peak, the rise was more marked in the first 24 h compared to the next 24 h. Significantly, the levels of abnormal hemoglobin in inherited hemoglobinopathy did not show rise on further incubation. Conclusion: Our data indicates that the simple approach adopted by us is useful in distinguishing between acquired and inherited ‘‘P3’’ peaks.

Topic: Thalassemia and Allied; Transfusion Medicine PO 85 Individual Donor Nucleic Acid Testing for Transfusion Safety in Thalassemia in a Tertiary Care Hospital from Eastern India 1

Dr. Dipanjan Haldar, 2Prof. Prantar Chakrabarti, Prof. Maitreyee Bhattacharyya, 1Dr. S. S. Roy, 1Dr. U. K. Nath, 3 Prof. Krishnendu Mukherjee 1

1

Institute of Haematology & Transfusion Medicine, Medical College, Kolkata, 2NRS Medical College, Kolkata, 3Department of Immunohaematology & Blood Transfusion Summary: Due to frequent transfusions, thalassemic patients are exposed to the risk of transfusion transmitted infections (TTIs). To the best of our knowledge, so far, no study has been carried out to assess the impact of NAT tested blood in preventing TTIs in thalassemic patients in India. Introduction: Blood safety is a challenging task in India; with a high prevalence rate of HIV (0.29 %), HBV (2–8 %) and HCV (&2 %) in general population. Individual donor nucleic acid testing (ID NAT) in blood donor screening has been implemented in many developed countries to reduce the risk of TTIs. Materials and Methods: We used third generation ELISA to screen blood samples of all blood donors. ID-NAT was done by transcription-mediated amplification (TMA).Routine serological testing for HIV, Anti HCV and Hepatitis B Surface antigen was done every 6 months on the 130 thalassemic patients enrolled in this study from March 2011 to August 2014. Results: Of the 5,003 donors tested by both ID-NAT and third generation ELISA, 11 were found to be NAT-positive but ELISA-

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 negative (NAT yield). The prevalence of NAT yield cases among routine donors was 1 in 454 donations (0.22 %). Out of these 11 samples, 1 was reactive for HIV, 3 were reactive for HCV only, 6 were reactive for HBV only, and there was a single case of HBV–HCV coinfection. Importantly no case of new seropositivity was documented on the recipients over this period. Conclusion/Diagnosis/Impression: NAT could detect HIV, HBV and HCV cases in blood donor samples that were undetected by serological tests. NAT is effective in preventing transfusion transmitted infections in thalassemia patients. Keywords Thalassemia, Individual Donor Nucleic Acid Testing

PO 86 A Study on the Cost Effective Methods of Screening of Beta-Thalassemia Trait in Children Aged 6 Months to 15 Years Dr. Fairy Susan Varghese, Dr. S. K. Agarwalla, Dr. Sameer Behera M.K.C.G. Medical College, Brahmapur, Odisha 760004 Summary: The present study highlights a set of cost effective screening tools in predicting beta-thalassemia trait (BTT) in microcytic cases, later confirmed by raised HbA2 on electrophoresis. Introduction: The management of patients with beta-thalassemia major, one of the most common inherited diseases worldwide, demands a cumbersome expenditure in view of the transfusion dependency, iron chelators and drugs to control infections. Effective screening of carriers is the backbone in prevention of birth of children with beta-thalassemia major. Early detection of the carrier status helps in preventing marriage with another carrier and paves way for partner screening and planning of prenatal workup for their offsprings especially in regions where consanguineous marriages are common. Materials and Methods: 100 paediatric cases in M.K.C.G. Medical College, Odisha with microcytosis (MCV \ 80 fl) included in the study period (Jan–June 2014) were screened for the presence of BTT by using 5 indices namely-A:(MCV)2X RDW/100 X Hb; B:MCV/ RBC; C:MCV X RDW/RBC; D:(MCV)2X MCHX 0.01; E:MCH/ RBC. Those cases which showed positivity for at least 2 indices suggestive of BTT were subjected to NESTROFT and Hb electrophoresis for confirmation. Results: The mean Hb, MCV, MCH, RBC count and HbA2 in BTT cases were 10.80 ± 2 g/dL; 68.10 ± 8 fL; 23 ± 4 pg; 4.8 ± 0.5 9 1012/L and 5 ± 0.09 %. 34 cases showed positivity for at least 2 of the indices suggestive of BTT and 25 among them showed an increased HbA2 ([3.5 %) confirming the diagnosis of BTT. Conclusion: The present study demonstrates the differentiation of BTT from non-BTT in microcytosis cases by routine hemogram and a set of cost effective screening indices. The final diagnosis is by HbA2 quantitation and electrophoresis. Keywords BTT (Beta-Thalassemia Trait), Microcytosis, NESTROFT, Electrophoresis

Topic: Free paper PO 87 Efficacy of Once Weekly Factor VIII Prophylaxis in the Prevention of Clinically Significant Bleeds in Patients with Severe Hemophilia A Dr. Deepak Charles, Dr. T. K. Dutta, Dr. P. Nalini, Dr. S. Mahadevan, Dr. Niranjan Biswal, Dr. Debdatta Basu, Dr. A. Ramesh JIPMER, Puducherry, India

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Summary: In this study we plan to assess the efficacy of once weekly low dose factor VIII prophylaxis in preventing clinically significant bleeds in severe hemophilia A. Introduction: The optimal regime for prophylaxis remains to be defined. Here we are reporting the preliminary 6-month result from an ongoing study. Materials and Methods: Children 1–12 years of age of severe hemophilia were divided into prophylaxis and on-demand groups. Prophylaxis group received factor VIII 20U/kg once a week. Results: The median age was 5(2–10) in the prophylaxis group and 9(5–10) in the on-demand group (p = 0.012). The total number of bleeds (i.e. during 6 months) were 2.3 ± 1.72 in the prophylaxis group and 4 ± 0.92 in the on-demand group (p = 0.01). The number of hemarthrosis per patient per month was 0.16(0.00–0.60) in prophylaxis group and 0.50(0.16–0.67) in the on-demand group (p = 0.002). The Clinical joint score (HJHS) was 0(0–3) and 1(0–2) at baseline (p = ns) and 0(0.0–4.0) and 1.5(1–4) at 6 months (p = 0.005) in the prophylaxis group and on-demand group respectively. Conclusion: Once weekly low dose factor VIII prophylaxis appears to be effective in reducing the number of joint and nonjoint bleeds in patients with severe hemophilia A. Keywords Severe hemophilia A, Factor prophylaxis, Joint bleed

Topic: Haemophilia & Haemostasis PO 88 Overview of a Global Clinical Trial Programme with Turoctocog Alfa, A New Recombinant Factor VIII: The GuardianTM Programme 1

S. Shankar, 2P. Laguna, 3V. Vdovin, 4L. Rageliene, L. Abad Franch, 6A. Lindblom

5 1

Senior Medical Advisor, Biopharm, Novonordisk India Pvt Ltd., Warsaw Medical University Warsaw, Poland, 3Hematological Center of Izmailovskaya Children Municipal Clinical Hospital Moscow, Russian Federation, 4Vilnius University Children’s Hospital Vilnius, Lithuania, 5Novo Nordisk AG Healthcare Zurich, Switzerland, 6Novo Nordisk A/S, Søborg, Denmark

2

Summary: Congenital haemophilia A is a rare bleeding disorder where bleeding can occur spontaneously or after minor trauma and result in serious complications. Aims: To present an overview of a global clinical trial programme (involving multiple centres and countries) assessing the pharmacokinetics (PK), safety and efficacy of turoctocog alfa, a new recombinant FVIII (rFVIII) for the treatment and prevention of bleeding in relevant patient populations with severe congenital haemophilia A (baseline FVIII B 1 %). Methods: Among seven, three trials investigated turoctocog alfa PK (two of these in Japanese subjects) and one also included a comparison with octocog (Advate). Two phase IIIa trials guardian 1 and 3 evaluated PK, safety/ efficacy of turoctocog alfa in the prevention and treatment of bleeds in previously treated adult/adolescent ([12 years of age) and paediatric (\12 years of age) patients, respectively. guardian [TRADEMARK] also comprised a surgical subtrial. The guardianTM an extension trial evaluated long-term safety/efficacy of turoctocog alfa who completed phase IIIa trials. GuardianTM, a phase IIIb study evaluating safety and efficacy in 60 previously untreated patients (PUP), with a follow up for C100 exposure days is ongoing. Results: The PK properties of turoctocog alfa studied in[60 patients are comparable to other rFVIII and plasma derived FVIII. The total turoctocog alfa clinical experience in more than 210 patients ([50 % on prophylaxis C18 months) from 18 countries is [50,000 exposure days and 340 patient years. Conclusions: This comprehensive programme investigating the PK, safety and efficacy of turoctocog alfa will provide an extensive clinical database.

S511 Keywords Guardian, Haemophilia

Turoctocog

alfa,

Recombinant

FVIII,

Topic: Haemophilia & Haemostasis PO 89 Structural Comparison of a New Recombinant rFVIII Molecule, Turoctocog Alfa, and Commercially Available FVIII Product 1

S. Shankar, 2A. K. Kristensen, 3M. Kjalke, 4N. K. Klausen, M. Ezban, 6K. Vad

5 1

Biopharm, Novonordisk India, Bangalore, 2Protein Characterization, Novo Nordisk A/S, Ma˚løv, Denmark, 3Haemophilia in Vitro Biology, Novo Nordisk A/S, Ma˚løv, Denmark, 4CMC Haemophilia, Novo Nordisk A/S, Gentofte, Denmark, 5Haemophila Biology, Novo Nordisk A/S, Søborg, Denmark, 6Haemophilia R&D, Novo Nordisk A/S, Søborg, Denmark Summary: Advate and Kogenate FS are full length gene products with varying length of B-domain attached to Heavy Chain (HC). For ReFacto AF, the B-domain consist of only 14 amino acids and for Turoctocog alfa, a new recombinant FVIII molecule (rFVIII), the B-domain consists of only 21 amino acids. Aim: To compare the structure and homogeneity of turoctocog alfa to commercially available rFVIII products. Methods: Advate, Kogenate FS, ReFacto AF and turoctocog alfa were analysed using SDS-PAGE with and without thrombin digestion followed by sequencing of excised bands, size exclusion (SE)-HPLC and reverse phase (RP)-HPLC. Results: SDSPAGE and SE-HPLC showed similar profile for turoctocog alfa and ReFacto AF containing the heavy chain (HC), light chain (LC) and a single chain (scFVIII). Several high molecular weights bands for HC were observed for Advate & Kogenate FS. After thrombin cleavage all rFVIII compounds contained the expected pattern of A1, A2 and activated LC, however, ReFacto AF also contained smaller size A2 domains. RP-HPLC profile demonstrated heterogeneity of HC for all rFVIII compounds. Turoctocog alfa had two components, one with C-terminal at the end of the A2 domain, and one containing the 21 amino acid B-domain. Advate and Kogenate FS exhibited several HC components due to the heterogeneously processed B-domains. A specific LC component containing part of the B-domain was observed for Advate. Conclusion: Turoctocog alfa is a highly homogeneous rFVIII product with varying length of the B-domain being the main difference between different products. Keywords Turoctocog alfa, Recombinant FVIII, Structure

PO 90 Life Threatening Rare Site Hemorrhages in Patients with Hemophilia: Case Series from a Single Center 1

Dr. Manoj Toshniwal, 1Dr. S. Chandrakala, Dr. Nilesh Wasekar, 2Dr. Farah Jijina, 3Dr. K. Ghosh

1 1

Department of Hematology, KEM Hospital, 2Hinduja Hospital and National Institute of Immunohaematology (NIIH), KEM Hospital, Mumbai, India

3

Introduction: Patients with congenital or acquired clotting disorders are at increased risk for experiencing spontaneous hemorrhage into unusual sites. Besides hemarthrosis that are frequently observed in Hemophiliacs, bleeding may also rarely occur in various soft tissues or solid organs not usually seen or reported. Objective: To report 12 patients of hemophilia from our Comprehensive Hemophilia Care

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Center in western India, presenting with bleeding at very rare and unusual sites and highlight diagnostic and management issues in them. Materials and Methods: We report 12 patients of hemophilia from our Comprehensive Hemophilia Care Center in western India presenting with bleeding at very rare and unusual sites. Results: Subdural hematomas and other central nervous system (CNS) hemorrhages are uncommon but represent a major cause of death and disability. Spontaneous spinal subdural hematoma is a rare subtype of CNS hemorrhage in patients with hemophilia, reported in only a handful of cases in the literature. Epidural hematomas have been reported somewhat more frequently, but are still rare. Diffuse alveolar hemorrhage has hardly been reported in hemophilia. Spontaneous hemothorax is also very rare in these patients.

Sr. no. Diagnosis

No. of Pts Management

Protein-C deficiency was present in one patient and Protein-S deficiency in 2 patients but the results were non-significant. The ATIII, Factor VIII and fibrinogen levels were comparable in both the cases and the controls. D-dimer was raised in 60 % of the cases (p = 0.011) and LA positivity was detected in 50 % cases (p = 0.033). Conclusion/Diagnosis/Impression: LA positivity and d-dimer positivity were found to be significantly associated with VTE in young patients. LA positivity is often not actively investigated for in VTE in pediatrics and study in a larger population is needed to determine its significance.

PO 92 Association Between Predicted FVIII Levels and Risk of Bleeding Episodes in Clinical Trials with Turoctocogalfa, A New rFVIII Product from Novo Nordisk

1

Diffuse Alveolar Hemorrhage 4

Conservative

2 3

Spontaneous Haemothorax 2 Spontaneous Splenic Rupture 1

Conservative Conservative

S. Shankar, M. C. Ozelo, D. Veljkovic, R. Klamroth, M. Misgav, R. Nielsen, A. Groth

4

Neck Muscle Hematoma

2

1-Conservative

Novo Nordisk India Pvt Ltd

2-Tracheostomy 5

Spinal Subdural Bleed

2

Conservative

6

Spinal Epidural Bleed

1

Conservative

Summary: Turoctocogalfa, a new rFVIII product was administered in severe haemophilia A patients at a prophylactic dose range 20–50 IU/ kg for adult patients (C12 years) and 25–60 IU/kg for paediatric patients (\12 years), in phase III clinical trials (guardian[TRADEMARK]). Introduction/Aims: To demonstrate the association between predicted FVIII: C and estimated bleeding rates for spontaneous and traumatic bleeding episodes in adult and paediatric patients. Methods: All patients (N = 214) reported detailed information about dosing and bleeding episodes (spontaneous and traumatic), including the time points of these events, in diaries. A subset of patients (N = 50) underwent pharmacokinetic assessments. A population pharmacodynamic analysis of association (correlation) between predicted FVIII:C and the risk of experiencing bleeding episode was constructed for each patient. FVIII: C profiles from nonbleeding patients (N = 67) were included in the analysis, whereas patients (N = 3) with insufficient reporting of dosing were excluded. Results: 211 patients were exposed to turoctocog alfa for 58.1 patient years for adults and 18.0 patient years for children and reported 550 bleeding episodes (326 spontaneous, 188 traumatic and 36 not categorised). FVIII:C levels 1–5 %, appears to protect paediatric patients, particularly against spontaneous bleeding episodes. Between-patient variability of bleeding risk at any specified FVIII:C was substantial in both age groups. Conclusion: Increasing FVIII:C appears more protective for paediatric versus adult patients, and for spontaneous versus traumatic bleeding episodes. The high variability between patients reflects that patients from different regions with varying economic resources and treatment histories participated in the trial.

Conclusion: The present case series highlights the need to be aware of rare presentations of bleeding disorders and their timely management, so as to give patients the best possible outcomes with the limited resources available to them.

PO 91 Prevalence of Thrombophila Markers in Young Patients with Deep Vein Thrombosis Aditya Kumar Gupta, Priyanka Bhagat, Madhu Jain, Vineeta Gupta, Jyoti Shukla Institute of Medical Sciences, Banaras Hindu University Summary: The predisposing factors for pediatric thrombosis have not been well studied. Lupus anticoagulant was found to be strongly associated with thrombosis in young in our study. Introduction: In the pediatric population and young adults the etiology of venous thromboembolism (VTE) is often multifactorial and the genetic thrombophilia states are more likely to be present in them. The present study was undertaken to study the various thrombophilia markers in young patients presenting with deep vein thrombosis. Materials and Methods: The study included 10 young patients presenting to our hospital with deep vein thrombosis and 10 age and sex matched healthy controls without any history of thrombosis. The thrombophilia work up done in the cases and controls included the prothrombin time (PT), Activated partial thromboplastin time (APTT), Platelet count, general blood picture (GBP), Protein-C levels, Protein-S levels, Antithrombin-III(AT III) assay, Fibrinogen levels, factor-VIII levels, Lupus anticoagulant (LA) and d-dimer. Results: There was no difference in the mean age in the case and control groups (range 6–21 years). The M:F ratio was 4:1 for both. Swelling of the extremity was the commonest presentation (80 %) followed by pain (60 %). Hemiparesis was present in one patient. The APTT values were significantly lower in the cases (p = 0.031) compared to the cases whereas the PT in both was comparable.

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Topic: Lab Hematology PO 93 Lupus Anticoagulant Testing by Dilute Russel Viper Venom Test (Drvvt)-Nims Experience Abid Hussain, Shantveer G. Uppin, T. Roshni Paul, Kalapala Stalin, Liza *Rajashekhar, **Amvr Narender Department of Pathology, *Rheumatology, **General Medicine, Nizams Institute Of Medical Sciences (NIMS) Introduction: Lupus Anticoagulants (LA) are antibodies directed against phospholipids/protein complexes. Anti cardiolipin antibodies

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 (ACA) and Lupus Anticoagulant (LA) constitute Anti Phospholipid Antibodies. Multiple screening tests are recommended (e.g.: APTT, dilute PT and dRVVT). The dRVVT is one of the most important screening procedures. Aims and Objectives: To evaluate utility of dRVVT in screening of LA. Materials and Methods: All patients tested for LAs by dRVVT from January 2012 to June 2014 were included. An integrated test involving screening and confirmatory tests using STACLOT DRVV SCREEN and STACLOT DRVV CONFIRM reagents was performed using fully automated coagulation analyzer. dRVVT values [1.2 were taken as positive. Results: During the study period there were 908 requests for Drvvt, of which 211 cases were positive for LA. These included 43 males & 168 females (M:F = 1:3.9), with age ranging from 6 to 82 years. The varied presentations included, SLE (37) Thrombotic episodes (20), Fever (18), Gangrene (16), Recurrent Abortions (13), Bleeding Tendencies (12), Rash (8) Miscellaneous (22). Discussion: Accurate and timely detection of lupus anticoagulants (LA) is of diagnostic and prognostic importance due to the association of persistent LA with thrombotic disease. Antibody heterogeneity and assay variability complicate LA detection and weak antibodies can go undetected. The LA is less frequently positive in APS and is thus regarded as a less sensitive but more specific test for detection of APL antibodies. So, a repeat testing after 12 weeks is to be performed to confirm the persistence of LA. Conclusion: Although there are a number of test systems designed to detect the presence of LA, no single test system is 100 % sensitive or specific. Because of the inherent heterogeneity of LA, Laboratories must be able to perform two or three screening tests to identify the presence of LA. Keywords Lupus anticoagulant, dRVVT, SLE

Topic: Clinical/Adult Haematology PO 94 Study of Fibrinolytic Parameters in Indian Patients with Venous Thrombosis Aniket Prabhudesai, Bipin Kulkarni, Shrimati Shetty, Kanjaksha Ghosh Department of Haemostasis and Thrombosis, National Institute of Immunohaematology (ICMR), KEM Hospital Campus, Parel, Mumbai, India Summary: Impaired fibrinolytic function is increasingly being reported to be the cause of several kinds of thrombotic diseases. The study is being done to establish co-relation between impaired or defective fibrinolytic system and venous thrombosis in Indian patients. Introduction: About 2/3rd of the patients with venous thrombosis are found to be negative for the conventional thrombophilia markers Protein C, Protein S, Antithrombin and Factor V Leiden, though many of them have strong history of recurrent thrombosis and thrombosis in unusual sites. The role of defects in the fibrinolytic system as causative factors for thrombosis in Indian patients with venous thrombosis is investigated in the study. Materials and Methods: 52 patients with an evidence of thrombosis at the time of presentation, who had stopped anticoagulants at least 15 days prior, were included in the study. Fibrinolytic parameters Plasminogen (PLG), Tissue Plasminogen Activator (TPA), Plasminogen Activator Inhibitor (PAI-1), Thrombin activatable fibrinolysis inhibitor (TAFI); Thrombomodulin (TM), Tissue factor pathway inhibitor(TFPI) along with Protein C, Protein S, Antithrombin were measured by ELISA. PAI-4G/5G and Factor V Leiden polymorphisms were studied by allele-specific PCR and direct sequencing. Results: 9 patients (17 %) were deficient for at least one of the

S513 conventional anti- coagulant proteins and 13 patients (25 %) were found to have defect in at least one fibrinolytic protein. 10 patients (19.23 %) showed grossly elevated levels of plasma PAI-1 levels, 5 patients (9.61 %) had the PAI-4G/4G genotype, whereas 2 patients (3.84 %) had elevated plasma TAFI levels and 1 patient (1.92 %) had low plasma TFPI level as compared to the Normal Pooled Plasma. Conclusion/Diagnosis/Impression: Though preliminary, the data shows that investigating for a combination of markers both in the anticoagulant system and in the fibrinolytic pathway will facilitate in a comprehensive study for the causative mechanisms for thrombophilia in Indian patients with venous thrombosis. Keywords Fibrinolysis, Venous thrombosis, Thrombophilia

PO 95 Thrombophilic Risk Factors are Uncommon in Young Patients with Retinal Vein Occlusion in North India J. Ahluwalia, R. Sandeep, 1S. Varma, 2A. Gupta, S. K. Bose, J. Masih, R. Das, N. Kumar, S. Naseem, P. Sharma, M. U. S. Sachdeva, N. Varma Departments of Hematology, 1Internal Medicine and 2Opthalmology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Objective: RVO is a cause of visual loss in the elderly and is often associated with dyslipidemia, hypertension and diabetes mellitus. Not much is known about the risk factors associated with RVO in the young. We present the laboratory data of thrombophilic risk factors in young patients RVO to assess their role as risk factors in this condition. Materials and Methods: The thrombophilia parameters listed above were analysed from the laboratory records of 50 patients with the clinical diagnosis of RVO, aged less than 50 years. Results: A single prothrombotic factor was seen in 2 (4 %) cases. The highest positivity was for the antiphospholipid antibodies (lupus anticoagulant in 6 %, anticardiolipin antibodies in 2 % and anti-beta 2 glycoprotein 1 in 10 % cases). Other than 1 case where antiphospholipid syndrome was confirmed, these were transient. One patient had antithrombin deficiency. Protein C, Protein S deficiency and Factor V Leiden mutation were not seen in this group. Conclusion: Our data suggests that these thrombophilia risk factors are not commonly associated with RVO in the young and there is a need for studies on other factors which contribute to the development of this condition. Keywords Retinal vein occlusion, Thrombophilia

Topic: Hematology PO 96 The External Quality Assessment Scheme in Coagulation: Five Year’s Experience as a Participating Laboratory Dr. Jayani D. Patel, Dr. Swapnil N. Patel Department of Pathology, Pramukhswami Medical College, Shree Krishna Hospital, Karamsad, Gujarat, India Objectives: The attainment of quality services in a laboratory requires a comprehensive Quality Assurance Programme which includes both Internal and External quality control material. External Quality Assessment Scheme (EQAS) programmes are accepted around the world as invaluable tools by laboratories to assess the performance of their testing systems. Results are objectively compared to other laboratories, using the same methodologies for every parameter. The goal of this study was to

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S514 review EQAS results from time to time in an effort to improve the performance of the laboratory. Materials and Methods: This is an observational study in which we have evaluated our EQAS test result of the past five years, from 2009 to 2013. We receive samples in lyophilised form in 4 vials which are stored at 2–8 °C in the refrigerator. The samples are reconstituted with 1 ml of distilled water at the time of procedure. The samples are received quarterly in a year. The test results of all the samples are analysed and documented. Results: Satisfactory results were obtained in all the cycles except four times. Discrepancy was observed in Prothrombin time INR (International Standardized Ratio). Root cause analysis was performed and necessary action taken. Conclusions: This participation in EQAS over the last 5 years has helped us significantly to improve our laboratory services in terms of performance evaluation, patient care and overall quality of laboratory practices. Keywords External quality assessment scheme, Quality control, Quality assurance

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 presented to our department with bleeding manifestations and no significant past or family history of bleeding were included in this study. All pts underwent a detailed hemostasis work up and investigations for underlying cause. Results: From 2010 to 2013, 6 patients were found to have acquired inhibitors. Out of six, 3 patients were having inhibitors against FVIII, two against Vwf and the remaining patient had inhibitors against factor XIII. Five patients received immunosuppression in the form of steroids and cyclophosphamide. Three patients received Rituximab, FEIBA and Novo 7 during treatment. Inhibitor titre decreased gradually. Five responded clinically of which three had complete resolution of inhibitors. One died due to unrelated cause. Conclusion: In patients with acquired inhibitors FEIBA or activated factor VII forms the mainstay of management. Rituximab appears to be an effective and safe treatment for patients with acquired FVIII inhibitors

Table 1 pt

PO 97

.

Age (Y)/ sex

Associated disease

55/M

No

VIII (6.2 %)

Factor level

Inhibitor titer, BU

Bleeding events

Replacement

Immunosuppressant

Response

30

10

F = 19,000 IU, N7 =15mg EACA,FFP

CP, MMF, P, R,MPS

Responded

Negative

5

F = 500 IU

At diagnosis

After t/t

57

VIII (\1 %)

665

kikuchi ds

Vwf

Disseminated Intravascular Coagulation and Death Due to Snake Bite

1

2

24/F

Post partum

Dr. Mirza Hammad Ali Baig, Dr. Syeda Heena Kauser

3

19/F

SLE with

4

62/F

No

VIII (3 %)

13

Negative

3

F = 2,000 IU, N7 =10 mg

MPS, P, R

Responded

5

71/M

ANA 4+

XIII (\1 %)

–

–

2

Cr = 20

CP, P

Responded

6

25/f

SLE

Vwf (\1 %)

66

Negative

2

F = 2,000

CP, P

Responded

General Medicine, Al Ameen Medical College Summary: Disseminated intravascular coagulation is a serious and life threatening systemic complication of snake bites that can cause death if the treatment is delayed. Introduction: Disseminated intravascular coagulation is a serious and life threatening systemic complication of snake bites that can cause death if the treatment is delayed. Materials and Methods: Ninety-eight patients who experienced snakebite were enrolled in this study. We divided all the patients into three groups by the ISTH DIC scoring system: the normal, simple coagulopathy and DIC groups. The coagulopathy group included both the simple coagulopathy and DIC groups. We then conducted a case– control study. Results: There was a significant decrease in the Hct, protein, albumin, ALP and cholesterol levels in the coagulopathy group, and only the cholesterol level was deceased in the DIC group Leukocytosis and rhabdomyolysis were significantly associated with coagulopathy, and hemolysis and rhabdomyolysis were associated with DIC The presence of rhabdomyolysis was considered a risk factor for coagulopathy). These conditions continued for up to 6–7 days after the snakebite. Conclusion/Diagnosis/Impression: Evaluation of coagulopathy with using these characteristics is helpful to properly manage the patients who experience snakebite. Keywords DIC, Snake bite

PO 98 Acquired Coagulation Factor Inhibitors: A Case Series *Nilesh Wasekar, *S. Chandrakala, *Toshniwal Manoj, *Farah Jijina, **K Ghosh *Dept. of Hematology, Seth GS Medical College & KEM Hospital, Mumbai, **National Institute of Immunohematology (ICMR), KEM Hospital Campus, Mumbai Introduction: Acquired inhibitors are auto-antibodies directed against the factor VIII and other coagulation factors. This disorder may be associated with conditions like pregnancy, malignancy, rheumatoid arthritis and other auto-immune disorders. We report five patients diagnosed in the Hematology Department, KEM Hospital Mumbai, from 2010 to 2013. Materials and Methods: Patients who

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(\1 %) VIII (\1 %)

CP, MMF, P, R, MPS

Responded

54

–

Died

CP–Cyclophosphamide, Cr – Cryoprecipitate, F–FEIBA, MMF - Mycophenolate mofetil, MPS - Methyl prednisolone, N7- Novoseven, P Prednisolone, R – Rituximab

Topic: Haematology PO 99 Primary Antiphospholipid Antibody Syndrome Dr. Noora Mohd. Ahmed, 1Dr. Vanajakshi, 1Dr. Amina Shaik, 2 Dr. Vemula Srikanth, 2Dr. Alok Ranjan, 3Dr. Mallikagoyal Department of 1Haematology, 2Neurology and 3Ophthalmology, Apollo Hospitals, Jubilee Hills, Hyderabad, India Objective: To establish the diagnosis of Primary antiphospholipid antibody syndrome in a 19y/o female with spontaneous unprovoked cerebrovenous sinus thrombosis. Methods: A 19 y/o female presented with severe headache, vomiting and photophobia. Fundoscopy showed papilloedema. Patient is a known case of hypothyroidism. Imaging studies revealed sagittal sinus and bilateral transverse sinus thrombosis which was managed by lumboperitoneal shunt and the patient was put on warfarin. Laboratory investigations done, included CBP with ESR, serum TSH levels, Antinuclear antibody Profile, Thrombophilia profile, Coagulation studies and Anticardiolipin antibody panel. Antiphospholipid antibody syndrome can occur as a primary disorder or as a secondary disorder when associated with some other autoimmune disorder such as SLE. For diagnosis, the patient must have at least one clinical criterion (either vascular event or pregnancy complication) and one laboratory criterion (aCL antibody or lupus anticoagulant positive). Results: CBP and ESR were normal.TSH levels were normal. Antinuclear antibody panel was reported as negative. Coagulation studies revealed increased PT and aPTT, which were not corrected by mixing with normal patient plasma. DRVVT assay showed increase in both screen time and confirmation time. Protein C activity was decreased. Protein S and antithrombin levels were normal. Anticardiolipin antibody panel done

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 showed positivity for IgM antibody. Factor V Leiden mutation was absent and anti-b2 glycoprotein I antibodies were negative. Conclusion: The above findings are suggestive of Primary Antiphospholipid antibody syndrome with decreased Protein C function secondary to warfarin administration or LA/aCL antibodies. The patient is stable with regular follow-ups. Keywords Pathology, Haematology, Antiphospholipid, Antibody, Thrombosis

Topic: Clinical/Adult Haematology PO 100 Microparticles as Potentialbiomarkers for Anticoagulant Therapy in Women with Pregnancy Loss

Department of Hemostasis and Thrombosis, National Institute of Immunohaematology, 13th Floor, New Multistoreyed Bldg, KEM Hospital Campus, Parel, Mumbai 400012 Summary: In a search for the cause and diagnosis ofunexplained pregnancy loss (PL), cell derived microparticles (MPs) seem tobe a promising new thrombophilia marker. It was observed that MPsdecrease significantly as anticoagulant therapy progresses duringpregnancy in such women and successful pregnancy outcome was observedwhere MP levels normalized. Introduction: Anticoagulant therapy has been found to be beneficial in womenwith PL. Recently, elevated MPs, a new prothrombotic marker, have beenfound associated with PL. The main aim of the study was to analyze the MPprofile during pregnancy in women with PL on heparin and aspirintherapy. Materials and Methods: The study comprised of 25 such patients alongwith 25 healthy pregnant women. Enumeration of circulating MPs, mainly annexinV along with endothelial (CD62e) and platelet (CD41a), was done by flowcytometry. Statistical significance was assumed at p \ 0.01, 99 % confidence interval and were calculated using paired Student’s t test. Results: 20 patients had exaggerated MPs at the onset of pregnancy. In 15, the MPs decreased as therapy continued and in 14, the levels normalized; theoutcome being successful pregnancy with live births. In 2 women only onaspirin, the exaggerated microparticle levels increased even more aspregnancy progressed. Both suffered from PL. Conclusion: Though preliminary, the data shows that MPs decreasesignificantly as therapy progresses and successful pregnancy outcome wasobserved where MPs normalized. It is possible that MPs may serve asimportant biomarkers for anticoagulation therapy and provide indications foradjustment of dosage. This is a novel finding which can be extendedto other thrombosis complicated diseases. All these studies could thusopen new areas of investigation for the understanding, follow up and therapeutichandling in such patients. Keywords Microparticles, Pregnancy loss, Uteroplacental thrombosis

PO 101 Acquired and Inherited Thrombophilia in Women with Recurrent Pregnancy Loss Rucha Patil, Kanjaksha Ghosh, Sonal Vora, Shrimati Shetty National Institute of Immunohaematology, 13th Floor, New Multistoreyed Bldg, KEM Hospital Campus, Parel, Mumbai 400012 Summary: Thrombophilia markers are important contributing factors in recurrent pregnancy loss (RPL) and should be screened in the order of their prevalence; i.e. among antiphospholipid antibodies–anticardiolipin antibodies (ACLA), followed by antiannexinV (AAnnVA), antib2

S515 glycoprotein1 (Ab2GP1) and lupus antibodies (LA); and among genetic thrombophilia-protein S (PS) followed by PAI-1 4G/4G polymorphisms and protein C (PC) deficiency. Introduction: 15 % of reproducing couples suffer from pregnancy loss (PL) and recurs in 2–3 %. The most frequently hypothesized cause of unexplained PL refers to a defective maternal haemostatic response leading to uteroplacental thrombosis. The present study aims to investigate the prevalence of different genetic and acquired thrombophilia markers in a large series of Indian women with RPL. Materials and Methods: The study comprised of 578 women with no apparent etiological causes of RPL and 115 healthy women with at least one live child and no history of PL or thrombotic episode. Genetic thrombophilia- PC, PS, antithrombin (ATIII), Factor V Leiden mutation (FVL) were done in 578 patients and EPCR 23 bp insertion, MTHFR polymorphism, PAI-1 4G/5G polymorphism in 488 patients. Acquired thrombophilia- LA and ACLA were done in 550 patients; Ab2GP1 and AAnnVA could be analyzed only in 532 patients. p values were calculated with two tailed Fisher’s exact test, and statistical significance was assumed at p \ 0.01, 99 % confidence interval. Results: Among genetic thrombophilia, the risk of PL was highest with PS deficiency (16 %, 99 % CI, p = 0.006) followed by PAI-1 4G/4G (19 %) polymorphisms, PC deficiency (6 %). Among antiphospholipid antibodies, the risk of PL was the highest in women with ACLA i.e. 24 and 2.6 % in patients and controls, respectively. This was followed by AAnnVA—18 % against 1.7 %, Ab2GP1—11 % against 1.7 % and LA—8 % against 0.86 % in patients and controls, respectively (99 % CI, p \ 0.01). Conclusion: Thrombophilia, both genetic and acquired, is an important contributing factor in RPL and women with unexplained PL should be screened in a cost effective method in the order of their prevalence.

Topic: Clinical/Adult Haematology PO 102 Phenotypic and Genotypic Characterization of Antithrombin (AT) Deficiency in Indian Patients with Thrombosis Rutuja Deshpande, Bipin Kulkarni, Kanjaksha Ghosh, Shrimati Shetty Department of Haemostasis and Thrombosis, National Institute of Immunohaematology (ICMR), KEM Hospital Campus, Parel, Mumbai, India Summary: Antithrombin (AT) deficiency is an autosomal dominant disorder which increases risk of thrombosis by 10- to 40-folds. The genetic screening of this gene has not been done in India and is important for counseling the patients as well as forming a database. We have found a deleterious mutation in 1 patient. This patient and his family members will be screened and counseled so as to take any precautionary measures if required. Introduction: Complete AT deficiency has lethal consequences and the heterozygous deficiency significantly increases (10- to 40-folds) the risk of thrombosis-recurrent venous thromboembolism (VTE), which includes both deep venous thrombosis (DVT) and pulmonary embolism and also arterial thrombosis. Molecular characterization of AT deficiency has not yet been carried out in India. Hence, this study will be carried out to provide a comprehensive picture of molecular defects in AT deficient cases in Indian population. Materials and Methods: 50 patients with DVT were screened for AT gene mutation. Direct sequencing of all seven exons of the AT gene was performed with the ABI 3130 sequencer to screen polymorphism and mutation in this gene. Results: Out of 50 patients, a novel variant i.e. pro305his (exon 5 of AT gene) was found in one patient. Herein, proline, a non polar amino acid was replaced by histidine, a polar amino acid. This mutation was found to be deleterious by software panther 8.0. Other polymorphisms detected

123

S516 are, heterozygous serpinc1T[C (rs78972925) in the promoter region and serpinc1C[G (rs677) found in homozygous state in 10 patients and in heterozygous state in 1 patient. Conclusion/Diagnosis/ Impression: Genetic counseling and screening of family members of this patient will be done which will help to provide precautionary measures if they are also found to have the deleterious mutation. More patients need to be screened for AT gene mutation in Indian population which will not only help give appropriate treatment and counseling to the patients but also help establish a database for the same. Keywords Antithrombin, DVT, Mutation

Topic: Non Hodgkins Lymphoma PO 103 Primary CNS Lymphoma with Steroid Effect: A Diagnostic Dilemma Y. Khonglah, V. Raphael, A. K. Jitani, *A. Kakoti, **P. Phukan Departments of Pathology, *Neurosurgery and **Radiology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences, Shillong, Meghalaya Summary: Primary CNS lymphoma is rare and maybe radiologically diagnosed as demyelination leading to steroid treatment with disastrous morphological results. It is prudent for the pathologists to be aware of such lesions and employ immunohistochemistry for a definitive diagnosis. Introduction: Primary CNS lymphoma is a rare neoplasm. Its incidence is 0.4 % in immunocompetent and 5 % in immunodeficient patients. It is extremely sensitive to corticosteroids which cause loss of distinctive histological findings or even total disappearance of lesions. Materials and Methods: A 55 year old immunocompetent man presented with headache and difficulty in speech for 4 months. MRI was suggestive of cerebral tumefactive demyelination, based on which he was started on a 6 weeks course of steroids. However, the patient deteriorated and a biopsy for intraoperative crush smear followed by a routine histopathological examination was sent. Results: Crush smears were cellular with numerous apoptotic/degenerated cells. A differential diagnosis of inflammatory/demyelinating lesion was given with a note that malignancy could not be ruled out. On routine H&E sections, a definitive morphological diagnosis could not be made. A panel of immunohistochemistry confirmed the diagnosis of Primary CNS lymphoma-Diffuse large B cell type. Conclusion: This case highlights that lymphoma should be considered in the differential diagnosis of intracranial space occupying lesions. As pathologists, it is prudent to be aware of the disastrous effect of steroid intake on the morphology of CNS lymphomas. Unless clinically indicated, intake of steroids should be avoided prior to performing a neurosurgical biopsy of intracranial lesions. Keywords Primary CNS lymphoma, Steroid effect

Topic: Laboratory Hematology PO 104 Hairy Cell Leukemia With Hypoplastic Marrow and Without Splenomegaly Masquerading Aplastic Anemia: Report of Two Cases Surabhi, Khaliqur Rahman, Manish K. Singh, Ruchi Gupta, Soniya Nityanand Department of Hematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Summary: Hairy cell leukemia presenting with pancytopenia, in absence of splenomegaly and concomitant hypoplastic marrow can mimic aplastic anemia. We present here two such cases that were diagnosed with the help of diligent morphological examination aided by immunophenotyping by flow cytometry. Introduction: Hairy cell leukemia (HCL) has a unique clinical, morphological and immunophenotypic features. Absence of splenomegaly and marrow hypoplasia is unusual. We present herein two such cases that were diagnosed with the help of immunophentyping. Materials and Methods: Detailed history and clinical examination was performed. Bone marrow aspiration and biopsy were carried out with all aseptic precautions. Smears were prepared and stained with May-grunwald-Giemsa stain and biopsy slides stained with H&E and reticulin stain. Immunophenotyping was done through flow cytometry. Results: Both the cases presented with easy fatigability and generalized weakness for past 3 months. The examination revealed pallor without organomegaly or lymphadenoapthy. Peripheral blood smear examination showed occasional atypical lymphoid cells with monocytoid nucleus and moderate amount of cytoplasm with projections. Bone marrow aspirate was particulate and hypo-cellular. Trails showed presence of atypical cells of similar morphology. Bone marrow biopsies in both the cases were hypoplastic with scattered atypical cells. Flow cytometry revealed the abnormal larger cell population to be positive for CD19/CD20 (strong)/ CD22/CD38 (dim)/CD25/CD123/CD11c and CD103 and weak lambda light chain restriction. Conclusion: HCL patients presenting with pancytopenia, in absence of splenomegaly with hypoplastic marrow can mimic aplastic anemia. A vigilant peripheral blood smear and bone marrow aspirate examination to look for any suspicious cells, and if present a subsequent immunophenotyping, can be helpful in differentiating these two entity. Keywords Hairy cell, Bone marrow hypoplasia, Flow cytometry

Topic: Haematopathology PO 105 Leukemic Phase of Mantle Cell Lymphoma (Blastoid Variant) Mimicking Acute Leukemia: A Series of 4 Cases with Review of Literature K. S. Archana, Vikrant Bhar, Narender Kumar, Shano Naseem, Jasmina Ahluwalia, Reena Das, *Subash Varma, Neelam Varma *Department of Haematology and Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh Introduction: Mantle cell lymphoma (MCL) is a mature B-cell neoplasm of naive B cells arising from the mantle zone of the lymph nodes. It accounts for about 3–10 % of all NHL. Blastoid variant is the uncommon one and occurs in 10–20 % of patients with MCL. It is a very aggressive neoplasm with a poor prognosis. Blastoid variant with leukemic conversion is much rarer entity and often confused with acute leukaemia. Here we report a 4 cases of MCL (blastoid variant) with leukemic conversion which on morphology mimics acute leukemia. Case Series: Materials and Methods: The retrospective analysis was done in Department of hematology, PGIMER, Chandigarh. Total 9 cases of MCL with leukemic conversion were retrieved in last 5 years. Out of these 9 cases, four showed blastoid morphology. These 4 cases were analysed extensively. Results: The mean age of these 4 cases is 63 years and all were males. All patients had high TLC with a mean of 89.2 9 109/L with 72 % of lymphoid cells. These cells showed blast like morphology on peripheral blood and bone marrow smears. On flowcytometry the cells were negative for immaturity markers such as Tdt and CD34. These cells showed expression of B lineage markers along with presence of CD5. All the four cases showed monclonality in the form of light chain expression. Cycin D1 postivity was

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 demonstrated on IHC. Conclusion: The Mantle cell Lymphoma (MCL)—blastoid variant is an uncommon entity. The leukemic conversion of this variant may be confused with acute leukemia. The Hematopathologist should keep this possibility as a differential diagnosis for acute leukemia. The flowcytometric Immunophenotyping/immunohistochemisry are absolutely necessary for confirmation or exclusion. Keywords Mantle cell lymphoma, Blastoid variant

PO 106 A Rare Case of Mantle Cell Lymphoma Without Lymphadenopathy and With CD23 Positivity 1

Ashutosh Jain, 2Vijai Tilak, 3I. S. Gambhir, 4Nilesh Kumar, Hema Goyal

5 1

Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh. 2Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh. 3Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh. 4Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh. 5Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh Summary: Mantle cell lymphoma(MCL) represents 2.5–7 % of all non Hodgkin’s lymphomas, usually presenting with lymphadenopathy and CD23 positivity on flow cytometry, however MCL is characterized by translocation t(11;14) (q13;q32), resulting in overexpression of cyclinD1. Introduction: Here we introduce a seventy five year old man persenting with complaint of progerssively increasing heaviness in left half of abdomen for past 10 months. History was negative foe fever, weight loss, night sweats. Physical examination was noteworthy for moderate pallor and massive splenomegaly. There was no lymphadenopathy. Materials and Methods: Investigations showed: hemoglobin 7.1, total leukocyte count 98400, nutrophils 04 % and lymphocytes 93 %. Peripheral blood smear showed markedly increased number of leukocytes with fair number of smudged cells. Bone marrow aspirate examination was suggestive of chronic lymphocytic leukamia (CLL). Results: Immunophenotyping results were disfavouring CLL as according to scoring system a score of 4 or 5 is reqired for diagnosis of CLL whereas here the score was 3. IgH/CCND1 fusion signal was detected in 92 % cells by flourescent in situ hybridization (FISH). Conclusion/Impression: The distinction between chronic lymphocytic leukamia (CLL) and mantle cell lymphoma (MCL) has important clinical implications. Typically CLL is CD23 positive whereas MCL is CD23 negative. The majority of MCL cases co-express CD20, CD5, BCL2 and cyclin D1. The distinguishing feature of MCL is juxtaposition of the cyclin D1 locus and regulatory regions of the Ig heavy chain gene via the t(11;14) (q13;q32), resulting in the overexpression of cyclin D1. Keywords Mantle cell lymphoma, Cyclin D1, Chronic lymphocytic leukamia, FISH, CD23

Topic: Lab Hematology PO 107 Bi-clonal Chronic Lymphocytic Leukemia: A Case Report Divya Shelly, Kiran Ghodke, Asma Bibi, Sitaram Ghogle, Y. Badrinath, Nikhil Patkar, P. G. Subramanian, Prathibha Amare, Sumeet Gujral, Prashant Tembhare Hematopathology Laboratory, Department of Pathology, Tata Memorial Hospital, Parel, Mumbai 400012

S517 Introduction: Demonstration of mono-clonality by surface kappa or lambda light chain restriction is the main feature of immunophenotypic diagnosis of B-cell non-Hodgkin’s lymphoma (NHL). B cells expressing both light chains are considered as polyclonal and nonneoplastic. However, rare cases of B-NHL with bi-clonal tumor cells are reported. We report a case of CLL with typical morphologic and immunophenotypic features but bi-clonal pattern of light chain expression. Materials and Methods: 65-year-old asymptomatic patient presented with high WBC count. CBC, peripheral smear (PS), immunophenotyping and cytogenetics study on peripheral blood was performed. Immunophenotyping was done using 8-color antibody panel on Navios (Beckman Coulter) and data was analyzed by Kaluza software. Results: CBC revealed WBC count of 28.9 9 109/L and PS showed 88 % atypical lymphocytes. Flow-cytometric immunophenotyping demonstrated abnormal B-cells expressing moderate CD19, dim CD20, moderate CD5, variable CD23, variable CD43, dim CD200, dim surface CD22, bright CD45. On evaluation of surface light chains, two subsets of abnormal B-cells were noted, one positive for kappa and the other positive for lambda. These findings were consistent with diagnosis of bi-clonal CLL. Cytogenetic analysis revealed 13q deletion. Conclusion: This report calls attention to uncommon entity of bi-clonal CLL and highlights an example in which light chain ratios can be deceiving leading to misdiagnosis. Careful attention to the morphologic features of the blood smear and the full immunophenotype are important for correct diagnosis. Keyword Bi-clonal chronic lymphocytic leukemia

Topic: Laboratory Haematology PO 108 Prognostic Value of Smudge Cell Percentages on Peripheral Blood Smear in Chronic Lymphocytic Leukemia 1

Prasanna Bhanshe, 1Kiran Ghodke, 1Mukund Sable, Nilesh Deshpande, 1Nikhil Patkar, 1P. G. Subramanian, 1 Sumeet Gujral, 1Sitaram Ghogale, 1Y. Badrinath, 2 Pratibha Aamre, 3Manju Sengar, 1Prashant Tembhare 1

1

Hematopathology Laboratory, Tata Memorial Centre, 2Cytogenetics Laboratory, Tata Memorial Centre, 3Adult Hemato-Lymphoid Disease Management Group, Department of Medical Oncology, Tata Memorial Centre Objectives: Smudge cells are ruptured chronic lymphocytic leukemia (CLL) cells appearing on the blood smears of CLL patients. Recently, GS Nowakowski et al. (JCO 2009) showed smudge cell percentage as an independent prognostic factor in CLL. In this study, we evaluated the role of smudge cell percentage in prediction of survival in CLL patients. Methods: Peripheral Blood smears of 57 CLL patients (age ranges 35–70 years and M:F ratio 3:1) analyzed for smudge cell percentages over a period of 5 years (2008–2012). The relationship between percentage of smudge cells, patient survival, and other prognostic factors was studied. Results: Of the 57 CLL patients 19.3 % had Rai stage 0 and 1 each, 17.5 % had stage 2, 12.3 % had stage 3 and 31.6 % had stage 4. The median smudge cell percentage was 20 % (range 2–60 %, mean 21). About 93 % cases belonging to early rai stage (0, 1 and 2) had more than 30 % smudge cells while 48 % cases were of stage 3 and 4 with less than 30 smudge cells (p \ 0.0001). Median follow up was 23 months (ranges 8–56 months). Correlation of Smudge cell percentage with Rai staging, cytogenetic data and overall survival will be statistically analyzed and presented in the meeting. Conclusion: In this study, premature analysis of data indicates the good prognostic value in CLL patients with Rai stage 0, 1 and 2 when

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S518 compared with Rai stage 3 and 4. However the final conclusion will be presented in the meeting. Keywords Chronic lymphocytic leukemia, Smudge cells

Topic: Role of CD200 in Chronic Lymphoproliferative Disorders PO 109 Role of CD200 in Chronic Lymphoproliferative Disorders Dr. Radhika Sekhri, Dr. Sonal Jain Rajiv Gandhi Cancer Institute and Research Centre Summary: CD200 is a relatively useful marker in differential diagnosis of mature B-cell neoplasms by multiparametric flowcytometry. Studies so far have demonstrated its utility in differentiating chronic lymphocytic leukemia (CLL) from mantle cell lymphoma (MCL). It has also been found to be useful in differentiating other chronic lymphoproliferative disorders (CLPDs). Introduction: The membrane glycoprotein MRC OX-2 (CD200) is expressed in a wide range of B-cell derived neoplasms and has diagnostic utility in their differential diagnosis. Materials and Methods: Peripheral blood samples of 25 patients of CLPDs from March 2014 to July 2014 were analyzed. CD200 expression was correlated with its complete immunophenotype. Its utility was evaluated and compared with respect to conventional scoring system. Results: Out of 25 cases examined, 18 were of CLL, 5 were of SMZL and 1 each of hairy cell leukemia (HCL) and its variant (HCL-v). Analysis of CD200 expression revealed 100 % immunoreactivity in CLL cases (18/18) while 60 % in SMZL (3/5). In addition, CD200 was expressed in HCL while was negative in HCL-v. Conclusion: CD200 may be a useful immunophenotypic marker in the evaluation of B-cell derived neoplasms, however its utility in the differential diagnosis of CLPDs has to be explored in a larger patient cohort. Analysis of its expression has several diagnostic and potentially prognostic applications in the flow cytometric evaluation of lymphoid malignancies. Keywords CD200, B-cell neoplasms, Chronic lymphoproliferative neoplasms

Topic: Laboratory Haematology PO 110 ‘Hepatosplenic Gamma–Delta T-Cell Lymphoma’ Involving Peripheral Blood: A Case Report 1

Dr. Reena Mittal, 1Dr. Anita Chopra, 2Dr. Rita Sood, 1 Dr. Rajive Kumar 1

Department of Laboratory Oncology, 2Department of Medicine, All India Institute of Medical Sciences, New Delhi Summary: Hepatosplenic gamma–delta T-cell lymphoma is a rare form of primary extranodal lymphomas, derived from cytotoxic T-cells of cd T-cell receptor type. It predominantly occurs in young males. Bone marrow involvement is common; however peripheral blood involvement is rare and often confused with acute leukemia. Here, we describe a case of Hepatosplenic gamma-delta T-cell lymphoma who presented with peripheral blood involvement and leucoerythroblastic picture. Flowcytometric immunophenotyping of peripheral blood confirmed the diagnosis of Hepatosplenic gamma-delta T-cell lymphoma. Case Report: A 21-year-old male presented with chief complains of fever, anorexia and abdominal pain for 1 month duration. Physical examination

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 revealed hepatosplenomegaly, bleeding manifestations, pallor and icterus. Baseline laboratory investigations showed Hb 7 g%, TLC 5,000/ cumm and platelets 45,000/cumm. Peripheral blood smear examination revealed leucoerythroblastic picture with 15 % atypical cells resembling blasts. Flowcytometric immunophenotyping of peripheral blood showed that these atypical cells express stronger CD45 positivity in CD45 versus side scatter plot as compared to normal blood T lymphocytes. These cells were CD3+, CD2+, CD7+, CD10+, CD57-, CD56-, CD16-, CD8-, CD4-, CD1a-, CD19-, CD34-, Tdt-, CD5-, CD13-, CD33-, CD65-, TCR ab- and TCRcd+. Subsequent bone marrow examination revealed near total replacement by atypical lymphoid cells which were positive for surface CD3 on immunohistochemistry. Conclusion: Hepatosplenic gamma–delta T-cell lymphoma is an aggressive form of extranodal lymphomas which rarely involves peripheral blood. In cases of peripheral blood involvement, immunophenotyping is very important for early diagnosis and treatment as the disease has fatal clinical course. Keywords Hepatosplenic T cell lymphomas, Gamma–delta T-cell, Flowcytometry

PO 111 Hairy Cell Leukemia: A Rare Case Report Dr. Sana Fatima, Dr. Zaheda Kausar, Dr. Naval Kishore, Dr. N. Ezhilarasi Upgraded Department of Pathology, Osmania Medical College, Hyderabad, Telangana Hairy cell leukemia is a rare clonal chronic lymphoproliferative disorder commonly seen in males in the middle years of life. A 60 year old male presented with shortness of breath, pedal edema and moderate splenomegaly. Flow cytometry confirmed the diagnosis. Details will be presented. Keywords Flow cytometry, Hairy cell leukemia, Splenomegaly, Lymphoproliferative disorder. PROF & HOD, Dr. N. EZHIL ARASI IAPM REG.NO.s: (1) AM-457/14 (2) LM-367 (3) LM-137 (4) LM-145

Topic: Hematopathology PO 112 Immunophenotypic Profile of Diffuse Large B Cell Lymphoma with Special Reference to Presence of c-MYC, CD5, and Ebstein Barr Virus 1

Y. Sharmila, 1Pampa C. Toi, 1Debdatta Basu, 2Biswajit Dubashi

Department(s) of 1Pathology & 2Medical Oncology, Jawaharlal Institute of Post Graduate Medical Education and Research (JIPMER), Puducherry, India Summary: Immunophenotypic classification of diffuse large B cell lymphoma (DLBCL) help us to predict the prognosis of the patient. Introduction: DLBCL is the commonest subtype of non Hodgkin lymphoma. Survival of DLBCL patients varies due to the molecular diversity of the tumor. Our study aimed at subclassifying DLBCL into germinal centre variant (GCV) and activated B cell variant (ABC) based on the Hans algorithm. We also studied the association of DLBCL with markers

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 like c-MYC, CD5, EBV and their impact on the clinical outcome of the patient. Materials and Methods: Diagnosed cases of DLBCL between Jan 2010 to June 2014 were included in the study. Morphologic subtyping was done and the cases classified as GCV and ABC variants using Hans algorithm. In addition, staining for CD5, c-MYC and EBV-LMP were also done and analysed to find the association of these markers with the clinical outcome. Results: 90 cases of DLBCL were included in the study. 64 cases (71 %) of ABC and 26 cases (29 %) of GCV were identified. 10 cases showed CD5 positivity, all cases were ABC subtype. c-MYC positivity was seen in 14 cases. We did not get a statistical signifance with clinical outcome both for immunophenotypic classification and for CD5 and c-MYC positive cases. EBV-LMP positivity was seen in only three cases. Conclusion: There was no significant difference in the clinical outcome of the patients between the two group. CD 5 and c-MYC positivity shows poor prognosis but was not statistically significant. Keywords DLBCL, cMYC, Hans algorithm

Topic: Hematology PO 113 A Case Series on Paediatric Lymphomas in a Tertiary Care Center Dr. J. S. Sukanya, Dr. J. Thanka, Dr. J. Julius Xavier Scott, Dr. N. Priyathersini Department of Pathology, Sri Ramachandra Medical College and Research Center, Chennai, India Summary: The incidence of paediatric lymphomas is raising throughout the world. In children lymphoma is the third most common childhood cancer. Introduction: Lymphoma, a common hematopoietic neoplasm has gained greater attention with the advent of various targeted chemotherapy. Lymphomas in children are generally aggressive malignancies such as Lymphoblastic lymphoma, Burkitts lymphoma, Large B cell lymphoma, and Anaplastic large cell lymphoma except Hodgkin’s lymphoma which has a favourable prognosis. Materials and Methods: We analysed all the cases of Paediatric Lymphomas coming to our hospital from 2009 till date. The histopathology, Immunohistochemistry and baseline investigations were reviewed. Result: In the past 6 years 25 paediatric patients were diagnosed with Lymphoma. Most of them had a male preponderance and fall within the age group of 4–17 years. 14 cases were diagnosed as Non-Hodgkin’s Lymphoma and 11 cases as Hodgkins Lymphoma including all the variants common in paediatric age group. Conclusion: The clinicopathological analysis of paediatric lymphomas in a tertiary care center showed predilection towards Non-Hodgkin’s lymphoma in paediatric age group. It is essential to identify them accurately as the cure rates for these patients are high. Keywords Paediatric lymphoma, Hodgkins, Non-Hodgkin’s, IHC.

Topic: Chronic Lymphoproliferative Disorder PO 114 Hairy Cell Leukemia-Variant: Clinical, Hematological and Immunophenotypic Profile of Six Cases from Single Tertiary Care Center of North India Swati Mahajan, Man Updesh Singh Sachdeva, Narender Kumar, Jasmina Ahluwalia, Reena Das, Neelam Varma Department of Hematology, PGIMER, Chandigarh

S519 Summary: Hairy-cell leukemia variant (HCl-v) is a rare, relatively ill-defined B-cell neoplasm. Distinguishing HCL-v from HCL-c and SMZL can be challenging, as clinical and pathologic findings may overlap, but accurate diagnosis is crucial for treatment and prognosis. We present 6 cases of HCLv from our center and discuss the clinical, haematological and immunophenotypic features of this rare entity. The study validates the use of the 4 core antigens, CD11c, CD25, CD103 and CD123, along with common B-cell antigens in establishing an accurate diagnosis. Introduction: Hairy-cell leukemia variant (HCL-v) is a distinct clinico-pathological entity with intermediate features between classical HCL (HCL-c) and B-cell prolymphocytic leukemia. Compared to HCL-c, HCL-v is a more aggressive disease and is no longer considered to be biologically related to HCL-c. Furthermore HCL-v is resistant to traditional HCL therapy, making accurate diagnosis imperative. Materials and Methods: Cases of HCL-v were selected from archives of Flow Cytometry Laboratory of Department of Hematology, diagnosed during 2007–2014. Review of demographic profile, clinical presentations, hematological investigations and immunophenotype was done. Results: Six cases of HCL-v were diagnosed in 7 years duration on the basis of classical immunophenotype, i.e. CD19+CD22+CD103+CD11c+CD25-CD123- with light chain restriction. All patients were males with mean age of 58 years. Four of 6 patients had massive splenomegaly. One patient was already on treatment and did not have enlarged spleen and another was postsplenectomy. Mean leukocyte count was 36.3 9 109/L (range 0.8–155 9 109/L). Only 1 patient had pancytopenia. Absolute monocytopenia was seen in 2 patients. The percentage of neoplastic lymphoid cells in bone marrow ranged from 10 to 77 %. Trephine biopsy revealed interstitial pattern of infiltration in all cases. Immunophenotyping showed CD20 positivity in 4 cases, lack of CD123 in all 6 cases and CD25 negativity in 4 with dim CD25 expression in 2 cases. Conclusion: HCL-v is a rare mature B cell neoplasm with distinct clinico-pathological profile. Its characteristic immunophenotype necessitates use of multicolour flow cytometry for a definite diagnosis, which is must for management of the patients. Keywords Hairy cell leukemia-variant, Flowcytometry, CLPD

Topic: Non-Hodgkin Lymphoma PO 115 Plasmablastic Lymphomas (PBL) in Immunocompromised Patients T. Sravani, Shantveer G. Uppin, T. Roshni Paul, Ashwani Tandon Dept. of Pathology, NIMS, Hyderabad Aims and Objectives: Plasmablastic Lymphoma is diffuse proliferation of large neoplastic cells that resemble lymphoma cells but have the immunophenotype of plasma cells.This work was done to study the clinical, morphologic and immunophenotypic profile of PBLs in immunocompromised patients. Materials and Methods: Immunocompromised patients with lesions at different sites and dignosed as PBLs between 2008 and 2014 were included in this study.The histomorphology of the cases were reviewed along with IHC panel. Results: There were 15 cases of proven PBLs in the study period. All were males with age ranging from 27 to 62 years. Sites of involvement were as follows—oral cavity (4), anorectal mass (1), abdominal lymphnode (1), cervical lymphnodes (4), chest wall swelling (1), scalp swelling (1), scrotal mass (1), nasal cavity growth (1), dorsal spine (1). The IHC profile was as follows LCA(9/15) positive, CD 138(12/13) positive, CD 3, CD 20 negative. Conclusion: Though PBLs are an uncommon lesion, the incidence is high in

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S520 immunocompromised patients. The sites involved are mainly extra nodal and IHC with CD 138 is essential whenever there is a strong suspicion. Keywords Plasmablastic lymphoma, Immunocompromised, CD138

PO 116 Analysis of Mature T-Cell Lymphomas with Bone Marrow Involvement Vikrant Singh Bhar, Man Updesh Singh Sachdeva, Narender Kumar, Prashant Sharma, Shano Naseem, Jasmina Ahluwalia, Reena Das, Pankaj Malhotra, Subhash Varma, Neelam Varma

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 histopathological examination of nodal or any extranodal site was included in the study. Results: Lymphoma cells were evident in 100 % of the trephine biopsy sections, 41.67 % of imprint smears and 33.33 % of aspiration smears. Interstitial pattern was seen in most of the lymphomas, either as a distinct pattern or in a mixed pattern (7/ 11). Diffuse pattern was seen equally as that of focal nodular pattern, as a distinct pattern and mixed pattern (4/11 each). Most of the cases with marrow involvement had bilateral bone marrow involvement. Only 16.7 % cases showed unilateral marrow involvement. To conclude, this study highlights the incidence and different patterns of involvement in the marrow. Keywords Non-Hodgkins lymphoma (NHL), Bone-marrow examination

PGIMER, Chandigarh

PO 118

Introduction: T- cell lymphomas are heterogeneous group of disorders with frequent bone marrow involvement by certain sub-types. Diagnosis of T-NHLs is relatively challenging even for experienced pathologist. This study aimed at comprehensive analysis of clinicopathological and immunophenotypic profile of T-NHL cases showing infiltration in bone marrow. Materials and Methods: T-NHL cases were retrieved from archives of Flow Cytometry Laboratory of Department of Hematology, PGIMER, Chandigarh. All cases diagnosed as T-NHL on immunophenotyping of bone marrow aspirate sample, over a period of 4 years (2010–2014) were selected. Cases were analyzed as per demographic profile, clinical features, peripheral blood and bone marrow findings and immunophenotype. Results: Thirteen cases of mature T-cell immunophenotype were retrieved, out of—cases who underwent flowcytometric immunophenotyping for CLPD in our institute. Mean age of the patients was 43.2 years (range 22–72 years). Majority of our patients (10/13) were male. All patients presented with one or more cytopenias. Ten had anemia and/or thrombocytopenia, 7 had bicytopenia and 2 cases had pancytopenia. Pattern of infiltration in the bone marrow was variable which included interstitial, diffuse, non-paratrabecular nodules and sinusoidal infiltration. Interestingly cases of hepatosplenic lymphoma mimicked acute leukemia often on morphology. On immunophenotyping all cases were positive for surface CD3. Conclusion: T-NHLs have varied clinical presentations. The patterns of bone marrow infiltration are also varied and immunophenotyping helps in diagnosis as well as sub-classification.

Primary Testicular Lymphoma: A Case Report V. Beshwanth Chowdary, Dr. Jyoti R. Kini, Dr. Hema Kini, Dr. Kausalya Sahu Department of Pathology, Kasturba Medical College, Manipal University, Mangalore Summary: Primary testicular lymphoma is a rare type of extranodal lymphoma affecting elderly men [60 years of age. It accounts for 1–2 % of all non-Hodgkin’s lymphomas and 5 % of testicular cancers. Introduction: Primary testicular lymphoma present usually with a unilateral testicular mass in 90 % of cases and bilateral involvement occurs in 35 % of cases. Materials and Methods: A 73 year old man presented with rapidly progressive painless swelling of right testis of 2 months duration. On examination, there was unilateral nontender testicular swelling with no other organomegaly or lymphadenopathy. Right high orchidectomy was performed. The specimen was sent for histopathological examination. Results: Histopathological examination showed features of testicular lymphoma. The subtype was confirmed by immunohistochemistry as diffuse large B-cell lymphoma. Conclusion: Primary testicular lymphoma constitutes 4 % of extranodal NonHodgkin’s Lymphoma. The most common histological type is diffuse large B-cell lymphomas. Rare subtypes are clinically important and must be recognized. Primary testicular lymphoma is a rare disease and needs high index of suspicion for early diagnosis.

PO 117 Haematological Profile and Bone Marrow Findings in Non-Hodgkin Lymphoma

Topic: Non Hodgkin’s Lymphoma PO 119

Dr. Anita Tahlan, Prof. Harsh Mohan, Dr. Tripti Kaur, Dr. Anshu Palta

Primary Bone Lymphoma: A Case Report of a Polyostotic Disease with Review of Literature

Government Medical College & Hospital, Chandigarh Introduction: Non Hodgkin lymphoma (NHL) is a heterogenous group of lympho-proliferative disorders originating in B, T or natural killer (NK) cells. In India its incidence is on the rise. Materials and Methods: To study the haematological findings in patients of NHL.To study the incidence and pattern of BM infiltration of various histological subtypes. To compare the diagnostic accuracy and relative efficacies of BM aspiration, BM biopsy imprints, BM trephine biopsy in NHL. The present study was conducted in the Department of Pathology, Government Medical College and Hospital, Chandigarh on consecutive patients undergoing bone-marrow examination for infiltration by NHL. Any patient who was confirmed NHL by

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E. Jajodia, J. Mishra, V. Raphael, Y. Khonglah, *A. Kakoti, **A. Handique, H. Nobin Department of Pathology, *Department of Neurosurgery, **Department of Radiology Summary: Primary bone lymphomas (PBL) account for nearly 7 % of primary malignancies of the bone. Only one-fourth of adult cases are polyostotic while one-half show soft tissue extension. We present a rare case of PBL of spine with polyostotic presentation and epidural extension. Introduction: PBL is defined as lymphoma that arises in bone with or without extension into adjacent soft tissue but without lymphoma elsewhere on staging. Fewer than 1 % of all the

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 lymphomas arise in bone. Even rarer is a polyostotic presentation involving the axial skeleton while sparing the limbs. Materials and Methods: A 65 years old male patient presented with tingling and weakness of both legs and difficulty in passing urine. Radioimaging showed multiple lytic-sclerotic rib and vertebral lesions with soft tissue component. No adenopathies were seen. A wide range of imaging tests, serum electrophoresis, FNAC and needle biopsies could not arrive to a diagnosis. So open biopsy specimens from two involved sites were submitted for histopathology. Results: With H&E, cytochemistry and immunohistochemistry panels we could rule out most of the differentials. A diagnosis of polyostotic primary bone lymphoma of the diffuse large B-cell lymphoma (DLBCL) type was finalised. Conclusion: Only 5 % of extranodal non-Hodgkins lymphoma occur in bones. However a consideration of polyostotic primary bone lymphoma should also be kept in mind in the differential workup of multiple lytic-sclerotic lesions in the axial skeleton. DLBCL is the most common among PBL. Keywords Primary bone lymphoma (PBL), DLBCL, Polyostotic, Epidural extension.

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S521 and IHC profiles. Their diagnosis and precise classification is difficult because of their rarity and complex biology. Theirrecognition is essential for establishing accurate diagnosis and adequate therapeutic strategy. Materials and Methods: Case report with clinopathological corelation with autopsy findings. Results: 42 year old retro-negative patient presented with a primary bone marrow lymphoma that resembled a DLCL with partial plasmablastic features but was CD20 and LCA negative with strong positivity for CD79a and showed weak lamda restriction. They tumour cells were negative for Kappa, TDT, CD99, PAX5, PanCK, CD30, CD43, CD56, CD4, ALK, CyclinD1, MUM1, CD138, LMP1, Anti MPO and bcl2. MIB index was low. CD79a, CD20 and MIB1 were repeated with the same outcomes.The unique IHC profile and clinical features prevented an accurate classification.The patient had a rapid downhill course and died.Autopsy showed fungal septecemia, multiorgan involvement and failure due to Fusarium Solani. There was no involvement of any major organ system or lymphnode by the tumour other than the primary site. Conclusion/ Diagnosis/Impression: We present a very rare case of a primary Bone Marrow DLBCL, with a unique IHC profile in a HIV negative individual. Unraveling the complex biology of such rare lesions will give more insight into the murky world of DLBCLs. Keywords Primary bone lymphoma, Plasmablatic lymphoma, Fungal septecemia

ALK-Positive Large B Cell Lymphoma: A Series of 3 Cases Dr. Gautami Kokil, Dr. Marie Therese Manipadam, Dr. Vikram Mathew Christian Medical College, Vellore Summary: ALK-positive large B cell lymphomas (ALK-positive LBCL) form \1 % of diffuse large B cell lymphomas. We present three cases with ALK-positive LBCL diagnosed in our institute in the last 5 years. Introduction: All three were male patients ranging in age from 22 to 58 years. Two of these presented with extranodal masses whereas one patient had cervical lymphadenopathy. Histopathological examination of the respective lesions showed high grade large cell lymphoma. Immunohisthochemistry confirmed the diagnosis. Materials and Methods: Routine tissue processing with haematoxylin and eosin staining and immunohistochemistry was done. Results: These three cases were diagnosed to have ALKpositive LBCL which was proven by immunohistochemistry. Conclusion/Diagnosis/Impression: ALK-positive LBCL are rare aggressive large B cell lymphomas. Despite multiagent chemotherapy the median survival of patients is 11 months. These tumours are usually negative for CD20 and hence are insensitive to rituximab therapy. Though rare, these tumours should always be in the differential diagnosis of CD20 negative large cell lymphomas.

Topic: Leukemia/Lymphoma PO 121 A Case of Unclassifiable Primary Bone Marrow Lymphoma: Clinicopathological and Autopsy Correlation with Brief Review of Literature V. Manu, P. Mayur, B. Sonia, Vikrant, R. Kapoor, Darmesh Department of Pathology, Armed Forces Medical College, Pune Summary: A rare case of Primary bone lymphoma with unique clinical features, presentation, and IHC profile, mimicking plasmablastic lymphoma in an immunocompetent patient with autopsy corelation. Introduction: Primary bone marrow lymphoma and plasmablastic lymphoma are rare entities with unique clinical features, presentation,

Topic: Infection PO 122 Disseminated Histoplasmosis in Immunocompetent Individual: A Single Centre Experience from a Tertiary Care Hospital in Eastern India Dr. De Dibyendu, Dr. Uttam K. Nath, Dr. S. S. Roy, Prof. M. Bhattacharyya Dept. of Haematology, Institute of Haematology and Transfusion Medicine, Medical College, Kolkata Introduction: Histoplasmosis is a rare fungal disease caused by dimorphic fungi Histoplasma capsulatum. Disseminated Histoplasmosis (DH) is defined as a clinical condition where fungus is present in more than one location. DH is the rarest and generally found in immunecompromised individual. Here we are presenting our experiences of the series of cases of Disseminated Histoplasmosis in immune-competent individuals who have been diagnosed in our institute in last 5 years. Materials and Methods: This is a single centre retrospective observational study, from May 2009 to April 2014. Only cases with Disseminated Histoplasmosis in otherwise healthy immune-competent individuals were included in the study. The Histoplasmosis is confirmed by either presence of Histoplasma in biopsy specimen from extra-pulmonary organ or by positive growth in fungal culture. Results: Total seven patients met the inclusion criteria. Five of the 7 patients presented with fever, six patients had weight loss, one patient had skin nodules, five patients had hepato-splenomegaly, and two patients had lymph-adenopathy.The laboratory investigation revealed anaemia in six out of 7 patients, and pancytopenia in 3 patients. Two patients had features of hemophagocytic syndrome. One patient with adrenal involvement died in hospital. The patient with skin nodule had recurrent relapses. The other patients had resolution of symptoms and clinically cured. Conclusion: Disseminated Histoplasmosis is not an uncommon etiology of fever of prolonged duration even in immuno-competent individual. Early bone marrow biopsy and fungal culture help in diagnosis. Imaging study to exclude adrenal involvement prevents case fatality in DH. Cytopenia may be due to secondary hemophagocytic syndrome. Keywords Histoplasmosis, Immuno-competent, Adrenal failure, Bone marrow

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Topic: Clinical Hematology PO 123 A Rare Presentation of Progressive Disseminated Histoplasmosis in an Immunocompetent Patient: Clinically Misdiagnosed as Leishmaniasis Dr. Megha Gupta, Dr. Anuj Sharma, Dr. Narayani Joshi Mahatma Gandhi University of Medical Sciences and Technology, Jaipur Summary: Histoplasmosis is an important systemic fungal infection in endemic areas. In India, the disease has been reported from several parts of the country, most cases being from eastern India considered to be endemic for the disease. We report a case of progressive disseminated histoplasmosis (PDH), which was clinically misdiagnosed as leishmaniasis. Introduction: Histoplasmosis caused by the dimorphic fungus Histoplasma capsulatum presents most commonly as pulmonary and progressive disseminated forms. Approximately 10 % of individuals infected with histoplasmosis may develop progressive disseminated histoplasmosis, which usually presents with fever, malaise, hepatosplenomegaly and lymphadenopathy. Progressive disseminated histoplasmosis is usually seen in immunocompromised patients such as persons with HIV infection. This case merits discussion due to the rarity of the presentation in an immunocompetent patient, clinically misdiagnosed as Leishmaniasis. Materials and Methods: We report a case of 60-year-old immunocompetent male patient, presenting with pancytopenia and continuous fever. Initially Patient was clinically diagnosed and treated for Leishmaniasis. Patient recovered but 2 months later, he came back with similar complains. All haematological investigations was repeated which indicated Pancytopenia. All other serum test for leishmaniasis was negative. Bone marrow aspiration was done to rule out the cause of Pancytopenia. Results: Bone Marrow Aspirate show foamy histiocytes filled with small intracytoplasmic yeasts morphologically in favour of H. capsulatum. Conclusion: PDH should always be considered in the differential diagnosis of pancytopenia, irrespective of the patient’s immune status. Keywords Histoplasmosis, Leishmaniasis, Immunocompetent

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 either sex presenting with complications of cirrhosis, (2) patients with liver cirrhosis diagnosed and ruled out all other causes of thrombocytopenia, (3) patients non reactive for HIV I and II, tuberculosis negative and not having any other disorder. Results: Study reveals 80 % of the patients were found to have abnormal heamatological indices. More than 50 % had a combination of cytopenias. Chronic anemia may contribute to a poorer outcome after any heamorrhagic episode. The platelet count varied around 1.5 lakh per cu mm in almost 73 % of cirrhotic cases. Conclusion: Liver plays an important role in the function and number of platelets, alterations in primary heamostasis and secondary heamostasis (coagulation). An increased intrasplenic platelet breakdown with variable roles of decreased platelet production and splenic pooling appear to be the most important determinants of thrombocytopenia. Keywords Thrombocytopenia, Cirrhosis, Cytopenias

PO 125 Anemia Due to Marching-March Hemoglobinuria Dr. Prabhakar Garg, Surender Mittal, Ankit Manglunia, D. P. Bansal, V. D. Maheshwari, G. N. Saxena Mahatma Gandhi College and Hospital, Jaipur

Dr. Nishath Khan

Introduction: March hemoglobinuria is a form of hemolytic anemia due to mechanically induced damaged to red blood cells, which result in hemoglobinuria. Repetitive impacts to the body may cause mechanical trauma (hemolysis) of red blood cells. It a rare abnormal condition characterized by presence of hemoglobin in the urine, occurs after strenuous physical exertion on prolonged exercise, such as marching or distance running. Case Report: We describe here a 24 year old male who presented with complain of generalized weakness, pain B/L lower limb and reddish brown discoloration of his urine after a religious march bare footed of about 180 km which continued for 4 days. On physical examination pallor was present and his urine examination revealed blood in urine. Blood picture showed Hb-6.2, MCV-113.6, RBC-1.34, reticulocyte count-3.5 %, LDH-410, ESR-110, Sr. bilirubin-2.7, uribilinogen1.5, haptoglobin 0.2 and direct coombs test was negative. The urine sample was negative for myoglobulin and other causes of hemolysis and hematuria was excluded. After 3 days there was resolution of his symptoms and investigation showed normal urine study. The association of mechanical trauma induced hemoglobinuria is being recognized. We however feel prevalence of such activities causes trauma to red blood cell causing hemolysis resulting in release of free hemoglobin from lysed cells which filtered into the urine. We hope to raise the awareness of trauma induced hemolysis in evaluation of patient with hemoglobinuria Keyword March hemoglobinuria

Deccan College of Medical Sciences, Kanchanbagh, Hyderabad, Owaisi Hospital and Research Centre, Hyderabad, Princess Esra Hospital, Hyderabad

PO 126

Topic: Clinical Hematology PO 124 Analysis of Platelet Count in Patients with Cirrhosis of Liver

Introduction: Cirrhosis is the development of regenerative nodules surrounded by fibrous bands in response to chronic liver injury that leads to end stage liver disease. Thrombocytopenia refers to decreased number of platelets (\1.5 lakh cells/cu mm) Objectives: (1) To know the incidence of thrombocytopenia in cirrhosis of liver. (2) To study the mutual relationship between platelets and liver cirrhosis due to different etiologies. (3) Analyzing the platelet count and its significance in the prognosis of liver cirrhosis. Materials and Methods: patient selection:60 patients from OHRC and PEH presenting with clinical features suggestive of complications of cirrhosis from September 2013 to September 2014. Diagnosis of cirrhosis was based on history, clinical examination, USG and UGI endoscopy. Controls: 40 healthy individuals of either sex presenting with no history of jaundice, anemia, any chronic disease and no history of alcoholism Inclusion criteria: (1) patients [ age 15 years of

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Fatal Hemolytic Anemia Associated with Metformin: A Case Report Dr. Prabhakhar Garg, Anchin Yadav, Surender Mittal, Surbhi Chaturvedi, G. N. Saxena, V. D. Maheshwari Mahatma Gandhi Medical College and Hospital, Jaipur Introduction: Metformin is a widely prescribed antidiabetic drug that has been implicated as a cause of hemolytic anemia. We report a case of fatal hemolysis that was associated with initiation of metformin as treatment for diabetes. Materials and Methods: Case report: A 54 year old male with type 2 diabetes mellitus was started on metformin to improve glycemic control, shortly after 7 days patient experienced easy fatigability and developed jaundice. There was no

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 history of fever, any other drug ingestion, alcohol intake, liver disease or blood transfusion. Clinically he had pallor and scleral icterus. No other clinical abnormality was seen. Laboratory studies showed severe hemolysis with a fall in Hb from 13.8 to 5.4 g/dl over 4 days, markedly elevated LDH, bilirubin and reticulocyte count and a low haptoglobin level. A direct coombs test was positive for anti Ig G and negative for anti C3. A peripheral blood flim showed no schistocytes, the glucose-6-phosphate dehydrogenase level was within normal range. Despite corticosteroid treatment, patient showed no improvement and further decline in hemoglobin to 3.3 g/dl was noted. Results: The serologic finding, in this case suggest autoimmune hemolytic anemia, caused either by drug induced auto anti body or a warm auto antibody. We proposed that metformin induced hemolysis is a strong possibility, physicians should be aware of potential side effect of metformin,as it is a widely used antidiabetic drug although this effect is infrequent. Conclusion/Diagnosis/Impression: Metformin induced autoimmune hemolytic anemia

PO 127 Value of CRP and ANC in Diagnosis of Bacterial Sepsis in BMT and Haematolymphoid Cancer Patients (TMP-032) Dr. Preeti Chavan, Dr. Vivek Bhat, Rajani Mohite, Swati Waykar, Asha Kumari, Shrutika Panchal, Nayana Baraskar Summary: This study was performed to determine the sensitivity and relevance of absolute neutrophil count (ANC) and levels of C-reactive protein (CRP) in predicting bacterial sepsis in cancer patients. 21 of 238 blood culture samples received were positive for infection and were tested for CRP, ANC. ANC was high in 33.3 % cases without leucopenia and low in 93.33 % cases with leucopenia. CRP significantly increased in all cases. CRP could be a more reliable indicator for infections than ANC and should be requested more frequently. Introduction: Infections are an important cause of morbidity and mortality in immunocompromised cancer patients. This study was performed to determine the sensitivity and relevance of absolute neutrophil count (ANC) and levels of C-reactive protein (CRP) in predicting bacterial sepsis in cancer patients. Materials and Methods: 238 blood culture samples were received between December 2013 and May 2014, of which 21 confirmed positive for infection by bacterial culture. All were tested for CRP and ANC. Results: Common organisms isolated included Klebsiella pneumoniae (14), E. coli (2), Staphylococci (2), Streptococcus (1), Acinetobacter (1) and Salmonella typhi (1). ANC was high in 33.3 % cases without leucopenia and low in 93.33 % of cases with leucopenia. CRP significantly increased in all cases. In 87.5 % of patients with leukopenia CRP levels were more than 9 mg/dl, in 83.5 % of cases without leukopenia CRP was 5.5 mg/dl or less. Conclusion: Though CRP is a non-specific marker for inflammation, especially in leukopenic and immunosuppressed patients, it could be a more reliable indicator for infections than ANC and should be requested more frequently.

PO 128 Successful Management of Chemotherapy Induced Hypertriglyceridemia in Acute Leukemia with Omega 3 Fatty Acids Shirali Agarwal, Manas Kalra, Nita Radhakrishnan, Anupam Sachdeva Pediatric Hematology Oncolgy Unit, Sir Ganga Ram Hospital, New Delhi, India

S523 Objective: To study the effect of presecribing omega 3 Fatty acids for treatment of hypertriglyceridemia in ALL instead of plasmapheresis and demonstrating its efficacy. Methods: Two patients who developed severe hypertriglyceridemia while on chemotherapy for ALL were prescribed Omega 3 Fatty Acids. Triglyceride and cholesterol levels were monitored sequentially on days 2,4, 6 and 10 along with clinical assessment for development of complications due to hypertriglceridemia. Results: A 15-year-old male child who was a case of T cell ALL on reinduction chemotherapy as per BFM 95 protocol, presented with symtoms of neuropathy and myopathy. His seum sample was lipemic. His S.Triglyceride levels were[4,870 mg/dl and cholesterol level was 655 mg/dl. He was started on Omega-3 capsules (300 mg) thrice a day. Triglyceride levels dropped to 1,980, 1120, 540, 326 and 250 mg/dl on days 2,4,6,10 and 12, respectively. His cholestrol levels decreased from 380 to \150 mg/dl within 10 days. The patient recovered clinically and his neurological as well as laboratory parameters remained stable. A 4 year old patient, who was a case of B cell ALL was being treated on BFM95 Protocol developed hypertriglyceridemia which was detected during routine sampling while on induction chemotherapy. His Triglyceride levels were equal to 2,489 mg/dl. He was started on omega 3 fatty acids. His triglyceride level decreased to 1500, 840 and 437 mg/dl and 250 mg/dl on days 2, 4, 6 and 10 following starting of therapy. We were able to avoid plasmapheresis in both these patients and successfully treat them conservatively. Conclusion: Severe hypertriglyceridemia may be conservatively managed with omega 3 fatty acids as an adjunct to diet. Plasmapheresis may be reserved for symptomatic patients with very high triglyceride levels not responding to conservative therapy. Omega-3 fatty acids, has demonstrated efficacy and safety and should prove to be valuable for the medical management of hypertriglyceridemia in ALL patients. Keywords ALL, Omega 3 fatty acid asparaginase, Hypertriglyceridemia

Topic: Proferred PO 129 Hemophagocytic Lymphohistiocytosis: An Unusual Presentation of Tuberculosis in Hemodialysis Patients 1

Sindhura Lakshmi Koulmane Laxminarayana, Shankar Prasad Nagaraju, 2Ravindra Prabhu Attur, 3 Chethan Manohar, 2Rajeevalochana Parthasarathy, 1 Brahmaiah Chari 2

1

Department of Pathology, Melaka Manipal Medical College, Manipal University, 2Department of Nephrology, Kasturba Medical College, Manipal Unversity, 3Department of Pathology, Kasturba Medical College, Manipal University Summary: We report a series of three patients on maintenance hemodialysis presenting with hemophagocytic lymphohistiocytosis (HLH) as an unusual manifestation of extra pulmonary tuberculosis. Introduction: HLH is a benign non Langerhans cell histiocytosis. HLH is an unusual manifestation of tuberculosis. There are only 5 reported cases of tuberculosis associated HLH in patients on hemodialysis. Materials and Methods: We present three patients who were middle aged males having fever, pancytopenia, and hepatosplenomegaly. Results: All of them had abnormal liver function tests, coagulopathy, increased serum ferritin and triglyceridesBone marrow examination revealed hemophagocytosis and caseating granuloma. Acid fast bacilli were demonstrated in two patients. The HLH-2004 diagnostic criteria suggested by the histiocytic society were followed to arrive at the diagnosis. All of them succumbed to death.

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S524 Conclusion: Aggressive diagnostic work-up must be done when hemodialysis patients present with fever and pancytopenia. Keywords Hemodialysis, Tuberculosis, Pancytopenia, Fever, Hemophagocy

PO 130 Methotrexate Induced Myelosuppression in Psoriasis: The Need for Close Haematological Monitoring Dr. Surender Mittal, Dr. Prakhar Garg, Dr. Surbhi Chaturvedi, Dr. G.N. Saxena Mahatma Gandhi Medical College and Hospital, Jaipur Introduction: Methotrexate (MTX) is an effective antipsoriatic agent. It has been widely used to treat severe psoriasis. The most important potential side-effect of methotrexate is acute myelosuppression and when used in chemotherapy it causes profound suppression of the bone marrow. Even at low doses it is often associated with bone marrow suppression and is the cause of most of the rare deaths attributed to the use of methotrexate therapy for psoriasis. A 65 year old female patient presented with eruptions all over body associated with itching and joint pain since 10 months. There was history of oral ulcers and mild grade fever since last 10 days. The patient was on long term methotrexate (15 mg oral dose weekly) since 10 months. On examination there was pallor, also multiple well defined erythematous plaques with psoriasiform scales, in symmetric distribution were present all over the body. Blood picture revealed pancytopenia. Bone marrow aspiration and biopsy revealed suppression of all three erythropoitic, granulopoitic and megakaryocytic series. The diagnosis of MTX related myelosuppression was made. Results: In MTX-treated psoriatic patients, the prevalence of haematological toxicity, including leucopenia, thrombocytopenia, megaloblastic anaemia and pancytopenia, is estimated to be 3 %. The extent of pancytopenia, a serious and unpredictable adverse effect of low-dose MTX, may be underestimated and needs attention. Keywords Methotrexate induced myelosuppression in psoriasis

Topic: Haematology PO 131 A Case Series of Kikuchi’s Disease: A Histopathological Perspective 1

Akanksha Malik, 2Sandhya Sundaram

Department of Pathology, Sri Ramachandra Medical College Chennai, India Summary: We analyzed the clinicohistopathological features of Kichuchi’s disease over a period of 2 years. The characteristic microscopic feature was necrotizing lymphadenitis and the usual clinical feature was fever with lymphadenopathy. However, it has to be differentiated from other clinical and histological mimickers. Introduction: Kikuchi’s disease or histiocytic necrotizing lymphadenitis is a rare, benign condition which affects young adults generally encountered in the Asian population. It commonly presents with cervical lymphadenopathy and mild fever. The exact etiology is unknown; however, infectious and autoimmune mechanisms have

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 been implicated. The main differential diagnosis includes tuberculous lymphadenitis, lymphoma and SLE.We report a case series from our hospital. Materials and Methods: The clinicopathological features of 16 patients diagnosed as Kikuchi’s disease at Sri Ramachandra Medical College Chennai over a period of 2 years is presented. The histopathology slides were reviewed and its salient microscopic features were noted. The close differentials were excluded by special stains and immunohistochemistry markers, where applicable. All baseline investigations were also performed. Results: Out of 16 patients, most (75 %) were female who presented with lymphadenopathy and fever. The group of nodes involved were predominantly cervical (68.75 %). The age ranged from 11 to 49 years, median age being 23.5 years. Two patients had mesenteric lymph node enlargement and one was positive for EBV virus. The diagnostic method of choice was excision biopsy of the node. The follow up was uneventful. Conclusion: Kikuchi Fujimoto disease is a rare, benign and self limiting condition involving the lymph nodes. Recognition and differentiation of this entity is necessary to rule out other simulating lesions. Keywords Kikuchi’s disease, Lymph node, Lymphadenitis

Topic: Oral Paper Session PO 132 Evaluation of Newonset Pancytopenia in Geriatic Population with Emphasis on Absolute Neutrophil Count (ANC) and Changes in Bonemarrow G. Amrutha, Debdatta Basu, Rakhee Kar, E. J. Sajini Department of Pathology, Jipmer Summary: 30 geriatric patients with pancytopenia are included in the study. The causes of pancytopenia were diverse with hematolymphoid malignancy being the commonest. Introduction: Geriatric population is associated with many comorbidities and identification of an accurate cause of pancytopenia helps in implementing appropriate therapy. Definition and labeling of pancytopenia varies in various study groups. Most of the studies use total leucocyte count (TLC) of \4,000/ll, while some use absolute neutrophil count (ANC) \1,800/ll in defining pancytopenia. Materials and Methods: It is a descriptive study of 30 geriatric patients [60 years of age, who presented with pancytopenia during the period of Oct 2013 to Sept 2014. The criteria for pancytopenia was Hemoglobin \10 g/ dl, TLC \4,000/ll, Platelet count \100,000/ll. In presence of normal TLC (4,000–11,000/ll), ANC \ 1,800/ll were included in the study. Clinical details, detailed hematological workup and bone marrow aspiration and trephine biopsy was done in all the cases. Results: The median age of presentation was 66 years, with male predominance. Most of the patients presented with generalized weakness, fever and gum bleeding. Dimorphic anemia was the predominant blood picture. Bone marrow aspiration was conclusive in 29 of the thirty cases. The commonest cause for pancytopenia was acute leukemia (24 %) followed by aplastic anemia (20 %). Lymphoma infiltration, myelodysplastic syndrome and megaloblastic anemia were seen in 10, 3, 6 % cases, respectively. Conclusion: Pancytopenia is associated with increased morbidity in the geriatric population and hematol-ymphoid malignancy accounts for the majority of cases. Keyword Pancytopenia

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Topic: Hematology PO 133 Hemophagocytic Lymphohistiocytosis in Kikuchi–Fujimoto Disease: Stranger than Fiction? 1

M. Deepak Nayak, 2Sushma V. Belurkar, 3Chethan Manohar, Mary Mathew, 5Anuradha C.K. Rao

4

Department of Pathology, Kasturba Medical College & Hospital, Manipal University, Manipal, Department of Pathology, Kasturba Medical College & Hospital, Manipal University, Manipal Objectives: Kikuchi–Fujimoto disease is a benign disease with a wellknown set of clinical features. Hemophagocytic lymphohistiocytosis on the other hand is characterized by marked histiocytic proliferation and subsequent pancytopenia; driven by immunologic activation. This paper aims to highlight the rare association between the two and also the challenges faced in diagnosis and treatment. Methods: A retrospective analysis of the 3 cases was done after retrieving information from the hospital database and patient records; placing importance on clinical features, treatment protocol and tissue biopsy characteristics. Results: The cases presented with cervical and axillary lymphadenopathy, high-grade fever and pancytopenia on the peripheral blood smear examination. The lymph node biopsy findings were consistent with Kikuchi disease in all 3 cases. The bone marrow aspirate and biopsy revealed significant phagocytosis of all three hematopoietic cell lines. Other laboratory data such as an elevated serum ferritin, liver transaminases, LDH and triglycerides along with hypofibrinogenemia substantiated the diagnosis of hemophagocytic lymphohistiocytosis. 2 cases responded to a combination regimen of corticosteroids and iv immunoglobulin. One case mandated etoposide therapy. Conclusion: Hemophagocytic lymphohistiocytosis is an often underrated entity in diagnostic hematology. This is further sidelined in conditions where an association is hitherto not described; such as Kikuchi disease. But an immediate recognition and treatment is imperative as sepsis-related mortality can be quite significant in these patients. Keywords Hemophagocytosis, Kikuchi disease, Pancytopenia

Topic: Morning PO 134 Pancytopenia with Atypical Cells in Peripheral Smear in a Case of Malaria: A Rare Presentation

S525 secondary to hemophagocytosis since it is a rare presentation. Methods: CBC, peripheral smear was done followed by antigen detection test for malaria. Bone marrow aspiration followed by workup for hemophagocytic syndrome like serum ferritin were done. Result: CBC showed pancytopenia with peripheral smear showing atypical cells. ICT was positive. Bone marrow showed normocellularity with malarial pigments and hemophagocytosis. Conclusion: Pancytopenia secondary to hemophagocytosis in vivax cases is rare. In endemic areas, malaria is the first differential diagnosis in all cases of fever that should be considered unless proved otherwise. Keywords Malaria, Pancytopenia, Hemophagocytosis

PO 135 A Very Rare Association in a Case of Myelodysplastic Syndrome: A Case Report Dr. Gurmeet Singh, Dr. M. Ravindranath, Dr. Shuddhasattwa Bhattacharjee Jawaharlal Nehru & Research Centre, Sector 9, Bhilai Introduction: MDS represents a spectrum of stem cell malignancies that manifests dysplastic and ineffective blood cell production compounded by a variable risk of transformation to acute leukemia. The term refractory anaemia with excess blasts is subdivided into two subgroups, patients with \10 % of marrow blasts or fewer than 5 % circulating blasts classified as refractory anaemia with excess blast-1 (RAEB1), and patients with 10–19 % of marrow blasts or more than 5 % of circulating blasts as RAEB2. Summary: A 57 year old male presented with complaints of weakness, low grade fever and loss of appetite. Clinically he had moderate degree of pallor. Serial haemograms showed low Hb, low platelet, and high reticulocyte counts along with haemolytic blood picture and high nucleated RBC count on peripheral smears examined. To rule out haemoglobinopathies, high performance liquid chromatography and haemoglobin electrophoresis were performed which showed presence of HbH, high percentage of HbF and HbS. Brilliant cresyl blue stained smear showed presence of HbH inclusions. Peripheral smear examination showed atypical cells and after bone marrow evaluation a diagnosis of RAEB-1 was made. Karyotype showed hyperdiploidy(63-80,XY[6]/ 46,XY[26]. Result: Finally the case was diagnosed as Alpha-thalassemia-myelodysplastic syndrome (ATMDS). Conclusion: ATMDS is a rare entity but there is no disease so rare that it does not deserve attention. Keyword Alpha-thalassemia-myelodysplastic syndrome (ATMDS).

*Gouranga Charan Nayak, **Sujata Naik, ***Manoj K. Patro, ***Atanu K. Bal, ***Anita Choudhury, ****Bodhisatwa Behera, ****J. Nayak, ****D. P. Mishra

Topic: Laboratory Pathology

Department of Pathology, M.K.C.G. Medical College, Berhampur, Odisha

PO 136

Introduction: Malaria contributes to significant morbidity and mortality in developing country of the tropics. World Health Organization (WHO) has estimated (WHO Malaria news release year 2010) that about 250 million cases of malaria occur every year worlwide causing 8,60,000 death. Malaria presents classically with febrile paroxysm with associated sweating and rigor. In endemic areas, malaria can have atypical manifestation like severe anemia, thrombocytopenia, acute kidney injury, acute respiratory distress syndrome, cerebral involvement and hepatic dysfunction. Pancytopenia with atypical cells is an uncommon presentation. We report a 22 year male patient presenting with fever, breathlessness and head reeling. Objective: To demonstrate plasmodium vivax malaria presenting as pancytopenia

Role of Quality Indicators in Haematology and Clinical Pathology Laboratory Dr. Jyoti Prakash Sapre, Dr. Vishva C. Patel Department of Pathology, Pramukhswami Medical College, Shree Krishna Hospital, Karamsad, Gujarat, India Objectives: Quality indicators are measurement tools, screens or flags that are used as guides to monitor, evaluate and improve the quality of patient care, clinical support services and organizational function that affect patient outcomes. The goal of this study was to design and review quality indicators from time to time in an effort to improve the performance of the laboratory. Settings and Design: Observational study done

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S526 at Pramukhswami Medical College, Shree Krishna Hospital, Karamsad from January 2011 to December 2013. Materials and Methods: This was a observational study conducted at Haematology and Clinical Pathology section of Central Diagnostic Laboratory, Shreekrishna Hospital, Karamsad during January 2011 to December 2013. Total 08 quality indicators were studied. The target for each quality indicator was set according to previous year’s performance. Results: Total 1,43,106 samples were received. Quality indicators like internal quality control, external quality assessment, critical alerts, turnaround time, specimen collection and storage, completeness of requisition form, specimen rejection and avoidance of transcriptional error in reporting from log book to computer were studied and showed continuous quality improvement. Conclusions: Quality improvement plan in haematology and clinical pathology is an effective tool for ensuring continuous quality improvement by the use of quality indicators. Keywords Quality improvement, Quality indicators, Haematology, Clinical pathology

Topic: Haematology (Pathology)

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Objectives: Pancytopenia is defined by reduction in all three formed elements of blood, below the normal reference range. Varieties of hematological and non- hematological disorders may affect bone marrow either primarily or secondarily, resulting in the manifestation of pancytopenia. The objective of this study was to find the cause and spectrum of clinical presentation and correlate hematological parameters with clinical findings in differentiating causes of pancytopenia. Methods: A 2 year study was conducted in Department of Pathology, Alameen Medical College, Bijapur. Fifty patients with pancytopenia were included in the study. Complete blood count, peripheral study, bone marrow aspirations were performed. Results: Males were affected more than females. Generalised weakness and pallor were noted in most of a cases. The commonest cause for pancytopenia was megaloblastic anemia, followed by iron deficiency anemia, leukemia. Conclusion: Large number of patients had reversible etiology. Hence complete work up including clinical details with hematological examination along with bone marrow study will lead to early and proper diagnosis of case followed by proper treatment. Keywords Pancytopenia, Adults, Megaloblastic anemia, Iron deficiency anemia

PO 137 Assessment of Haematological Parameters in Patients with Liver Cirrhosis Dr. N. Kiruthiga, Dr. Anita Ramdas Department of Pathology, Pondicherry Institute of Medical Sciences, Pondicherry 605014 Summary: Haematological parameters of cirrhotic patients were compared with severity of the liver disease based on Child–Pugh and Model for End Stage Liver Disease (MELD) scoring. Introduction: Haematological abnormalities in cirrhotics include anaemia, thrombocytopenia and increased PT/INR. Spur cell anemia is seen in advanced cases. Our study aimed at correlating haematological parameters to the severity of liver cirrhosis. Materials and Methods: 50 patients with diagnosed cirrhosis irrespective of age, sex, etiology or treatment were included over a period of 18 months. Complete blood count with peripheral smear and coagulation parameters were noted along with relevant patient history. Variations in these were compared with the scoring system (Child–Pugh and MELD). Results: 50 patients were included. M:F ratio was 9:1 with mean age of 52 years. In 80 % cases the etiology of cirrhosis was alcoholism. 56 % of cirrhotics had Hb 5–10 g/dl. Macrocytic anemia and spur cell anemia was seen in 22 and 4 patients, respectively. 9 patients had PT/INR [ 2.3, of whom 8 had Child–Pugh Class C, indicating severe disease and 6 had MELD score 30–39 (3 months mortality 52.6 %). Thrombocytopenia was noted in 22 patients. Of all parameters haemoglobin level alone showed significant correlation (P \ 0.05) with severity of liver disease. Conclusion/Diagnosis/Impression: Although a number of haematological abnormalities are seen in cirrhotic patients, haemoglobin level alone showed significant correlation with severity of liver disease. Keywords Haemoglobin, PT/INR, Cirrhosis

Topic: Haematology PO 138 Pancytopenia: Clinico-Haematological Study Dr. Nadia Shafi, Dr. Ashok M. Patil, Dr. Y. Saeed, Dr. Sarita Al-Ameen Medical College, Bijapur

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PO 139 Apoptosis Induction in CML cell K562 by Diospyros melanoxylon Bark Extract (DMBE) Nilanjana Deb, Biswajit Chatterjee, Chinmoy Chowdhury, Snehasikta Swarnakar, Shila Elizabeth Besra Drug Development/Diagnostic & Biotechnology Division, Chemistry Division, CSIR, Indian Institute of Chemical Biology, Raja S.C. Mullick Road, Kolkata 700032, West Bengal, India Summary: Identification of cytotoxic compounds that induce apoptosis has been the mainstay of anti-cancer therapeutics. Study revealed that DMBE possesses potent anti-proliferative activity. It merits consideration and further investigation as a therapeutic option for the treatment of leukemia. Introduction: The plant Diospyros melanoxylon is widely used in Indian traditional medicine as a broad spectrum medicine. This encouraged us to investigate the anti-leukemic effect of the bark of Diospyros melanoxylon in human leukemic CML cells K562 and delineate the apoptotic pathways. Materials and Methods: Using MTT based cell cytotoxicity assay and trypan blue based cell viability assay, we evaluated the cytotoxic potential of the bark on K562 cells. Cell morphology was determined by Fluorescence (A.O & EtBr) and confocal microscopy (Annexin V FITC). DNA fragmentation was studied by Agarose gel electrophoresis. To examine whether cell death occurred via apoptosis or necrosis, double staining Flow cytometry was performed. Results: DMBE significantly inhibited the cell viability and cytotoxicity (MTT) in a time and concentration dependent manner. Fluorescence microscopy showed characteristic features of membrane blabbing, chromatin condensation, the sign of apoptotic changes in the cells after treatment with DMBE. Gel electrophoresis study showed fragmented DNA in the form of ladder. The flow cytometric analysis confirmed the presence of apoptotic cells. Moreover the externalization of phosphatidylserine by confocal microscopy is indicative of apoptosis. Conclusion/Diagnosis/ Impression: The findings suggest that DMBE mediates Cytotoxicity in leukemic cells via apoptosis. Further mechanistic studies are going on to determine the different pathways contributing to apoptosis.

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 PO 140 Thiamine-Responsive Megaloblastic Anemia Syndrome in a 7 Years Old Female Child: A Case Report Dr. Nishath Khan, Dr. Zakia Abid, Dr. Atiya Begum, Dr. Nabila Afsar Department of Pathology, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad Thiamine-Responsive Megaloblastic Anemia Syndrome is a rare condition characterized by sensori neural hearing loss, non-type 1 diabetes mellitus and megaloblastic anemia. Age of onset is from infancy to adolescence. It has been reported in approximately 30 families worldwide. It is inherited in an autosomally recessive pattern. Here we are reporting a case from Owaisi Hospital of a 7 years old female child, weighing 16 kg, with H/O diabetes mellitus, seizures and deafness. She presented to the hospital with fever since 2 days, respiratory distress since 1 day and abdominal distention. After investigations, treatment and follow-up, the child was diagnosed to have thiamine-responsive megaloblastic anemia syndrome.

Topic: Hematopathology PO 141 A Case Report of Adult Onset Niemann–Picks Type C Disease

S527 Introduction: HLA-B27 is a MHC class I molecule whose principle function is to present cell-derived peptides to CD8+ cytotoxic T lymphocytes, as part of the adaptive immune response. The strong association of the HLA-B27 with ankylosing spondylitis (AS) was first discovered independently by groups in London and California in 1972. Yet, despite nearly four decades of extensive research, the exact role that HLA-B27 plays in pathogenesis remains unknown. No laboratory tests are specific and are often more helpful to exclude other diagnoses rather than confirming AS. There may be normochromic normocytic anaemia of chronic disease. ESR or CRP level may correlate with disease activity but these are less useful for monitoring activity than in other inflammatory arthritis such as rheumatoid arthritis. Objectives: This is a retrospective 2 years study from September 2012 to august 2014. Patients suspected for spondyloarthrosis or those with complaints of backache were analysed for there HLA B27 status and sample were collected for other investigations. Here, we have tried to see the association between HLA B27 and other laboratory parameters. Results: Total 393 patients were analysed out of which 141 (35.8 %) patients were found positive for HLA B27 while 252(64.2 %) were negative. These results were analysed for association with other laboratory parameters. Conclusion: Strong association is established between ankylosing spondylitis and HLA B27. Also, c-reactive protein and erythrocyte sedimentation rate are associated with activity of disease. Here, we have tried to see association of HLA B27 with other laboratory parameters. Keywords HLA B27, Spondyloarthrosis, Lab parameters

1

S. Mishra, 2K. Karan, 2K. Dutta, 1D. Nag, 2D. Basu, 2P. Sengupta

1

Department of Pathology, Medical College & Hospital, Kolkata. Department of Neuro-Medicine, Medical College & Hospital, Kolkata

2

Introduction: Niemann–Pick type C disease (NPC) is a rare autosomal recessive lipid storage disorder caused by impaired cellular functions in processing and transporting low-density lipoprotein–cholesterol. The disease may be under-diagnosed due to its highly heterogeneous presentation and there are no fixed criteria for diagnosis of this disease yet. NP-C is characterised by visceral, neurological and psychiatric manifestations that are not specific to the disease and that can be found in other conditions. Here we present an adult onset case. Case Report: A 23 year old male patient presented with gradually progressive dysphagia, dysarthria, Parkinsonian like dystonia with vertical gaze palsy. He also showed decreased cognition and psychiatric problems. There were no visceral signs and symptoms. Detailed history was taken. Several investigations were done. Most striking result was presence of sea-blue histiocytes in bone marrow aspirate. Discussion: Our patient presented with wide array of neurological and psychiatric signs and symptoms. The onset was late and gradually progressive in nature. On investigating him we found that he was suffering from a rare lysosomal storage disorder. A strong suspicion is always warranted in such cases even in absence in visceral symptomatology. Even though there are some limitations in our study we can negate other causes on the basis of history and investigational findings. Conclusion: This case is presented to show the range of presentation of the disease. It may help in setting diagnostic criteria of this disease in future. Keywords Niemann–Picks disease, Adult onset Type-C Niemann– Picks disease, Neuro

PO 142 Correlation of HLA B27 and Other Laboratory Parameters Pratibha Maan, Gurdeep Buxi, A. S. Nigam, Sadhna Marwah, Vijay Kumar Department of Pathology, PGIMER and Dr. RML Hospital, New Delhi

Topic: Hematopathology PO 143 Double – Trouble: Hematalogic and Non-Hematalogic Collision Tumour - A Report of Two Rare Cases Ranjith. A. R, Debdatta Basu, A. Jhansi Rani, Sajini Elizabeth Jacob, Rakhee Kar Department of Pathology, Jawaharlal Institute of Postgraduate Medical Education & Research (JIPMER), Puducherry, INDIA Summary: A report of two cases with the rare occurrence of concurrent presence of a hematological and a non-hematological malignancy in the same site is presented. Introduction: Second malignancies are newly developed malignant neoplasms present either synchronously or metachronously in a patient with a known malignancy. Collision tumour is defined as two histologically distinct neoplasms occuring concurrently in the same location. Materials and Methods: Two cases of double malignancies diagnosed in our department are included in the study. Results: Case 1: A 9 year old male after 10 months postchemotherapy for stage IV neuroblastoma showed 47 % myeloblasts in a follow-up peripheral smear and 87 % myeloblasts positive for CD34, MPO and CD117, in the bone marrow aspirate. A diagnosis of therapy related AML with MLL gene translocation was made. Interestingly, the bone marrow trephine biopsy in addition to the myeloid leukemia, showed a focus of residual neuroblastoma. Case 2: A 56 year old male was diagnosed to have chronic lymphocytic leukemia (CLL) on bone marrow aspirate and biopsy. Lymph node biopsy showed adenocarcinoma cells positive for CK7 and negative for CK20 in the background of CLL (CD20+, CD5+, CD23+). Final diagnosis metastatic of adenocarcinoma, possibly from upper aerodigestive tract, in a lymph node harbouring CLL/SLL was made. Conclusion: Two rare cases of double malignancies present in the same organ—collision tumour in a lymph node and simultaneous presence of a therapy related metachronous tumour with a residue of the original tumour in bone marrow is presented.

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S528 Keywords Collision tumour, Double malignancy, Therapy related AML

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Topic: Hematopathology PO 146

Topic: 1

Langerhans Cell Histiocytosis(LCH) of Tonsil: An Uncommon Disease at an Unusual Site

PO 144

K. Siddartha, A. Faiq, K. Rachna, M. Bharathi, S. Sudha Murthy, M. V. T. Krishna Mohan

A Case of Myelodysplastic Syndrome Dr. Shabana Tabbassum Department of Pathology, Deccan College of Medical Sciences, Hyderabad Summary: Myelodysplastic Syndrome are now considered the most common form of leukaemia and in some cases deserve immediate intervention. Introduction: The Myelodysplastic syndrome are clonal bone marrow disorders that lead to underproduction of normal blood cells. The consequent cytopeanias result in infection and bleeding complications. Materials and Methods: A 55 year old male presented with vague ill health. On investigation CBP shows atypical cells, case was extensively evaluated with Bone marrow Aspiration, Bone marrow biopsy, Flowcytometry etc. Conclusion: Turned out to be Myelodysplastic syndrome, Case is under follow up. Keywords Myelodysplatic syndrome, Leukemia, Flow cytometry

Topic: Hematopathology PO 145 A Case Report of Adult Onset Niemann–Picks Type C Disease 1

S. Mishra, 2K. Karan, 2K. Dutta, 1D. Nag, 2D. Basu, 2P. Sengupta

1

Department of Pathology, Medical College & Hospital, Kolkata. Department of Neuro-Medicine, Medical College & Hospital, Kolkata

2

Introduction: Niemann–Pick type C disease (NPC) is a rare autosomal recessive lipid storage disorder caused by impaired cellular functions in processing and transporting low-density lipoprotein– cholesterol. The disease may be under-diagnosed due to its highly heterogeneous presentation and there are no fixed criteria for diagnosis of this disease yet. NP-C is characterised by visceral, neurological and psychiatric manifestations that are not specific to the disease and that can be found in other conditions. Here we present an adult onset case. Case Report: A 23 year old male patient presented with gradually progressive dysphagia, dysarthria, Parkinsonian like dystonia with vertical gaze palsy. He also showed decreased cognition and psychiatric problems. There were no visceral signs and symptoms. Detailed history was taken. Several investigations were done. Most striking result was presence of sea-blue histiocytes in bone marrow aspirate. Discussion: Our patient presented with wide array of neurological and psychiatric signs and symptoms. The onset was late and gradually progressive in nature. On investigating him we found that he was suffering from a rare lysosomal storage disorder. A strong suspicion is always warranted in such cases even in absence in visceral symptomatology. Even though there are some limitations in our study we can negate other causes on the basis of history and investigational findings. Conclusion: This case is presented to show the range of presentation of the disease. It may help in setting diagnostic criteria of this disease in future. Keywords Niemann–Picks disease, Adult onset Type-C Niemann– Picks disease, Neuro

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Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad Summary: LCH is a disease characterized by proliferation of dendritic cell. Incidence of it is 5 per million population per year. Though there are preferential locations in case of solitary LCH, bone and adjacent soft tissue are most commonly involved. Less common site of predilection are the lymph nodes, skin and lung. Tonsillar involvement per se is very rare. We here present a case of 40 years male who presented with left neck swelling of 4 months duration associated with dysphagia. CT scan revealed tonsillar swelling. Patient underwent tonsillectomy. Histopathological examination and Immunohistochemistry rendered a diagnosis of LCH. Introduction: Langerhans Cell Histiocytosis is neoplastic clonal proliferation of Langerhans type cell. The disease can be localized or a more disseminated with multisystem involvement. Unifocal disease is usually seen in older children or adults. Its occurrence in tonsil as primary isolated manifestation is very rare. Materials and Methods: Received excised bilateral tonsillectomy specimen which was subjected to routine histopathological evaluation along with Immunohistochemistry using antibodies LCA, CD1a, CD68, S100. Results: Microscopic examination revealed tonsillar tissue with nodules of closely packed lesional histiocytic cells amidst numerous eosinophils and plasma cells. The histiocytic cells exhibit characteristic irregular shaped nuclei, few being reniform, notched, with coffee bean shaped nuclear grooves. IHC reconfirmed the neoplastic cells to be histiocytes which showed membranous immunoreactivity to CD1a, cytoplasmic positivity for CD68 and both cytoplasmic and nuclear positivity for S100. Further staging workup revealed bone marrow free of disease. Conclusion: Tonsillar involvement in LCH is quite unusual. Histopathology with Immunohistochemistry is cornerstone in diagnosis of LCH.

Topic: Afternoon Session PO 147 An Interesting Case of Fever, Pancytopenia and Hyperpigmentation Subhransu Kumar Hota, S. K. Behera, D. P. Mishra Department of Pathology, M.K.C.G. Medical College, Brahmapur, Odisha, India Objective: Vitamin B12 is a rare cause of fever of unknown origin. We describe here a case of 24 year old pure vegetarian male who presented us as pyrexia of unknown origin (PUO) and hyperpigmentation. Methods: Meticulous evaluation and exclusion of infective, inflammatory, endocrine causes done. Results: After exclusion of all the cause of illness was attributed to vitamin B12 deficiency. There was swift and dramatic recovery after parenteral vitamin B12 supplementation. Conclusion: Vitamin B12 deficiency should be born in mind where as evaluating a febrile patient particularly with pancytopenia and hyperpigmentation as it is one of the rare but easily correctable and reversible cause of PUO

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Keywords Hyperpigmentation, B12 deficiency, Pancytopenia

PO 148 SLE Associated Macrophage Activation Syndrome with Disseminated Histoplasmosis Masking HIV Dr. Tapas Ranjan Mishra, Dr. P Mohanty, Dr. P. Pradhan Department of Pathology, SCB Medical College & Hospital, Cuttack, Odisha, India Summary: Macrophage activation syndrome (MAS) is rarely associated with SLE and can be triggered by HIV and Histoplasmosis. Rarely SLE can mask HIV thereby creating a diagnostic inaccuracy which is reflected in the present case. Introduction: MAS is a life threatening complication of rheumatic disease, frequently associated with systemic onset juvenile idiopathic arthritis (SOJIA) and adult onset still disease, characterised by pancytopenia, liver insufficiency, coagulopathy and neurologic symptoms. It is thought to be caused by activation and uncontrolled proliferation T lymphocytes and macrophages leading to haemophagocytosis and cytokine overproduction. We report a case of SLE associated MAS with disseminated histoplasmosis and HIV, that was repeatedly negative. Materials and Methods: A 42 years female presenting with low grade irregular fever for 1 month followed by oral ulceration & bleeding P/R. Investigations: Peripheral smear revealed pancytopenia. ICT for malaria, widal test, viral serology for dengue, Hepatitis B, C, and HIV were all -ve. Bone marrow aspiration showed marked depression of haemopoisis and proliferation of macrophages stuffed with yeast form of Histoplasma capsulatum along with F/O haemophagocytosis. She had marked elevation of serum ferritin, triglyceride, liver enzymes. P/H revealed that she was being treated for SLE with omnacortil for last 8 months. Repeat HIV(ELISA) test was strongly positive with a HIV-1 (RNA Load of 1 crore copies/ml) and CD4 count of 12/cmm. She developed altered coagulation profile and DIC, then encephalopathy and finally succumbed 20 days after hospitalization. Results: With these features she was diagnosed as Macrophage activation syndrome with disseminated Histoplasmosis and AIDS. Conclusion: The coexistence of SLE and Histoplasmosis has been reported as a consequence of immunosuppression. But there are a few reports showing co-existence of HIV and SLE; interestingly SLE masking AIDS. Haemophagocytic syndrome (HPS) may be the first manifestation of HIV infection and MAS can be triggered by all three predisposing factors namely SLE, HIV, and Histoplasmosis. Keywords SLE, Histoplasmosis

Topic: Clinical Hematology PO 149 A Rare Case of Haemophagocytic Lymphohistiocytosis in an 18 Year Old Patient Dr. Tashi Agarwal, Dr. Shashi Bansal, Dr. Upendra Sharma Department of Pathology, Mahatma Gandhi Medical College and Hospital, Jaipur (Raj) Summary: An 18 year old patient presented with fever, pancytopenia and hepatosplenomegaly. Bone marrow aspirate showed marked haemophagocytosis. After diagnosing the condition as Haemophagocytic lymphohistiocytosis, she was treated with cor-

S529 ticosteroids, etoposide and cyclosporine followed by good response. Introduction: Haemophagocytic lymphohistiocytosis is a rare and potentially fatal syndrome characterised by multisystem inflammation resulting in an uncontrolled and ineffective immune response. It usually presents in children less than 1 year of age as unexplained peripheral blood pancytopenia with history of fever and presence of hepatosplenomegaly. Materials and Methods: An 18 year old girl presented with 15 days of generalised weakness, fatigue and high grade fever with chills and rigors. On examination there was presence of pallor with pronounced hepatosplenomegaly. Bone marrow aspirate showed an increase in marrow macrophages with marked haemophagocytosis. Results: After diagnosing the condition as Haemophagocytic lymphohistiocytosis according to the established criteria of International Histiocytic Society, she was treated with corticosteroids, etoposide and cyclosporine followed by good response. Conclusion: This report adds to the limited adult cases of Haemophagocytic Syndrome reported in literature as well as emphasizes that the prompt diagnosis of HLH may be an important strategy to optimizing the clinical approach and outcome. Keywords Hemophagocytic lymphohistiocytosis, Familial haemophagocytic lymphohis

Topic: Haematology PO 150 Evaluation of Serum Hepcidin as a Marker of Iron Status and Carotid Atherosclerosis in Patients with Chronic Kidney Disease Vipra Malik, Satendra Sharma, Om Prakash Kalra, Neelam Wadhwa, Shuchi Bhatt University College of Medical Sciences and GTB Hospital, Dilshad Garden, Delhi 110095 Background: Anemia is the most common complication of CKD. Hepcidin has been implicated in altered iron metabolism, endothelial dysfunction and resistance to erythropoietin stimulating agents (ESAs) in CKD. Thus, assessment of serum hepcidin may help therapeutically and also as a marker for assessing atherosclerosis. Aim: To evaluate the role of serum hepcidin as a marker of body iron status and carotid atherosclerosis in patients of CKD. Methods: This was a cross sectional, case–control study and comprised of 40 CKD patients and 40 healthy controls. Assessment of body iron status included serum ferritin, serum iron and total iron binding capacity. Serum ferritin, serum hepcidin and hsCRP were measured using ELISA kit. Carotid doppler study for the assessment of carotid intima media thickness (CIMT) was also done. Results: Iron deficiency anemia was seen in 12/40 (30 %) CKD patients. Patients of CKD with iron deficiency anemia had significantly higher ESR and hsCRP level. Serum hepcidin levels were found to be significantly higher in patients with CKD and the levels increased from stage 3 to stage 4 to stage 5. Serum hepcidin levels were also found to be more in CKD patients on hemodialysis than those not on hemodialysis, however no significant association could be documented. Serum hepcidin levels were found to show a significant positive correlation with serum ferritin, serum creatinine levels and CIMT measurements. Conclusion: Serum hepcidin was significantly associated with anemia in CKD patients. Association of hepcidin with carotid atherosclerosis indicates a possible role of hepcidin in the causation of cardiovascular morbidity and mortality. Keywords Hepcidin, Chronic kidney disease, Atherosclerosis

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Topic: Haematology PO 151 Antimalarial Activity of Imatinib: An In-Vitro Study Vrushali Pathak, Kanjaksha Ghosh, Roshan Colah Department of Haematogetics, National Institute of Immunohematology, KEM hospital campus, Parel, Mumbai 400012 Summary: Antimalarial activity of commercially available tyrosine kinase inhibitor (imatinib) was tested in in vitro culture system for five different laboratory strains and found to cause dose dependent inhibition of parasite maturation. Introduction: Development of the antimalarial drug resistant strains has currently become a major public health challenge. There is an urgent need to develop new antimalarial drugs. Tyrosine kinase inhibitors are receiving increasing attention as they play a very important role in parasite development and can be used in the antimalarial therapy. Here we investigated the in vitro antimalarial activity of commercially existing tyrosine kinase inhibitor, imatinib mesylate (Gleevac 400, Novartis Pharmaceuticals). Materials and Methods: The modified WHO microtest technique was used to measure the inhibition of schizont maturation by microscopy. Five different strains of Plasmodium falciparum (3D7, 7G8, Dd2, MRC2 and RKL9) were used in this in vitro study. IC50 values were determined for imatinib mesylate (Gleevac 400, Novartis Pharmaceuticals) for each strain. Final concentrations were ranged from 1 to 10 lM for Imatinib. IC50 values were calculated by probit analysis. Results: The IC50 values (drug concentration at which 50 % of cell growth inhibition occurs) for P. falciparum strains were given in the Table.

Strain

IC50 (lM/L)

3D7

2.291

7G8

2.291

RKL9 MRC2

2.089 1.445

Dd2

1.184

Conclusion: In this in vitro study Imatinib showed dose dependant inhibition of parasite maturation. Synergistic effect of Imatinib in combination with other antimalarial drugs can also be studied by this in vitro system. Keywords Imatinib, Antimalarial activity, Plasmodium falciparum

PO 152 Anti-Leukemic Activity of Relafen Against U937 and K562 Human Myeloid Cell Lines Shila Elizabeth Besra, Subhadeep Roy, Nilanjana Deb Drug Development/Diagnostic & Biotechnology Division, CSIR, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata-700032, West Bengal, India. Tel: 91-33-2413-3223, Fax-9133-2473-5197 Summary: Cancer is a complex multifactorial disease of the cell. There has always been a close relationship between inflammation and cancer. Some anti-inflammatory chemo preventive agents have

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been found to suppress growth and proliferation of malignant cells through induction of programmed cell death. It is likely that antiinflammatory agents may possess anti-cancer activity and vice versa. Relafen is a non-acidic NSAID, rapidly metabolized in the liver to 6-methoxy-2-naphthyl acetic acid. Relafen’s active metabolite inhibits cyclooxygenase enzyme and blocks COX-2 activity. Introduction: In hematopoietic malignancies, chemotherapeutic approaches are widely applicable. Nowadays, several therapeutic approaches have been taken to overcome the complexities of cancers. We have studied Cytotoxic and apoptogenic effect of Relafen on human myelogenous leukemic cells. Materials and Methods: We performed Cell viability study by MTT assay; Morphological study was done by florescence (A.O and EtBr) and confocal microscopy (PI), DNA laddering assay by Agarose gel electrophoresis, flow-cytometric assay (Annexin V FITC and PI) and Caspase (9 and 3) study. Results: The IC50 values were found in U937 and K562 cell line. The morphological analysis and DNA laddering assay confirm apoptosis in acute and chronic myelogenous cells significantly. Flow-cytometric analysis also confirms early & late apoptosis at the four different quadrants and cell cycle arrest at G1/ G0 phase of U937 & K562 cell lines. Apoptosis is mediated through activation of Caspase—9 and 3. Conclusion/Diagnosis/Impression: The findings suggest that Relafen shows apoptosis mediated through Caspase cascade. It can therefore be considered as a good therapeutic agent in cancer chemotherapy.

Topic: Laboratory Pathology PO 153 Analysis of Critical Values in NABL (National Accreditation Board for Testing and Calibration Laboratories) Accredited Hematology and Clinical Pathology Laboratory Dr. Mauli M. Joshi, Dr. Het B. Thanky Department of Pathology, Pramukhswami Medical College, Shree Krishna Hospital, Karamsad, Gujarat, India Objectives: Reporting of laboratory critical values has become an issue of national attention as illustrated by recent guidelines described in the National Patient Safety Goals of the Joint Commission on Accreditation of Healthcare Organizations. The aim of this study was to improve effectiveness, patient safety and operational efficiency by improving laboratory outcome measurement. : All data were obtained from reports generated by hematology and clinical pathology laboratory that have been recorded into critical call back register Results: During the period of the study, the hematology and clinical pathology laboratory reported 19,423 critical values. The majority of critical callbacks (78.4 %) resulted from testing performed in the hematology laboratory. The analytes most commonly called back were Hemoglobin and Urine Ketone. We have recorded maximum 52.7 % call back from inpatient department followed by emergency department 34.2 % and outpatient department 13.1 %. Critical value calls were prominent 59.6 % in morning shift and minimum 2.9 % in night shift. The mean time between entering value in the critical callback register and conveying the information to the patient location or ordering clinician was 21 min for IPD, 30 min for OPD and 20 min for ED. Conclusions: Every laboratory should have at its disposal a procedure to notify critical results. A consensus should be reached with clinicians to establish a specific list of critical limits according to the type of patient and the timeliness of laboratory test. Keywords Critical call back, Turn around time, Laboratory information system

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Topic: Proffered PO 154 Association of Factor XIII A&B Subunit Polymorphism with Acute Myocardial Infarction in Indian Patients Sunil Kumar, Ravi Ranjan, Rahul Sharma, Sudha Sazawal, Renu Saxena Hematology & All India Institute of Medical Sciences and Research New Delhi Summary: Acute Myocardial infarction (AMI) is a major cause of death and disability worldwide. Several studies have documented the role of coagulation factor XIII (FXIII) polymorphisms in the pathogenesis of AMI. In view of this, we aimed to study the correlation of the polymorphisms of FXIII A&B subunit (Val34Leu and His95Arg) with the plasma FXIII levels in AMI patients as well as healthy controls in India. Plasma FXIII levels and polymorphism of FXIII were identified by ELISA and PCR–RFLP respectively. The elevated levels of FXIII did not show any significant association with the polymorphisms of FXIII. Objective: The aim of the study was to determine the association of FXIII A&B subunit polymorphism (Val34Leu and His95Arg) with the plasma factor XIII level in AMI patients as well as healthy control in India. Method: Total 50 consecutive patients with AMI and 50 age and sex matched healthy controls were the study subjects. Plasma levels of FXIII were identified by ELISA and the Polymorphisms of FXIII A&B subunit was detected by PCR–RFLP respectively. Result: Plasma levels of FXIII were found to be significantly higher in AMI patients (22.2 ± 2.5 SD) than controls (17.2 ± 2.1 SD), (P \ .001). The frequency distribution for his95arg polymorphism among the AMI patients and healthy control was as follows: His/His, His/Arg, Arg/ Arg genotypes were 88, 12, 0 % and 86, 12, 2 %, respectively (P = 0.766), For Val34Leu polymorphism, Val/Val, Val/leu, leu/leu genotype were 68, 32, 0 % and 82, 16, 2 %, respectively (P = 0.100). Conclusion: The elevated level of FXIII did not show any association with the polymorphisms of FXIII gene. A study with larger sample size is required to find out the association of phenotype with the genotype Keywords Acute myocardial infarction, Coagulation factor XIII, Polymorphisms

S531 possibly leads to hemorrhagic or thromboembolic events despite careful dosage monitoring. Several polymorphisms in VKORC1, CYP2C9, CYP4F2, EPHX1, Calumenin, GGCX, factor IX, factor II and factor VII have been shown to be associated with warfarin dosage and other associated adverse events Materials and Methods: 89 warfarin treated patients were genotyped for VKORC1 haplotype by direct sequencing method 250 warfarin treated patients were genotyped for VKORC1 and CYP2C9 genotype using PCR–RFLP method 200 warfarin treated patients were genotyped for EPHX1 T113C (rs1051740) and CYP4F2 rs2108622: G[A polymorphism using allele specific PCR method 73 warfarin treated patients were genotyped for CALU c.*4A[G (rs1043550) polymorphism using allele specific PCR method. 50 patients were screened for GGCX 12970 C[G genotype using allele specific PCR method. 34 patients were screened for Factor VII c. -401G[A and Factor VII c. -402G[T genotype using direct sequencing method Results: VKORC1 haplotype H1 and H2, VKORC1-1639G[A, and CYP2C9*3 were found significantly associated with therapeutic warfarin dose requirement and risk of overanticoagulation. VKORC1 haplotype found strongly linked with VKORC1-1639 G[A polymorphism, hence addition of this haplotype does not add more information. CYP4F2 G[A (rs2108622), EPHX1 T113C (rs1051740), and CALU c.*4A[G(rs1043550) SNPs added minor contribution in warfarin pharmacogenomics. No contribution was noted for FVII promoter region SNPs. GGCX 12970 C[G variant was not found in Indian patients. Conclusion: VKORC1-1639 G[A, CYP2C9 genotype, CYP4F2 G[A (rs2108622), EPHX1 T113C (rs1051740), and CALU c.*4A[G (rs1043550) polymorphisms will be beneficial to formulate warfarin pharmacogenetic algorithm in Indian patients. Skipping of four VKORC1 haplotype tag SNP’s, Factor VII promoter region polymorphism and GGCX 12970 C[G genotype can make warfarin pharmacogenetics more cost effective in Indian population. Key words Warfarin, Pharmacogenetics, India

Topic: Plenary Symposia PO 156 A Study of the Clinicopathologic Features of Multiple Myeloma in Young Patients

Topic: Clinical/Adult Haematology Anshu Palta*, Anita Tahlan*, Asif Baliya*, Kisley Dimri** PO 155 Warfarin Pharmacogenetic Studies in Indian Population Tejasvita Gaikwad, Shrimati Shetty, Kanjaksha Ghosh Department of Haemostasis and Thrombosis, National Institute of Immunohematology (ICMR), KEM Hospital, Parel, Mumbai, India Summary: Warfarin is an anticoagulant, widely prescribed for prevention of thrombosis. Several polymorphisms have been found to be associated with dose requirement and adverse effects. In the present study, we found that, VKORC1 and CYP2C9 polymorphisms are clearly the most important genetic factors for warfarin response, but inclusion of EPHX1, CYP4F2 and CALU genotypes will be beneficial for accurate warfarin dosage prescription in Indian population. No contribution was noted for VKORC1 haplotype tag SNP’s, Factor VII promoter region polymorphisms and GGCX 12970 C[G genotype; hence skipping these polymorphisms in the screening algorithm can make it more cost effective in Indian population. Introduction: Warfarin (CoumadinÒ) is a widely used anticoagulant to prevent thromboembolic disorders. Management of anticoagulation therapy is difficult as there is a wide inter- and intra-individual variability that

*Dept. of Pathology, **Dept. of Radiotherapy; GMCH-32, Chandigarh. *Assistant Prof., *Associate Prof., *PG student, **Assistant Prof. Objectives: To study the clinicopathologic features of young patients diagnosed with MMo To study response to therapy in these patients Materials and Methods: This is a retrospective observational study of clinicopathologic features of patients diagnosed with multiple myeloma in GMCH-32, Chandigarh from June 2006–June 2014. The detailed clinical data, radiological features and slides of serum electrophoresis, bone marrow aspirate and trephine biopsies were retrieved from archival material. Results: Of the 122 patients diagnosed with multiple myeloma, 4 patients (3 males and 1 female) were less than 40(30–39) years of age, constituting 3.2 % of all cases. All the patients presented with complaints of backache. Three of the patients (75 %) had anemia; impaired renal function was seen in 2 patients (50 %) and hypercalcemia in 1 patient (25 %). Extramedullary plasmacytomas were found in 2 patients (50 %). Radiological examination revealed lytic bony lesions in all the 4 patients. Serum electrophoresis was positive for M band in 2 cases (50 %). On bone marrow examination, all cases had [10 % plasma cells (17–58 %). Three patients (75 %) responded well to therapy: M P regime (one),

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S532 M PT regime (two) and are doing well on follow up. One patient who received M PT regime expired during the course of therapy because of aggressive disease. Conclusion: Presenting features of young patients with MM are similar to those in older age group except for the higher incidence of extramedullary involvement. The survival of these patients is longer than that of older patients. Keywords MM: Multiple Myeloma, M: Melphalan, P: Prednisolone, T: Thalidomide

PO 157 Bisphosphonates Related Osteonecrosis of Jaw (BRONJ) in a Myeloma Patient: A Case Report Dr. Neelesh Jain, Dr. Shilpa Bhartia, Dr. Amrita Chakrabarti, Dr. Joydeep Chakrabartty Apollo Gleneagles Hospital, Kolkata 700054 Background: Bisphosphonates use has become the standard of care in the treatment of myeloma because of their inhibitory effect on osteoclastic activity, reduction in the risk of pain and fractures, thereby increasing quality of life for the patient. Osteonecrosis of the jaw is an emerging problem which is mainly associated with the long term use of bisphosphonates, especially second and third-generation (nitrogen-containing), including zoledronate and pamidronate. Case details: A 76-year old male patient was diagnosed as IgG Kappa multiple myeloma with no lytic lesions on skeletal survey in July 2012, and received multiple chemotherapy regime till Feb 2014 when he improved remarkably. An initial dental assessment and extraction was done prior to starting zolendronic acid (4 mg IV monthly). After receiving three injections of zolendronic acid patient developed left mandibular toothache for which a dentist’s opinion was taken and pain was subsided with analgesics. In this period the zolendronic acid was stopped for about 10 months and again restarted. After having four monthly doses patient again complained of toothache, this time the pain was more severe with left mandibular swelling for which the patient was referred to a specialist dentist. USG and MRI was done which showed soft tissue inflammation but not definite osteonecrosis. Subsequently a cone beam computed tomography (CBCT) confirmed it as mandibular osteonecrosis stage-I disease. This was treated with long term broad spectrum antibiotics and after 6 months off treatment involved dental extraction and surgical debridement was done. Now after 2 months of this surgical intervention patient is doing very well and pain free. In conclusion, there is always a risk of osteonecrosis of jaw while using a long term bisphosphonates in myeloma patients, and a regular monitoring and care is required to identify the problem at right time. Never hesitate to refer these patients to a specialist dentist in case of any dental issues prior to or during the therapy.

Topic: Haematology, Oncology PO 158 Clinical Study of Multiple Myeloma with Special Reference to its Staging Vineet Kumar Todi*, Pegu A. K.**, Kakoti S.*** *PGT Medicine, **Associate Professor Department of Medicine, ***Professor & HOD Department of Medicine Department of Medicine, Assam Medical College & Hospital Dibrugarh-786002, University-Srimanta Sankaradeva University of Health Sciences Assam

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Summary: Multiple myeloma is a common haematological malignancy, but need strong suspicion for the diagnosis. Correlation exists between age, hemoglobin, serum creatinine, bone marrow plasma cell, severity of symptoms with staging of the disease. Introduction: Multiple myeloma is a malignant proliferation of plasma cell derived from single clone, or morphologically abnormal plasma cells. As presentation is variable, under-diagnosis may be a possible explanation for low incidence rate in India. Materials and Methods: Hospital based observational study done at AMCH Dibrugarh, during last 1 year. Clinical-laboratory data gathered on 56 untreated symptomatic myeloma patients. Staging done by ISS. Results: Most common age 50–60 years, Male: Female ratio 6:4; backache and easy fatiguability are most common symptom followed by neurological symptoms. Mean Hemoglobin 10 g/dL, creatinine level 1.1 mg/dL, Sb2M 3.8 lg/mL, albumin 3.4 g/dL, and bone marrow plasma cell 41.0 %. 44 % of patients had three or more bone lesions, and 24 % had pathologic fractures. Numbers of patients in different stages were 16.5, 50, and 33.5 % in stage I, II, III respectively. There is significant correlation between staging and observed patient variables. More than ninety percent patient with creatinine [2 mg/dl were in stage 3 Impression: In our study age of presentation was one decade earlier than in western literature which correlates with other studies in India. Maximum numbers of patients were in later stages and stages correlate with patient variable, so strong clinical suspicion is required for diagnosis of disease at early stages and help patient to live a better life. Keywords Sb2 M—Serum b2 microglobulin, ISS—International Staging system

Topic: Haematology PO 159 A Study of Hemostatic Abnormalities in Patients with Multiple Myeloma Aarti Gogia, Meera Sikka, Usha Rusia, Satendra Sharma Department Of Pathology, University College of Medical Sciences, Delhi Introduction: Varied hemostatic abnormalities have been reported in patients with multiple myeloma. Though these patients are asymptomatic for the coagulopathy, its presence confers a poor prognosis as it occurs usually in the terminal stages. This study aimed to ascertain the nature and frequency of hemostatic abnormalities in patients with myeloma. Materials and Methods: Tests of hemostasis (platelet count, PT, APTT, TT, factor VIII assay, plasma fibrinogen and D-dimer) were performed in 30 patients of myeloma and 30 age matched controls. Results: Thrombocytopenia was identified in 8.5 % patients. Prolonged PT, APTT and TT were observed in 48.3, 68.9 and 34.5 % patients respectively. Reduced fibrinogen and FVIII were seen in 37.5 % patients each. One or more laboratory parameter of hemostasis was abnormal in all (100 %) patients. An isolated prolonged APTT was the most frequent abnormal screening test. Prolonged APTT and reduced fibrinogen together were present in 14 (70 %) patients. Prothrombotic markers (D-dimer, elevated FVIII and fibrinogen) were seen in 32.7 % patients. None of these patients had clinical features of bleeding/thrombosis. Conclusion: In this study, abnormality in the tests of coagulation were observed frequently. As coagulopathy confers a poor prognosis all patients with myeloma should be screened for hemostatic abnormalities at diagnosis. Keywords Multiple myeloma, PT, TT, APTT

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Topic: Haematology PO 160 A Pilot Study of Myeloma Over a Period of 3.5 Years at SVIMS Y. C. Spoorthy Rekha1, M. Alekhya1, N. Rukmangadha1, V. Siva Kumar2, Manickavasagam3, T. Asha1 1 Departments of Pathology, 2Nephrology, 3Medical Oncology, Sri Venkateswara Institute of Medical Sciences (SVIMS), Tirupathi

Summary: Bone marrow analysis is an important element in establishing the diagnosis of multiple myeloma regardless of the indicative immunology or radiology findings. Introduction: Plasma cell myeloma is a bone marrow based, multi-focal plasma cell neoplasm. The annual incidence is 4 per 100,000 and it represents approximately 1 % of all malignant diseases. The aim of the study is to estimate the percentage of plasma cells in bone marrow aspiration, their pattern of infiltration in bone marrow biopsy and to correlate them with clinicopathological parameters based on ISS classification. Materials and Methods: Forty-one multiple myeloma patients diagnosed between 2011 and 2014 were included in the present study, of which data could be retrieved for 23 cases, from the Medical Records Department. The percentage of plasma cells, plasmablasts in bone marrow aspiration and their pattern of infiltration on trephine biopsy was correlated with clinical stage using Pearson’s test Results: Most of the patients presented in 5th and 6th decades with a mean age of presentation being 53.6 years. Plasma cell percentage on bone marrow aspiration was calculated in all the cases, few of which showed presence of plasmablasts also. Plasma cell percentage on marrow aspirations correlated significantly with stage (p \ 0.01), presence of plasmablasts and pattern of infiltration on trephine biopsy was correlated with clinical stage (p \ 0.01). Electrophoresis showed M-Band in majority. Imageologically many showed presence of lytic lesions (skull, vertebra, sternum). Conclusion/Diagnosis/ Impression: There was significant statistical correlation between the percentage of plasma cells in bone marrow aspiration and its pattern of infiltration in trephine biopsies in patients with plasma cell dyscrasias. Keywords Plasma cell dyscrasia, Plasmablasts, ISS classification

Topic: Morning Session on 7th November PO 161 Plasma Cell Neoplasms: Comparison of Bone Marrow Aspirates and Biopsies with Additional Role of CD138 Immunostaining Dr. Gala Roshani Hansraj, Dr. Sonal Jain Department of Laboratory services, Rajiv Gandhi Cancer Institute and Research centre, New Delhi, India Summary: Bone marrow involvement in Plasma cell neoplasms can be focal with preservation of areas showing normal haematopoiesis causing discordance between results of Bone marrow aspirates and biopsies. In addition these infiltrates can be missed on routine H&E staining which are highlighted CD138 immunostaining. Introduction: Plasma cell neoplasms constitute a broad spectrum of diseases characterized by clonal proliferation and accumulation of B cells producing monoclonal immunoglobulins (M component). Assessment of plasma cells in bone marrow forms an important part of diagnosis and prognosis in these entities. Materials and Methods: Patients suspected to have a plasma cell neoplasm and being treated for symptomatic multiple myeloma from July 2013 to July 2014 were selected. Bone marrow aspirates and biopsies of all these patients were analysed. Percentage of plasma cells in aspirates and nature of infiltration in bone marrow biopsies was assessed. This included 85 aspirates and biopsies. CD138 staining was performed in many cases. Results:

S533 Suspected cases of myelomas included 49 cases. Out of which 15 cases showed discordant results between findings of Bone marrow aspirates, H&E stained biopsies and CD138 immunostaining(30.6 %). 36 Bone marrows were analysed for assessing response to treatment in symptomatic myelomas. 12 of which showed discordant results between aspirate, H&E stained biopsies and CD138 immunostaining(33.33 %). Conclusion: Bone marrow biopsies should be routinely performed both for initial diagnosis of Plasma cell neoplasms and for assessment of treatment response in symptomatic multiple myelomas. Also CD138 immunostaining should be performed to correctly assess the percentage of plasma cells in bone marrows. Keywords Plasma cell neoplasms, Bone marrow biopsy, CD138

PO 162 Value of Absolute Lymphocyte Count and Absolute Lymphocyte Count/Absolute Monocyte Count (ALC/AMC) in the Prognostication of Multiple Myeloma Moumita Bakshi1, Mukund Sable1, Avinash Pandey2, Komal Walunj1, Shashikant Mahadik1, Nikhil Patkar1, PG Subramanian1, Hari Menon2, Pratibha Amre3, Sumeet Gujral1, Prashant Tembhre1 1

Laboratory of Hematopathology, Pathology department, Tata Memorial Centre, Mumbai-12, 2Department of Medical Oncology, Tata Memorial Centre, Mumbai-12, 3Department of Cytogenetics, Tata Memorial Centre, Mumbai-12 Introduction: Multiple myeloma (MM) is a malignant neoplasm of plasma cells with median duration of survival less than 5 years. However, there is considerable variability from one patient to another. Many clinical and laboratory parameters are being used for prognostication in MM. Recently, absolute lymphocyte count (ALC), absolute monocyte count (AMC) and ALC/AMC ratio in peripheral blood has been reported to be an independent prognostic factor in multiple myeloma (MM). Materials and Methods: One hundred and forty one cases of MM diagnosed in TMH from 2004 to 2013 were studied for ALC and ALC/AMC ratio in peripheral blood at diagnosis. CBC was performed on LH750 and ADVIA2120i. The results were further correlated with other laboratory and clinical prognostic factors. Statistical analysis was performed by using SPSSv16. Results: On univariate analysis, low ALC (\1,400 cells/lL) and low ALC/AMC ratio (\2.9) were not correlated with worse overall survival (OS) (p = .541, and p = .637, respectively) and progression free survival (PFS) (p = .838, and p = .925, respectively). Low ALC/AMC ratio (\2.9) were associated with poor prognostic factors such as low hemoglobin level (p = .02), and high b2-microglobulin (p = 0.002). There was no significant association of low ALC with other prognostic factors. Conclusions: Our study demonstrated that ALC and the ALC/AMC ratio are not significant independent prognostic factors. This in concordance with published data may be due to variation in the treatment protocol. Keywords Multiple Myeloma, ALC, AMC

PO 163 Serum Protein Electrophoresis: NIMS Experience Dr. M. Noorjahan1, Priscilla Abraham Chandran1, B. Yadagiri1, G. Sadashivudu2, D. Sri Bhushan Raju3, M. Nageshwar Rao4 Department of Biochemistry, 2Medical Oncology, 3Nephrology, General Medicine, Nizam’s Institute of Medical Sciences, Hyderabad 1

4

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S534 Summary: Serum protein electrophoresis (SPE) is an easy to perform laboratory test which can be used for detection and quantification of monoclonal gammopathy. Along with paraproteinemia, other SPE patterns which were found in our study were chronic inflammation pattern, acute inflammation and moderate to marked polyclonal gammopathy which reflect an underlying condition. Introduction: SPE is a commonly used laboratory examination to identify patients with multiple myeloma (MM) and other disorders of serum protein. Electrophoresis separates proteins based on their physical properties and the subsets of these proteins display reasonably predictable changes in response to acute and chronic inflammation. It is included in one of major criteria for diagnosis of MM, according to International Myeloma Working Group diagnostic criteria, 2010. Aim: To statically analyse and identify SPE pattern in 1160 cases in tertiary care centre. Materials and Methods: Retrospective analysis was done of 1160 new cases received in past 2 years in the department of Biochemistry, NIMS. 100 follow-up cases were excluded. SPE was done with agarose gel. Results: In our study we found polyclonalgammopathy (332, 28.6 %), paraproteinemia (137, 11.8 %), other SPE patterns were Protein losing syndrome (116, 10 %), acute inflammation (86, 7.4 %), chronic inflammation pattern (17, 1.5 %) hypogammaglobinemia (16, 1.4 %) and Nephrotic syndrome (13, 1.1 %). Rest of 380 (32.7 %) samples showed normal pattern. Conclusion: Commonly seen pattern in our group of patients was of polyclonalgammopathy. Majority of them are from department of Nephrology. Followed by monoclonal gammopathies indicating the importance of SPE in diagnosis of MM. Along with the pattern of SPE, clinical setting, physical findings and relevant laboratory tests would be helpful in guiding clinician in right direction.

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 PO 165 Multiple Myeloma: A Five Year Hospital Based Retrospective Study Dr. Suvarna Inumella, Dr. Lalit Kumar, Dr. Rajeshwari S. Handigund, Dr. P. R. Malurm, Dr. M. S. Khanpet Department of Pathology, KLE’s Dr. PK hospital & MRC, Belgaum Summary: Our study revealed high frequency of Multiple Myeloma cases in our hospital. The risk increased with increasing age and a significant male preponderance was observed. Most cases presented with anaemia, backache and deranged renal function tests. Introduction: Multiple Myeloma is a haematological malignancy characterized by abnormal proliferation of plasma cells that produce a monoclonal immunoglobulin (M protein). The incidence in the Asian population is relatively low as compared to the industrialized world. Aim: To estimate the frequency of Multiple Myeloma cases presenting in our hospital. Materials and Methods: The study included all the Multiple Myeloma cases diagnosed at the Hi-Tech Laboratory of KLE’s Dr. PK Hospital, Belgaum, between January 2009 and July 2014. The relevant clinical data was obtained from the hospital records and analysed. Results: A total of 1446 bone marrow aspirations were performed during the study period. 255 haematological malignancies were diagnosed out of which 53 Multiple Myeloma cases (20.78 %) were detected. 23 out of 53 cases (43.39 %) were detected in patients above 60 years of age. Marked male predominance was noted, with male to female ratio of 3.1:1. Majority of the patients presented with anaemia, backache and had elevated serum urea, creatinine and ESR levels. About a third of all cases presented with lytic lesions of the bones as well as fractures. Conclusion: More extensive studies should be undertaken to establish the incidence of Multiple Myeloma in different parts of our country.

Topic: 2014 PO 164 Hepatic Sol, Lymphadenopathy and Monoclonal Gammopathy: A Diagnostic Challenge Shilpy, Pranati Mohanty, D. K. Aggarwal*, R. K. Jena, B. P. Das S.C.B. Medical College and Hospital, *A.H.R.C.C. Objective: Hepatic SOL & lymphadenopathy not a common presentation of myeloma. Methods and Results: 60 years old male presented with pain abdomen.USG revealed multiple SOL in the liver upper GI endoscopy and colonoscopy were normal. CT scan revealed multiple SOL in liver & also in one of the ribs with periportal & peripancreatic lymphadenopathy. Thus, clinically suspected as metastatic carcinoma. Guided FNAC and biopsy from hepatic SOL revealed features of lymphoplasmacytic lymphoma/plasmacytoma. On further investigation Mband positive. Bone marrow revealed 4 % plasma cells. Quantitation& immunofixation & SFLC assay could not be done Conclusion: Though patient presented with lytic lesion of bone, positive M band & tissue plasmacytosis pointing towards diagnosis of myeloma but at the same time atypical features like hepatic SOL & lymphadenopathy were seen which are extremely uncommon. Further study required to differentiate lymphoplasmacytic lymphoma, Waldenstroms macroglobulinemia from MGUS & myeloma. Keywords Hepatic SOL, Myeloma, Gammopathy

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PO 167 Safety and Tolerability of Dasatinib in Patients with Chronic Myeloid Leukemia (CML) and Philadelphia Chromosome– Positive Acute Lymphoblastic Leukemia (ph + all): Pooled Analysis of Over 2,400 Patients Hemant Singh Bhadauria*, Giuseppe Saglio1, Philipp le Coutre2, Jorge Cortes3, Jirˇ´ı Mayer 4, Philip Rowlings5, Milayna Subar6, Jeanette Preston7, Neil Shah8 *Bristol-Myers Squibb, India, 1University of Torino, Ospedale San Luigi Gonzaga, Orbassano-Torino, Italy, 2Charite´, Campus Virchow Klinikum, Universita¨tsmedizin Berlin, Berlin, Germany, 3The University of Texas MD Anderson Cancer Center, Houston, Texas, United States, 4Department of Internal Medicine, Hematology and Oncology, University Hospital Brno, Brno, Czech Republic, 5Calvary Mater Newcastle Hospital, University of Newcastle, Waratah, New South Wales, Australia, 6Bristol-Myers Squibb, Lawrenceville, New Jersey, 7Bristol-Myers Squibb, Hopewell, New Jersey, 8UCSF School of Medicine, San Francisco, California, United States Introduction: The objective of this analysis is to report clinical safety data from a large adult population of dasatinib treated pts (n = 2440) derived from 9 single-arm or comparative clinical trials of CML or Ph + ALL resistant or intolerant to imatinib (n = 2182) or newly diagnosed CML in chronic phase (n = 258). Materials and Methods: All pts who received C1 dose of dasatinib are included. AEs that were determined by the investigator to have a certain, probable, or

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 possible relationship to the study drug were defined as drug-related. Very common AEs and AEs of interest were analyzed. Results: The median duration of therapy was 37 months in pts with newly diagnosed CML-CP and 15 months in pts with IM resistant or intolerant CML or Ph + ALL. Minimum follow-up was 3 years and 5 years in the first-line DASISION (n = 258) and second-line (n = 662) CMLCP trials, respectively. On-study drug related AEs with frequency C10 % (any grade; n = 2440) were diarrhea, pleural effusion, headache, dyspnea, rash, fatigue, nausea, peripheral edema, musculoskeletal pain, hemorrhage, pyrexia, vomiting, abdominal pain, infection and cough. Drug-related AEs led to discontinuation in 16 % of the 2,440 pts. Fluid-related AEs (drug-related, any grade) were reported in 44 % of pts. Cardiac AEs, including ischemic AEs, rhythm abnormalities, and congestion were infrequent. All-cause cerebrovascular accidents and transient ischemic attacks were reported in 0.6 % and 0.4 % of pts, respectively, and were rarely drugrelated. Summary/Conclusion: Analysis of a large pooled dataset from dasatinib clinical trials did not identify new safety signals. Although pleural effusions are considered common, they are generally reversible. AEs those are potentially irreversible, including vascular occlusive AEs, were rarely reported in dasatinib-treated pts.

PO 168 Tyrosine Kinase Inhibitor (TKI) Switching: Experience from Simplicity: A Prospective Observational Study of ChronicPhase Chronic Myeloid Leukemia (CP-CML) Patients in Clinical Practice Dr. Rahul Bargaje (Bristol-Myers Squibb, India), Ru¨diger Hehlmann1, Jorge Cortes2, Carlo Gambacorti-Passerini3, Stuart L. Goldberg4, H. Jean Khoury5, Michael Mauro6, Mauricette Michallet7, Hesham Mohamed8, Ron Paquette9, Bengt Simonsson10, Milayna Subar8, Michelle Turner11, Teresa Zyczynski8 1

Universita¨t Heidelberg, Mannheim, Germany; 2MD Anderson Cancer Center, TX, USA; 3University of Milano Bicocca, San Gerardo Hospital, Monza, Italy; 4John Theurer Cancer Center at Hackensack University Medical Center, NJ, USA; 5Emory University, GA, USA; 6Memorial Sloan-Kettering Cancer Center, NY, USA; 7Centre Hospitalier Lyon Sud, Lyon, France; 8BristolMyers Squibb Co, Princeton, NJ, USA; 9UCLA Medical Center, CA, USA; 10Uppsala Universitet, Upsala, Sweden; 11ICON Plc, San Francisco, CA, USA Introduction: To investigate patterns of, and reasons for, TKI switching in patients with CP-CML who discontinued first-line treatment. Materials and Methods: The primary study objective is to understand TKI management patterns in clinical practice. The study includes three prospective cohorts of pts treated with imatinib (IM), dasatinib (DAS) or nilotinib (NIL) as initial therapy. Data on treatment discontinuation and treatment switching in all pts with C12 months of follow-up are presented for prospective cohorts. Results: 949 pts were enrolled. Median follow-up was 1.6 years. Across all regions, 733 of 949 pts were followed for C12 months after initiation of first-line TKI (IM: n = 375, DAS: n = 178 or NIL: n = 180). Of these, 30.1, 17.4 and 22.8 % of patients discontinued IM, DAS and NIL, respectively, within a year of initiation. The main reason for TKI discontinuation was physician-reported intolerance (IM: 62.8 %, DAS: 80.6 %, NIL: 87.8 %). Physician-reported primary resistance, leading to discontinuation, was only noted in IM-treated pts (8.8 %). Median time to first discontinuation varied by TKI (IM: 127 days, DAS: 129 days, NIL: 56 days). A proportion of pts who discontinued first-line TKI within 12 months switched to a second-line TKI (IM: 70.8 %, DAS: 54.8 %, NIL: 58.5 %). Conclusion: The proportion of pts discontinuing first-line treatment for CP-CML, and reasons for discontinuation, vary by

S535 TKI. While intolerance was the primary reason for treatment discontinuation in all TKI cohorts during the first 12 months, primary resistance was reported in IM-treated pts only.

Topic: Clinical Haematology PO 169 Priapism: Rare Presenting Manifestation of Chronic Myeloid Leukemia and its Management: Case Series of 5 Cases Dr. Rajesh Patil, Dr. S. Chandrakala, Dr. Farah Jijina, Dr. Nilesh Wasekar, Dr. Manoj Toshniwal, Dr. Shailesh Bamborde Dr. J. C. Patel Dept Of Haematology, KEM hospital, Parel, Mumbai, 400012 Introduction: Priapism is defined as a persistent penile erection that continues hours (4 h) beyond, or is unrelated to sexual stimulation. Haematological conditions are the cause of 20 % of cases of priapism. CML accounts for half of these cases. Materials and Methods: Patients presenting as priapism and later diagnosed to have CML at our centre in last 5 years are studied. The management and complications in these patients are discussed. Results: (1) Priaprism was seen in 5 out of 317 (1.52 %) of our CML patients at presentation. (2) Patients were aged from 25 to 30 years with median age of 28 years. (3) Two of our patients presented within 24 h of symptoms onset while rest 3 presented later. (4) Presenting total leucocyte count ranged from 2,84,000 to 6, 07,000 cu mm. (5) All 5 patients had splenomegaly ranging from 2 to 15 cm while only one patient had hepatomegaly. (6) Due to failure of intercavernous aspiration and phenylephrine injections, 4 of our patients had undergone surgical shunt procedure. (7) All of them were initially given hydroxyurea along with supportive treatment. All 5 were diagnosed as CML-CP and started treatment in form of TKI inhibitor, Imatinib. (8) All of them achieved complete hematologic response (CHR) and cytogenetic response while one lost CHR to Imatinib and one progressed to blast crisis. (9) Erectile dysfunction developed in 3 out of 5 patients. Conclusion: Priapism in CML needs prompt diagnosis and treatment. Diagnosis of underlying CML and its treatment can avoid recurrence of priapism and further consequences. Keywords Priapism, Chronic myeloid leukemia, Erectile dysfunction

Topic: 2014 PO 170 Avascular Necrosis of Femoral Head: A Rare Complication in CML with Co-existant Beta Thalassaemia Trait Shafqat Bano, Pranaty Mohanty, Rabindra Kumar Jena*, Bidyut Prava Das Department of pathology, Clinical Haematology, *S.C.B. Medical College and Hospital Objective: Avascular necrosis is a common complication of sickle cell anaemia. It is extremely rare in Thalassemia syndrome though bone marrow involvement in CML is seen in 3–5 % of cases, AVN is not a complication. Here we report AVN developing during the course of therapy in CML in a known case of beta thalassemia trait-a rare occurence Methods and Case History: A 42-year female, known case of beta thalassemia trait presented with pain & distension of abdomen with generalized weakness. She was diagnosed with CML in chronic phase from peripheral smear, bone marrow &

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S536 confirmed by BCR-ABL FISH study. Imatinib 400 mg/day was started & she was doing well &attained complete remission. After 4 years the patient complained of limping & pain in right hip joint. X-ray & MRI revealed AVN right femoral head (stage 4).further investigation revealed normal coagulation profile & sickling negative & other thrombophilic states ruled out. Conclusion: Possibility of AVN be thought of while dealing with CML patients presenting with osseous complications in presence of normal coagulation profile hyperviscosity resulting from leucocytosis and thrombocytosis may be responsible for vaso occlusion in and end artery leading to AVN Keywords CML, AVN, BETA Thalassemia

PO 171 The Enigmatic Chronic Myelomonocytic Leukemia Dr. R. Archanadevi, Dr. Jyoti R. Kini, Dr. Pooja K. Suresh, Dr. M. Nirupama, Dr. Urmila N. Khadilkar, Dr. B. Prashanth Departments of Pathology & Hematology, Kasturba Medical College, Mangalore, Manipal University Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder characterized by absolute persistent monocytosis ([1 9 109 L) and is classified as myeloproliferative neoplasm/myelodysplastic syndrome by 2008 WHO classification of hematopoietic neoplasm. The clinical, haematological and morphological features vary from predominantly myelodysplastic to myeloproliferative neoplasm. Since CMML is a rare entity, we attempted to study the clinicopathological and molecular parameters of CMML in our institution. Materials and Methods: Records of bone marrow aspirate were reviewed for all the cases diagnosed as chronic myelomonocytic leukemia over a period of 20 months from January 2013 to August 2014 in Kasturba medical college, Mangalore. The history of the cases, clinical findings, peripheral smear, bone marrow findings and flow cytometry were evaluated along with all other investigations. Results: A total of 5 cases were diagnosed as CMML based on the morphological criteria on bone marrow aspirates. The average Absolute monocyte count was 1.3 9 109 L. The age group ranged from 21 to 70 years with M:F = 2:3. The most common presenting symptom was fatigue and generalized weakness. Biopsy was available in 4 cases, which confirmed CMML in 3 cases, 1 case was diagnosed as myeloid sarcoma with blastic transformation based on immunohistochemistry. Flow cytometry was done in one case, which was diagnosed as acute myelomonocytic leukemia based on [20 % blasts. Cytogenetic evaluation such as BCR-ABL, monosomy 7 and deletion 20 were negative in all the cases. Conclusion: The diagnosis of CMML rests on a combination of morphological, histopathological and chromosomal abnormalities in the bone marrow. It is important to exclude other myeloproliferative neoplasms and infections/autoimmune conditions that can cause monocytosis. Keywords Chronic myelomonocytic leukemia, Monocytosis

Topic: Hematology PO 172 Prevalence of JAK2 (V617F) Mutation Among Polycythemic Subjects Dr. S. Damodhar, Prof. Vinod Kumar Paniker, Assoc. Prof. R. Krishnamoorthy Department of Immunohematology and Transfusion Medicine, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Chennai

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Objectives: To determine the prevalence of JAK2 mutation among 94 polycythemic subjects referred to our department for therapeutic venesection. Materials and Methods: This retrospective study was carried out in our Department of Transfusion Medicine, Sri Ramachandra Medical College and Research Institute, Chennai between January 2013 and August 2014. Polycythemic individuals who visited our dept for therapeutic venesection were reviewed from previous records and JAK2 mutation status was obtained from previous records and JAK2 mutation status was obtained from computerised laboratory registry and analysed. During the study period 94 polycythemic individuals were referred to our department for therapeutic venesection. Results: All the individuals were subjected to JAK2 mutation testing. 5.30 % of polycythemic individuals showed JAK2 V617F mutation. Out of which 2 % were male and 3 % female. Conclusion: Prevalence of JAK2 mutation among polycythemic individuals was very minimal. All of them tested positive showed JAK2 V617 mutation. Irrespective of JAK2 mutation status therapeutic venesection remains the main stay of treatment in polycythemia. Keywords Polycythemia, Therapeutic venesection, JAK2 V617F mutation

Topic: Laboratory haematology PO 173 Spectrum of BCR-ABL Kinase Domain Mutations Seen in Patients of Chronic Myeloid Leukemia at Tata Memorial Centre Kiran Ghodke1, Shruti Chaudhary1, Prashant Tembhare1, P. G. Subramanian1, S.K. Hasan1, Bhausaheb Bagal2, Uma Dangi2, Hasmukh Jain2, Manju Sengar2, Navin Khattry2, Hari Menon2, Sumeet Gujral1, Nikhil Patkar1 Hematopathology Laboratory, Tata Memorial Centre, 2Adult Hemato-lymphoid Disease Management Group, Department of Medical Oncology, Tata Memorial Centre 1

Objectives: We analyzed the ABL1 kinase domain for mutations in chronic myeloid leukemia (CML) patients with suspected resistance to tyrosine kinase inhibitor (TKI) therapy. We describe single point, compound/polyclonal mutations, insertions/deletions as well as 8 novel mutations as a mechanism of resistance to TKI. Materials and Methods: Consecutive patients of CML with suspected resistance to TKI therapy were accrued. A nested PCR using cDNA was done followed by Sanger sequencing with overlapping primers to ensure that every base substitution was confirmed at least twice. Results: 136 patients were sequenced [age ranging from 7 to 70, male predominant (4:1)]. 73 (53.7 %) patients had a detectable abnormality in the ABL KD. Four classes of mutations: single point mutations (16.9 %), polyclonal point mutations (18.4 %), insertions and deletions other than alternative splicing events (8.1 %), alternative splice variants (28.7 %) were seen. Amongst base substitutions, 20.5 % mutations were in P loop, 24.7 % in Imatinib binding region, 1.4 % in the catalytic domain and 4.1 % in the activation domain. The commonest genetic aberration was deletion of ABL exon 7 (p.R362fs*21/c.1086_1270 del 185, 28.7 % of patients). The intron-derived insertion/truncation mutation was noted in 8 (5.9 %) patients (p.C475fs*11; c.1423_1424ins35). The commonest known pathogenic mutation was p.T315I (10.3 % of patients). Eight novel mutations described include p.H396L, p.V506E, p.V299A, p.Q477X, p.V338G, p.G442E, p.Ala424Glyfs*3 (c.1271_1342del71) and c.1103_1116delins216. Conclusion: It is widely believed that only point mutations in the kinase domain contribute to TKI resistance, the presence of in-del type of mutations must be taken into account as demonstrated by this study. Keywords Chronic myeloid leukemia, ABL1 kinase domain

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Topic: Deligate PO 174 Philadelphia Positive Adult Type CML in Childhood: A Rare Case Report Manamohan Biswal, Aparajita Mishra, Kalpalata Tripathy, Bidyut Prava Das Dept. of Pathology, S.C.B. Medical College, Cuttack, Odisha Summary: A 3-year old male child presents of fever, pallor, hepatomegaly for 3-months duration. Haematological investigation, fetal haemoglobin, and normal karyotype are done & is diagnosed as philadelphia chromosome positive CML (adult type). It is a rare entity in childhood & responding well to therapy. Introduction: Philadelphia positive adult type CML in childhood is very rare. It is to be distinguished from Juvenile myelomonocytic leukaemia (JMML). Modem of information is available in the form of case report. Materials and Methods: Haemoglobin profiles:- Hb-6.9 g/dl, TLC110 9 109/L TPC-620 9 109/L. DC-N-29 %, E-05 %, B-15 %,Ll11 % M-06 %. Myelocyte-14 %, Metamyelocyte-15 %, Blast-5 % Fetal haemoglobin & normal karyotype –Philadelphia chromosome positivity. Result: The case is diagnosed as Philadelphia chromosome positive CML (adult type). Conclusion: Philadelphia chromosome positive adult type of CML though rare is possible in childhood. Since its course and prognosis is similar to adult type of CML so a definitive diagnosis if done can show excellent response to therapy. Keywords Childhood, CML, Karyotype, Philadelphia

Topic: Oral paper presentations PO 175 Retrospective Analysis of Clinical Correlates of Three BCR-ABL Kinase Domain Mutations (t315i, m351t and f311l) in Chronic Myelogenous Leukemia (CML) Patients Treated with Imatinib Mesylate (TKI) Dr. Mukul A Gharote1, Dr. Harsha P. Panchal2, Dr. Asha Anand2, Dr. Apurva P. Patel2, Dr. Sonia K. Parikh3, Dr. Rakesh Rawal4, Dr. Krupa Shah5 1

3rd Year Resident Medical oncology (DM), Gujarat Cancer & Research Institute (GCRI); 2Professor, Department of Medical Oncology, GCRI; 3Associate professor, Department of Medical Oncology, GCRI; 4Professor, Department of molecular biology GCRI; 5Fellow, Department of molecular biology GCRI Summary: BCR-ABL kinase domain mutation leads to Imatinib resistance, these mutations may preexist. Introduction: BCR-ABL Kinase domain mutations represent the most important disease-related factor in CML resistance. Highly resistant clones may preexist and emerge rapidly. Patients with CML can acquire more than one BCRABL1 mutation, which may result in increased oncogenicity. Materials and Methods: Retrospective analysis of 50 patients of Imatinib resistance was done in GCRI, from January 2014 till May 2014. Response to Imatinib was defined according to European leukemia net 2009 (ELN) criteria. Allele specific oligonucleotide–polymerase chain reaction (ASO–PCR) was performed on genomic DNA, extracted from peripheral blood mononuclear cells (PBMCs). Results: Average age was 40.75 years, 33 were males and 17 females. 47(94 %) were in Chronic phase, 2(4 %) in accelerated phase, 1 (2 %) in blastic crisis. 29/50 were having low EUTOS score, whereas SOKAl score was low in 20, intermediate in 21 while only 9

S537 had high SOKAL at presentation. Median duration of Imatinib was 48 months. 43/50 had one or more than 1 mutation. T315I mutation in 5 (10 %) patients. M351T in 32 % (16/50), F311L in 8. Conclusion: Clinical correlates, speculate, that these mutation were pre-existing and were selected by standard dose Imatinib continued for median 2 years, detection of pre-existing mutation is not the current recommendation. We report low cytogenetic response (25 %) and durability of response to 600 mg of Imatinib, even in M351T mutation, after 400 mg of Imatinib for median period of 2 years. Keywords Imatinib resistance, Pre-existing mutations, BCR-ABL Kinase Domain mut

Topic: haematology PO 176 Serum Vascular Endothelial Growth Factor (VEGF) in Patients with Acute and Chronic Leukemias Neha Bedi, Usha Rusia, Meera Sikka, Sunil Gomber Department of Pathology, University College of Medical Sciences, Delhi Introduction: Angiogenesis plays an important role in the pathogenesis of hematological malignancies. Vascular endothelial growth factor (VEGF) is a key cytokine involved in tumor angiogenesis and its levels have been reported to be elevated in leukemias and are associated with poor prognosis. Aim: To study serum VEGF levels in acute and chronic leukemias and their correlation with other hematological parameters and Bone Marrow (BM) VEGF immunoexpression. Materials and Methods: A case control study was undertaken on 90 newly diagnosed patients of leukemia (CML = 52, AML = 21, CLL = 10, ALL = 7) and 90 healthy controls. VEGF levels were measured in the serum using a commercially available ELISA kit. VEGF immunoexpression was studied in BM biopsies of 90 patients (10 normal BM biopsies used as controls). Results: The mean serum VEGF levels were significantly (p \ 0.001) higher in patients (318.34 ± 218.66 pg/ml) as compared to controls (31.9 ± 7.97 pg/ml). Serum VEGF levels were higher in CML & AML (326.136 ± 106 pg/ml) as compared to CLL and ALL (63.63 ± 27 pg/ml). Significant correlation was found between serum VEGF values and TLC in each leukemia and also between serum VEGF in CML and percentage of blasts, percentage of basophils, splenomegaly and the hasford prognostic score. Serum VEGF levels showed a significant (p \ 0.001) positive correlation with BM VEGF expression. Conclusions: A significantly higher level of serum VEGF as seen in this study and its correlation with BM VEGF suggest its role in the pathogenesis of hematological malignancies. Its correlation with other parameters indicates that it may be used as an independent prognostic marker. Keywords VEGF, Leukemia, Angiogenesis

PO 177 Expression of the Uptake Drug Transporter hOCT1 is an Important Clinical Determinant of the Response to Imatinib in Chronic Myeloid Leukemia: AIIMS Experience Sunita Chhikara, Sudha Sazawal, Rekha Chaubey, Rahul Sharma, Pravas Mishra, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi, India

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S538 Summary: Expression level of hOCT1 is a critical determinant of outcome in imatinib-treated CML. To identify potential imatinib failures, we investigated the expression of imatinib uptake transporter hOCT1. Introduction: Human organic cation transporter (hOCT1), which is a member of solute carrier 22 (SLC22) super family and is a multispecific organic cation transporter, is one of these influx transporters. An altered expression of hOCT1 may be an important factor in determining intracellular levels of the drug and, hence, affecting cell response to imatinib. Materials and Methods: Total RNA was extracted from peripheral blood mononuclear cells. Complementary DNAs (cDNAs) were synthesized and RT-PCR was performed. Results: A total of 56 CML-CP patients were analysed for expression of hOCT1 gene. CML patients aged between 20 and 60 years (mean 40 years), 44 males and 12 females with mean follow up of 36 months. Patients were classified as expressing either high (n = 22) or low hOCT1 mRNA (n = 34). Patients with high pre-treatment hOCT1 expression had higher progression-free survival and overall survival (B0.038 and B0.021). Patients who had low expression of hOCT1 mRNA (8/34), progressed to AP/BC. Moreover, 2 (12.5 %) of the 34 living patients in this group had their therapy changed to another TKI because of a loss of molecular response. Lower expression of hOCT1 can reduce the intracellular concentration of imatinib inside leukemic cells, resulting in the development of resistance to imatinib. Conclusion/Diagnosis/Impression: The expression of hOCT1 is important in determining the clinical response to imatinib.

PO 178 Thrombohemorrhagic States in Myeloproliferative Neoplasms: A Prospective study on Selected Prothromotic Factors & Contributing Role of Platelets Yatendra Parashar, Kamal Agrawal, Ashutosh Kumar, Rashmi Kushwaha, A. K. Tripathi King George’s Medical University, Lucknow Summary: In summary it should be noted that, whereas some individuals with MPN exhibit a pattern exclusively of hemorrhagic or thrombotic events, many others have both of these during the course of disease. Pathogenesis of these events is multifactorial and involves the abnormalities of platelets, erythrocytes and leukocytes arising from MPN clonal population and abnormalities of endothelial cell. Introduction: Thrombohemorrhagic episodes are directly related to morbidity and mortality in MPN patients; their early detection is essential for optimum treatment outcome and reduction of mortality and morbidity from such complications. In this study we are reevaluating the prothrombotic factors and platelet functional activity in MPN patients. Materials and Methods: This study will be conducted at Department of Pathology King George Medical University, Lucknow in year 2013–2014. 50 MPN cases and 30 age-sex matched controls were taken. Complete clinical history and examination was done. Further bleeding time, aPTT, PT, protein C, protein S and platelet aggregation with ADP was studied in these patients. Results: We found decreased activity of protein C and protein S in MPN patients. Aggregation studies show a decrease in aggregation with loss of secondary wave of aggregation with ADP in MPN cases. Values of bleeding time, PT, aPTT were increased in MPN cases as compare to controls. Conclusion/Diagnosis/Impression: Thrombotic and hemorrhagic complications both can be seen in MPN patients which increases morbidity and mortality, concern about these with early diagnosis can be helpful.

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 PO 179 Study of 24-hour Serum and CSF Methotrexate Levels in Children with ALL Receiving High Dose Methotrexate and its Correlation to Relapse Smitha HV*, Nita Radhakrishnan, Veronique Dinand, Tanvi Khanna, Anupam Sachdeva Pediatric Hematology Oncology Unit, Sir Ganga Ram Hospital, New Delhi Introduction: High dose methotrexate is a major component of most contemporary ALL treatment protocols as a CNS-directed therapy. Materials and Method: Cases of ALL diagnosed between January 2007 to December 2013 who received high dose methotrexate (MTX) were included in the study. The patients were followed up for occurrence of CNS and systemic relapse till September 2014. Children who had CNS positive status at diagnosis were excluded. Patients received 3–4 cycles of High dose methotrexate (dose 5–6 g/ m2/cycle) depending on the treatment regimen. Serum and CSF MTX levels were measured at the end of 24 h following infusion. Folinic acid rescue was instituted at 36 and 42 h based on the individual regimen and continued till serum MTX reached \0.25 micromoles/L. Results: 108 patients fulfilled the criteria with a median age at diagnosis of 4 years (1–18) and M:F ratio of 2.3:1. 88.9 % children were B cell ALL and remaining TALL. A total of 338 cycles of high dose MTX were analysed. 67 patients have completed ALL treatment and are on follow up where as the rest are in maintenance phase of chemotherapy. Median follow up of patients after completion of therapy is 58 months. 7 deaths and 6 relapses (5.9 %) were observed in the study group during this period. Of the relapses, 1 had CNS relapse, 2 combined relapse (bone marrow + testicular) and rest 3 had medullary relapse. CSF MTX levels [1 micromoles/l in all cycles of methotrexate infusion was achieved in 79.6 % cases. No correlation was found between CSF MTX levels and CNS relapse. 24 h serum Methotrexate levels of \60 micromoles/L had a significant bearing with relapse and poor 5 year EFS in 2nd and 3rd cycle of methotrexate infusions (p \ 0.001). Conclusion: No correlation was observed between CSF methotrexate levels and CNS relapse. Serum MTX levels\60 micromoles/l had statistically significant relapse and poor 5 year EFS.

Topic: Proffered PO 180 Hemophagocytic Lymphohistiocytosis Associated with Inflammatory Myopathy: A Rare and Challenging Case Afreen Karimkhan, S. Sitalakshmi, Shanthala Devi, Pradeep, Gayathri*, Latha Fathima, Sivaganesh, T. C. Yasha* Department of Haematology and Transfusion medicine, St. John’s Medical College and Hospital, Bangalore, *Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore Introduction: Secondary hemophagocytosis, a disease due to dysregulated immune system, has been rarely seen with inflammatory myopathy. This unique presentation requires early identification, to prevent mortality. Here we report a case of hemophagocytosis, secondary to inflammatory myopathy. Case: 15 year old boy was evaluated at St. Johns’ Medical College, for fever of one month duration and generalized swelling. Physical examination revealed pallor and bilateral non pitting pedal edema. Investigations showed Hemoglobin—8.8 g/dL, Total count—1.85 9 103/lL with peripheral

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 smear showing normocytic normochromic blood picture with leucopenia. Bone marrow revealed mildly hypocellular marrow with normoblastic erythroid hyperplasia, with increase in megakaryocytes and prominent hemophagocytosis. Plasma fibrinogen, serum triglycerides, serum ferritin, S. LDH and CPK levels were markedly elevated. Ultrasonography showed bulky muscles in forearm, thigh and neck with no subcutaneous edema. Electromyography showed myopathic changes and muscle biopsy revealed inflammatory myopathy. Thus, diagnosis of Hemophagocytic lymphohistiocytosis secondary to inflammatory myopathy was established. Child was started on IVIG. He improved and was discharged. Conclusion: Hemophagocytosis, a rare disorder of immune system, can be secondary to infections, malignancies or connective tissue disorders. It is rarely associated with myopathy, with approximately 10 cases reported worldwide. This report aims to highlight such rare triggers for HLH. This is associated with good prognosis, if recognized early and treated adequately. Keywords Hemophagocytosis, Inflammatory myopathy

Topic: Proffered PO 181 Spectrum of Hematological Malignancies in Children with Down’s Syndrome: A Tertiary Care Centre Experience Shirali Agarwal, Nita Radhakrishnan, Veronique Dinand, Tanvi Khanna, Anupam Sachdeva Paediatric Haematology Oncology and Bone Marrow Transplant Unit, Sir Ganga Ram Hospital, New Delhi Introduction: Children with down syndrome (DS) are at increased risk of developing both acute myeloid and lymphoblastic leukemia. In contrast to the superior outcome of AML in DS, ALL fares worse when compared to children without DS. Objective: To review the clinical features, treatment and outcome of hematological malignancies in children with DS. Design: A retrospective analysis of 14 children with DS and malignancy presenting between January 2006 and September 2014 was done. Data including history, clinical examination, diagnosis and treatment was analyzed. Results: 10 cases of leukemia and 4 cases of transient myeloproliferative disorder were diagnosed at our center during this period. In those 10 leukemia patients there were 4 males and 6 females. Median age was 1.5 years, ranging from 2 months to 5 years. Presenting features included pallor (n = 10), fever (8), petechiae (n = 4), hepatosplenomegaly (8), and respiratory distress (2). None of the patients had CNS or testicular involvement. Among the cases with leukemia, 7 had AML, 2 had Pre B ALL and one had JMML. 5 out of 7 patients with AML had M7 subtype. Bone marrow cytogenetics was available for 7 patients. Apart from trisomy 21, other common abnormalities identified were trisomy 8 and complex cytogenetic abnormalities. The 2 patients of Pre B ALL were treated on standard ALL protocol. One patient had progressive disease and another child opted for indigenous medicines and both finally died. Patients with AML were treated with COG AML protocol for DS. Out of 6 AML patients who opted for treatment, 3 are alive at a median follow up of 4.4 years, 2 died due to sepsis and 1 due to disease progression. Of the 2 AML patients with complex cytogenetics, one had refractory disease and the other is in remission and is presently doing well at the last follow-up 1 month back. The JMML patient opted not to take treatment. All 4 TMD showed spontaneous resolution. Thus amongst all those treated, the overall mortality is 57 %. Conclusion: Spectrum of hematological malignancies in DS include AML, TMD, ALL and JMML. A careful scrutiny of peripheral blood film and a close follow-up is warranted.

S539 PO 182 Clinicohematological Profile and Response to Treatment in Diamond Blackfan Anemia D. Tarangini1, Nita Radhakrishnan1, Sabina Langer Kumar2, Amrita Saraf2, Jasmita Dass2, Manorama Bhargava2, Anupam Sachdeva1 1 Pediatric Hematology Oncology and Bone Marrow Transplant Unit, Institute of Child Health, Sir Ganga Ram Hospital, New Delhi, 2 Department of Hematology, Sir Ganga Ram Hospital, New Delhi

Aims/Objectives: To study the clinical and haematological profile of children with DBA and assess response to treatment as per criteria used by DBA registry. Materials and Methods: It is a retrospective analysis of DBA cases presented to the Department of Pediatric Hematology Oncology, SGRH, between 2000 and 2014. All patients fulfilled DBA consensus criteria. Case records of all patients were analysed and follow up done telephonically. Treatment response was assessed as per criteria used by the DBA registry. Results: A total of 19 patients were diagnosed as DBA after bone marrow examination. Males:Female = 1.7:1 (12 males and 7 females). History of consanguinity was present in 2 children and family history was absent in all. Major anomalies were observed in 3 patients. 1 had anorectal malformation, 1 had clinodactyly and 3 had characteristic facies. 3 children had short stature. Hepatomegaly observed in 8 (42 %). Birth weight was available only in 13 children (mean 2.84 (2–3.6 Kg)). Mean age at 1st symptom was 6.84 months (0.5–50) and at diagnosis was 13.84 months (2–60). Mean age at first transfusion was 8.7 months (0.5–54). Presenting symptoms in all the cases was anaemia requiring blood transfusion with mean haemoglobin of 5.27 g/dl (2.8–8.9), Reticulocyte count 0.67 % (0–2.2) and MCV of 88.7 fL(81–104). HbF level was available in 11 patients with mean of 3.08 % (1.2–5.2). Parvovirus testing could only be done in one patient (negative) due to monitory constraints. High platelet count was seen in 7 (36.8 %). All patients were started on steroids. 5 patients are being followed actively at our centre and 7 were telephonically contacted. 2 showed complete response, 4 had no response, 7 were partial responders to Prednisolone at 6 weeks follow up and 6 patients were lost to follow up. Of the 4 patients who showed no response, 1 underwent MSD transplantation and is in remission, 1 expired, 1 progressed to aplastic anemia and 1 is still on prednisolone and transfusion dependent. 7 patients with partial response are transfusion independent and steroid dependent. Response to initial steroids (complete and partial) was observed in 9 (47.3 %). Conclusions: DBA shows good response to steroids. However, patients are often dependent on steroids and HSCT offers complete cure. Keywords Diamond Blackfan anemia, Steroids, HSCT

PO 183 Che´diak–Higashi Syndrome Dr Neha Khaitan, Dr. Lalita Y Patil, Dr. Kanchan Kothari, Dr. Daksha Prabhat Seth G.S Medical College, Mumbai Objectives: Che´diak–Higashi syndrome is a rare autosomal recessive disorder that arises from a mutation of a lysosomal trafficking regulator protein. The decrease in phagocytosis results in recurrent pyogenic infections, partial albinism and peripheral neuropathy The exact incidence is unknown. Less than 500 cases have been reported worldwide in the past 20 years. Light skin and silvery hair (partial albinism) and frequently complain of solar sensitivity and photophobia neuropathy are common. Materials and Methods: Clinical

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S540 history of 2 year old female child 2nd baby by birth order, Came with complaints of high grade fever on and off, Decreased intake of feed since 2 months, with h/o recurrent episodes of infections. Confirmed by bone marrow, prenatally by examining a sample of hair from a fetal scalp biopsy or testing leukocytes from a fetal blood sample, light microscopy and polarized light microscopy along with clinical history of the patient. Results: Manifestations of Che´diak–Higashi syndrome as neutropenia the most common. Associated with oculocutaneous albinism. Bone marrow smears shows ‘‘giant inclusion bodies’’ in the cells that develop into white blood cells (leukocyte precursor cells). CHS can be diagnosed prenatally by examining a sample of hair from a fetal scalp biopsy or testing leukocytes from a fetal blood sample. Under light microscopy the hairs present evenly distributed, regular melanin granules, larger than those found in normal hairs. Under polarized light microscopy these hairs exhibit a bright and polychromatic refringence pattern. Conclusion: Che´diak– Higashi syndrome is a rare disorder. Clinical correlation with lab investigations helps to reach the diagnosis. Keywords Che´diak–Higashi syndrome

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Objectives: Immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterised by a low circulating platelet count caused by destruction of antibody-sensitised platelets in the reticuloendothelial system. Eosinophilia has been reported in patients of hypertension, bronchial asthma, HIV infection, schizophrenia, rheumatoid arthritis and parasitic infection. The aim of the study was to see incidence of eosinophilia in patients of ITP. Materials and Methods: This prospective study was carried out in the Department of Pathology, PGIMS, Rohtak, Haryana over a period of two years i.e. January 2012–December 2013. A total of 1669 bone marrow aspirates were evaluated, out of which 86 patients (0.5 %) have evidence of ITP on bone marrow aspiration examination. All other causes of eosinophilia were ruled out. Results and conclusions: Thirty one patients (36 %) were male and 55 (63.5 %) were female. Majority of patients (84 %) belonged to first three decades of life. Bone marrow aspirates showed increased eosinophilic precursors in 34 patients (39 %) and 52 patients (61 %) showed normal count of eosinophilic precursors. Peripheral blood eosinophilia was observed in 12 cases. Few case reports of incidence of eosinophilia in ITP has been reported in the literature reviewed. Keywords Eosinophilia, Immune thrombocytopenic purpura

PO 184 Study of Platelet Functions and Antithrombotic Factors in Cerebrovascular Accidents: A Prospective Study Poonam Rani1, Ashutosh Kumar2, Rashmi Kushwaha3, Mili Jain4, Mohd. Yusuf5, U.S. Singh2 1

Junior Resident, Department of Pathology, KGMU, Lucknow, 2 Professor, Department of Pathology, KGMU, Lucknow, 3Assistant Professor, Department of Pathology, KGMU, Lucknow, 4Lecturer, Department of Pathology, KGMU, Lucknow, 5Research Associate, Department of Pathology, KGMU, Lucknow Introduction: Hemostasis is a delicately balanced normal physiological process which keeps the blood in fluid state while it circulates within the vascular system. It maintains a balance between excessive clotting at one end and clotting inhibitory process on other end. If this fine balance is disturbed, this may lead to abnormal enhanced clotting process which may be due to presence of large amount of activated clotting factors or deficiency of any of the natural inhibitors of coagulation. Materials and Methods: A total of 50 diagnosed cases of ischemic and haemorrhagic stroke over a period of 1 year were included in the study. An evaluation of PC, MPV, and Platelet aggregation, Protein C, Protein S and Homocysteine were done in these cases. Results: MPV was significantly higher in ischemic infarct cases (p \ 0.001) while PC was reduced (p = 0.012). Platelet aggregation was reduced in ischemic as well as haemorrhagic stroke cases. Reduced Protein C & S was found in younger age groups. Increased Homocysteine values were observed in both ischemic as well as haemorrhagic stroke cases however values were significantly higher in ischemic stroke. Conclusion: We conclude that the assessment of MPV, protein C & S provides a useful diagnostic clue in cases of ischemic infarct. Keywords Platelet count, Mean platelet volume

PO 185 Eosinophilia in Immune Thrombocytopenic Purpura Dr. Sant Prakash Kataria, Dr. Abha, Dr. Gajender Singh, Dr. Sanjay Kumar, Dr. Monika Gupta, Dr. Narender Dahiya Department of pathology, PT. BD Sharma PGIMS, Rohtak, Haryana

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Topic: Transfusion Medicine PO 186 Flow Cytometric Analysis of Platelet Allo-Immunization in Multiply Transfused Bone Marrow Transplant Patients Dr. Shashank Ojha, P. Nagaraju, M. Kamble, B. Bagal*, N. Khattry* Dept. of Transfusion Medicine, *Dept. of Medical Oncology, Tata Memorial Center-ACTREC, Kharghar, Navi Mumbai 410210 Summary: Thirty patients undergoing allogeneic bone marrow transplant (BMT) and receiving single donor platelets (SDP) were assessed for alloimmunization by FlowPRA screening test during pretransplant, peritransplant and post transplant phases. Though incidence of panel reactive antibody (PRA) positivity was high, there was no correlation with number of single donor platelets (SDP) transfusion. Introduction: Alloimmunization is characterized by increasingly poor recovery and survival of platelets in multiply transfused patients. Data from India on platelet alloimmunization in such patients are scarce. The current study was planned to know the same in allogeneic BMT patients Materials and Methods: Thirty patients were tested for anti-HLA antibodies using FlowPRA screening test. The samples were collected from pre transplant, following 4 weeks, 45 and 60 days after bone marrow transplant. All samples were stored at -80 °C mechanical freezer till analysis. Patients were categorized into three groups based on PRA levels. Each groups were further sub categorized into pretransplant, peritransplant (from hematopoietic stem cell infusion up to 21 days after transplant) and post transplant phases. All patients received leucodepleted SDP. Results: Total 30 patients were analyzed, of which 3 had low (\10 %),17 had moderate (11–50 %) and 10 had high ([50 %)PRA levels respectively. The mean SDPs received in low PRA group was 11.66 (range 4–12), in moderate 12.98 (range 0–24) and in high 10 (range 0–12) respectively. No SDP was transfused in pre transplant phase of low PRA patients. Conclusion: The incidence of alloimmunization in patients receiving multiple platelet transfusions in BMT is high. However there is no correlation of level of alloimmunization and SDP transfusion. Keywords Platelet Alloimmunization, PRA, Allogeneic BMT

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PO 187

PO 189

Effect of Carica Papaya Leaf Extract Capsule on Platelet Count in Patients of Dengue Fever with Thrombocytopenia

Non- Hemolytic Transfusion Reactions: A Dilemma

Dr. Pankaj Tantia1, Dr. Ritika Agrawal2, Dr. R. P. Agrawal1, Dr. Ajeet Garhwal1 Department of 1General Medicine and 2Pathology, Sardar Patel Medical College, Bikaner, India Objective: Thrombocytopenia in dengue fever is a common and serious complication. However, no specific treatment is available for dengue fever induced thrombocytopenia. In few countries the leaf extract of Carica papaya have been effectively used for thrombocytopenia. So, the study is planned to access effect of Carica papaya leaf extract on platelet count in dengue fever patients. Materials and Methods: All participants were randomised into two groups, study group and control group; the study group was given papaya leaf extract capsule of 500 mg once daily and routine supportive treatment for consecutive 5 days. The controls were given only routine supportive treatment. Daily complete blood counts, platelet counts and hematocrit level, liver function test, renal function test of both groups were observed. Result: On the first 2 days platelet count of study group was comparable to control group, but on 3rd, 4th and 5th day platelet count of study group was significantly higher than control group. Average hospitalization period and average platelet transfusion requirement of study group was less than control group. Conclusion: It is concluded that Carica papaya leaf extract increases the platelet count in dengue fever without any side effect and prevents the complication of thrombocytopenia. So, it can be used in dengue fever with thrombocytopenia patients. Keywords Thrombocytopenia, Dengue, Carica papaya

Topic: Proffered PO 188 Application of ITP-BAT Bleeding Score in Clinical Practice Meet Kumar, Shyamali Datta, Maitryee Bhattyacharyya, Shuvra Neel Baul, Prakas Kr Mandal, Tuphan Kanti Dolai, Rajib De, Prantar Chakrabarti

Dr. Rajeshwari G, Dr. Amrit Kaur Kaler; Associate Professor & Blood Bank Incharge MVJ Medical College and Research Hospital, Hosakote, Bangalore Summary: A non-hemolytic transfusion reaction (NHTR) is associated with RBCs destruction caused by possibly thermal injury, osmotic injury, mechanical injury, infection, drugs and congenital and acquired hemolytic anemia etc. The non- hemolytic reactions can be allergic reaction or febrile transfusion reaction. Febrile non-hemolytic transfusion reactions are the most common type of transfusion reaction, occur in 3–7 % of patients receiving Red Blood Cells and are precipitated by leucoagglutinins present in the recipient plasma. Allergic reaction to an unknown component in donor blood is common, usually due to allergens in donor plasma or to the antibodies from allergic donor. Introduction: NHTR accounts for less 4 % of the total reactions. These may vary from mild reactions to those which rarely assume life threatening proportions. Fever, rigor, nausea, vomiting and dyspnea are the common manifestations of these reactions. Most of these transfusion reactions are of immediate type occurring within 1 h. This study is done to understand the possible causes of Non hemolytic transfusion reactions. Materials and Methods: A retrospective and prospective analysis was done in Blood Bank for the total number of transfusion reactions to blood components and on per unit basis was calculated. Proper sequential history of the blood transfusion and clinical history of the patient was taken. All the blood parameters were noted down. Samples like pre and post transfusion blood sample of the patient was collected to rule out hemolytic transfusion reaction. The urine sample was also checked for haemolysis. Results: The incidence of transfusion reactions to Whole Blood and PRBC is 0.77 and 0.8 % respectively. There was no transfusion reaction to FFP and PC. Total number of transfusion reactions per unit basis of whole Blood is 0.37 % and PRBC showed 0.23 %. Most of the transfusion reactions were febrile non hemolytic reactions in PRBCs, while reactions to whole blood showed more of allergic reactions. The onset of reaction was more of immediate type in case of whole Blood than in PRBC. Conclusion/Diagnosis/ Impression: The pre-existing infection leads to the majority of the reactions in our blood bank. It reflects the potential risk of transfusion reaction in patients. Despite the limitations, this study provides insight into risks of adverse reactions regarding transfused patients.

NRS Medical College & Hospital, Kolkata-700014 (West Bengal) Objectives: Many treatment modalities are available for treatment of ITP patients. Response to these therapies is usually monitored by platelet count increments, although it is an inadequate parameter for making therapeutic decisions. We used the recently available bleeding tool, ITP-BAT, version 1.0, for assessment of response to steroids in ITP patients. Materials and Methods: Patients of persistent and chronic ITP (Aug 2013 to Dec 2013) were analyzed for bleeding as per ITP-BAT, version 1.0 by IWG. They were monitored for bleeding scores and platelet counts at baseline and after steroids. Primary endpoints were to assess the ability of the bleeding score to estimate effectiveness of steroids and its correlation with platelet counts changes. Results: A total of 20 patients of persistent and chronic ITP were enrolled and evaluated. We found that the improvement in bleeding score after institution of therapy correlated with the increments in platelet counts. However, the score is time consuming for routine use (approximately 20 min per patient). Conclusion: The current bleeding score correlates well with platelet counts increments after steroid therapy, but is of limited utility in clinical practice due to time-consuming format. Keywords Bleeding score, ITP, Steroids

Topic: Transfusion Medicine PO 190 Assessment of Direct Antiglobulin Test Positivity in Neonates: A Tertiary Care Hospital Based Study R. Chauhan, S. Pahuja, S. Nangia*, P. L. Jyotsna Departments of Pathology & Pediatrics*, Lady Hardinge Medical College & Kalawati Saran Children’s Hospital*, New Delhi Introduction: ABO blood group (BG) incompatibility and Rh isoimmunization can lead to haemolytic disease of newborn (HDN). Direct antiglobulin test (DAT) is used as a screening test for HDN. Objectives: To assess feto-maternal blood group incompatibility and DAT positivity in newborns and to correlate with requirement of therapeutic intervention Material and Methods: A cross-sectional study over a period of 6 months including 1,020 newborns was done. Blood grouping of the newborns, mother along with DAT, hemoglobin (Hb) & bilirubin on cord

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S542 blood was performed. Results: Out of 1,020 cases, neonates and mothers with BG A/B/0/AB were 22.6/33.7/36.3/7.2 % and 19.3/25.9/45.6/9.1 % respectively; Rh D(+)/Rh D(-) were 92.5/7.5% and 88.3/11.6 % respectively. Occurrence of ABO incompatible feto-maternal pair was 27 % (276/ 1,020) and Rh D incompatible pair was 6.4 % (66/1,020). DAT was positive in 34 neonates (prevalence 3.33 %) including 10/66 (13.6 %) RhD incompatible pairs and 24/275 (7.28 %) ABO incompatible pairs. Two newborns with RhD(+) and non ‘O’ BG mother and one Rh(D) negative newborn also had DAT positivity. Frequency of trace + DAT was 1.1 % (mean bilirubin = 1.5 mg/dl), 1+ was 1.1 % (mean bilirubin = 2.7 mg/ dl), 2 + was 0.5 % (mean bilirubin = 3.9 mg/dl), 3+ was 0.4 % (mean bilirubin = 6.7 mg/dl) and 4+ was 0.2 % (mean bilirubin = 12.5 mg/dl). Also, 14.7 % of DAT + cases with mean Hb = 10 g/dl, bilirubin = 10.2 mg/dl (DAT ranging from 3+ to 4+) required therapeutic intervention, i.e. phototherapy/exchange transfusion while rest 85.3 % DAT + cases with mean Hb = 13.8 g/dl, bilirubin = 3.3 mg/dl (DAT trace + to 2 +) were managed conservatively. Conclusion: Determination of feto-maternal incompatible pairs and their follow up with DAT screening helps to predict cases that require therapeutic intervention. Not only BG ‘O’/Rh D(-) but all mothers should be screened for presence of incomplete antibodies to prevent complications related to isoimmunisation with ABO, Rh, and other minor BG antigens. Keywords Neonates, Blood group incompatibility, Direct Antiglobulin Test

PO 191 Platelet Transfusion Practices During a Dengue Epidemic: Empirical or Evidence Based? Col. (Dr.) S. Tripathy*, Lt. Col. S. Tripathy#, Dr. Q. S. Mahapatro$ *MH Kirkee, #CH(SC), Pune, $ACMS Delhi Objective: Thrombocytopenia is common in dengue infection & hemorrhagic manifestations are one of its dreaded complications. Data on the relationship between platelet count and bleeding manifestation are conflicting. Definite criteria for giving platelet transfusion in Dengue patients are not yet well documented. Materials and Methods: This retrospective study was done on 116 serologically confirmed dengue patients admitted in a tertiary care Hospital, during the dengue epidemic, who received platelet transfusion during their stay in the hospital. Clinical data, platelet count, platelets transfusion were retrieved and analyzed. We evaluated the role of platelet transfusion in patients admitted with dengue infection with special emphasis on the relationship between hemorrhagic manifestations and thrombocytopenia. Results: Out of 945 patients, with suspected dengue infection, 145(15.3 %) patients received platelet transfusion. Among them, 116 had serological evidence of dengue infection. Thrombocytopenia (platelet count \1,00,000 cu mm) was detected in 109(93.96 %) patients. Of these, 56(48.2 %) patients had platelet count \20,000 cu mm; 29(25 %) and 24(20.7 %) patients had platelet count of [20,000; \50,000 cu mm and [50,000 but \1,00,000 cu mm respectively. Out of 52 patients who had hemorrhagic manifestations, 23(44.2 %) had platelet counts of \20,000 cu mm, 16(30.7 %) had count between 20,000 and 50,000 cu mm and 12(23 %) patients had platelet count between 50,000 and 1,00,000 cu mm. One patient with platelet count of [1,00,000 cu mm also had hemorrhagic manifestations. Conclusion: Specific guidelines for the use of platelets in Dengue are lacking. There was no co-relation between platelet count and bleeding manifestation. The exact indications for platelet transfusion in Dengue infection have to be standardized. Keywords Thrombocytopenia, Dengue, Platelet transfusion

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Topic: Transfusion Medicine PO 192 Transfusion Support in Chemotherapy Induced Anemia Dr. Deepak Mathias, Dr. Vinod Kumar Panicker, Dr. R. Krishnamoorthy Sri Ramachandra Medical College & Research Institute, Porur, Chennai 600 116 Objective: Red cell transfusion support in chemotherapy induced anemia in a tertiary care hospital was analyzed. Most of the patients showed an improvement in Hb levels. Introduction: Anemia a common condition in patients with cancer (60 % among patients with solid tumours and 70–90 % in patients receiving chemotherapy and radiotherapy). Indication for transfusion mainly depends on patient’s clinical condition and lab investigations. Materials and Methods: A study was carried out at Sri Ramachandra Medical College and Research Institute, Chennai in the Department of Transfusion Medicine during the period of January 2014 to June 2014. Data regarding blood components transfused were collected from patient records and the outcome analyzed. Results: Percentage of patients who received transfusion are as follows. Carcinoma Ovary 19.4 % being the highest followed by carcinoma breast-15.0 %, carcinoma colon-12.0 %, carcinoma rectum-3.7 %, carcinoma gastric-8.0 %, carcinoma endometrium-8 %, molar pregnancy-3.7 %, carcinoma oesophagus-3.7 %, neuroblastoma-3.7 %, carcinoma sigmoid colon-3.7 %, hepatocellular carcinoma-3.7 %, primitive neuro ectodermal tumour (PNET)8.0 %, carcinoma bladder-3.7 % and retroperitoneal sarcoma-3.7 %. Out of the 29 patients transfused, 20 patients showed an improvement in hemoglobin and 9 patients showed no improvement, despite transfusion. Conclusion: Blood transfusion support is essential in all cases of chemotherapy induced anemia. Transfusion triggers and patient’s clinical condition should be taken into consideration before ordering a transfusion. Keywords Chemotherapy, Carcinoma, Transfusion

PO 193 Blood Component Transfusion in Neonates Dr. Manjiri Hawal, Dr. Ramesh Chavan Department of Pathology, JNMC, KLE University, Belgaum Summary: This study was undertaken in KLESH and MRC to assess the frequency, indications and multiplicity of blood component transfusion in neonates. Multiple platelet component packs were most commonly transfused, followed by packed cell (PC) and fresh frozen plasma (FFP). Sepsis secondary to pneumonia, meningitis was the most frequent indication for transfusion of various components. Introduction: Blood component transfusion is an integral part of neonatal intensive care provided to preterm and term infants for anemia, thrombocytopenia and other complications. Exchange transfusion is required for effective treatment of ABO and Rh incompatibility. Neonates form a very sensitive patient group. Hence, it is important to study the transfusion practices in neonates to find out the practice patterns and indications. Materials and Methods: This is a cross sectional study conducted in 2014. Neonates admitted in NICU requiring blood transfusion were included. The various indications, age, blood group and the component transfused were noted. Single or multiple transfusions of one or different components in the same patient were recorded. The number and indications of various blood components transfused were subsequently analysed Results:

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 Platelets, PC, FFP were mostly requested. The commonest asked was platelets followed by PC and FFP. Platelets also had maximum number of multiple transfusions. The major indications for platelet transfusion were thrombocytopenia secondary to sepsis due to pneumonia, meningitis Conclusion/Diagnosis/Impression: In our tertiary center, platelet packs was common blood component used for sepsis.

PO 194 To Evaluate the Role of Platelet Rich Plasma in Biologic Enhancement of Healing in acute Fractures of Femur

S543 mainly in camps by voluntary blood donors. Donations in camps: voluntary—11,544 (81.5 %). Donations in blood bank: voluntary— 1,067(7.53 %), replacement—1,555(10.97 %) Results: Overall 330 AE’s occurred in a total of 14,166 donors, resulting in an AE rate of 2.33 %. Most common AE was presyncopal or vasovagal reaction (73 %). Syncopal minor reactions associated with transient loss of consciousness comprised 14.3 %. Severe AE’s formed a very small fraction accounting for 1.16 %. Conclusion: AE analysis helps in identifying the donors at risk & adopting environmentally appropriate measures to reduce risk & improve donor satisfaction. Keywords Blood donation, Outdoor camps, Adverse events, Vasovagal reactions.

Dr. P. K. Sehgal, Dr. Rama, Dr. Jeetesh, Dr. Roop l, Dr. Bimla Pt. B.D. Sharma PGIMS, Rohtak Objective: Lower limb fractures account for approximately one third of all fractures. PRP treatment do have positive role in treatment of acute fractures of femur. In present study we have found additive role of PRP in healing of fracture femur. New research is focusing on the use of natural growth factors to increase the effect of bone fracture healing. The easy preparation protocols, biosafety and versatility of platelet rich preparation(PRP) and their reduced cost have encouraged their therapeutic use for stimulation of tissue healing and bone regeneration. Method: Platelet rich plasma (PRP) was prepared in Department of Blood Transfusion using the standard preparation techniques on the day of application from the patient’s own blood. The activated PRP was taken in a syringe and injected at the fracture site under radiological control in closed intramedullary nailing case. Results: Patients who were operated with closed intramedullary nailing and PRP injection at the fracture site under radiological control showed a statistically significant difference in the cortex to callus ratio. Conclusion: we conclude that PRP does have an osteogenic potential in the early stages of bone healing. Keywords Fracture femur, PRP, Healing

Topic: Blood Transfusion PO 195 A Retrospective Study of Adverse Events in Blood Donors from MGM Hospital, Kamothe Dr. Ruhi Mehra, Dr. Seema Gupta, Dr. Ujwala Maheshwari, Dr. Atul Jain Department of Immunohematology & Blood Transfusion, MGM Medical College & Hospital, Kamothe, Navi Mumbai Summary: All medical procedures including blood donation carry some risk of adverse events (AE). Although blood donation is generally safe, approximately 2–6 % of donors experience some complications, mostly mild, that resolves promptly but are still unpleasant for donors & blood services have an obligation to minimize them. The aim of this study was to estimate the frequency & severity of AE’s occurring in whole blood donors, especially in outdoor camps & to suggest measures to minimize them. Introduction: Occasionally adverse events do occur which have negative effect on donor health, donor satisfaction & blood donor return rate (BDRR). Various studies have quoted an AE of 2–6 % in donors but only 0.08–0.3 % have a syncopal reaction where there is loss of consciousness. In order to have an adequate & regular supply of blood it is not only important to recruit new donors but also to retain them as repeat donors. Materials and Methods: This is a 6-year retrospective study on whole blood donors (2008–2013). Our blood collection is

Topic: Blood Transfusion PO 196 A2 Blood Group in an Individual with Anti A1 Antibodies Sazal Aggarwal, Anjali Kelkar, Amit Nisal, Parineeta Shelke Bharati Vidyapeeth College & Hospital, Dhankawadi, Katraj, Pune-411043 Summary: Our patient, a 26-year old female, G2P1L1, came for routine antenatal check-up at 4 months of gestation, had no history of prior transfusion or surgery. Blood group of the patient was found to be A2 positive with anti A1 antibodies by tube method which was confirmed by gel card method. Introduction: ‘Anti-A1’ antibodies react only with ‘A1’ and ‘A1B’ cells and are found in 1–8 % of group ‘A2’ and 25–50 % of group ‘A2B’ individuals. There may be posttransfusion reactions in the carriers of such antibodies, who are transfused donor Red Cells containing the antigen ‘A1’, which is particularly significant when it reacts at 37 °C. Materials and Methods: Blood group by tube method (Antisera from Tulip Diagnostics) & gel card method (Ortho BioVue.) was done. Results: Blood group by tube method showed delayed agglutination with ‘A’ antisera in forward grouping and strong agglutination with B cells (4+) as well as weak agglutination with ‘A’ cells (1+) in reverse grouping. Also, by gel-card method, the sample showed agglutination with ‘B’ and ‘A1’ cells. Conclusion: This highlights the importance of carrying out blood grouping by test tube or gel-card method. The rare occurrence of ‘anti-A1’ antibodies in a person with A2 blood group can only be detected by these methods which will prevent transfusion related complications.

Topic: Miscellaneous PO 197 Transfusion Support to Haemato Oncology Unit in a Tertiary Care Hospital Dr. R. A. Shanmugha Priya, Dr. Vinodkumar Panicker, Dr. R. Krishnamoorthy Department of Transfusion Medicine, Sri Ramachandra Medical College and Research Institute, Porur, Chennai 600 116 Objectives: To determine blood transfusion support to haemato oncology unit in a tertiary care hospital. Introduction: Blood transfusion constitutes an important part of patient management in Haemato Oncology. Indications for blood use must be clear in the mind of ordering clinicians to avoid its misuse and also to avoid unnecessary exposure of the patient to donor blood antigens, adverse

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S544 reactions and transfusion transmissible diseases. Materials and Methods: This Retrospective study was carried out at The Department of Transfusion Medicine, Sri Ramachandra Medical College and Research Institute Chennai, between January and June 2014. Blood products issued to Haemato-oncology unit were reviewed from previous records and patient’s laboratory parameters from computerized in-patient registry and analysed. Results: From January to June 2014, 76 Haemato Oncology patients received transfusion support. Among them, 9 % were cases of Thalassemia, 1 % Fanconi Anaemia, 3 % Aplastic Anaemia, 7 % Idiopathic Thrombocytopenic Purpura, 53 % Acute Leukemias, 7 % Chronic Leukemias, 10 % Lymphomas, 5 % myeloma and 5 % myelodysplastic syndrome. 51 % were male and 49 % were female patients. 47 % were adults and 53 % children. Total number of issues were 282, out of which 49 % were packed Red Cells, 39 % random donor platelets, 7 % Fresh Frozen Plasma, 4 % leukofiltered packed Red Cells and 1 % other blood components. On an average 60 % received packed Red Cells, 6 % platelets, 5 % fresh frozen plasma and 29 % received multiple blood components. Among the 76 patients, 67 % patients gained expected improvement in laboratory parameters. Transfusion reaction occurred in 4 of the 138 packed cell transfusions. Conclusion: Packed Red cells was found to have a higher issue percentage and also higher transfusion reactions. Good quality transfusion support warrants appropriate selection and use of blood components. Keywords Transfusion, Packed Red Cells, Platelets, Leukemias, Blood components

PO 198 Study of Seropositivity in Voluntary and Replacement Blood Donors Dr. Shilpa Bhimalli, S.S. Devarmani, Dr. Sainath, K. Andola Department of pathology, Mahadevappa Rampure Medical College Gulbarga Summary: Blood safety comprises the provision of safe, adequate and quality blood components to the needy patients. A total of 21,796 donations were analysed. Introduction: Transfusion of blood and its components is life saving as well as it has life threatening hazards. A majority of known cases of post transfusion diseases have been caused by HIV, HBV, HCV, Treponema Pallidum and Malaria parasites. The aim of the present study was to find out prevalence of transfusion transmitted infections (TTI) in voluntary and replacement donors. Materials and Methods: A total of 21,796 donors were analyzed for the prevalence of Transfusion Transmitted Diseases over a period of 2 years from July 2012 to June 2014. Results: A total of 21,796 donors were enlisted in the study. Of these 16.07 % (3504) were voluntary and 83.93 % (18,292) replacement donors. Majority of donors were in age range of 26–35 years. Males 97.67 % (21,287) outnumbered females with M:F 41.9:1. The prevalence of HIV was noted only in replacement donors 0.15 % (32 cases).The seroprevalence of HBsAg was higher in replacement donors 0.76 % (160 cases) compared to voluntary donors (7 cases). HCV seroprevalence was noted in 11(0.05 %) cases of replacement donors. The seroprevalence for syphilis and malaria was 4 (0.01 %). Conclusion: To conclude, with the implementation of firm selection norms of donor as per the guide lines laid down for the blood banks in the gazette notification by the Government of India and use of sensitive laboratory screening tests, it is achievable to decrease the occurrence of seropositivity of TTI and improve the blood product safety. Keywords Voluntary, Replacement, Transfusion transmitted infections

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546

Topic: Transfusion Medicine PO 199 Cold Agglutinin in a Healthy Blood Donor: A Case Report Dr. V. Vidya, Dr. Vinodkumar Panicker, Dr. R. Krishnamoorthy Transfusion Medicine Department, Sri Ramachandra Medical College, Chennai Introduction: Cold agglutinins may be detected during a routine pretransfusion testing of blood sample and the results prompts further testing within the immunohematology laboratory. Case report: A 46-year old male donor came for blood donation. His predonation Hb was 15.4 g/dl. Vitals were normal. He was asymptomatic and healthy and was accepted for blood donation. 450 ml of blood was collected from him. When pretransfusion tests were carried on his blood sample, grouping discrepancy was observed following which cold agglutinin disease was suspected and workup was done. Materials and Methods: Forward and reverse grouping were done by the tube technique. There was discrepancy between the forward and reverse grouping results. Blood grouping was repeated by the pre-warm technique to resolve the discrepancy. Result: At room temperature there was a grouping discrepancy, Direct Coombs test, Autocontrol and Antibody screening were positive. Antibody titre was 1:2 at room temperature and 1:16 at 4 °C. After pre-warming at 37 °C, blood group was found to be ‘‘O’’ positive. Antibody screening, autocontrol and Direct Coombs test were negative. Conclusion: It is thought that many people who develop cold agglutinins remain asymptomatic or become only subclinically unwell. Keywords Cold agglutinin, Grouping discrepancy, Antibody titre

PO 200 Wilm’s Tumor-1 Gene Mutations in Acute Myeloid Leukemia: A Descriptive Analysis A. Senthamizhselvi1, Ajay Abraham1, Savitha Varatharajan1, Sreeja Karathedath1, Nancy Arthur1, J. Ashok Kumar1, Vivi M. Srivastava2, Aby Abraham1, Auro Viswabandya1, Biju George1, Alok Srivastava1, Poonkuzhali Balasubramanian1, Vikram Mathews1 Department of Haematology & 2Cytogenetics Unit, Christian Medical College Vellore

1

Wilm’s tumor 1 (WT1) is a zinc finger transcription factor shown to be overexpressed in various leukemias particularly in acute myeloid leukemia (AML). WT1 mutations are reported to occur in approximately 10 % of normal karyotype AML and are considered independent predictors of poor outcome. WT1 gene mutations majorly occur in the 4 Cys-His zinc finger domains on exons 7 and 9, and could result in the formation of truncated non-functional proteins. The present study aims to evaluate the incidence of WT1 mutations and determine its coexistence with other molecular markers in a large cohort of adult AML patients diagnosed at the Department of Haematology, Christian Medical College-Vellore. Adult AML patients (n = 328) diagnosed between June 2009 and February 2014 were included in this study (patient demographics Table 1). WT1 exon 7 and 9 mutation detection was done using genomic DNA sample at diagnosis by PCR followed by GeneScan analysis. This mutation was further characterized by sequencing. Presence of other relevant molecular (FLT3, NPM1, N-RAS, JAK2, cKIT and CEBPA) and cytogenetic markers were also evaluated in WT1 mutant patients. Fourteen patients (4.26 %) were identified with either exon 7 or 9 mutations, of which 13 had mutations in WT1 exon 7. The respective

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 WT1 mutations are as shown in the Table 2. As reported earlier, WT1 mutations were associated with young age [29 years (21–43 years)] and not confined to any FAB subtype, but seen only in intermediate or adverse cytogenetic groups (Table 1). Six out of 14 patients with WT1 mutations had cytogenetically normal karyotype. Of the 14 patients with WT1, coexistence of CEBPA mutations were found in 5, NPM1 in 3, FLT3–ITD in 2 and N-RAS in one patient. FLT3–TKD, JAK2 and cKIT mutations were absent in WT1 mutated patients. Our study for the first time has come up with a detailed analysis of WT1 exon 7 and 9 mutations in a large set of AML patients and its association with various pre-treatment parameters. Table 1 . Patient characteristics (n = 328) Gender

Male = 198 Female = 130

Age

41 years (15–75 years)

Cytogenetics risk group (*available in n = 301) Favorable

42 (14 %)

Intermediate

178 (59.1 %)

Adverse

81 (26.9 %)

WT1 mutations 14/323 (4.26 %) WT1 Exon 7 mutations

13/14 (93 %)

WT1 Exon 9 mutations

2/14 (14.3 %)

Favorable risk Intermediate risk

0/14 9/14 (64.3 %)

Adverse risk

5/14 (35.7 %)

Other mutations with WT1 CEBPA

5/14 (35.7 %)

NPM1

3/14 (21.5 %)

FLT3-ITD

2/14 (14.3 %)

N-RAS

1/14 (7.2 %)

Pretreatment parameters for WT1 mutated patients WBC

12,550 (500–329,100)

Blast %

50(24–98)

LDH

796.5 (354–4060)

CD33 expression (%)

94.15 (57.6–99.6)

CD34 expression (%)

73.05 (2.2–95.7)

PO 201 Primary Renal Burkitt Leukemia Presenting as Acute Renal Failure Dhwanee Thakkar1, Nita Radhakrishnan1, Jasmita Dass2, Kanav Anand3, Pallav Gupta4, Manas Kalra1, Anupam Sachdeva1, Megha Saroha1 1

Pediatric Hematology Oncology and Bone Marrow Transplant Unit, Institute of Child Health, Sir Ganga Ram Hospital, New Delhi, 2 Department of Haematology, Sir Ganga Ram Hospital, New Delhi, 3 Pediatric Nephrology Unit, Institute of Child Health, Sir Ganga Ram Hospital, New Delhi, 4Division of Histopathology, Sir Ganga Ram Hospital, New Delhi Introduction: Primary renal involvement in Burkitt Lymphoma is extremely rare in children. Case Report: 2-year old girl presented to

S545 us with history of fever followed by generalized oedema and oliguria. She had pallor, anasarca, hypertension with no organomegaly or lymphadenopathy. She has anemia (Hb: 10.5 g/dl), leukocytosis with plasmacytoid lymphocytes in peripheral smear. She presented in renal failure (creatinine: 2.8 mg/dl) with raised LDH and uric acid. USG abdomen showed B/L bulky kidneys. She required dialysis with inotrope support and invasive ventilation. Bone marrow aspiration biopsy was done which revealed extensive infiltration by monomorphic abnormal lymphoid cells. Immunophenotypic analysis revealed abnormal cells expressing bright CD19, CD20 with co-expression of CD10 and bright CD38, bright HLA DR and a moderate cytoplasmic CD79a expression with surface Kappa light chain restriction. CT chest and abdomen showed bilateral bulky kidneys with no other mass or lymphadenopathy. In view of the absence of any significant primary site, renal biopsy was planned to rule out kidneys as a possible primary site. Renal biopsy showed diffuse infiltration of renal interstitium with atypical lymphoid cells. Tumor cells were positive for CD20, CD10, bcl-6 suggestive of Burkitt lymphoma. CSF was positive for malignant cells. Diagnosis of stage IVBurkitt lymphoma with BM and CNS involvement was made and she was started on COP chemotherapy. She had a stormy course with sepsis and coagulopathy and could not be salvaged. Conclusion: Primary renal involvement is a relatively uncommon clinical presentation of Burkitt Lymphoma. Renal failure, enlargement of kidneys without obstruction, absence of other organ or nodal involvement should prompt a renal biopsy to establish the diagnosis.

PO 202 Kawasaki Disease with Autoimmune Hemolytic Anemia: An Unusual Association Dhwanee Thakkar, Nita Radhakrishnan, P. K. Pruthi, Anupam Sachdeva Pediatric Hematology Oncology Unit, Sir Ganga Ram Hospital, New Delhi Introduction: The association of AIHA is seldom reported with Kawasaki disease. We report a case of KD associated with AIHA not related to IVIG administration. Case Report: A 7-month-old male child, presented with fever for 1 month and maculopapular rash with erythema of palms and soles. On admission, he had fever, severe anemia, mild icterus, lymphadenopathy andhepatosplenomegaly. Hemoglobin was 3.6 gm % with corrected reticulocyte count of 3.4 %.Peripheral smear examination showed features of hemolysis. DCT was positive with mixed pattern (IgG 3?, IgM 3?, C3d 3?). Mycoplasma IgM was positive. In view of the prolonged fever, erythematous rash and desquamation over extremities, 2DECHO was done which showed aneurysms of the main coronaries. Hence he was diagnosed as AIHA with underlying KD. He received IVIG (2 g/kg) and aspirin (80 mg/kg/day) for KD and prednisolone (2 mg/kg/day) for AIHA. Child became afebrile with treatment and hemoglobin remained stable after transfusion. He was discharged on aspirin and is on tapering doses of steroids. Discussion: The association of AIHA with KD has been reported previously, but as a complication following IVIG infusion. In our patient, onset of AIHA was prior to IVIG administration. A mixed pattern of antibodies was observed with strong positivity for IgG, IgM and C3d which is observed in patients with rheumatological conditions. Mycoplasma infection has been reported with KD and AIHA. Patients of AIHA with unusual features such as prolonged fever, skin rash, mixed antibody response in DCT may be evaluated for underlying KD. Conclusion: AIHA is a co-morbidity observed rarely with KD, and its etiology can be multifactorial. Early identification and management will help in reducing complications of both problems.

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S546 PO 203 Acquired Bleeding Disorder Due to Secondary Antiphospholipid Antibody Syndrome (APS) in a Young Female Siddharth Samrat1, Saurabh Chopra2, Madhukar Rai3, L. P. Meena4

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S448–S546 present with bleeding manifestation rather than thrombosis. Although acquired factor inhibitors are more common in older age group, Younger Patients of APS with bleeding manifestation should be suspected for acquired factor inhibitor. Keyword Antiphospholipid antibody syndrome, APS, Lupus anticoagulant, LAC, SLE, Secondary APS, Acquired Factor inhibitor, Acquired bleeding disorder.

1,2

General Medicine Resident, Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh. 3Prof., Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh. 4 Assistant Prof., Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh Antiphospholipid antibody syndrome (APS) occurs in 1–5 % of general population, while one third patient of SLE possess these antibodies. Patient of APS present with venous or arterial thrombosis and/or pregnancy morbidity. In Vitro APS patient had prolonged phospholipid dependent assay (APTT, PT and RVVT).In Vivo bleeding in patients of APS are very rare presentation. Methods/ Presentation: Sixteen year unmarried female came to us with history of menorrhagia since menarche (2 year). For last 2 month she had continuous menstrual flow. She doesn‘t have history of hemoptysis, malena, epistaxis, petechiae, easy brusability, gum bleed, fever, rash, joint pain, hair fall, photo sensitivity, jaundice. Her siblings and parents had no similar illness. She had been transfused with 3 units packed RBC in last 2 year. On examination she had marked pallor; there was no icterus, lymphadenopathy, fundal bleed and hepatosplenomegaly with normal sexual maturity index. Results: Her Hb7.6, Tlc-5000 lac/dl, Plt-1.89 lac/dl, Mcv-92 with normal differentials. In urine R/M- alb 1?, sugar/RBC-Nil. RFT, LFT, FSH, LH, Prolactin and Thyroid profile was normal; she was Retro Negative. Her PT-INR 13.8/22.3-1.61, APTT-29.7/43.7. she latter underwent for Mixing studies which surprisingly shown raised APTT even with normal and adsorbed plasma so possibility of factor inhibitor in patient plasma suspected. Collagen profile (Dated 17/9/2013) shown raised ANA (6.4, N \ 1) and anti dsDNA (585 IU/ML, N \ 40). Her plasma also showed presence of Lupus anticoagulant (LAC) and raised both anti Cardiolipin IgM(48, N \ 10)/IgG(37, N \ 10). Conclusion: In view of raised ANA and anti dsDNA along with presence of LAC and raised anti Cardiolipin IgM/IgG diagnosis of secondary APS with Acquired bleeding disorder made. She was given oral Prednisolone 1 mg/kg for 4 week then dose tapered. At 12 week her ANA-5.5, anti dsDNA-168, PT-INR 12.5/13.6-1.08, APTT-29.3/ 31, LAC-Negative, Anti Cardiolipin antibody IgM(45)/IgG(27).Now She is totally asymptomatic with normal cycle and taking Prednisolone 5 mg alternate day. Discussion: The prevalence of LAC was very high in younger patients (63 % in juvenile SLE as Compared with 4 % in patients aged more than 16).Rarely these patient may

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PO 204 NHL and CML Simultaneously in a Patient: A Rare Occurence N. Gaur N, P. S. Ghalaut, S. Atri, J. K. Pehalajani, R. Singh, H. K. Dahiya, J. Duhan Pt. B.D. Sharma, PGIMS, Rohtak Summary: Chronic myeloid leukaemia (CML) is a clonal stem cell disorder characterized by the acquisition of an oncogenic BCR/ABL fusion protein and by proliferation of granulocytic elements at all stages of differentiation. Patients with CML in CP PHASE has Lymph nodes palpable in around 60–70 % but rarely they are more than 1 cm in largest diameter.NHL patient usually presents with lymphadenopathy with hepatosplenomegaly, the peripheral blood picture may shows anaemia and pancytopenia due to bone marrow involvement Introduction: NHL and CML have different etiopathogenesis so the finding of both the disease in same patient simultaneously is highly unlikely. Materials and Methods: We came across a patient. 43 year male patient presented with complaints of generalised weakness since last three months which was also associated with mild grade fever associated with significant weight loss. Patient also noticed a small lump in his bilateral axilla and neck region Results: Examination revealed mild pallor and generalised lymphadenopathy, rest general physical examination was normal on abdominal examination patient was having liver Palpable 2 cm below right costal margin with a span of 13.5 cm patient also has spleen palpable around 3 cm below left 9th costal cartilage, rest systemic examination was normal. Lymph node biopsy was done which showed NHL, mixed small and large T-cell types CD-3 and CD-5 positive. On his routine investigation haemogram showed Hb 8.2 g/dl, TLC showed 23,000 myelocyte metamyelocyte promyelocyte in peripheral blood film. In view of this Patient was investigated for Myeloproliferative disorder, to our surprise Bcr-Abl positive was in 100 % cells by Rt-Pcr. Conclusion/Diagnosis/Impression: Thus patient was diagnosed as a case of NHL with CML. Patient was given CHOP regimen with Imatinib therapy patient’s lymph node regressed after 2 weeks of first cycle. Simultaneous presence of two haematological malignancies are rare so this case is being presented here.