Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 DOI 10.1007/s12288-013-0302-z

ABSTRACTS

Haematacon 2013 (54th Annual Conference of Indian Society of Haematology & Blood Transfusion)

Ó Indian Society of Haematology & Transfusion Medicine 2013

Acute Myeloblastic Leukaemia (AML) & Myeloid Sarcoma

mutations. Use of this high sensitivity assay permitted us to detect mutations in patients with low level mutant clones.

Abstract No. 1 Abstract No. 2 Molecular Risk Stratification of Patients with AML: Results of FLT3-ITD, NPM1, CEBPA Mutations Using a High Sensitivity Technique Vishwakarma Devanand, Patkar Nikhil1, Hasan Syed Khizer, Tembhare Prashant, Sengar Manju, Bagal Bhausaheb, Menon Hari, Gujral Sumeet, Khattry Navin, PG Subramanian Hematopathology Laboratory, Tata Memorial Centre, Mumbai Introduction: Acute myeloid leukemia (AML) with normal karyotype (NK-AML) is a biologically heterogeneous entity. Mutations in FLT3 (FMS-like tyrosine kinase 3), NPM1 (nucleophosmin), and CEBPA (CCAAT/enhancer-binding protein alpha) genes have profound prognostic significance. In this study we screened all consecutive AML (except AML-M3) patients for these mutations using a high sensitivity technique to obtain baseline frequencies. Materials and Methods: 47 patients (August 2012–March 2013) diagnosed with AML according to the WHO 2008 criteria were accrued. DNA was used in a multiplex fluorescent PCR that amplified a 330 bp internal tandem duplication site in wild type FLT3 gene as well as a 170 bp site in the exon 12 of wild type NPM1 gene where (type A-F) mutations occur. Similarly the transactivation 1 & 2(TAD1, TAD2) and leucine zipper domains (b-ZIP) of the CEBPA gene were amplified. These amplicons were analyzed by capillary electrophoresis for any duplication, insertion and/or deletion. Results: FAB subtypes: AML-M0 (6.1 %), AML-M1 (8.2 %), AML-M2 (61.2 %), AML-M4 (4.0 %) & AML-M5b (4 %) & inv16, t(8:21), v(11q23) were seen in 4.2, 23.4 and 4.2 % of patients respectively. FLT3-ITD mutations were seen in 27 % of patients [FLT3+/NPM1-/ CEBPA-(12.8 %), FLT3+/NPM1+/CEBPA-(6.4 %), FLT3+/ NPM1-/CEBPA+ (2.1 %)], NPM1 (FLT3-/NPM1+/CEBPA-) & CEBPA mutations (FLT3-/NPM1-/CEBPA+) accounted for 10.6 %. Biallelic mutations (33.3 % of all CEBPA mutations) occurred in the TAD1 & ZIP domain. FLT3-ITD allelic ratio varied from 0.04 to 3.14 whereas FLT3ITD % ranged from 3.1 to 75.8 %. No mutation was detected in 55 % of patients. Conclusion: The frequencies of these mutations are comparable with published data. As almost all mutations are of in-del type, fragment length analysis can be used to detect

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Role of Matrix Metalloproteinases and Its Inhibitors in Hematological Malignancies AK Chaudhary, A Nadkarni, K Ghosh, BR Vundinti, S Chandrakala National Institute of Immunohaematology (NIIH), ICMR Institute, KEM Hospital Campus, 13th Floor, NMS Bldg., Parel, Mumbai, 400012 Summary: In this study we have tried to look at the effect of molecular markers of the MMPs and TIMPs on clinical outcome in haematological malignancies. We observed that in-vitro BM-MNCs continuously secret the MMP-9 & its mRNA expression was increased. Hence it can be used for development of good prognostic and predictive marker. Introduction: Matrix metalloproteinases (MMPs) are family of Zn + 2 dependent endopeptidases which play an important role in angiogenesis, tumor invasion and metastasis. The main function of MMPs is the degradation of extra cellular matrix (ECM). MMPs participate in the turnover of ECM in the hematopoietic microenvironment, regulating the release of hematopoietic stem cells and mature leucocytes from bone marrow (BM) to peripheral blood (PB). The aim of the present study was to look at the polymorphic association and expression of MMP-2, MMP-9, TIMP-1 & TIMP-2 in haematological malignancies. Materials and Methods: Total 41 cases were enrolled in this study. They comprised of Myelodysplastic Syndrome (MDS; n = 20), Acute Myeloid leukemia (AML; n = 10), acute lymphoblastic leukemia (ALL; n = 06) and chronic Myeloid leukemia (CML; n = 05) confirmed cases and 45 healthy individuals served as control group. 5 ml of peripheral blood (PB) and 5 ml of bone marrow (BM) aspirates were processed for the isolation of genomic DNA. The detection of polymorphic variation was studied using PCR-RFLP & direct DNA sequencing. cDNA was used for mRNA expression studies also the cultured mononuclear cells (MNCs) supernatant was used for gelatin zymography analysis. Results: The genotypic frequency of 0 T’ allele of MMP-2 (-1306C/ T) did not showed statistically significant difference among the all patients (p = 0.56), while ‘T’ allele of MMP-9 (-1562C/T)

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 polymorphism showed significant association (p = 0.001) in all the cases as compared to healthy controls. The TIMP-1 (372C/T) and -2 (-418G/C) did not show any association with disease. Zymographic analysis of BM-MNCs-conditioned medium showed release of MMP9 with Mr 92,000 gelatinolytic activity in all subjects studied, while MMP-2 with Mr 72,000 gelatinolytic activity was found only in patients and not in controls. We have reconfirmed the activity of the MMP-2 and -9 via mRNA expression using TaqMan chemistry. We found that mRNA expression of MMP-2 had 1.5 fold while MMP-9 had 5.5 fold increased expression in all cases compared to the healthy controls. Conclusion: This study showed that BM-MNCs continuously secrete MMP-9. This was also confirmed by positive mRNA expression. We also reported polymorphic association of MMP-9 (-1562C/T) with severity of disease, while MMP-2 (-1306C/T), TIMP-1 (372C/T) and -2 (-418G/C) polymorphism did not show any association.

279 among AML patients. IC50 of Ara-C ranged 0.24–80 lM with a median of 5.64 lM. Totally 7 SNPs were identified in the regions screened in RRM1 gene. The allelic frequencies of these SNPs in AML patients and normal controls were similar (Table). RRM1 expression was significantly higher in Ara-C sensitive samples compared to Ara-C resistant samples (Median 193; range 21–23097, vs. Median 93; range 23–960; p = 0.0373). RNA expression of patients with mutant or heterozygous variant genotype for a synonymous SNP rs17850106 had a higher RRM1 expression than those with wild type genotype (p \ 0.05). No association between any of the RRM1 genetic variants with ara-C IC50 was observed. To conclude, RRM1 along with other genes involved in metabolism of Ara-C might have impact on Ara-C cytotoxicity in AML patients and contribute to the variation in treatment outcome. The effect of RRM1 RNA expression and genetic variants on treatment outcome in this cohort is currently being analyzed and will be presented.

db SNP id

Gene

Location

Abstract No. 3 Identification of Ribonucleotide Reductase-1 Genetic Variants and Their Influence on mRNA Expression and Invitro Cytotoxicity in Acute Myeloid Leukemia Ajay Abraham, Keshav Bharadvaj, Savitha Varatharajan, Sreeja Karathedath, Biju George, Alok Srivastava, Vikram Mathews, Poonkuzhali Balasubramanian Department of Haematology, Christian Medical College, Vellore Summary: Altered drug metabolism is one of the mechanisms by which cancerous cells are able to alleviate the cytotoxic effect of chemotherapy. Acute myeloid leukemia (AML), a clinically and genetically heterogeneous disease is treated with Cytarabine (Ara-C) and daunorubicin. Wide inter-individual variation in terms of treatment outcomes and the toxic side effects of treatment exist among patients with AML. Ribonucleotide reductase (RR) is a holoenzyme (dimeric protein comprising of large subunit RRM1 and smaller subunit RRM2) which converts ribonucleotides (NTPs) to 20 deoxynucleotides (dNTPs) and provides necessary precursors for both synthesis and repair of DNA. dNTPs generated by RR acts as competitive inhibitor against the active metabolite Ara-CTP and prevents it from incorporating into the DNA. The prognostic significance of genetic variants in RRM1 on gemcitabine chemotherapy has been studied in various solid tumors including pancreatic cancer, nonsmall-cell lung cancer, breast cancer, however, there is no report on the role of RRM1 genetic variants in AML patients treated with cytarabine. Objectives of the study: To examine the effect of genetic variants in ribonucleotide reductase M1 (RRM1) gene on mRNA expression and invitro cytotoxicity of Ara-C in AML patients. Materials and Methods: A total of 136 adult patients with de novo AML (excluding AML-M3) at diagnosis before the initiation of therapy were enrolled after informed consent. Total cellular RNA was extracted using Tri Reagent and cDNA was synthesized. mRNA expression of RRM1 relative to housekeeping gene GAPDH was analyzed using Taqman based gene expression assays. In vitro cytotoxicity was assessed using MTT cell viability assay and IC-50 was calculated. Based on Ara-C IC50, samples were categorized as sensitive and resistant (IC50 \5 lM and [5 lM for Ara-C). In addition, 93 healthy volunteers were used as controls for RRM1 genotyping as well as gene expression studies. Based on previous studies, significant polymorphisms (SNPs) located in 50 UTR, exon 9 and exon 19 of RRM1 gene were screened by PCR and subjected to automated sequencing using Applied Biosystems 3130 genetic analyzer (CA, USA). Sequences were aligned and SNPs/indels were identified using SeqScape software. Results and Conclusion: RRM1 mRNA expression showed wide variation (median 112; range 21–23098)

rs11030918

RRM1

50 UTR

SNP/indel Amino acid change

Nucleotide change

NA

AF AMLs Wt

Variant

AF normal controls Wt Variant

-524C[T

0.581

0.418

0.57

0.43

rs12806698

RRM1

5 UTR

NA

-37C[A

0.624

0.376

0.569

0.431

rs183484

RRM1

Exon 9

Arg284Arg

C[A

0.649

0.350

0.688

0.312

rs17850106

RRM1

Exon 19

Thr741Thr

A[G

0.680

0.319

0.756

0.244

rs17850107

RRM1

Exon 19

Ala744Ala

A[G

0.888

0.111

0.854

0.146

rs1042858

RRM1

30 UTR

NA

T[A

0.902

0.097

0.838

0.152

rs866902

RRM1

30 UTR

NA

del T

0.884

0.116

0.872

0.128

0

Abstract No. 4 Fms like Tyrosine Kinase (FLT3) Mutations in De Novo Acute Myeloid Leukemia (AML) Patients from Tertiary Referral Center in Eastern India S Kishore Kumar, Uttam Nath, SS Roy, Sambit Samanta, Prantar Chakrabarti, Utpal Choudhuri Institute of Haematology & Transfusion Medicine, Kolkata, India Summary: Mutations in FLT3 and NPM1 are important prognostic factors in AML, influencing outcome. A clear data on the incidence of these mutations is lacking in our Indian patients especially from the east India. We have analysed incidences of FLT3/ITD and D 835 mutations in 67 patients with de novo AML using PCR and a comparison is made with clinical and laboratory parameters. Introduction: Though cytogenetics is a major prognostic factor in AML, the two molecular markers which have revolutionized the prognostication of normal karyotype AML patients is NPM1 and FLT-3 mutation studies. To our knowledge, there are no existing data on FLT3 mutations in eastern Indian patients. Materials and Methods: Newly diagnosed 67 adult patients with acute myeloid leukemia were screened for the presence of FLT3/ITD and D835 mutations using PCR. We tried to examine the correlation between incidence of FLT3 mutations and clinical and hematological prognostic factors in AML like age (\ vs. [ 50 years), sex (male vs. female), TLC (\ vs. [ 11,000 cells/mm3), lactate dehydrogenase LDH (\ vs. [ 400 U/L) and peripheral blast percentage (\ vs. [ 50 %). Results: 67 cases were screened for mutations. FLT3/ITD mutations were detected in 12 (17.9 %) AML and 835D were detected in 4(5.9 %).None had both expressed. The incidence of FLT3 mutations was significantly higher in those with elevated LDH, TLC and peripheral blasts (p = \ 0.002, 0.007, \ 0.001). There was no difference in incidence of mutations between sexes. 55 % AML patients had normal karyotype. Conclusion: Our data concurs with previously published reports

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280 and may reflect similar biology of AML in Indian and western patients.

Abstract No. 5 Correlation of Immunophenotypic Features (FCM) with Morphology and Cytochemistry in AML Annapurna Saksena, AP Dubey1, N Gupta2, N Khurana, T Singh Departments of Pathology, Paediatrics and Medicine, Maulana Azad Medical College, New Delhi-110002 Summary: 38 AML cases presenting to Department of Pathology, MAMC, were evaluated using morphology, cytochemistry and immunophenotyping (by Flow cytometry).Flow cytometry was found to be a useful adjunct for diagnosis of AML and its subtypes especially AML M0 and biphenotypic acute leukemia. Introduction: Role of immunophenotyping in AML, especially in under resourced laboratories, is not clearly defined. The present study was conducted to evaluate efficacy of morphology and cytochemistry with immunophenotyping for diagnosis of AML and its subtypes. Materials and Methods: 38 cases, consisting of 37 cases of acute myeloid leukemia (AML) and 1 case of biphenotypic leukemia were studied. The cases were diagnosed based on morphology and cytochemistry (MPO, NSE, PAS) into FAB AML subtypes. Flow cytometry (FCM) was performed on all cases using CD45, CD34, HLADR, CD13, CD33, CD117, CD11c, CD2, CD7, CD10, CD19, CyCD3, CD 38 and CyCD 22. Results: Morphology and cytochemistry diagnosed AML in 34/38 cases. Of the remaining cases, 3 cases were acute non myeloid/ undifferentiated on morphology and cytochemistry as MPO and PAS was negative. 2 of these cases showed CD33/13 positivity and were CD19 and CD 3 negative on FCM and hence diagnosed as AML M0. The third case was inconclusive even on flow as blasts were only CD34 positive; however CD61 was positive on IHC and hence diagnosis of AML M7 was clinched. The fourth case was diagnosed as AML M1 on morphology and cytochemistry but on FCM also expressed lymphoid markers & hence found to be. biphenotypic leukemia by FCM. Conclusion: Flow cytometry is a useful adjunct to morphology and cytochemistry for diagnosis of acute myeloid leukemia and its subtypes. It plays a vital role in diagnosis of AML M0, biphenotypic leukemia and AML M7.

Abstract No. 6 Lineage Infidelity Markers in Acute Myeloid Leukemia: Correlation with Morphology and Cytogenetics Sanjeev, Neha Singh, Soniya Nityanand, Ruchi Gupta Department of Hematology, Sanjay Gandhi Post Graduate, Institute of Medical Sciences, Lucknow, Uttar Pradesh Summary: Discovery of flow cytometry has revolutionized the management of acute myeloid leukemia in the current era. It is commonly used as a standard diagnostic procedure in diagnosis and management of acute myeloid leukemia. Introduction: Flow cytometry is the preferred method of immunophenotyping in acute myeloid leukemia, especially in undifferentiated subtypes and cytogenetics has independent significance in risk stratification of AML patients. We analyzed the expression of aberrant lymphoid antigens in various FAB subtypes of AML and correlated the immunophenotypic profile with cytogenetics and FLT3 mutations, wherever available. Materials and Methods: This was a retrospective study carried out

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 from January 2011 to June 2012 in the Department of Hematology at SGPGI. Complete hematological profiles and bone marrow aspiration slides were reviewed. Three colour flow cytometry was performed in all cases. Cytogenetic studies including conventional cytogenetics, RT-PCR, and FLT3 mutation analysis were carried out in all patients who underwent treatment. Results: A total of 175 patients of AML were evaluated. FAB subtype AML M2, was found in majority of the cases (46 %). CD19 was the most frequently expressed aberrant marker (22.8 %), followed by CD7 (16 %) and CD2 (8 %). CD7 correlated with high TLC at presentation (mean TLC-68.3/ll). Cytogenetic data was available in 54 cases, of which normal cytogenetics was seen in 28 cases. 13/14 cases of APML, showed t(15;17), 6 cases tested positive for t(8;21), and one each for inv3 and inv16. 4/12 cases of AMLM2 with dysplasia showed t(8;21). 2 of 6 pediatric patients tested positive for t(8;21) and FLT3. In total, FLT3ITD mutation was seen in 5/54 cases, all of which presented with pancytopenia. Conclusion: Cytogenetics is the sole predictor of treatment outcome in AML, however, it is strongly integrated with abnormal antigen expression and morphology.

Abstract No. 7 CD 9 Expression in Acute Myeloid Leukemia: Does It Predict any Cytogenetic Subgroup? Sanjeev Kumar Gupta1, Anita Chopra1, Saroj Singh1, Smeeta Gajendra1, Sameer Bhakhshi2, Atul Sharma2, Lalit Kumar2, Rajive Kumar1 Laboratory Oncology Unit1, and Medical Oncology2, Dr. BRA IRCH, All India Institute of Medical Sciences (AIIMS), New Delhi Summary: This study attempts to establish if the expression pattern of CD9 is useful in predicting any specific cytogenetic group of AML, particularly AML with t(8;21). Introduction: CD9 is a tetraspanin molecule involved in cell adhesion and motility. Absence or low expression of CD 9 has been reported to be fairly accurate for predicting t(12;21) translocation (ETV6/RUNX1) in B cell ALL. This raises the possibility of CD9 being a transcriptional target of RUNX1 since the fusion protein ETV6/RUNX1 exerts a dominant negative effect on normal RUNX1 function. Based on this premise, we analysed our AML cases to ascertain whether a differential CD9 expression can discriminate AML with t(8;21) (RUNX1/RUNX1T1) from other AML subtypes since the fusion protein RUNX1/ RUNX1T1 also results in repression of normal RUNX1 function. Materials and Methods: 62 consecutive cases of AML (42 males; age 2 months–74 years (Median 11 years)) were analysed for CD9 expression on FC-500 (Beckman Coulter) flow cytometer in last six months. The cases were (FAB) M0—2, M1—3, M2—36, M3—7, M4—3, M5—6, M6—2 and M7—3 cases. Cytogenetic data available in 35 cases was normal—13, t(15;17)—7, t(8;21)—5, Inv16, trisomy 8, del Y, t(7;11), hyperdiploidy & complex karyotype in one case each and unsatisfactory in 4 cases. Results: CD9 expression was low/ absent in 50 % (1/2) M0, 33.3 % (1/3) M1, 50 % (18/36) M2, 14.3 % (1/7) M3, 33.3 % (1/3) M4, 16.7 % (1/6) M5, however, none of the two cases of M6 and three cases of M7. Among the various cytogenetic subgroups, CD9 was low/absent in 60 % cases (3/5) with t(8;21), 46.2 % (6/13) with normal cytogenetics, and 14.3 % (1/7) with t(15;17). Two of the other six cases (33.3 %) were also negative for CD9. Conclusion: Low or absent CD9 expression was more common in AML with t(8;21) compared to cytogenetically normal AML or other AML subtypes. A larger dataset analysis, however, is desirable before concluding whether CD9 expression is useful in predicting AML with t(8;21).

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Abstract No. 8 Cytogenetic and Molecular Profile of Acute Myeloid Leukaemia in India Jeevan Kumar, Vandana Arya, Anupam Sachdeva, Shyam Aggarwal, Manorama Bhargava Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi-110060 Summary: In this prospective study on acute myeloid leukemia (AML) patients, the frequencies of FLT3-ITD and NPM1 mutations were lower than from western studies but comparable to other studies from India and other Asian countries. Introduction: Cytogenetic and molecular analyses have recently been used to risk-stratify AML patients. There is limited number of Indian studies on cytogenetic and molecular analysis of AML, more studies are warranted to elucidate the mechanisms of leukemogenesis and to better plan the post remission therapy. Materials and Methods: 44 newly diagnosed and previously untreated AML patients attending OPD/indoor of Sir Ganga Ram Hospital, New Delhi were studied. After obtaining their clinical profile, they were investigated for hematological parameters, karyotyping and molecular profile [NPM1 (nucleophosmin 1), FLT3ITD (FMS-like tyrosine kinase-3 gene internal tandem duplication) and FLT3-TKD (tyrosine kinase domain) mutations]. Analyses of FLT3 (ITD and TKD) mutations was done by polymerase chain reaction (PCR) with agarose gel electrophoresis and analyses of NPM1 gene exon 12 mutations was done using PCR and high resolution melting analysis on Light Cycler 480 (Roche Diagnostics, Germany). Results: The median age of patients was 36 years, ranging from 6 months–85 years. AML M5 was the most common subtype (25 %), followed by AML M3 (20.5 %). Normal cytogenetics were present in 42.5 % patients. FLT3-ITD and NPM1 mutations were present in 15.91 and 11.36 % patients respectively. FLT3-TKD mutations were not detected in any of the patient in the present study. FLT3-ITD and NPM1 mutations were most frequent in AML M3. In normal cytogenetics patients FLT3-ITD and NPM1 mutations were detected in 11.76 % patients each. In non-acute promyelocytic leukemia patients FLT3-ITD and NPM1 mutations were more common in patients with normal cytogenetics than in abnormal cytogenetics but the difference was not significant. Both FLT3 ITD and NPM1 mutations were not significantly associated with age, sex, hepatosplenomegaly, lymphadenopathy, median hemoglobin, TLC, platelet count and normal cytogenetics. FLT3 ITD mutations were significantly associated with AML M3 (p = 0.002), higher peripheral blood (p = 0.034) and bone marrow blast count (p = 0.034). Conclusion: The frequencies of FLT3-ITD and NPM1 mutations in present study was lower than from western studies but comparable to other studies from India and other Asian countries. Keeping in view the above findings and limited number of Indian studies, more ongoing larger studies are warranted to elucidate the cytogenetic and molecular pathogenesis of AML.

Abstract No. 9 Morphologic, Immunophenotypic and Cytogenetic Profile Along Their Correlation in Cases of Acute Myeloid Leukemia Neha Seth, Khaliqur Rahman Rajiv Gandhi Cancer Institute & Research Centre, Delhi Summary: AML is considered a heterogenous disease and a few reports have analyzed immunophenotype profile of AML in North Indian population. We thus analyzed the immunophenotypic markers of newly diagnosed AML patients in order to determine the incidence of anomalous markers and their correlation with morphologic and

281 cytogenetic findings. Introduction: French American British (FAB) utilised the morphological features of blast along with their differentiation pattern to classify acute myeloid leukemia (AML). The new WHO classification widely utilises the morphological, immunophenotypic and cytogenetic features for the diagnosis and classification of acute myeloid leukemia. This ongoing study was designed to see morphologic, immunophenotypic and cytogenetic profile along their correlation in cases of AML. Materials and Methods: Morphological evaluation has been done from Leishman and MGG stained peripheral smears and bone marrow aspirates. Special relevant cytochemical stains like MPO, PAS have been obtained. Multiparameter flow cytometry was used for immunophenotyping. Cytogenetic studies were done using standard G banding technique. Results: The most common AML subtype irrespective of age & gender was FAB AMLM5 (26 %) followed by AML M2 (22 %) and AML M4 (20 %). The highest WBC counts were seen in AML M5 and lowest in AML M0. Progenitor cell markers HLA-DR, CD117 and CD 34 expressed at an overall rate of 82, 86 and 72 % respectively. However, none of the cases of AML M3 expressed CD34 and HLA-DR. Amongst the lymphoid markers, CD19 was expressed in four cases, three of which showed t(8;21) translocations. CD4 is expressed in 14 cases of which 12 were morphologically AML-M5. Cytogenetic studies revealed 22 cases to be having a normal karyotype. Recurrent genetic abnormalities were seen in 10 cases, t(8;21) in four cases, inv 16 in four cases and t(15;17) in 2 cases. Cases with t(8;21) showed CD19 expression (75 %) along with CD34/HLADR/CD13/CD117/MPO expression. CD56 was seen in only 1 case (25 %). Both the cases with t(15;17) were consistently negative for progenitor markers (CD34/ HLADR/CD117). CD7 was expressed in 10 cases, 6 of them showed non recurrent cytogenetic abnormalities and remaining 4 had a normal karyotype. Conclusion: Immunophenotypic analysis in AML is required for diagnosis and classification. Morphological features and cytogenetic findings are related to the aberrant antigenic expression in the myeloid blasts.

Abstract No. 10 Spectrum of Recurrent Genetic Abnormalities in Acute Myelogenous Leukemia Cases in a Tertiary Care Centre Priti, Jogeshwar Binota1, Shano Naseem1, N Varma1, Pankaj Malhotra2, RK Marwaha3, S Varma2 Post Graduate Institute of Medical Education and Research, Chandigarh Summary: Incidence of various fusion transcripts of AML in PGIMER, Chandigarh. Introduction: The reported incidence of common recurrent genetic abnormalities in Acute Myeloid Leukemia (AML) is around 40–45 %. Few of these translocations e.g. t(15;17), t(8;21) and inv(16) have a favorable response to therapy. The aim of this study was to note the spectrum of these common fusion transcripts presented in our institute during the last 3 years. Materials and Methods: A total of 270 AML cases diagnosed on bone marrow, cytochemistry and flow-cytometry over a period of 3 years were enrolled in the present study. Bone marrow or peripheral blood samples in EDTA were processed for molecular studies and multiplex Reverse Transcriptase Polymerase Chain reaction (RT-PCR) assay was carried out using the primers specific to detect fusion transcripts resulting from translocations t(15;17), t(8;21) and inv(16). Results: Of the total 270 cases, male: female ratio was 1.66: 1. 240 (89 %) were adult AML cases and 30 (11 %) were pediatric cases. A total of 84/270 (31.3 %) AML cases showed positivity for various fusion transcripts of which 65/240 (27 %) were adults and 19/30 (63 %) were pediatric cases. Out of the 65 positive adult cases, 36/65 (55.3 %) were positive for t(15;17), 24/65 (36.9 %) for t(8;21) and

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282 5/65 (7.6 %) for inv(16). Of the 19 positive pediatric cases, 10/19 (52.6 %) had t(8;21), 6/19 (31.5 %) had t(15;17) and 3/19 (15.7 %) had inv(16). Conclusion: In this study the overall incidence of various fusion transcripts of AML was found to be 31.25 %. In adults the most common was t(15;17) followed by t(8;21) and inv(16) whereas in pediatric cases it was t(8;21) followed by t(15;17) and inv(16). The identification of these transcripts provides useful prognostic and diagnostic information to the treating clinician.

Abstract No. 11 Hyperdiploid Acute Myeloid Leukemia: A Distinct Cytogenetic Sub Group Akshay P Gore, Mayur Parihar, Anurag Gupta, Arun SR, Anil K Yadav, Anupam Chakrapani, Saurabh Bhave, Deepak K Mishra, Reena Nair, Mammen Chandy Tata Medical Center, Kolkata Summary: A cytogenetic study of hyperdiploid acute myeloid leukemia along with clinicopathological correlation. Introduction: Cytogenetics is the most important prognostic factor in the risk stratification of AMLs. Patients are classified into favorable intermediate and unfavorable groups with complex karyotypes carrying the worst prognosis. Hyperdiploidy a rare occurrence in AML is defined as presence of C 47 chromosomes and is associated with an intermediate prognosis. We present clinicopathological and cytogenetic features of five patients with hyperdiploid AMLs. Materials and Methods: Karyotyping and FISh was carried out on bone marrow aspirates cultured overnight using standard protocols. The karyotypes were reported as per the ISCN 2009/2013. Results: Of the 134 cases of AML karyotyped from 2011 to 2013, five patients had hyperdiploidy with modal chromosomal number ranging from 48 to 53. There were 2 males and 3 females with age ranging from 46 to 62 years. Chromosome 21 was the most commonly gained chromosome (5 patients) while chromosome 8, 9, 14 and 22 were gained in 2 patients each. One case had three additional copies of chromosome 8. Two cases were associated with a complex karyotype while one case showed simultaneous occurrence of inv(16). Conclusion: The pattern of gains of chromosomes is similar to that reported and the incidence of hyperdiploid AML higher than that reported in literature. One of our patients had an inversion 16 along with hyperdiploidy, an association never reported before in literature. FISH analysis to look for cryptic structural abnormalities in these cases is recommended.

Abstract No. 12 The der(1;7)(q10;p10) in Myeloid Neoplasia Poonam Jain1, S Yuvarani1, Usha Sitaram 2, Marie-Therese Manipadam4, Abhijeet Ganapule3, Aby Abraham3, Auro Viswabandya3, Biju George3, Vikram Mathews3, Alok Srivastava3, Vivi M Srivastava1 Christian Medical College, Vellore Introduction: The der(1;7)(q10;p10) is an unbalanced whole-arm translocation which results in gain of the long(q) arm of chromosome 1 and loss of chromosome 7q. This translocation is seen in myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and rarely, in myeloproliferative neoplasms and has been reported to be associated with eosinophilia, trilineage dysplasia and a previous history of chemo- or radio-therapy.1 It usually occurs as a solitary abnormality. Associated abnormalities include trisomy 8 and, less commonly, trisomies 9 and 21 and deletion 20q.1–3.

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Patients and Methods: G-banded karyotypes of all patients with the der(1;7)(q10;p10) seen between 2003–2013 in the Department of Hematology, Christian Medical College, Vellore were analysed and correlated with blood and bone marrow findings. Results: The der(1;7)(q10;p10) was seen in nine patients (median age 52 years, range 26–68; male: female ratio, 3.5:1). The median haemoglobin was 7.9 g/dl (range: 4.8–13.5), median total leucocyte count, 3.8 9 109/L (range: 1.3–82.9), median platelet count, 40 9 109/L (range: 3–154), and median peripheral blood eosinophil count, 1 % (range 0–7 %). Five had MDS; three, AML and one, myelofibrosis. The der(1;7) accounted for 0.67 % (5/741) of MDS and 0.16 % (3/ 1842) of AML. All nine had dysmegakaryopoiesis, with bi- or trilineage dysplasia in two (22 %) each. Reticulin was increased in seven patients. Seven patients had additional cytogenetic abnormalities, with complex karyotypes (three or more abnormalities) in four. These included trisomies 8 (four cases), 9 and 21 (two each). Conclusions: Our findings are similar to the literature with respect to the male preponderance, the frequency of the der(1;7) in AML and its association with trisomy 8. We noted differences with respect to the median age (52 vs. 58–68 years), the frequency of the der(1;7) in MDS (0.67 vs. 1.5–6 %), trilineage dysplasia (22 %), additional abnormalities (77 vs. 36–42 %) and complex karyotypes (44 vs. 12 %) and the absence of deletion 20q and eosinophilia.

Abstract No. 13 Acute Myeloid Leukemia with Ph Chromosome and BCR-ABL Positivity: AIIMS Experience Sudha Sazawal, Deepti Mutreja, Sonal J Malhotra, Sunita Chhikara, Rekha Chaubey, Manoranjan Mahapatra, Pravas Mishra, Tulika Seth, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi-110029 Summary: BCR/ABL1 fusion transcript was identified from available RNA samples in 9/213 cases of AML after exclusion of MPAL and CML-BC. At a median follow up of 15 months 5/8 (62.5 %) patients achieved a complete remission (CR) which was lower than CR in 47/62 (75 %) of Ph-AML during the same period. De novo Ph+ AML appears to be a distinct clinical entity with an unfavourable prognosis. Screening for BCR-ABL gene may be included in the initial diagnostic molecular panel in cases of AML. Introduction: Expression of the BCR/ABL1 fusion gene is characterised by the Philadelphia chromosome (Ph) is most commonly associated with chronic myeloid leukaemia (CML). Little is known about the morphological, immunological and clinical features of the minority of acute myeloid leukaemia (AML) that carry t(9;22) translocation. Materials and Methods: Ph+ AML patients were identified from available RNA samples by reverse transcriptase polymerase chain reaction (RT-PCR) at the time of diagnosis. Patient medical records were reviewed for Ph+ AML patients and a cohort of Ph-AML for their treatment and clinical outcome and FLT3-ITD and NPM1 gene mutations were correlated. Mixed phenotype acute leukemia (MPAL) and de novo CML/BC were excluded from the study. Results: Out of a total of 213 patients of AML, nine (4.2 %) showed the existence of p210 BCR-ABL fusion transcript with b3a2 expression in five cases and b2a2 in four cases. NPM1 mutation was positive in one case and FLT3 ITD was absent in all the cases. The median follow up for Ph+ AML cases was 15 months (range 2-34 months). Eight patients received intensive chemotherapy regimens, five (62.5 %) achieved a complete remission (CR) which was lower than CR in 47/62 (75 %) of Ph-AML patients who received similar chemotherapy in whom follow up data

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was available. Conclusion: De novo Ph+ AML appears to be a distinct clinical entity with an unfavourable prognosis. Screening for BCR-ABL gene may be included in the initial diagnostic molecular panel in cases of AML.

Abstract No. 15

Abstract No. 14

Christian Medical College, Vellore

Acute Myeloid Leukemia with the t(1;22)(p13;q13)—Report of Three Cases

Introduction: Rearrangements of the core binding factor beta (CBFß) gene on chromosome 16, band q22 are associated with a good prognosis in acute myeloid leukemia (AML) [1]. The inversion (16)(p13q22) is the typical abnormality in this group and results in the formation of a CBFß/MYH11 fusion gene [2]. It accounts for *4 % of the AMLassociated abnormalities and is usually associated with AML M4 with eosinophilia or M2 [3]. Trisomy 22 is the most common secondary change [4]. Materials and Methods: G banded karyotypes of all patients with the inversion 16 seen in the Department of Haematology between January 2003 and December 2012 were correlated with blood and bone marrow findings. Results: There were 23 patients with the inversion 16 among 1842 AMLs seen during this period (1.2 %). The median age was 33 years (range 1–58 years). There were 11 males. The median haemoglobin was 8.7 g/dl (1.6–14.4 g/dl), the median WBC count, 64.9 9 109/L (range 1–244.3 9 109/L) and the median platelet count, 26 9 109/L (range 7–192 9 109/L). The FAB subtypes were AML M4 (11 patients), M2 (six), AML not otherwise specified (four), M1 (two). The bone marrow blast percentage ranged from 5–91 %. Abnormal eosinophils were seen in nine patients with AML M4 and M2 accounting for four each. The inversion 16 was solitary in 13 (57 %), associated with a single additional abnormality in three (13 %) and with two or more additional abnormalities in seven (30 %). The common additional abnormalities were trisomy 8 (seven patients), trisomy 22 (four) and trisomy 9 (two). Conclusion: Our data is similar to the literature except for trisomy 8 being the most common secondary change. The inversion 16 can be overlooked unless the morphology of the metaphases is good. FISH analysis or molecular testing is helpful for confirmation of this abnormality, especially if the bone marrow shows myelomonocytic leukemia or abnormal eosinophils or if a solitary trisomy 22 is present.

J Meena, A Nancy1, U Sitaram2, A Ganapule1, A Abraham1, A Viswabandya1, B George1, V Mathews1, A Srivastava1, VM Srivastava3 Christian Medical College, Vellore Introduction: The t(1;22) is a rare translocation which is typically associated with AML M7 in infants and young children and an intermediate to poor prognosis [1]. This translocation is seen in 0–3 % of paediatric AML and results in the formation of a fusion gene, RBM15/ MKL1 [2,3]. In the WHO classification (2008) of AML with recurrent genetic abnormalities, AML with the t(1;22) has been included as a separate subtype [4]. Fewer than 50 cases have been described till date [5]. We describe the cytogenetic and morphological features of three children with the t(1;22). Materials and Methods: Karyotypes of AML with the t(1;22) seen between January 2003 and July 2012 were correlated with peripheral blood and bone marrow findings. For each patient, at least 20 G-banded metaphases were analysed from unstimulated overnight cultures of bone marrow. Results: There were 1694 AML seen during the period of study of which three (0.18 %) showed the t(1;22). The age of the patients was 9 months to two years. Two were males. The haemoglobin ranged from 7.5–10.3 g/dl, WBC count, 1.3–30.9 9 109/L, and platelet count, 5–129 9 109/L. One of the three children had received treatment elsewhere and his marrow showed acute leukemia with 40 % blasts; further morphological subtyping was not possible. The other two patients presented at diagnosis. In one, the bone marrow morphology was typical of AML M7. The marrow of the third patient showed scattered immature cells resembling megakaryoblasts with focal marked reticulin fibrosis. All three children had hyperdiploid karyotypes (chromosome number 47–56). The additional abnormalities included gain of the derivative chromosome 1 and trisomy 6 in two patients. Conclusion: AML M7 may be mistaken for a non-haematopoietic tumour due to the primitive appearance of the blasts [1]. Therefore, it is important to be aware of this translocation which is diagnostic of AML M7. References 1. Bernstein J, Dastugue N, Haas OA, Harbott J, Heerema NA, Huret JL, Landman-Parker J, LeBeau MM,Leonard C, Mann G, Pages MP, Perot C, Pirc-Danoewinata H, Roitzheim B, Rubin CM, Slociak M, Viguie F.Leukemia. 2000 Jan;14(1):216–8. 2. Carroll A, Civin C, Schneider N, Dahl G, Pappo A, Bowman P, Emami A, Gross S,Alvarado C, Phillips C, et al. Blood. 1991 Aug 1;78(3):748–52. 3. Ma Z, Morris SW, Valentine V, Li M, Herbrick JA, Cui X, Bouman D, Li Y, Mehta PK, Nizetic D, Kaneko Y, Chan GC, Chan LC, Squire J, Scherer SW, Hitzler JK. Nat Genet. 2001 Jul;28(3):220–1. 4. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, Harris NL, Le Beau MM, Hellstro¨m-Lindberg E, Tefferi A, Bloomfield CD. Blood. 2009 Jul 30;114(5):937-51. doi: 10.1182/blood-2009-03-209262. Epub 2009 Apr 8. 5. Huret JL. t(1;22)(p13;q13). Atlas Genet Cytogenet Oncol Haematol. June 2001.

The Inversion 16 in Acute Myeloid Leukemia M Manoj, A Nancy1, U Sitaram2, A Ganapule1, A Abraham1, A Viswabandya1, B George1, V Mathews1, A Srivastava1, VM Srivastava3

References 1. Delaunay, J., et al., Prognosis of inv(16)/t(16;16) acute myeloid leukemia (AML): a survey of 110 cases from the French AML Intergroup. Blood, 2003. 102(2): p. 462–9. 2. Liu P, Tarle´ SA, Hajra A, Claxton DF, Marlton P, Freedman M, Siciliano MJ, Collins FS. Fusion between transcription factor CBF beta/PEBP2 beta and a myosin heavy chain in acute myeloid leukemia. Science. 1993 Aug 20;261(5124):1041–4. 3. Le Beau MM, Larson RA, Bitter MA, Vardiman JW, Golomb HM, Rowley JD. Association of an inversion of chromosome 16 with abnormal marrow eosinophils in acute myelomonocytic leukemia. A unique cytogenetic-clinicopathological association. N Engl J Med. 1983 Sep 15;309(11):630–6. 4. Xu, W., et al., Trisomy 22 as the sole abnormality is an important marker for the diagnosis of acute myeloid leukemia with inversion 16. Onkologie, 2008. 31(8–9): p. 440–4.

Abstract No. 16 Five Year Survival Analysis of All Patients Treated by MCP 841 Protocol: A Retrospective Study Akhil Kapoor1, Ashok Kalwar2, Surender Beniwal2, S Narender, Satyanarayan1, Raj K Nirban1, M Sitaram1, Mukesh Singhal1,

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284 Daleep Singh1, Puneet Bagri1, Shankar L Jakhar1, Neeti Sharma1, HS Kumar1, Ajay Sharma1, MR Baradia1, Sandeep Jain1, SK Bishnoi1 Department of Radiation Oncology1, Department of Medical Oncology2, Acharya Tulsi Regional Cancer Treatment & Research Institute, Sardar Patel Medical College, Bikaner, Rajasthan 334003 Summary: This retrospective analysis of 310 patients of ALL treated with MCP-841 protocol was undertaken to study the patterns of relapse and 5 year survival analysis of ALL patients in terms of T cell versus B cell ALL, presence of blasts in PBF on day 15 of induction, TLC at presentation [1 lakh/mm3, age at presentation, CALLA positivity and completion of protocol within prescribed time. Introduction: A dramatic improvement in survival of ALL patients in last three decades has been observed. MCP 841 protocol is an effective tool with tolerable toxicities. BFM 90 and GMAL protocols are more useful for adult ALL but rarely used in countries with limited resources. Materials and Methods: 310 ALL patients from North West India willing for treatment and regular follow up upto 5 years were accrued in this study. 54 % patients were \15 years and 69 % were male. All patients were uniformly treated with MCP 841 protocol. Statistical calculations were performed using MedCalc for windows version 12.7. Results: Relapse free survival at 5 years by Kaplan Meir analysis for B cell ALL was 65 % while for T cell 30 % (p = 0.0012).In patients with day 15 PBF without blast, relapse free survival was 65 % while with persistent blast, it was 45 % (p = 0.0007). Patients with TLC \1 lakh at presentation, relapse free survival was 70 % and [1 lakh had 40 % survival (p \ 0.0001). CALLA negative patients had 95 % survival while positive had only 20 % (p \ 0.0001). For \3 years age group, survival was 30 %; [20 years 15 %; 3–10 years and 11–15 years had 65 % survival each and for 16–20 years, it was 60 % (p \ 0.0001). Conclusion: MCP 841 protocol is a useful tool for ALL treatment of children especially for centres with limited resources

Abstract No. 17 Clinical Profile and Outcome of Pediatric Acute Myeloid Leukemias Rajesh Kota, Muralidhar Gullipalli, K V Krishnamani, Vijay Gandhi Linga, Sadashivudu Gundeti, Lakshmi Srinivas Maddali, Ragunadharao Digumarti Nizam’s Institute of Medical Sciences, Hyderabad, Andhra Pradesh Introduction: Approximately 20 % of childhood leukemias are of myeloid origin, the majority of them being acute myeloid leukemias (AML). There is a paucity of data addressing the outcome of pediatric AML from Indian subcontinent. We analyzed the clinical profile and outcome of pediatric AML from our center. Materials and Methods: We retrospectively studied the case records of all pediatric AML patients (1–18 years) from January 2007 to December 2012 for demographic and clinical details, treatment and outcomes. Results: Sixty three children were registered, but only 45 took treatment during the study period. Median age was 14 (range 2–18), males constituted 39 and females 24. Most common presenting symptom was fever (87 %), followed by fatigue (73 %) and bleeding manifestations (38 %). Acute Promyelocytic Leukemia (APML) was the most common subtype (30 %), followed by AML-M2 (25 %). None of the children had any inherited disorder. Of all the children who took treatment, 66 % (30/45) achieved complete remission following standard induction regimens (All trans retinoic acid + Daunorubicin for APML and 3 + 7 regimen for other AMLs), whereas 22 % patients failed induction. The remission rates after induction for APML were significantly higher at 88 % as compared to 54 % for other AMLs. Five out of 45 patients abandoned treatment before the

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 completion of the planned chemotherapy. Median follow up of the study is 13 months (range 1–60), the median OS being 15 months. Conclusion: Treatment of childhood AML, unlike ALL, is still a challenge for the Pediatric Oncologist. Though the survival rates for APML are encouraging, little success has been achieved in the treatment of other AMLs.

Abstract No. 18 Profile of Pediatric Acute Myeloid Leukemia (AML)—Single Centre Study from Eastern India Sandeep Saha, Tuphan Kanti Dolai, Prakas kr Mandal, Basab Bagchi, Ashutosh Panigrahi, Meet Kumar, Maitreyee Bhattacharyya, Shyamali Dutta, Rajib De, Malay Ghosh Hematology Department, NRS Medical College and Hospital, Kolkata Summary: Median ages of presentation were 10 (3–17) years. Most of them presented with fever and pallor. 60 % of pediatric AML had cytogenetic abnormalities. AML BFM 93 protocol regimen had worser survival than 3 + 7 induction in short follow up period. Introduction: Objective was to determine the demographic, clinical profile & treatment of pediatric AML patients from Eastern India. Materials and Methods: Prospective demographic, clinical, investigations and treatment protocol data were collected from patients attending hematology services of NRS Medical College, Kolkata. Period: July 2011 to July 2013. Results: Among the 10 AML (non APL) 6 were male and 4 female. Median ages of presentation were 10 (3–17) years. At presentation, 100 % (10/10) with fever, 90 % (9/10) with pallor, 10 % (1/10) with infection(septicemia), 20 % (2/10) proptosis (chloroma), 40 % (4/ 10)bone pain and 30 % (3/10) with bleeding manifestation. Six (60 %) patients presented with hepatomegaly and 5(50 %) with splenomegaly, 4 with purpura and ecchymosis, 4 (40 %) had nontender cervical lymphadenopathy. One (10 %) had no peripheral lymphadenopathy (clinically) which were revealed by imaging. Two (20 %) presents with TLC [ 50,000, 9(90 %) with anemia and 4 (40 %) with thrombocytopenia. The common AML type were M2 (20 %), M4 (30 %). Two (20 %) patients with 459 & -Y, 3 (30 %) had t(8;21) and 1 (10 %) had monosomy 7 at diagnosis. One patient was FLT3 ITD positive. One patient died due to septicemia with DIC before receiving chemotherapy. Six received chemotherapy according to AML BFM 93 protocol and CR rate was 83.33 %. All patients completed therapy. Five patients relapsed within 6 months of completion of chemotherapy and all except 1 died after within 3 months after 2nd line of chemotherapy. Three patients received 3 + 7 induction followed by three cycles Hidac and two of them are in CR. Two completed therapy and on CR 1.5 years of follow up. Conclusion: 3 + 7 induction regimen had better survival than AML BFM 93 protocol in short follow up period.

Abstract No. 19 Clinical Outcome of Allogeneic Stem Cell Transplant for Acute Myeloid Leukemia: A Single Centre Experience Abhijeet P Ganapule, Chepsy Philip, Aby Abraham, Auro Vishwabandya, Kavitha N Lakshmi, Biju George, Alok Srivastava, Vikram Mathews Department of Haematology, Christian Medical College, Vellore Summary: This is retrospective analysis of clinical outcome of 254 Acute Myeloid Leukemia (AML) patients who have undergone allogeneic stem cell transplant (SCT) at our centre. Introduction: Allogeneic

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 SCT is a curative option for patients with diagnosis of AML in CR1 and beyond. There is limited data of the clinical outcome following an allogeneic SCT for AML from India. Materials and Methods: Between 1986 till 2013, 254 allogeneic SCT for AML have been done at our centre. There were 162 (63.7 %) males and the median age at transplant was 33 years (range: 4–63). SCT was done in CR1, CR2, CR3 and residual disease in 127 (50 %), 43 (16.9 %), 6 (2.4 %) and 78 (30.7 %) respectively. The myeloablative conditioning regimens were used in 149 (58.6 %) cases. The donors were either matched related 220 (86.2 %) or matched unrelated 34(13.4 %) for patients. Conventional graft versus host disease (GVHD) prophylaxis was used. However in 6 (2.4 %) patients GVHD prophylaxis was not started as there was active disease at the time of transplant. The stem cell source was peripheral blood in 216 (85 %) cases and bone marrow in the rest. Results: Total 242 (95.2 %) engrafted. The median time for platelet, neutrophil engraftment was 14 days (9–25) and 18 days (1–62) respectively. TRM at 100 days was 64 (25.19 %). Acute GVHD was seen in 134 (52.8 %) patient, of which 55(21.65 %) had grade III/IV GVHD. Chronic GVHD was seen in 149 (58.7 %) patients, of which extensive GVHD was seen in 41(16.1 %) patients. The EFS at 5 years among the CR1, CR2, CR3 and residual group was (54.8 ± 5.6 %), (47.7 ± 7.8 %), (33.3 ± 19.2 %) and (21.8 ± 4.9) respectively. Conclusion: Allogeneic stem cell transplantation for AML is associated with durable remission even for patient beyond CR1. However it should preferably be done in patients in complete remission prior to transplant.

Abstract No. 20 Case Report of Hypocellular Acute Myeloid Leukemia S Bariya Jaydeep, Biren Parikh, Dhaval Jetly, Manoj Shah Pathology Department, The GCRI, Civil Campus, Ahmedabad, Gujarat Summary: Hypocellular Acute Myeloid Leukemia is a very rare disease and mostly occurs in old age patients. It is critical to differentiate it from disease having similar clinical presentation which are hypocellular MDS and aplastic anemia for selecting appropriate therapy and prognosis. Introduction: Acute Myeloid Leukemia is relatively uncommon hematological malignancy consisting 15 % of all hematological malignancies and hypocellular AML is even more rare disease consisting of less than 10 % of all cases of AML. Materials and Methods: A patient named Ravjibhai Dabhi, 50 years male, came to the GCRI with complains of bone pain, dizziness and weakness since 1 month, having mild pallor and no splenomegaly. CBC, bone marrow aspiration And bone marrow biopsy were done for definite diagnosis of the patient. Results: On bone marrow aspirate and biopsy examination patient was diagnosed as hypocellular acute leukemia and on immunophenotyping it was diagnosed as Acute Myeloid Leukemia. Conclusion: Hypocellular AML is a very rare hematological malignancy and bone marrow aspiration, bone marrow trephine biopsy with the aid of immunophenotyping are required for definite diagnosis.

Abstract No. 21 Hypocellular Acute Leukemia—Case Series from a Tertiary Care Center Tushar Sehgal, S Naseem, N Kumar, N Varma, R Das, J Ahluwalia, MUS Sachdeva, P Sharma, P Malhotra1, S Varma1 Departments of Hematology and 1Internal Medicine, PGIMER, Chandigarh

285 Summary: We present our institutional experience including clinical, morphological and flow cytometric analysis in 8 cases of hypocellular acute leukemia (HAL). It may pose a challenge with regards to its diagnosis and treatment. Introduction: Hypocellular acute leukemia is defined as having C20 % blasts in peripheral blood or bone marrow with a bone marrow cellularity of less than 20 %. The incidence varies from 5–12 % of all acute leukemias. HAL usually present in elderly people and can simulate hypoplastic myelodysplastic syndrome (MDS) or aplastic anemia (AA). HAL almost always have a myeloid phenotype, however rare cases with lymphoid differentiation have also been reported. Materials and Methods: We analysed all our cases of freshly diagnosed acute leukemia from May 2012 to 2013 and retrieved cases which showed hypocellularity. Details of clinical presentation, intake of drugs, hemogram, bone marrow aspirate findings were recorded. Trephine biopsies were seen for percentage cellularity. Immunophenotyping was done in all cases for subtyping, by flow cytometry in 5 cases and by immunohistochemistry in 3. Results: During the study period there were 316 cases of acute leukemia, of which 8 (2.5 %) were of HAL. Median age was 31 years (range, 16–50 years) with M:F ratio of 1.7:1. Symptomatic anemia and fever were the commonest presentations, followed by bleeding, lymphadenopathy and splenomegaly. All patients had with cytopenias—bicytopenia and pancytopenia in 5 and 3 cases respectively. Circulating blasts were seen in 6 cases and dysplasia in none. Bone marrow showed \20 % cellularity in 6 cases while in 2 cases cellularity was between 20–40 %. Conclusion: Presentation of acute leukemia with hypocellular marrow is rare. In this study it was seen in 8/316 (2.5 %) cases of acute leukemias. Cytopenia was seen in all cases of HAL. AML constituted 87.5 % and ALL 12.5 % cases of HAL. In addition to the importance of identification and differentiation of HAL from AA and hypoplastic MDS, it is also important to categorize them as a distinct morphologic entity of acute leukemia, as recent studies have shown a different therapeutic approach to their treatment with use of less intensive chemotherapy and hematopoietic growth factors having a beneficial effects in these patients.

Abstract No. 22 Clinical-Hematological Profile of Acute Erythroleukemia from a North Indian Tertiary Care Centre Tushar Sehgal, Prashant Sharma, Man Updesh Singh Sachdeva, Jasmina Ahluwalia, Reena Das, Shano Naseem, Narender Kumar, Pankaj Malhotra, Subhash Varma, Neelam Varma Department of Hematology and Adult Clinical Hematology Unit, Department of Internal Medicine (PM, SV), Postgraduate Institute of Medical Education & Research, Sector 12, Chandigarh 160012 Summary: We present our institutional experience, including clinical, morphological and immunophenotypic findings in 43 cases of acute erythroleukemia, an enigmatic neoplasm that often overlaps with myelodysplasia. Introduction: Acute erythroleukemia (AEL) is a morphologically distinctive entity. It may pose a diagnostic challenge by overlapping with other malignancies like the myelodysplastic syndromes and AML with myelodysplasia-related changes. The 2008 WHO classification recently refined diagnostic criteria for these entities. Materials and Methods: Clinical data, laboratory records and archival pathological material from 43 morphologically-diagnosed AEL cases were retrieved. Presence of antecedent causes was ascertained. Detailed morphological findings including dyshemopoietic changes were reviewed. Flow cytometric immunophenotyping was done in 16 cases. Results: AEL comprised 3.9 % of all AML. Forty two cases were erythroleukemia, erythroid/myeloid while one was pure erythroleukemia. Median age was 31 years with a bimodal distribution. M:F ratio was 2.1:1 Symptomatic anemia, fever and bleeding were the commonest

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286 presentations. Lymphadenopathy, hepatomegaly, splenomegaly and jaundice were present in 1, 18, 12 and 3 patients respectively. Prior MDS, cytotoxic chemotherapy, MPN (likely CML) and bone marrow failure syndrome were seen in 7 patients. Bone marrow showed multi-lineage dysplasia (C50 %) in 4 cases. Immunophenotyping revealed myeloid marker expression in 14 cases. Blasts in 2 cases expressed only glycophorin. Monocytic markers were seen in 3 cases. Conclusion: Refinements in criteria make AEL partly a diagnosis of exclusion as 11 of our patients would fit into other WHO 2008 categories. Characteristic clinical and morphological findings however, merit the continued recognition of AEL as a distinctive subtype of AML.

Abstract No. 23 Myeloid Sarcomas: A Single Centre Experience from India Aditya Kumar Gupta, Nita Radhakrishan, K Anand Kumar, Vasant Chinnabhandar, Veronique Diand, Anupam Sachdeva Department of Pediatric Hemato-Oncology and BMT, Sir Ganga Ram Hospital, Old Rajinder Nagar, New Delhi-110060 Summary: Myeloid Sarcomas (MS) were present in 12.6 % of all AML patients at our centre. Of 7 treated patients, 3 are alive. Orbital MS and t(8,21) have favourable outcome. Introduction: MS are rare extramedullary manifestations of AML. MS are reported in 2.5–9.1 % of AML patients. MS are characterized by distortion of extramedullary tissue due to proliferation of blast cells. A review of the clinical and laboratory profile, with outcomes of MS patients at our centre is presented here. Materials and Methods: We retrospectively reviewed recorded data of 71 children with AML and/or MS at our centre, from January 2006 to December 2012. Results: Nine (6 males) of 71 (12.6 %) AML patients had MS at presentation. Their age ranged from 9 months–18 years. Four had orbital MS. The other patients had MS of the maxilla, mandible, porta-hepatis and urinarybladder whereas an 18 year girl with paraplegia had a rare presentation with disseminated MS (involving the spinal-cord, paravertebral muscles, pre-sternal and retro-sternal regions, uterus and the craniofacial sinuses). Bone marrow was involved in 8 patients. AML-M2 was the commonest associated subtype. Immunohistochemical staining with Myeloperoxidase was positive on the biopsies. Genetic work-up was done in 7 showing t(8;21) in 3 patients, complex cytogenetics in 2 and normal cytogenetics in 2. Two patients—one with a mandibular MS and another with an orbital MS refused treatment due to financial reasons, and both have expired. Out of 7 treated patients one died with pseudomonal necrositing fasciitis post chemotherapy. Three patients had refractory disease died. Two with orbital MS and t(8,21) are alive and in remission at 10 and 34 m respectively. The child with Maxillay MS and t(8;21) is in clinical remission at 6 months. Conclusion: MS are rare tumors with varied presentation. Favorable cytogenetics of t(8;21) and orbital MS respond well to therapy and have favorable outcome.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Sarcoma in Thyroid in a Chronic Myeloid Leukaemia patient is very rare. Discontinuation/irregular intake of chemotherapeutic drugs, may complicate such a disease. The present case is reported due to its rarity and stress upon the need for compliance with chemotherapeutic drugs. Introduction: A Myeloid Sarcoma is a tumour mass consisting of Myeloid blasts with or without maturation occurring at an anatomical site other than the Bone Marrow. Myeloid Sarcoma of Thyroid is a very rare entity. Materials and Methods: A 50 year aged woman presented with weakness, abdominal discomfort, intermittent fever and pain in neck for 1 month. Patient was a diagnosed case of Chronic Myeloid Leukaemia on oral Imatinib therapy since November 2011. However, she was non-complaint for the last one year. Results: Her haemogram show Hb-6.2g/dl, TLC-1.1 lakh/cmm, blasts-06 %, Basophils-04 %, Platelet 1.7 lakhs/cmm, USG Abdomen Hepatosplenomegaly, USG Neck multiple nodules along with calcification in Right lobe of Thyroid. FNAC Neck-Myeloid Sarcoma of Thyroid. Conclusion: Myeloid Sarcoma of Thyroid in a case of Chronic Myeloid Leukaemia is a very rare entity. Discontinuation of chemotherapy may lead to such complication. Hence, increased morbidity and mortality may result in such a case. Keywords Myeloid Sarcoma, Thyroid, Chronic Myeloid Leukaemia

Abstract No. 25 Granulocytic Sarcoma of the Uterus Vidhya Manohar, Shubhangi V Agale, AG Valand, SR Bijwe, Varsha Bhatia, Diana Grant Government Medical College, Byculla, Mumbai

Abstract No. 24

Summary: A 33 year old primigravida, delivered a healthy baby came with complaints of severe bleeding. Post-mortem examination revealed a tumour involving the female genital tract with no history or complaints of any other haematological malignancy. The tumour was highly aggressive and a diagnosis of granulocytic sarcoma was given. Introduction: Granulocytic sarcoma (GS) is a rare solid tumour of immature myeloid cells involving an extramedullary anatomic site. GS only occurs in 32.3 % of patients with granulocytic leukemia, being clinically evident in less than 1 %. Most common sites include the soft tissue, lymph nodes and skin but uncommonly also involves the gynaecologic tract, of which ovary is the most common. Materials and Methods: 33 year old primigravida, delivered a healthy baby came with complaints of severe bleeding and died within hours of admission in the critical care unit. An autopsy was performed and revealed nothing significant on thorough gross examination. On gross examination the wall of the uterus and cervix was thickened. Results: Microscopic examination of the sections studied from uterus and cervix revealed a tumour comprising of sheets of discohesive cells. The cells were moderately pleomorphic with eosinophilic cytoplasm having hyperchromatic nuclei with prominent nucleoli. Sections studied from heart, liver, kidney, spleen, adrenal, bilateral fallopian tubes and ovaries shows tumour infiltrates with the above histomorphological features. Immunohistochemistry confirmed the diagnosis of granulocytic sarcoma. Conclusion: GS of the uterus is a rare entity with a poor prognosis. No patient surviving more than 5 years has been reported in literature.

Myeloid Sarcoma of Thyroid in a Case of CML—Report of a Rare Case

Abstract No. 26

Praveen, Palash Kumar Mondal, Samrat Dutta, Mamata Guha Mallick Department of Pathology, North Bengal Medical College, Sushrutanagar, Darjeeling, WB Summary: Chronic Myeloid Leukaemia is a common haematological malignancy of elderly person. However, development of Myeloid

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Myeloid Sarcoma of Maxillo-Ethmoidal Sinus: An Uncommon Presentation of Acute Myeloid Leukemia Amar Ranjan, RK Chandoke, Pranay Tanwar, Ashutosh Kumar, MD Ray Employee State Insurance Hospital, Basaidarapur, New Delhi

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Introduction: Myeloid Sarcoma (MS) is a rare localized extramedullary tumor of myeloid precursor cells. Short Clinical History: An eleven year old male presented with nasal bleed, 2 days; breathlessness & loss of appetite, one month and bilateral nasal blockade for 2 months. There was no fever. On clinical examination bilateral cervical lymphadenopathy & hepatosplenomegaly found. On opening mouth, a pink mass hanging behind the soft palate noted. Magnetic Resonance Imaging of Head and Neck revealed a nasopharyngeal mass in maxillo-ethmoidal sinus. Gross Pathology: Excised mass was greenish pink, measuring 2 9 2 9 2 cm. Microscopic Examination: Hemogram revealed total leukocyte count 1.2 lac/ cmm, Hemoglobin 10.1 g %, & Platelet count 41,000/cmm. Differential count showed 24 % blast with marked left shift. Bone Marrow Aspirate revealed 27 % blasts. No auer rod seen. Cytochemistry showed Myeloperoxidase (MPO) positivity. Trephine Biopsy showed myeloid hyperplasia with excess blasts. Histopathological (HPE) examination of Lymph Node & Soft tissue showed leukemic infiltration. Immunohistochemistry showed blasts positive for MPO & negative for CD3. On Multicolour Flowcytometry blasts were positive for MPO, CD34 & CD45 and negative for CD3 & CD79a. The diagnosis; Myeloid Sarcoma with AML was made. Discussion: It is rare in children (1 month–89 years). Any site of body most commonly skin, lymph node, bone etc are affected. Prediction of first appearance of MS or AML was difficult in this case. MS may progress to AML simultaneously or may remain localized, never progressing to AML. Conclusion: Any extramedullary tumor showing myeloid precursor cells, should be investigated for MS, easily misdiagnosed as solid tumors.

Myelodysplastic Syndrome (MDS) Abstract No. 27 Aberrant Methylation of p15INK4B, SOCS-1, Calcitonin And FHIT Genes in Patients With MDS And Their Prognostic Implications Rekha Chaubey, Sudha Sazawal, Sunita Chhikara, Manoranjan Mahapatra, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi-110029 Summary: In the past few years transcriptional inactivation of tumor suppressor genes by promoter CpG island methylation has been a subject of intense interest as a causal factor in haematologic malignancies. We studied methylation status four genes in 100 MDS patients by methylation specific PCR. It was found that at least one gene methylation is associated with transformation, disease progression and survival. Introduction: Myelodysplastic syndromes (MDS) are clonal haematologic disorders characterized clinically and morphologically by ineffective haematopoiesis. During the past decade, a growing number of genes that are inactivated by aberrant methylation have been reported in patients with MDS but no such study has been reported from India. Materials and Methods: Peripheral blood/bone marrow samples were taken from the 100 MDS patients. DNA was extracted and modified by bisulphate treatment. Methylation specific PCR was employed to examine the methylation status of four genes: p15, SOCS-1, Calcitonin and FHIT. Results: Of the 100 MDS patients studied, forty patients (40 %) showed p15 INK4b gene methylation. Fifty three patients (53 %) showed SOCS-1 gene methylated. Fifty eight patients (58 %) showed calcitonin gene methylation. Forty three patients (43 %) showed FHIT gene methylated. Disease progression (n = 21) was observed significantly more frequent in patients with methylated p15INK4b gene, SOCS-1 and

287 FHIT gene in comparison to the patients with unmethylated gene. There was a significant difference observed between the progression free survival of patients with methylated and unmethylated p15 INK4b gene, SOCS-1 and FHIT. The median overall survival was significantly shorter in patients with methylated SOCS-1,calcitonin and FHIT genes when compared to the patients with unmethylate gene. In multivariate analysis, only p15INK4b gene methylation was found as an important predictor for progression of disease in MDS patients. Conclusion: Aberrant methylation is a frequent event in Indian patients with MDS and appears to confer a worse prognosis.

Abstract No. 28 Cytogenetic and Molecular Study of Myelodysplastic Syndrome in Indian Population Seema Korgaonkar, S Chandrakala, Nikesh Kawankar, Kanjaksha Ghosh, BabuRao Vundinti National Institute of Immunohaematology (ICMR), 13th Floor, New Multistoried Building, K.E.M. Hospital, Parel, Mumbai-12 Introduction: The myelodysplastic syndromes (MDS) are a group of clonal disorders of the hematopoietic system characterized by the presence of ineffective hematopoiesis, peripheral cytopenias, and an increased risk of transformation to acute myeloid leukemia. Cytogenetic studies reported 30–70 % chromosomal abnormalities in MDS. We have carried out a study to detect chromosomal abnormalities and molecular mutations in MDS from Indian population. Materials and Methods: We have carried out a study on 225 patients with MDS. The MDS were sub grouped according to WHO classification. The cytogenetic study using GTG banding and fluorescence in situ hybridization (FISH) was carried out using locus specific probes, EGR1 (5q31), C-MET (7q31), PTPRT (20q12) and centromeric alpha satellite DNA probe for chromosome 8. DNA was isolated and mutation analysis of TET2, ASXL1 gene was carried out using specific primers and sequencing was done according to standard protocol. Results: In our study among 225 MDS patients, the MDS subgroups were RA (9.3 %), RARS (3.5 %), RCMD (12.8 %), RAEB-I (5.3 %), RAEB II (3.1 %) according to WHO classification. The cytogenetic abnormalities were detected in 87 (38.6 %) patients. The deletions 7q (12.9 %), 20q (8.9 %) and Trisomy 8 (7.1 %) were frequent in our study group. The molecular study of TET2, ASXL1 genes is being carried out and the mutation data will be presented. Conclusion: The FISH including conventional cytogenetics is important in the prognosis of the disease. The molecular mutations such as TET2 and ASXL gene play a important role in understanding the progression of the disease.

Abstract No. 29 The Clinico-Pathologic Spectrum of Myelodysplastic Syndromes Afaq Ahmad Khan1, Mir Sadaqat Hassan Zafar1, SP Yadav2, Meena Lal3, IC Verma3, Anupama Jaggia1, Shyam Aggarwal4, Manorama Bhargava1 1

Department of Hematology, 2Department of Pediatric Hematology, Centre of Medical Genetics, 4Medical Oncology, Sir Ganga Ram Hospital, New Delhi 3

Summary: Given the fact that the diagnosis of MDS is a challenging one and the paucicity of data on Indian patients, we studied 25 patients of MDS with regard to their clinical, morphological and cytogenetic profiles. Introduction: Clinical variability of

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288 Myelodysplastic Syndromes, diversity of cytomorphological phenotypes and genetic heterogeneity can make the diagnosis and assignment of prognosis a challenge. Materials and Methods: This study was conducted in the Department of Hematology at the Sir Ganga Ram Hospital, New Delhi. This 2 years prospective observational study included 25 patients with myelodysplastic syndromes diagnosed as per the WHO classification, 2008. Their clinical profile, bone marrow and cytogenetics (conventional karyotyping/FISH) were analyzed. Results: The median age was 61 years, ranging from 6–77 years. The males were 17 and females 8. The commonest presentation was anemia (96 %), followed by fever (32 %) and bleeding (24 %). Splenomegaly was seen in 8 % and hepatomegaly in 4 % of patients. 40 % of the patients had a lag in diagnosis of more than 6 months due to decreased awareness among the clinicians, poor economic status, poor educational background, resorting to ayurvedic medications, refusing bone marrows and neglecting mild symptoms. The median hemoglobin was 7.7 g/dl, median TLC 3.7 9 109/L and median platelet count 58 9 109/L. The mean MCV was 95.5 fl. CBC revealed anemia in 88 %, neutropenia in 64 %, thrombocytopenia in 64 %, pancytopenia in 44 %, bicytopenia and unicytopenia in 28 % cases each. Peripheral Smear showed anisocytosis in 96 %, macrocytosis in 68 %, pseudopelger Huet anomaly in 40 % and blasts in 12 % of patients. Bone Marrow was normocellular in 52 %, hypercellular in 28 % and hypocellular in 20 % of patients. The erythroid maturation was megaloblastic in 44 % of patients. Erythroid dysplasia was seen in 76 %, myeloid dysplasia and megakaryocytic dysplasia in 80 % of cases each. ALIPS were seen in 20 % of patients. RAEB was the commonest WHO subtype (48 %) seen. Bone marrow cytogenetics revealed cytogenetic abnormality in 9 cases (36 %). Complex cytogenetics was observed in 8 %, del20q in 8 %, while as hyperploidy, del5q,del12p; del5q,del1q; del9q;del5q in 1 case each. Trisomy 10 with Inversion 17, a rare cytogenetics in MDS was seen in 1 case. Conclusion: The younger age of onset, RAEB as commoner presentation, lower yield of cytogenetics, somewhat different cytogenetic profile, possible different etiological factors, lag in diagnosis due to reasons mentioned above characterized our subset of MDS patients in this study.

Acute Promyelocytic Leukaemia (APL) Abstract No. 30 Molecular Transcript Profile of t(15:17) Positive Acute Promyelocytic Leukemia Jogeshwar Binota, Shano Naseem, Neelam Varma, Deepak Bansal, Pankaj Malhotra, Subash Varma Post Graduate Institute of Medical Education and Research, Chandigarh Summary: The frequency of t(15;17) positivity in our study was around 16 %. BCR-1 type of transcript was most commonly noted in both adult and pediatric age group. Introduction: Correct diagnosis of acute promyelocytic leukemia (APML) is necessary as it has the best outcome of all acute myeloid leukemias (AMLs) if treated early. Depending upon the breakpoint regions, three transcripts are noted in t(15:17) and include BCR1 (long isoform), BCR2 (variable isoform) and BCR3 (short isoform). The aim of the present study was to highlight the incidence of molecular transcript profile of t(15:17) translocation in both adult and paediatric APML cases. Materials and Methods: Correct diagnosis of acute promyelocytic leukemia (APML) is necessary as it has the best outcome of all acute myeloid

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 leukemias (AMLs) if treated early. Depending upon the breakpoint regions, three transcripts are noted in t(15:17) and include BCR1 (long isoform), BCR2 (variable isoform) and BCR3 (short isoform). The aim of the present study was to highlight the incidence of molecular transcript profile of t(15:17) translocation in both adult and paediatric APML cases. Results: Out of total 270 cases, 240/270 (88.9 %) were adult and 30/270 (11.1 %) were paediatric. 84/270 (31.1 %) AML cases showed positivity for various fusion transcripts, of which 65/240 (27.1 %) were adult AML cases and 19/30 (63.3 %) paediatric cases. Positivity for t(15:17) breakpoints was noted in a total of 42/270 (15.6 %) cases. Out of the 42 positive APML cases, 36 were adults and 6 were paediatric. BCR1 type of t(15:17) transcript was seen in 25/36 (69.4 %) and BCR3 in 11/36 (30.1 %) adult APML cases. Two (5.6 %) cases showed a fusion product of both BCR1 and BCR2 transcript. In paediatric cases, BCR1 positivity was seen in 4/6 (66 %) and BCR3 in 2/6 (33 %). Conclusion: In this study, the most common transcript was BCR1 (long isoform) in both adult and paediatric t(15:17) positive APML. Few of the studies quote a higher incidence of BCR3 transcript, while others show a higher incidence of BCR1 positivity in APML cases.

Abstract No. 31 Morphological and Immunophenotypic Profile of t(15;17) Positive Cases of Acute Promyelocytic Leukemia Sandeep, Man Updesh Singh Sachdeva, Shano Naseem, Jogeshwar, Parveen Bose, Neelam Varma Department of Hematology, PGIMER, Chandigarh Summary: Acute promyelocytic leukemia (APL) is a clinically and biologically distinct form of acute myelogenous leukemia. While in the majority of classic APL cases the immunophenotypic pattern is distinctive, the features of variant cases are more heterogeneous and nonspecific. Retrospective analysis of t(15;17) positive cases of APL showed non-classical morphology as well as immunophenotypic profile in around 20 % of all cases. Hence, a multimodal approach is must for accurate diagnosis of such cases. Introduction: Acute promyelocytic leukemia (APL) is a highly aggressive disease requiring prompt diagnosis and specific early intervention. Since demonstration of t(15;17) takes time, flow cytometry is often used to facilitate early diagnosis. The immunophenotype of APL typically displays marked autoflourescence, increased side scatter, lack of HLA-DR & CD34 expression, bright and homogenous CD33 and heterogenous expression of CD13. This study aimed to assess utility of above mentioned immunophenotypic-profile in t(15;17) positive cases of APL. Materials and Methods: Thirty cases of APL showing presence of t(15;17) on RT-PCR were selected for further analysis. Morphological evaluation was done on May Grunwal-Giemsa stained bone marrow aspirate slides. Expression profile of CD34, HLADR, CD33 and CD13 was reviewed from the archival FCS files, available in flow cytometry laboratory of Department of Hematology, PGIMER, Chandigarh. Results: Classical hypergranular promyelocytes were seen in 80 % (24/30) cases. Three cases each showed microgranular and blast like morphology. Typical immunophenotypic pattern of HLA-DR-/CD34-/CD33+ (bright & homogenous)/ CD13+/- (heterogenous) was seen in 76.6 % (23/30) cases. CD34 expression was seen in 10 % (3/30) cases and HLA-DR positivity was seen in 13.3 % (4/30). Remarkably, one case with morphologically dual population of blasts, showed expression of both B-lineage & myeloid antigens. Conclusion: Around 20 % cases of t(15;17) positive APL remain diagnostic challenge on morphology and flow cytometry and an accurate diagnosis requires a multimodal approach.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Abstract No. 32 Acute Promyelocytic Leukemia—Clinical Features & Diagnosis in De Novo & Secondary Variants Rajesh Mohanty, Minakshi Mishra, Radhika Narayan, Sreedevi Jakka, Farah Rana Department of Pathology Tata Main Hospital, Jamshedpur Summary: Clinical and pathologic features of secondary Acute promyelocytic leukemia is similar to that of denovo types. Introduction: Acute promyelocytic leukemia (APL) is a relatively common form of acute myeloid leukemia (AML) that has an excellent prognosis. In contrast, secondary acute myeloid leukemias, including therapy-related AML and AML with myelodysplasia-related changes, have a relatively poor prognosis. Materials and Methods: We diagnosed 67 cases of acute leukemia from 2010 January–2013 June. 38 cases (56.7 %) were identified as Acute myeloid leukemia. Out of those 38 cases, 22 (57.8 %) were labelled as Acute Promyelocytic leukemia based on morphology, immunophenotyping & cytogenetic studies. In most cases, the panel included antibodies specific for CD45, CD71, HLA-DR, CD33, CD13, CD2, CD3, CD5, CD7, CD19, CD22, CD10, CD34. Results: The 22 new diagnoses of APL included 13 females and 9 males with an average age of 47.5 years (range, 15–83 years). The diagnosis of APL was verified by cytogenetic and/ or molecular methods in all cases; 1 case had a variant translocation t(11;17) and the remainder had t(15;17). Of the patients, 19 (86 %) had de novo APL with no history of treatment for another malignancy, chronic immunosuppression, or MDS. The patients with de novo APL had an average age of 46.8 years (range, 15–83 years) and included 12 females and 1 males. Out of 3 cases (14 %) of secondary APL, there was a history of solid neoplasm in 2 cases that was treated with cytotoxic chemotherapy and/or radiation therapy. A 69-year-old woman with newly diagnosed APL had been given a diagnosis of MDS with a normal karyotype approximately 1 year before the leukemia developed. The patient was treated supportively with transfusions, but did not receive chemotherapy. Conclusion: We report that the abnormal promyelocytes in secondary APL are morphologically and immunophenotypically indistinguishable from abnormal promyelocytes of de novo APL and that there was no difference in the occurrence of additional cytogenetic abnormalities. In addition, in this limited series, the prognosis of patients with therapy related APL (t-APL) was not significantly different from the prognosis of patients with de novo APL. These data suggest that secondary APL is similar to denovo APL and this should be considered distinct from other secondary acute myeloid neoplasm.

Abstract No. 33 Acute Promyelocytic Leukemia—Factors that Predict Differentiation Syndrome with Arsenic Trioxide Delon D’Souza, Sanjukta S Rao, Abdul Mateen Athar, Rathnamala Chaudhari, Cecil Ross St. John’s Medical College Hospital, Bangalore Summary: A study of 40 patients who were treated with arsenic trioxide induction has shown that older age and WBC count were independent predictors for development of differentiation syndrome in APML. Introduction: Arsenic trioxide is being used as first line therapy for Acute Promyelocytic Leukemia (APML) in developing countries as a financially viable option. Development of differentiation syndrome is not as well studied with arsenic as with All Trans Retinoic Acid. This study attempts to identify factors that predict the risk of ATRA like syndrome with Arsenic induction. Materials and

289 Methods: A cross sectional study was conducted on 40 consecutive patients diagnosed with APML at our centre. Variables at baseline and during induction with arsenic trioxide were compared in patients who developed differentiation syndrome with those who did not. Chi square and student t tests were used as applicable. Results: Of the patients studied 60 % were males and 40 % were females. 14(36.8 %) of patients developed differentiation syndrome. Mean duration of arsenic therapy prior to development of differentiation was 11 days. Older age and a higher leukocyte count on day 8 of induction were found to be predictive of differentiation syndrome by univariate analysis. Mean age of these patients was 42 years as against 32 years in the latter group and total leukocyte count was 8820 cells/mm3 compared to 2,854 cells/mm3. Sex, duration of symptoms at onset and presence of comorbid illnesses, febrile neutropenia, promyelocyte percentage in peripheral blood/bone marrow use were not statistically significant contributing factors. There was no difference in outcomes between the two groups. Conclusion: Pre-existing risk factors might help predict the development of differentiation syndrome in APML patients treated with arsenic. Though commonly encountered with arsenic induction, ATRA like syndrome does not appear to influence the outcome of therapy.

Abstract No. 34 Patterns of Immune Reconstitution in Patients with Acute Promyelocytic Leukemia Treated with Single Agent Arsenic Trioxide and Its Impact on Time to Molecular Remission Ansu Abu Alex, Asma Banu, Saravanan Ganesan, Ezhilarasi Chendamarai, Kavitha M Lakshmi, Poonkuzhali Balasubramanian, Biju George, Aby Abraham, Auro Viswabandya, Alok Srivastava, Vikram Mathews Department of Haematology, Christian Medical College, Vellore Summary: There is a substantial data to suggest that immune response to acute promyelocytic leukemia (APL) contributes significantly to achieving durable remission. The impact of arsenic trioxide (ATO) on immune reconstitution, when used to treat APL, has not been studied. Objective: We undertook a prospective study to evaluate the pattern of immune reconstitution in patients with newly diagnosed APL treated at our center with a single agent ATO regimen. Materials and Methods: Peripheral blood samples were obtained from patients before treatment, on day 15 after starting ATO, post induction, pre consolidation, maintenance cycle 2 (6 months from diagnosis) and maintenance cycle 6 (10 months from diagnosis) for flow cytometry analysis. Briefly cells were labeled using a panel of monoclonal antibodies to CD3, CD4, CD8, CD56, CD16, CD19, CD45RO, CD45RA directly conjugated with FITC, PE, PerCP or APC followed by red cell lysis and cells were then washed and analyzed using FACS Calibur. Cells were gated using forward versus side scatter dot-plots. Lymphocyte subset percentages were calculated from the lymphocyte gate and absolute lymphocyte subset counts were calculated for the analysis. Results: Following treatment there was differential pattern of immune reconstitution in different lymphocyte subsets (summarized in Fig. 1). The earliest recovery to normal range was seen in the CD8 subset and CD4CD45RO (memory T cells) (Fig. 1) while the mean CD4 subset reached the normal range only at 3 months from diagnosis. A normal CD4 to CD8 ratio had not been reached even at the last follow up in this study (10 months from diagnosis). There was a significant delay in the immune reconstitution of NK cells (CD56+CD3–) and naı¨ve T cells (CD4+CD45RA+) where it had not reached normal levels even at maintenance cycle 6 (10 months from diagnosis). The number of relapses in this cohort was too few to make any association between relapse and the pattern of immune reconstitution. However, we had previously reported that

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 RT-PCR negative versus those who were positive (5.9 ± 4.1 9 107/ Lt vs. 3.9 ± 3.7 9 107/Lt; P = 0.041) (Fig. 2). None of the other subsets showed a similar association. Conclusion: This study demonstrates that there is heterogeneity in the pattern of immune reconstitution in different lymphocyte subsets post treatment of APL with ATO. Modulation of immune recovery and response could potentially improve leukemia clearance and maintenance of remission.

Abstract No. 35 APL Presenting with Gangrene And Skin Necrosis Successfully Treated Without Chemotherapy Anusree, Pankaj Malhotra, S Verma, Alka Khadwal, Gaurav Prakash, Rishi Dhawan PGIMER, Chandigarh

Fig. 1 Immune reconstitution pattern of T cells, B cells and NK cells post therapy with single agent ATO in patients with APL

Fig. 2 Comparison of absolute counts of NKT cells at end induction with patients who are RT-PCR+ve versus RT-PCR-ve at end induction RT-PCR positivity at the end of induction was a significant independent risk factor for subsequent relapses with this regimen. Among the 52 cases available for evaluation at the time of completion of induction and documentation of hematological remission 27 (51.9 %) were RT-PCR negative. At this time point there was a significantly higher number of NKT cells (CD56+CD3+) in those who were

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Summary: Acute promyelocytic leukemia (APML) is now considered as one of the curable hematological malignancy. However, early mortality in this condition has not decreased despite improvement in therapeutic advancements. We present a young lady who presented in emergency room with bilateral symmetrical gangrene of both upper and lower limbs and later diagnosed with high risk APML. She was successfully treated with a combination of arsenic trioxide and All trans retinoic acid. Introduction: Bilateral symmetric gangrene of the limbs occur when there is a block in the arterial supply of the limbs simultaneously. The most common causes are bacterial sepsis with DIC, autoimmune disorders like systemic lupus erythematosus, procoagulant states including protein C, S deficiency and APLA syndrome and use of vasopressors like noradrenaline. We present a case of a young lady who for the first time presented with arterial gangrene involving all four limbs following a brief diarrhoeal illness. Materials and Methods: A 28 years old lady presented with fever, loose stools and vomiting of 1 week duration and blackish discolouration of both hands and feet, cheeks and tip of nose of 5 days duration. At admission her PR was 120/min, BP 110/50 mmHg, RR 21/min. She had pallor, gangrene involving all fingers of left hand, middle 2 fingers of right hand, both feet extending upto 1/3rd of calf with absent pulses in both dorsalis pedis and posterior tibial arteries and left ulnar artery. Systemic examination was unremarkable. Initial possibilities considered were sepsis with DIC, SLE with APLA syndrome and acute gastroenteritis with vasopressor induced gangrene. Results: Investigations revealed Hb 6.3 g/dl, TLC 33200/c.mm, Platelet 39000/c.mm, PBF 81 % promyelocytes and 3 % blasts, PT 20 s, INR 1.36, aPTT 31 s. Subsequently BM examination, flow cytometry and real time PCR PML RAR alpha were done and she was diagnosed with high risk APML. She was started on arsenic trioxide 0.15 mg/kg and ATRA 45 mg/m2 along with hydroxyurea and prophylactic prednisolone 1 mg/kg. During hospital stay she developed cellulitis of the involved limbs, bilateral maxillary fungal sinusitis and catheter related blood stream infection. With timely and appropriate management she achieved complete hematological, BM and molecular remission. After improvement in her platelet count to more than one lakh, she was started on aspirin. After two months of therapy, she developed clear line of demarcation of gangrene and bilateral below knee amputation and amputation of all left fingers were done. Currently she is on maintenance therapy with intermittent ATRA, daily 6 mercaptopurine and weekly methotrexate. Conclusion: Acute promyelocytic leukemia is a medical emergency with high incidence of disseminated intravascular coagulation. Patients can present with atypical manifestations and index of suspicion should be kept high. Early treatment can lead to gratifying results as was seen in the present case.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Abstract No. 36 Concurrent Presentation of Sickle Cell Disease with Acute Promyelocytic Leukemia: A Rare Case Report Sima Chauhan, Prabodha K Das Department of Hematology, I.M.S. & SUM Hospital, Bhubaneswar Summary: A 23 year old female presented with breathlessness for one month, diagnosed to be a case of Sickle cell disease, Bone marrow examination revealed APML. Introduction: Very few articles have highlighted the occurrence of Acute Leukemia after 10–15 years of treatment for Sickle cell disease as a consequence of Hydroxyurea. However no case has been reported in the literature of concurrent presentation of such two pathologies involving two Haematopoietic cell lineages. We report a highly rare case. Materials and Methods: CASE REPORT: A 23 years old female presented with breathlessness for last one month, low grade fever and whole body swelling for one week. By the time the patient came had received three units of Blood Transfusion O/E hepatomegaly, mild Splenomegaly. Routine hematological investigation revealed HB-8.2 g/%, TLC-680/mm3, TPC-68,000/mm3, DC-70 % neutrophil, HB electrophoresis revealed Hbs. 55 %. Results: She was diagnosed to be a case of Sickle Cell disease. But Sickle cell disease is usually associated with leukocytosis with left shift & thrombocytosis. But our hematological picture was pacytopenia. Finally bone marrow aspiration was done and showed highly cellular aspirate with total replacement of the marrow by the abnormal hypergranular promyelocytes with classical faggot cells. The trephine biopsy showed near total replacement by sheets of immature myeloid cells. A diagnosis of APML was made. PML-RARA testing by gel PCR was positive for bcr 1 isoform. The patient was treated with ATRA. Now the patient is doing well. Conclusion: To the best of our knowledge this is the first case in literature of concurrent presentation of Sickle cell disease and APML and highlights the important of bone marrow examination in Hemoglobinopathy patients presenting with pancytopenia.

Abstract No. 37 A Case of Acute Hypogranular Promyelocytic Leukemia On Complete Remission Following Ayurvedic Therapy Vijai Tilak, Vaidya Balendu Prakash Department of Pathology, Institute of Medical Sciences, B.H.U., Varanasi & V.C.P. Cancer Research Foundation, Mandir Marg, Turner Road, Clement Town, Dehradun Summary: Allopathic modality of treatment of acute promyelocytic leukemia(AML M3) using all-trans retinoic acid(ATRA) is costly, is associated with dreaded complications & is of prolonged duration. Ayurvedic therapy is emerging as a cost effective, time saving & safe alternative especially suited for India with a fund crunch. A case of 35 years old male in complete remission following Ayurvedic therapy is presented. Introduction: Treatment of AML-M3 with ATRA is often associated with life threatening complications like ATRA syndrome & resistance. It works only in the classical form of t(15;17). And therefore the limelight has started shifting from ATRA to other safer therapeutic strategies. Ayurvedic therapy provides one such safe alternative. Paucity of work in this area in India prompted us to report this case. Materials and Methods: A 35 years old male presented with on & off bleeding from the gum of two months duration & on & off fever for a similar duration. There was no hepatosplenomegaly or lymphadenopathy. Complete blood count, blood smear examination & bone marrow examination was undertaken. Results: A diagnosis of variant form of hypergranular promyelocytic leukemia was made. The

291 patient was put on Ayurvedic therapy ‘‘Navjeevan’’ (containing ‘‘purified’’ silver, non-volatile mercury, sulphur, arsenic trisulphide, three herbs & serpentine stone) & is on complete remission (CR). Conclusion: Ayurvedic therapy is emerging as a cost effective, time saving & safe alternative especially suited for third world countries like India with a fund crunch. It is specially useful in poor patients who cannot afford the high cost of allopathic treatment & who will invariably die because of coagulopathy in the absence of treatment

Acute Lymphoblastic Leukaemia (ALL) Abstract No. 38 Epidemiologic Shift of Pediatric Acute Lymphoblastic Leukemia (ALL) in India: Lessons Learnt Over a 25-Year Period Seema Gulia, Brijesh Arora, Girish Chinnaswamy, Gaurav Narula, Mani Subramanian1, Sumit Gujral1, Prathibha Amre Kadam2, Shripad Banavali Departments of Paediatric Oncology, Hematopathology1, Cytogenetics2 and Hematopathology3,Tata Memorial Hospital, Mumbai Summary: Epidemiologic pattern of childhood ALL in a population is dependent upon the environment and relates particularly to socio-economic circumstances (McGrath hypothesis). As the socioeconomic status improves, the incidence of T-ALL decreases and precursor B-ALL especially the favorable genotypes predominates. Methods: We undertook a retrospective review of ALL cases diagnosed at our centre from 1986-2010 (25 years). The data was extracted by retrieving case records of ALL patients who were registered during the study period and treated on our uniform institutional protocol (MCP-841). The abstracted information included demographic profile, Immunophenotype of ALL and cytogenetic details. Results: A total of 1930 ALL patients were treated at our institution during this period. The male to female ratio was 2.68:1; median age was 6.8 years (range 4 months– 25 years); median WBC count was 13,860/cumm; median hemoglobin was 8.4 g/dl, and median platelet count stood at 400,000/cmm. Furthermore, the disease burden at presentation quantified by degree of splenomegaly and lymphadenopathy decreased over time. The incidence of Pre-B ALL was 68 % during initial eight years (1986 to 1993) while it was 72.5 % in the time period from 1994 to 2010. The incidence of T-ALL was 20 % in initial period and 23.3 % in later time period. The percentages of TEL-AML1 cases increased from 7 to 11.5 % during the study period. Similarly there was a change in percentages of MLL-rearrangement, BCR-ABL and E2A-PBX1 positive cases from 4 to 2.7, 5–7 and 7–8 % respectively in this period. Conclusion: The epidemiology of childhood ALL has changed over last 3 decades at our centre with greater proportion of patients with precursor B cell ALL, lower WBC at presentation and decreased leukemia burden. Although, there is modest increase in favorable TELAML mutated subset but frequency of unfavorable genotypes is still high compared to west. Chemotherapy with TKI’s improves outcome in Ph positive ALL although the toxicity in our cohort is quite significant.

Abstract No. 39 Biological Characteristics of Acute Lymphoblastic Leukemia Patients: Experience of a North India Tertiary Care Medical Centre Nosad Husen, Nishad Dhakate, Ruchi Gupta and Soniya Nityanand

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292 Department of Hematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow Summary: We did a retrospective analysis of the clinical features, immunophenotypic profile, and frequency of aberrant antigen expression in 148 patients of ALL who presented to us between Oct 2011 to March 2013, and correlated these with the cytogenetic and molecular characteristics of the malignancy. Introduction: ALL accounts for approximately 70–80 % of childhood and 20 % of adult malignancies. Although immunophenotyping by flow cytometry is essential for the diagnosis of acute leukemia, cytogenetics is a strong independent indicator of treatment response. We thus analysed and correlated these two major components to understand the biology of the disease. Materials and Methods: A total of 148 cases of pediatric and adult ALL were included in the study. A complete haematological work-up, followed by immunophenotyping using 3 colour flow cytometry was performed in all patients. Conventional cytogenetics and molecular analysis by RT-PCR for Philadelphia chromosome was carried out in patients who opted for treatment. Results: Majority of our patients were adults (58 %), possibly due to a referral bias. Immunophenotyping revealed that 78 % had B-ALL, 19.5 % T-ALL, and 2.5 % Mixed phenotype acute leukemias (MPAL). Risk stratification revealed that 72 % of the patients were in medium/high risk, 10 % in standard risk and in 18 % the risk was unknown (on induction therapy or died during induction). Aberrant expression of myeloid antigens was seen in 60 % cases. Cytogenetic data was available in 96/148 cases, and was abnormal in 40 % patients including 7.3 % incidence of philadelphia chromosome. Complete remission with induction therapy could be achieved in 93 % pediatric and 78 % adult ALL patients. Conclusion: Majority of our patients presented with high tumor burden being in medium/high risk groups, yet induction remission rates were high. Incidence of T-ALL (19.5 %) was relatively higher than reported in Western series (11–14 %), but comparable with Indian studies. The incidence of Ph chromosome (7.3 %) was lesser than reported in the West (18.9 %) but comparable with Indian data (8.3 %). Though the incidence of aberrant myeloid antigen expression was high, yet it had no correlation with induction outcome.

Abstract No. 40 Anti Leukemic Activity of cd T Cells in T-Acute Lymphoblastic Leukemia Patients Gauri Mirji, Shripad Banavali1, Manju Sengar1, Shubhada Chiplunkar2 Chiplunkar Lab, Advanced Centre for Treatment Research & Education in Cancer, Kharghar, Navi Mumbai-410 210, India, 1Tata Memorial Hospital, Dr. E. Borges Road, Parel, Mumbai-400 012 Summary: cd T cells exhibit potent cytotoxicity against zoledronate treated leukemic blasts due to better immune synapse formation, expression of perforin and activation markers. Introduction: cd T cells are involved in the immune defence against various solid tumors, but their role in leukemias is not clear. In present study, we aimed at characterising the anti-leukemic functions of cd T cells in T-ALL patients. Materials and Methods: Multiparametric flow cytometry was used for studying cd T cell subsets, activation markers and memory markers in T-ALL patients. Expansion of cd T cells from peripheral blood mononuclear cells (PBMCs) was monitored by coculture with leukemic blasts. Cytotoxic potential of cd T lymphocytes against leukemic blasts was analysed using 51Cr release assay. Immune synapse formation between cd T cells and leukemic targets was studied using laser confocal microscopy. Results: Co-culture of leukemic blasts with PBMCs led to expansion of Vd2 T cells. CD166

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 and phosphotyrosine were involved in immune synapse formation between leukemic blasts and cd T cells. F-actin polarization was observed at the immune synapse. cd T cells exhibited potent cytotoxicity against zoledronate treated leukemic blasts. Patients at remission showed increased effector memory cd T cells, increased Vd2 and Vd1 T cells and higher expression of activation markers (CD25, CD69) and perforin. Conclusion: cd T cells exhibit robust anti-leukemic activity by killing the leukemic blasts through efficient immune synapse formation. Adoptive transfer of cd T cells may be a promising immunotherapeutic approach for treatment of T-ALL patients.

Abstract No. 41 Immunophenotypic Profile of BCR-ABL Positive B-Lineage Acute Lymphoblastic Leukemia Parveen Bose, Man Updesh Singh Sachdeva, Jogeshwar, Shano Naseem, Neelam Varma PGIMER, Chandigarh Summary: BCR-ABL positive and negative cases of B-ALL were retrieved for comparison of their immunophenotypes on flow cytometry. No difference was noted between the expressions of CD19, CD10, CD20, cytoplasmic-CD79a, CD3, CD4, CD7, CD8, CD13, CD33, CD117, HLADR, CD38 and CD45. Only CD34 and CD22 showed significant difference in expression pattern of these two groups. Introduction: BCR-ABL positive B-lineage acute lymphoblastic leukemia is a hematological malignancy with distinctly poor outcome. Diagnostic tests for confirmation of this entity short of molecular studies/cytogenetics are not common in routine diagnostic facilities. This study aimed at analyzing immunophenotypic profiles of bcr-abl positive B-ALL in comparison to bcr-abl negative cases for better understanding of this neoplasm. Materials and Methods: Eighteen cases of B-ALL positive for BCR-ABL rearrangement on RT-PCR were selected for evaluation of their immunophenotype. Equal number of BCR-ABL negative cases was chosen for comparative analysis. Archival FCS files available in the flow cytometry laboratory of department of Hematology, PGIMER, Chandigarh, were reviewed for expression pattern of myeloid-, B-, T-lineage and immaturity markers, and findings of all 36 cases were tabulated in structured format for further analysis. Results: No difference was noted between the expressions of CD19, CD10, CD20, cytoplasmicCD79a, CD3, CD4, CD7, CD8, CD13, CD33, CD117, HLADR, CD38 and CD45. CD34 was more frequently positive (15/18) in BCR-ABL positive group as compared to BCR-ABL negative group (10/18). CD22, however, was more frequently seen in BCR-ABL negative B-ALL cases. Conclusion: BCR-ABL positive B-ALL shows a higher CD34 and a lower CD22 expression when compared with BCR-ABL negative B-ALL, however, molecular/cytogenetic analysis is must for confirmation of BCR-ABL positive leukemia.

Abstract No. 42 Early T-Cell Precursor Leukemia: A Subtype of Very High-Risk Acute Lymphoblastic Leukemia G Vimal Kumar, M Deenadayalan, Sri Karuna, Hemalatha, V Lakshmanan, Madhan Kumar, Jose Easow, Revathi Raj Pediatric Blood and Marrow Transplantation Unit, Apollo Speciality Hospital, Chennai, India Introduction: T-lineage Acute Lymphoblastic Leukemia (T-ALL) is known to have poorer outcomes when compared to B-lineage ALL.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 However with advances in diagnostics, chemotherapy, supportive care and monitoring of Minimal Residual Disease (MRD), outcomes in T-ALL have also improved significantly. Over the last decade a small subset of T-ALL patients have been identified to belong to a very high risk group called Early Precursor T-ALL. These children showed CD1a, CD8 negativity, weak expression of CD5 with one or more stem-cell or myeloid marker positivity in flowcytometry. AIMS: To assess the outcome of children with Early T-cell precursor leukemia treated in our unit from 2007 to 2013. Materials and Methods: We did a retrospective analysis of 158 children treated for ALL in our unit from 2007 to 2013 based on flowcytometry. Results: There were 34 children diagnosed with T-ALL, out of which 8 children demonstrated immunophenotypic features consistent with Early Precursor T-ALL. All the children were treated as per UK MRC protocol. Minimal Residual Disease (MRD) at day 28 was positive in 6 of the 8 children while 2 children were MRD negative. The 2 children who were MRD negative were continued with chemotherapy and currently in maintenance phase and in remission. Among the 6 children who were MRD positive at day 28, bone marrow transplantation following high dose chemotherapy was offered to 3 children, out of which 2 children are in remission, roughly 18 months after transplantation while one child died 6 months following transplantation due to disease relapse. The three children who were continued on chemotherapy had persistent MRD positivity and died due to disease relapse. Conclusion: ETP-ALL is a distinct, previously unrecognized entity that confers a poor prognosis with use of standard intensive chemotherapy. Its early recognition, by use of immunophenotypic criteria outlined here, is essential for the development of an effective clinical management strategy. This study has been done to raise awareness amongst physicians treating children with ALL as chemotherapy schedules can be modified and upfront Stem Cell Transplantation can be offered to these children to improve outcomes.

Abstract No. 43 T-ALL with Eosinophilia with Aberrant Expression of CD13 Rare Entity and CD33 Ashu Singh, Parul Gautam, Tejinder Singh, AP Dubey Maulana Azad Medical College, New Delhi-110002 Summary: T cell acute lymphoblastic leukemia with eosinophilia is an uncommon entity. B cell acute lymphoblastic leukemia is more commonly associated with aberrant expression of CD13 and CD33. However this aberrant expression was found in this case of T cell ALL. A close hematological work up is required in such cases. Introduction: T cell acute lymphoblastic leukemia with eosinophilia is a rare entity. Also, the aberrant expression of CD13 and CD33 is more frequently observed in B cell lymphoblastic leukemia. We present here a case of T-ALL with eosinophilia and aberrant expression of CD13 and CD33. Materials and Methods: A 12 years old female child presented with fever for 10 days and respiratory distress for 2 days. Physical examination revealed bilateral cervical lymphadenopathy, hepatosplenomegaly and petechiae all over the body. Chest X-ray revealed mediastinal widening. FNAC cervical lymph node showed features of lympho-hematopoietic malignancy. On hemogram the total leucocyte count was raised with diminished platelets. On peripheral blood examination 40 % blasts were seen. Blasts were large with high N:C ratio, minimal to moderate amount of pale blue cytoplasm, opened up nuclear chromatin, inconspicuous to 1-2 prominent nucleoli. Smear also showed eosinophilia (22 %). On cytochemistry, blasts were negative for Myeloperoxidase (MPO) but positive for Periodic acid schiff (PAS) stain. On flow cytometry, the blasts were positive for cyCD3, CD13, CD33 and negative for CD19, CD34, HLA-DR and CD11c. Results: Based on the above

293 hematological findings and the result of flow cytochemistry a diagnosis of T-ALL with eosinophilia with aberrant expression of CD13 and CD33 was made. Conclusion: Though T-ALL with eosinophilia is rare, it may represent as a distinct clinic-pathological entity with a high risk of subsequent myeloid neoplasia. Hence, a close hematological work up is advised.

Abstract No. 44 Cytogenetic Profile of 188 Cases of Acute Lymphoblastic Leukemia: A Single Center Experience from East India Mayur Parihar, Anurag Gupta, SR Arun, Anil K Yadav, Akshay P Gore, Anupam Chakrapani, Arpita Bhattacharyya, Saurabh Bhave, Deepak K Mishra, Reena Nair, Mammen Chandy Tata Medical Center, Kolkata Summary: Cytogenetic profile of 188 cases of acute lymphoblastic leukemia (ALL) using karyotyping and Fluorescent in situ hybridization (FISH) is described. Hyperdiploidy was the commonest abnormality in paediatric ALL and t(9;22) in adult ALL. Introduction: Cytogenetics is the most important prognostic factor in ALLs. The little data available on cytogenetic abnormalities in ALL from India suggests lower incidence of good risk cytogenetics in pediatric ALL. Materials and Methods: From September 2011 to June 2013 karyotyping and FISH was carried out on bone marrow aspirates of ALL patients using standard protocols. Results: Of the 188 ALL patients, 136 were pediatric and 52 were adult with 126 males and 62 females. On immunophenotyping, 158 were precursor B-ALL, 28 T-ALL and 2 cases had mixed phenotype. Overall 66 % (125) of patients had abnormal cytogenetics. Among the pediatric ALLs hyperdiploidy was seen in 46 (34 %) patients. MLL gene rearrangements and t(12;21) were seen in 6 patients each. The other recurrent cytogenetic abnormalities include t(9;22) and hypodiploidy: 2 patients, t(1;19): 5 patients and deletion 6q: 7 patients. Among the adult ALL t(9;22) was seen in 25 % of patients. Hyperdiploidy and hypodiploidy were seen in 2 patients each. The short arm of chromosome 9 (9p) was involved in 20 patients (13 pediatric and 6 adult). Conclusion: The cytogenetic profile of ALL in our series is similar to the western literature in contrast to previous reports from India with respect to hyperdiploidy, t(9;22), MLL gene rearrangements and hypodiploidy with a lower incidence of t(12;21). Almost 10 % of both pediatric and adult ALL showed structural abnormalities involving 9p.

Abstract No. 45 The t(8;14) and Its Variants in ALL Meenu Angi, S Yuvarani3, U Sitaram2, S Nair2, A Ganapule1, A Abraham1, A Viswabandya1, B George1, V Mathews1, A Srivastava1, VM Srivastava3 Christian Medical College, Vellore Introduction: The t(8;14)(q24;q32) and its variants—the t(2;8)(p12;q24) and t(8;22)(q24;q11) are associated with B cell neoplasia, notably Burkitt lymphoma/leukemia (ALL-L3). These translocations involve the MYC gene on chromosome 8q24 and one of the immunoglobulin genes—IgH on 14q32, (the most common translocation), the IgK on 2p12 or IgL on 22q11 leading to the formation of a MYC/Ig fusion gene [1,2]. Secondary changes (structural abnormalities of chromosome 1q and 13q3, trisomies 7 and 12) are seen in *60 % of patients [3, 4]. Materials and Methods: G-banded karyotypes of all patients with ALL and the t(8;14) and its variants

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294 seen in the Department of Haematology, Christian Medical College, Vellore, between January 2003 and December 2012 were correlated with blood and bone marrow findings. Results: The translocations were seen in 26/1235 (2.1 %) patients with ALL. The t(8;14) was seen in 19 patients (73 %), t(8;22) in six (23 %) and t(2;8) in one (4 %). The majority (23, 88.4 %) of these patients were males and adults (21, 80.7 %, median age 37 years, range 1–58). The median hemoglobin was 7.9 g/dl (range: 4.3–12.6); median WBC count, 9.6 9 109/L (range: 2.1–245.8); median platelet count, 45 9 109/L (range: 1.6–243), and the median BM blast percentage, 78.5 (range 36–96 %). ALL L3 was seen in 16, L2 in one, L1 in two and ALL not otherwise subtyped in seven. Six patients had extra medullary disease with involvement of nodes (four) or stomach or kidney (one each). Four had Burkitt lymphoma and two, high grade B cell lymphoma. The majority (19/26, 73.1 %) of patients had complex karyotypes (two or more additional abnormalities). One had ALL-L3 and nodal high grade B cell lymphoma with both t(8;14) and t(14;18)—doublehit lymphoma. Structural abnormalities of chromosome 1q accounted for 42 % of the secondary abnormalities followed by deletions 9q, 11q (11.5 % each) and 6q (7.7 %), addition 13q34 (7.7 %), trisomies 7 and 12 and deletion 17p (3.8 % each). Conclusion: The frequency of these translocations in our series is slightly lower than what has been reported (2 vs. 3–5 %). We also had a higher number of t(8; 22)—23 vs. 10 %. The association with 1q abnormalities is similar to the literature while 13q3 abnormalities and trisomies 7 and 12 were seen in fewer patients (7.7 and 3.8 vs. 15 %). References 1. Bilhou-Nabera C. t(8;14)(q24;q32); t(2;8)(p12;q24); t(8;22) (q24;q11). Atlas Genet Cytogenet Oncol Haematol. February 1999. 2. M R Velangi, M M Reid, N Bown, et al. J Clin Pathol 2002 55: 591–595. 3. EG Boerma, R Siebert, PM Kluin and M Baudis. Leukemia (2009) 23, 225–234. 4. Johansson B, Mertens F, Mitelman F. Cytogenetic evolution patterns in non-Hodgkin’s lymphoma. Blood 1995;86:3905.

Abstract No. 46 Molecular Genetic Analysis in Acute Lymphoblastic Leukemia— A North Indian Tertiary Care Centre Experience Shano Naseem, Neelam Varma, Jogeshwar Binota, Deepak Bansal1, Pankaj Malhotra2, Subhash Varma2 Departments of Hematology, 1Pediatrics and 2Internal Medicine; PGIMER, Chandigarh Summary: In this study we analyzed the molecular genetic profile in newly diagnosed cases of acute lymphoblastic leukemia by multiplex RT-PCR for TEL-AML1, BCR-ABL1, E2A-PBX and MLL-AF4 fusion transcripts. Introduction: Acute lymphoblastic leukemias (ALL) are characterized by distinctive chromosomal/molecular abnormalities, some of which have important diagnostic and prognostic implications. Identification of these abnormalities identifies prognostically relevant subgroups. Many western studies quote the incidence of fusion transcripts to be 30–35 % in ALL. However the data from Indian sub-continent is scarce. Materials and Methods: The present study was carried in the department of Hematology, PGIMER, Chandigarh from May 2010 to July 2013. Cases diagnosed as ALL by bone marrow morphology and flow cytometry

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 immunophenotyping were enrolled in the study. Common fusion transcripts, including TEL-AML1, BCR-ABL1, E2A-PBX and MLLAF4 were tested with a single multiplex RT-PCR assay by using primers specific to the transcript being tested. Results: During the study period 397 cases of ALL were tested for above mentioned fusion transcripts, of which 127 (32 %) were adults and 270 (68 %) children, with a male:female ratio of 1.9:1. Overall, 70 (17.6 %) cases were positive for any of the fusion transcripts; of these, 25 were adult and 45 pediatric patients. The most common fusion transcript was TEL-AML1, seen in 28 (7.1 %) cases, followed by BCR-ABL seen in 27 (6.8 %) cases and E2A-PBX and MLL-AF4 in 11 (2.8 %) and 4 (1 %) cases respectively. Adult ALL—Of the 127 cases, 25 (19.7 %) were positive for one of the fusion transcript, with BCR-ABL being most common, seen in 14/127 (13.7 %) cases, followed by TELAML1 in 6 (5.9 %), E2A-PBX in 3 (2.4 %) and MLL-AF4 seen in 2 (1.6 %) cases. Pediatric ALL—Of the 270 cases, 45 (20 %) were positive for any of the fusion transcript, TEL-AML1 was most common seen in 22 (8.2 %) cases, followed by BCR-ABL in 13 (5.8 %) cases and E2A-PBX in 8 (3 %) and MLL-AF4 in 2 (0.7 %) cases. Conclusion: In this study on 397 ALL cases—(i) the overall incidence of fusion transcript was 17.6 % with most common being TEL-AML1 and BCR-ABL seen in approximately 7 % cases each. (ii) No significant difference was found in the overall incidence of occurrence of recurrent genetic translocations between adult and pediatric cases. However, BCR-ABL was most common transcript in adults while TEL-AML1 was in children.

Abstract No. 47 Detection of Balanced Translocations in Acute Lymphoblastic Leukemia by a Novel Multiplex Reverse Transcriptase RT-PCR in Indian Population—A Pilot Study MG Manoj, J Kotwal, A Sachar, Vibha Dutta, R Kapoor, V Nair Armed Forces Medical College, Pune Summary: Multiplex RT-PCR is a novel and rapid method to detect known translocations. Analysis of fusion transcripts showed the common occurrence of t(9;22) ALL cases who can benefit from Imatinib and the consistent presence of CD25 in these patients which can be used as a surrogate marker. Introduction: Fusion-transcripts detection is essential for subtyping and diagnosis of ALL. This enables institution of appropriate therapy and provides a parameter to monitor disease progression and response to therapy. This study endeared to detect and analyze various balanced translocations known in ALL by using a novel PCR method. Materials and Methods: 16 cases of ALL were studied. Diagnosis was established after subjecting blood/bone marrow aspirate samples to morphological examination, immunophenotyping and detection of fusion transcripts by multiplex RT PCR using Hemavision Kit. Results were analyzed by correlating with the age, sex, signs, symptoms, morphology, immunophenotype and response to therapy. Results: 43 % (7 cases) showed balanced translocations, with all 7 cases being B-ALL. and t(9;22) being the most common. There was a consistent association of CD25 with cases with t(9;22). Analyses with relation to other parameters were as expected by their respective WHO 2008 subtype. No significant correlation in terms of survival benefit was seen between cases with and without balanced translocations (p = 0.7472). Conclusion: The study demonstrated the utility of Multiplex RT-PCR in initial evaluation, subtyping and monitoring MRD in ALL cases with balanced translocations, thereby guiding both therapy and prognosis. The consistent association of CD25 in cases of t(9;22) ALL indicated that CD25 could be used as a surrogate marker.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Abstract No. 48 Rare BCR-ABL Fusion Transcripts in Acute Lymphoblastic Leukemia M Sathya, Neeraj Arora, A Senthamizhselvi, J Priyadarshini, Vivi M Srivastava, Biju George, Alok Srivastava, Vikram Mathews, B Poonkuzhali Department of Haematology, and Cytogenetics Unit, Christian Medical College, Vellore Introduction: The Philadelphia (Ph) chromosome, a shortened chromosome 22, results from a t(9;22)(q34;11) reciprocal translocation, allowing the fusion of the 30 region of the proto-oncogene c-ABL (9q34) with the 50 -region of the BCR (breakpoint cluster region) gene on chromosome 22q11. Approximately 5–40 % of patients with acute lymphoblastic leukemia (ALL) are Ph+. Most cases of ALL have a p190-encoding BCR-ABL gene resulting from fusion of exon a2 from the ABL gene to exon e1 when m-bcr is involved (called e1a2 transcript) and rarely p210 due to fusion of either of exon b2 or b3 when BCR is broken at M-bcr (called b2a2 or b3a2 transcripts). Rare variants of BCR–ABL fusion transcript has been reported depending on the position of the breakpoint in BCR and/or ABL but most of these reports have been described in chronic myeloid leukaemia. We report four rare BCR-ABL fusion transcripts detected in patients with ALL diagnosed at our centre. Materials and Methods: RT-PCR for BCR-ABL fusion transcript was carried out in all newly diagnosed patients with ALL in the Department of Hematology, Christian Medical College, Vellore. Patients were diagnosed and sub classified according to the WHO classification 2008 based on morphology, immunophenotyping and cytogenetic analysis. Total cellular RNA was extracted from leucocytes (QIAamp1 RNA blood mini kit, Qiagen) and cDNA was synthesized (Superscript, Invitrogen). The type of BCRABL1transcript was determined in patients’ samples by nested qualitative RTPCR based on the previously published Biomed Concerted Action Protocol. DNA sequencing was performed using cycle sequencing kit on ABI 3130 Genetic Analyzer. Results: Of 834 cases of ALL tested for BCR-ABL transcript by RT-PCR from January 2009 to

Table 1 . No

Age

Sex

Haematological parameters

Karyotype

Immunophenotyping

FISH

BCR-ABL transcript

Treatment

1

33

F

Hb-5.9, TLC-800, Plts128,000

46, XX, t(9;22) (q34; q11.2), -12, -12, +16, 19, 20, +21, +22, +mar, +mar [6]/46, XX [14]

Pre B ALL (CD20 = 70.6 %, HLA DR = 86.3 %, CD34 = 14.3 %)

NA

e6a2

Modified BFM-98 ALL in remission

Hb-7, TLC3,200, Plts21,000, blasts (30 %)

46, XY [20]

Pre B All (HLADR = 96.1 % CD13 = 93.9 %, CD34 = 98.1 %, TdT = 54 %

80 % Fusion positive

e8a2

Hb-9.5, TLC7,800, Plts457,000 Blasts (63 %)

NA

NA

NA

ela3

Hb-8.6, TLC3,600, Plts26,000 blasts (95 %)

46, XY, t(2;20)(q21; p13), t 9;22)(q34;q11.2) [19]/47, idem, + der(22)t(9;22) (q34;q11.2) [1]

2

3

4

37

40

40

M

M

M

(pl85 kDa)

(p210 kDa)

(pl90 kDa)

Pre B ALL (HLA DR = 99.6 % CD13 = 78.0 % CR34 = 99.4 %)

73 %

e14a3 (p210 kDa)

Modified BFM-98 ALL relapsed, PBSC transplant, Well 1 year post transplant Hyper CVAD and Imatinib No follow up available Did not receive treatment no follow up available

295 December 2012, 108 (12.9 %) cases were positive for BCR-ABL. Atypical rare transcripts were documented in 4/108 (3.7 %) cases. The demographics of these cases is described in Table 1. Conclusion: Ph+ acute lymphoblastic leukemia (Ph+ ALL) is a high-risk acute leukemia with poor prognosis, affecting primarily adults and the elderly. We report here 4 unusual variant BCR-ABL transcripts in ALL diagnosed at our centre. The identification of these transcripts in ALL is relevant not only for diagnosis but also for prognosis, therapeutic staging, and for follow-up of minimal residual disease (MRD). To ensure the detection of the rare BCR-ABL fusion transcripts, different approaches should be attempted, especially if unusual or a slightly different size product is noticed.

Abstract No. 49 Treatment Outcomes in Ph+ ALL—A Single Centre Experience Uma Dangi, M Sengar, H Menon, P Amare, P Subramaniam, S Laskar, N Khattry, B Bagal, R Nair Summary: Philadelphia (Ph) positive acute lymphoblastic leukemia (ALL) constitutes about 11–29 % of all adult ALL. The incidence increases with age and is associated with lower chance of achieving a CR and a worse prognosis when compared with Ph negative ALL. Use of BCR-ABL-specific TKI’s with chemotherapy has been associated with higher CR rates without additional toxicity. This has resulted in improved outcomes in this set of patients. Here we analyze the outcomes of adult and AYA patients with Ph positive ALL treated with chemotherapy and TKI’s with or without allogenic stem cell transplant (ASCT) treated at our center. Materials and Methods: This is a retrospective, single center study. All patients registered at our centre between January 2009 to October 2012 aged more that 14 years and diagnosed with Ph positive ALL treated with TKI and chemotherapy were included in the analysis. Case records were reviewed for demography, details of chemotherapy, grade ’ toxicities (CTCAE version 4) and response. Overall and progression free survival were calculated using the Kaplan Meier method. Results: A total of 586 patients with ALL were registered of which 96 were Ph positive. Sixty five patients were treated at TMH and analyzed for outcomes. Median age was 28 years (range 15–53) with a male preponderance (8:2). Median total leucocyte count (TLC) was 33.5 9 109/cmm with 23.5 % patients having CNS 2 or 3 disease. Additional chromosomal abnormalities were seen in 15 %, aneuploidy in 71 % and Ph duplication was seen in 26.2 % of patients. Fifty patients received MCP-841 protocol along with Imatinib, 6 patients received BFM90 with Imatinib, 1 each received Dasatinib and 5 patients did not receive any TKI. ASCT was done in 6 patients. Response was assessed in 46 patients of which 42 achieved a morphological complete remission (CR). Cytogenetic remission was available in 33 patients of which 30 achieved a cytogenetic CR. Grade ’ hematological toxicity was seen in almost all patients. Commonest grade 3–4 non hematological toxicity was hepatic seen in 55.4 %. We had a very high incidence of fungal infections with more than 66 % patients requiring antifungals at some time during therapy. Thirty patients died and 24 deaths were treatment related most of whom were during induction (17). The median follow up for our cohort was 25 months (range 3–48). The 2 year overall survival (OS) and relapse free survival (RFS) were 29.5 and 30.4 % respectively. There was no difference with respect to age more or less than 30 years or TLC more or less than 30 9 109/cmm. For patients achieving CR, 2 year OS and RFS were significantly better at 44.4 and 45.8 % respectively (P \ 0.001).

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Abstract No. 50 Improved Outcome of Philadelphia Positive Acute Lymphoblastic Leukemia Using Aggressive Chemotherapy with Imatinib in Children: A Retrospective Analysis from Tata Memorial Hospital Mumbai Pankaj Dwivedi, Brijesh Arora, Vijay Patil, Avinash Pandey, Girish Chinnaswamy, Gaurav Narula, Siddharth Laskar1, Seema Medhi2, Mani Subramanian3, Sumit Gujral3, Prathibha Amre Kadam4, Shripad Banavali Departments of Paediatric Oncology, Hematopathology1, Radiotherapy2, Radiology3, Cytogenetics4 and Hematopathology5, Tata Memorial Hospital, Mumbai Summary: Philadelphia positive acute lymphoblastic leukemia (Ph+ve ALL) is a very high risk subset of childhood ALL with historically poor outcomes without stem cell transplantation before the advent and use of Imatinib Mesylate. There is higher incidence of Ph+ve ALL in approximately 7 % of all ALL cases at our centre compared to 2-3 % in west. There is paucity of data on outcome of Ph+ve ALL from India where stem cell transplantation is not affordable for most patients. We conducted a retrospective analysis of pediatric patients treated with intensive chemotherapy with or without Imatinib. Materials and Methods: We audited records of 54 pediatric patients diagnosed between January 200-December 2010 with Ph+ve ALL treated with institutional ALL protocol (MCP-841) with or without Imatinib. No patient underwent stem cell transplantation. Patient’s disease status till last follow up was recorded. PFS and OS were calculated from date of diagnosis to date of relapse/progression and date of last follow up respectively. Univariate and multivariate analysis was done to find out the prognostic factors. Results are analyzed with SPSS 19. Results: Median age of presentation was 11.3 years (range 3–17.7 years). The presenting WBC counts ranged from 870 to 642,000 cells/mm3 (Median 118,000 cells/mm3). Thirty one had CNS I status, four were CNS II and nine had CNS III status. Of 54 patients, thirty five patients received Imatinib during their treatment; 12/35 received Imatinib during induction and 23/35 received Imatinib post induction. 14 didn’t receive Imatinib any time during treatment. Median overall survival (OS) of the entire cohort was 18.33 months and estimated 5-year OS was 17.6 %. Median OS for patients who received Imatinib was 35.2 months as compared to 6.9 months for patients who didn’t received Imatinib. Similarly, 2-year OS in Imatinib group was 49 % compared to 0 % in the non-Imatinib group. Patients who initiated Imatinib during induction did poorly (median OS-10.9 months) due to increased toxic deaths compared to the patients who started it after induction (median OS not yet reached). Projected 5-year in the group starting Imatinib post-induction is 66 %. In multivariate analysis, use of Imatinib and initiation of Imatinib after induction were found to be favourable prognostic factors. Conclusions: Outcome of Ph+ ALL without Imatinib and stem cell transplantation is dismal. However, combined therapy including aggressive chemotherapy and Imatinib improves outcome. Imatinib should preferably be initiated after induction therapy to minimize treatment related toxicity and death.

Abstract No. 51 Administration of Pegylated Asparaginase Results in Higher Asparaginase Activity at the End of Induction as Compared to Unmodified Asparaginase in Acute Lymphoblastic Leukemia Deepak Bansal, Ritu Aggarwal, Manila Salaria, RK Marwaha Pediatric Hematology-Oncology unit, Advanced Pediatrics Centre and Department of Immunopathology, Post Graduate Institute of Medical Education and Research, Chandigarh

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Summary: The principle of asparaginase therapy is the enzymatic cleavage of the amino acid asparagine into aspartic acid and ammonia, with consequent depletion of asparagine levels. Serum asparaginase activity can be used as a surrogate parameter for asparagine depletion. Introduction: The aim of the study was to evaluate if administration of pegylated asparaginase (PEG-ASP) or unmodified Escherichia coli asparaginase (ASP) results in higher asparaginase activity at the end of induction in children with ALL. Materials and Methods: The large majority of children with ALL in the unit receive ASP. Option of PEG-ASP was offered to patients whose parents could purchase the expensive medication. Patients with ALL-B lineage received 3-drug induction with dexamethasone, vincristine and ASP or PEG-ASP, besides intrathecal methotrexate. Patients with ALL-T lineage received Daunorubicin in addition. ASP was administered as 6,000 IU/m2 9 9 doses, IM and PEG-ASP as 1,000 IU/m2, IM 9 2 doses. Serum asparaginase activity was measured by medac-Asparaginase-Aktivita¨ts-Test (MAAT) (medacHamburg-Germany) assay at the end of induction. Study was approved by Institute ethics committee and funded by Department of Science and Technology, Chandigarh. Results: Twenty five patients received ASP and 9 PEG-ASP. Mean age of patients in the ASP and PEG-ASP group was 6.1 ± 3.2 and 7.3 ± 2.3 years, respectively (p = 0.3). Number of patient with T-lineage ALL in the ASP and PEG-ASP group were 6 (24 %) and 1 (11 %), respectively (p = 0.66). Mean serum asparaginase activity at the end of induction in the ASP and PEG-ASP groups was 18.5 ± 42.7 and 193.7 ± 273.7 U/L, respectively (p = 0.0033). The corresponding median serum asparaginase activity was 1.92 and 65 U/L, respectively. This indirectly indicates greater asparagine depletion in the PEG-ASP group. Effect on long-term survival cannot be commented from the study. Small sample size is a limitation. Conclusion: Administration of PEG-ASP resulted in higher asparaginase activity at the end of induction as compared to ASP in children with ALL.

Abstract No. 52 Hydroxyurea for Hyperleucocytosis in Pediatric Acute Lymphoblastic Leukemia (ALL) Ankita Goel, Jagdish Chandra, Bhavna Dhingra, Sunita Sharma, Anu Gulati, Deonath Mahato, Varinder Singh Kalawati Saran Children Hospital and Lady Hardinge Medical College, New Delhi Summary: Hyperleucocytosis is a medical emergency in leukemia requiring urgent cytoreduction. We found hydroxyurea to be safe and effective treatment in ALL with hyperleucocytosis. Introduction: Hyperleucocytosis (WBC [ 100,000/cu mm) is associated with increased morbidity and mortality due to leucostasis and tumour lysis syndrome (TLS). The objective of this study was to assess the leucocytoreductive effect of hydroxyurea in hyperleucocytotic acute lymphoblastic leukemia (HAL). Materials and Methods: Fifty eight children with ALL, treated in our hospital from July 2011–June 2013, were studied retrospectively for incidence of hyperleucocytosis, associated risk factors, TLS and induction mortality. Hydroxyurea was administered to children with HAL and its efficacy and side effect profile was noted. Results: Out of 58, hyperleucocytosis was seen in 11 patients (18.9 %), with age [10 years (36 vs. 8.5 %) and T cell ALL (27 vs. 6.3 %) being significant risk factors though no difference was found for male sex (72 vs. 68 %). The incidence of both induction mortality (36 vs. 6.3 %) and Laboratory TLS was higher in HAL (27 vs. 15 %) with 1 case of clinical TLS at presentation. Hydroxyurea, given to 9 patients in a dose of 50 mg/kg divided 12 hourly, reduced WBC count to \50,000 in mean time of 5.77 + 2.27 days and induction chemotherapy could be

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 started at 6.55 + 2.0 days. No signs/symptoms of leucostasis or side effects attributable to the drug were observed. Conclusion: Although described previously for AML and CML with hyperleucocytosis, hydroxyurea can be safely used in treatment of HAL to decrease the incidence of leucostasis.

Abstract No. 53 Anti-leukemic Activity of Trichosanthes Dioica Root Extract on Human Leukemic Cell Lines & ALL Patients’ Cell Sudipta Chanda1, Subarta Pal1, Santanu Basu2, Shila Elizabeth Besra1 1 Drug Development/Diagnostic & Biotechnology Division, CSIR, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata-700032, 2Employee State Insurance Hospital Sealdah, 301/3, A.P.C. Road, Kolkata-700009

Summary: Anti-leukemic activity of methanolic extract of Trichosanthes dioica root (TDR) has been studied on human leukemic cell lines (K562, MOLT-3) & ALL PBMNCs. Introduction: In the present scenario, the demand for herbal products is growing exponentially throughout the world. This enthusiasm seems to be a result of people all over the world looking to various alternative systems of medicine, especially herbal drugs which are claimed to be safe, equally effective in comparison to allopathic drugs and which provide some answer to some of the chronic diseases like cancer. Materials and Methods: Cell growth inhibition study by trypan blue exclusion method, cytotoxicity study by MTT assay, morphological study was determined by Fluorescence microscopy. Agarose gel electrophoresis study was performed. Toxicity study has been done on normal human lymphocyte. Results: TDR showed significant cytotoxic activity in human leukemic cell lines K562 and MOLT-3 having IC50 of 74.86 and 60.25 lg/ml respectively, as well as on PBMNCs of ALL patients having IC50 62.5 lg/ml doses. TDR treated leukemic cells showed formation of DNA ladder, chromatin condensation and apoptotic body formation indicating apoptosis. Conclusion: The present study reveals that methanolic extract of Trichosanthes dioica root (TDR) has potent anti-leukemic activity.

Abstract No. 54 The Benefit of the Morphology Assessment, in Routine Post Induction Day-8 Bone Marrow Examination and Routine Bone Marrow Examinations During Maintenance Therapy in the Treatment of Childhood Precursor b-Acute Lymphoblastic Leukaemia KS Pathirage, I Dharmasena, WM Dayakumara, HM Perera, DKJ Thanthree National Cancer Institute Maharagama-Sri Lanka Summary: In the absence of Minimal Residual Disease (MRD) monitoring, morphological assessment by routine bone marrow (BM) assessment is a poor predictive factor of risk stratification and relapse in childhood precursor B-ALL. Introduction: Risk stratification using cytogenetics and monitoring of MRD in childhood precursor B-ALL have remarkably improved the disease prognosis. Unfortunately, these facilities are not freely available across the globe. This study was done to analyze whether this can be overcome by assessment of BM morphology at Day 8 post induction and every 2/3 months during the maintenance therapy. Materials and Methods: A group of 45 precursor B-ALL children who were treated in the National Cancer Institute—Maharagama which is the centre of

297 excellence for cancer in Sri Lanka, were subjected. Their Day 8 BM morphological results were analyzed to identify any relationship with future relapses. As the second step, on demand BM morphological findings were compared with the routine BM biopsies during the maintenance therapy. Results: The results revealed that there is no significant relationship between Day-8 morphology and relapse predicting power. Further compared results proved that there is no advantage on routine BM biopsies over on demand BM biopsies during the maintenance therapy. Conclusion: (1) Omission of post induction Day—8 marrow morphology as it hasn’t shown significant impact on plan of management in this study in the absence of MRD monitoring. (2) To stick to on demand BM biopsies instead of routine BM biopsies in every 2/3 months during the maintenance.

Abstract No. 55 Mid-induction MRD Assessment in Peripheral Blood Samples of Pediatric B-ALL Patients Man Updesh Singh Sachdeva, BK Karthik Bommannan, Parveen Bose, Neelam Varma, Deepak Bansal, RK Marwaha Postgraduate Institute of Medical Education & Research, Chandigarh Summary: Minimal residual disease (MRD) levels were assessed on day-15 of induction in pediatric B ALL patients on their paired bone marrow (BM) and peripheral blood (PB) samples, by flow cytometry. A direct correlation between PB-MRD and BM-MRD (p \ 0.000) was seen. BM-MRD levels were 7 times higher than the PB-MRD and the most likelihood of PB-MRD being positive was when BM-MRD was C0.31 %. Hence, an assessment of isolated PB-MRD alone might yield clinically relevant prognostic information for B-ALL pediatric patients on therapy. Introduction: There is a strong correlation between minimal residual disease (MRD) levels in bone marrow (BM) and the risk of relapse in acute lymphoblastic leukemia (ALL). This study was aimed to assess possibility of doing an isolated peripheral blood (PB)-MRD analysis as a surrogate for BM-MRD, for further management of the patient. Materials and Methods: Forty newly diagnosed pediatric B-ALL patients were assessed for MRD levels on their paired mid-induction (day-15) PB and BM samples. Lyse-stain-wash technique was used to prepare a single six colour tube comprising of SYTO13/CD34PE/CD20PerCP/CD19 PECy7/ CD10APC/CD45APCH7 for each sample. The processed samples were run on BD FACS Canto II and analysed with BD FACS Diva software. MRD of C0.01 % was considered positive. Results: Among 40 pairs of day 15 PB and BM samples, 16 pairs (40 %) had PB-MRD and BM-MRD co-positivity, 9 pairs (22.5 %) had isolated BM-MRD positivity and 15 pairs (37.5 %) were MRD negative in both PB and BM samples. Overall analysis of MRD positive cases showed a direct correlation between PB-MRD and BM-MRD (p \ 0.000) and BM-MRD levels were 7 times higher than the PBMRD. In addition, ROC analysis showed that the most likelihood of PB-MRD being positive was when BM-MRD was C0.31 %. Conclusion: Mid induction PB-MRD positivity indicates that there is a minimum BM-MRD of 0.31 %. Since literature emphasises the prognostic significance of mid-induction BM-MRD at levels C1 %, an assessment of isolated peripheral blood MRD alone might yield clinically relevant prognostic information for further management.

Abstract No. 56 CSF Flow Cytometry for Detection of CNS Disease in Acute Lymphoblastic Leukemia Patients at Diagnosis

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298 A Dayama, J Dass, P Mishra, M Mahapatra, HP Pati, R Saxena All India Institute of Medical Sciences, New Delhi Introduction: Acute Lymphoblastic Leukemia (ALL) is a rapidly progressively leukemia and is fatal if untreated. ALL has a predilection to involve Central nervous system (CNS) which is conventionally detected by examination of cytocentrifuged CSF samples. Study Objectives: To detect CNS involvement in ALL patients at diagnosis by flow cytometry and comparing it with cytopathology. In addition, a correlation of both flow and cytopathology was performed with known risk factors for CNS disease like total leucocyte count, elevated levels of Lactate dehydrogenase, cytogenetics, mediastinal mass, immunophenotype (T-ALL), BCR-ABL1 gene fusion in B-ALL. Materials and Methods: We studied 42 patients of ALL of which 33 were B and 9 were T-ALL. In all these patients, CSF counts, cytopathology and flow cytometry was performed at diagnosis. For CSF flow cytometry, a cluster of at least 25 events forming a distinct population was considered positive. Baseline parameters were recorded for all cases. Results: CSF cytopathology was positive in 5 patients (11.9 %) while flow cytometry detected CNS disease in 11 patients (26.14 %). The 5 cytopathology positive cases were also positive by flow cytometry. The use of flow cytometry detected an additional 6 cases. We observed a statistically significant association between CSF cell count and cytopathology positivity as expected. Also, when flow cytometry and CSF cytopathology results were combined, a statistically significant association was observed with serum LDH and CSF cell counts. All others high risk factors showed a non-statistical significant association. Conclusion: CSF flow cytometry detects CNS disease in ALL patients at diagnosis with a rate double than cytopathology alone. The disease thus detected is statistically associated with an elevated LDH level which is a known risk factor for CNS involvement in ALL.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Results: We had analysed 55 cases in which CSF was negative and cytospin was negative in all of them. We had found CSF positivity at diagnosis (in a clear CSF where RBCs were not found) in 8 cases at diagnosis. Except for one, all these cases were morphologically reported as ‘‘Negative’’ for blasts in the cytospin analysis. We had 9 cases which had CNS stage 3 disease. Among these 1 case had CNS disease at diagnosis and rest of a cases had isolated CNS relapse and followed up. This patient (I) had cranial nerve seen at diagnosis and was treated as CNS disease. Rest of cases had manifestations of CNS relapse, suspected clinically by symptoms such as deterioration of consciousness, seizures, headache etc. Among these 9 patients, all of them had (see Table 1) more than 1,000 events detected by FCM over a period of 300 s. We have found that in those patients when the positive events (per 300 s) were less than 2000, almost in all instances, the blasts were not detected by cytospin methods by the pathologist (Table 2). Examination by cytospin alone would have missed certainly 2 cases (and was suspicious in 3 other cases) and an early therapeutic intervention would not been possible. Conclusion: We have convincing evidence that flowcytometry has much higher sensitivity than cytospin from our studies. we also speculate that persistence of blasts beyond 4th sitting may indicate resistant disease (similar to persistence of MRD in bone marrows), which however may need to be confirmed by larger cohort of patients.

Abstract No. 58 Vancomycin Induced Impaired Renal Clearance of Methotrexate: Report of Two Children with Acute Lymphoblastic Leukemia Swati Dash, Manish Taneja1, Girish Bhardwaj2, Rajan Kapoor3, Vishal Sondhi2 Armed Forces Medical College

Abstract No. 57 Usefulness of CSF Flowcytometry in Detection and Follow Up of CNS Involvement in ALL Suthanthira Kannan Ramamoorthy, R Vishnu Kumar, L Veera Pappammal G Kuppusamy Naidu Memorial Hospital, Coimbatore 641037 Summary: We have used CSF analysis by Flowcytometry in all our patients with Acute lymphoblastic leukemia which has helped us to find out CNS 2 and CNS 3 diseases and the proportion of cases detected were much higher than the routine cytospin analysis. Since most of the ALL patients have aberrant markers and also any reactive pleocytosis of CSF will have only normal T cells, it is easy to dilineate blasts by flow cytometry. Introduction: Flow cytometry (FCM) is a highly sensitive technique capable of accurately detecting malignant cells, even in samples with very low cell counts. Flow cytometry allows for the earlier detection of NM before the onset of clinical symptoms and CSF pleocytosis and therefore may enable more effective treatment Not only that, we also postulate that early clearance of CSF by flow cytometric technique is an indicator of response to therapy and long term outcome. Materials and Methods: We used three or four color single gating technique and limited our exercise in cases of Pre B ALL, with characteristic CD 19 and CD 10 population and T ALL in which surface CD 3 was negative. We used combination of CD 45, CD 3, CD 7 and CD 5 in those T ALL patients. We had one patient with ALL L3 (burkitts) in which the combination of CD 45, CD 20 with kappa restriction was used.

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Summary: Two patients of acute lymphoblastic leukemia (ALL) being treated with high dose methotrexate as part of BFM-2002 protocol developed serious methotrexate toxicity due to concomitant vancomycin administration. Introduction: Methotrexate is an antifolate antimetabolite that forms the backbone of most chemotherapeutic protocols. High dose methotrexate is the standard of care for patients with acute lymphoblastic leukaemia (ALL). Vancomycin forms part of treatment protocol of febrile neutropenia. Both the drugs are excreted primarily by kidneys. We report two children with ALL who were administered high dose methotrexate within 5 days of vancomycin administration and developed methotrexate associated toxicity. Materials and Methods: Two patients with ALL were being managed with high dose methotrexate (5 g/m2) in accordance with the BFM-2002 protocol. During the febrile neutropenic episode, both the patients were administered intravenous vancomycin along with cefoperazone and amikacin. The patients showed a gradual improvement with subsidence of fever and improvement in neutrophil counts. After improvement in general condition, subsidence of fever, and improvement in blood counts, they were administered high dose methotrexate. The children were administered methotrexate 3 and 4 days after stoppage of vancomycin. In accordance with protocol, they were started on leucovorin rescue after sending their serum sample for methotrexate level. Results: The serum methotrexate levels were noted to be 8.7 and 7.2 lmol/L respectively. Both the patients subsequently developed grade IV mucositis. They were managed with intravenous leucovorin. At 72 h, serum methotrexate levels were noted to be \0.2 lmol/L. DTPA scan done 4 weeks later demonstrated a decreased GFR, which returned to normal at 12 weeks. Conclusion: Vancomycin may impair renal clearance of methotrexate and manifest with increased toxicity.

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Abstract No. 59 Clinical, Hematological, Molecular and Follow-Up Profile of BCR-ABL Positive Pediatric Acute Leukemias Prateek Bhatia, Deepak Bansal, Amita Trehan, Neelam Varma, RK Marwaha Post Graduate Institute of Medical Education and Research, Chandigarh Summary: Overall BCR-ABL positivity frequency in our pediatric acute leukemias was around 7 %. The p210 transcript positivity was more as compared to p190 and Imatinib treatment in addition to chemotherapy resulted in higher remission rates. Introduction: Data on frequency and profile of BCR-ABL positive pediatric acute leukemias from Indian sub-continent is limited. The present study highlights the clinico-hematological and molecular profile of BCRABL positive pediatric acute leukemias. Materials and Methods: Pediatric acute leukemia cases positive for BCR-ABL transcript by RT-PCR over a period of one year (2011–2012) were selected and their data analyzed from record files. Results: Of total 81 pediatric acute leukemia cases, 66 (81 %) were B-ALL, 12 (15 %) AML and 3 (4 %) MPAL. The BCR-ABL frequency noted was 6/81 (7.4 %), with 5/66 (7.5 %) positive B-ALL and 1/3 (33 %) positive MPAL cases. None of the AML case was BCR-ABL positive. Of 5 positive B-ALL cases, all were male with age range from 0.9 to 11 years. All cases had hepato-spleenomegaly, high WBC count ([50 9 109/L) and very low platelet count (\30 9 109/L) at presentation (p \ 0.05). All cases (100 %) showed aberrant myeloid marker expression (CD13 & 33). The BCR-ABL transcript type was p210 in 3/5 (60 %) cases, while p190 in 2/5 (40 %) cases. 4/5 (80 %) cases received Imatinib in addition to induction regimen. 3/4 (75 %) had rapid early response at D14 check marrow and 100 % complete hematological remission (CHR) at post induction. However, sustained CHR was noted in 1/4 (25 %) case only at 6 months follow-up. Two died of febrile neutropenia and sepsis post consolidation, while one had disease relapse and left further treatment. The 1 positive MPAL case was also male child and presented with high TLC and low platelet count. The immunophenotype was B/Myeloid (with 40 % MPO positivity). The BCR-ABL transcript was p210 (b3a2). The patient received Imatinib and was in CHR at 6 months follow-up. Conclusion: BCR-ABL frequency in our study is similar to that quoted in western studies. BCR-ABL positive acute leukemias usually present with organomegaly, high TLC and low platelet count and this association was significant in our study when compared with non-BCR-ABL positive cases. It is mandatory to look for expression of both transcripts (p210 & p190) as positivity for p210 was high in our study. Imatinib should be added to induction regimen as remission rates are higher, however long term outcome studies are needed.

Abstract No. 60 Clinical Profile of Relapsed Acute Lymphoblastic Leukemia Patients: A Single Center Experience Reashma Roshan, Punit Jain, Biju George, Auro Viswabandya, Aby Abraham, Abhijeet Ganpule, Alok Srivastava, Vikram Mathews Department of Haematology, Christian Medical College, Vellore Summary: A single center retrospective analysis (Jan 2005 to Dec 2010) of clinical, demographic and treatment parameters of relapsed acute lymphoblastic leukemia (ALL) patients is reported in this abstract. Introduction: Despite high cure rates, relapse is a significant hindrance in cure of ALL, more so in adult ALL. There is scarce data

299 from developing nations concerning pattern, determinants and clinical outcome following relapse. Materials and Methods: Retrospective analysis of 573 patients including 331 adult and 242 pediatric patients who were diagnosed as ALL was done. The diagnosis was based bone marrow examination and immunophenotyping was used to classify into B and T subtypes. The risk stratification was done as per conventional cytogenetic analysis and molecular markers. All the patients were treated on BFM based protocols. Pediatric patients (age \15 years) were treated with BFM Pediatric protocols as per the risk stratification, while the adult patients ([15 years) were treated with Adult GMALL protocol (data previously reported). Results: Of the 573 patients who were treated for ALL, 148 (25.8 %) patients relapsed. The median age of relapsed patients was 18.0 years (range: 1–67). The male/female ratio was 2.4:1. Out of 331 adult ALL patients, 94 relapsed (28.4 %). The median age of relapsed adult patients was 28.5 (range: 15–67) while male/female ratio was 2:1. Of these 94 patients, 69 (73.4 %) were standard risk while as 24 (34.78 %) were high risk at diagnosis. 69 (73.4 %) patients had very early relapse, 13 (13.8 %) early and 12 (12.8 %) had late relapse. Out of adult relapsed patients, only 15 (16 %) were treated with intensive chemotherapy post relapse while 74 (78.7 %) opted for palliative therapy. Out of 242 pediatric ALL patients, 54 relapsed (22.3 %). The median age of relapsed patient was 6 years (range: 1–14) while male/ female ratio was 3.5:1. The risk categorization as standard, intermediate and high was 13 (24.1 %), 34 (63.0 %) and 7 (13.0 %) respectively. There were 19 (35.2 %) very early, 24 (44.4 %) early and 11 (20.4 %) late relapses. Intensive curative intent chemotherapy was given to 17 (31.5 %) patients while 37 (68.5 %) opted for palliation. Conclusions: This abstract summarizes the clinical and demographic profile of relapse ALL patients. Unfortunately majority of the patients who relapsed, due to financial constraints, opt for palliation following a relapse.

Abstract No. 61 ‘‘Granular ALL’’, A Morphological Mimicker of Acute Myeloid Leukaemia Surekha, Gonmei Jenny, Kumar Vijay, Marwah Sadhna, AS Nigam, Buxi Gurdeep Department of Pathology, PGIMAR, Dr. RML Hospital, New Delhi Summary: A 40 years old female presented with complaints of Low grade intermittent fever since 2 months, Bone pains, decreased appetite and weight. Patient had mild hepatosplenomegaly and periportal lymphadenopathy. Introduction: Granular ALL (Acute lymphoblasic leukaemia) is a morphological mimicker of acute myeloid leukaemia due to presence of granules in the cytoplasm of the lymphoblasts. Granular ALL accounts for 2 to 8 % of all ALL cases in children but is very rare in adults. Materials and Methods: Peripheral smear, Cytochemistry-Myeloperoxidase and Periodic acid Schiff0 s (PAS). Flow cytometry was performed for further characterization. Results: Peripheral smear showed 48 % blasts with granules in 32 % of blast, Morphologically suggestive of myeloid subtype. Cytochemistry was performed and blasts were found to be negative for Myeloperoxidase and Periodic acid Schiff0 s (PAS). Flow cytometry was performed for further characterization and will be discussed. Conclusion: Granular CALLA positive B cell lymphoblastic leukemia (Granular’ ALL) is an important morphological entity and pose difficulty in distinction from myeloid differentiation. This may lead to an erroneous diagnosis of acute myeloid leukaemia particularly in adult age group. This case highlights the limitation of morphology on one hand and utility of flowcytometric immunophenotyping for characterization of blasts and correct diagnosis of leukaemia. It is pertinent for appropriate management of the patient.

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Abstract No. 62 Extra-Medullary Hematopoiesis and Leukemic Infiltration in Lymphnode in Adult ALL: A Rare Coexistence and Pitfalls in Diagnosis

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 leukemias are rare. The mechanism may be due to overwhelming immune response. In most cases the disease relapses and respond poorly to treatment.

Pradyumn Singh, Namrata Awasthi, Nuzhat Husain

Abstract No. 64

Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow

Posterior Reversible Encephalopathy Syndrome in a Child with Acute Lymphoblastic Leukemia

Summary: Extramedullary hematopoiesis in lymph node is well documented, however its co-occurrence with leukemic infiltration is extremely rare. Here we present this unusual case of adult ALL and discuss the diagnostic pitfalls on cytological examination as well as some other uncommon clinical and lab findings noted. Introduction: A 26 year old male presented with progressive debilitating illness of 4 months duration and back pain for past two days. Clinically, he had severe pallor, cervical and axillary lymphadenopathy, mild hepatosplenomegaly and tenderness over D10-12 vertebral region. Materials and Methods: Investigative work up was done to exclude lymphoproliferative disorder and tuberculosis. He was referred to us for FNAC of cervical lymphnode and complete blood counts. Bone marrow examination and immunophenotyping by flow cytometry were also done. Results: X-ray of spine showed compression fracture of D11 vertebra. Laboratory tests revealed pancytopenia with severe anaemia and leucoerythroblastic blood picture. Bone marrow examination showed features of Acute Leukemia. Immunophenotyping by flow cytometry revealed B-Acute Lymphoblastic Leukemia, CALLA positive with co-expression of CD13 and CD33. Conclusion: Uncommon presentations of adult ALL may pose diagnostic challenges both for the clinician as well as the pathologist. Documenting and discussing these would help in better understanding of the disease course and approach to correct diagnosis.

Abstract No. 63 Spontaneous Remission in Acute Lymphoblastic Leukemia—A Case Report S Venkatesan, S Venkatesan Department of Hematology, All India Institute of Medical Sciences, New Delhi-110029 Summary: Spontaneous remission in hematolymphoid malignancy is uncommon though not rare. Usually it is of short duration and in majority of patients the disease relapses. Here we report a case of acute lymphoblastic leukemia in which the spontaneous remission and subsequent relapse were documented. Introduction: Spontaneous remission is defined as the complete or partial disappearance of malignancy in the absence of chemotherapy. The remission is usually short lived and may be associated with infection and blood transfusion. Materials and Methods: 46 year old male presented with fever, malena, breathlessness and cough for two weeks. Ejection systolic murmer found in aortic area. Blood examination, blood culture and echocardiography were done. Results: Peripheral blood smear showed 20 % blasts. Blood culture grew Acinetobactor. Echocardiography revealed features of infective endocarditis. CT scan showed features suggestive of fungal pneumonia. Bone marrow aspirate revealed 90 % blasts with IPT features of B-ALL. In view of infections, chemotherapy was deferred and treated with antibiotics and antifungals. After two weeks there was normalization of blood counts and a repeat BMA revealed no increase in blasts compatible with remission. In view of spontaneous remission, close follow up was planned. After nine weeks, PS and bone marrow showed 80 % blasts. He was started on chemotherapy but succumbed to fungal pneumonia during induction. Conclusion: Spontaneous remissions in acute

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Nishad Dhakate, Sneha Tandon, Soniya Nityanand Department of Clinical Hematology, SGPGIMS, Lucknow Summary: Posterior reversible encephalopathy syndrome is a known complication in childhood leukemia. We report a case of acute lymphoblastic leukemia who presented with seizures, altered sensorium and visual hallucinations in the third week of Induction chemotherapy. Her blood pressure was normal. MRI showed white matter hyperintensity suggestive of PRES. She improved with antiepileptics & supportive care. Introduction: PRES is characterized by seizures, headache, altered mental status and typical transient lesions on neuroimaging. It is a known abnormality leading to seizures & altered sensorium in children with leukemia. Typical MRI findings include bilateral cortical and subcortical edema with a predominant posterior distribution. Management includes control of seizures & treatment of precipiting cause. Materials and Methods: 8 years female with ALL on induction chemotherapy presented with right focal seizures with secondary generalization followed by altered sensorium. Her blood pressure was 108/68 mmHg. She also had irrelevant talk, visual hallucinations & nystagmus. She was treated with antiepileptic drugs. CT head revealed multiple hypodensities in frontal, parietal and occipital region. MRI showed presence of multifocal areas in T2 and FLAIR cortical and subcortical white matter hyperintensity in the bilateral fronto-parietal, right occipital region and bilateral postero-medial thalami suggestive of PRES. (MRI Images available). Results: Patient showed complete clinical recovery and was discharged on antiepileptics. She was restarted on chemotherapy two weeks later. After 7 months of follow up patient is asymptomatic without any neurological sequelae & bone marrow is in remission. Conclusion: Awareness of PRES as a complication of childhood leukemia may prevent unnecessary diagnostic procedure & treatment.

Abstract No. 65 Refractory T Lymphoblastic Leukemia Presenting as Bilateral Breast Masses in an Adolescent Girl: A Case Report Nilanjan Sinha, S Kishore Kumar, Uttam Nath, SS Roy, Sambit Samanta, Prantar Chakrabarti, Utpal Choudhuri Institute of Haematology & Transfusion Medicine, Kolkata Summary: Hematological malignancies are considered to be the commonest reason for secondary deposits in the breast. Mostly NonHodgkins lymphoma’s infiltrate the breast followed by Acute myeloid leukemia. Involvement of bilateral breast with Acute T lymphoblastic leukemia (ALL) is a relatively rare occurrence. Introduction: This case highlights the rare extramedullary breast involvement in refractory T lymphoblastic leukemia along with clinical, immunophenotypical and cytogenetics features. Materials and Methods: A 16/female K/C/O Intermediate risk ALL on induction phase of BFM 2002 ALL protocol. About day 30 of her induction she developed frequent dry cough along with tenderness over the left breast. Over the next 3–4 days her symptoms progressed and she complained of nodular tender swellings over both the breasts. A well defined mass

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 was palpable in the lower quadrant of the left breast measuring about 5 9 4 cm and a deep seated ill defined tender mass was noted in the right breast. CT Thorax revealed extensive anterior mediastinal lymphadenopathy. Simultaneously she developed left UMN type of facial nerve palsy. CT brain was unremarkable but CSF examination revealed Blasts. Results: The Fine needle aspiration done from both the breast masses revealed sheets of blasts. These have been immunotypically similar to the initial bone marrow blasts except for aberrant expression of CD79a. The bone marrow done now shows less than 5 % blasts. This confirms the extramedullary involvement of both the breasts along with mediastinal lymph node and Central nervous system. Conclusion: This case signifies the fact that when adolescent girls with Acute lymphoblastic leukemia present with fibroadenoma like masses, in spite of benign radiological appearance and bone marrow remission status, its important to rule out extramedullary relapse of Leukemia

Chronic Myeloid Leukaemia (CML) Abstract No. 66 CD34+ CML Cells have Low hOCT1 RNA Expression and Truncated RNA But Does not Determine Response to Imatinib Therapy Sreeja Karathedath, Sukanya Ganesan, K Vinodhini, A Senthamizhselvi, MR Bharathi, Aby Abraham, Auro Viswabandya, Biju George, Alok Srivastava, Vikram Mathews, Poonkuzhali Balasubramanian Department of Haematology, CMC, Vellore Chronic Myeloid leukemia (CML) is a clonal malignancy of the hematopoietic system which renders the cells with constitutive tyrosine kinase activity. The tyrosine kinase inhibitor Imatinib has tremendously improved the treatment outcome in CML patients. Despite this therapeutic success, resistance and intolerance to Imatinib has hampered the clinical outcome. Overexpression of efflux transporters (ABCG2, ABCB1), decreased expression of influx transporters (hOCT1) and polymorphisms in these transporters and drug metabolizing enzymes have been shown to influence Imatinib therapy. The reduced expression and activity of hOCT1 in CML primitive progenitor population is also a marker of long term disease persistence. We evaluated the role of RNA expression and polymorphisms in Imatinib influx and efflux transporters in newly diagnosed patients with CML; ratio of influx to efflux transporters: dCt hOCT1/dCt ABCB1*dCTABCG2, referred to as resistance index (RI), was lower significantly in patients with MMR compared to those who did not achieve MMR. We developed a predictive model with RNA expression pattern and genotype that could identify CML patients who would achieve MMR on Imatinib therapy (ASH annual Meeting abstracts 2012-120: Abstract 2785). The expression status of these transporters in CML CD34+ population, a major contributor to disease progression, could predict the molecular response with better precision. The aims of the present study was to identify differential RNA expression of transporters hOCT1, ABCB1, ABCG2 in CML CD34+ population vs. total cellular expression and the potential association with molecular response to Imatinib. Bone marrow samples from Imatinib naı¨ve patients with CML in chronic phase (n = 50) was collected after informed consent. RNA from the total cells as well as the cells enriched for CD34 (EasySep, Stem cell technologies, Canada) was isolated by Trizol extraction followed by cDNA synthesis. The expression of ABCB1, ABCG2 and hOCT1 was identified by qRT PCR using Taqman gene expression assays and the normalization was done against GAPDH. The expression of these

301 genes in CD34+ and total CML cells was compared. There was a significant increased expression of efflux transporters ABCG2 (p = 0.001) and ABCB1 (p = 0.007) and decreased expression of hOCT1 (p B 0.001) in CD34+ fraction compared to the total cellular RNA. This reduction in OCT-1 mRNA was also observed in CD34+ cells derived from healthy donors (data not shown). To determine whether these parameters may be predictive of clinical responses to IM, RNA expression of these transporters in the CD34+ cells was compared with molecular response to Imatinib therapy. There was no significant association between RNA expression of these transporters and molecular response, though the number of samples for which the CD34+ cell fraction was available was very less (n = 50). We further tested if the decreased expression of hOCT1 in CD34+ fraction was due to the presence of short transcript of hOCT1 resulting in decreased functional activity and found that it is indeed true. We are currently evaluating the gene expression profiles of CD34+ fractions from patients with good or poor response to Imatinib therapy to predict a molecular signature of Imatinib response in CML.

Abstract No. 67 SLC22A1 (hOCT1) Genetic Variants Influence Molecular Response to Imatinib in Patients with Chronic Myeloid Leukemia MR Bharathi, Preetha Markose, K Vinodhini, Sukanya Ganesan, K Sreeja, A Senthamizhselvi, Aby Abraham, Auro Viswabandya, Biju George, Alok Srivastava, Vikram Mathews, Poonkuzhali Balasubramanian Department of Haematology, Christian Medical College, Vellore Targeted therapy in CML with tyrosine kinase inhibitor (TKI) Imatinib has resulted in significant improvement in outcome. However, resistance and intolerance to Imatinib have seriously limited the success of this therapy, with only a proportion of patients achieving major molecular response (MMR). It is important to identify predictors of response to Imatinib, since early switch to second generation TKI has been shown to improve outcome in nonresponders. Polymorphisms in the Imatinib influx and efflux transporter genes and drug metabolizing enzymes have been shown to influence Imatinib therapy. We evaluated the role of polymorphisms in Imatinib influx transporters hOCT1 (SLC22A1) on molecular response to Imatinib therapy in newly diagnosed patients with CML at our centre. In addition, we screened hOCT1 SNPs in normal healthy volunteers to compare the genotypic frequencies between CML patients and normal controls. From December 2009 till August 2012, 134 newly diagnosed CML patients with written informed consent and serial follow-up were included in the study. BCR-ABL transcript levels were monitored in CML patients at diagnosis, 3, 6, 9, 12, 18, 24 and 30 months after start of Imatinib. Molecular response to Imatinib after a minimum of 12 months was calculated using the ratio of BCR-ABL to ABL transcript expressed in International scale. Patients were classified to have MMR (BCR-ABL/ABL \0.1) or no MMR (BCR-ABL/ABL [0.1) at around 12 months from the start of Imatinib therapy, and resistant (BCR-ABL/ABL [10; no cytogenetic response even after Imatinib dose was increased), or intolerant (severe cytopenia or skin toxicity requiring frequent dose reductions). All coding exons of hOCT1 gene were screened using genomic DNA samples by PCR followed by direct sequencing. SNPs were identified using SeqScape software. We identified nine coding nonsynonymous variants including a novel exon2 variant Gly-165-Cys and 5 intronic variants in SLC22A1 upon sequencing. The genotype frequencies of the polymorphisms in hOCT1 in CML patients and healthy volunteers were comparable and are given in Table. A in/del polymorphism in intron 7, which was in complete linkage disequilibrium with the exon

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6 coding variant (rs628031) results in a splice variant due to aberrant truncation. Both patients and normal controls with this polymorphism in the mutant state did not produce full length hOCT1 transcript while the heterozygotes showed both truncated and full length transcripts and the wild type did not show the truncated transcript upon RT-PCR. Out of the 134 patients, 56 achieved MMR, 43 did not achieve MMR; and 19 were intolerant to Imatinib; 16 patients progressed. We then compared the influence of SNPs in candidate genes with molecular response to Imatinib. Among these SNPs, Pro341Leu (rs628031) in exon 6 (p = 0.015) and -TGA del polymorphism resulting in frame shift in exon 7 (p = 0.02) were significantly associated with decreased molecular response. None of the screened SNPs were particularly different in the intolerant patients. Further studies are ongoing to compare the Imatinib pharmacokinetics in relation to the hOCT1 genetic variants. Our study suggests that hOCT1 genetic polymorphisms may contribute towards inter-individual variations in response to Imatinib treatment in CML patients.

SNPs

Genotype frequency in CML

Genotype frequency in normal controls

wt

wt

het

het

mt

mut

rs1867351T[C

84

43

7

66

30

3

rs12208357C[T

124

9

1

95

4

0

rs683369C/G

whereas chronic-phase CD34+ cells migrated normally to this chemokine. Down-regulation of CXCR4 may have important implications in chronic myelogenous leukemia pathogenesis. Materials and Methods: PB/BM sample from CML patients in chronic, accelerated and blast crisis phase were collected in EDTA vial and processed within 24 h for flow cytometry. Monoclonal antibodies combination of CD45 (PerCP-Cy5.5)/CD34 (PE-Cy7)/CD38 (APC)/ CXCR4 (PE) were applied in each of the patients for flow cytometric assessment. CXCR4 expression was analyzed by sequential gating strategy as CD45+/CD34+/CD38-/CXCR4+ in the sample processed through direct stain, Lyse and wash method. Percentage CXCR4 expression as well as their MFI was interpreted in BD-FACS Diva software. Results: A total of 40 CML patients (26 males and 14 females), median age 40 years (Range 20–60 years), Chronic phase (n = 28) accelerated phase (n = 6) blast crisis (n = 6) were enrolled in this study. Progenitor cells from newly diagnosed CML-CP patients expressed markedly high CXCR4 levels on cell surface. Although, the reactivity of CXCR4 was variable among samples, there was no significant difference between chronic phase CML and accelerated phase, CD34+ CD38-cells (P \ 0.4). In contrast, level of CXCR4 was constantly lower in blastic-phase CML CD34+ CD38cells compared with chronic phase CML CD34+ CD38-cells (P B 0.02). Conclusion: In CML—Blast crisis, high p210 BCR-ABL can induce a transcriptional silencing of CXCR4, leading to abnormal chemotaxis that result in the release of blast cells in the blood and the colonization of non hematopoietic tissues.

97

32

4

57

38

3

rs201942835G[T

121

12

0

93

4

0

rs4646277C/T

133

0

0

99

0

0

rs4646278C[T

131

2

0

98

1

0

rs77092743G-[A

92

38

3

86

10

0

Megaloblastoid Erythropoiesis in Imatinib Treated Chronic Myeloid Leukemia (CML) Patients—Does It Have any Significance?

rs7762846C/T TGTTCCGCA(c[t) GCCGCGCC-

108 132

22 1

3 0

83 96

16 3

0 0

T Roshni Paul, Shantveer G Uppin, Megha S Uppin, Ashwani Tandon, Rachel T Jacob, D Raghunadha Rao*

rs2282143C/T

92

38

3

86

13

0

rs628031A/G

14

55

64

18

52

29

rs72552763-/GAT

1

35

97

74

21

4

rs4646281

14

55

64

17

51

31

rs9457843C[T

47

8

2

38

7

0

Abstract No. 68 P210 BCR-ABL Transcript Load Down Regulates CXCR4 Expression in CML Blast Crisis Sunita Chhikara, Sudha Sazawal, Rahul Sharma, Rekha Chaubey, Ravi Kumar Pravas Mishra, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi Summary: Chronic myelogenous leukemia (CML) is driven by constitutively activated Bcr-Abl tyrosine kinase, which causes the defective adhesion of CML cells to bone marrow stroma. The overexpression of p210 Bcr-Abl was reported to down-regulate CXCR4 expression, and this is associated with the cell migration defects in CML—Blast crisis patient. Introduction: p210BCR-ABL significantly impairs CXCR4 signaling. The overexpression of p210Bcr-Abl was reported to down-regulate CXCR4 expression, in blast crisis,

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Abstract No. 69

Nizam’s Institute of Medical Sciences, Hyderabad Summary: Megaloblastoid erythropoiesis is rarely reported in Imatinib treated CML patients. In 2.3 % of post-Imatinib treatment CML patients, megaloblastoid erythropoiesis was noted. As few patients had progressive disease, there is a possibility that this megaloblastosis is part of dyspoietic changes before transformation or conversion to MDS/acute leukemia. Introduction: Imatinib mesylate is the drug of choice for CML. Megaloblastosis was reported in patients on hydroxyurea, however is rare with Imatinib. Whether the megaloblastoid change is a part of nutritional deficiency or a possible forerunner of a myelodysplasia following Imatinib remains to be elucidated. Nevertheless, megaloblastosis as a part of MDS with further progression to AML in Ph negative CML has been reported in literature. We have tried to concentrate on the megaloblastoid change in treated CMLs for its possible explanation. Materials and Methods: Cases of CML treated with Imatinib, showing marrow megaloblastosis were retrieved from Hematopathology record files (Pathology Department, NIMS-4 year study). The morphology was correlated with the clinical details, cytogenetics & molecular studies; and follow-up obtained from Medical Oncology records. Results: Of 1,515 post-therapy CML marrows, 35 (2.3 %) showed megaloblastosis. The patients ranged from 16–67 years (median age-37 years) and a male:female ratio of 1.7:1. Vitamin B12 and folate levels were available in only one patient (high); not done in others. Seven patients were previously treated with Hydroxyurea. Nineteen patients had progressive disease in the form of accelerated and blast crisis. Additional cytogenetic abnormalities like Trisomy 8, 19, monosomy 7, additional Ph chromosome and hypoploidy were also noted. Conclusion: Patients with megaloblastoid change should be put on

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 close follow-up, as there may be a risk of progression. In Indian settings an underlying nutritional deficiency; however has to be ruled out.

Abstract No. 70 Automated Hematology Analyzer: Derived Neutrophil Population Indices Distinguish Chronic Myeloid Leukemia from Reactive Neutrophilic States Balan Louis Gaspar, Prashant Sharma, Neelam Varma, Reena Das, Ishwar Bihana, Sudershan Kumar Sharma Department of Hematology, Level 5, Research Block A, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012, India Introduction: Leukocyte volume, conductivity and scatter (VCS) data were analyzed with an aim to develop software-based flagging algorithms to distinguish chronic myeloid leukemia (CML) from reactive neutrophilic (leukemoid) states. Materials and Methods: Blood count and VCS data from four patients groups were obtained from LH750 automated hematology analyzers (Beckman Coulter Inc.). Group 1: CML patients, either newly diagnosed (n = 38) or on Imatinib therapy (n = 65); Group 2: patients with reactive neutrophilia (n = 58); Group 3: pregnant women (n = 100); and Group 4: healthy controls (n = 99). Receiver-operating-characteristic (ROC) curves was plotted to compare the diagnostic efficiencies of VCS parameters and their combinations using MedCalc software (v.12.7.0). Areas-under-curve were compared. Results: Patients with CML displayed significantly greater mean neutrophil and monocyte volumes (MNV and MMV) and higher mean lymphocyte scatters (MLS) along with increased variability in neutrophil volume and conductivity (NE-V-SD and NE-C-SD) and in lymphocyte conductivity (LY-C-SD) (p \ 0.0001 for all). Parameters most successful in distinguishing untreated CML from reactive neutrophilia were MNV (AUC 0.956, sensitivity 89.5 %, specificity 90.6 %), NE-SD-C (AUC 0.989, sensitivity 94.7 %, specificity 95.6 %) and MLS (AUC 0.983, sensitivity 94.7 %, specificity 94.0 %). A combination of MNV [163.0 + NE-SD-C [12.69 showed 89.5 % sensitivity and 100 % specificity for CML. Both parameters declined post-therapy to reactive neutrophilia ranges. Conclusion: VCS indices (MNV, NESD-C and MLS) accurately distinguish CML from reactive neutrophilia. Admixture with immature granulocytic precursors in the neutrophil region is likely to cause altered MNV and NE-SD-C while basophilia might result in increased lymphocyte scatter. Decline of VCS indices after therapy warrants their further study as tools to monitor CML patients.

Abstract No. 71 Varieties of CML at Chromosomal Level Bani Bandana Ganguly1, Nitin N Kadam1, Shouvik Mandal1, MK Ghosh2, M Bhattacharyya2, TK Dolai2, D Banerjee3, S Chandra3, MB Agarwal4 1 MGM Center for Genetic Research & Diagnosis, MGM Institute of Health Sciences, Navi Mumbai 410209, India, 2Hematology Department, NRS Medical College, Kolkata, India, 3Clinical Hematology Services, Kolkata, India, 4Hematology Department, Bombay Hospital & Research Centre, Mumbai, India

Summary: Variant translocations, additional acquired translocations and constitutive rearrangements have been detected in 20 Ph+ CML following G-banding and FISH techniques. Additional acquired and

303 genomic aberrations were differentiated through PHA-stimulated blood culture. Constitutive aberrations have been confirmed retrospectively, which was important for family members for genetic counseling. Half yearly and annual follow up study of these cases presented secondary clones and poor or no response to Imatinib. Involvement of extra aberrations might have played a different mechanism to Imatinib action. The study highlights the importance of molecular investigation of such complex situation and pharmaceutical alteration of tyrosine kinase inhibitors (TKI) accordingly. Introduction: Involvement of a third or fourth chromosome has been reported in 5–10 % of CML. The presence of additional cytogenetic abnormalities is rarely reported in CP-CML and has merely been discussed for prognostication. However, management of leukemia varies considerably with the involvement of additional chromosome(s). The effect of the alteration in third chromosome and its fusion with der(9) in CML and/or clonal expansion has not yet been understood due mainly to limited cases registered. Materials and Methods: Bone marrow samples were processed for conventional and FISH studies. Result: Three-way translocations, involving a variable third chromosome in 10 cases; additional abnormalities, including (4;11), t(7;22), t(8;17), etc.; and constitutive aberrations, including t(1;12), inv(9), t(14;X)etc. have been detected in addition to Phchromosome. Conclusion: Additional acquired or de novo aberrations lead to poor prognosis in Ph+ CML. Hence, molecular mechanism in such situation needs to be understood for a different formulation of tyrosine kinase inhibitors.

Abstract No. 72 Monitoring in Chronic Myeloid Leukaemia (CML) Patients with Stem Cell Transplantation (SCT) by Quantitative Real Time PCR on the International Scale: Our Experience in Three Cases of Post Stem Cell Transplant Patients in a Tertiary Care Centre in India BK Chakrabarty, P Rout, J Kotwal, R Kapoor, V Nair, V Dutta Armed Forces Medical College, Pune Summary: Regular serial monitoring in Chronic Myeloid Leukaemia patients with stem cell transplantation (SCT) is now recommended by quantitative real time PCR on the International Scale (IS) for better sensitivity and redefining criteria’s of molecular response. Introduction: In Imanitib era there is rapid decline in the number of allogenic-SCT for CML. However relapse remains an important clinical problem in transplanted patients, therefore serial Bcr-Abl RQPCR monitoring is recommended. Materials and Methods: The tests done by using Ipsogen BCR-ABL Mbcr kit containing IS-MMR calibrator, aligned with (IS) and single plasmid standards for both BCR-ABL and ABL. Three post SCT patients were monitored. Results: Initially patients were monitored by RQPCR using M BCR fusion Quant kit. The sensitivity of test is 4 log reduction (0.01 %). Tests showed two patients in CMR and one patient, who was on TKI had relapsed with Bcr-abl/abl transcript ratio 0.30 %. As the sensitivity of new test has been increased to 5 log reduction (0.001 %) we found one of our earlier CMR cases detected to have Bcr-abl/abl transcript ratio 0.0025 % and one continued in CMR. Relapsed case had transcript ratio 0.287 %. Conclusion: As per review of literature ‘Complete Molecular Response’ (CMR 5.0 C5 log reduction from baseline; B0.001 %IS) is now termed as ‘Molecular Response’ (MR 5.0 C5 log reduction from baseline; B0.001 %IS). Therefore the earlier ‘CMR case’, now being reported as ‘In major molecular remission as per sensitivity of the RQ PCR’ as better sensitivity of kit changed the CMR status. The patient is not considered as ‘relapse’ but advised to undergo regular monitoring for better predictive outcome.

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Summary: This study evaluates the hematologic response to Imatinib mesylate in CP-CML patients, documenting sequential hematologic parameters (peripheral blood and bone marrow) at initiation of therapy and during prolonged therapy ([6 months), adverse drug reactions and calculate Hasford score for outcome of CP-CML on Imatinib. Introduction: Chronic myeloid leukaemia (CML) is a clonal hematopoietic stem cell disorder characterised by reciprocal translocation between chromosome 9 and 22, generating bcr-abl fusion protein having constitutive tyrosine kinase activity. The tyrosine kinase inhibitor, Imatinib mesylate has become the prototype of molecular targeted therapy in the treatment of CML. Materials and Methods: The study sequentially followed up 75 Ph+ CML-CP patients, receiving 400–600 Imatinib over a period of 18 months. Hematologic parameters at admission, 2 weeks, one month, 3 months, 6 months, 12 months and the morphologic changes in bone marrow aspirate and biopsy were evaluated at admission, 6 and 12 months of treatment in accordance with NCCN guidelines. Results: In post therapy patients complete hematologic response (CHR) was at one month (47.1 %), 3 months (80 %), 6 months (85.4 %), 12 months (90.4 %). Bone marrow aspirates and biopsies showed reduction in cellularity and myeloid precursors with regeneration of erythroid precursors, decrease in myelofibrosis (p value \ 0.04). Anemia was most common hematologic adverse effect (80 %) and edema (37.5 %) and musculoskeletal pain (37.5 %) most common non hematologic adverse effect. Hasford score decreased with low score in 90.4 % patients at 6 months and 85.7 % patients at 12 months. Conclusion: Imatinib is a safe and effective first line therapy for CP-CML patients especially with respect to myelofibrosis, reducing its grade and preventing its development if treatment is started early in course of CP-CML. It downgraded the Hasford score indicating adequate response and better overall progression free survival. It is generally a well-tolerated drug, the adverse effects are generally mild not requiring discontinuation of therapy.

levels and molecular response. We aimed to determine correlation of plasma Imatinib and its metabolite desmethyl Imatinib levels as well as intracellular Imatinib levels (after ex-vivo incubation of primary CML cells with Imatinib) with molecular response in CML. Imatinib naı¨ve CML patients (n = 135) treated with standard dose of Imatinib with serial follow-up after Imatinib therapy were included in this study [Chronic phase (n = 117), Accelerated phase (n = 12), Blast crisis (n = 6)]. After obtaining informed consent, peripheral blood samples were collected from patients 24 h post first Imatinib dose and on Day 28 (steady state). Imatinib and desmethyl Imatinib levels were measured using HPLC with UV detection. Molecular response to Imatinib was measured by RQ-PCR for BCR-ABL fusion transcript from peripheral blood samples collected once in three months with a minimum follow-up of 12 months post therapy. The results were expressed in the International Scale (Poonkuzhali et al., Acta Haematol, 2012). The median plasma concentration of Imatinib and its metabolite desmethyl Imatinib at 24 h was 593 ng/mL (74–2,177) and 86 ng/mL (12–609) while on Day 29 it was 1,072 ng/mL (106–3,694) and 206 ng/mL (31–613) respectively. Based on therapeutic response after 12 months, patients were divided into complete molecular response [CMR (n = 17)], major molecular response [MMR (n = 40)], no molecular response (n = 42); in addition, patients were classified as intolerant (n = 20) and those with disease progression (n = 16). The plasma Imatinib concentration (24 h) was significantly higher in patients who achieved CMR/MMR when compared to those with no molecular response (P = 0.02) and intolerance. Interestingly, desmethyl Imatinib (Day 29), but not Imatinib levels were significantly high in the intolerant group than those who achieved CMR/ MMR (P = 0.02). In addition, desmethyl Imatinib levels in the intolerant group is higher when compared with No MMR group though not reaching statistical significance. Comparison of the intracellular Imatinib levels, though statistically insignificant, displayed increasing median values in the three groups (CMR \ MMR \ no molecular response). When plasma Imatinib levels were further divided into quartiles, higher proportion of patients who achieved CMR/MMR were in the highest quartile suggesting that higher the level of plasma Imatinib concentration, higher is the probability of obtaining better response. Though the number of patients included is small, this study suggests that monitoring plasma and intracellular Imatinib levels will help predict treatment outcome as well as timely dose increase in Imatinib to improve clinical response.

Abstract No. 74

Abstract No. 75

Association of Plasma and Intracellular Imatinib Levels with Molecular Response in Patients with CML

Six-Year Follow-Up of Patients with Imatinib-Resistant or Intolerant Chronic-Phase Chronic Myeloid Leukemia (CML-CP) Receiving Dasatinib

Hematologic Response to Imatinib in Patients with Chronic Phase CML Neha Chopra, Usha Rusia Department of Pathology, University College of Medical Sciences, Delhi, India

Sukanya Ganesan, Preetha Markose, Savitha Varatharajan, Ezhilpavai Mohanan, Vinodhini Kumaraswamy, Senthamizhselvi Anandan, Auro Viswabandya, Biju George, Alok Srivastava, Vikram Mathews, Poonkuzhali Balasubramanian Department of Haematology, Christian Medical College, Vellore Targeted therapy with Imatinib mesylate, a selective inhibitor of BCR-ABL kinase activity, has revolutionized the treatment response and survival in patients with chronic myeloid leukemia (CML). Therapeutic drug monitoring (TDM) for Imatinib has been suggested to improve efficacy, assess compliance, and evaluate drug-drug interactions. Imatinib steady-state plasma levels, measured following the first month of treatment with the standard 400-mg/day dose, has been shown to be correlated with long-term complete cytogenetic and molecular responses, as well as long-term event free survival (EFS). There is limited report from India on the association between Imatinib

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S Shankar, NP Shah1, H Kantarjian2, DW Kim3, A Hochhaus4, G Saglio5, F Guilhot6, C Schiffer7, JL Steegmann8, H Mohamed9, D Dejardin10, D Healey11, J Cortes2 Bristol-Myers Squibb India Pvt Ltd. Objective: This ongoing study in patients with Imatinib-resistant/intolerant CML-CP provides results of the longest follow-up (6 years) of a second-generation BCR-ABL inhibitor. Materials and Methods: 670 CP-CML patients with resistance, intolerance or suboptimal response to Imatinib were randomized to dasatinib 100 mg once daily (QD), 50 mg twice daily (BID), 140 mg QD, or 70 mg BID. Results: After a minimum follow up of 6 years, 31 % remained on study therapy with Progression-free survival (PFS) of 49 %, overall survival (OS) of 71 and 6 % transformation rate to advanced disease for patients receiving Dasatinib 100 mg QD (n = 165). In a landmark analyses, 6-year PFS & OS (64 & 83 %) were higher in patients

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 whose BCR-ABL B10 vs. 26 & 59 % respectively for patients with BCR-ABL [10 % at 3 months irrespective of the presence or absence of mutations and baseline responses(Partial Cytogenetic Response and Complete Hematologic Response). For dasatinib 100 mg QD, nonhematologic adverse events (AEs; all grades) generally first occurred in \24 mos with cumulative incidence of headache (47 %), diarrhea (41 %), fatigue (37 %), and pleural effusion (25 %). Incidence of grades 3/4 neutropenia and thrombocytopenia were 36 % & 24 % respectively with Dasatinib 100 mg QD. These generally first occurred in \12 mos. AEs were managed by dose/schedule modifications. Conclusion: After a Six-yr followup, Dasatinib 100 mg QD was well-tolerated in the second line setting with low rates of transformation to advanced phases and high rates of PFS and OS.

Abstract No. 76 Dasatinib v Imatinib in Newly Diagnosed Chronic Myeloid Leukemia in Chronic Phase (CML-CP): DASISION 3-Year Follow-Up

305 kinase induced signal transduction by Imatinib mesylate can lead to hematological remissions. ATO appears to sensitize ph+ cells to activity of Imatinib mesylate. Materials and Methods: Thirty adult patients of CML in accelerated/blast phase were given Imatinib mesylate at a dose of 400 mg/d and arsenic trioxide 0.15 mg/kg/d for 15 days and after that Imatinib was continued in the same dose for 6 months. Complete hemogram, bone-marrow, BCR-ABL % levels and all routine investigations including chest X-ray, ECG, B. urea, S. creatinine, B. sugar, s. electrolytes and liver function tests (SGOT/ SGPT, S. Bilirubin) were estimated weekly for first 15 days and monthly thereafter. Hematological response was assessed at 1 month and 6 months. Results: Thirteen (43 %) cases achieved a complete hematological response whereas 15 (50 %) cases had incomplete hematological response at the end of 6 months of Imatinib therapy. Two cases expired during the study. There was significant regression of liver and spleen size. Significant improvement was also seen in hemoglobin values. A significant reduction in blast, TLC and BCRABL% was seen in 13 cases after treatment. Conclusion: ATO+ Imatinib Combination therapy can be used in refractory or nonresponsive cases to Imatinib therapy alone, in CML accelerated or blast phase.

S Shankar Bristol-Myers Squibb India Pvt Ltd. Objective: We update the results of 3 year follow up of DASISION trial. Materials and Methods: Patients were randomized to receive dasatinib 100 mg once daily (QD; n = 259) or Imatinib 400 mg QD (n = 260). Results: At a minimum follow-up of 36-months; MMR (BCR-ABL B0.1 %) 68 vs. 55 %, P \ 0.0001; MR4 (BCRABL B0.01 %) 35 vs. 22 %, P = 0.00635; MR4.5 (BCR-ABL B0.0032 %) 22 vs. 12 %, P = 0.00069 were higher for dasatinib v Imatinib. MMR rates were higher for Dasatinib v Imatinib in all Hasford risk groups (high 61 vs. 43 %; intermediate 65 vs. 57 %; low 83 vs. 65 %). In an ITT analysis, fewer Dasatinib patients (n = 11; 4.2 %) transformed to advanced phase v Imatinib (n = 16; 6.1 %). 36-month OS (93.7 vs. 93.2 %) and PFS (91.0 vs. 90.9 %) for dasatinib v Imatinib were similar. 84 % patients achieved B10 % BCRABL within 3 months of Dasatinib initiation vs. 64 % with Imatinib. Exploratory analysis revealed that achieving B10 % BCR-ABL within 3 months of either TKI treatment correlates to higher PFS and OS at 3 years. Few additional adverse events (AEs) were reported between 24 and 36 months in both arms. For dasatinib v Imatinib, 29 vs. 31 % discontinued treatment for drug-related AEs (11 vs. 6 %), progression (7 vs. 7 %), failure (3 vs. 5 %), unrelated AEs (2 vs. \1 %), death (2 vs. \1 %), and other (5 vs. 10 %). Conclusion: Pts receiving dasatinib had a lower transformation rate and higher molecular responses v pts receiving IM, supporting the use of dasatinib in newly diagnosed CML-CP.

Abstract No. 77 Role of Arsenic Trioxide in Accelerated/Blast Phase in Chronic Myeloid Leukemia Patients Refractory to Imatinib Therapy

Abstract No. 78 Radioiodine and CML: Is There any Relation? Akhil Jain, Apurva Patel, Asha Anand, Sandip Shah, Shailesh Talati, Harsha Panchal, Sonia Parikh, Bhavesh Parekh, Shilin Shukla Department of Medical and Pediatric Oncology, The Gujarat cancer & Research Institute, Ahmedabad Introduction: Increased risk for occurrence of second primary malignancy had been described after exposure to radioiodine for the treatment of thyroid carcinoma. Majority of these are solid malignancies and development of leukemia is a rare event. Leukemias have been reported with the incidence of 2 % after 131I therapy. Acute leukemia constitutes the majority of cases. Though the casual relationship is questionable, case reports do exist for the patients developing chronic myeloid leukemia (CML) after radioiodine treatment. Case Report: We report a case of 27 year old female patient who was treated with total thyroidectomy and 110 mCi therapeutic 131I radioiodine for papillary thyroid carcinoma. After a period of 28 months patient developed splenomegaly with TLC of 2.5 lac. Bone marrow examination revealed chronic myeloid leukemia under chronic phase and FISH was positive for BCR-ABL fusion. Presently patient is on Imatinib therapy. Discussion: Many authors have reported cases depicting development of leukemia in patients treated with radioiodine. Wang and colleagues (2005) reported a case of CML after 131I treatment. CML can develop from as early as 10 months to the longest reported period of 13 years with the dose ranging from 30 mCi to 850 mCi. Our patient was exposed to 110 mCi 131I and developed CML after 28 months of exposure

PS Ghalaut, J Ahuja, R Singh, S Goyal, M Sharma Pt B D Sharma, PGIMS, Rohtak Summary: Thirty adult patients of CML in accelerated/blast phase, refractory to Imatinib therapy alone, were given Imatinib and arsenic trioxide and were followed for 6 months and it was found that this combination is effective with complete hematological response in 43 % patients. Introduction: Arsenic trioxide induces apoptosis in BCR-ABL + cell lines and reduces the proliferation of CML blasts but not of CD34+ progenitors. Inhibition of BCR-ABL tyrosine

Abstract No. 79 A Case of Imatinib Resistant CML with Bcr Abl Kinase Domain Mutation Megha Pradipbhai Shah, Krishna Patel, Killol Desai, Mustafa Ranapurwala, Keyuri Patel, Menka Shah M. Patel Centre for Medical Care and Education, Pramukhswami Medical College, Shree Krishna Hospital, Karamsad-388325, Gujarat

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306 Summary: Resistance to Imatinib mesylate caused by kinase domain mutation is common in patients with Chronic Myeloid Leukemia (CML). We describe a 66 years old female with CML on Imatinib, who developed resistance on prolonged treatment, in whom BCRABL kinase domain mutation has been detected. Introduction: Imatinib, a targeted tyrosine kinase inhibitor is popular for treatment of CML. Treatment with Imatinib sometimes fail due to drug resistance—primary and secondary. Most common mechanism of resistance is mutation in BCR-ABL kinase domain. Materials and Methods: A 66 years female with CML, was on treatment with Imatinib. BCR-ABL kinase mutation is detected by sequencing based test from peripheral blood. Results: BCR-ABL1 kinase domain mutation detected. The mutation detected is homozygous E255K. Conclusion: Patient of CML, having treatment failure, relapse or sub optimal response to Imatinib therapy should undergo BCR-ABL kinase domain mutation analysis, as resistance to Imatinib due to this mutation is currently emerging.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Introduction: CML is often discovered incidentally in the chronic phase, when CBC reveals leucocytosis or splenomegaly is found on general examination. Splenomegaly is the most common physical finding in CML patients. However, absence of splenomegaly does not rule out a diagnosis of CML as is seen in this case. Case report: A 50 year old female patient presented with fever, orbital cellulitis, erythematous rash and neutrophilic leucocytosis with shift to left. A NAP score was performed which was found to be low. Cytogenetics showed positivity for Philadelphia chromosome. On the basis of history, clinical examination, complete blood count, peripheral blood smear, bone marrow examination and biopsy, NAP score and cytogenetics, patient was diagnosed as CML. The patient is on follow up. Conclusion: This case highlights that high suspicion at the pathologist’s end based on peripheral blood findings can help in diagnosing CML cases even in absence of clinical suspicion.

Abstract No. 82 Abstract No. 80 Extramedullary Blast Crisis in Chronic Myeloid: A Report of Two Cases Parul Sobti, Annapurna Saxena, Shipra Aggarwal, Tejinder Singh, Naresh Gupta Department of Pathology and Medicine, Maulana Azad Medical College, New Delhi Summary: We present two cases of EMBC in cases of CML one of which was in remission on Imatinib therapy. Subsequently both developed BC in other organs despite aggressive therapy. Clinical and biological characteristics of EMBC in patients in remission need further study. Introduction: The occurrence of extramedullary blast crisis (EMBC) in patients in chronic phase of chronic myeloid leukemia is rare. The most common sites are lymph nodes and meninges, with other less common sites such as orbit, skin, tonsils and pleura. Materials and Methods: Case 1: A 27 year old male patient a known case of CML in remission on Imatinib therapy presented with hepatosplenomegaly and lymphadenopathy. Lymph node was effaced by sheets of blasts, which stained with myeloperoxidase. Subsequently he developed deposits in spleen and meninges and succumbed to the disease. Case 2: A 35 year old male presented with multiple skin coloured nodules all over the body for 2 months. Skin biopsy revealed a dermis infiltrated by sheets of medium sized blasts. Hematological examination revealed chronic myeloid leukemia. Lymph node revealed infiltration by blasts. The patient was started on methotrexate and daunorubicin therapy. Results: Extramedullary blast crisis in patients on Imatinib therapy is a new entity. The above cases presented with BC in lymph node, meninges, skin and spleen. Both were given aggressive treatment with methotrexate and daunorubicin. Conclusion: BC can present in patients in haematological remission, complete cytogenetic response or major molecular response. The presentation of BC can vary and requires aggressive management. Treatment protocols for these patients need further evaluation.

Childhood CML: A Case Report and Review of Literature Manveen Kaur, Anshu Palta, Kana Ram Jat Government Medical College and Hospital, Sec-32, Chandigarh Summary: Chronic Myeloid Leukemia is a clonal stem cell disorder characterised by the acquisition of an oncogenic BCR/ABL fusion protein, usually resulting from a reciprocal translocation (9; 22). It is a very rare disease in children and adolescents. Introduction: CML in children and adolescents accounts for \10 % of all cases of CML and 2–3 % of all leukemias. The annual incidence is 1 case per million children. The presenting features include asthenia (50–60 %), symptoms related to splenomegaly (20–30 %), weight loss (15–20 %) and bleeding (10 %). The total leucocyte count is generally higher than seen in adult patients, with a median WBC count of 2.42 9 106/ ll at the time of diagnosis. Materials and Methods: A 12 year old female presented with complaints of lethargy and abdominal distension since two months. Systemic examination revealed splenomegaly with a span of 20 cm, firm in consistency. Hemogram findings were: Hb-9.8 g/dl, TLC-2, 25,000/ll, platelet count-5.5 9 105/ll. DLC showed shift to left including 29 % myelocytes, 16 % metamyelocytes and 7 % blasts. In view of these findings with high suspicion of CML, bone marrow examination and bilateral trephine biopsies were advised. Results: Bone marrow examination showed hypercellular marrow with 9 % blasts and M: E ratio of 25:1. Trephine biopsy showed hypercellular marrow spaces with preponderance of myeloid series showing cells in varying stages of maturation including few scattered immature cells along with increased reticulin (3+). The diagnosis of CML was confirmed by presence of Philadelphia chromosome by cytogenetics. Conclusion: In children presenting with splenomegaly and the above mentioned complaints, a diagnosis of CML should be considered.

Myeloproliferative Neoplasms (MPN) Abstract No. 83

Abstract No. 81 An Unusual Case of CML

Vascular Endothelial Growth Factor Expression and Its Correlation with Angiogensis in Chronic Myeloproliferative Neoplasms

Palak Agarwal, Gurdeep Buxi, Sadhna Marwah, AS Nigam, Vijay Kumar

Amita Jain, Tejinder Singh, Nita Khurana, Naresh Gupta

PGIMER, Dr. Ram Manohar Lohia Hospital, New Delhi

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

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Summary: A prospective study was done in 2010–2012 on 40 cases of myeloproliferative neoplasms. Mean VEGF-IRS score, percentage of cells expressing VEGF, intensity of staining and angiogenesis (as assessed by Microvessel Density) were increased in the cases studied as compared to the controls. Introduction: Malignant hematological disorders are dependent on vascular support in bone marrow for their growth and proliferation. The extent of angiogenesis in such tumours demands for a delicate equilibrium between antiangiogenic and proangiogenic factors. A prospective study was carried out to assess the expression of VEGF (vascular endothelial growth factor), the degree of angiogenesis in cases of MPN (myeloproliferative neoplasms) and the correlation between the above stated parameters. Materials and Methods: The study included 40 newly diagnosed cases of MPN (myeloproliferative neoplasms) as per WHO classification (2008). Bone marrow biopsies were taken from all patients. Microvessel density (using CD34) was calculated as suggested by Weidner et al. VEGF expression was studied by immunohistochemistry and IRS (Immunoreactive score) was calculated as per Remmele et al. Serum VEGF levels were also estimated in 25 cases by ELISA. Results: Mean VEGF-IRS score, percentage of cells expressing VEGF, intensity of staining and MVD were increased in all 40 cases of MPN compared with controls, though no correlation was found between the VEGF expression and MVD. Multivariate analysis was carried out and significant correlation was observed between MVD, percentage of cells expressing VEGF and serum VEGF levels in 25 cases of MPN. Conclusion: VEGF contributes to endothelial cell proliferation in cases of MPN. Larger number of MPN cases and for a longer duration need to be followed up to evaluate the exact impact of drugs on neoangiogenesis in these cases. An urgent need is felt for cases resistant to tyrosine kinase inhibitors, whether addition of antiangiogenic drugs will be of benefit in this scenario.

Abstract No. 84 Polycythemia Vera: Correlation of JAK2V617F Mutation with Clinicohematologic Characteristics: A Single Tertiary Care Centre Experience Deepti Mutreja, S Sazawal, S Chhikara, M Mahapatra, P Mishra, T Seth, R Saxena All India Institute of Medical Sciences Summary: A cross sectional study done on 81 cases of erythrocytosis who underwent JAK2V617F mutation testing showed Polycythemia vera (PV) to be the leading cause of erythrocytosis (n = 57; 70.3 %). JAK2V617F mutation was found positive in 51 (89.6 %) patients of PV with 17 (33.3 %) cases showing homozygosity. JAK2V617F mutation correlated with distinct clinicohematologic phenotypes of PV patients, namely older age, higher leucocyte and platelet counts, and greater rates of splenomegaly. Introduction: Janus kinase2 (JAK2) V617F mutation in myeloid cells has been reported from the majority of patients with Polycythemia vera (PV). To our knowledge, this is the first Indian study on PV. Materials and Methods: Clinical records of 81 cases of erythrocytosis who were evaluated for JAK2V617F mutation by tetra primer Amplification Refractory Mutation System polymerase chain reaction at our institution were reviewed. The significance of this mutation in the context of clinical & haematological features in cases that fulfilled World Health Organisation (WHO) 2008 diagnostic criteria for PV was investigated. Results: Fifty seven (70.3 %) patients fulfilled the WHO diagnostic criteria for PV. Erythrocytosis could be attributed to secondary causes in 18 (22.2 %) cases and was idiopathic in six (7.4 %) cases. The JAK2V617F mutant allele was detected in 51 of the 57 PV patients (89.5 %) with 17 (33.3 %) showing homozygosity. A comparison between JAK2 V617F mutation positive and JAK2 V617F

307 wild type PV patients at presentation revealed that the JAK2 V617F mutation positive patients were older (p = 0.0261), with significantly more splenomegaly (p = 0.006). Mean values of haemoglobin and hematocrit were significantly higher in the JAK2V617F negative group (p = 0.0261) while median values of total leukocyte count (p = 0.0014) and platelet count (p = 0.02) were significantly lower in the JAK2V617F negative groups. However, compared with heterozygote counterparts, JAK2V617F homozygotes did not reveal any significant differences. Conclusion: The prevalence of JAK2V617F mutational status is 89.5 % in PV patients with 33.3 % homozygosity and correlates with distinct clinicohematologic phenotypes of PV patients, namely older age, higher leucocyte and platelet counts, and greater rates of splenomegaly.

Abstract No. 85 Clinicohematological Profile of JAK2 (V617F) Positive Myeloproliferative Neoplasms Puttaiah Parimala2, Karuna Rameshkumar2, Cecil Ross1 Department of Clinical Pathology and Hematology1, St John’s Medical College Hospital, Bangalore Summary: Detection of jak2 mutation may have a significant role in diagnosing BCR-ABL negative CMPD and in identification of subsets who would respond to JAK2 inhibitor therapy. Introduction: Identification of JAK2 mutation in myeloproliferative neoplasms (MPN) has now been considered to have a fundamental role in the pathogenesis of Polycythemia Vera (PV), idiopathic myelofibrosis (IMF) and essential thrombocythemia (ET). Its frequency in West has shown remarkable consistency, but very few studies are reported on Indian population. Materials and Methods: To study prevalence, clinical and laboratory features of Philadelphia chromosome negative and JAK2 (V617F) positive myeloproliferative neoplasms. All cases of JAK2 (V617F) mutation positive MPN’s from January 2011 to July 2013 who fulfilled 2001 WHO criteria of MPN were included. Clinical features and laboratory profile including complete hemogram, bone marrow aspiration and biopsy were analysed. JAK2 mutation analysis was done by allele specific PCR. Results: Among 48 patients diagnosed as JAK2 positive MPN, Polycythemia Vera was most frequent (63 %) followed by idiopathic myelofibrosis (16 %) and essential thrombocythemia (21 %). Male predominance was noted (68 %). Overall presence of JAK2 mutation was associated with higher WBC count (16.6 9 109/L).Between the subtypes PV had higher Hb level (18.1 g/dl). Splenomegaly was seen in all cases of IMF. Thrombosis was seen in 23 % cases of PV. Two of PV patients presented with acute hemorrhage. Conclusion: In view of high frequency of JAK2 mutations in PV, screening for this mutation in cases of erythrocytosis will help to exclude secondary erythrocytosis. This study supports the recommendation that mutation screening be incorporated in the initial workup to identify MPN’s. Keywords JAK2 (V617F) positive, Myeloproliferative neoplasms, Polycythemia vera.

Abstract No. 86 A Case Series of Interesting Cases of ‘‘Hypereosinophilia’’ Shreeniwas S Raut, Bhavesh B Parekh, Shailesh S Talati, Sonia K Parikh, Asha S Anand, Sandip A Shah, Harsha P Panchal, Apurva A Patel, Shilin N Shukla Department of Medical and Pediatric Oncology, Gujarat Cancer and Research Institute, Ahmedabad

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308 Summary: This interesting case series of hypereosinophilia represents various causes, presentations and associations of hypereosinophilia in a tertiary care oncology institute. Introduction: Hypereosinophilia most of the times burdens an extensive work up and many times the cause remains unclear. Materials and Methods: Design: Retrospective. Setting: Gujarat cancer and research institute, Ahmedabad, Period: Jan 2012–July 2013. A case series of patients presenting with/developing hypereosinophilia. Results: We report a case series of patients with hypereosinophilia presented at oncology tertiary care institute. These are (1) An elderly couple (husband 65 year, wife 60 year) presenting with hypereosinophilic syndrome where no cause could be detected including infectious and environmental causes. (2) Hypereosinophilia associated with myeloproliferative disease with myeloblastic crisis and splenomegaly in a 9 year old child. (3) Hypereosinophilia with hereditary Philadelphia chromosome in a 19 year old female with her father having chronic myeloid leukemia. (4) DRESS syndrome (Drug rash, eosinophilia, systemic symptoms) secondary to prolonged vancomycin treatment for acute meningitis in a 30 year old male who had recently undergone hematopoietic stem cell transplantation. (5) chronic eosinophilic leukemia in an 18 month old child. (6) Recurrent hypereosinophilia in a 7 year child with acute lymphoblastic leukemia due to recurrent/persistent worm infestation. Conclusion: Hypereosinophilia can be a presentation as a primary disease, secondary to underlying disease, drug, parasites, malignancy or unexplained.

Abstract No. 87 Idiopathic Hypereosinophilic Syndrome—A Rare Case Report Sainath Andola, Murlidhar S Rao, JS Kintan

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Abstract No. 88 Juvenile Myelofibrosis—A Rare Case Presentation Varsha Mahesh Baldwa, Vasudha Mahendra Bhagat, Bharti Mahendru Jha Government Medical College, Surat Summary: We intend to present a rare case of juvenile myelofibrosis in six year old female child to evaluate clinical features and findings in peripheral smear, bone marrow aspirate and bone marrow biopsy who presented with facial puffiness and difficulty in breathing since three days and pallor. Peripheral blood picture showed presence of myeloid precursors along with 30 nucleated red blood cells per 100 white blood cells. Bone marrow aspiration yielded dry tap and biopsy showed coarse reticulin fibers with collagenisation. Introduction: Myelofibrosis is uncommon condition in children and in juvenile myelofibrosis females outnumber males. Various degrees of peripheral blood abnormalities may be present at diagnosis and patients usually have one or more of the following changes: Normocytic anemia, thrombocytopenia or leucocytosis (with left shift). Anisopoikilocytosis, dacrocytes, nRBCs, myelocytes and promyelocytes are usually seen in peripheral blood. Bone marrow aspirate is hypo cellular (‘‘dry tap’’) and biopsy demonstrates increased reticulin staining. Cytogenetic studies ensure exclusion of other myeloid neoplasm that may be associated with marrow fibrosis (e.g. BCR-ABL 1). Materials and Methods: Geimsa staining was done on peripheral smear and bone marrow aspirate smears. Bone marrow biopsy was stained using Haematoxylin and Eosin, Reticulin and Masson Trichrome stains. Results: Reticulin and Masson trichrome stains highlighted the presence of coarse reticulin fibers with collagenisation on biopsy and aspirate was hypocellular. Conclusion: Based on presence of coarse reticulin fibers on biopsy and hypocellular aspirate, diagnosis of Myelofibrosis was offered. As patient did not turn up for follow up, cytogenetic evaluation was not done.

Department of Pathology, M.R. Medical College, Gulbarga-85105, Karnataka Summary: Idiopathic Hypereosinophilic syndrome (IHES) is a rare hematological disorder with clinical heterogeneity. It is characterized by peripheral blood eosinophilia of unknown origin exceeding 1.5 9 109/L persisting at least for 6 months. Present case was diagnosed as IHES based on clinical features and hematological findings and being presented for its rarity. Introduction: Idiopathic hypereosinophilic syndrome IHES) is defined as eosinophilia [1.5 9 109/ L persisting for at least for 6 months, for which no underlying cause can be found and associated with signs of organ involvement and dysfunction. It is a rare entity. Materials and Methods: A 55 year male presented with abdominal discomfort, hepatosplenomegaly. No history of parasitic infection, drug reaction or allergy. Stool examination: No parasites.; Hemogram done on day 1 and day 21 and inbetween Diethyl carbamezine was given. Results: Hemogram showed TLC: 86 9 109/L & 67 9 109/L with Neutrophils 37 and 40 %, Lymphocytes 09 and 08 %, Eosinophils 42 and 38 %, Monocytes 01 and 02 %, myelocytes 06 and 07 %, metamyelocytes 04 and 04 %, myeloblasts 01 and 01 %. Platelets 284 9 109 and 262 9 109/ L and Absolute Eosinophil count 36.1 9 109 and 25.5 9 109/L. Hemogram done before 1 year showed total count of 56 9 109/L, eosinophils 39 % and Absolute eosinophil count 21.8 9 109/L. Conclusion: IHES is a rare multisystem disorder with invariable involvement of BM & PB and hepatosplenomegaly in 30–50 % cases, the true incidence of which is not known. Hereby we are presenting a rare case of IHES with diagnostic feature of persistent eosinophilia ([1.5 9 109/L) and hepatosplenomegaly without any underlying cause.

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Abstract No. 89 Diagnostic Dilemma? Chronic Myeloproliferative Disorders N Priyathersini, Febe Renjitha Suman, S Suresh1, Anitha Ramesh1, S Rajendiran Department of Pathology and 1Medical Oncology, Sri Ramachandra University, Chennai Summary: The myeloproliferative diseases/neoplasms include chronic myeloproliferative disorders and overlap myelodysplastic/ myeloproliferative syndromes (MPDS). The aberrant activation of tyrosine kinase signaling pathway is identified among the myeloproliferative diseases. Their diagnosis and classification require correlation of morphology with clinical, hematologic and molecular genetics findings. Introduction: The traditional classification of the common MPDS is based on, which cell line is most proliferative and the amount of marrow fibrosis. We present a case of 76 year old man who had features of chronic myeloid leukemia and myelofibrosis. Materials and Methods: A 76 year old male presented with features of acute left ventricular failure. On examination he had a moderate hepatosplenomegaly. His total count was 41,230/cumm, Hb 9.9 g/dl and platelets were 2.57 lakhs/cumm. The peripheral smear showed a picture of chronic myeloid leukemia—stable phase. Patient underwent bonemarrow aspirate, biopsy and material was sent for FISH analysis for Philadelphia chromosome. Results: Bone marrow aspirate was diluted. Bone marrow biopsy showed grade 3 myelofibrosis. Many bizarre forms of megakaryocytes were seen. By FISH analysis

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Philadelphia chromosome was negative. Conclusion: There was a diagnostic dilemma between Philadelphia negative CML with secondary myelofibrosis and primary idiopathic myelofibrosis. However the absence of leukoerythroblastic picture and high TC with CML features, Philadelphia negative CML was favoured. JAK2 mutation study was requested, yet to be performed. The patient was put on Imatinib and in spite of bcr-abl negativity clinically there is improvement, with counts coming to baseline and reduction in spleen size.

Chronic Lymphocytic Leukaemia (CLL), Hairy Cell Leukaemia (HCL) & Other Chronic Lymphoproliferative Disorders (CLPD) Abstract No. 90 Immunogenetics of Chronic Lymphocytic Leukemia (CLL)—A Pilot Study Demonstrating a Geographical Bias Shruti Chaudhary, Patkar Nikhil, Deshpande Prashant, Aamre PA, Tembhare Prashant, Menon Hari, Sengar Manju, Bagal Bhausaheb, Gujral Sumeet, PG Subramanian Hematopathology Laboratory, Tata Memorial Centre, Mumbai Introduction: The B cell receptor (BCR) contains the immunoglobulin (IG) molecule as a key component. Alterations in the BCR form a foundation on which CLL immunogenetics is based upon. This pilot study addresses the immunogenetics of CLL by analyzing three unique clinically important variables; identification of immunoglobulin heavy chain variable (IGHV) gene mutations, skewed IGHV gene usage & cytogenetics. Materials and Methods: 16 consecutive cases of CLL (diagnosed on basis of WHO 2008/2001 criteria) were prospectively accrued from May to June 2013. Peripheral blood DNA was subjected to a multiplex PCR using fluorescently labelled primers (VH1-VH6 and JH) followed by capillary electrophoresis to identify clonal B cell rearrangement, the framework & VH family used. Following bidirectional Sanger sequencing, the data was compared to the closest germline sequences on NCBI & IMGT databases. Interphase FISH was performed according to standard techniques. Results: All patients showed clonal VH gene rearrangements. 75 % of cases showed presence of somatic hypermutations, rest were unmutated. A biased VH usage was seen towards VH1 & VH3 (31.25 % each). The commonest gene used was IGHV1-2 (31.2 %) followed by IGHV330 (18.75 %). The median CDR3 length was 17 amino acids. FISH (done in 75 %) revealed del13q as commonest abnormality, followed by trisomy12 &del 17p (16 % each). Conclusion: The immunogenetics of Indian CLL is unique (highly biased VH1-2 usage)as compared to published data (VH1-69, VH3-23,VH4-69 are common in other studies) highlighting a geographical difference. This is the first data from India on the immunogenetics of CLL.

Abstract No. 91 IGHV Gene Usage in Indian Patients with CLL Nitin Mathur, Lata Rani, G Smeeta, Atul Sharma, Lalit Kumar, Ajay Gogia, Ritu Gupta All India Institute of Medical Sciences, New Delhi

309 Summary: Immunoglobulin variable heavy chain (IGHV) gene rearrangements influence prognosis in chronic lymphocytic leukemia (CLL) and since geographic variations in IGVH gene usage has been reported; we analyzed the status and configuration of IGHV gene and report frequencies similar to western population. Introduction: Mutation status of the IGHV gene has significantly improved risk prediction in CLL and is has been established as a crucial prognostic factor. Thus the present study aimed at the analysis of IGHV gene family usage in Indian patients with CLL. Materials and Methods: Peripheral blood mononuclear cells from 126 B-CLL patients were assessed for IgVH mutation status as per BIO-MED-2 protocol and evaluated using Ig-BLAST. Results: 60 % patients (n = 76) belonged to the Mutated (M) subset, whereas 40 % (n = 50) belonged to the unmutated (UM) subset. In M subset, the range of mutations varied from 2.1–20.7 % (median-5.9 %). The most frequently expressed IGHV subgroup was IGHV3 (50 %), followed by IGHV4 (23 %), IGHV1 (19 %), and IGHV2 (5 %), with only 2, 1 and 1 cases in IGHV5, IGHV6 and IGHV7 subgroups respectively. IGHV3 (n = 59 %) and IGHV4 (n = 83 %) subgroups were frequently associated with mutated while IgHV1 was predominantly associated with UM IGHV (67 %). The most frequently used IGHV genes were IGHV3-23, IGHV3-48, IGHV3-30, IGHV3-7 and IGHV1-69. There was no overrepresentation of IGHV3-21 (n = 2) in Indian patients as opposed to western population. Conclusion: Similar to the western data, the hierarchy of mutations in Indian patients with CLL is IGHV3 [ IGHV4 [ IGHV1 with IGHV3-23 being the most frequent gene rearrangement.

Abstract No. 92 DNA Methylation Pattern in Chronic Lymphocytic Leukemia Ritu Gupta, Lata Rani, Nitin Mathur, Atul Sharma, Lalit Kumar, Ajay Gogia All India Institute of Medical Sciences, New Delhi Summary: The contribution of epigenetic silencing of genes by aberrant methylation has been reported to be important in the pathogenesis of Chronic Lymphocytic leukemia (CLL). The present study shows distinct DNA methylation patterns in prognostically relevant subgroups of CLL underscoring the prognostic importance of epigenetic events. Introduction: Our main aim was to investigate and compare the genome-wide DNA methylation profiles of different CLL prognostic subgroups for identification of the differentially methylated genes that can play a role in CLL pathogenesis. Materials and Methods: Peripheral blood samples from treatment naı¨ve, CLL patients were assessed for IgVH mutation. Methylation status was assessed in 12 CLL patients by utilizing 1 9 244k methylation array (27,627 CpG islands, Agilent technologies) and analyzed using Agilent Genomic Workbench (ver.7.0) software. To identify differentially methylated genes, unpaired t-test was performed and the genes having p \ 0.05, log2 FC [ 1.0 were considered. Results: Genome wide DNA methylation profiles were compared between newly diagnosed IGHV-M (n = 5) and IGHV UM (n = 7) CLL cases. Array data revealed 1,099 differentially methylated promoters in unmutated cases compared to the mutated. Differentially methylated genes included the genes involved in cell cycle regulation (CCND1, CDK6, DNM2, E2F4), apoptosis (BCL2, CASP9, FADD), epigenetic regulation (DNMT1, DNMT3, HDAC1, HDAC10) etc. Conclusion: Our results show distinct DNA methylation patterns in different CLL subsets which suggest importance of epigenetic alterations in prognosis of CLL.

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Abstract No. 93 Complex Rearrangement Involving Chromosomes 9 and 22 Resulting in Chronic Myeloid Leukemia in a Case of Chronic Lymphocytic Leukemia Harboring TP53 Deletion Anil Kumar Yadav, Mayur Parihar, Anurag Gupta, SR Arun, Akshay R Gore, Anupam Chakrapani, Saurabh Bhave, Deepak K Mishra, Reena Nair, Mammen Chandy Tata Medical Center, Kolkata Summary: Coexistence of CML and CLL in the same patient is a rare event. We present a rare case of chronic myeloid leukemia developing as a result of complex rearrangements involving chromosomes 9, 15 and 22 in a case of chronic lymphocytic leukemia harboring TP53 deletion following treatment with single agent cyclophosphamide. Introduction: Concurrent manifestation of a myeloid and lymphoid neoplasm is of rare occurrence and is reported in approximately 1 % of the patients. Only 10 cases of sequential occurrence of CML and CLL had been described in literature where CML developed after the diagnosis of CLL. We present one such case. Results: A 57 year old male presenting with weakness, lymphadenopathy and hepatosplenomegaly, was diagnosed as CLL based on high lymphocyte count and immunophenotyping. Cytogenetic studies showed TP53 deletion. On treatment with cyclophosphamide, the patient showed symptomatic response. One year post diagnosis, the patient showed high WBC counts with 2 % blasts, myeloid shift to left, 3 % basophils and 40 % lymphocytes. Karyotype showed absence of Philadelphia chromosome with a t(9;15). RT PCR and FISH confirmed the diagnosis of CML. showing a complex rearrangement with BCR/ABL fusion transcript on derivative chromosome 9 with normal chromosomes 22. The patient was treated with Imatinib and on 3 months follow up shows CML responding with persistent CLL. Conclusion: Cyclophosphamide has been implicated as a causative agent in secondary CML. The two clones are distinct and independent in this case, the CML being in cytogenetic remission in response to Imatinib, in presence of an active CLL.

Abstract No. 94 Efficacy and Toxicity of Bendamustine in CLL, Low Grade Lymphoma and Mantle Cell Lymphoma Krishnamani Kalpathi, Sadashivudu Gundeti, Vijay Gandhi Linga, Raghunadharao Digumarti Nizam’s Institute of Medical Sciences, Hyderabad Summary: CLL and low grade lymphomas are indolent in nature and incurable. Bendamustine has emerged as a frontline agent in the treatment of these disorders with excellent outcomes and acceptable safety. Introduction: The aim of this study was to evaluate the Progression free survival (PFS), overall survival (OS), overall response rate (ORR) and toxicity profile of Bendamustine in the first or second line setting in patients with CLL, low grade lymphomas and mantle cell lymphoma treated in our Institute between 2004–2012. Materials and Methods: All patients of CLL, follicular lymphoma and mantle cell lymphoma treated with Bendamustine either in the first line or second line setting were retrospectively analyzed for response rates and toxicity. IPSS version 19 was used for analysis. Results: A total of 39 patients were eligible for evaluation. The number of CLL, follicular and mantle cell lymphoma patients were 23 (59 %), 12 (30.7 %) and 4 (10 %) respectively. Male to female ratio was 2.8:1. Bendamustine was used upfront in 24 (61 %) patients and as second line in 15 (39 %) patients. The average number of cycles received was 5.2. Mean time to hematologic response in CLL patients was 41 days. The ORR in our study

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 was 87 %. CR was seen in 28 (71.7 %) patients. Median OS and PFS was not reached. Median time to follow up was 20 months. Estimated OS at 3 years was 59.8 %. Five patients progressed on bendamustine. PFS was 95.8 % in the first line setting and 73.3 % when used after another agent. Estimated PFS at 8 months in upfront use is 95 % and is 64.8 % at 15 months in second line setting. OS in the upfront bendamustine group was 91.7 and 66.7 % in the second line group. Median OS was not reached in the upfront setting. Projected OS at 8 months in upfront use is 90.9 %. Projected OS at 36 months in second line is 39.5 %. Commonest toxicities observed were skin toxicity in 12 (30.7 %) patients followed by TLS and FN in 2 patients and one respectively. Commonest skin toxicity seen was maculopapular rash associated with pruritis in 3 subjects followed by Herpes zoster and erythema multiformae in 2 patients each. Most of the skin toxicities were seen during cycle 3 and 4. Six deaths were seen in our study. Three patients passed away due to cardiac failure presumably secondary to anthracyclines used as first line treatment. Conclusion: Bendamustine is a safe and effective agent in the management of CLL and low grade lymphomas with excellent safety. The response rates in our study are comparable to other published studies in literature. Efficacy of bendamustine in the first line setting is better. Median OS and PFS were not reached in our study probably owing to shorter duration of follow up and lesser events. Further studies with larger numbers and longer duration of follow up are necessary.

Abstract No. 95 Hairy Cell Leukemias: Comprehensive Analysis in a Tertiary Care Haematology Center Monali Gupta, Chanchal Das, Sambhunath Banerjee, Subhajit Brahma, Saurabh Bhave, Anupam Chakrapani, Mayur Parihar, Deepak Mishra, Reena Nair, Mammen Chandy Tata Medical Center, Kolkata Introduction: Classical Hairy cell Leukaemia (HCL) is a rare indolent B-cell chronic lymphoproliferative disorder characterized by splenomegaly, pancytopenia, presence of hairy cells in peripheral blood/bone marrow. HCL-v encompasses cases of B-chronic lymphoproliferative disorders that resemble classical HCL but exhibit variant cytohematologic features, variant immunophenotype and lack of dramatic response to purine analogues. We present here 4 cases of HCL diagnosed and treated in Tata Medical Center from May 2011– May 2013. Materials and Methods: 4 patients presented with cytopenias and splenomegaly. CBC, differential count, peripheral smears, bone marrow aspirates and biopsies were studied. Annexin A1 and DBA. 44 immunohistochemical stains were done on BM biopsies. Flowcytometry was performed on bone marrow using CLPD panel on BD Facs Canto II. Allele specific PCR for detection of BRAF V600E mutation was standardized by extracting genomic DNA from the archived bone marrow aspirate slides of all these 4 patients. Results: The peripheral smears, bone marrow aspirates and bone marrow biopsies of all 4 patients displayed classical hairy cells. The hairy cells were positive for Annexin A1 and DBA.44 except for one which was negative for Annexin A1. Flow cytometric analyses exhibited characteristic HCL immunophenotype (CD20/CD22/ CD11c/CD103/CD25 positive; CD5/CD10/CD23 negative) in 3 cases except one which showed FMC7 positive and CD25 negative. So, a diagnosis of HCL-variant was suggested in this case though the hairy cells showed classical morphology. All three cases of classical HCL showed mutation BRAF V600E while the HCL-v did not. A fifth case diagnosed HCL and received 2 months of Cladirabine elsewhere was confirmed to have BRAF V600E mutation on PB. Immunohistochemical staining for DBA.44 on his BM biopsy showed residual interstitial hairy cell infiltrates post 3 months of Cladirabine.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Conclusion: HCL-v is important in the differential diagnosis of HCL as it is a more aggressive disease and shows resistance to conventional HCL therapy. BRAF V600E mutation was established as a signature marker of classical HCL. Immunostaining for DBA.44 and BRAF V600E are extremely useful to monitor the response to Cladirabine in HCLs.

Abstract No. 96 The Study of Hairy Cell Leukemia at the Gujarat Cancer & Research Institute—18 Cases Kazoomi Patel, Biren Parikh, Bina Brhambhatt, Vibha Vyas, Jyoti Sawhney, Manoj Shah Pathology Department, The Gujarat Cancer & Research Institute Ahmadabad, Gujarat Summary: Hairy cell leukemia is a rare, low grade B-cell neoplasm with characteristic and morphologic and immunophenotypic profile. Most common in middle aged male, present with pancytopenia and, splenomegaly. Introduction: Hairy cell leukemia (HCL) is an uncommon chronic B-cell lymphoproliferative disorder. It affects primarily elderly men and presents with splenomegaly, pancytopenia and monocytopenia. It has to be differentiated from various chronic lymphoproliferative disorders (CLPDs) because of different protocol of treatment and, clinical course. Materials and Methods: This study included 18 cases diagnosed for a period of 4 years (2009–2012) out of 300 cases of CLPDs at GCRI. We have studied clinical history, complete blood count, bone marrow aspiration, bone marrow biopsy, immunophenotyping and, immunohistochemistry. Also, studied treatment details and follow up. Results: This study included 18 cases, age group of 35–69 years, with a median age of 51 years, with a male predominance. M:F ratio is 6:1. HCL is 6 % of all CLPDs. Patients were presented with a complains of weakness, fever and, abdominal pain. On clinical examination commonest finding were pallor, splenomegaly, hepatomegaly and, abdominal lump, On laboratory investigation, 17 cases had anemia, 4 cases had leucocytosis, 9 cases had leucopenia and 11 cases had pancytopenia. Dry tap was observed in 75 % of cases with 10–70 % non-erythroid nucleated cells. On bone marrow biopsy we found diffuse infiltration of the marrow. CD19 gated leukemia cells show, 100 % patients showed co expression of CD103, CD11c and CD25. We found a single case of CD5 positive hairy cell leukemia variant. Conclusion: Most common presentation of a HCL was present in middle aged male with pancytopenia and, splenomegaly, few patient presented with unusual clinical features like absence of a palpable spleen, generalized lymphadenopathy, leucocytosis and expression of CD5 by leukemic cells.

Abstract No. 97 Clinico-Hematological Profile of Hairy Cell Leukemia: A Report of 4 Cases Jyoti R Kini, Deepa Adiga, Nirupama M, Urmila N Khadilkar, Prashanth B, Krishna Prasad Kasturba Medical College, Mangalore, Manipal University, Karnataka Summary: The purpose of the study is to evaluate the clinicohematological features of cases diagnosed as hairy cell leukemia in our institution. Introduction: Hairy cell leukemia is a rare, indolent, B-cell neoplasm characterized by small mature lymphoid cells with oval nuclei and abundant cytoplasm with ‘‘hairy’’ projections involving peripheral blood and diffusely infiltrating the bone marrow

311 and splenic red pulp. Materials and Methods: The present series included four cases diagnosed over a period of six years. Clinical presentation, complete blood count, bone marrow aspirate and biopsy features were reviewed along with the flow cytometry findings. Treatment and follow up details were noted. Results: Four cases of hairy cell leukemia, all males, and aged 42–69 were studied. The clinical presentation included generalized weakness in all four, easy fatigability in two and weight loss in two. Splenomegaly was consistently seen in all patients and two patients had cervical lymphadenopathy. Three patients presented with low leukocyte count and one patient presented with markedly leukocytosis. A dry tap was obtained on bone marrow aspiration in two patients with atypical lymphoid cells seen in the aspirates of all patients. Bone marrow biopsy and flow cytometry were confirmatory. Conclusion: Morphological and clinical correlation is essential for accurate diagnosis and optimal therapy of hairy cell leukemia.

Abstract No. 98 Unusual Presentation of T-Prolymphocytic Leukemia: A Case Report Girish Badarkhe, S Kishore Kumar, Uttam Nath, SS Roy, Sambit Samanta, Prantar Chakrabarti, Utpal Choudhuri Institute of Haematology & Transfusion Medicine, Kolkata, India Summary: T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-cell lymphoproliferative disorder. Common cytogenetic abnormalities in T-PLL usually include 14q11.2, chromosome 8 rearrangements, 11q abnormalities leading to the deletion of the ATM and MLL genes, and abnormalities on 12p, 5q, 6q and 13q. The presence of a combination of these fairly unique structural genetic abnormalities makes chromosome analysis very crucial and extremely helpful to get an accurate and definitive diagnosis of T-PLL along with immunophenotypic and morphological data. Introduction: Herein, we present a distinct and diagnostic challenging case of T-PLL that emphasize the heterogeneity of this disease but showed cytogenetic aberrations that helped to confirm the diagnosis of T-PLL. Materials and Methods: 59 years old male non-hypertensive & non-diabetic presented with the history of erythmatous nodule (M: 2 9 2 cm) over left forearm since 4 months, which was non pruritic & non-scaly in nature. Similar lesions had appeared over chest, upper part of back, both arms & left sheen. There were no associated B symptoms. On examination patient was having generalized lymphadenopathy. There was no hepato-splenomegaly. Results: On Investigations patient was found to have high WBC count with lymphocytosis. Skin biopsy: Hyperkeratosis of epidermis & dermis infiltrated by sheets of lymphoid cells mixed with few lymphocytes & histiocytes. Immunohistochemistry on skin biopsy: Cells were positive for CD3, CD2, CD5, CD7 & negative for CD20, CD34, Tdt, Pax-5, CD117, CD56, Granzyme B, CD30 Immunophenotyping: Cells positive for CD2, CD5, CD7, CD4 & 8. Diagnosis of T-PLL favoured. FISH: Showed no evidence of 9p21 deletion, monosomy 11 or loss of MLL gene. But FISH showed TCR rearrangement for the c and d chains. Patient was started on single agent Gemcitabine (1,000 mg/m2) on D1 & D8 in a 21 days cycle. Conclusion: The uniqueness of this case is absence of organomegaly, though he had skin lesion & generalized lymphadenopathy.

Abstract No. 99 Splenomegaly an Enigma: Case Report Deepti Varshney, Gurdeep Buxi, Sadhna Marwah, AS Nigam, Vijay Kumar, Jyoti Garg

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312 PGIMER, Dr. Ram Manohar Lohia Hospital, New Delhi Summary: A 52 year old female patient presented with splenomegaly and persistent lymphocytosis for 6 years. She was otherwise asymptomatic. The cause of lymphocytosis could not be established. At her most recent visit FCM immunophenotyping was done on her peripheral blood using CLPD panel. Flowcytometry revealed a B cell lineage of cells. On reviewing peripheral blood smear villous lymphocyte and few prolymphocyte were observed. Based on indolent clinical course, FCM and peripheral blood morphology, a diagnosis of SLVL was made. Introduction: Splenomegaly can be seen in many nonneoplastic and neoplastic disorder including chronic lymphoproliferative disorder. SLVL is a rare disorder comprising less than 1 % of lymphoid neoplasm. It is characterised by splenomegaly, moderate lymphocytosis and villous lymphocyte on peripheral blood. Conclusion: This case emphasizes the importance of careful morphological evaluation of awareness of subtle morphological features of SLVL. Cases of low grade CLPD with subtle morphological variation should be further evaluated by immunophenotyping.

Hodgkin Lymphoma (HL) Abstract No. 100 Prognostic Impact of CD20 Expression in Advanced Stage Classical Hodgkin Lymphoma (CHL) Treated with ABVD Chemotherapy: A Retrospective Analysis Hasmukh Jain, Manju Sengar1, Epari Sridhar2, Hari Menon1, Uma Dangi1, Tanuja Shet2, Sumeet Gujral2, Hasmukh Jain1, Siddhartha Laskar3, Nehal Khanna3 1

Department of Medical Oncology, 2Department of Pathology, Department of Radiation Oncology

3

Summary: CD20 expression on Hodgkin and Reed-Sternberg (HRS) cells is seen in approximately 5–58 % of CHLs. The prognostic relevance of CD20 expression in CHL remains conflicting. Given the efficacy of rituximab in CHLs in a few studies, we felt it relevant to further investigate its prognostic role. Herein we report the prognostic impact of CD20 expression in patients with advanced stage CHL treated at our centre with ABVD chemotherapy. Materials and Methods: The electronic medical records of newly diagnosed advanced stage (stage IIB, III and IV) CHL patients (14 years or more) registered at our centre from January 2008 to December 2010 were reviewed for baseline disease characteristics, international prognostic score (IPS), treatment, response, death, progression or relapse. CD20 expression (focal or diffuse membrane positivity in HRS cells) was noted. Patients receiving more than 2 cycles with evaluable response were analysed for overall survival (OS) and progression free survival (PFS). Results: A total of 143 patients (males 112) were analysed. Median age was 28 years (range 14–70 years). 73 % of patients had stage III/IV disease. B symptoms, bulky disease, IPS C4 and bone marrow involvement was seen in 75, 38, 22 and 15 %, respectively. Post 6 cycles of ABVD 88 % achieved complete response whereas 8 % experienced disease progression. CD20 positivity was noted in 22 % (evaluable in 112 patients). CD15 was positive in 31/92 patients. Baseline disease characteristics were similar in the CD20+ and negative subset. The 3-year PFS and OS were 80 and 95 % respectively. 3-year PFS was 65.5 and 87 % (p = 0.035) respectively in CD20+ and negative subset, while OS was 88 and 97 % respectively (p = 0.06). CD20+, IPS C4 was associated with inferior PFS both in the univariate and multivariate analysis. Absolute lymphocyte and monocyte ratio and CD15 expression did not impact PFS or OS. Conclusion: In our study CD20 expression did affect progression free survival. However there was no

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Abstract No. 101 Bone Marrow Involvement in Nodularlymphocyte Predominant Hodgkin Lymphoma—Patterns and Impact on Behavior Poonam Panjwani, Sridhar Epari, Manju Sengar, Siddhartha Laskar1, Hari Menon2, Tanuja Shet Department of Pathology, Radiation Oncology1 and Medical Oncology2, Tata Memorial Hospital, Mumbai-40012 Summary: Bone marrow involvement in Nodular lymphocyte predominant Hodgkin’s lymphoma (NLPHL), though rare, presents usually as diffuse involvement with fibrosis. Patients with pancytopenia fare poorly as opposed to those without them. Introduction: Amongst Hodgkin’s lymphoma, Nodular lymphocyte predominant Hodgkin’s lymphoma has a propensity for bone lesions. Also marrow is not done in patient with Stage I disease. This study was an attempt to evaluate if upfront occult marrow involvement occurs in NLPHL. Materials and Methods: A total of 90 cases of Nodular Lymphocyte Predominant Hodgkin Lymphoma were retrieved from archives and reviewed. The bone marrow involvement in these cases was also studied. Of these, 24 cases were excluded due to change in diagnosis. CD20 and CD3 were done on all cases to check if occult involvement was seen. Results: A total of 8/66 (12.12 %) patients with confirmed NLPHL were found to have marrow involvement. Of these five showed diffuse involvement, replacing marrow elements with fibrosis. A marrow preserving partial involvement was seen in one case, while one case had interstitial clusters of large cells and one patient showed a paratrabecular cluster of large cells only. Of these, two patients died of disease. The nodal disease showed T cell rich nodules in 6/8 cases while two cases had a typical NLPHL like morphology. Patients with pancytopenia fared poorly as opposed to those without them. Conclusion: Upfront bone marrow involvement is rare in NLPHL and it is usually diffuse and obvious so there is no need to tests this patient for occult involvement.

Abstract No. 102 FDG-PET After Two Cycles of Chemotherapy Predicts Treatment Failure and Progression-Free Survival in Advanced Pediatric Hodgkin Lymphoma Amol Dongre, Venketesh Rangarajan1, Shripad Banavali, Girish Chinnaswamy, Gaurav Narula, Siddharth Laskar2, Seema Medhi3, Sneha Shah1, Sumit Gujral4, Tanuja Seth4, Sajid Quereshi5, Brijesh Arora Departments of Paediatric Oncology, Nuclear Medicine1,Radiotherapy2, Radiology3, Surgery4 and Pathology5, Tata Memorial Hospital, Mumbai Summary: Risk-adapted Hodgkin lymphoma treatment in children requires early and precise assessment of prognosis. Starting from January 2005, newly diagnosed patients with advanced Hodgkin’s lymphoma (HL) were enrolled to assess the prognostic role of an early interim 2-[18F]fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) scan and the other prognostic factors in advanced HL, treated with conventional ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) therapy. Materials and Methods: One hundred and two consecutive, newly diagnosed children underwent FDG-PET at staging, after two-three cycles of chemotherapy (PET-2), and after completion of chemotherapy. No

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treatment change was allowed on the basis of the PET-2 results. Results: The median age was 9.2 years and median follow-up was 40 months. After two cycles of chemo-therapy, 70 (69 %) patients had negative FDG-PET scans and 32 (31 %) patients had positive scans. Twelve of 32 FDG-PET–positive patients progressed and seven of 70 FDG-PET–negative patients progressed. Survival analyses showed strong associations between interim FDG-PET and PFS (P \ .002) and OS (P \ .006). The OS and PFS was 93 and 91 % for patients with negative PET-2 whereas OS and PFS was 72 and 68 % respectively for slow early responders. None of the patients with stage II or stage III without B symptoms or bulky disease and negative PET-2 relapsed. For prediction of PFS, interim FDG-PET was as accurate after two-three cycles as later during treatment and superior to computerized tomography (CT) at all times. No patient in metabolic CR after 2 cycles showed progression at the end of therapy. In regression analyses, early interim FDG-PET was stronger than the established prognostic factors. Conclusion: Early interim FDG-PET is a strong and independent predictor of PFS in advanced HL in children. After demonstration of CR on interim PET, there is no incremental value of repeating the scan after completion of therapy.

The EFS and OS of the cohort was 78 and 97 %, respectively. The EFS in TG-1, 2 and 3 was 91, 100 and 67 %, respectively. The mean survival time was 26 months. Twelve episodes of febrile neutropenia requiring hospitalization were observed: one patient died. One patient, each in TG-1 and 3, relapsed at 9 and 15 months, respectively. One patient in TG-3 had progressive disease and another had secondary AML. None of the 20 patients in whom the re-evaluation PET was normal, relapsed, as compared to 3 (25 %) with inadequate response (p = 0.07). Conclusion: A re-evaluation PET-CT performed after 2 courses of chemotherapy is predictive of outcome and permits reduction of therapy in early-stage (TG-1/2) HD with excellent short-term outcome. The EFS in TG-3 was however suboptimal, contributed in part by misinterpretation of PET in the early period of the study.

Abstract No. 103

Maya Prasad, Shripad Banavali, Girish Chinnaswamy, Gaurav Narula, Siddharth Laskar1, Seema Medhi2, Sneha Shah3, Sumit Gujral4, Tanuja Seth4, Sajid Quereshi5, Brijesh Arora

Non-ABVD Chemotherapy and PET-CT Based Strategy for Treating Hodgkin Lymphoma in Children: PGIMER, Chandigarh Experience Sonali Mohapatra, Sonali Mohapatra, Deepak Bansal, Amita Trehan, BR Mittal1, Anish Bhattacharya1, Rakesh Kapoor2, SC Sharma2, Radhika Srinivasan3, Arvind Rajwanshi3, Nandita Kakkar4, Ashim Das4, RK Marwaha Hematology-Oncology Unit, Department of Pediatrics, Advanced Pediatric Center, Department of 1Nuclear Medicine, 2Radiotherapy, 3 Cytology and 4Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh Summary: Reduced-anthracycline and non-bleomycin protocols are being evaluated to reduce long-term toxicity in Hodgkin disease (HD). Introduction: Short-term outcome of patients treated for HD from October 2010 to February 2013 in the Pediatric HematologyOncology unit, PGIMER, Chandigarh was analyzed retrospectively. Materials and Methods: Management strategy was adapted from the ongoing Euronet-PHL-C1 study-protocol. A PET-CT was performed at diagnosis. SUV of the involved sites, in comparison with mediastinal blood pool SUV and the size was considered for assessing PETCT response after 2 courses of chemotherapy. Treatment group (TG) 1: (IA/B, IIA); plan: 2 OEPA ? PET-CT. TG-2: (IEA/B, IIEA, IIB, IIIA); plan: 2 OEPA ? PET-CT ? 2 COPDAC. TG-3: (IIEB, IIIEA/B, IIIB, IVA/B); plan: 2 OEPA ? PET-CT ? 4 COPDAC. Radiotherapy recommended after chemotherapy if inadequate PET response after OEPA-2. OEPA: vincristine, etoposide, prednisone, doxorubicin. COPDAC: cyclophosphamide, vincristine, prednisone, dacarbazine. Results: Thirty-three patients were treated. The mean age was 7.6 ± 2.5 years (range: 4–12), with a M:F ratio of 5.6:1. The number of patients in TG-1, 2 and 3 were 14, 6 and 13, respectively. The mean duration of follow-up was 16.4 ± 8.3 months (range: 0.330). Inadequate response in re-evaluation PET-CT was observed in 12 (38 %) patients. Radiotherapy was administered to 2 patients (both TG-2). Radiotherapy was replaced by 2 courses of COPDAC in 4 patients (all TG-1), due to physician preference. Of the remaining 6 patients (TG-1: 3; TG-3: 3) in whom the re-evaluation PET was not normal, radiotherapy or additional chemotherapy was not administered due to misinterpretation of PET in the early period of the study.

Abstract No. 104 Clinical Characteristics and Outcome of Relapsed Pediatric Hodgkin Lymphoma: Experience from a Tertiary Care Centre in India

Departments of Paediatric Oncology, Nuclear Medicine1, Radiotherapy2, Radiology3, Surgery4 and Pathology5, Tata Memorial Hospital, Mumbai Summary: The treatment of relapsed Hodgkin lymphoma (HL) is challenging, more so in low-income countries. We present the clinical characteristics and outcome of relapsed pediatric HL patients from our centre. Materials and Methods: A retrospective analysis was performed on children (0–18 years) with relapsed HL who were treated with salvage chemotherapy ± HSCT (haematopoietic stem cell transplant) at Tata Memorial Hospital from January 2005 to December 2012. Results: There were 67 children (70 % male) with median age of 11 years (range 4–18 years). Sixty-three (91.3 %) were classical HL and 5 (7.46 %) were Nodular Lymphocyte Predominant. Majority (71.6 %) presented in advanced stages and had received ABVD based regimens. Twenty patients (29.8 %) had multiple ([2) relapses. Thirty-five (52.24 %) were either primary refractory or early relapsers (\12 months in remission). Salvage chemotherapy included GDP (gemcitabine, dexamethasone, cisplatin) in 27 (45.7 %), MINE (mitoxantrone, ifosfamide, etoposide) in 16 (27.1 %) and others in 16 (27 %). 33 patients were consolidated with HSCT and six patients with Involved field RT; 8 patients refused any therapy. At a median follow-up of 34 months (6–132 months), 24 (35.8 %) were in complete remission, 30 (44.8 %) had either progressed or relapsed, and 11 (16.2 %) were dead. 3 year overall survival was 44.8 % and 3 year relapse free survival was 38 %. Independent prognostic factors included multiple relapses (P = 0.01), extranodal disease at relapse (P = 0.009), primary refractory disease (P = 0.001) and poor response on Interim-PET evaluation (P = 0.009). Patients with high risk relapse (adverse factors: early relapse/primary refractory disease with extranodal/advanced stage relapse) had a better outcome with HSCT (P = 0.01); 37.5 vs. 18.2 % without BMT. No advantage was found in intermediate risk patients (only one above adverse factor) treated with/without HSCT (62.5 vs. 50 %; P = 0.33). Conclusion: Multiple relapses, primary refractory disease/early relapse, extranodal relapse and poor initial response to salvage chemotherapy are predictive of poor outcome in relapsed paediatric HL. Intermediate risk patients do well with chemotherapy but high risk patients benefit with HSCT.

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Abstract No. 105

Abstract No. 106

Treatment Results in Advanced Stage Hodgkin’s Lymphoma: A Retrospective Study

Brentuximab-Bendamustine Combination Demonstrates Good Clinical Activity in Refractory-Relapsed Classic Hodgkin Lymphoma

Hasmukh Jain1, Manju Sengar1, Epari Sridhar2, Hari Menon1, Uma Dangi1, Reena Nair1, Tanuja Shet2, Sumeet Gujral2, Siddhartha Laskar3, Nehal Khanna3 Department of Medical Oncology, 2Department of Pathology, Department of Radiation Oncology

1

Vivek Sulekha Radhakrishnan, Vishvdeep Khushoo, Sriram Ravichandran, Saurabh Jayant Bhave, Anupam Chakrapani, Reena Nair, Mammen Chandy

3

Tata Medical Center, Kolkata

Introduction: Hodgkin’s lymphoma represents 15 % of all cancers in young adults. It is a curable malignancy, however up to one-third of the patients fail to be cured with the current chemotherapy regimens. The epidemiology of this disease is different in the Asian population, mixed cellularity being the commonest histology and a greater proportion of patients present with advanced stage disease. However very scant literature is available on the outcomes of advanced stage Hodgkin’s lymphoma from developing countries, less so with ABVD regimen, which is the standard of care in most of the centers. Hence we conducted this retrospective study to analyze the outcomes of patients with advanced stage Hodgkin’s lymphoma from our centre. Materials and Methods: One hundred and twenty five treatment naive patients with advanced stage Hodgkin’s lymphoma aged C15 years, who were treated at Tata Memorial hospital from January 2004 to December 2007 were included in this analysis. Results: A total of 165 patients of advanced stage Hodgkin’s lymphoma were registered in our centre between 2004–2007, out of which 125 patients received at least one cycle of chemotherapy and were included in the analysis. The median age was 32 years (range 15–65 years). Seventy seven percent (96/125) of the patients were males. Seventy five percent (94/125) of the patients had B symptoms at presentation. Stage III disease was seen in 63 % (79/125) and stage IV in 37 % (46/125). International prognostic score (IPS) could be estimated in 112 patients. IPS score B4 was seen in 85 % (95/112) of the patients. Ninety four percent of the patients were treated with ABVD (118/125), while six percent patients received COPP regimen (7/125). Ninety percent (112/125) of the patients received at least six cycles of chemotherapy. Thirty six percent (45/125) of the patients received radiation for either bulky sites or residual disease at the end of chemotherapy. A total of 125 patients were treated; response data was available in ninety percent (112/125) of the patients. Complete remission (CR) was seen in seventy six percent (95/125), partial remission seen in ten percent patients (13/125), and three patients had disease progression on treatment. There were nineteen deaths, seven deaths were related to progressive disease, 8 were related to toxicities, and 4 were due to unrelated causes. There were forty two patients with progressive/relapse disease; Forty percent (55/125) of patients remain on follow up till the time of analysis (December 2013). With a median follow up of 28 months (range: 2–100 months), the 5 years overall survival (OS) is 60 % and the progression free survival (PFS) is 58 %. On multivariate analysis, PFS was affected by international prognostic score and response to initial therapy while the OS was affected by age, IPS and response to therapy. Conclusion: This retrospective study from single centre showed comparable results to the reported literature with respect to the outcomes in advanced Hodgkin’s lymphoma treated with ABVD regimen. However, the major limitation of the study is significant number of post treatment losses to follow-up. This lead to shorter median follow up and difficulty in assessing long term disease status—an inherent flaw of retrospective analysis.

Introduction: Among patients diagnosed with Hodgkin lymphoma, 30 % will experience primary refractory or relapsed disease. Brentuximab vedotin (SGN-35; AdcetrisÒ) is an anti-CD30 antibody conjugated via a protease-cleavable linker to the potent anti-microtubule agent monomethyl auristatin E. It was approved by the US FDA in Aug 2011. In Hodgkin lymphoma, it is indicated after failure of autologous stem cell transplantation (auto-SCT) or after failure of at least two prior multi-agent chemotherapy regimens (in auto-SCTineligible candidates). Bendamustine is a hybrid alkylating agent with a purine-like benzimidazole ring component, and is found to exhibit low or incomplete cross-resistance with other alkylating agents due to its unique cytotoxic properties. It has been found to be effective in relapsed/refractory Hodgkin lymphoma in phase-II studies (Moskowitz et al., JCO 2013). A novel combination of Brentuximab and Bendamustine in salvage therapy of relapsed/refractory Hodgkin Lymphoma has not been reported in literature. Materials and Methods: We report our experience with 2 patients in whom this novel combination was used due to clinical compulsions of poor ‘initial’ symptomatic response with Brentuximab. Case 1: A 19 year old girl diagnosed with Hodgkin Lymphoma Stg IIB (IPS-Favourable) in Jun 2012 developed progressive disease after 4 cycles of ABVD. She received 2 cycles of GDP regimen and progressed further at the primary site and developed B symptoms. She was switched to single agent Brentuximab (1.8 mg/kg, q21d) and was not relieved of B symptoms even after 1 week following therapy. She then received Bendamustine (100 mg/sq m, 2 days) additionally. B symptoms settled and she received 3 further cycles of the combination and achieved PET-CT Complete Response (CR) at the end of 3rd cycle. No serious adverse events related to the combination therapy were observed. She received consolidation therapy with High-dose chemotherapy (BEAM) followed by autologous stem cell rescue. She is in CR on last follow up in Jul 2013. Case 2: A 19 year old boy with Relapsed Hodgkin Lymphoma (post-autologous stem cell transplant) presented to us in Aug 2012 with refractory Stg IVB disease post salvage therapy with a combination of Rituximab and Oxaliplatin-Cytarabine chemotherapy. His prior therapies were ABVD, and GDP followed by LACE (high dose chemotherapy with autologous stem cell rescue) at primary diagnosis (Oct 2009), and first relapse (Apr 2011) respectively. He received one course of Brentuximab followed by one dose of Bendamustine 1 week later, owing to poor symptomatic improvement. A partial response and complete disappearance of B symptoms were observed. He developed grade-2 mucositis and grade4 neutropenia with sepsis, which recovered. He discontinued treatment after the first cycle, had a PFS of 4 months and an OS of 7 months. Conclusion: Brentuximab in combination with Bendamustine is an effective combination therapy in patients with Refractory/Relapsed Hodgkin Lymphoma. Further phase-II studies are required to determine feasibility of this novel combination regimen.

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Abstract No. 107 Lenalidomide Induced Good Clinical Response in 3 Patients with Multiple Relapsed and Refractory Hodgkin’s Lymphoma R Jetani Amit, A Shah Sandip, N Shukla Shilin, S Anand Asha, S Talati Shailesh, P Panchal Harsha, A Patel Apurva, K Parikh Sonia, B Parekh Bhavesh Department of Medical and Pediatric Oncology, Gujarat Cancer Research Institute, Ahmedabad Summary: Three patients were diagnosed with nodular sclerosis Hodgkin’s lymphoma in clinical stage II, and combination chemotherapy followed by radiotherapy was initiated. During the following 3 years, the disease progressed despite several standard therapeutic approaches, including autologous stem cell transplantation. Introduction: Relapsed or refractory classical Hodgkin lymphoma remains a clinical challenge, with limited effective treatment options available after stem cell transplantation. Lenalidomide, a thalidomidederivate, belongs to a novel class of immunomodulatory drugs approved for the treatment of Multiple Myeloma and Myelodysplastic Syndrome. Lenalidomide has multiple modes of action, including direct induction of apoptosis in tumour cells, antiangiogenic effects and the activation of immune cells, such as Natural Killer cells and T-cells. Apoptosis resistance, increased neoangiogenesis and impaired immunity critically contribute to HL. We therefore hypothesized that single agent lenalidomide might be active in HL patients who have failed conventional chemotherapy. Materials and Methods: Relapsed disease was conformed by biopsy and imaging. Lenalidomide (25 mg daily) on 1–21 days of 28 days treatment was then initiated. Positron emission tomography scans were performed during lenalidomide treatment. Hematologic and laboratory values, as well as physical condition were also assessed before and during lenalidomide treatment. Results: In 2 patients with four months after continuous lenalidomide treatment, tumor load was significantly reduced, B symptoms had resolved, and the patient’s physical condition had improved, allowing them to resume normal daily-living activities. One out of these 2 patients is in complete remission on positron emission tomography scan, and he has reached 11th month of his Lenalidomide treatment. One patient has completed 1 month of treatment only, and his tumor load was significantly reduced, B symptoms have resolved, and the patient’s physical condition has improved. Treatment was well tolerated, allowing the patients to remain on regular dosing. Conclusion: Long-term lenalidomide treatment provided clinical benefit and was well tolerated in patients with relapsed, advanced classic Hodgkin’s lymphoma.

Abstract No. 108 Haematopoietic Stem Cell Transplantation for Hodgkin’s Lymphoma: A Single Institute Experience Akhil Jain, Sandip Shah, Asha Anand, Apurva Patel, Kamlesh Shah, Kinnari Patel, Shailesh Talati, Harsha Panchal, Sonia Parikh, Bhavesh Parekh, Shilin Shukla Department of Medical and Pediatric Oncology, The Gujarat cancer & Research Institute, B.J. Medical College, Ahmedabad-380016 Summary: Haematopoietic stem cell transplantation plays an integral role in the treatment of patients with relapsed Hodgkin’s lymphoma. H SCT is currently the optimal treatment for patients who fail chemotherapy and radiation therapy. Introduction: The treatment of recurrent or high risk lymphoma patients with high dose chemotherapy and autologous transplantation has greatly expanded over the past few years. Owing to use of better supportive care measures the

315 morbidity & mortality rates associated with this therapy have decreased with successful results and increase in disease free survival rates. Materials and Methods: Design: Retrospective. Setting: The Gujarat Cancer & Research Institute. Period: Sept 1999–June 2013. To analyse & study the outcome of haematopoietic stem cell transplantation (HSCT) in Hodgkin’s lymphoma patients at GCRI. Retrospective analysis of case records of 30 Hodgkin’s lymphoma patients treated with HSCT at GCRI since 1999 is done to study the outcome of HSCT in Hodgkin’s lymphoma. Results: Out of 30 Hodgkin’s lymphoma patients there were 9 female and 21 male patients. All patients were heavily pretreated with chemotherapy ranging from 2 to 5 lines of regimen. At the time of transplantation 3 (10 %) patients were in 2nd complete remission (CR), 4 (13 %) had 3rd CR and 23 (76 %) patients had persistent disease. All patients were treated with BEAM (BCNU/CCNU, etoposide, cytosar arabinoside and melphalan) conditioning regimen followed by autologous HSCT. Treatment was successful in 27 patients (90 %) that achieved CR at 100 days post-HSCT. Presently, 22 (73 %) patients are alive and under CR. Median disease free survival (DFS) is 24 months. 3 year DFS and 5 year DFS are 60 and 37 %, respectively. Two patients (6 %) expired due to transplant related mortality. Six patients had expired within 1 year of treatment; 5 because of relapse (17 %) that occurred in period of 1 month to 11 month post HSCT and 1 (3 %) because of infection after 10 months. Four (13 %) patients have still not completed 3 year period of post transplant but are in remission at present. Conclusion: Autologous transplantation is a safe and successful treatment option in relapsed Hodgkin’s lymphoma patients and should be done as early as possible.

Abstract No. 109 Acute Anthracycline Cardiotoxicity in a Child with Hodgkin Lymphoma: A Miraculous Escape from Death K Anand Kumar1, Anil Sachdev2, Vasant Chinnabhandar1, Nita Radhakrishnan1, Anupam Sachdeva* 1

Pediatric Hematology Oncology, 2Interventional Radiology—Sir Ganaga Ram Hospital Summary: Early onset cardiotoxicity with a clinical heart failure is very rare particularly even rarer in pediatric patients. Here we describe one such case with a favorable outcome. Introduction: Overall incidence of clinical heart-failure due to anthracyclines is 16 % and most of it is late-onset but early-onset cardiotoxicity can occur (\1 %). There are no reports of survival in a child after acute anthracycline cardiotoxicity. Materials and Methods: We report a child with acute anthracycline toxicity admitted at our center in June 2012. Results: 7-year-old female with Hodgkin lymphoma was admitted for 2nd cycle of ABVD chemotherapy. Within 12 h of receiving chemotherapy she developed signs and symptoms of congestive heart failure. She was given fruesimide and shifted to PICU. She developed revived after aggressive and prolonged cardio-pulmonary resusitation for 45 min. During resuscitation cardiogenic shock leading to cardiac arrest. She had multiple episodes of cardiac arrest and was along with standard measures IV magnesium (0.1 ml/ kg of 50 % MgSo4) was given She had persistent lactic acidosis (pH6.8) for [2 h, required support of multiple inotropes, vasopressors and mechanical ventilation. Sepsis workup and viral PCR for possible myocarditis was negative. We gave her IVIG empirically 2 g/kg. Her cardiac evaluation showed she had non-specific ECG changes, 2Decho showed ejection fraction \20 %. She developed multiorgan hypoxic ischemic insult. Appropriate support with fluid, electrolytes and blood components was provided. After 2 weeks on mechanical ventilation her organ functions improved and she was weaned off with intact neurological outcome and she is doing well at 10 months

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316 follow up with no deficits. Conclusion: Acute-anthracycline cardiotoxicity even though rare can occur and should be considered in differentials of a child with congestive heart failure. Along with standard resuscitative measures IV magnesium might help to improve cardiac function.

Abstract No. 110 Cryoglobulinemia Presenting as Palpable Purpura in a Case of Hodgkin Lymphoma Shuddhasattwa Bhattacharjee, M Ravindranath, K Manisha, Raju Bhaisare, S Gurmeet, M Bhalla Department of Pathology, Jawaharlal Nehru Hospital and Research Centre, Sector 9, Bhilai Summary: A 58 years old female had joint pain involving small joints of hand and feet, non blanchable purpura over feet, and ulcer over right leg of one year duration which showed exacerbation during winter. On skin biopsy and haematological tests diagnosis of cryoglobulenemia was made. After a symptom free interval of four years patient presented with oral ulcer, tonsillar mass and on and off fever. Histopathology and Immunohistochemistry confirmed a diagnosis of Hodgkin’s lymphoma. Introduction: Cryoglobulenemia is a disease mediated by cryoglobulin with the property to precipitate below 37 °C. Cryoglobulin are immunoglobulins that at low temperature, (4 °C) in vitro, have the property of forming precipitates, which at normal body temperature (37 °C) are dissolved. Materials and Methods: Skin biopsy of leg ulcer showed eosinophilic amorphous material in dermal vessels suggesting cryoglobulin. For cryoglobulin test patient’s serum was incubated at 37 and 4 °C. Serum incubated at 4 °C showed cryoprecipitates which redissolved on incubating the serum at 37 °C. Patient’s plasma also showed similar phenomenon. CBC, LFT, RFT, ESR were normal and M band electrophoresis, and RA factor, ANA, viral markers for HBV and HCV were negative. On histopathology and Immunohistochemistry of tonsillar mass, a final diagnosis of Hodgkin’s lymphoma was made. Results: Patient was finally diagnosed as a case of cryoglobulenemia and subsequently presented with Hodgkin’s lymphoma of oral cavity, an unusual site for Hodgkin’s disease. Conclusion: Presentation and late complications of cryoglobulenemia can be varied, as this case showed an unusual presentation as Hodgkin’s lymphoma.

Non Hodgkin Lymphoma (NHL) Abstract No. 111 Mutations Occurring in the PEST Domain of the NOTCH1 Gene are Seen in Nearly 10 % of Mantle Cell Lymphoma Ghodke Kiran, Patkar Nikhil1, Mascarenhas Russel, PG Subramanian, Tembhare Prashant, Menon Hari, Sengar Manju, Bagal Bhausaheb, Gujral Sumeet Hematopathology Laboratory, Tata Memorial Centre, Mumbai Introduction: Recently mutations of NOTCH1 gene have been described in MCL & CLL with varying frequencies. In this study we characterized the baseline frequency of NOTCH1 mutations in MCL with de novo leukemic presentation or bone marrow involvement in our center. For the commonly occurring mutation we developed a high sensitivity fluorescent PCR technique. Materials and Methods:

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 31 cases of MCL (diagnosed on basis of WHO 2008/2001 criteria) were retrospectively accrued from medical records from 2007 to 2013. Genomic DNA was extracted from stained bone marrow aspirate smears by a modified silica membrane-column based technique. A high sensitivity PCR using fluorescently labeled primers followed by capillary electrophoresis was carried out in all cases to identify a commonly occurring frameshift mutation in the PEST domain of the NOTCH1 gene. In all cases additional mutations in the same region were screened for using bidirectional Sanger sequencing. Results: Twenty-five cases had secondary involvement of PB and BM with lymph node being the most common primary site of disease (n = 23). Extra nodal involvement was found in 2 patients. Six patients presented with a de novo leukemic presentation. Fluorescent PCR detected a 2 bp deletion P2515Rfs*4 (c.7544_7545delCT) in 3 patients (9.7 %). These mutations were confirmed and other rare mutations were ruled out by Sanger sequencing. Conclusion: The frequency of these mutations is no different in Indian population than what has been published in the literature. The common mutation in MCL (c.7544_7545delCT) is identical to the NOTCH1 mutation in CLL. Development of a high sensitivity technique allows for rapid screening and detection of small mutant clones.

Abstract No. 112 Lymphoplasmacytic Lymphoma/Waldenstr m Macroglobulinemia (LPL/WM) Shows High Frequency of Somatic Hypermutations, IGHV-3 Usage and MYD88 L265P Mutation Deshpande Prashant, Patkar Nikhil1, Mascarenhas Russel, PG Subramanian, Tembhare Prashant, Menon Hari, Sengar Manju, Bagal Bhausaheb, Gujral Sumeet Hematopathology Laboratory, Tata Memorial Centre, Mumbai Introduction: LPL/WM is a rare B cell lymphoma that harbours a mutation in MYD88 gene (MYD88 L265P). Important immunogenetic variables include IGHV somatic hypermutations (SHM), VH gene usage & length of the complementarity determining region 3 (CDR3). The aim of this study was to study prevalence of MYD88 gene mutation and immunogenetic profile (SHM, VH gene usage & CDR3 length) of LPL/WM in our institute. Materials and Methods: 33 cases of LPL/WM (diagnosed on basis of WHO 2008/2001 criteria) were retrospectively accrued from 2007-2013. DNA was subjected to an allele specific oligonucleotide PCR using fluorescently labelled primers followed by capillary electrophoresis was done in all. Immunogenetics was assessed in 30/33 patients. FR1 and FR2 regions of the VH gene amplified clonally products which were sequenced. Results: MYD88 L265P mutation was found in 84.8 % (28/33) of patients. The immunogenetic results here pertain only to samples with productive IGHV gene rearrangement [22/31 (*71 %) cases]. An overwhelming 96 % of cases showed presence of somatic hypermutations. 59 % of cases showed a biased use for the VH3 gene followed by VH4 (22.7 %) and VH1 (18.2 %). The commonest gene used was IGHV3-7 (27.3 %) followed by IGHV1-18 (18.2 %). The median CDR3 length was 14 amino-acids. Conclusion: MYD88 L265P mutation has a high frequency but is not diagnostic of LPL/WM. The immunogenetics of LPL/WM as compared to other common lymphomas such as CLL is unique in demonstrating a post GC origin as demonstrated by SHM & biased VH usage as demonstrated by VH3 usage. Relatively high frequencies of LPL/WM show IGHV3-7 usage & almost all cases showed a short CDR3 sequence. This is the first data from India on immunogenetics of LPL/WM.

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Abstract No. 113 BCL10 Expression in Gastric MALT Lymphomas Sushant Vinnarakar, Sridhar Epari, Manju Sengar, Hari Menon, Siddharatha Laskar, Tanuja Shet Tata Memorial Hospital, Parel, Mumbai-400012 Summary: This study was an attempt to evaluate gastric MALT lymphomas for bcl10 expression at our institute. BCL 10 was not helpful in prognostication of gastric MALT lymphoma in present study however clinical indices and stage of disease were effective in predicting relapse free progression of patients with gastric MALT lymphoma. Introduction: A small group of Gastric lymphomas are associated with bcl10 translocations leading to nuclear expression of the protein. Nuclear expression of BCL 10 is being studied as prognostic marker to predict H pylori independent status, presence of specific translocations and poor prognosis in gastric MALT lymphoma. Materials and Methods: A total of 61 gastric lymphomas were accessioned during study period and 52 cases with follow up were selected for thorough histology analysis and bcl10 immunohistochemistry. Results: The incidence of gastric MALT lymphoma in our institute was 73 % of all the GIT MALT lymphoma and 23.6 % of all the primary gastric lymphomas during the study period. The median age of presentation was 58 years with prevalence of gastric MALT lymphoma in older age group and male predominance with male to female ratio of 3:1. Though MALT lymphoma is low grade lymphoma and has indolent course about 17 % cases in the study had bone marrow involvement. Most of the cases had a multifocal site of involvement in stomach. Only 38.5 % cases showed concurrent H pylori infection and 4 cases showed nuclear bcl10 expression. However, no significant co relation between Bcl 10 and disease free survival (DFS) or higher stage of disease or presence of large cells was seen in the study. Factors effecting disease free survival (DFS) were stage of disease, depth of infiltration, IPI and complete response to primary therapy. Conclusion: Bcl10 expression in MALT lymphoma is uncommon in our study pand prognosis of gastric MALT lymphoma is more commonly affected by stage of disease. H pylori was documented in less number of cases chiefly because inadequate sampling of normal mucosa and most patients presenting with advanced stage disease.

Abstract No. 114 A Clinicopathological Study with Bone Marrow Pattern Evaluation in Cases of Hepatosplenic Gamma Delta T-Cell Lymphoma Afshan Khan, PG Subramanian1, Pratibha Amare2, Shweta Bhavsar, Nikhil Patkar1, Prashant Tembhare1, Sitaram Goghale1, Ashok Kumar1, Y Badrinath1, Tanuja Shet, Epari Sridhar, Shripad Banavali3, Brijesh Arora4, Gaurav, Narula4, Hari Menon3, Manju Sengar3, Uma Dangi3, Navin Khattry3, Sumeet Gujral Department of Pathology, 1Hematopathology Laboratory, 2 Department of Cytogenetics, 3Department of Medical Oncology, 4 Department of Paediatric Oncology, Tata Memorial Hospital (TMH), Mumbai-400012 Summary: Hepatosplenic gamma delta T-cell lymphoma (HSGDTCL) is a rare malignancy accounting for \1 % of all Non Hodgkin lymphomas (NHLs). Bone marrow (BM), reportedly involved in nearly all cases, is difficult to diagnose due to lymphocytic morphology and subtle tissue pattern. Introduction: HSGDTCL is a distinct T cell lymphoma, often confused with the commoner

317 T-cell acute lymphoblastic leukemia (T-ALL). The study aims to evaluate clinicopathological features and morphological patterns in BM biopsy of HSGDTCL. Materials and Methods: Clinicopathological findings, FCI (4–8 color, FACS CantoII), BM, hepatosplenic evaluation with immunohistochemistry (IHC), cytogenetic aberrations (FISH) and follow-up were noted in 23 HSGDTCL cases (Jan 2006– Dec 12). BM biopsy was categorised on infiltration pattern as intrasinusoidal, interstitial, both and diffuse. Results: HSGDTCL cases were 0.2 % of all NHLs. Age range was 4–57 years. M:F ratio was 2.3:1. Lymphadenopathy (42.1 %) was also seen with hepatosplenomegaly. Patients presented with anemia, leukocytosis and thrombocytopenia. FCI showed aberrant T cell immunophenotype with positive CD3, TCR cd and negative CD4, CD34, Tdt. 31.6 % cases expressed CD8. Commonest BM biopsy pattern (at presentation) was both intrasinusoidal and interstitial (12/23 cases) with mainly intrasinusoidal pattern on sequential post chemotherapy (CT) BM. Isochromosome7q abnormality(87.5 %) was noted. Multiple CT protocols (CHOP/DHAP/ SMILE) were given with median follow-up of 3 months and maximum 18 months survival. Conclusion: High suspicion of HSGDTCL was raised on blast-like cells expressing surface CD3 with negative Tdt in cases mostly referred morphologically as T-ALL. Along with clinicopathological correlation, CD3 IHC was mandatory to highlight subtle infiltrates in liver, spleen and BM biopsies.

Abstract No. 115 Incremental Value of PET and Role of Early Interim PET on Response Prediction & Outcome of Pediatric B-NHL Nirav Thakar, Venketesh Rangarajan1, Shripad Banavali, Girish Chinnaswamy, Gaurav Narula, Siddharth Laskar2, Seema Medhi3, Sneha Shah1, Sumit Gujral4, Tanuja Seth4, Brijesh Arora Departments of Paediatric Oncology, Nuclear Medicine1, Radiotherapy2, Radiology3, and Pathology4, Tata Memorial Hospital, Mumbai Introduction: PET scan has proven to be more sensitive and specific than contrast enhanced CT for initial staging, response assessment and prognostication in adult patients with high grade NHL but its role in pediatric NHL is still unclear. We sought to study the incremental value of baseline PET over bone marrow biopsy for staging and role of early interim PET-CT scan for predicting response and final outcome. Materials and Methods: Fifty eight newly diagnosed children with B-NHL underwent FDG-PET at staging, after two cycles of chemotherapy (PET-2), and after completion of chemotherapy if necessary from January 2009 to December 2011. No treatment change was allowed on the basis of the PET-2 results. Patient’s disease status till last follow up was recorded. PFS and OS were calculated from date of diagnosis to date of relapse/progression and date of last follow up respectively. The relation of CR and PR in interim PET/CT scan with PFS and OS was analyzed using Kaplan–Meier survival analysis. Results: The median age of presentation was 8 years (3–18) with a median follow up of 21.4 months (3–43). 46 (79.3 %) patients had advanced disease (stage III/IV). All the BM positive patients (8/8) had uptake on PET; however 5(8.6 %) patients had multifocal BM uptake only on PET, leading to upstaging of the disease. PET-2 showed CR (metabolic + morphological) in 39/58 (67.2 %) of patients, PR in 17/58 (29.30 %) of patients and progressive disease in 2/58(3.4 %). There was a 100 % concordance between the metabolic and morphologic response (CT component of PET/CT) in all patients. All the patients who were in CR in interim PET continued to be so at end of treatment. Response at interim PET could predict progression free survival (PFS) (p \ 0.000) and overall survival (p \ 0.003). Conclusion: PET-CT scan in pediatric NHL picks up additional sites

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318 of bone marrow involvement leading to upstaging of disease. Interim PET/CT significantly predicts the PFS and OS in NHL making it a useful tool for response assessment, prognostication and a risk adapted strategy in future.

Abstract No. 116 Comparison of Treatment Outcomes with EPOCH-Rituximab Versus CHOP-Rituximab in Patients with De-Novo Intermediate and High Risk International Prognostic Index (IPI) Diffuse Large B Cell Lymphoma (DLBCL): A Single Center Retrospective Analysis Hasmukh Jain1, Manju Sengar2, Hari Menon2, Uma Dangi3, Bhausaheb Bagal4, Navin Khattry4, Sridhar Epari5, Tanuja Shet5, Sumeet Gujral6, Venkatesh Rangarajan7, Siddhartha Laskar8

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 (32 %) on R-EPOCH and 14 (33 %) on R-CHOP. LDH was elevated in all but two patients on R-EPOCH compared to 37 (88 %) patients on R-CHOP. Complete response was seen in 60 %, and disease progression in 18 % patients on R-EPOCH, compared to 59 %, and 20 % on R-CHOP respectively. There were 5 deaths on R-EPOCH, 3 due to toxicity and 2 due to disease progression, and in comparison there were 4 deaths on R-CHOP, all of them due to disease progression. With a median follow up of 6 months, the estimated OS at 1 year is 74 % and progression free survival (PFS) is 62 % for patients on R-EPOCH. For patients on R-CHOP, with a median follow up of 31 months, 1 year OS is 68 % and PFS is 64 %. Conclusion: Our retrospective analysis indicates that treatment with R-EPOCH regimen resulted in similar results as with R-CHOP regimen. However patients treated with R-EPOCH had more adverse features in terms of disease bulk, poor performance status and high IPI score. A prospective randomized comparison is warranted between these two regimens.

1

Hemato-Oncology Disease Management Group, Tata Memorial Hospital, Mumbai, India; 2Hemato-Oncology Disease Management Group, Tata Memorial Hospital, Mumbai, India; 3Medical Oncology, Tata Memorial Centre, Mumbai, India; 4Bone Marrow Transplant Unit, ACTREC, Tata Memorial Centre, Mumbai, India; 5Department of Pathology, Tata Memorial Centre, Mumbai, India; 6Department of Pathology, Tata Memorial Hospital, Mumbai, India; 7Department of Nuclear Medicine, Tata Memorial Centre, Mumbai, India; 8Radiation Oncology, Tata Memorial Centre, Mumbai, India Introduction: Poor prognosis DLBCL, including intermediate and high risk disease according to IPI accounts for approximately 20 % of new cases of DLBCL. The addition of rituximab to conventional chemotherapy (CHOP) has improved the outcomes in this subset, with a 2-year overall survival (OS) of about 50 %. However, 40–50 % of these patients still have either primary refractory disease or relapse after an initial response. Rituximab-EPOCH (R-EPOCH), an infusional regimen has a dynamic dose adjustment strategy based on the hematopoietic nadir in previous cycle to achieve an optimal drug concentration. Phase II studies with R-EPOCH in untreated DLBCL with intermediate and high risk IPI have reported improved outcomes, with an estimated 2-year OS of 75 % which appears superior to that of R-CHOP. Hence we analysed the outcomes of patients with de-novo, poor prognosis (intermediate and high risk IPI) DLBCL who received R-EPOCH and compared it to the historical cohort of patients who were treated with R CHOP at our centre. Materials and Methods: Treatment-naı¨ve patients of DLBCL with intermediate or high risk IPI, registered at our centre between November 2011 to June 2013, who received R-EPOCH regimen, were included for the analysis. Case records were reviewed for—demography, histology, stage, bulk of disease, extranodal sites, performance status, IPI, LDH, albumin, details of chemotherapy, grade ’ toxicities (CTCAE version 4) and need for hospitalization. Responses were evaluated at mid and end of chemotherapy. Overall and progression free survival were calculated. Similar analysis was done for poor prognosis DLBCL patients treated with R-CHOP between Jan 2007 to December 2010. Results: Baseline characteristics and treatment outcomes of 32 patients (males 24, females 8) treated with R-EPOCH were compared to 42 patients (males 28, females 14) who received R-CHOP. Median age in R-EPOCH group was 47 years (range 20–75 years) versus 55 years (23–72 years) in R-CHOP. Performance status C2 was seen in 47 % in R-EPOCH as compared to 28 % in R-CHOP group. Significant proportion of patients in R-EPOCH had bulky disease (81 vs. 16 %) and stage III/IV disease (90 vs. 81 %) as compared to R-CHOP. Patients with IPI of two represented 8 (25 %), IPI of three, 11 (34 %), and IPI of four and five, 10 (32 %) on R-EPOCH compared to 21 (50 %), 19 (45 %) and 2 (5 %) on R-CHOP, respectively. Serum albumin \3.5 g/dL was seen in 10

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Abstract No. 117 Retrospective Analysis to Assess the Feasibility of Replacing Anthracycline with Oral Etoposide in Treatment of Elderly Diffuse Large B-Cell Lymphoma (DLBCL) MV Chandrakant, Manju Sengar1, Tanuja Shet2, Epari Sridhar2, Hari Menon1, Uma Dangi1, Sumeet Gujral2, Hasmukh Jain1, Siddhartha Laskar3, Nehal Khanna3 1

Department of Medical Oncology, 2Department of Pathology, Department of Radiation Oncology

3

Summary: Treatment of elderly DLBL remains a challenge due to several factors like-comorbidities, compromised organ reserve and lack of treatment guidelines. Adverse biology further compounds the impact of compromised therapy on outcomes. Anthracycline based therapy, the standard of care in young patients, can lead to impaired cardiac function and mucositis in elderly. Given the activity of etoposide in DLBL, we analyzed its efficacy in elderly patients. Materials and Methods: Outcomes of de-novo elderly DLBL treated with CEOP (oral etoposide 65 mg/m2 day 1–3) with or without rituximab based therapy from Jan 2010 to December 2011 were analyzed retrospectively. Demography, stage, bulk of disease, aaIPI, grade 3/4 toxicities, responses, relapse and death were recorded. Overall and progression-free survivals were calculated with Kaplan– Meier method. Log-rank test was used to assess the effect of rituximab on overall survival. Immunohistochemistry (CD10, Bcl-6, MUM-1, Bcl-2) was used to classify the germinal center and nongerminal center phenotype in 45 cases. Results: A total of 106 patients were registered and planned for CEOP based therapy. However only 82 patients (males-57, female-25) started treatment at our centre and were evaluable for response and toxicity. The median age was 65 years (range 50–82 years) and 52 % patients had comorbidities. Advanced stage, bulky disease, extranodal involvement and raised LDH were seen in 50, 67, 60 and 80 % respectively. 56 % had high or high intermediate aaIPI. 45 % of patients received rituximab. 75 % percent of DLBL were non-germinal center type and 56 % were bcl-2 positive. EBER was positive in 1 case. Complete responses were higher in patients who received rituximab (67 vs. 51 %,p = 0.03). 2-year overall survival and progression-free survival were 55 and 44.5 % respectively. The overall survival of patients treated with rituximab (37 patients) was significantly better (70 vs. 43.5 % p = 0.02). 3 patients died due to treatment related toxicity and grade 3/4 toxicities were seen in 25 % patients. Conclusion: Etoposide based combination chemotherapy appears to be a reasonable alternative in elderly patients and should be tested prospectively.

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Abstract No. 118 Immunochemotherapy with Rituximab Plus Temazolamide for Primary CNS Lymphoma De Dibyendu, SS Roy, S Samanta, UK Nath, P Chakrabarti, U Chowdhury Institute of Hematology and Transfusion Medicine, Medical College, Kolkata Summary: Treatment for primary CNS lymphoma (PCL) with high dose methotrexate and alkylating agent based therapy is associated with major toxicity including myelosuppression, sepsis, and mucositis. We treated a patient of refractory PCL with Rituximab plus Temazolamide with intrathecal Rituximab resulting in partial response without any toxicity and good quality of life. Introduction: Common treatment options for primary CNS lymphoma includes high dose methotrexate and alkylating agent based therapy. The most common toxicity with these regimens includes myelosuppression, sepsis, and severe mucositis, which often results in dose reduction. Also old age, poor performance status, and other comorbidities leads to therapy interruptions. Immune-chemotherapy with Rituximab and Temazolamide provides an alternative regimen for refractory primary CNS lymphoma patient who can not tolerate or resistant to conventional high dose Methotrexate therapy. We are providing our experience of using this new regimen for an elderly refractory primary CNS lymphoma patient. Materials and Methods: Patient: We had a 56 year male, non immunocompromised, with hypertension, diabetes, presented with left sided hemiplegia and vomiting, without organomegaly or lymphadenopathy. The MRI brain revealed multiple periventricular enhancing lesion. The stereotactic biopsy revealed Diffuse large B cell lymphoma with CD 20 +ve. The PET scan revealed multiple FDG avid areas involving right basal ganglia, posterior parietal region and middle cerebral peduncle. He received high dose methotreaxate therapy, but experienced severe mucositis and grade 4 neutropenia. The repeat PET scan after 3 cycles showed less than 25 % reduction of previous PET avid areas and development of new areas in bilateral cerebellar peduncle and left cerebellar hemisphere with clinical deterioration. We treated this patient of refractory PCL not responding to high dose methotrexate therapy with Rituximab plus Temazolamide chemotherapy. Rituximab 375 mg/m2 iv on day 1, and Temazolamide 200 mg/m2 oral on day 1–5, along with Rituximab intrathecally on day 1. The cycles are repeated every 28 days. Results: After 1 cycle patient had rapid improvement of power of left lower limb and of cognitive function. The repeat CT scan after 2 cycles revealed complete regression of posterior fossa lesions and more than 50 % regression of right parietal region. Patient tolerated the chemotherapy well without any significant toxicity. Patient is planned to receive 2 more cycles of Rituximab + Temazolamide followed by Temazolamide maintenance therapy.

Abstract No. 119 Nodal Marginal Zone Lymphoma—From Wastebasket to Diagnostic Sense Katha Kanthe, Sridhar Epari, Manju Sengar, Hari Menon, Siddharatha Laskar, Tanuja Shet Tata Memorial Hospital, Parel, Mumbai-400012 Summary: Our study of 67 nodal marginal zone lymphoma revealed that only 20 cases were actually NMZL. NMZL is indeed a wastebasket diagnosis and expanded immunopanel along with flow cytometry in cases is essential for correct diagnosis. Accurately defined NMZL indeed had excellent prognosis. Introduction: This

319 was a retrospective analysis of 67 cases of nodal marginal zone lymphoma (NMZL) with a view to define morphologic and immunophenotypic features of NMZL. Materials and Methods: Besides negativity for CD10/CD23/bcl6/cyclinD1, an extended panel of MNDA1/MUM1/IGD/HCAM was used. Results: Out of the 67 cases of NMZL, on review, only 20 could be reclassified as NMZL which included a case of pediatric NMZL. The other cases were reclassified as SLL/CLL (small lymphocytic lymphoma), follicular lymphoma with marginal zone differentiation (FLMZD), splenic marginal zone lymphoma (SMZL) and reactive pathologies(Castleman’s disease, and IgG4 related disease). The median age group of the study was 56 years with age ranging from 35 to 76 years. None of the lymphomas, other than NMZL presented with stage I disease. Most patients had no B symptoms and good performance status. The 19 case of NMZL showed varied morphologic and cytologic features. Plasma cells were consistently identified in all cases of NMZL giving a clue to diagnosis. The major reason of misdiagnosis of SLL/CLL was the atypical morphology and immunohistochemistry. We observed that flow cytometry helped in picking up these misdiagnoses cases. Failure to include bcl6 in immunopanel and lack of follicular growth lead to misdiagnosis of FLMZD. The new immunopanel (MNDA, MUM-1, CD44, IgD) attempted was not able to delineate one disease from the other. As compared with the misdiagnosed entities NMZL had superior prognosis. Conclusion: NMZL is indeed a wastebasket diagnosis and hence adequate IHC along with flow cytometry in cases with marrow involvement is essential for correct diagnosis. Accurately defined NMZL indeed had excellent prognosis.

Abstract No. 120 Story of Anaplastic Large Cell Lymphoma (ALCL) Survival: Sometimes Much More than ALK Status Komal Agarwal, Tanuja Shet, Sridhar Epari, Sumeet Gujral, Manju Sengar, Hari Menon, Siddharatha Laskar, Brijesh Arora, Shripad Banavalii Tata Memorial Hospital, Mumbai-400012 Summary: Though ALK is an important prognostic factor in ALCL—In our study of 102 patients we encountered a poor survival and factors like stage and IPI were more effective in prognostication. Introduction: ALK status is thought to be the golden factor affecting survival in all patients with anplastic large cell lymphoma(ALCL). Materials and Methods: We report a study of 102 ALCL from a cancer referral institute in Mumbai. Results: Systemic ALCL comprised 2.7 % of all non-Hodgkin lymphomas and 26.9 % of T cell non-Hodgkin lymphoma in our institute during our study period. Out of 100 patients of ALCL, 39 patients (39 %) had nodal disease at presentation, 12 patients (12 %) had pure extranodal disease and remaining 49 patients (49 %) had nodal as well as extranodal disease. Out of the 99 patients where Ann Arbor stage could be recorded, only 19 had Stage I disease and 42 (42.4 %) had stage IV disease The cases studied included 75 cases of common variant, 2 small cell variants, 3 lymphohistiocytic variants, 5 tumors with large nucleolated cells, 13 tumors with admixture of the above patterns and 4 ALCL NOS. Of the 101 cases with ALK-1 immunohistochemistry, ALK-1 positivity was seen in 70.3 % cases The 2 year overall survival (OAS) rate for the ALK positive patients was 62.6 % and for the ALK-negative patients the 2 year overall survival rate was 59.1 % The 2 year disease free survival (DFS) rate for the ALK positive patients was 55.7 % and for the ALK negative patients it was 41.5 %. Amongst factors affecting both OAS and DFS were the Ann Arbor stage, ECOG performance status, IPI and Histological subtype (small cell vs. others). Presence of lesser numbers of hallmark cells was associated with poor prognosis and extent of surrounding reactive

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320 inflammatory component also negatively impacted DFS. In the multivariate analysis using Cox-regression model, Histological subtype was the only significant factor. The ALK status did not impact either DFS/OAS. Conclusion: Thus ALK is not the final word in ALCL and factors like stage, IPI and histological features impact survival in the patients with more adverse clinical features.

Abstract No. 121 Leukemic Presentation of ALK1 Positive Anaplastic Large Cell Lymphoma Anurag Gupta, Mayur Parihar, Arun SR, Arpita Bhattacharyya, Deepak K Mishra, Mammen Chandy Tata Medical Center, Kolkata Summary: We present a rare case of leukemic ALK1 positive anaplastic large cell lymphoma (ALCL) presenting with very high white blood cell (WBC) counts and circulating large cells diagnosed on cytogenetic analysis. Introduction: Leukemic presentation of ALK1 positive ALCL is uncommon with near to 20 cases described in literature and can be a diagnostic enigma. Commonly associated with small cell histology, we present a 2 year old female patient presenting with respiratory distress, high counts and large circulating cells. Materials and Methods: Case Report: The patient presented with a 3 week history of fever and abdominal distension and respiratory distress. Examination revealed tachypnoea, generalized lymphadenopathy and hepatosplenomegaly. Hemogram revealed low hemoglobin, high WBC count (TLC: 224,000/cmm) with 38 % large atypical monocytoid cells, shift to left in the myeloid series and low platelet counts. Bone marrow aspirate and trephine showed a hypercellular marrow with atypical cells which were positive for CD3, CD30 and CD43. Flowcytometric analysis suggested a lymphoproliferative disorder. Cytogenetic analysis showed a neoplastic clone characterized by a balanced translocation t(2;5)(p23;q35), resulting in fusion of ALK gene on chromosome 2 with NPM1 gene located on chromosome 5 along with trisomy 7. Biopsy of the right axillary lymph node was consistent with the diagnosis of ALK1 positive ALCL. After an initial response to disease the patient succumbed to the disease. Conclusion: Leukemic presentation of ALCL can be a diagnostic enigma and requires a high index of suspicion. We emphasize the importance of multidisciplinary approach and cytogenetic analysis in diagnosing these cases.

Abstract No. 122 Lymphoma Developing Concurrently in Patients with Myeloproliferative Neoplasm: Report of 2 Cases K Subramaniam, Marie T Manipadam1, Sheila Nair1, Aby Abraham2, Biju George2, Rekha Pai1, B Poonkuzhali2, Vivi Srivastava3 Department of General Pathology1, Department Clinical Haematology2, Department of Cytogenetics, Christian Medical College, Vellore Summary: Lymphoma occurring as a synchronous malignancy with myeloproliferative neoplasm is a rare entity. We present two patients with myeloproliferative disorder concurrently associated with lymphoma. Introduction: Case 1: A 25 year old lady presented with right lower limb weakness and on evaluation found to have soft tissue lesion involving the right sacral ala-S1–S3, with cortical erosions and involvement of spine and on biopsy diagnosed as anaplastic large cell lymphoma with bone marrow involvement. Clonal population of T

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 cells on T-Cell gamma chain rearrangement confirmed the diagnosis. During the course she was evaluated for leukocytosis and was diagnosed as chronic myeloid leukemia (BCR-ABL positive, t(9;22) and RTPCR:b3a2 positive). Case 2: A 69 year old gentleman diagnosed as JAK-2 positive myelofibrosis on treatment showed response to treatment. After one year, he presented with increase in spleen size. Radiologically he was found to have periportal, subcarinal and hilar lymph nodes. Follow-up bone marrow biopsy revealed Hodgkin lymphoma involving marrow, confirmed by immunohistochemistry. Materials and Methods: Routine tissue processing with hematoxylin and eosin staining and Immunohistochemistry was done. FISH, conventional cytogenetics, RTPCR and molecular analysis for T-cell rearrangement were performed for confirmation. Results: 2 Cases of MPN was concurrently diagnosed to have lymphoma which was proven by Immunohistochemistry and molecular studies. Conclusion: Development of lymphoma following myeloproliferative neoplasm is rare. Literature shows that patients with myeloproliferative neoplasm have a higher risk of developing lymphoid neoplasm than the general population. We report two cases of MPN presented with concurrent lymphoma.

Abstract No. 123 Extra Nodal Lymphoma: A Six Year Retrospective Analysis Meraj Fatima, M Ravindranath, K Manisha, Raju Bhaisare, S Gurmeet, M Bhalla Department of Pathology, Jawaharlal Nehru Hospital and Research Centre, Sector-9, Bhilai Summary: The incidence of extra nodal lymphoma (ENL) is rising throughout the world. Lymphomas arising in extra nodal sites are intriguing. The types of lymphomas encountered vary widely from one extra nodal site to another. Introduction: A substantial percentage of non-Hodgkin’s lymphoma (NHL) arises from tissues other than lymph nodes. Lymphomas arising primarily in extranodal sites can be diagnostically challenging because of lack of uniformity in definition and diagnostic criteria. Extranodal lymphomas have been reported to originate from any anatomic site. Materials and Methods: We analysed all the cases of lymphomas coming to our department in last six years (2007–2013). Paraffin embedded Haematoxylin and eosin (H and E) stained tissue sections were analysed by minimum of two pathologists. For Immunohistochemical (IHC) analysis, slides and paraffin embedded blocks were sending to an authorized laboratory Cases were reclassified based upon morphologic and immunophenotypic criteria according to World Health Organization 2008 classification. Results: A total of sixty nine cases of lymphoma were retrieved out of these twenty five (25/69; 36.2 %) were confirmed to be of extra nodal origin. The biopsy materials from these twenty five patients with ENL (15 Female, 10 Male; F: M = 3:2) were further analysed. The mean age of presentation was 54.8 years. Gastrointestinal system constituted the most common extra nodal site (7/25; 28 %) followed by oral cavity (6/25; 24 %). Diffuse large B-cell lymphoma was the most common histological type observed. Conclusion: Incidence of extra nodal lymphoma was 36.2 % in our study and gastrointestinal system was most commonly involved site of ENL and most common histological subtype is DLBCL.

Abstract No. 124 Crizotinib as a Targeted Therapy for Paediatric ALCL—A Case Report (Exploring an Unapproved Option)

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Palanki Satya Dattatreya Omega Hospitals, A Unit of Hyderabad Institute of Oncology Case report: A 4 years old female child presented in February 2013 with intermittent fever, weight loss, skin lesions, cutaneous abscesses and subcutaneous nodules (10–14 days duration). Excision of one of the skin abscess and a lymph node revealed CD 30 positive, ALK positive, Anaplastic Large Cell Lymphoma. A whole body PET-CT scan performed on 8th February 2013 showed multiple metabolically active supraclavicular, axillary, retroperitoneal and pelvic lymphadenopathy with many metabolically active soft tissue nodules. She was initiated on the BFM NHL 90 protocol (Seidemann et al. in Blood 2001; 97:3699–3706). Cytoreductive prephase was initiated on 8th February 2013 followed by course AA from 18th February 2013; course BB from 8th March 2013. A whole body PET-CT scan (4th April 2013) revealed complete metabolic and morphologic response to treatment. Hence, the protocol was continued (course CC from 5th April 2013, course AA from 27th April 2013, course BB from 18th May 2013 and course CC from 9th June 2013). She recovered fully from the cytopenic complications of course CC by July 2013. She was told to come for a clinical review in the first week of August 2013. However, she again became symptomatic in the last week of July, 2013 and a complete clinico-radiologic re-evaluation on 31st July 2013 was remarkable for multiple metabolically active cervical, axillary, mediastinal, retroperitoneal, iliac and inguinal lymphadenopathy with involvement of spleen. Additionally, the peripheral blood showed high WBC count with large numbers of atypical lymphocytes. A bone marrow examination could not be performed in view of her poor general condition. She was initiated on the oral ALK inhibitor, Crizotinib on the same day (31st July 2013) and she responded dramatically. Her performance status improved in a couple of days, the peripheral blood parameters normalized in a week and a PET-CT performed after four weeks of Crizotinib (27th August 2013) was completely normal, morphologically and metabolically. Conclusion: This case report signifies the advantage of Crizotinib as a targeted therapy to treat ALK positive Anaplastic large cell lymphoma.

Abstract No. 125 Unusual Presentation of Non Hodgkin Lymphoma Deepak Mulchandani, Sujata Sharma,Vishal Jadhav, Priyashree Mukherjee, Ratna Sharma, Mamta Manglani Division of Pediatric Hematology-Oncology, Lokmanya Tilak Municipal Medical College & General Hospital, Sion, Mumbai Introduction: Lymphoma generally presents with lymphadenopathy and hepatosplenomegaly. However unusual presentations may occur sometimes. This may delay the diagnosis, which may affect outcome. Some of the unusual presentations of lymphoma like pleural effusion, septic arthritis, pott’s spine etc have been mentioned in literature. Hence we report five cases of Non Hodgkin Lymphoma with unusual presentations. Case 1: A 2.5 years old female child presented with tibial swelling of around 2 9 2 cm along with pelvic mass involving retroperitoneum, pancreas, left ovary & bilateral kidneys. Biopsy of tibial swelling confirmed B cell lymphoblastic lymphoma. Additionally she also had right atrial mass on CT chest, suspected to be metastasis of lymphoma which resolved on chemotherapy. Case 2: 6.5 years old female child presented with progressive left eye proptosis over last 6 months with multiple convulsions. CT brain was suggestive of retroorbital mass. Biopsy of retroorbital mass proved to be tdt positive B cell lymphoma. CSF cytospin showed lymphomatous involvement. Case 3: 11 years male child with HIV infection presented with multiple fractures at neck and epiphyseal region of

321 bilateral femur. MRI bone was suggestive of nonhomogenous mass involving both femurs which on biopsy proved to be Non Hodgkin lymphoma. Case 4: 7 years old male child came with a non healing wound over left leg since one month, which required amputation. Histopathology of amputed leg was suggestive of Non Hodgkin Lymphoma. Case 5: 12 years old male child presented with left sided massive pleural effusion. Pleural fluid cytology was confirmative of T cell lymphoblastic lymphoma. Conclusion: Hence high index of suspicion and early workup is required to avoid upstaging of lymphoma in children.

Abstract No. 126 Case Report of Six Patients of Mature B-Cell Lymphoma Treated with LMB 96 (IR) Protocol Saurabh Prasad, Harsha Panchal, Bhavesh Parekh, Asha Anand, Shailesh Talati, Apurva Patel, Sonia Parikh, Shilin N Shukla Department of Medical and Paediatric Oncology, Gujarat Cancer and Research Institute, Ahmedabad Summary: Mature B-cell NHLs in children are most typically BL (including atypical Burkitt lymphoma [aBL]) and DLBCL. BL is relatively common (about 40 %), where as DLBCL accounts for only 15–20 % of all childhood NHL. Introduction: Cure rate of childhood mature B-cell lymphoma have significantly improved over the last 25 years, largely due to prospective studies including LMB and BFM. FAB/LMB 96 trial have demonstrated a four year EFS of 93.4 % for intermediate risk group B defined as not resected stage I/II and CNS negative advanced stage III/IV. Materials and Methods: We here report the profile, response rate, toxicity and short follow up of 6 patients with intermediate risk Mature B-cell lymphoma (BC, BLL & DLBCL), stratified by stage defined by Murphy at St Jude Children Research Hospital, treated on LMB 96 PROTOCOL between Oct’12– Mar’13 at our institute. Results: The age range of patients was between 5–13 years. All patients were male, four were of BL and two were of DLBCL. The most common primary site was abdominal. Two patients presented with intestinal obstruction. All five patients achieved CR before second cycle CYM. At a median follow-up of five months (range 2.5–7.5 months) there is one relapse, who is being treated with R-ICE. Most common toxicity was neutropenia, thrombocytopenia and fever. There was no mortality. Conclusion: LMB96 protocol have good response rate with manageable toxicity in intermediate risk Mature B-cell lymphoma but long term follow up is required.

Abstract No. 127 Importance of Clinical Correlation in Diagnosis of Angioimmunoblastic Lymphoma: A Case Report Tathagata Adhikari, Subhalakshmi Mukhopadhyay Medical College, Kolkata Summary: Angioimmunoblastic lymphoma is peripheral T cell lymphoma of germinal center T helper cell origin. Clinical presentation of this tumour is like a complex systemic disease condition. Lymphadenopathy is generalized but the size of the affected lymph node is usually less than 3 cm in size and they remain mobile. Constitutional symptoms include fever, polyclonal gammopathy. Presence of circulating immune complex and autoantibodies, autoimmune haemolytic anaemia, proneness to certain infections like Mycobacteria, Cytomegalovirus, Aspergillus, Pneumocystis carinii. A case of angioimmunoblastic lymphoma diagnosed by typical but

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322 bizarre clinical features, a missed histological diagnosis by first biopsy of lymphnode and proved by another biopsy on a later time after some progression of the disease and then proved by immunohistochemical positivity for CD 5, CD3, CD 10 is being reported here as it is as important case for a clinician and pathologist worth noticing. Clinical features included tuberculous meningitis and gangrene of fingers, along with other features. Conclusion: Awareness of the complex presentation of this variety of non-Hodgkin lymphoma is important for reaching the diagnosis.

Abstract No. 128 Primary Hepatic Lymphoma—A Rare Case Report Abhishek Purohit, Mukul Aggrawal, AK Dinda, P Mishra, R Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi-110049 Summary: Primary hepatic lymphoma (PHL) is a very rare malignancy representing approximately 0.016 % of all cases of nonHodgkin’s lymphoma (NHL) and 0.4 % of extranodal NHL. Because of its rarity, non-specific clinical symptoms, laboratory and imaging performance, PHL was often misdiagnosed as hepatitis, primary liver cancer or metastatic tumor. In this case report, we present a patient with pathologically confirmed primary hepatic diffuse large B cell lymphoma. Introduction: PHL, which is defined as a lesion or lesions confined to the liver without involvement of other organ or lymph nodes, is extremely rare. Diagnosis of PHL requires a liver biopsy compatible with lymphoma and absence of lymphoproliferative disease outside the liver. Materials and Methods: A 58 year old female, presented with complaints of high grade fever, epigastric discomfort and non bilious vomiting of five months duration and jaundice of one month duration. On examination, she had tender hepatomegaly of 10 cm below the costal margin. Results: On investigations, her liver function tests were markedly deranged. Ultrasonography and contrast enhanced computed tomography of abdomen revealed a large hypoechoic lesion in the left lobe of liver. Histopathologic examination of liver biopsy revealed features of diffuse large B cell lymphoma. Bone marrow examination showed normal hematopoietic elements without lymphoma deposit. Conclusion: PHL, though a rare disease, should be considered in an elderly who presents with liver mass or infiltration. Although hepatocellular carcinoma or metastatic diseases are more common, absence of elevated levels of CEA and AFP and hypoechoic liver mass should indicate a search for presence of PHL for which a biopsy is mandatory.

Abstract No. 129 A Rare Case of Primary Hepatic T cell Lymphoma Sanjay Mishra, AK Tripathi, Deependra K Yadav, Ayush Shukla, Yogendra Yadav King George’s Medical University, Lucknow Summary: Primary hepatic lymphoma (PHL) is rare and represents approximately 0.016 % of all cases of non-Hodgkin’s lymphoma (NHL). The majority of these are B-cell NHL of diffuse large B-cell type. Primary T-cell lymphoma constitutes approximately 5–10 % of all PHLs arising in the liver, 90 % being B-cell type. Peripheral T-cell lymphoma, cd hepatosplenic T-cell lymphoma and ab hepatosplenic T-cell lymphoma are the common T-cell lymphomas

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 involving hepatic parenchyma. We encountered a case presenting with gross hepatomegaly extending beyond umbilicus, mild ascites, pedal oedema, icterus and dyspnoea. Haemogram showed moderate anaemia with counts. Bone marrow aspiration showed erythroid hyperplasia with dimorphic anaemia. There was no evidence of atypical lymphoid cells in peripheral blood of bone marrow. We present a rare case of primary T-cell lymphoma presenting as primary liver involvement without splenomegaly, lymphadenopathy, bone marrow or peripheral blood involvement. Introduction: Primary T-cell lymphoma is rare, and few cases have been reported in the literature, particularly from the Indian Subcontinent. Early diagnosis can be made by high suspicion index and by simple screening methods such as fine needle aspiration cytology and serological tests. Primary T-cell lymphoma carries a poor response to therapy, but in our case the patient is in progression-free survival for the last year. Materials and Methods: A 40-year-old woman, belonging to a low socioeconomic group, presented with progressive distension of abdomen primarily in the right hypochondrium for 1 month duration, and for the previous week she had developed progressive icterus, dyspnoea and pedal oedema. She also had mild pain over her right hypochondrium. She had no fever, night sweats, bony pain, bleeding from any site, malaena, altered bowel habits, cough, haemoptysis or nodular swelling elsewhere in body. On examination, she was looking ill and dyspnoeic. She had massive hepatomegaly which was tender and extended beyond umbilicus. Also, she had mild ascites and pedal oedema. There were no splenomegaly, lymphadenopathy, any other palpable organomegaly or skin lesions. She did not have any significant/relevant medical or family history. Results: Her complete blood count showed haemoglobin of 10.4 g/dl, total leucocyte count of 9,000/mm3 with granulocytes constituting 65 %. Platelets were 2.5 9 105/mm3. General blood picture and bone marrow aspiration/biopsy showed no atypical lymphocytes or infiltration. Her biochemical investigations revealed progressive jaundice (bilirubin went up to 6.7 mg/dl), alanine transaminase 98 IU/l, aspartate aminotransferase 258 IU/l, serum alkaline phosphatase 2513 IU/l and serum lactate dehydrogenase (S-LDH) 688 IU/dl. Serum protein was found to be 4.5 g/dl with albumin being 2.4 g/dl. Serum uric acid was raised to 7.2 mg/ dl. a-Fetoprotein was found to be 0.68 ng/ml (normal value \10 ng/ ml). Her viral profile of hepatitis A virus, hepatitis B virus (HBV) and hepatitis C virus (HCV) was negative. There was no contributory evidence of autoimmune hepatitis. Fine needle aspiration of the mass showed sheets of atypical lymphoid cells with moderate pleomorphism (Fig. 1). There were few small clusters of hepatocytes, showing fatty changes, interspersed within these atypical lymphoid cells. A provisional diagnosis of lymphoma was established. Thorough search for evidence of lymphadenopathy was made both clinically and by imaging studies. Contrast-enhanced CT and plain CT of whole abdomen and thorax showed gross hepatomegaly measuring upto 30 cm in size with well-preserved hepatic architecture, homogeneous parenchyma with normal homogeneous attenuation value. Spleen and lymphnodes were within normal limits. Histopathological examination revealed disorganised architecture of liver parenchyma with diffuse infiltration of medium-sized round to oval cells mainly concentrating around the biliary ductules and infiltrating the sinusoids (Fig. 2). The hepatic cells show fatty changes and bile stasis. Immunohistochemistry showed positivity for CD 3, CD 5, T-cell (UCHL1) and it was negative for cd T-cell receptors (cd-TCRs) and ab TCR,CD20, CD 23, CD 10, CD 30, CD7, CD 56, Bcl2 and cyclin D1. Conclusion: Primary T-cell lymphoma of liver is very rare, although secondary involvement of liver in lymphoma is very common. (1) Primary T-cell lymphoma of liver can be very aggressive and require early diagnosis and therapeutic intervention. (2) When treated early response may be better than reported in various studies.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Abstract No. 130 Primary Hepatic Burkitt’s Lymphoma: A Case Report and Literature Review Vashista P Maniar, K Parikh Sonia, N Shukla Shilin, S Talati Shailesh, S Anand Asha, A Shah Sandip, P Panchal Harsha, A Patel Apurva, B Parikh Bhavesh, Goyal Divesh, Gohel Vandana, Raut Shreeniwas Department of Medical and Paediatric Oncology, GCRI, Ahmedabad Summary: We report a 21 year old young female referred to our institute with a working diagnosis of liver metastasis with unknown primary. Her examination revealed isolated gross hepatomegaly. On biopsy, histopathology was suggestive of Burkitt’s lymphoma (BL), which was confirmed with immunohistochemistry and mib index. We report our experience in treating her with a short duration, highly intensive pediatric inspired protocol (LMB 96). Introduction: Primary hepatic lymphoma (PHL) is a very rare malignancy, and constitutes about 0.01 % of all cases of non-Hodgkin’s lymphoma. Fewer than 150 cases of PHL have been reported in the Literature Worldwide. Regarding histological distribution of PHL, incidence of primary hepatic Burkitt’s lymphoma (PHBL) is only 3 % of all primary hepatic lymphomas. We report one such rare case and our experience in managing it. Materials and Methods: A case report— retrospective analysis of a rare presentation of an aggressive malignancy and review of literature. Results: We successfully treated the patient with short duration, highly intensive paediatric inspired protocol, LMB 96. Patient is in complete remission and on regular follow up since 6 months. Conclusion: Burkitt’s Lmphoma, is an aggressive mature B-cell NHL, and follows a rapid clinical course. It is fatal within months if left untreated. Because of its rarity, the diagnosis of PHBL is often not suspected until histopathological examination. Primary hepatic lymphoma should be considered as one of the differential diagnosis of a liver sol especially in young adults.

Abstract No. 131 Prostatic Non-Hodgkin Lymphoma—A Rare Case Report Muralidhar Gullipalli, Rajesh Kota, KV Krishnamani, Vijaygandhi Linga, Sadashivudu Gundeti, Lakshmi Srinivas Maddali, Raghunadharao Digumarti

323 PSA, CD3 were negative. Ki67 labelling index-47 %), features were suggestive of B-Cell type Non Hodgkin lymphoma. Staging evaluation with CECT neck and chest was normal. Bone marrow not involved. LDH was raised (1,250 IU/L). His international prognostic index (IPI) score was 3. We started him on standard CHOP chemotherapy. Patient improved symptomatically after first cycle. Conclusion: It should always be kept in mind that adenocarcinomas are not the only malignant entities encountered in the prostate gland and a thorough histological examination is the only safe way, in order to avoid diagnostic errors, especially in the absence of elevated PSA levels.

Abstract No. 132 Primary Prostatic Non Hodgkin Lymphoma: A Rare Presentation of Non Hodgkin Lymphoma Pradeep Kumar, Soniya Nityanand Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow Summary: We present a case of primary prostatic Non Hodgkin Lymphoma (NHL) diagnosed during work-up for symptoms of prostatomegaly. He was treated with systemic chemotherapy (R-CHOP) and continues to be in remission after 2 years of treatment. Introduction: Prostatic-NHL, whether primary or secondary, is a rare condition, and is usually diagnosed on biopsy. Here we report a case of primary prostatic NHL who was successfully treated with R-CHOP chemotherapy. Materials and Methods: This 63 years male patient presented to a surgeon with symptoms of an enlarged prostate and retention of urine. A TURP was done and biopsy was suggestive of NHL which was CD 20 and LCA positive. On further work-up he was staged as IE and he was treated with 6 cycles of systemic chemotherapy (R-CHOP). Results: He responded very well and post chemotherapy PET scan was suggestive of complete remission and continues to be in remission after 2 years of treatment. Conclusion: Involvement of the prostate by NHL may be one of the causes for prostatic enlargement and it may present without any other glandular enlargement or organomegaly as was in our case. The diagnosis is established by a biopsy of the prostate. Our patient achieved a complete remission with R-CHOP which thus may be a standard treatment for this type of NHL also.

Nizam’s Institute of Medical Sciences, Hyderabad, Andhra Pradesh Introduction: Lymphomas involving the prostate gland are very rare. Case report: We report a case of primary prostatic NHL in a 61 year old gentleman who presented with dysuria, hematuria and poor urinary stream of 3 months duration. He also reported intermittent fever, anorexia and a 10 kg weight loss over the preceding three months. His general physical and systemic examination was normal except for right lower limb edema. Digital rectal examination revealed a firm nontender enlarged prostate. Transrectal ultrasound scan showed a 88 9 76 9 75 mm (volume 260 cc) lobulated ill-defined, hypoechoic mass arising from the right lobe of prostate. Serum PSA was within normal limits. Contrast enhanced CTscan of the abdomen and pelvis showed a 13 9 10 cm, large heterogeneously enhancing solid mass involving the prostate, extending to right iliac fossa, right lateral pelvic wall, infiltrating posterior and lateral walls of the urinary bladder. Rest of the abdomen and pelvic structures were normal. Transrectal ultrasound guided biopsy from the prostatic mass was done. Histopathological and immunohistochemistry (LCA, CD20 were positive in lesional cells, whereas Cytokeratin, chromogranin,

Abstract No. 133 Primary Anaplastic Large Cell Lymphoma of Ilio Psoas Muscle a Rare Presentation Manveen Kaur, Md Zia ur Rehman Khan, Monal Dayal, CH Mohana Vamsy, P Satya Dattatreya Department of Pathology, Omega Hospitals, Hyderabad Introduction: Primary anaplastic large cell lymphoma (ALCL) of skeletal muscle is very rare. Case Report: We report a case of ALCL arising from the right ilio psoas muscle. A 60-year-old male presented with pain in the lower back. CT scan revealed a large heterogeneously enhancing mass in right ilio psoas muscle, eroding iliac bone. In view of the tumor’s location and the patient’s age, soft tissue tumors such as rhabdomyosarcoma were initially considered. However, histopathological examination and subsequent immunohistochemistry with LCA, ALK1, CD 30 yielded a diagnosis of ALK positive anaplastic large cell lymphoma.

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Abstract No. 134 Unusual Presentation of Precursor B Cell Lymphoblastic Lymphoma—A Case Report Gohel Vandana, Talati Shailesh, Shukla Shilin, Parikh Sonia, Anand Asha, Shah Sandip, Panchal Harsha, Patel Apurva, Parekh Bhavesh B, Maniar Vashishth, Goyal Devesh, Raut Shrinivas Department of Medical and Pediatric Oncology, Gujarat Cancer & Research Institute, Ahmedabad, Gujarat Summary: Unusual presentation of Precursor B cell lymphoblastic lymphoma—a case report. Introduction: Cutaneous swelling as presenting symptom of neoplasia in children is observed in neuroblastoma, histiocytosis, leukemia and lymphoma. Precursor B type lymphoblastic lymphoma with primary cutaneous involvement is very rare (\1 % of non-Hodgkin’s lymphoma). Materials and Methods: A 2 year old female child presented with 6-month history of asymptomatic scalp swelling without any B symptoms. On examination, right parietal region had a 3 9 3 cm sized firm, nonfluctuant, non-tender and smooth surfaced swelling without signs of inflammation. There was no peripheral lymphadenopathy and hepatosplenomegaly. Complete blood count and blood chemistries were unremarkable. Brain magnetic resonance imaging revealed soft tissue swelling in right parietal region without evidence of bone or nervous system involvement. Biopsy from swelling showed diffuse infiltrate of atypical small round lymphoid cells in the entire dermis. On immunohistochemistry atypical lymphoid cells were positive for CD10, CD79a, terminal deoxynucleotidyl transferase (TdT) and leukocyte common antigen, but negative for CD3, CD20, and CD34 suggestive of precursor B-cell type. Computerized tomography of neck, chest, abdomen, and pelvis revealed no lymphadenopathy or organ involvement. Bone marrow aspiration and trephine biopsy also showed no involvement, confirming isolated involvement of skin as presentation. Results: She was started on standard ALL treatment (MCP-841). Her tumour had clinically regressed at end of induction and at present she is on maintenance since 12 months and in complete remission. Conclusion: Primary cutaneous involvement of scalp without lymphnode involvement in child of precursor B cell lymphoblastic lymphoma is rare (18 cases reported) and may present a diagnostic challenge to treating physician.

Abstract No. 135 Spontaneous Rupture of Spleen in Hepatosplenic Non Hodgkin’s Lymphoma Sheetal Mahajan, AG Valand, Shubhangi V Agale, Varsha Bhatia, Diana Shah, SR Bijwe Grant Government Medical College, Byculla, Mumbai Summary: A 18 years male was brought to the hospital with complaints of fever with chills and breathlessness since 15 days. He was declared dead on admission and postmortem was performed. Liver and spleen were enlarged with spleen showing tears on inferior surface. Microscopic examination of spleen revealed non-Hodgkins lymphoma. Introduction: Spontaneous pathologic rupture of the spleen in hematologic malignancies is rare. The common malignancies in which the pathological rupture can occur are acute leukemias, non-Hodgkin’s lymphoma and chronic myelogenous leukemia. It seems that, apart from splenic infiltration by a hematologic disease, splenic infarcts and coagulation disorders are important pathophysiologic factors leading to rupture. Hepatosplenic T-Cell Lymphoma (HSTCL) is an uncommon neoplasm that comprises 5 % of peripheral

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 T-cell lymphomas. We present a case of spontaneous rupture of hepatosplenic lymphoma. Materials and Methods: A 18 years, male was brought to the hospital with complaints of fever with chills since 15 days and breathlessness. He was declared dead on admission and postmortem was performed. On gross examination, spleen was enlarged, pale, and soft with multiple grayish white tiny nodules. There were three tears over inferior surface of spleen. Results: On microscopic examination, splenic red pulp showed monotonous population of atypical lymphoid cells with large areas of haemorrhage and of necrosis. Liver showed congestion, with presence of atypical lymphoid cells in sinusoids and portal triads. IHC confirmed the diagnosis of hepatosplenic lymphoma. Conclusion: Pathological rupture of spleen is uncommon but is seen in hematological malignancies such as non-Hodgkin’s lymphoma.

Abstract No. 136 A Case Series of Plasmablastic Lymphoma with AIDS Ravikumar Narayan Wategaonkar, Bhavesh Parekh, Harsha Panchal, Asha Anand, Shailesh Talati, Apurva Patel, Sonia Parikh, Shilin N Shukla Department of Medical and Paediatric Oncology, Gujarat Cancer and Research Institute, Ahmedabad Summary: Plasmablastic lymphoma (PBL) has a predilection for oral cavity involvement, a feature not typically seen in other lymphoid malignancies with aggressive behavior. As patient may have poor survival with CHOP chemotherapy, more intensive therapies are recommended. Introduction: PBL is a distinct subtype of DLBCL, characterized by its aggressive nature and plasmacytic differentiation observed in the setting of HIV disease. PBL is a rare entity and commonly arises in the oral cavity of HIV-positive patients. PBL remains a diagnostic and therapeutic challenge given its peculiar morphology and an immunohistochemistry (IHC) similar to plasma cell myeloma and clinical course characterized by a high rate of relapse and death. Materials and Methods: Design: case series. We are reporting three cases of PBL in HIV patients with CD4 count of less than 200. All three patients presented with mass in oral cavity. Biopsy of mass lesion showed high grade lymphoma and IHC was positive for LCA, CD79a, CD138 and MUM1 and negative for CD20 suggestive of plasmablastic lymphoma. Two patients were treated with dose adjusted EPOCH regimen along with HAART. Third patient was treated with radiotherapy followed by CVP along with HAART. Results: One patient achieved more than partial response after three courses and in other two one course has been completed with good subjective response. All patients tolerated chemotherapy well without major complication. All patients are on chemotherapy at the time of evaluation. Conclusion: PBL has a predilection for oral cavity involvement with aggressive behavior. In view of the poor outcomes and no prospective therapeutic trials for PBL, new and aggressive therapies must be developed.

Multiple Myeloma (MM), Waldenstrom’s Macroglobulinemia (WM), Amyloidosis & Allied Disorders Abstract No. 137 Ki-67 Immunostaining and Its Correlation with Microvessel Density in Patients with Multiple Myeloma Himani, Meera Sikka, Usha Rusia

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi Summary: In our study involving 30 patients Ki-67 and MVD are found to correlate significantly with prognosis in multiple myeloma. Introduction: Multiple myeloma is a neoplastic plasma cell disorder. Ki-67 is a proliferative marker. We correlated Ki-67 with microvessel density and other prognostic markers in multiple myeloma. Materials and Methods: This study was conducted on 30 patients with newly diagnosed multiple myeloma (18 males and 12 females, median age 55 years). Proliferative activity of myeloma cells was analyzed by Ki67 antibody and correlated with Microvessel density (MVD) as assessed by CD34, plasma cell number, morphology, stage of disease, renal function, hematological and biochemical parameters. Clinical staging was done according to Durie Salmon criteria. Results: Ki-67 positivity was significantly higher (p \ 0.0001) in patients (range 35–80 %, mean 60.1 %) than controls (range 8–25 %, mean 18.1 %) and increased progressively with increasing stage of myeloma. MVD/ mm2 was significantly (p \ 0.0001) higher in patients (range 62–237/ mm2, mean 178.0/mm2) than controls (range 5.2–50/mm2, mean 18.3/ mm2). Ki-67 correlated significantly with MVD (r = 0.729, p \ 0.001). Ki-67 showed a statistically significant negative correlation with Hb, RBC count, platelet count, serum albumin and positive correlation with blood urea and LDH. MVD showed a statistically significant negative correlation with Hb, RBC count and positive correlation with B urea, LDH, S creatinine, b2 microglobulin and skeletal lesions. Ki-67 and MVD were significantly higher in patients with diffuse pattern of infiltration of plasma cells as compared to those with nodular and interstitial pattern. Conclusion: Ki-67 and MVD both are increased and are indicators of poor prognosis in myeloma.

Abstract No. 138 Plasma Cell Dyscrasias: A Diagnostic Approach, Army Hospital (Research & Referral) Experience Rigvardhan, Prosenjit Ganguli, P Sengupta, Bhushan Asthana, Brig R Bharadwaj, Brig Ajay Sharma, Maj Gen Velu Nair, UD Gupta, Rehan Ahmed

325 confirmative diagnosis and monitoring of therapy, a comprehensive analysis of all parameters (SPEP, IFE, sFLC, BM) should be done.

Abstract No. 139 Role of Bone Marrow Biopsy in Diagnosis and Prognosis of Multiple Myeloma Roopal Rathi, Tejinder Singh, N Gupta Department of Pathology and Medicine, Maulana Azad Medical College, New Delhi-110002 Summary: Bone Marrow Biopsy (BMB) plays crucial role in diagnosis of Multiple Myeloma (MM) with hypocellular and fibrotic marrow. Introduction: MM is commonest hematologic malignancy of old age. Diagnosis requires demonstration of increased monoclonal plasma cells in bone marrow. We aim to highlight role of BMB in diagnosis of MM. Materials and Methods: BMB of 30 patients of multiple myeloma were examined for pattern of involvement, cellularity, differentiation of plasma cells, residual haematopoiesis, fibrosis. CD38, CD138, kappa, lambda antibodies were used for diagnosis and assessment of clonality. 10 control cases with 10–15 % plasma cells were taken. Results: Age of patients ranged from 30-82 years. Pattern of involvement was diffuse in 9, focal in 14, interstitial in 7 cases. Morphology of plasma cells was classified (Griepp et al.) mature in 15, intermediate in 10, plasmablastic 5 cases. Residual haematopoiesis was adequate in 20 while inadequate in 10 cases. Of 8 cases with unsatisfactory bone marrow aspirate (BMA), 4 cases showed significant fibrosis. In other 4 cases, marrow cellularity was low (20–30 %) and tumor deposit was focal, thus, BMA was inadequate. 8 of 14 cases with focal deposits on BMB couldn’t be picked up on BMA. Monoclonality was demonstrable using CD38, CD138, kappa, lambda. Two cases with non-specific symptoms were diagnosed Smoldering Myeloma on basis of [10 % clonal plasma cells on BMB. Control cases showed polyclonality of plasma cells. Conclusion: Bone Marrow Biopsy is particularly useful for diagnosis of MM in cases with low cellularity and marrow fibrosis where BMA is inadequate.

Army Hospital (Research & Referral), New Delhi Summary: We wish to present a series of 733 cases of plasma cell dyscrasias who were subjected to serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE) and bone marrow studies in various permutations and combinations and to assess diagnostic accuracy in a given subset. Introduction: The measurement of biological markers for multiple myeloma serum and urine specimen has proven to be invaluable in detection and monitoring of disease progression. Newer modalities include nephlometric determination of serum free light chains (FLC) and more recently intact Ig subsets; IgGj, IgGk, IgAj and IgAk. Both of these markers have exhibited apparent value as diagnostic and prognostic indicators. As our understanding of the utility of these markers deepens, we achieve better understanding in how to most effectively use these markers in combination with other methods for monoclonal protein detection. Current international guidelines for identifying monoclonal gammopathies (MG) include serum protein electrophoresis, immunofixation electrophoresis and serum free light chain immunoassays with derived kappa/lambda ratios. Materials and Methods: Past five years data was revisited to ascertain the cases of plasma cell dyscrasias who had undergone SPE/IFE or both and results were tabulated. Results: Retrospective analysis of past five years cases revealed and interesting mix of diagnostic accuracy depending on the modalities employed which will be disclosed at the conference. Conclusion: For

Abstract No. 140 Emerging Role of Serum Free Light Chain (SFLC) Assay & Ratio in Plasma Cell Disorders: Experience in a Tertiary Care Hematology Laboratory T Sethumadhavan, J Kotwal, R Kapoor, V Dutta, V. Nair Hematology Section, Department of Pathology, AFMC, Pune Summary: Serum Free light chain (SFLC) assay is a diagnostic modality which has currently been advocated by International Myeloma Working Group (IMWG) as an essential component of the primary screening algorithm for suspected Monoclonal plasma Cell Disorders (PCD). We evaluated SFLC utility in diagnosis and monitoring of PCD with special reference to Quality control in laboratory. Introduction: SFLC assay has been included recently in the screening algorithm for suspected PCD. SFLC should be evaluated to know its utility in diagnosis and monitoring of PCD. Materials and Methods: We measured SFLC in 100 suspected cases of PCD over a period of one year by nephelometric method in AFMC, pune. Results: SFLC measurement helped in diagnosing 40 of 100 suspected cases of PCD. Among the 40 cases, 30 cases were symptomatic myeloma, 05 cases were Monoclonal gammapathy of undetermined significance (MGUS), 03 cases were Smouldering

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326 Myeloma (SM) and 02 cases were non-myeloma with raised SFLC. All the negative cases were tested again with serial dilution (Delta check), which then showed positivity in 02 cases, indicating antigen excess ‘Hook effect’. The 30 symptomatic myeloma cases were followed up while on a standard therapy for a period of one year with sFLC, which showed alteration in the SFLC levels, indicating its role in monitoring treatment response and prognosis. Conclusion: SFLC measurement is a valuable test in diagnosing and monitoring of PCD. Laboratory standardization and quality control of the test is of extreme importance for its utility.

Abstract No. 141 Phase (Ph) I/II Study of Elotuzumab Plus Lenalidomide/ Dexamethasone (Len/dex) in Relapsed/Refractory Multiple Myeloma (RR MM): Updated Ph II Results and Ph I/II Long Term Safety S Shankar, Sagar Lonial1,2, Sundar Jagannath2,3, Philippe Moreau4, Andrzej J Jakubowiak2,5, Marc S Raab6, Thierry Facon7, Ravi Vij2,8, Darrell White9, Donna E Reece10, Lotfi Benboubker11, Jeffrey Zonder2,12, Wei Deng13, Eric Bleickardt14, Anil K Singhal13, Paul G Richardson2,15, On Behalf of the 1703 Study Investigators Bristol Myers Squibb India Pvt. Ltd Summary: This study included a dose finding Ph I cohort (N = 28) and a Ph II cohort (N = 73). Here we update Ph II data and provide long term safety data from both cohorts. Materials and Methods: Patients (pts) treated with C1 (Ph I) or 1–3 (Ph II) prior therapies received Elo + Len/dex as described previously (Lonial JCO 2012; Richardson ASH 2012) until disease progression, unacceptable toxicity, or death. All pts received a premedication regimen including methylprednisolone, diphenhydramine or equivalent, ranitidine or equivalent, and acetaminophen to mitigate infusion reactions. Adverse events (AEs) in Ph I/II pts occurring B18 months (mo) (N = 98) were compared to AEs with a [18 mo onset in a subgroup of pts treated [18 mo (n = 49). This safety analysis excluded 3 Ph I pts treated with Elo 5 mg/kg. Results: In the Ph II cohort (median 63 years), objective response rate (ORR) was 84 %; 92 % with 10 mg/kg (n = 36) and 76 % with 20 mg/ kg (n = 37). At a median follow-up of 20.8 mo, median progression free survival (PFS) was not reached (10 mg/kg) and 18.6 mo (20 mg/ kg). Most common treatment emergent grade C3 AEs were lymphopenia (19 %), neutropenia (18 %), thrombocytopenia (16 %) and anemia (14 %). 15 pts discontinued due to AEs; none after 18 mo of treatment. There were 4 second primary malignancies; none were reported after 18 mo. Conclusion: Elo 10 mg/kg + Len/dex was generally well tolerated and resulted in a high ORR and encouraging PFS in pts with RR MM. AEs emergent after 18 mo of therapy were consistent with AEs during the initial 18 mo.

Abstract No. 142 Bortezomib Plus Dexamethasone Induction Followed by Autologous Stem Cell Transplantation in Multiple Myeloma: A Study from India Jeevan Kumar, RK Saran, Manorama Bhargava, Shyam Aggarwal Summary: In this retrospective analysis of Indian patients with newly diagnosed multiple myeloma (MM) Bortezomib plus dexamethasone induction followed by autologous stem cell transplantation (ASCT) had significantly improved the post-induction and post-transplant

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 response rates without affecting stem cell collection. Introduction: The use of novel agents for induction prior to ASCT has considerably improved the complete response (CR) rate in MM patients. There are very few studies from the developing countries on the use of novel agents like bortezomib followed by ASCT. Here, we report a retrospective evaluation of the efficacy and response rates of induction with bortezomib (Velcade) plus dexamethasone (VD regimen) followed by ASCT in Indian patients. Materials and Methods: Ten patients with newly diagnosed, symptomatic MM who had received four cycles of VD induction before stem cell collection were evaluated. VD induction comprised of bortezomib (1.3 mg/m2) and dexamethasone (40 mg) administered weekly. Stem cell mobilization was done by administration of granulocyte stimulating factor (G-CSF); 300 ˇlg twice daily for 5 days. High dose melphalan (200 mg/m2) was given for conditioning followed by stem cell transfusion. Thalidomide or lenalidomide was used as post-transplantation maintenance treatment. Results: Post VD induction, the overall response rate (ORR) was 90 % including 20 % CR, 40 % very good partial response (VGPR), and 30 % partial response (PR). These response rates were similar to observed in previous studies with the VD regimen. Post ASCT, the ORR was 100 %, including 80 % CR and 20 % VGPR. These response rates were higher than that observed in other similar studies. These variations may be due to less number of total patients in the present study. The 5-year overall survival and progression free survival rates were 65.6 and 57.1 %, respectively. Adverse events which included infections, peripheral neuropathy, herpes zoster and thrombocytopenia, were manageable. Conclusion: The VD induction regimen was effective and well tolerated in this retrospective analysis of Indian patients with newly diagnosed MM. It significantly improved the post-induction and posttransplant response rates without affecting stem cell collection.

Abstract No. 143 Efficacy and Tolerability of Bortezomib Plus Dexamethasone Induction in Newly Diagnosed Multiple Myeloma Mir Sadaqat Hassan Zafar1, Afaq Ahmad Khan1, Anupama Jaggia1, RK Ganjoo2, Shyam Aggarwal3, Manorama Bhargava1 1

Department of Hematology, Sir Ganga Ram Hospital, New Delhi, Department of Internal Medicine, Sir Ganga Ram Hospital, New Delhi, 3Department of Medical Oncology, Sir Ganga Ram Hospital, New Delhi 2

Summary: Bortezomib plus dexamethasone was given to newly diagnosed patients of multiple myeloma with the aim to assess the efficacy and tolerability of this induction regimen. As compared to the previous data, higher response rates and better safety profile was seen. Introduction: Bortezomib, a proteasome inhibitor has been investigated as part of a number of different induction regimens. Significantly higher response rates with an added advantage of being effective in patients with renal failure and adverse cytogenetics have been observed. Materials and Methods: This study was conducted in the Departments of Hematology and Medical Oncology at Sir Ganga Ram Hospital New Delhi. Four cycles of bortezomib plus dexamethasone induction were given to 25 patients of newly diagnosed multiple myeloma and monitored for response and safety. All the patients received prophylactic acyclovir. Results: Our study population comprised of 17 males and 8 females (50–80 years). Overall 32 % of patients attained complete response (CR), 56 % attained very good partial response (VGPR), 4 % attained partial response (PR) and 8 % showed no response. Forty four percent of patients (11/25) had renal failure at presentation. Amongst the renal failure patients, 45.45 % were in VGPR, 36.36 % in CR, 9 % in PR and 9 % showed

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 no response. Out of 11 patients of renal failure, 8 patients showed complete normalization of serum creatinine (72.27 %) and 3 patients showed [50 % reduction in serum creatine from baseline. Twenty four percent of patients developed bortezomib induced neuropathy (grade 1 = 16 %, grade 2 = 4 %, grade 3 = 4 %, grade 4 = none). In our study, 56 % of patients showed hematological side effects, out of which thrombocytopenia was seen in 32 % (grade 1), anemia in 16 % (8 % grade 2 and 8 % grade 3) and leukopenia in 8 % (grade 1). Other side effects seen were mild nausea (52 %), vomiting (20 %), fever (12 %), diarrhea (8 %) and constipation (4 %). None of the patients showed activation of herpes zoster. Conclusion: Bortezomib plus dexamethasone induction regimen showed higher response rates in all the ISS (International Staging System) categories and was well tolerated even in elderly and renal failure patients.

Abstract No. 144 Plasma Cell Leukemia in a Very Young Patient: Management Issues Sriram Ravichandran, Vishwadeep Khushu, Monali Gupta, Saurabh Bhave, Anupam Chakrapani, Reena Nair, Mammen Chandy Tata Medical Center, Kolkata, West Bengal Summary: Plasma cell leukemia is a rare plasma cell disorder in young patients with a very poor prognosis. Case report: We report a case of plasma cell leukaemia in a young patient and the deliema’s in its management. A 24 years old nurse presented with back pain, nausea, vomiting, abdominal pain and distension of 4–5 days duration. She was noted to have anaemia, leucocytosis and renal failure (urea-238 mg/dl, creatinine-9.9 mg/dl, uric acid-21.3 mg/dl). Peripheral blood film showed atypical plasmacytoid cells. BM aspiration/biopsy was consistent with plasma cell dyscrasia. She had complex karyotype along with gains of 1q and losses of 1p in the neoplastic clone. IFE suggested M band and the free light chain ratio was 137.5. Treatment: Patient was commenced on dialysis and was started on IV steroids, TLS Prophylaxis and definitive treatment with Bortezomib, Thalidomide, and Dexamethasone (VTD). Response post 3 cycles was VGPR and in sCR post 6 cycles. She underwent consolidation with cytoreductive autologous HSCT followed by maintenance Thalidomide. Follow up: IFE 4 months posttransplantFaint M band, SPE-Definite M band at 6 months. Peripheral blood11 % circulating plasma cells. What next? Hyper CVAD to attempt second remission followed by Haplo transplant (No matching donor). Conclusion: Most patients will relapse with standard chemotherapy and median survival remains poor.

Abstract No. 145 Autologous Peripheral Blood Haematopoietic Stem Cell Transplant in Post Kidney Transplant Multiple Myeloma Rishi Dhawan, Gaurav Prakash, Alka Khadwal, Vivek Kumar, Ritambhara Nada, Neelam Varma, Subhash Varma, Pankaj Malhotra Post Graduate Institute of Medical Education and Research, Chandigarh, India Summary: Autologous stem cell transplantation for multiple myeloma achieves superior response rates compared to chemotherapy alone. We present a patient who underwent kidney transplant for end stage renal disease. He subsequently developed multiple myeloma, 3 years later with kappa light chain deposition disease in transplant

327 kidney. He underwent an autologous hematopoietic stem cell transplant at our institute. This case is described for its rarity. Introduction: Multiple myeloma occurring after kidney transplantation is exceedingly rare. Further, there are only a few cases that had undergone autologous hematopoietic stem cell transplantation following renal transplantation. Materials and Methods: A 44-years old gentleman underwent allogeneic renal transplantation for end stage renal disease at our institute in June 2009. Post renal transplant he received immunosuppression with sirolimus and prednisolone. His renal functions were normal and he did not have any rejection episodes. He presented in June 2012 with weakness, easy fatigability and weight loss. Serum creatinine was 2.96 mg/dl. The possibility of graft rejection was considered and allograft kidney biopsy was done that showed kappa light chain deposition disease. Skeletal survey showed no lytic lesions. Serum protein electrophoresis showed M band of 2.36 g/dl in gamma region. Beta 2 microglobulin was 7.3 g/dl. Bone marrow aspiration and trephine showed 8 % plasma cells. His serum calcium was 9.2 g/dl and haemoglobin was 13.5 g/dl. He was started on VTd regimen (Bortezomib, Thalidomide, weekly dexamethasone). After 4 cycles, he achieved stringent CR and his serum creatinine came down to 1.6 g/dl. Results: He underwent autologous peripheral blood stem cell transplant on 30/5/13. Conditioning regimen used was Melphalan 140 mg/m2. It was administered in two fractionated doses of 70 mg/m2 given 12 h apart. Neutrophil and platelet engraftment occurred on Day + 9. His post transplant clinical course was uneventful. Tacrolimus was given throughout the course of transplant. Day + 30 renal function assessment showed serum creatinine: 1.3 mg/dl. Conclusion: Multiple myeloma is a rare malignancy in the post kidney transplant period. The patient described has been successfully treated with high dose chemotherapy followed by autologous hematopoietic stem cell transplant.

Abstract No. 146 Smoldering Multiple Myeloma-Report of 6 Cases Suniti Pahwa, Tejinder Singh, Naresh Gupta Maulana Azad Medical College, New Delhi Summary: Smoldering myeloma is asymptomatic myeloma and defined by diagnostic criteria. Six cases diagnosed in our department in last 3 years were taken. Introduction: Smoldering multiple myeloma is an indolent form of multiple myeloma. International myeloma working group gave the diagnostic criteria: M-protein in serum at myeloma levels (30 g/L) AND/OR 10 % or more clonal plasma in marrow. No end organ damage. Materials and Methods: The hemogram, bone marrow aspirate and marrow biopsy findings in the six cases were correlated with radiological findings, serological values. Results: The 6 patients were in the age group of 50–70 years presented with weakness and lassitude. Hemograms findings: hemoglobin 6–11 g/ml, TLC-6,000-11,000/ml, Platelets 70,000–1 lac/ml. PERIPHERAL SMEAR showed normocytic normochromic red blood cells with rouleaux formation. BONE MARROW ASPIRATE was adequate in 4 cases and in 2 cases it was diluted. In the 4 cases plasma cells constituting 15–28 % of marrow cells. BONE MARROW BIOPSY Biopsy in all 6 cases showed aggregates of plasma cells amongst hematopoietic cells. Plasma cells were CD38/CD138 positive and 4 cases showed kappa positivity while two cases were lambda positive. M protein levels were in range of 32–46 g/l. SKELETAL SURVEY and KIDNEY FUNCTION TEST were normal. A diagnosis of smoldering multiple myeloma was made. Conclusion: Bone marrow biopsy is essential in suspected smoldering myeloma cases to confirm plasma cell infiltration and monoclonality.

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Abstract No. 147

Abstract No. 149

Multiple Myeloma Presenting as Pancytopenia: A Case Series 1

2

2

2

Myelomatous Pleural Effusion: A Case Report

Jaimin Panchal , HB Sridevi , KS Pooja , Sharada Rai , MS Somesh3

Hardik Khandelwal, Jyoti R Kini, Ashok Prabhu, Krishna Prasad, K Alok Shetty

1

Kasturba Medical College, Manipal University, Mangalore, Karnataka

Department of Pathology, Yenepoya Medical College, Mangalore, Department of Pathology, Kasturba Medical College, Mangalore, 3 Department of Anatomy, Srinivas Medical College, Mangalore 2

Summary: Multiple myeloma can present as pancytopenia. The diagnosis is established with a proper correlation of clinical history, laboratory evaluation and confirmation with bone marrow. Introduction: Multiple myeloma presenting as pancytopenia is reported in many case studies. Generally, marrow infiltration by malignant plasma cells is the cause of pancytopenia in these cases. The present study was conducted to identify the clinical-hematological profile of multiple myeloma patients presenting with pancytopenia. Materials and Methods: A prospective and retrospective study of cases of multiple myeloma presenting as pancytopenia were evaluated clinically along with hematological and other laboratory parameters for a period of 2 years. Results: A total of 10 cases of myeloma presented with pancytopenia with a mean age of 66.3 years & M:F: 4:1. The average ESR was 108 mm/h. The mean values of hemoglobin, total leucocyte count and platelet count were 7.7 mg/dl, 2,898 and 68,200 cells/cumm respectively. The diagnosis of multiple myeloma was confirmed by bone marrow examination with average plasma cells of 62 %. A high-resolution serum electrophoresis showed a dense, sharp and wide M band in the gamma globulin region. Conclusion: The study shows that multiple myeloma can present with pancytopenia. An elderly patient with non-specific complaints and a raised ESR is the usual presentation in multiple myeloma. The presence of pancytopenia may be misleading in such cases. Hence, pathologists and clinicians must keep a differential of myeloma in cases presenting with pancytopenia as it can be easily diagnosed by bone marrow examination & protein electrophoresis.

Abstract No. 148 Plasmacytoma: A Case Report A Jaisri, M Ravikumar, SVO Chandrakumar, D Dhanalakshmi, TE Ramesh, Sainath Reddy, Kalanidhi, D Mitra, Sare Paul Southern Railway Headquarters Hospital, Perambur, Chennai 23 Summary: A 68 year old male patient presented at the physician’s office with backache and paraparesis. He was then referred to the Orthopedician. Traumatic fracture of D10 was diagnosed. Posterior stabilisation and laminectomy was done. Plasmacytoma was diagnosed on histopathological examination. Introduction: A 68 year old male patient presented with backache at the physician’s office and subsequently developed paraparesis. He was referred to the Orthopedician and diagnosed with traumatic fracture at D10 vertebra. Materials and Methods: A biopsy was taken from D10 vertebra and sent for histopathological examination. A posterior stabilisation and decompression of D9–D11 was done by laminectomy. Results: Histopathological examination showed numerous plasma cells and atypical plasma cells; so a serum protein electrophoresis and immunohistochemistry was suggested. Conclusion: Protein electrophoresis showed monoclonal gammopathy with M spike and immunomarkers were positive therefore a diagnosis of plasmacytoma was established.

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Summary: We report the case of a 75 year old man with multiple myeloma presenting with myelomatous pleural effusion, 2 months following remission after chemotherapy. Introduction: Malignant myelomatous pleural effusion are rare, occurring in less than 1 % of cases with multiple myeloma. Materials and Methods: A 75 year old man presented with breathlessness and right sided chest pain. Radiological examination revealed right sided pleural effusion which was tapped. Results: Cytological examination of aspirated fluid showed numerous singly scattered abnormal cells with eccentric nuclei and binucleate cells. A diagnosis of myelomatous pleural effusion was made and confirmed by monoclonal M band in the gamma region on pleural fluid protein electrophoresis. On obtaining further history, it was revealed that the patient was a diagnosed case of multiple myeloma, 2 months post chemotherapy. The patient succumbed to his illness within 1 week of cytological diagnosis. Conclusion: Myelomatous pleural effusion predicts a poor prognosis as seen in our patient. A high index of suspicion is required to diagnose patients on chemotherapy with myelomatous effusion.

Abstract No. 150 MONEY MATTERS! Economic Burden of Multiple Myeloma and Its Impact on Patients Undergoing Chemotherapy in Tertiary Care Centers—A Cross Sectional Study K Alok Shetty, Krishna Prasad, Jyoti R Kini Kasturba Medical College, Manipal University, Mangalore Introduction: The incidence of cancer including hematological malignancies is increasing worldwide and so are the economic costs associated with its management. There is limited scientific information available on the actual expenses borne by patients and the perceived impact of costs of cancer treatment. Multiple myeloma, a commonly managed hematological malignancy in our setup was opted for the study. Understanding the economical factors would aid in economically reviewing the current modalities of diagnosis and therapy of multiple myeloma & provide optimum patient care. Materials and Methods: Study population: Multiple myeloma patients who were undergoing chemotherapy in the months of July and August, 2011. Sample size-30 patients; Inclusion criteria: Patients who had finished at least one cycle of chemotherapy prior to the time of interview Methodology: Questionnaire based interview with aid of patient records. Results: 33 % (10 out of 30) of the patients were insured for treatment and could opt for more expensive chemotherapy regimens. All patients spent less than Rs. 10,000 for the diagnostic workup. Expenses for chemotherapy varied from regimen to regimen, with bortezomib containing regimens being the most expensive. Complications of the disease such as renal failure and pathological fractures significantly contributed to the expenses borne. About 60 % (18 out of 30) of the patients and families were distressed due to the expenses borne. 80 % of the patients who were insured were generally not distressed due to the costs. Conclusion: Patients who were insured had many benefits most importantly lesser distress caused by the expenses borne for chemotherapy. The expenses borne for investigations prior to diagnosis were relatively minimal in most patients.

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Abstract No. 151 Cryoglobulinemic Vasculitis as a Presenting Features of Multiple Myeloma Sumitra Sivakoti, Shantveer G Uppin, T Roshini Paul, Megha S Uppin, Liza Rajshekar Nizams Institute of Medical Sciences(NIMS), Hyderabad Summary: Cryoglobulins(CG) are immunoglobulins that have tendency to precipitate in temperatures below 37 °C and dissolve with rewarming. Here we present a case of 50 year male who presented with poly arthralgia of two month duration and haemorrhagic vesicles over the feet for 15 days. skin biopsy was done, diagnosed as Cryoglobulinemic vasculitis. Further investigation confirmed multiple myeloma. Introduction: Type I CG accounts for 10–15 % of CG and is most commonly encountered in patients with a plasma cell dyscrasia such as multiple myeloma or Waldestrom macroglobulinemia. These cases are typically associated with hyperviscocity syndrome and small vessel vasculitis. Materials and Methods: Clinical and Investigative findings were studied. Results: A 50-year-old male presented with poly arthralgia of two month duration and haemorrhagic vesicles over the feet for 15 days. Laboratory investigations revealed haemoglobin 8.9 g/dl with normal WBC count and platelets. Mild rouleaux was noted on peripheral smear. Serum creatinine was 1.8 mg/dl. Vasculitic work up was negative and viral markers were negative. Skin biopsy was done. Biopsy showed eosinophilic PAS positive deposits in the lumen and on the wall of the vessels with evidence of inflammatory infiltrate in wall and endothelial swelling suggestive of vasculitis. In view of these findings, serum electrophoresis and bone marrow evaluation was sought for. Serum electrophoresis revealed monoclonal M protein spike of IgG kappa type. Bone marrow aspiration cytology confirmed marrow plasmacytosis (55 %). Conclusion: The association of cryoglobulinemia with multiple myeloma is rare. The cutaneous manifestations are present in most patients varying from purpuric lesions to severe necrotizing lesions. Knowledge of MM related CG is necessary for early diagnosis. It requires prompt and effective treatment and plasmapheresis to achieve a rapid control of CG-related symptoms and to avoid disease relapse.

Abstract No. 152 Waldenstrom Macroglobulinemia: An Experience in a Tertiary Centre Priyanka Samal, SS Roy, S Samanta, UK Nath, P Chakrabarti, U Chowdhury

329 evaluated for all patients. Results: Of the 6 patients, 3 received cycles of Bortezomib + Dexamethasone + Rituximab. 1 received methyl prednisolone + Rituximab.1 was treated with Bendamustine + Bortezomib + Dexamethasone + Rituximab along with plasmapheresis. 1 received Bendamustine + Bortezomib + Dexamethasone 1 cycle followed by Bortezomib + Dexamethasone + Rituximab. All these patients had received blood products support during the treatment. 5 patients are symptomatically better and have achieved a reduction in IgM levels after chemotherapy while 1 had no response. 3 patients are dead while 3 are still on therapy. Conclusion: A precise therapeutic algorithm for therapy of WM remains to be defined given the paucity of randomized clinical trials. Combination therapy, particularly with rituximab, has been associated with improved clinical outcomes.

Abstract No. 153 Bone Marrow Trephine Biopsy in Diagnosis of Amyloidosis M Murari, V Bharani Department of Pathology, SGPGIMS, Lucknow Summary: Systemic amyloidosis is a protein conformation disorder associated with a clonal plasma cell dyscrasia. Accumulation of amyloid progressively disrupts the normal tissue structure and ultimately leads to organ failure. In addition to abdominal fat pad aspiration, bone marrow trephine biopsy may be useful for tissue diagnosis in amyloidosis. Introduction: Amyloidosis is a widespread systemic disease with variable manifestations. Bone marrow aspiration and biopsy is done in patients with amyloidosis for the diagnosis or exclusion of plasma cell proliferative disorder. Bone marrow involvement in amyloidosis is an infrequently reported finding. Materials and Methods: Bone marrow trephine biopsy findings in five patients are described. Marrow cores were fixed in B5 and paraffin embedded sections were examined using HE and PAS stains. Congo red staining with polarized microscopy was done for confirmation. Serum and/or urinary electrophoresis were done in all cases. Results: All patients were males, age ranged from 43 to 79 years. Presenting clinical features included general anasarca with gastrointestinal manifestations, renal failure and backache. Marrow trephine biopsy showed three patterns of amyloid deposition, vascular/perivascular, interstitial, and diffuse pattern. Amyloid deposits could be identified in marrow aspirate in only two of the five cases. Concomitant plasma cell proliferation and serum monoclonal protein were not seen in all patients. Other organ biopsies showed amyloidosis in four patients. Conclusion: Marrow trephine biopsy is useful for demonstration of amyloid, is preferable to marrow aspirates. Use of slightly thicker sections facilitates better demonstration of congophilia and apple-green birefringence under polarized light.

Institute of Hematology and Transfusion Medicine, Medical College, Kolkata Summary: Waldenstrom macroglobulinemia is an uncommon cause of hematological malignancy. We described the clinical features and management of 6 patients with Waldenstrom Macroglobulinemia. Introduction: Waldenstrom macroglobulinemia is a distinct clinicopathologic entity resulting from accumulation of clonally related lymphocytes, lymphoplasmacytic cells and plasma cells in bone marrow that secrete a monoclonal IgM protein. Materials and Methods: Patients: A total of 6 cases of Waldenstrom Macroglobulinemia were taken up and analysed in our study. 5 patients had IgM kappa on SPEP & IFE while 1 had 2 monoclonal bands of IgM & IgA. The diagnosis of Waldentrom Macroglobulinemia was based on Bone marrow biopsy with Immunohistochemistry and Serum protein electrophoresis & Immunofixation. Prognostic scoring was done based on IPSS for WM. The clinical features and treatment records were

Paediatric Haemato-Oncology & Rare Malignancies Abstract No. 154 Immunophenotypic and Cytogenetic Features in Infant Leukemia Sabina Langer, Sabina Langer, Nita Radha Krishnan, Veronique Dinand, Amrita Saraf, Meena Lall, Anupam Sachdeva, Manorama Bhargava Sir Ganga Ram Hospital, New Rajinder Nagar, New Delhi 110060 Summary: Of the 20 cases of infant leukemia, 5 had AML and 15 ALL. Amongst the Acute Lymphoblastic Leukemia, all the cases were B-cell ALL. The MLL gene re-arrangement was seen in 4 of the

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330 15 cases which were immunophenotypically CALLA negative with aberrant expression of myeloid antigens. In AML the commonest FAB subtype was AML-M7 which included two cases of Down’s Syndrome. Introduction: Infant Leukemia is a rare disease with specific biological features and poor outcome. This includes subsets of both acute lymphoblastic leukemia and acute myeloid leukemia with higher incidence of AML. It is characterized by high incidence of MLL gene rearrangements. The infant acute lymphoblastic leukemia are shown to have well defined prognostic factors like hyperleukocytosis, very young age at presentation, lack of CD10, CNS involvement and MLL gene rearrangement. Immunophenotypically MLL-rearrangements are associated with CD10 negativity, frequent expression of myeloid antigens like CD15, CD65. Materials and Methods: From 450 cases of acute leukemias in the paediatric age group received in our department from 2003 to 2013, 20 cases were characterized as infant Leukemia. The flow cytometric and cytogenetic patterns were analyzed. Results: Of the 20 patients there were 15 boys and 6 girls from 2 day old to 1 year of age. Five of these infants belonged to the very young age subgroup (\3 months) and five presented with hyperleukocytosis. Immunophenotypically of the 15 cases of ALL, 7 were CALLA negative. The MLL gene rearrangement was found in 4 of the seven cases which also had aberrant expression of myeloid antigens like CD15. Amongst the AML patients four patients had AML-M-7 and one had AML-M1. Two of the patients with AML-M-7 had trisomy 21. The patient with AMLM1 had complex cytogenetics. Conclusion: Of the 15 cases of ALL four had shown CD10 negativity and MLL gene rearrangement with aberrant expression of myeloid antigens like CD15. The AML cases were mainly AML-M7 two amongst which had Down’s Syndrome.

Abstract No. 155 Infants with Leukemia: Survival in a Developing Country Nita Radhakrishnan, Veronique Dinand, Sabina Langer, Aditya Kumar Gupta, Manorama Bhargava, Anupam Sachdeva Pediatric Hematology Oncology & BMT Unit, Center for Child Health, Sir Ganga Ram Hospital, New Delhi & Department of Hematology, Sir Ganga Ram Hospital, New Delhi Summary: Leukemias occurring in infants have very different clinical, biological and survival characteristics compared to those in older children. The present study is a retrospective analysis of clinical presentation and outcome of infants presenting with acute leukemia to our center in the last 10 years. Clinical data of all infants diagnosed as acute leukemia from January 2003 to December 2012 was analyzed for the present study. 16 infants of ALL and 5 with AML were identified. Among the ALL patients, 6 were CD 10 negative and 4 had MLL rearrangement. WBC count [1 lakh at presentation was seen frequently in CD 10 negative subset. Only 11 patients opted for treatment. After exclusion of LFU, 2-year OS was 43.7 ± 18.8 % and EFS 42.9 ± 18.7 %. Among infants that were treated, OS was significantly better in infant ALL than in AML (60.0 vs. 0 %, p = 0.047). To conclude, the results are suboptimal even in those patients opting to continue treatment. Introduction: Leukemias occurring in infants have very different clinical, biological and survival characteristics compared to those in older children. Clinically both acute lymphoblastic (ALL) and acute myeloid leukemia (AML) in infants are characterized by high white cell count, bulky extramedullary disease and poor cytogenetic features. Hence they do not respond as well to therapy despite risk adapted chemotherapeutic regimens. The present study is a retrospective analysis of clinical presentation and outcome of infants presenting with acute leukemia to our center in the last 10 years. Materials and Methods: Clinical data of all infants diagnosed as acute leukemia from January 2003 to

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 December 2012 was analyzed for the present study. Diagnosis of all cases was established by bone marrow aspiration. Immunophenotyping and cytogenetic analysis was performed wherever possible. All patients underwent lumbar puncture as part of initial evaluation. Patients diagnosed as CD 10 negative ALL were treated as per Interfant protocol (Interfant 99 or 2006). Those diagnosed as CD 10 positive ALL were treated on high-risk arm of BFM 95 protocol. Down syndrome with AML was treated with COG 2971 protocol and denovo AML on MRC AML 10 protocol. Results: Twenty-one cases of infant leukemia (below 15 months) were diagnosed in the defined study period. This included 14 boys and 7 girls. AML was seen in 5, including 2 babies with Down syndrome. Out of 16 ALL, immunophenotyping done in 15 showed precursor B cell CD 10 negative ALL in 6, precursor B cell CD 10 positive ALL in 8 and T-cell ALL in 1. High white cell count ([100,000/mcl) was present in the same proportion of ALL and AML (40 % each), but more frequently in CD 10 negative ALL (50 %) vs. CD 10 positive (0 %). MLL gene rearrangement was present in 4 out of 7 tested. CSF was positive at diagnosis in one patient with ALL. Hepatosplenomegaly was noted in all cases. Myeloid sarcoma was seen in one child with AML. Out of the 21 cases only 11 infants opted for treatment. On follow up by telephonic interview, we have ascertained that all of the 10 patients, who refused treatment, have died in due course. Among the 11 patients who opted for treatment, 6 cases were CD 10 negative infant ALL and they were treated as per Interfant protocols. (3 were further lost to follow up during the course of therapy, 1 has completed therapy, 1 undergoing therapy, 1 died). Among CD 10 positive cases, 2 opted for therapy, and have completed chemotherapy and are in CR1. Among AML cases, only 3 opted for therapy and all have died in due course. After exclusion of LFU, 2-year OS was 43.7 ± 18.8 % and EFS 42.9 ± 18.7 %. On including LFU as an event, 2-year EFS was noticed to be 15.8 ± 8.4 %. Among infants that were treated, OS was significantly better in infant ALL than in AML (60.0 vs. 0 %, p = 0.047). Conclusion: In infant leukemias, only around 50 % cases opted for treatment. Even in those who were treated, the results are poor.

Abstract No. 156 Changing Trends in Pediatric Acute Leukemias: Hospital Based Study Monika Singh, Ashutosh Kumar, US Singh, Archana Kumar, Rashmi Kushwaha King George’s Medical University, Lucknow, Uttar Pradesh Summary: In our study we categorised pediatric acute leukemias on the basis of morphology and immunophenotyping and observed an increase in proportion of AML as well predominance of B cell type ALL. Introduction: Our study aimed at identification of patients with AML and ALL (in 0–14 years age group) by means of morphological assessment for both and immunophenotyping (B and T cell type) for ALL along with study of current trends of acute leukemia in above age group. Materials and Methods: The study included 426 patients during a period of 5 years (June 2008–June 2013) out of which 288 patients were of ALL and 138 of AML. Of the total 288 patients of ALL, 114 were subtyped by flowcytometry due to limited resources. Patients of AML were diagnosed on the basis of morphology and were classified according to WHO classification. Patients of ALL were diagnosed on the basis of morphological features in all and immunophenotyping in a limited number of patients. Results: Out of 426 patients studied 67.6 % were of ALL and 32.4 % of AML. Out of 114 patients of ALL, 84 (73.6 %) were of B cell type and 30 (26.4 %) were T cell type. Most common type in AML patients was Acute myeloid leukemia with maturation (50 %) with Acute myeloid

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 leukemia without maturation (30.4 %) being the second most common. Additionally we found an increasing trend in the number of pediatric patients of AML through the 5 year period of study. Conclusion: In our study the percentage contribution of B and T cell subtype in patients with ALL and M2 type in AML matched the western literature. We found an increase in proportion of patients of AML during the 5 year period of study.

Abstract No. 157 A Girl with Mixed Phenotype Acute Leukemia: B/T Subtype—A Rare Variant B Aasha, Sunita Sharma, Preeti Rai, Richa Chauhan, Jagdish Chandra Department of Pathology and Pediatrics, Lady Hardinge Medical College and Kalawati Saran Children Hospital, New Delhi Summary: Mixed phenotype acute leukemia (MPAL) is a rare entity which is grouped under ‘Acute leukemia of ambiguous lineage’. We report a case of pediatric B/T-cell subtype of Mixed Phenotype Acute Leukemia and its clinical course. Introduction: A small subset of leukemias cannot be classified as AML or ALL by immunophenotyping and are categorized as ‘leukemia of ambiguous lineage’. MPAL constitutes less than 2 % of all acute leukemias. Most common subtypes include B/myeloid, T/myeloid and rarely B/T mixed phenotype. Materials and Methods: A 5 year old girl presented with fever, petechial rashes, pallor and progressive weight loss. On examination she had generalized lymphadenopathy and hepatosplenomegaly. Results: Complete blood count showed Hemoglobin 78 g/L, Total Leukocyte Count 12.1 9 109/L, Platelet count 25 9 109/L. Peripheral blood smears revealed l % blast. Bone marrow aspirate smears showed 97 % blasts of L1 morphology. Blasts were negative for MPO, PAS, Sudan Black B, and NSE. Immunophenotyping performed on the bone marrow aspirate by multicolor flowcytometry showed 96 % blasts on CD45/side scatter gating. Blasts were positive for CD19, CD79a CD22, cytoplasmic CD3 and CD7 and negative for Tdt, HLA-DR, CD34, CD117, CD13, CD14, CD33, CD10 and cytoplasmic MPO. Thus a diagnosis of B/T cell MPAL was made. The patient received ALL treatment regimen and achieved complete remission. Conclusion: B/T subtype of MPAL is a very rare variety accounting for 4 % of all MPAL. No treatment strategy for MPAL has been established. It was observed in some studies, that pediatric MPAL may carry a better prognosis than adults.

Abstract No. 158 Improvement in Paediatric Cancer Survival in a Developing Country Through Collaboration with Non Governmental Organizations Priti Mehta, Priti Mehta, Sujata Sharma, Rajesh Shah, Bhooshan Holey, Ghosha Makwana, Ratna Sharma, Mamta Manglani Division of Pediatric Hematology-Oncology, Lokmanya Tilak Municipal Medical College & General Hospital, Sion, Mumbai Introduction: Lokmanya Tilak Municipal General Hospital is a municipal corporation run tertiary care hospital catering to the population from the lowest socioeconomic strata. Maintaining Personal Hygiene by patients and their relatives is a big issue due to both, low economic status as well as poor education. Above this, making them understand the gravity of prognosis and duration of treatment is a big challenge to clinicians as well as councilors. With this background,

331 we studied the survival of pediatric cancer patients at our centre after funding for personnel by Non-governmental organization (NGO). Aim: To evaluate the improvement in cancer survival in paediatric patients at our centre through funding for personnel by a nongovernmental organization(NGO). Methods and Materials: A retrospective analysis of case records enrolled. Study sample: Children with cancers enrolled between 2005 to 2011. A comparison was made pre (2005–2008) and post (2009–2011) collaboration with JivDaya foundation (a NGO based in USA). Through JivDaya foundation we got extra personnel exclusively for pediatric oncology patients (4 staff nurses, 1 medical social worker, 1 data manager and 1 out reach worker) and we evaluated the impact of these personnel on patient care and outcome. Results: Children abandoning treatment reduced from 60.6 % (n = 66) in 2005 to 2008 to around 24.07 % (n = 55) in each year from 2009 to 2011 (p \ 0.001). Additionally the number of children completing treatment increased from 8 (n = 66) in 2005–2008 to 26 (n = 55) in 2011 (p \ 0.001). The overall survival rate has improved from 20 % in 2005–2008 to 51.85 % in 2011(p \ 0.001). These results show a significant impact on patient management and outcome. Conclusion: Improvement in overall outcome of cancer depends on the infrastructure which includes manpower and financial aid and collaboration with such NGO’s such as JivDaya Foundation had a very positive impact on patient care and survival benefit to children with cancers.

Abstract No. 159 Infants with Malignancy: A Tertiary Care Hospital Experience Geeta Mandhani, Priyashree Mukherjee, Sujata Sharma, Shivani Patel, Sneha Fernandes, Ratna Sharma, Priti Mehta, Mamta Manglani Division of Pediatric Hematology-Oncology, Lokmanya Tilak Municipal Medical College & General Hospital, Sion, Mumbai Introduction: Infants with cancer often have a different clinical presentation from older children with same disease. Their response to therapy also differs indicating unique biologic characteristic of cancer in infants. The Spectrum of malignancies in infants has been quite variable. Aims and objective: (1) To evaluate the incidence of infantile malignancies, (2) study of the type of malignancies and (3) determine their outcomes. Methods and materials: Retrospective analysis from case records. Study Period: January 2008–December 2012. All children with malignancies were screened and infants (\1 year age) with malignancies were included in the study. Details of their symptoms and signs, final diagnosis and outcome were documented and descriptive analysis was performed in the data. Results: A total of 306 children were diagnosed with malignancies in the study period. Of these 28 (9.1 %) were infantile malignancies. Amongst 28 infantile malignancies, 9 (32 %) infants had haematological malignancies and 19 (67.8 %) had solid tumours. Amongst haematological malignancies ALL was most common (5 cases—17.8 %), followed by AML (2 cases—7.1 %), 1 (3.5 %) case of JMML and 1 (3.5 %) case of Langerhan Cell histiocytosis. Amongst solid tumours, commonest was Wilm tumour 6 cases (21.4 %), followed by 4 (14.2 %) neuroblastoma, 3 (10.7 %) hepatoblastomas, 2 (7.1 %) brain tumour, 2 cases (7.1 %) of germ cell tumours like retroperitoneal teratoma and sacrococcygeal teratoma and 1 (3.5 %) each of lipoblastoma, paraganglioma and Ewing sarcoma. Treatment was initiated in 19 patients (67.8 %). In the remaining 9 patients, 4 patients (14.2 %) expired and 3 (10.7 %) patients did not agree for the treatment and 2 (7.1 %) were lost to follow up. While on treatment 7 (25 %) patients expired, 5 (17.8 %) were lost to follow up, 1 patient (3.5 %) discontinued treatment against medical advice. We have 6 patients (21.4 %), all of solid tumours who had a successful outcome. Conclusion: Incidence

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332 of Infantile Malignancies in our centre is 9.1 %. Outcome of malignancies in infant is usually poor.

Abstract No. 160 Peripherally Inserted Central Venous Catheters (PICC) in Pediatric Oncology Patients: A Single Centre Experience K Anand Kumar1, Arun Gupta2, Hinnabhandar1, Shiny1, Nita Radhakrishnan1, Anupam Sachdeva1 Pediatric Hematology Oncology, 2Interventional Radiology—Sir Ganaga Ram Hospital 1

Summary: PICCs are a very convenient mode of delivery of chemotherapy in pediatric oncology patients, but this does not come with a flip side to it. The most common complication being infections which may add on to the burden of these patients. Pain relief was the major benefit and a tradeoff for increased risk of infection. Introduction: To determine common complications in peripherally inserted central catheters (PICCs) among pediatric oncology patients in a single center in northern India. Methods and materials: Retrospective analysis of data of patients in whom PICC line was inserted at our centre from January 2011 to January 2013. Results: A total of 46 PICCs were inserted in 42 patients over the study period among 246 newly diagnosed cancer patients. Four patients had PICCs inserted twice. Thirty-three patients were males (71.7 %) and 13 were females (28.3 %) with a mean age of 6.7 years (range 0.8–18 years). 42 (91.3 %) had hemato-lymphoid malignancies [ALL (38 out of 42) was the commonest] and 4 (8.7 %) had solid tumors [neuroblastoma (3) was most common]. The basilic vein was used most frequently (n = 38; 82.3 %) for PICC placement. The commonest indication for PICCs was chemotherapy and majority were inserted during induction-phase chemotherapy. The total duration in-situ for the 46 PICC in our cohort was 2,023 catheter-days (median-time 52.5 days; range: 4–120 days). PICC-associated blood-stream infection, in 20 PICCs was the most common complication in these patients (43.5 %, n = 46; 9.88 episodes/1,000 catheter-days) with Gram-positive organisms isolated in 35 % cases, Gram-negative organisms in 30 % and fungi in 25 % cases. Well-recognized hospital-acquired pathogens like Stenotrophomonas, Pseudomonas, Enterococci, Acenetobacter, Candida and Penicillium constituted 50 % of isolated organisms. Other complications noted were catheter-occlusion (8.7 %), accidental displacement (4.3 %) and catheter-associated venous thrombosis (2.1 %). No mortality attributable to PICCs was noted. Conclusion: Despite significant complications, PICCs are a relatively safe and cost-effective method of establishing long-term central venous access in pediatric-oncology patients. Catheter associated blood-stream infection (CABSI) remains a major morbidity in this patient cohort, often necessitating catheter removal.

Abstract No. 161 Megakaryoblastic Leukemia in One Year Old Child—A Case Report Ruchi Agrawal, JJJ Falliero, RK Pasale

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 marrow following two cycle of chemotherapy demonstrated persistent disease. Introduction: Acute megakaryoblastic leukemia (AML-M7) is a rare type of pediatric AML. It represents approximately 1 % of all leukemia during childhood and has an incidence of 0.5 % per million per year. In young children with down syndrome, AML-M7 is the most common type of AML. Materials and Methods: Case report of one year old child admitted to pediatric ward in Dhiraj General Hospital. Diagnostic method included blood analysis, bone marrow aspirate and biopsy, flow cytometry and cytogenetic study. Results: PS demonstrated marked anemia with leukoerythroblastosis and dysplastic changes and bone marrow aspirate show absence of hematopoiesis and increased blasts with megakaryoblastic features. The bone marrow biopsy was fibrotic with an atypical mononuclear cell infiltrate. Flow cytometry study showed 14 % of total cells expressing CD61. Cytogenetic evaluation of the bone marrow aspirate demonstrated hyperdiploid karyotype with 50 chromosomes and t(1;10) translocation; no constitutional trisomy 21. These findings, together with the clinical presentation, were suggestive of acute myeloid leukemia of megakaryoblastic lineage (FAB M7). Conclusion: The prognosis of acute megakaryoblastic leukemia is poor, particularly in non-Down Syndrome patients.

Abstract No. 162 Kaposiform Hemangioendothelioma in a 3 Month Old Infant—A Case Report G Vimal Kumar1, M Deenadayalan1, Hemalatha1, Karuna Sri1, Radhakrishnan Satheesan2, Ramya2, Revathi Raj1 Apollo Speciality Hospital, Chennai1; Apollo Children’s Hospital, Chennai2 Introduction: Kaposiform hemangioendothelioma is a rare, highly vascular and aggressive soft tissue tumor frequently associated with Kasabach-Merritt phenomenon, usually seen in early infancy. It needs to be distinguished from juvenile infantile hemangioma due to treatment and prognostic implications. Early diagnosis by means of MRI and tissue biopsy portends a better outcome. Treatment includes surgical excision when feasible and medical management with steroids, propranolol and supportive treatment for coagulopathy. Case Report: We report a 3 months old infant who was diagnosed with invasive Kaposiform hemangioendothelioma in our unit. A 3 months old, developmentally normal female infant presented with an ill defined, nontender, firm and immobile mass in the right paravertebral region measuring 3 9 4 9 2 cm along with symptoms of consumptive coagulopathy due to Kasabach-Merritt phenomenon. Imaging revealed a very ill defined mixed echoeic mass lesion in the retroperitoneum encasing the abdominal aorta and inferior vena cava. Coagulopathy was managed by blood products along with intravenous steroids, propranolol and immunoglobulin. Open biopsy confirmed the diagnosis of Kaposiform hemangioendothelioma. Child was then started on chemotherapy. The swelling resolved in size with chemotherapy and the child is in complete remission at present. This case has been presented for its rare nature in infancy and the need to diagnose it early to reduce the high mortality associated with the disease.

S.B.K.S.M.I. & R.C., Piparia, Vadodra Summary: A Case of one year old child presented for routine checkup and was found to be anemic and thrombocytopenic. Mother had noticed occasional ‘‘dots’’ on the toddler’s skin for few days. Peripheral smear examination, bone marrow, flow cytometry and cytogenetic studies helped in making the diagnosis of megakaryoblastic leukemia. Evaluation of patient’s peripheral blood and bone

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Abstract No. 163 A Rare Case of Histiocytic Sarcoma Following T Cell Acute Lymphoblastic Leukaemia Gautami Kokil, Marie Manipadam, Sheila Nair, Alok Srivastava

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Departments of General Pathology and Clinical Haematology, Christian Medical College, Vellore Summary: Histiocytic sarcoma is a rare tumour of mature tissue histiocytes. We present a case of histiocytic sarcoma which developed in a patient on maintenance chemotherapy for T cell acute lymphoblastic leukaemia. Introduction: A 6 year old male was a diagnosed case of T cell acute lymphoblastic leukemia in October 2010 who was on relapse protocol because of poor prednisolone response. While on maintenance chemotherapy, he developed multiple painful nodules on the chest wall. Imaging revealed pleural thickening with lytic lesions in the ribs which were biopsied. Materials and Methods: Routine tissue processing with haematoxyline and eosin staining and immunohistochemistry was done. Results: Histopathological examination revealed histiocytic sarcoma which was confirmed by immunohistochemistry. Conclusion: Histiocytic sarcoma can occur as a rare second malignancy in haematological neoplasms.

Abstract No. 164 Dendritic Cell Neoplasm—A Rare Case Presentation Mamata V Yargop, Abhijit P Ahire, VP Antia Jaslok Hospital and Research Centre Summary: The objective of this presentation is to evaluate the clinical features, prognostic factors, and efficacy of treatments in patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN) with a leukemic presentation. We present a rare case of 78-year old man who was a known case of ulcerative colitis in remission. He presented with giddiness, generalized weakness and tendency to fall. Peripheral blood findings: Hb-7.7 g/dl, wbc-3420/cmm, platelet count-70,000/cmm, ESR-86 at end of 1 h at 20 % atypical lymphoid cells in peripheral blood film. LDH was 273 (normal). Introduction: Among ‘‘AML and related precursor neoplasms’’ in 2008 WHO classification, BPDCN is a rare subtype of acute leukemia characterized by clonal proliferation of precursors of plasmacytoid dendritic cells, known as interferon-producing cells/plasmacytoid monocytes. BPDCN is characterized by an aggressive behavior with rapid systemic dissemination, despite the often indolent clinical presentation, with isolated cutaneous involvement in form of solitary or multiple lesions. Rarely, patients show features of acute leukemia with systemic involvement without skin manifestations. These patients present with cytopenia, associated with variable rates of bone marrow infiltration. Materials and Methods: Immunophenotyping of bone marrow sample was done by flow cytometer using all myeloid, lymphoid and dendritic cell markers using FSC/SSC & CD45 gating strategies and the sample was biopsied as well. Results: Flowcytometry findings s/o cell cluster in blast region on SSC/CD45 gate. 67 % cells display variable FSC atypical cells were CD4+, CD56+, CD123+, dim CD33+, HLADR+ Conclusion: Based on the positivity of CD4, CD123, CD56, HLADR & biopsy report i/v/o High grade haematolymphoid neoplasm related to myeloid precursor cells origin a diagnosis of blastic plasmacytoid dendritic cell neoplasm is favoured.

Aplastic Anaemia & Allied Disorders Abstract No. 165 Telomerase Activity and Telomere Gene Mutations in Aplastic Anemia Dolly Joshi, S Chandrakala*, Kanjaksha Ghosh, BabuRao Vundinti

333 National Institute of Immunohaematology (ICMR), 13th Floor, New Multistoried Building, K.E.M. Hospital, Parel, Mumbai-12, Department of Haematology, KEM Hospital, Mumbai Summary: The study was carried out in 60 idiopathic aplastic anemia patients. Telomerase activity was reduced in aplastic anemia patients as compared to age matched controls. Telomerase activity and telomere gene mutations play an important role in aplastic anemia. Introduction: Idiopathic Aplastic anemia is a bone marrow disorder which contains few hematopoietic cells and consists mainly of fats. Aplastic anemia is usually observed to be associated with exposure to toxins such as benzene, or with the use of certain drugs such as chloramphenicol. In western countries the incidence of aplastic anemia is approximately 2 cases per million per year, but the count increases two to three times higher in Asia. Mutations of the genes involved in human bone marrow failure syndrome have been identified in components of the telomerase and telomere associated genes. We have carried out the study to detect telomere gene mutation and analyze the telomerase activity in idiopathic aplastic anemia patients from Indian population. Materials and Methods: The study was carried out in sixty (60) patient’s with idiopathic aplastic anemia. The telomere length was studied using quantitative fluorescence in situ hybridization (Q-FISH). Mononuclear cells were collected and lysate was prepared for the telomerase activity. Telomerase from cell lysate add telomeric repeats onto substrate oligonucleotide and the resultant product is amplified by quantitative real time PCR. Telomere gene mutations (TERT, TERC and TINf2) were studied using exon specific primers by direct sequencing method. Results: In our study, no difference in telomere length was observed between aplastic anemia patients and controls by Q-FISH. Telomerase activity was reduced in aplastic anemia patients (55,492 mol/reaction) as compared to age matched controls (303,555 mol/reaction). The telomere gene mutations in the TERT, TERC and TINf2 gene will be presented. Conclusion: The genetic factors such as telomere gene mutation and telomerase activity could contribute to bone marrow failure and development of aplastic anemia.

Abstract No. 166 Hematological Diversity of Pancytopenia—A Study of 390 Cases Sridevi HB1 1

Department of Pathology, Yenepoya Medical College, Mangalore, Department of Pathology, Kasturba Medical College, Mangalore, 3 Department of Anatomy, Srinivas Institute of Medical Sciences, Mangalore 2

Summary: The causes of pancytopenia varied from infections like malaria where the presentation was transient to diseases like aplastic anemia where the bone marrow production of the blood cells was primarily affected. Hence identification of the cause is important for right intervention. Introduction: Pancytopenia is defined by simultaneous presentation of anemia, leucopenia and thrombocytopenia. The present study was conducted to assess the entire spectrum of etiological pattern, the clinical-hematological profile of patients presenting with pancytopenia. Materials and Methods: A total of 390 cases of pancytopenia were evaluated clinically along with hematological parameters over a period of 30 months. Bone marrow study was done when the peripheral smear examination was non diagnostic. Results: The mean age of patients presenting with pancytopenia was 34.46 years with male predominance (M:F::1.4:1). Malaria (21.28 %) was the commonest cause of pancytopenia followed by megaloblastic anemia (19.74 %). Other causes included hypersplenism (19.23 %), retroviral disease (8.46 %), acute leukemia (7.43 %), hypoplastic marrow (5.89 %), sepsis (4.87 %), certain infections (3.84 %)

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334 myelodysplastic syndrome (3.07 %), lymphoma (2.30 %), myelofibrosis (2.05 %) and chronic kidney disease (1.28 %). A case each of Thalassemia (0.25 %) and mucolipidosis (0.25 %) presenting with pancytopenia were also encountered. Weakness was the commonest symptom and pallor, the commonest sign. Conclusion: The present study shows that pancytopenia is relatively a common hematological entity with a wide variety of differential diagnosis. The presentation can be transient in some cases but can also be a striking feature of many serious and life threatening disorders. Thus identification of correct cause by a proper systematic approach will definitely aid in implementing the right kind of therapy in such patients.

Abstract No. 167 A Study of Etiological Spectrum of Pancytopenia in Adults and Children in a Tertiary Care Hospital in Mumbai Varsha Bhatia, Shubhangi V Agale, AS Vyas, AG Valand, Shweta Bhavsar, Aditi Garud Grant Government Medical College & Sir J.J. Group of Hospitals, Byculla, Mumbai Summary: To evaluate the etiological and clinico hematological profile in adults and children with pancytopenia. Introduction: Pancytopenia is not a disease entity but a triad of finding that usually results from various disorders. Pancytopenia is defined as reduction in all of the cellular elements of blood, that is, red cells, white cells and platelets. The severity of pancytopenia and the underlying pathology determine the management and prognosis. Thus, identification of the correct cause will help in implementing appropriate therapy. Materials and Methods: The present study was conducted at Grant Govt Medical college, Mumbai, in the department of Pathology over a period of 5 years, i.e. from June 2008 to June 2012. The inclusion criteria for analysis were taken as Hb \ 13.5 g/dl in females, total leucocyte count \4,000/cumm, platelet count \1,500,000/cumm. During this time there were total number of 96 cases (out of 446) which fulfilled the criteria for pancytopenia, The age distribution was from 6 months to 75 years. Majority of these cases were of adult age group whereas eight cases were of pediatric age group. Out of 96 cases, 57 were males and 39 were females. Ninety four patients were subjected to bone marrow aspiration and in 79 patients trephine biopsy was available examination. Results: The commonest cause for pancytopenia in adults was megaloblastic anemia (24.25 %), followed by hypoplastic/aplastic anaemia (19.40 %) and mixed nutritional deficiency (8.73 %). The commonest cause of pancytopenia in paediatric cases was megaloblastic anemia (25.0 %). Conclusion: According to our study pancytopenia is more common in adult age group and has male preponderance.

Abstract No. 168 Clinical, Haematological and Cytogenetic Profile of 42 Cases of Aplastic Anaemia Subhajit Das, V Tilak, V Gupta, A Singh, M Kumar, AK Rai Institute of Medical Sciences, Banaras Hindu University, Varanasi Summary: Evaluation of 42 cases of aplastic anaemia reveals more severity of disease manifestation in children. No cytogenetic abnormality was detected in any of them. Introduction: Aplastic anaemia, characterized by suppression of haematopoiesis, and is diagnosed by identification of reduced bone marrow cellularity, in absence of increased marrow fibrosis. This study was carried out to find out the difference in clinical profile in paediatric and adult aplastic anaemia

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 patients, to establish the necessity of bone marrow biopsy for its diagnosis, and to detect any chromosomal abnormality in them. Materials and Methods: 22 paediatric and 20 adult cases were diagnosed combining the clinical findings, peripheral blood counts and bone marrow examination. Out of them, 24 cases were evaluated by karyotyping. Results: Occurrence of severe anaemia and frequency of leukopenia and absolute neutropenia were more common in children. The degree of leukopenia and absolute neutropenia was more severe in children. Because of frequent occurrence of blood tap and dry tap, diagnosis in many cases depended solely on biopsy findings. All the core biopsy specimens floated after proper decalcification. Lymphocyte culture failed in 9 cases, and rest 15 cases showed no cytogenetic abnormality. Conclusion: Bone marrow biopsy is mandatory for diagnosing aplastic anaemia. The findings of core biopsy floatation and its correlation with marrow cellularity, and absence of chromosomal abnormality in aplastic anaemia need larger studies to give any statistically significant opinion.

Abstract No. 169 Efficacy and Safety of Equine Antithymocyte Globulin Along with Cyclosporine in Patients with Aplastic Anaemia Farah Jijina1, MB Agarwal2, Pankaj Malhotra3, Sharat Damodar4, Sandip Shah5, Cecil Ross6 Department of Haematology, Seth GS Medical College & KEM Hospital Introduction: The aim of this study was to confirm the efficacy and safety of indigenous Equine Antithymocyte Globulin (eATG) in Indian patients with aplastic anaemia of any severity. Materials and Methods: Patients with very severe(VSAA), severe (SAA) and non severe (NSAA) aplastic anaemia from 6 hospitals were enrolled in the study between 2011 to 2013 after approval from Institutional Ethics Committee. Equine ATG at a dose of 40 mg/kg/day was infused for 4 days after sensitivity test. Cyclosporine (4–6 mg/kg/day) was given and adjusted to maintain trough levels between 200–400 ng/mL for 6 months along with standard supportive treatment. Modified IntentTo-Treat population for efficacy included patients who completed ATG treatment and followed-up on day 90 and/or 180. Lymphocyte subsets (CD 2, 3, 4 and 8) were tested on days 0, 3, 5, 7, 14 and 21. Complete response (CR) was defined as—transfusion independence, haemoglobin C11 g/dL, absolute neutrophil count (ANC) [ 1.5 9 109/l and platelet C150 9 109/l; partial response (PR) was transfusion independence, haemoglobin C8 g/dL, ANC [0.5 9 109/l and platelet C20 9 109/l; non responders were transfusion dependent. Safety analysis was done on the intention to treat population. Results: Thirty patients were enrolled in the study (7 NSAA, 18 SAA, 5 VSAA). Nineteen patients completed day 90 visit and 17 completed day 180 visit. Of the remaining 11 patients, 2 died on days 12 and 39 (septicemia, unresolved pneumonia), 1 withdrew consent and 8 were lost to follow-up. The mean age of 30 patients was 32.4 ± 16.8 years and weight was 55.5 ± 16.6 kg. Mean baseline Hemogram values were hemoglobin 8.1 ± 2 g/dl, platelet count 21.5 ± 22.1, ANC 429.8 ± 429 and ALC 1348.7 ± 855.5. On day 90, 10 of 19 patients responded (CR 1, PR 9) and the number of responders increased on day 180 to 14 of 17 (CR 1, PR 13). These 14 responders were 5/5 NSAA, 7/10 SAA, 2/2 VSAA. Lymphocyte subset values decreased between day 3 to 7 and returned to baseline level between day 14 to 21, picturing the effect of eATG on lymphocytes. Thirty-seven of total 93 adverse events reported during the study period were related to eATG (23 during infusion of eATG). Two patients died before day 90 visit and were included only in the safety analysis. Other SAEs included one patient each with acute renal failure and febrile neutropenia both of whom recovered. None of the patients relapsed

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 during the 6 months end of study. Conclusion: Equine ATG was safe and adverse events reported were same as reported in literature. Along with cyclosporine, the efficacy was documented in 82.3 % of patients who completed day 180 of treatment.

Abstract No. 170 A Randomized Study to Compare Cyclosporine with Cyclosporine and Androgen in the Treatment of Acquired Aplastic Anemia Avinash Kumar Singh, Avinash Kumar Singh1, Suman Kumar1, Sanjeev Sharma2, Pravas Mishra1, Tulika Seth1, Seema Tyagi1, M Mahapatra1, Renu Saxena1 1

All India Institute of Medical Science, 2B.L. Kapoor Memorial Hospital, New Delhi

Summary: Present randomised study of cyclosporine alone versus cyclosporine and menabol in acquired aplastic anemia establishes that cyclosporine alone is as effective and better tolerable than combination. Introduction: Acquired aplastic anemia is a clinical syndrome characterized by pancytopenia associated with hypocellular marrow without abnormal cell or fibrosis. Cyclosporine with or without androgen is a better option for patients, who lack matched donor or can’t afford Anti Thymocyte globulin (ATG). Materials and Methods: Design—Randomized controlled trial, single institution based. Duration of study—one and half years. Patients of newly diagnosed acquired aplastic anemia without HLA matched donor or who can’t afford ATG fulfilling inclusion and exclusion criteria. Oral cyclosporine was used in dose of 5 mg/kg/day in two divided doses in both arm and oral androgen (stanazolol) in dose of 2 mg/kg/day in three divided doses was added in combination arm. Follow up duration—6 months, Primary end point was hematological response at 6 months. Cyclosporine level were not done routinely. Results: Total 158 patients were randomized in two groups. The responses were as follows (Table 1): Except for G.I. upset, which was higher in combination arm (p = 0.006), there was no statistically significant difference in side effects in two groups. The blood transfusion requirement and use of antibiotic and antifungal were same in both groups. Response (n = no of evaluable patients) Combination arm (n = 59)Cyclosporine alone arm (n = 62) P = 0.501 Complete response 5 (8.47 %) 4 (6.45 %) Partial response 22 (37.29 %) 16 (25.81 %) Minimal response 5 (8.47 %) 6 (9.68 %) No response 27 (45.76 %) 36 (58.06 %). Conclusion: Cyclosporine alone is as effective as combination of cyclosporine and androgen. The use of androgen in combination not only leads to more side effects but patient has to bear extra cost for it, without having any benefit.

Abstract No. 171 Pure Red Cell Aplasia (PRCA): A Clinico-Pathological Study of 22 Cases J Saumya, Tara Roshni Paul, Shantveer G Uppin, Megha S Uppin, AVM Narendra, A Krishna Prasad, VR Srinivasan Nizam’s Institute of Medical Sciences Summary: Multiple factors are associated with development of Pure Red Cell Aplasia (PRCA). The study period included 22 diagnosed cases of PRCA. Peripheral smears, marrow cytology and trephine sections were reviewed. Etiologies included congenital (2), collagen vascular disease (1), parvo virus (1) and thymoma (4). Introduction: Pure red cell aplasia (PRCA) is a rare disorder characterized by

335 normochromic normocytic anemia, reticulocytopenia and a paucity of erythroblasts in the bone marrow. Materials and Methods: Cases diagnosed as PRCA during 2002–2013 were retrieved from hematology case files. The clinical and surgical details, serological tests were obtained from the medical records. The peripheral smears, marrow cytology and trephine sections were reviewed. Results: A total of 22 cases were diagnosed during the period of 11 years, age ranging from 5.5 months to 60 years with M:F ratio of 1:1.2. The patients presented with easy fatigability, dyspnea and anemia not responding to treatment. The causes included congenital (2), collagen vascular disease (1), parvo virus associated (1) and thymoma (4). The underlying cause was not found in the rest of the cases. Conclusion: PRCA has varied etiological factors. The diagnosis should be suspected in patient presenting with anemia, reticulocytopenia, with no physical abnormality and lack of erythroid precursors in the marrow. It also requires correlation with mediastinal imageology, previous surgeries, serological parameters, infections and intake of drugs.

Abstract No. 172 B19 Parvovirus Induced Severe Aplastic Anemia in Two Normal Pediatric Patients Without Underlying Disease Kuntal Patel, JJJ Falleiro, RK Pasale S.B.K.S.M.I. & R.C., Piparia, Vadodara Summary: We report two cases of aplastic anemia in a previously healthy pediatric patients having asymptomatic infection with the B19 virus. Laboratory examination initially showed thrombocytopenia, leucopenia in the peripheral blood, and severe hypoplastic bone marrow showing marked erythroblastopenia with very large pronormoblast having ‘’dog ear’’ inclusions and megaloblastic changes. Since pancytopenia developed and worsened progressively, i.v. immunoglobulin therapy was given, resulting in a complete remission. Despite the lack of an infectious prodrome histological analysis revealed an underlying infection with the B19 virus. Introduction: Human B19 Parvovirus, is the only member of the parvovirus family, which selectively replicates only in erythroid progenitor cells. Hematological consequences of B19 arise due to direct cytotoxic effect on erythroid progenitors in bone marrow with interruption of erythroid production and leads to pure red cell aplasia (PRCA) in patients with an underlying hemolytic anemia. Materials and Methods: We report two cases of severe parvovirus-induced aplastic anemia in two boys, admitted to Pediatric ward in Dhiraj General Hospital, with no underlying hematologic disorder. Results: There was marked erythroblastopenia with very large pronormoblast having ‘‘dog ear’’ inclusions and megaloblastic changes associated with parvovirus infection. The patients’ rapid hemoglobin recovery after treatment with i.v. immunoglobulin further supports this form of therapy for children with parvovirus-induced anemia. Conclusion: B19 virus infection must be considered one of the causes of aplastic anemia in patients without underlying hemolytic anemia and an apparent episode of the viral infection.

Abstract No. 173 Evaluation of Serum Alpha Fetoprotein and Serum Albumin in the Diagnosis of Fanconi Anemia: A Tertiary Care Center Experience Avinash Kumar Singh, Tulika Seth, Nita Radhakrishnan, Suman Kumar, Mrinalini Kotru, Divya Bansal, Vandana Jain, Vishnubhatla Sreenivas, Anand Kumar, Pravas Mishra, Manoranjan Mahapatra, Hara Prasad Pati

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336 Department of Hematology, All India Institute of Medical Sciences, New Delhi Summary: Present study establishes the role of serum AFP and serum albumin in the diagnosis of Fanconi anemia. Introduction: Fanconi anemia is a rare inherited bone marrow failure syndrome characterized by aplastic anemia, phenotypic malformations and a high risk to develop malignancy. It is diagnosed by stress cytogenetics with mitomycin C and/or diepoxybutane (DEB), which is the gold standard but has several limitations. Materials and Methods: All patients of Fanconi anemia presenting to hematology OPD or admitted under hematology department were enrolled in the study. Patients of acquired aplastic anemia and normal healthy subjects (age and sex matched) served as controls Study design prospective, single institution based study. Duration of study One and half years. Aim of the study—to evaluate the role of serum AFP and serum albumin in the diagnosis of Fanconi anemia. Results: A total of 31 patients were enrolled with a mean age of 12.9 years. The level of serum AFP was significantly higher in Fanconi anemia patients compared with normal healthy and acquired aplastic anemia controls (Mean 7.02 vs. 2.41 vs. 1.95 ng/ml). With 2.95 ng/ml cut off of AFP, this method showed sensitivity of 80.65 % and specificity of 80 % in the diagnosis of Fanconi anemia. The serum albumin value among Fanconi anemia patients were significantly higher compared with acquired aplastic anemia patients (4.38 vs. 4.17 g/dl, p = 0.002), while there was no difference among Fanconi anemia and normal healthy controls (mean 4.38 vs. 4.65 g/dl, p = 0.207). The sensitivity and specificity of serum albumin with cut off of 4.5 g/dl were 80 and 73.3 % respectively. When serum AFP and albumin together were analysed for diagnosis of Fanconi anemia, the effect of albumin was nullified and AFP value still correlated significantly. The comparison of several clinical and laboratory parameters between patients with Fanconi anemia with high and normal AFP revealed no statistically significant differences. Conclusion: Present study will help clinicians to diagnose patients of Fanconi anemia and to differentiate it from acquired aplastic anemia in less time, minimal cost and with good sensitivity and specificity.

Abstract No. 174 Fanconi Anemia—A Rare Case Report Mallikarjun A Pattanashetti, Ramesh Chavan, Ganga S Pilli Department of Pathology, KLE University’s Jawaharlal Nehru Medical College and Dr. Prabhakar Kore Charitable Hospital and Research Centre, Nehru Nagar, Belgaum-590010, Karnataka Summary: Fanconi anemia (FA) is rare & most common form of inherited constitutional aplastic anemia. This case was diagnosed on history, clinical features and haematological findings. Introduction: There are currently 13 known FA subtypes, all autosomal recessive except one which is X linked recessive. Incidence of FA is 1 in 1.6 million. Materials and Methods: A 8 year-old female presented with generalised weakness, breathlessness, fever and physical abnormalities. She was first full term born of consanguineous healthy parents. Past history of epistaxis and easy bruising present. On examination patient had short stature, generalised hyperpigmentation with areas of hypopigmentation, flat thenar eminence, absent right thumb and rudimentary left thumb, triangular face, microcephaly, hypertelorism, abnormal pinna, low set ears with narrow ear canal, wide 1st webspace of both feet, developmental delay and learning disability. Results: Hb-5.5 g/dL, PCV-15 %, RBC Count-1.4 million/cumm, Total WBC count-2,400/cumm, Platelets-34,000/cumm, MCV— 107 fl, PS shows: Pancytopenia, Bone marrow aspiration and biopsy shows Hypocellular bone marrow. Conclusion: FA is rare disorder diagnosed with physical abnormalities as detailed above, pancytopenia on PS, bone marrow showing aplastic anemia with supporting

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 history of consanguinity which is common in South Indian states like Karnataka, Tamil Nadu and Andhra Pradesh. Further workup with Cytogenetic quantitation of chromosomal breakage in response to diepoxybutane or mitomycin C, Immunoblot assay of FANCD2 protein monoubiquitination, Determination of complementation groups and Molecular Genetic testing is mandatory. Management is mainly conservative & BM transplantation.

Stem Cell Transplantation Abstract No. 175 Engraftment Kinetics: Correlation with Stem Cell Culture P Ganguli, Rehan Ahmed, Brig Reena Bharadwaj, Renu Madan, Sanjeevan Sharma, Velu Nair, UD Gupta Department of Pathology and Molecular Medicine, Army Hospital (R&R), Delhi Cantt-110010 Introduction: Haematopoietic Stem Cell (HSC) Transplantation is the most important therapy now days for the treatment of blood malignant diseases in the field of haematology including oncology. Since their introduction more than 30 years ago, colony assays have been used extensively to evaluate the hematopoietic proliferative potential of bone marrow, cord blood and mobilized peripheral blood samples for clinical transplantation. Aims: In vitro Clonogenic Assay as a marker of progenitor cell status in Haematopoietic Stem Cell Transplantation. Objectives: To study the Clonogenic potential of Haematopoietic Stem Cells harvested for allogenic and autologous stem transplantation. To correlate the engraftment potential with the Clonogenic Assay. Materials and Methods: It is proposed to include 30 patients in the study. (1) Patients diagnosed having haematological malignancies and Bone marrow failure syndromes undergoing HSC transplants will be included. (2) All the patients included will undergo a stem cell collection procedure comprising bone marrow harvest or peripheral blood stem cell (PBSC) collection. (3) Mononuclear cells will be separated. Viability will be assessed by Trypan blue, CD34 enumeration and 7AAD viability will be done flow cytometrically as per ISHAGE protocol. (4) Clonogenic assay for CFUGM,GEMM,CFU(E) will be done using commercially available methyl cellulose culture plates (Stem Cell Technologies, Canada). (5) In Autologous HSC transplant the clonogenic assay will be done immediately post harvest and post thaw prior to infusion. (6) In Allogenic HSC transplant the clonogenic assay will be done post collection. (7) Response will be measured as days to engraftment (neutrophils and platelets). (8) Analysis will be done on following (a) Correlation of colony counts with days to engraftment (neutrophils and platelets). (b) Clonogenicity of stem cells pre storage and post thaw. Observations & Results: Our correlation study proves the hypothesis of engraftment kinetics is earlier & healthy as observed invitro culture with high potential. Conclusion: Clonogenic culture method can be used as important tool in stem cell transplantation setup to evaluate the engraftment.

Abstract No. 176 Population Pharmacokinetics of a Generic Formulation of Intravenous Busulfan (BUCELON 60TM) in Patients Undergoing Hematopoietic Stem Cell Transplantation Ezhil Pavai Mohanan1, John Carl Panetta, D Salamun, M Sathya, S Gopinath, Biju George, Vikram Mathews, Alok Srivastava, Poonkuzhali Balasubramanian

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

337

Department of Haematology, Christian Medical College, Vellore

Table 1 Patient characteristics Parameters

Q24H (N = 73)

Q6H (N = 22)

Diagnosis

AML-38

Thal-22

ALL-02 APML-01 CML-08 CMML-01 JMML-02 MDS-13 Pre B-ALL-02 Ph+ ALL-02 PNH-03 AA-01 Age (years)

32 (1–61.4)

4 (1–14)

Sex (M/F)

45/28

13/9

Body weight (kg)

58.6 (8.8–104.2)

14.4 (4.4–31)

GST M1

Day 1 AUC (µ moles*min)

Introduction: High-dose intravenous Busulfan (i.vBu) in combination with cyclophosphamide or fludarabine is widely used conditioning regimen prior to hematopoietic stem cell transplantation (HSCT) for various hematological disorders. Targeted i.v Bu has resulted in reliable systemic exposure [due to its predictable pharmacokinetics (PK)] while minimizing toxicity and treatment failure. Since 2010, a generic form of i.v Bu (BUCELON 60TM, Celon Labs, India) with targeted dose adjustment has been used at our center, for HSCT with all Bu based conditioning regimen. There is no report on the PK of Bucelon in patients undergoing HSCT. This study aims at evaluating the POPPK of Bucelon, compare the systemic exposure to i.v Bucelon versus previous reports in patients with various hematological diseases and report the toxicity and outcome in our patient cohort. Materials and Methods: Seventy three out of 95 and 22/95 patients receiving once daily (Q24H) or 6H dose (O6H) i.v Bu respectively between June 2010 and July 2013 was included. Patient demographics, PK, and outcome are shown in Table 1. Peripheral blood samples were collected for PK and PG analysis. An AUC of 5000-5500 lmoles in Q24H or 900–1,350 lmoles in Q6H was targeted. A POPPK modeling analysis was performed to evaluate the effect of factors influencing the PK/systemic exposure of i.v Bucelon. Results and Discussion: Out of the 8 polymorphisms in GSTA1,

P = 0.0122

2000

1500

1000

500

0 l

s

1

ul

Po

1

M

ST

G

N

M

ST

G

Fig. 1 GSTM1 polymorphism on Bu AUC

GSTM1, GSTT1, GSTP1, CYP2B6*6, CYP2C19*2, CYP2C19*3, CYP3A4*1B analyzed for their effect on 1st dose AUC, GSTM1 positive patients in Q6H showed a significantly lower median AUC, 589 lmoles min (307–1,456) than GSTM1 null patients (median AUC, 963 lmoles min (670–1,240) (p = 0.0122) Fig. 1). None of the other polymorphisms were found to influence the 1st dose Bucelon AUC in patients receiving Q24H or Q6H dose. However, the median day 1 AUC is found to be much less compared to previous report on patients with thalassemia major receiving Q6H i.v Bu: 599.5 lmoles min (307–1,456) vs. 971 lmoles min (630–1,621) as well as in malignant conditions receiving Q24H dose of i.v Bu: 4180 lmoles min (1,713–10,456)vs. 5,113 lmoles min (2,796–9,355). There was no significant difference in the cumulative AUC between those who develop toxicities and those who did not. Conclusion: We report the POPPK of i.v Bucelon, a generic formulation of Bu for the first time in patients undergoing HSCT. Since the systemic exposure to Bucelon seems to be lower in similar age, disease matched cohort, this may mean that targeted adjustment of Bu is necessary when we use i.v Bucelon as conditioning regimen for HSCT. The lower systemic exposure could be due to population difference in PK or due to the drug formulation, or a combination of both, which needs to be evaluated.

BSA (m2)

1.63 (0.38–2.11)

0.61 (0.47–1.08)

Bu 1st Dose

210 (32–275)

11.8 (8.4–24.6)

Clearance (L/hr)

41.83 (2.47–144.84)

4.19 (0.81–21.55)

Marrow Donor Registry India (MDRI)-Strategies and Data

Vol. of distribution (L)

156.3 (6.5–454.51)

17.56 (2.51–243.9)

Leenam Dedhia, V Manasi, P Shruti, M Pooja

1st Dose AUC (lmoles min)

4180 (1713–10456)

599.5 (307–1456)

Regimen related toxicity

SOS

10 (10.5 %)

GVHD (Gr III–IV)

13 (13.7 %)

HC

3 (3.2 %)

Rejection

8 (8.4 %)

Relapse

5 (5.2 %)

SOS, sinusoidal obstruction syndrome; GvHD, graft vs. host disease; HC, hemorrhagic cystitis

Abstract No. 177

Marrow Donor Registry India Summary: Unrelated Indian donors where divided into linguistic groups demonstrated unique haplotypes along with few common haplotypes that are prevalent in Indian nationals. These unique haplotypes suggest the significance of the deep seated endogamous and consanginous social behaviour in India. These haplotypes are guiding factors while searching for an unrelated donor for patients of Indian origin in need of bone marrow transplantation. Introduction: Marrow Donor Registry India (MDRI) is a database of voluntary unrelated marrow donors and facilitates marrow and peripheral blood stem cell

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338 transplants for patients with life-threatening diseases such as leukemia, aplastic anemia etc. It recruits donors, maintains a registry of potential donors and initiates searches for Human Leukocyte Antigen (HLA) typed marrow donors for a patient requiring a transplant. Materials and Methods: DNA extracted from EDTA-blood sample of recruited donors, and HLA typing done using Luminex XMAP technology and SSP at low resolution. STATISTICAL ANALYSES USED: Arlequin software, version 3.1 for haplotype frequency analysis. Results: Our data demonstrates that each linguistic group has unique haplotypes along with a few common haplotypes thus creating a unique HLA gene pool. A*33-B*44-DRB1*07 and A*01-B*57DRB1*07 were the most common haplotypes across all linguistic groups. There were unique haplotypes such as A*33-B*14-DRB1*01 in parsis, A*26-B*08-DRB1*03 in punjabis etc. Conclusion: In order to adequately represent the Indian population on the registry, each linguistic group should be targeted for donor recruitment. This would enable a better chance for any patient to find a match for bone marrow transplant.

Abstract No. 178 Comparison of CD34+ Stem Cell Counts of Cord Blood, Bone Marrow and Peripheral Blood Stem Cell Harvest Samples Neena Verma, Sonia Vij, Pradeep Malik, Laxmi, Deepshikha, Rakesh Ojha, Meetu Shrikhande, Ravneet Kaur SRL Limited, Fortis Hospital, Noida Summary: CD34+ stem cells were enumerated by flowcytometry in cord blood, bone marrow and peripheral blood stem cell harvest (PBSCH) samples. PBSCH gave the highest yield. Introduction: Hematopoietic Stem Cell Transplantation is a life saving procedure. Historically HSCs were collected from the bone marrow. Recently, other sources like mobilized peripheral blood and umbilical cord blood are being increasingly used. The CD34+ cell count of the graft is necessary for dose calculation in stem cell transplantation and as a predictor of engraftment success. Flowcytometry is widely used to enumerate the CD34 cells in the graft. Materials and Methods: CD34+ stem cells were enumerated by flowcytometry in 348 cord blood samples, 17 preprocessed bone marrows, 37 post-processed bone marrows and 17 PBSCH samples. Single platform ISHAGE protocol was used on BD FacsCalibur. Results: The highest yield of CD34+ stem cells/lL was obtained from PBSCH. Average count was 2482.6/lL, highest: 5145/ lL from a 28 year old, lowest: 1039.5/lL in a 61 year old. The next best yield was obtained from post processed bone marrow; average: 651.3/ lL, highest: 3961.78/lL from a 1 year old, lowest: 25.13/lL from a 61 year old. Cord blood yielded an average of 87.11/lL, highest: 832.6/ lL, lowest: 3.68/lL. Pre processed bone marrow gave an average count: 76.6/lL, highest: 360.4/lL in a 21 year old, lowest: 3.35/lL in a 55 year old male. Conclusion: Mobilized peripheral blood obtained through stem cell apheresis gives the highest yield of CD34+ stem cells/ lL of the sample.

Abstract No. 179 HLA DRB1 Genotyping in Selected Random Population from Mumbai, India Priya Manathkar, Archana Kumari, U Shankarkumar, Abhay Choudhary, Bani Ganguly MGM School of Biomedical Sciences, Mumbai Summary: A study conducted on unrelated individual belonging to different linguistic groups from Maharashtra region for their HLA

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 DRB1 locus antigen profiles. HLA DR2 subtype 15, 16 DRB*16:01:01, DRB1*15:17 alleles were observed and DRB1*150101 were predominantly increased. The haplotype 2DRB1*13/14DRB3*01/03 susceptible for systemic lupus erythrmetosis was also found frequently in the population. Introduction: Indian Population exhibits not only a wide variety of ethnic but also great cultural and linguistic diversity. Being the most highly polymorphic genetic system, HLA has been used to great advantage for the definition of various racial groups, their migration pattern, possible admixture etc. Population specific distribution of HLA alleles is necessary and interesting both in population genetics and in HLA disease association studies. Materials and Methods: In the study 60 unrelated individuals belonging to different linguistic groups from Maharashtra were studied for their HLA DRB1 locus antigen profiles. The HLA antigens were identified using commercially procured PCR-SSP typing kits. The genotype frequency, haplotype frequency and Linkage disequilibrium estimates were calculated by using the standard method. Results: These alleles were predominantly observed in the study—HLA DR2 subtype 15, 16 DRB*16:01:01, DRB1*15:17 and DRB1*150-101 were predominantly increased. These alleles are susceptible for pulmonary tuberculosis which might explain the susceptibility of Tuberculosis in general population of India. The haplotype DRB1*13/14–DRB3*01/03 susceptible for systemic lupus erythrmetosis was also found frequently in the population. Conclusion: The association of HLA types with disease is well known however it could also serve at the means of understanding drug susceptibility in general population. The observed antigen frequencies, haplotype frequencies and linkage disequilibrium in the random population suggest the influence of genetic drift caused by selection, geography and culture. Further the study reveals that the population of India cannot be considered as a single panmictic population due to vast allelic diversity and immense heterozygosity in haplotypes.

Abstract No. 180 HLA Typing of Donors in Renal Transplant Setting: A Trend Analysis P Ramila, J Suchita, R Sawant, A Deshpande HLA Laboratory, Department of Laboratory Medicine, P D Hinduja National Hospital & Medical Research Centre Introduction: Kidney transplantation is the optimal treatment for chronic renal failure patients. HLA antigens of A, B, C (Class I) and DR, DQ (Class II) loci are particularly relevant in renal transplantation. The frequency of living kidney donation has increased over the past decade worldwide. In India, live related kidney donations account for [95 % of the total number of transplants. Aims: To analyze the degree of HLA haplotype matching of kidney donors with reference to their relationship with the patients. Materials and Methods: A retrospective analysis of demographic data and HLA test results of 1,136 prospective kidney donors screened at our centre over a 13 year (2001–2013) period was carried out. HLA typing was carried out using serology method and molecular method. (a) Serology typing was done using microlymphocytotoxicity (MLC) assay. (b) Molecular based typing was done using polymerase chain reaction-sequence specific primers (PCR-SSP) assay. The donor and recipient haplotypes were grouped based on degree of HLA antigen matching. The extent of HLA haplotype match was analyzed in specific relation wise category. HLA haplotype match from 2nd degree relatives (grand parents, uncle, aunty, niece, nephew) was compared with that of unrelated donors. Trend analysis of unrelated donors over the study period was performed. Results: The prospective donors included parents, siblings, spouse, children, second degree relatives and unrelated donors. The commonest donor-recipient

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Table 1 HLA haplotype matching in first degree relations Relation

Single haplotype match

Haplotype missmatch

Both haplotype match

Brother (N = 139)

(79) 56.83 %

(40) 28.78 %

(20) 14.39 %

Sister (N = 104)

(64) 61.54 %

(14) 13.46 %

(26) 25 %

Mother (N = 343) Father (N = 126)

100 % 100 %

– –

– –

Table 2 HLA haplotype matching in unrelated kidney donors & 2nd degree related kidney donors Relation

Single haplotype match

Haplotype missmatch

Both haplotype match

Spousal (N = 232)

(25) 10.77 % (207) 89.23 % –

2nd degree relatives (N = 44)

(17) 38.64 % (27) 61.36 %

Unrelated (N = 124)

(9) 7.25 %

–

(115) 92.75 % –

relations encountered were Parents (41.3 %), Siblings (21.4 %) and spouse (20.4 %). Majority of the kidney donors (50 %) belong to the 30–50 years age group. In spousal donation, wife to husband (87.1 %) was significantly higher than husband to wife (12.9 %). donation HLA-A2 (28.3 %), A11 (28.3 %), A24 (29.9 %), B35 (26.3 %), B40 (26.9 %), DR15 (44.5 %) & DR7 (25.9 %) were the most common HLA antigens observed in donors and HLA-A74 antigen which is rare in Indian population was observed in one donor (Tables 1, 2). Conclusion: The possibility of haplotype match was better with sisters as kidney donors, in our experience. This needs to be observed further. Second degree related donors is a better alternative to unrelated transplants from the HLA haplotype matching perspective. Further study to correlate degree of haplotype match with transplant outcome and graft survival is needed.

Abstract No. 181 Experiences with Autologous Peripheral Blood Stem Cell Harvest (PBSC) Sudha Ranganathan, Satish, Shyamala Sesikeran, Vanajakshi, AVS Suresh Apollo Hospitals, Hyderabad Summary: The yield of a good PBSC harvest depends on a pre collection CD 34 counts Although recombinant growth factors do increase the CD 34 counts in the patient, the harvest is done after the 5 day dose of 10 lg/kg in the patient. This study enables PBSC harvest based on the pre CD 34 counts. Introduction: Autologous PBSC collection with the use of a cell separator has been a routine practice for the past two decades. Patients are given recombinant growth factors for five days with or without chemotherapy to enable a good harvest. This study was aimed to identify a suitable pre CD34 count for a good PBSC harvest, yield of the product complications arising during the harvest and the time of

339 neutrophilic engraftment. Materials and Methods: 15 patients (Non Hodgkin’s lymphoma = 5 and Multiple Myeloma = 10) underwent autologous PBSC harvest over a period of 19 months between June 2012 to January 2013. The age of the patients ranged from 9-71.7 patients had a weight of \60 kg and 8 patients [60 kg. All patients were given 10 lg/kg/day of rGSF for 5 days before the harvest. The pre harvest and the product CD 34 counts and viability were done on the flowcytometer (Beckman Cytomix FC500 Germany). PBSC harvest was done using the Haemonetics plus (USA) cell separator using a central venous access. The average processed volume for every harvest was 10 L. Multiple harvests were done in patients to achieve a target of 2–3 9 106/kg. The ACD:blood ratio was set to 1:14. Adverse reactions like vasovagal reactions or those related to citrate toxicity were observed for. Results: For patients who were \60 kg a precollection CD34 count of 20/ll and a count of 30/ll for patients [60 kg would enable a harvest of approximately 2–3 9 106/kg in a single harvest. The average CD34 cells of the product was 3.25 9 106/kg body weight of the patient with 97 % viability.Perioral anaesthesia and cramps were observed in two patients and both were \60 kg in weight. The neutrophilic engraftment occurred between 8–12 days with an average of 10.9 days. Conclusion: This study enables us to plan PBSC harvest based on the CD34 count. A count of 20 cells/ll for patients \60 kg and 30 cells/ll for patients [60 kg enables a good collection of approximately 3 9 106/kg. Setting a preharvest CD34 count as recommended would enable PBSC harvest in patients who have achieved the recommended counts and would prevent unnecessary harvests and injections of growth factors which would have cost implications.

Abstract No. 182 Autologous Peripheral Blood Stem Cell in Children Weighing less than 15 kg with Solid Tumors: Technical Report of Two Cases Samantha Kumarage, Rajan Kapoor, Vishal Sondhi, Aditi, Joseph Phillip, P Mallhi, T Chatterjee, Velu Nair Armed Forces Medical College, Pune Summary: Peripheral blood stem cell (PBSC) harvesting in the smallest children (weight *15 kg) using separators is complicated by specific problems. The volume of the separation set exceeds 25 % of the total blood volume and the vascular access is generally not sufficient. Therefore, a simple manual technique for PBSC harvesting was developed. Introduction: Children with high risk neuroblastoma are generally treated with high dose sequential chemotherapy followed by autologous stem cell rescue. Use of PBSC is associated with faster recovery and less of tumor contamination. However PBSC harvest in small children is risky and much more complicated than adults. We describe a simple manual PBSC harvest technique in small children, which we have used in two children at our centre. Materials and Methods: Two children (one girl and one boy)of high risk neuroblastoma were subjected to PBSC harvest prior to administration of high dose chemotherapy. Both children weighed less than 15 kg. PBSC harvests were started after mobilization using G-CSF when the peripheral blood CD34 cell count exceeded 50/ll. PBSCs were collected using a continuous flow blood cell separator (Fresenius) after femoral vein cannulation. Prior to the procedure apheresis machine was primed with group specific irradiated packed red cells. Anticoagulation was done with combination of Heparin and ACD (1:10). Continuous Calcium infusion was administered. Results: Both children achieved the target PBSC collection in single sitting. There were no complications during mobilization or PBSC harvest. CD 34 dose of the PBSC product in girl child was 10 9 106/kg, while it was 17.9 9 106/kg for the boy. PBSC products were subsequently

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340 cryopreserved. Girl child subsequently underwent high dose chemotherapy followed by reinfusion of PBSC. She had uneventful recovery with neutrophil engraftment on D + 9 & platelet engraftment on D + 11. Conclusion: PBSC harvest is feasible in small children with minimal complications. However it requires technical expertise for successful procedure.

Abstract No. 183 A Treosulphan Based Reduced Toxicity Conditioning Protocol for Children with Thalassemia Major—Experience from a Tertiary Referral Centre in India G Vimal Kumar, M Deenadayalan, Karuna Sri, Hemalatha, Madhan Kumar, Jose Easow, V Lakshmanan, Revathi Raj Paediatric Blood and Marrow Transplantation Unit, Apollo Speciality Hospital, Chennai, India Aims: To assess efficacy and safety of treosulphan based conditioning protocol for children with Thalassemia major. Materials and Methods: We present a retrospective analysis on the use of a novel conditioning protocol for beta thalassaemia major in patients treated in our unit from 2009 to 2013. Sixty four children aged between 9 months and 15 years with a 6/6 HLA matched sibling donor were treated using a conditioning protocol with thiotepa 8 mg/kg, treosulphan 42 g/m2 and fludarabine 160 mg/m2. Data was analysed for Lucarelli class, mucositis, blood product requirement, need for parenteral nutrition, engraftment and transplant related outcome. In infants the treosulphan dose was reduced to 36 g/m2. Results: We had 8 class I, 34 class II and 12 class III patients. Mucositis was seen in 20 of them with only 5 children requiring total parenteral nutrition. Less than 4 units of red cells and less than 6 units of platelets were required in these children on average except the Class III patients. A peculiar macular rash was noticed in all our patients treated with treosulphan. All the children engrafted and 14 children had graft versus host disease requiring steroids. There were two deaths, one was a child with massive splenomegaly due to platelet refractoriness and intracerebral bleed. The other child had sepsis with consolidation lungs with pericarditis. Five out of the sixty four children had early graft rejection before day 100. No child had sinusoidal obstruction syndrome. Conclusion: We conclude that this treosulphan based regimen is well tolerated and results in durable engraftment even in Class III thalassaemia major children with no major toxicity or mortality. The cost involved in procuring the medication is balanced well by the reduced toxicity following conditioning as the need for antibiotics, blood products and parenteral nutrition is negligible.

Abstract No. 184 Sibling Cord and Bone Marrow to Cure Thalassaemia Major G Vimal Kumar, M Deenadayalan, Karuna Sri, Madhan Kumar, V Lakshmanan, Jose Easow, Hemalatha, Revathi Raj Blood and Marrow Transplantation Unit, Apollo Speciality Hospital, Chennai Introduction: There are 10,000 new births of beta thalassaemia major in India each year. The burden of long term transfusion and chelation in such children is huge and the majority of such patients in our country face early death in their teenage due to cardiac or liver haemosiderosis. Private cord blood banks have been operational in India since 2002. AIMS: Does cord blood make a difference to the outcome in children undergoing fully matched sibling allograft for thalassaemia major? Materials and Methods:

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 We did a retrospective analysis of a series of 13 children with thalassaemia major who have been cured of their disease by their saviour sibling. All 13 children were transfusion dependent with age ranging from 2 to 11 years. Nine children received thiotepa, treosulphan and fludarabine conditioning and four had busulphan and cyclophosphamide. The cord was thawed and infused first followed by bone marrow harvested from the sibling. The sibling donors were between 7 months to 3 years of age. Bone marrow was used in addition to cord for two reasons—cord nucleated cell count alone was inadequate in all our 17 cases and data was not yet available in our country regarding post thaw nucleated count from private cord banks. Results: Total nucleated cell count ranged from 0.5 9 106/kg to 1 9 107/kg in these eight children and the CD 34 ranged from 0.18 9 104/kg to 1.8 9 105/kg. Bone marrow harvest yielded 1 to 7 9 106/kg CD 34 count after harvesting less than 5 ml/kg marrow of the recipient body weight. There was on average 38 % cell loss after thawing from different private cord blood banks in India. All patients engrafted between days 12 to day 17 with persistent donor chimerism between 85 to 100 % after more than a year follow up. One child rejected his graft after initial engraftment but has been subsequently transplanted successfully using the donor’s bone marrow stem cells. One Class 3 child died of sinusoidal obstruction syndrome. Graft versus host disease was mild in two children and no cytomegalovirus reactivation was seen in any of these children. Conclusion: We conclude that the use of cord and bone marrow helps cure thalassaemia with the benefit of durable engraftment with a trend towards lower incidence of graft versus host disease. The donor needs to donate far lower doses of stem cells which enables us to plan transplantation early. Cord is an ideal source of stem cells for transplantation and should be used even if the cell dose seems suboptimal and balanced with addition of cells from bone marrow.

Abstract No. 185 Estimation of Serum Ferritin is a Useful Marker to Predict Adverse Outcome in Unrelated Cord Blood Transplantation in Children G Vimal Kumar, M Deenadayalan, Hemalatha, Karuna Sri, Madhan Kumar, V Lakshmanan, Jose Easow, Revathi Raj Blood and Marrow Transplantation Unit, Apollo Speciality Hospital, Chennai Introduction: Inflammatory cytokines play a key role in engraftment syndrome and graft versus host disease in unrelated transplantation. Mismatched cord units are particularly known to produce a peculiar engraftment syndrome with swinging fever, diffuse macular rash, capillary leak syndrome with respiratory distress. This is seen between days 5–25 after infusion of the cord unit. Early therapy with methylprednisolone at 2–4 mg/kg/day and fluid restriction are the mainstay of therapy. A complete lack of this immune response may be an early predictor of non engraftment and help plan the request for a back up cord unit. Hypercytokinaemia seen during engraftment has also been linked as a trigger for graft versus host disease. Serum ferritin is a good marker of acute inflammation. Aims: To assess whether serum ferritin is a useful marker to predict adverse outcome in unrelated cord blood transplantation in children. Materials and Methods: From April 2007 to July 2013 we have performed 30 unrelated cord blood transplants at our centre. We had prospectively performed serum ferritin in a serial manner in all our patients. The primary diagnosis was relapsed/high risk leukaemia (n = 13), Fanconi anaemia (n = 6), primary immune deficiency (n = 5), pure red cell aplasia (n = 1), sickle cell anaemia (n = 1) and Hurler syndrome (n = 3), thalassaemia major (n = 1). Results: Serial ferritin data is available on 24 out of 27 patients. Ferritin levels remained less than

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 500 in the two children who failed to engraft. Marked elevation of serum ferritin from baseline to up to 194,041 was noticed in our patients within the first 28 days of graft. Children who had a serum ferritin of over 50,000 died early due to steroid refractory graft versus host disease or sepsis. Conclusion: These results in a small series highlight the value of serial estimation of serum ferritin to predict engraftment failure, brisk engraftment syndrome and possible grades 3–4 graft versus host disease. Possible interventions like early introduction of TNF alpha blockade could help reduce the high mortality seen in our series.

Abstract No. 186 Acute Graft Versus Host Disease Following Sibling Donor Transplantation for Thalassemia Major—Risk Factors and Outcome PN Nisham, Vikram Mathews, Auro Viswabandya, Aby Abraham, Kavitha N Lakshmi, Abhijeet Ganpule, Alok Srivastava, Biju George Department of Haematology, Christian Medical College, Vellore Summary: Consecutive 321 patients who underwent allogenic haematopoietic stem cell transplant (HSCT) for Thalassemia Major with a matched related donor between Jan 1991 and Dec 2011 were studied for occurrence of acute GVHD. Clinical and Laboratory parameters were studied to understand the occurrence and outcome of Acute GVHD. Introduction: Acute GVHD (aGVHD) remains a major challenge in transplants for thalassemia transplant and is associated with significant morbidity. The incidence of aGVHD is linked to several patient and treatment related factors. Materials and Methods: Retrospective analysis of 321 patients who underwent allogeneic HSCT for Thalassemia Major at our centre between Jan 1991 and Dec 2011. Results: 321 patients (205 males and 116 females) with a median age of 7 years (range: 2–24) were analysed. Patients who died prior to 2 weeks or had primary graft rejection were excluded. Categories included 6.9 % with Lucarelli risk class I, 36.4 % with Class II, and 56.7 % with Class III. Acute GVHD (Grade I–IV) was seen in 125 patients (38.9 %) with a 12.7 % incidence of Grade III–IV GVHD. Type of conditioning regimen (p = 0.033), number of doses of methotrexate (p = 0.00) and time to neutrophil engraftment (P = .027) had impact on aGVHD. No other pre-or peri-transplant variables showed significant correlation. The 3 year overall survival was 80 ± 2.3 % in patients with GVHD compared to 81.1 ± 2.9 % in patients without GVHD (p = 0.422). Conclusions: Acute GVHD is seen in 39 % of patients undergoing transplant for thalassemia but it does not impact on overall survival.

Abstract No. 187 Safety and Efficacy of Eltrombopag in Post Hematopoietic Stem Cell Transplantation (HSCT) Thrombocytopenia Shreeniwas S Raut, Sandip A Shah, Kamlesh M Shah, Kinnari A Patel, Asha S Anand, Shailesh S Talati, Harsha P Panchal, Apurva A Patel, Sonia K Parikh, Bhavesh B Parekh, Shilin N Shukla Department of Medical and Pediatric Oncology, Gujarat Cancer and Research Institute, Ahmedabad Introduction: Eltrombopag is that induces proliferation and from hematopoieticprogenitor Design: Retrospective. Setting:

a thrombopoietin receptor agonist differentiation of megakaryocytes cells. Materials and Methods: Bone marrow transplantation unit,

341 GCRI, Ahmedabad, Period: Jan 2012–July 2013, Inclusions: All patients satisfied following: (1) Patients who have undergone HSCT, (2) Improved total leucocyte counts (post engraftment) and (3) Thrombocytopenia needing platelet transfusion with unavailability of donors for platelet transfusion OR ineffective platelet transfusion OR secondary fall in platelet count after initial rise with high risk of bleeding. Exclusions: Other secondary causes of thrombocytopenia (drugs, cytomegalovirus, infection). Results: Eleven adult patients (median age 32 years) were started on Eltrombopag 25–50 mg per day for post HSCT thrombocytopenia. Ten patients were having primary thrombocytopenia after HSCT and 1 patient had secondary fall in platelet count after achieving normal platelet count. No patient had other secondary cause for thrombocytopenia. Two patients were allogenic subsets (1 Acute myeloid leukemia i.e. AML and 1 aplastic anemia), and 9 were autologus transplants (4 multiple myeloma, 4 lymphoma and 1 AML). Eight patients were males, 3 were females. The median time of starting Eltrombopag was 21 days post stem cell infusion at a median platelet count of 9,000/cmm. The median duration for treatment was 25 days. Median total dose of 775 mg was received by patients and they had a median platelet increment of 33,000/ cmm. We observed that there were no adverse effects in these patients and there was a gradual increase in platelet count so that none of the patients had any complication due to thrombocytopenia. The cost of treatment was less than the cost of extended hospitalization and irradiated single donor platelet transfusions. Conclusion: 25–50 mg once daily dosing of Eltrombopag to enhance platelet recovery for post-HSCT thrombocytopenia is well tolerated, appears efficacious and offers transfusion independence.

Abstract No. 188 Steroid Refractory Acute Graft Versus Host Disease (SR-aGVHD)—Predictors and Outcome—A Tertiary Care Center Experience Sandeep Anil Nemani, Biju George, Vikram Mathews, Auro Viswabandya, Aby Abraham, Abhijeet Ganpule, Alok Srivastava Department of Haematology, Christian Medical College, Vellore Summary: Three hundred and seventy-six consecutive patients who underwent allogenic haematopoietic stem cell transplant (HSCT) from Jan. 2010 to Dec. 2012 were studied. Clinical and demographic parameters were studied to understand the occurrence and outcome of SR-aGVHD. Introduction: Acute GVHD (aGVHD) remains one of the major limiting factors in successful HSCT. Response to steroids is seen in approximately 50 % of the patients and those who fail to respond, have high mortality rates. Materials and Methods: Retrospective analysis of 376 patients who underwent allogeneic HSCT at our centre between Jan 2010 and Dec 2012. Results: Of the 346 patients analysed (30 excluded for graft failure/early deaths), 104 developed aGVHD (30 %). Of these, 46 patients (44.2 %) had SRaGVHD. A significant statistical association was found between grade of acute GVHD (grade III/IV) and steroid refractoriness (p \ 0.0005). No other pre-transplant or peri-transplant parameters showed any significant correlation. SR-aGVHD was treated with Cyclophosphamide, Basiliximab, Mesenchymal stromal cells and Myfortic acid derivatives, used in various combinations, but without significant difference in outcome. CMV reactivation was significantly higher in patients with SR-aGVHD (75.6 %) compared to steroid sensitive patients (32.2 %) (p \ 0.0005). The 3 year overall survival (OS) was significantly inferior in the SR group (29.3 + 6.9 %) compared to the steroid sensitive group (75.7 + 6.3 %; p \ 0.005). Conclusion: Our findings were similar to the data reported internationally including

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342 mortality rates. The prognosis remains dismal in SR-aGVHD irrespective of second line treatment options used.

Abstract No. 189 CMV Reactivation Following Allogeneic Stem Cell Transplantation Anup J Devasia, Vikram Mathews, Auro Viswabandya, Aby Abraham, Abhijeet Ganapule, Alok Srivastava, Biju George Department of Hematology, Christian Medical College, Vellore Summary: Retrospective analysis of consecutive transplants done in the Department of Haematology, CMC Vellore between January 2008 and December 2012. Recipient and donor demographics along with the pre, peri and post transplant data were recorded. Patients were monitored for CMV reactivation by CMV DNA PCR in the first 100 days post SCT. Introduction: Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality following allogeneic stem cell transplantation (SCT). We studied the incidence of CMV infection in a population where previous exposure to CMV is high. Materials and Methods: Retrospective analysis of consecutive transplants done in the Department of Haematology, CMC Vellore between January 2008 and December 2012. Results: Four hundred and seventy five cases were included at a median age of 21 years (range: 1–59). CMV reactivation (PCR positive) occurred in 36.6 % at a median time of 41 days post SCT (range: 10–100). Biopsy proven CMV disease was seen in 8 patients (1.68 %). (4 colitis, 2 esophagitis, 1 duodenitis, 1 pneumonitis). Donor age [40 (p = 0.037), unrelated donor (p = 0.000), male donor to female recipient (p = 0.030), HLA mismatch (p = 0.000), neutrophil recovery \15 days (p = 0.004), occurrence of acute GVHD (p = 0.000) and steroid refractory GVHD (p = 0.028) were predictive for CMV reactivation on univariate analysis. On multivariate analysis male donor to female recipient (p = 0.042), HLA mismatch (p = 0.006), early neutrophil recovery (p = 0.049), acute GVHD (p = 0.000) and steroid refractory GVHD (p = 0.021) remained significant. Most of the patients were treated with gancyclovir for a median duration of 16 days. 5 year overall survival was significantly lower in patients with CMV reactivation (58.2 ± 4.9 %) compared to those without reactivation (68.9 ± 3.7 %, p = 0.004). Conclusion: CMV reactivation after SCT is seen in 36 % of transplant recipients and is associated with a lower overall survival.

Abstract No. 190 Incidence of Invasive Fungal Infections in Patients Undergoing Allogeneic Stem Cell Transplantation Gaurav Dixit, Vikram Mathews, Auro Viswabandya, Aby Abraham, Abhijeet Ganpule, Alok Srivastava, Biju George Department of Haematology, Christian Medical College, Vellore Summary: Three hundred consecutive patients who underwent allogenic haematopoietic stem cell transplant (HSCT) from January 2010 to June 2012 were studied. Clinical and demographic parameters were studied to understand the occurrence and outcome of invasive fungal infections. Introduction: Invasive fungal infection remains one of the major limiting factors for a successful HSCT. Except for candida species, invasive fungal infections are most often caused by molds, like aspergillus. An increased incidence of mold infections has been reported from developing countries like ours in post transplant setting, with high mortality and morbidity. Materials and Methods: Retrospective analysis of 300 patients who underwent allogeneic

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 HSCT at our centre between January 2010 and June 2012. Results: Sixty four patients (21.3 %) developed invasive fungal infections. Of these, 58 (19.3 %) had possible, 2 (0.7 %) had probable and 4 (1.3 %) had proven fungal infections as per EORTC-MSG criteria. Only 4.7 % had previous fungal infections. Fungal infections were more common in patients with acute GVHD (30.9 %) compared to those without GVHD (18.1 %; p = 0.02). The use of corticosteroids was also more commonly associated with IFI compared to patients who did not require steroids during HSCT (29.5 vs. 14.3 % p = 0.002). Overall survival in patients with fungal infection was significantly lower than patients who did not develop fungal infection during HSCT [34 vs. 72 % (p = 0.000)]. Conclusion: There is a high incidence of fungal infections in patients undergoing allogeneic HSCT especially in the presence of acute GVHD. Better prophylactic and therapeutic strategies need to be considered in view of the higher mortality associated with these fungal infections following HSCT.

Abstract No. 191 A Case Report: Allogenic Hematopoietic Stem Cell Transplant from Hemophilic Donor, in a Case of Aplastic Anemia R Jetani Amit, A Shah Sandip, S Anand Asha, A Patel Apurva, N Shukla Shilin, S Talati Shailesh, P Panchal Harsha, K Parikh Sonia, B Parekh Bhavesh Department of Medical and Pediatric Oncology, Gujarat Cancer Research Institute, Ahmedabad Summary: We report a case of 19 year old female, diagnosed with severe aplastic anemia, who received Allogenic hematopoietic stem cells from her hemophilic brother. Recipient’s factor VIII level remained normal post allogenic stem cell transplant. Introduction: Factor VIII is generated in liver and endothelial cells of renal vessels & it is not related to bone marrow hematopoietic cells or peripheral hematopoietic stem cells. Therefore, hematopoietic stem cells from hemophilic donors can be used for Allogenic hematopoietic stem cell transplantation. There is no case reported of stem cell transplantation with hemophilic donor in Centre of International Bone marrow Transplantation Registry (CIBTR). Materials and Methods: A nineteen year old female patient with bone biopsy proven severe aplastic anemia was given Fludarabine and cyclophosphamide as conditioning regimen. She received Allogenic hematopoietic stem cells from her brother who had Hemophilia A, with factor VIII levels of 8 units per liter. Factor VIII level was assessed in recipient at 1 month after Allogenic hematopoietic stem cell transplantation, and then every 3 monthly. Results: Recipient’s blood counts recovered within 15 days of stem cell transplantation without any major complication. Chimerism (100 %) was documented on day 30 post transplant and recipient’s factor VIII level was 150 units per liter after 1 month of HSCT. Factor VIII level, after 4 months of stem cell transplantation, was 158 units per liter. Conclusion: Hematopoietic stem cell transplantation from hemophilic donor is safe and effective in aplastic anemia or any other hematological conditions.

Bleeding Disorders Abstract No. 192 Evaluation of Patients with Mucocutaneous Bleeding in a Tertiary Care Hospital in India S Sri Gayathri, Jennifer, S Kumar, V Kamath, S Nair Christian Medical College, Vellore, Tamilnadu

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Summary: Mucocutaneous bleeding occurs due to several causes and treatment depends on accurate diagnosis. This study emphasizes the role of history and algorithmic approach for judicious use of available screening and confirmatory tests in a stepwise fashion to arrive at a diagnosis. Introduction: Mucocutaneous bleeding disorders are characterized by easy bruising, gum bleeding, epistaxis, mennorhagia and bleeding after trivial injury. In general, they are the hallmark of disorders involving platelet-vessel wall interaction. Materials and Methods: A retrospective study of patients with mucocutaneous bleeding referred to our institution, during the period of seven months from January to July 2013 was performed by reviewing their records. Evaluation in the laboratory includes a detailed history and examination followed by screening tests of coagulation and complete blood counts. The subsequent tests are decided after reviewing these results. Results: Out of 170 patients reviewed, 30 (18 %) were diagnosed to be von Willebrand disease (VWD), 23 (14 %) Glanzman thrombasthenia, 13 (8 %) Acquired Platelet function defect with Eosinophilia (APDE), 15 (9 %) Platelet Function defect (not classifiable), 2(1 %) Bernard Soulier syndrome (BSS), 2 (1 %) Borderline low level VWD with risk of bleeding, 2 (1 %) Storage pool disorder, 1 (0.5 %) Platelet type VWD, 1 (0.5 %) Scott Syndrome and 76 (44 %) had no intrinsic haemostatic defect. Of the 30 VWD, 4 were type 1, 5 type 2 A/2 M, 1 type 2B, and 20 type 3. Conclusion: von Willebrand disease is the most common diagnosis among patients who presented with mucocutaneous bleeds, followed by defects in platelet function. A significant percentage of patients could not be given a definite diagnosis even after performing all available tests.

Abstract No. 193 A Comparative Study of Various Clinico-Hematological Parameters of Congenital Bleeding Disorders Shweta Puri, Suresh Hanagavadi, B Nikethan J.J.M. Medical College, Davangere Summary: The clinico-hematological study of congenital bleeding disorders on 58 patients presenting with history of prolonged/spontaneous bleeding evaluated by clinical history including consanguinity and laboratory methods was carried out in the Department of Pathology J. J. M. Medical college shows the predominance of Hemophilia-A followed by Hemophilia-B and von Willebrand’s disease. Rare disorders included deficiency of factor XI, XIII, fibrinogen & functional platelet disorder. Most of them manifested in the early age. Complete Hemogram and Basic Coagulation profile done to diagnose the appropriate disorder. Introduction: Congenital bleeding disorders are heterogenous group of diseases which reflects abnormalities of blood vessels, platelets and deficiency of coagulation factors. The clinical presentation includes prolonged bleeding from umbilical cord and joints, bleeding of gums, subcutaneous hematomas and severe epistaxis. Materials and Methods: 58 patients, referred from Karnataka Hemophilia Society to the Department of Pathology in the age group of 4 months to 49 years with prolonged bleeding history were included. Detailed Clinical examination, history, routine hematological tests, basic coagulation profile were performed to diagnose the underlying defect. Results: Among the Congenital bleeding disorders observed, Hemophilia-A accounted for 53 %, factor IX deficiency 22 %, von Willebrand disease 12 %. Rarer disorders included deficiency of factor XIII, factor XI, hypofibrinogenemia and functional platelet disorder. Conclusion: Congenital bleeding disorders is common in age group between 11–20 years. Hemophilia-A is predominant in age group of 1–5 years. Routine hematological investigations and basic coagulation profile remains the first panel towards the approach to diagnosis.

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Abstract No. 194 Molecular Pathology of Haemophilia A in Patients from Western India Preethi Satish Nair, Shrimati Shetty, Kanjaksha Ghosh National Institute of Immunohaematology (ICMR) Summary: Much Haemophilia A (HA) cases in India go undiagnosed and although Restriction Fragment Length Polymorphism is a feasible method, it has many limitations. Mutation screening adds on to the existing diagnosis methods to give more accurate results. We identified mutations in 91 HA cases (14 familial, 77 unrelated) in a cohort of 109 intron 1 and 22 inversion negative cases. Introduction: Despite increased awareness and diagnostic facilities, about 70–80 % of HA patients still remain undiagnosed. India, with a huge population (1.2 billion) is genetically heterogeneous due to several thousands of years of migration, colonization and genetic admixture. Hence studies on prevalent mutations on HA from this country will be educative. Materials and Methods: Intron 1 and 22 inversion negative HA patients with severe to mild-moderate clinical manifestations were included in the present study. Conformation Sensitive Gel Electrophoresis and DNA sequencing techniques were incorporated for mutation detection. Results: In a cohort of 109 inversion negative cases, we identified causative mutations in 91 cases (14 familial and 77 unrelated). Double mutations were identified in some, raising the tally to 95. 62 individual mutations comprising of 26 novel and 36 recurrent mutations were observed. Of these, 38 were missense, 7 nonsense, 11 deletions, 5 insertions and 1 splice site mutation. A classical case of female HA was also identified belonging to a family with strong history of moderate HA and consanguinity. Another female bleeder was identified with a heterozygous missense mutation. Mutations known to predispose towards CRM positive and inhibitor formation were also identified. Conclusion: A sizeable number of novel mutations, a few double mutations and recurrent mutations were detected. 6 % of mutations were CRM positive and 1.2 % patients were complicated by development of inhibitors. In spite of low prevalence of inhibitors observed probably due to less aggressive prophylactic treatment, few predisposing mutations were identified.

Abstract No. 195 Rare Site Hemorrhages in Patients with Hemophilia Manoj Toshniwal, S Chandrakala1, R Mahesh1, Farah Jijina 2, K Ghosh3 Department of Hematology, KEM Hospital, 2Hinduja Hospital, National Institute of Immunohaematology (ICMR), KEM Hospital, Mumbai 1

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. 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 is hardly been reported in cases of hemophilia. We report nine cases of hemophilia from our comprehensive hemophilic care center of western India presenting with bleeding at very rare and unusual sites. Four cases presented with

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344 severe anemia, jaundice, dyspnea and hemoptysis with HRCT chest suggestive of diffuse alveolar hemorrhage. All were managed conservatively with adequate factor replacement and supportive treatment. Two young adult patients had history of jerk while riding bike in village and after 3–4 days presented with paraparesis of subacute onset with bowel and bladder involvement with MRI spine showing spinal subdural hematoma, managed with factor replacement. Another 5 year old boy presented with acute onset weakness in both lower limbs after trivial trauma whose MRI spine revealed large epidural hematoma. This was treated with adequate and timely factor replacement leading to complete neurological and functional recovery. A young patient presented first time with swelling of neck, rapidly progressive dyspnoea and stridor after mild trauma to the neck by slipping from staircase. On examination had neck muscle hematoma and ultimately needed tracheotomy as life saving measure. A 22 year male presented with fever and pain in abdomen, USG was s/o splenic rupture with perislpenic hematoma with left sided pleural effusion. Patient factor assay was factor VIII levels \1 %. He was managed conservatively.

Abstract No. 196 Emergencies in ‘People with Hemophilia’ A Krishna Prasad, M Shetty, AMVR Narendra, AC Upadhyay, M Nageswar Rao Nizam’s Institute of Medical Sciences, Hyderabad, AP Introduction: People With Hemophilia (PWH) will present with various bleeding emergencies. Management in resource poor setting is a challenge because majority cannot afford. Situation warrants usage of factor concentrates in minimum effective thrombotic doses. Aim: Study of various emergencies encountered in PWH. Materials and Methods: Case records of PWH admitted to our department over one year, with various bleeding emergencies, were collected. Information was analyzed regarding type of emergency, treatment extended and outcome. Results: Total 32 PWH were admitted in 1 year with 40 bleeding episodes. Average age was 22.7 years. Hemophilia A in 35 and rest hemophilia B. Average body weight was 46.3 kg. Mild hemophiliacs were 3, moderate 11 and severe 26. Spontaneous bleeding episodes were noted in 29. Precipitating factors for bleeding were noted as injury in 8, intra-muscular injection and seizures in 1 each. Average duration of bleeding was 1.4 days. Acute hemophilic arthropathy was seen in 5, acute-on-chronic arthropathy in 4, acute intra-muscular hematoma in 13, Intra cranial bleed in 5, active GI bleed in 6, hemoperitonium in 2, hematuria in 1, hemoptysis in 1 and bleeding wounds in 3. Factor percentage was raised to 20–40 % depending on clinical problem except in certain severe and life threatening bleeds. Clotting factor concentrates were given in 12 and activated factor VII in 2. Rest received factor concentrates and blood components. Packed red cell transfusion was needed in 8. Factor inhibitors were positive in 3 and anti hepatitis C antibodies in 1. All patients improved with the management and there was no mortality. Conclusion: Minimum effective factor concentrates can bring gratifying results in majority. In certain situations higher doses are needed to stop the bleeding. Activated factor VII is an effective alternative in some PWH with inhibitors.

Abstract No. 197 Bone Mineral Density in People with Hemophilia in India Sushil Selvarajan, Abraham Sunder2, Joseph Bondu4, Shubhanker Mitra1, Aby Abraham2, Biju George2,

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Vikram Mathews2, Joe Fleming4, Samuel George Hansdak1, Simon Rajaratnam3, Auro Viswabandhya2, Thomas Paul3, Alok Srivastava2 Christian Medical College, Vellore Introduction: Bone Mineral Density (BMD) is known to be reduced in hemophilia due to several reasons. This study was undertaken to (i) evaluate the prevalence of low BMD among adult people with hemophilia in India and (ii) to study the factors possibly associated. Results: Fifty consecutive people with hemophilia (median age: 25 years, range: 16–48) had their BMD measured by a DXA scan. Forty-one had severe hemophilia A or B. The prevalence of low BMD, defined as the age appropriate Z score of B-2.0 at either the lumbar spine or at the hip was found to be 52 %. Among the factors evaluated, Body Mass Indices were found to be significantly lower in the group with low BMD (mean: 20.127 vs. 24.439 kg/m2; p = 0.005). Also, decreased physical activity (assessed with the International Physical Activity Questionnaire) was associated with low BMD (mean: 1544.85 vs. 2414.67 MET-minutes/week; p = 0.024). Among the laboratory parameters, Vitamin D was low in 76 % of the study group (mean: 14.93 ng/ml, range: \3.0–33.8 ng/ml). However, there was no significant difference in Vitamin D levels between the low and normal BMD groups. The serum Beta Crosslaps, a marker of bone resorption, was found to be significantly elevated in the low BMD group (mean: 1319.42 vs. 956.96 pg/ml; p = 0.045). No statistically significant correlation was noted between serum calcium, phosphate, parathormone levels and BMD. Conclusion: Overall, this shows the high prevalence of low BMD in adult people with hemophilia in our country. Serum bone markers may be a useful tool for the assessment and follow up of this. Appropriate physical activity needs to be encouraged among people with hemophilia and other therapeutic interventions need to be further evaluated.

Abstract No. 198 Intron 22 and Intron 1 Inversions of the F8 Gene as Predisposing Risk Factors for Inhibitor Development in Indian Severe Haemophilia A Patients Patricia Pinto, Kanjaksha Ghosh, Shrimati Shetty Department of Haemostasis & Thrombosis, National Institute of Immunohaematology (ICMR), Mumbai Summary: Inversions in the F8 gene (in intron 22 and intron 1) are the causative mutations in around 50 % of severe haemophilia A patients and are thought to be important genetic risk factors for the development of ‘FVIII inhibitors’ in certain populations. The present study was undertaken to study the association between these F8 inversions and inhibitor development, and the results suggest that intron 22 inversions are an important risk factor for FVIII inhibitor development in Indian severe haemophilia A patients. Introduction: Development of ‘FVIII Inhibitors’ in congenital haemophilia A patients is a serious complication of FVIII replacement therapy, and influenced by various genetic and non-genetic risk factors. Earlier studies in Indian haemophilia A patients, have shown IL10 and TNFA rs1799724 promoter polymorphisms (but not FVIII haplotypes), to be significantly associated with inhibitor development. Our aim was to analyse the association of inversions in introns 22 and 1 in the F8 gene with inhibitor development in Indian severe haemophilia A patients. Materials and Methods: 150 Indian severe hemophilia A patients, i.e. 60 consecutive inhibitor positive, 20 concordant/discordant family members, and 70 consecutive inhibitor negative patients ([10 years, [10 treatment product exposures) were included in the study after informed consent. Intron 22 Inversion was analysed by the modified I-PCR (Rossetti et al. 2005), and Intron 1 Inversion was

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 analysed by the method described by Bagnall et al. (2002). Results were analysed for statistical significance by Fisher’s exact test. Results: Intron 22 inversions were observed in 50 % inhibitor positive and 28.57 % inhibitor negative patients. Intron 1 inversions were observed in 3.33 % inhibitor positive and 5.71 % inhibitor negative patients Conclusion: Intron 22 Inversions were significantly higher in the inhibitor positive HA patients (P: 0.0184, OR: 2.500, 95 % CI: 1.211–5.161, Fisher’s exact test), and among the inhibitor concordant family members, suggesting that intron 22 inversions are an important risk factor for inhibitor development. Intron 1 inversions in the two groups were not significantly different. Association studies with other risk factors could provide further insights into the FVIII immune response.

Abstract No. 199 Prevalence of Inhibitors in Indian Haemophilia Patients Patricia Pinto, Tejashree Shelar, Vidhya Nawadkar, Darshana Mirgal, Alfiya Mukaddam, Kanjaksha Ghosh, Shrimati Shetty Department of Haemostasis & Thrombosis, National Institute of Immunohaematology (ICMR), Mumbai Summary: A serious complication of replacement therapy in patients with bleeding disorders is the development of specific antibodies or ‘inhibitors’ to deficient coagulation factors. This prevents efficient clinical management and also leads to an increase in the morbidity and mortality, as well as the cost of treatment. This study was planned (funded by NovoNordisk India Pvt. Ltd.) to analyse the prevalence of inhibitors in Indian haemophilia patients and examine contributing risk factors if any. Introduction: The mechanism of inhibitor development in patients with bleeding disorders is multi-factorial and cannot be predicted, but a prompt and accurate diagnosis is critical as early therapy can be life-saving. The aim of this study was to diagnose patients with bleeding disorders in India and screen them for inhibitors, and subsequently analyse and compare the prevalence of inhibitors in different regions in India. Materials and Methods: Patient details were recorded, samples from 3,332 patients in different cities in India were collected in sodium citrate vacutainers, after informed consent. Coagulation and inhibitor screening, and Bethesda assays (quantification of inhibitor titre) were performed. Results: Of the 3,332 samples screened, 2,694 were Haemophilia A patients (181 FVIII Inhibitor positive), 430 Haemophilia B patients (3 FIX inhibitor positive), 37 patients with rare bleeding disorders (without inhibitors), and 117 von Willebrand Disease (vWD) patients. Conclusion: The state-wise highest incidence of FVIII:C Inhibitors was seen among the Pondicherry samples (11.94 %), followed by Jammu & Kashmir (9.90 %), Tamil Nadu (9.09 %), and Maharashtra (8.46 %). Other regions showed an Inhibitor incidence \8 %. The overall FVIII:C Inhibitor incidence in the samples studied was 6.72 % (181/2694). FIX:C inhibitors were detected in samples from Maharashtra (1.10 %), Kerala (1.37 %), and Uttar Pradesh (3.13 %). The overall incidence of FIX:C inhibitors was 0.70 % (3/430). This study may have implications for the therapeutic management of these patients.

Abstract No. 200 Low Dose Factor VIII Prophylaxis in Children with Severe Hemophilia Shailendra Prasad Verma1, TK Dutta2, S Mahavevan3, P Nalini3, Niranjan Biswal3, Debdatta Basu4, A Ramesh5

345 Clinical Haematology, JIPMER, 2Division of Clinical Haematology, JIPMER, 3Department of Pediatrics, JIPMER, 4Department of Pathology, 5Department of Radiodiagnosis 1

Summary: Major morbidity in severe hemophilia is mainly due to recurrent joint bleeds. Joint bleeds and subsequent joint damage can be prevented by regular factor prophylaxis and is routinely practiced in Western countries. This involves high factor doses (25 units/kg thrice a week) and high cost, not feasible in developing countries. Our randomized study showed that low dose factor prophylaxis (10 units/ kg twice a week) is very effective in preventing joint bleeds, it is safe and overall cost effective. Introduction: Joint bleeds are the major cause of co morbidity and poor quality of life in hemophilia patients. Preventing joint bleeds with factor prophylaxis is the ideal treatment for hemophilia children. Severe hemophiliacs (factor level \1 %) may be converted to moderate hemophiliacs (factor level 1–5 %) with factor prophylaxis and spontaneous bleeds can be prevented. High dose factor prophylaxis practiced in developed countries is not feasible in developing countries like India. Low dose factor prophylaxis has not been studied systematically in India and worldwide. Joint bleeds are the major cause of comorbidity and poor quality of life in hemophilia patients. Preventing joint bleeds with factor prophylaxis is the ideal treatment for hemophilia children. Severe hemophiliacs (factor level \1 %) may be converted to moderate hemophiliacs (factor level 1–5 %) with factor prophylaxis and spontaneous bleeds can be prevented. High dose factor prophylaxis practiced in developed countries is not feasible in developing countries like India. Low dose factor prophylaxis has not been studied systematically in India and worldwide. Objectives of the study were (1) To assess the efficacy of low dose factor prophylaxis in children with severe hemophilia A. (2) To compare the amount of factor used and safety issues in Prophylaxis and Episodic groups. Materials and Methods: Twenty one Children with severe hemophilia A in age range of 1–10 years without measurable inhibitors were selected in study. They were randomly assigned to Prophylaxis group and Episodic group. Prophylaxis group children received factor VIII concentrate 10 units/kg body weight on two fixed days a week on OPD basis through peripheral veins. Episodic group received factor concentrate after having bleed in doses of 25 units/kg body weight till bleeding subsided and symptoms improved. Children in Prophylaxis group who had break though bleed were treated like episodic group. Both the groups were evaluated on monthly basis for number of bleeds and other records. Total study duration was 6 months. Results: Eleven children were assigned to prophylaxis and 10 to episodic group. Children on prophylaxis had 17 overall bleeds (7 joint bleeds) in comparison to 40 bleeds (25 joint bleeds) in episodic group. Median number of overall events per patient per month and median number of haemarthrosis per patient per month were 0.16 (range 0.0–1.2) and 0.1 (0–0.5) in prophylaxis group in comparison to 0.58 (0–1.6) and 0.41 (0–0.83) in episodic group (p \ .05). Total factor VIII consumption was 90 and 50 units/kg/month in prophylaxis and episodic group respectively. Average hospital emergency visits were \1 per month in prophylaxis group and 3 in episodic group (p = \0.05). Median days of absenteeism from school were 2 days/month (range 0.5–5 days) in episodic group and 0.5 days (0–1.6 days) in prophylaxis group(p = .002). None of patients required central venous catheter placement in prophylaxis group. No significant venous injury or complications were noted in prophylaxis group. Only one patient in prophylaxis group had discontinuation of prophylaxis for one week due to poor veins. No dropouts occurred during study period and compliance was 97 %. Conclusion: Low dose Factor VIII prophylaxis is efficacious, cost effective and safe method of preventing joint bleeds and consequent joint damage in children with severe hemophilia.

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Abstract No. 201 Orthopedic Surgeries in Patients with Hemophilia: Experience From a Comprehensive Hemophilia Care Center of Western India Manoj Toshniwal, S Chandrakala, R Mahesh, Nilesh Wasekar1, Farah Jijina2, K Ghosh3 Department of Hematology, KEM Hospital, 2Hinduja Hospital, National Institute of Immunohaematology (ICMR), KEM Hospital, Mumbai

1 3

Introduction: Management of patients with hemophilia (PWH), in resource poor developing countries remains a major challenge to the care givers. The problems are further compounded in patients requiring surgeries. Thus even orthopedic surgeries in hemophilia patients require much more planning and efforts than in non hemophiliacs. Several studies have shown that osteoporosis is a universal phenomenon in hemophiliacs all over the world. Magnitude of osteoporosis is much more in developing countries and hence the complications and fractures of long bones are also much higher. Materials and Methods: Over the past many years, our comprehensive hemophilia care center has carried out various orthopedic surgeries in PWH. Thirty different types of surgeries from fracture to amputation were carried out in 26 PWH over the period of last 10 years, age range of 8–62 years (Avg age 27.04 years). Of these 21 had hemophilia A (2 moderate, 19 severe), 5 patients had hemophilia B and 2 hemophilia A patients had inhibitors. The Factor concentrate used was optimized from our previous experience, along with generous use of antifibrinolytic drugs, oral, parenteral and local. Results: Of these 30 surgeries six cases were of complex hemophilic pseudotumor presented very late, seven patients needed amputation of limb or digit. Six patients underwent replacement surgery for major joints under meticulous factor replacement. The factor concentrate used in 30 surgeries ranged from 2,000–27,200 IU (mean 15847.24 IU), 366.17 IU/kg. Two patients with inhibitors received modest dose of FEIBA ? factor VIIa. Most of them received parenteral and local antifibrinolytic drugs. Three patients developed inhibitors post operatively. One patient had severe bleeding and wound infection requiring prolonged blood product and antibiotic support. Factor support was given for 7–10 days in all patients. Most of them discharged with good functional activity except one elderly patient who developed massive pulmonary embolism on 2nd post operative day and succumbed to the complication. Summary: The present study clearly shows that major orthopedic surgery in PWH can be accomplished with only 40–50 % of total factor concentrate as compared to international guidelines for factor concentrate prophylaxis for PWH undergoing surgery. There is need to define lower limits of factor replacement for the developing world so that the limited resources may be used optimally for the cohort of PWH. There are limited data from the developing world with regard to PWH undergoing surgical procedures. In our experience our low-dose protocols are effective, reduce factor consumption by almost one third, and are not associated with a significantly increased risk of delayed hemorrhage. This has major implications for hemophilia care not only in developing countries but also in the developed countries where the health systems are strained due to the very high cost of lifelong hemophilia care.

Abstract No. 202 Challenges in the Molecular Diagnosis of Von Willebrand Disease (VWD) Priyanka Kasatkar, Shrimati Shetty, Kanjaksha Ghosh National Institute of Immunohematology

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Summary: Large size of the gene, heterogeneous nature of mutations and presence of a highly homologous pseudogene region are the major impediments in the genetic diagnosis of VWD. Using a combination of several techniques, 92 % patients were characterized at the molecular level. This is the largest series of mutation analysis in type 3 VWD patients reported till date. The limitations of different techniques, methods adopted for achieving the highest sensitivity for mutation detection is being discussed. Introduction: VWD is the autosomal bleeding disorder caused by defects in VWF, a large multimeric multifunctional glycoprotein involved in hemostasis. Type 3 VWD is the commonest subtype reported from India, as many of the milder variants of VWD are underdiagnosed. Materials and Methods: 135 well characterized type 3 VWD patients (68 males and 67 females) from 100 families were included in the study. We adopted strategy to screen for CG-dinucleotide mutational hotspots, CSGE, sequencing and MLPA. Results: Standardization of stringent PCR conditions for the exclusion of pseudogene, multiplex PCR for exons with similar PCR conditions, different gel running conditions for CSGE, screening of hot spot mutations by PCR-RFLP, MLPA for larger deletions or duplications and direct sequencing for exons harboring multiple polymorphisms were some of the strategies adopted for an efficient detection of VWF mutations in the present analysis. Overall, mutations could be detected in 92 % type 3 VWD patients. 70 different types of mutations were identified, 40 of which were novel. Conclusion: The co-amplification of VWF pseudogene is a major impediment to genetic diagnosis of VWD. The problem is exacerbated because of the presence of polymorphisms in the pseudogene which mimics the hotspot mutations in the original gene. Though the identification of mutations in VWF gene is complicated, using an effective strategy like the one mentioned above, large majority of the patients could be characterized at the molecular level.

Abstract No. 203 A Reverse Dot Blot Assay for Quick Detection of Arginine Hot Spot Mutations in Von Willebrand Disease Bipin P Kulkarni, Priyanka Kasatkar, Shrimati D Shetty, K Ghosh National Institute of Immunohaematology (I.C.M.R.), 13th Floor, MS Building, KEM Hospital Campus, Parel, Mumbai 400012 Summary: 20 % of the women in the reproductive age group suffer from menorrhagia. Mutation detection in vWF gene is challenging. CGA arginine codons in the vWF gene are likely to mutate due to unequal methylation. The transition of C to T in 11 hot-spots CGA codon results in nonsense mutations, which account for 20 % of vWD cases. Detecting these 11 Hot-Spot mutations by RDB hybridization will facilitate in quickly obtaining information about 20 % of mutations that can cause vWD. Introduction: One fifth of the women in the reproductive age group suffer from menorrhagia. Nonsense mutations at arginine residues cause severe vWD. These variations were detected by PCR followed by reverse dot blot hybridization, which will facilitate a much quicker diagnosis. Materials and Methods: Biodyne C Transfer membrane 0.45 EDAC 1, Maleic acid, Blocking agent, NBT, BCIP, Streptavidin-AP conjugate. For pilot study, 3 patient samples typed for 2 mutations were blinded and taken for study. NH2 linked oligonucleotide probes complementary to normal and mutant DNA were blotted on Biodyne C membrane (1 ll, 10 pmols). vWD patient DNA was amplified using biotin-labeled primers and hybridized to the membrane strip at 38 °C O/N bearing the oligoprobes. An enzyme-linked system (streptavidin-alkaline phosphatase) was used for detection. Results: In all patients studied, following mutations were picked up correctly: Pt 1: Exon 31, c.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 5335T/T, Pt 2: Exon 31, c. 5335C/T, Pt 3: Exon 43, c. 7300T/T. Conclusion: This study is expected to provide us a faster, more cost effective method to identify 11 commonly found mutations in the vWF gene, which will facilitate quicker diagnosis and will be helpful in offering prenatal diagnosis to these families in shorter time.

Abstract No. 204 Type 2B VWD vs. Platelet-Type VWD: A Diagnostic Challenge—A Tertiary Care Centre’s Experience Vandana Kamath, J Mammen, S Singh, S Nair Christian Medical College Hospital, Vellore Summary: RIPA mixing test is a mandatory test to differentiate between almost similar, yet dissimilar type 2B VWD and PT-VWD disease entities. Introduction: Though Platelet-type von Willebrand disease (PT-VWD) and type 2B have different etiologies they share almost similar clinical bleeding and basic laboratory phenotype. The global incidence of platelet type VWD is approximately 15 % of 2B. Both these disease entities are a result of gain-of function mutation that leads to enhanced binding between platelet ligand and VWF molecule. The defect is at VWF gene in 2B, and at the platelet GPIBA in PT-VWD. As treatment decisions are critical, these have to be differentiated. The discriminatory test used is the Ristocetin induced platelet aggregation (RIPA) mixing study. Materials and Methods: In our institution over a period of 8 years (2006–2013), 301 patients were diagnosed as VWD. 18 showed an exaggerated response to low dose Ristocetin (0.5 mg/ml) were subjected to RIPA mixing to differentiate between 2B and PT-VWD. Results: All the 18 patients which showed an exaggerated response to low dose Ristocetin were subjected to RIPA mixing of which 3 showed defective platelets (PTVWD) and 15 showed defective VWF molecule (2B). In our study the incidence of type 2B is 5 % of the VWD and incidence of PT-VWD is 20 % of 2B. Conclusion: There is ongoing concern that most people with PT-VWD are misdiagnosed as type 2B. Differentiation between these two entities has to be made as treatment decisions play a critical role. Therefore we promote RIPA-mixing test as a mandatory phenotypic test for provisional identification of PT-VWD. Nevertheless, genetic testing is the confirmatory test for a definitive diagnosis.

Abstract No. 205 Role of Von Willebrand Factor (VWF) in Bleeding and Thrombosis Patients

347 through interaction with platelet receptor glycoprotein Ib 2. The binding and stabilization of FVIII (anti haemophilic factor) for secretion and transport in plasma. VWF and FVIII are two distinct proteins that circulate as a complex in plasma. Quantitative and qualitative abnormalities of VWF is seen in VWD. There are 3 forms of VWD Inherited, Acquired and Pseudo or platelet type. Inherited: Inherited are 3 types Type I seen in about 70–80 %. Its inheritance is dominant. Bleeding symptoms can be mild or moderate Type II seen in about 15–20 %. Inheritance is dominant or recessive. Bleeding symptoms can be mild or severe. Type II have four subtypes: Type 2A, Type 3B, Type 2 M and 2 N (Normandy) Type III is more severe form VWD. Its inheritance is autosominal recessive. Very low FVIII is present. Both parents are carriers of gene. Bleeding can be severe. Acquired: This occur in patients with autoantibodies. Structural or functional defects of VWF that are secondary to autoimmune, lymphoproliferative or myeloproliferative, malignant, cardiovascular, patients with aortic stenosis leading to gastrointestinal bleeding (Heyde’s syndrome), Wilm’s tumour, Hypothyrodism and mesenchymal dysplasia or other disorders. Thrombocythemia is another cause of acquired VWD. VWF abnormalities can be result from: (1) Antibody-mediated clearance or functional interference. (2) Absorption to surfaces of transformed cells or platelets. (3) Increased shear stress and proteolysis. Platelet Type: Also called as pseudo platelet type. It is autosomal dominant type of VWD caused by gain of function mutations of the VWF receptor as platelet like alpha chain of GP Ib receptor. Aim: Our aim was to study bleeding and thrombosis in patients. VWF was screened in patients with bleeding and susceptibility to thrombosis Inclusion Criteria: Patients with bleeding history and some who had underlying predisposition to thrombosis like CAD, MI, AF and elevated lipid profile were screened. Materials and Methods: The study was done at tertiary hospital in Mumbai, India. 120 patients were screened with different history of bleeding especially mucosal type and susceptibility to thrombosis. Patients who were on medicines like Aspirin or other nonsteroidal antiinflammatory drugs (NSAID), Clopidogrel, warfarin and heparin were not included in this study. The blood was taken in 3.2 % buff. Na Citrate of 0.109 M vacutainer (BD). Blood to anticoagulant ratio was 9:1. The blood was mixed gently and then centrifuged (2,000 g for 30 min). Plasma was separated, aliquotted, frozen at -20 °c until testing. The tests were carried out in batches. No sample was kept beyond 25 days. Samples were thawed for 5 min in water bath (37 °c) and kept at room temperature for 20 min before assay. Quantitative determinations of VWF antigen (Vwf Ag) in plasma with an immuno-Turbidimetric assay (STA-Liatest VWF Diagnostica stago, France) kits were used and analysed on stago-compact instrument. Results: 120 Samples were analysed.

Enna M Lourenco, VP Antia Breach Candy Hospital, Mumbai Summary: VWF is the factor that corrects the bleeding time defects in Von Willebrand disease as haemostasis maintains blood fluidity in the vascular system while allowing for rapid thrombus formation to prevent excessive haemostatic mechanism. Von Willebrand factor (VWF) is also considered a reliable marker of endothelial damage as thrombosis is a pathological extension of the normal hemostatic mechanism, occurring when the unwanted clot formation develops in certain pathological situations. Elevated vWF can be risk factor for thrombosis. Introduction: Von Willebrand is named after Finnish paediatrician Dr. Eric Adolf Von Willebrand. VWF is a multimeric protein encoded on the short arm of chromosome 12. It is produced in endothelial cells and megakaryocytes. In endothelial cells, the storage organelles for Von Willebrand factor are Weibel-Palade bodies and in platelet, stored in alpha granules. Two critical functions of VWF 1. Its involvement in the process of platelet adhesion and aggregation

N = 15 controls (Healthy patients)

N = 105 Patients taken who had bleeding history and susceptibility to thrombosis

From which 3 patients with bleeding history had low VWF, 28 patients who had underlying susceptibility to thrombosis like CAD, MI, AF and elevated lipid profile had very high VWF, The percentages were as follows: Low VWF 2.5 % associated with bleeding, High VWF 23.3 % associated with predisposition to thrombosis, Normal VWF 76 % Normal. (Our Normal range for VWF was 57–160 %.) Note: VWF is blood group specific. Individuals with blood group ‘O’ have lower level of VWF than non-O individuals and this should be considered when interpreting borderline results. Conclusion: From our studies we have found that VWF is very important test for bleeding disorder which helps in diagnosis of Von

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348 Willebrand disease (VWD) and an excellent biomarker for thrombosis. Key Message: Low VWF levels \57 % confer a risk of bleeding and High VWF [250 % confers a risk of thrombosis who are also prone for future bleeding as high VWF indicates endothelial damage.

Abstract No. 206 Inherited Factor X (Stuart-Prower Factor) Deficiency & Its Management in Transfusion Medicine AK Biswas, T Chatterjee, Joseph Philip, RS Mallhi Department of Immunohaematology & Blood Transfusion, AFMC, Pune Summary: Factor X is an extremely rare autosomal disorder that presents with variable bleeding tendency, prolonged PT and aPTT. We described a case that had severe inherited factor X deficiency (\1 %) and who was subsequently managed with fresh frozen plasma. Introduction: Factor X is a vitamin K dependent, liver produced serine protease that serves as a pivotal role in coagulation cascade. It can be inherited or acquired. It occurs with a frequency of one in 2 million in general population and so far only fifty cases have been reported worldwide. Materials and Methods: We present a case of one year old child who presented with complaints of easy brusiability and haemarthrosis, (Rt) knee since 4–5 months. Complete haemogram, red cell indices and BT were within normal limits. PT, aPTT, TT& dRVVT were prolonged, which showed correction after mixing studies with normal pooled plasma. He was further evaluated for Vit K deficiency by doing assays of Vit K dependent factors and then individual factor assays using factor X deficient plasma. Results: Results showed isolated deficiency of Factor X (\1 % of the normal value) the patient was diagnosed as a case of severe Factor X deficiency and treated accordingly. Presently he is on follow up and is advised to take Fresh Frozen Plasma (FFP) in case of severe bleeding. Conclusion: Factor X is an extremely rare autosomal disorder that presents with variable bleeding tendency, prolonged PT and aPTT and which gets corrected with 1:1 mix with normal pooled plasma. We described a case that had severe inherited factor X deficiency (\1 %) and was subsequently managed with FFP.

Abstract No. 207 Prenatal Diagnosis in FXIII Deficient Families Sharda Shanbhag, Shrimati Shetty, Kanjaksha Ghosh Department of Thrombosis and Haemostasis, National Institute of Immunohaematology, Parel, Mumbai-400012 Summary: Prenatal diagnosis in the first trimester of pregnancy in case of Haemophilia is offered on a routine basis at our centre. Two families of severe FXIII deficiency were offered first trimester prenatal diagnosis by direct DNA sequencing. If the causative mutation is known, the diagnosis can aid early intervention, and thus assist prevention of birth of severely FXIII deficient children. Introduction: Congenital Factor XIII (FXIII) deficiency is a rare autosomal recessive bleeding disorder which affects 1 in 1–5 million individuals caused mostly due to mutations in the F13A on chromosome 6 and rarely due to F13B defects on chromosome 1. Bleeding diathesis ranges from neonatal umbilical stump bleed to impaired wound healing, prolonged bleeding, intracranial hemorrhage, menorrhagia and recurrent miscarriages in women. Materials and Methods: 2 families were offered first trimester antenatal diagnosis. Genomic DNA was purified from citrated blood samples and chorionic villus

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 samples. F13A defects were screened by PCR and direct sequencing using ABI 3130XL sequencer. Results: Antenatal diagnosis in case of severe FXIII deficiency was offered to 2 families using direct sequencing technique. The propositi with severe FXIII deficiency carried the c.1241C[T (Ser413Leu) mutation in the exon 10 and c.58T[C (Ser19Pro) mutation in exon 2 of F13A. Parents of both the patients were found to be heterozygous carriers for the respective mutations. Chorionic villus sampling was done in the first trimester and both the fetuses were found to be unaffected. Conclusion: We report prenatal diagnosis of severe FXIII deficiency for the first time in India using direct sequencing technique. Once the causative mutation is identified in the index case, using direct sequencing method, carrier diagnosis and antenatal diagnosis can successfully be offered to the affected families.

Abstract No. 208 The Unlucky XIII for a Clan in North India K Anand Kumar, Vasant Chinnabhandar, Nita Radhakrishnan, Anupam Sachdeva Pediatric Hematology Oncology-Sir Ganaga Ram Hospital Summary: Coagulation factor XIII deficiency is one of the rare bleeding disorders. The most threatening and devastating complication is intracranial bleeding. The diagnosis can be delayed as the standard coagulation profile is normal. It needs a high index of suspicion for diagnosis. Introduction: To describe the spectrum of bleeding manifestation in factor-XIII deficiency. Although rare, with incidence of 1 in 3 million, here we describe 5 cases in a single clan with consanguineous marriages. Failure to diagnose can lead to mortality by intracranial or body-cavity bleeds Materials and Methods: Retrospective analysis of medical records of all children who were diagnosed/treated at our unit in Sir Ganga Ram Hospital between January 2001 to September 2011. Outcomes and adverse events noted in this set of patients. Results: All 5 cases belong to the fourth generation of a clan that migrated from Pakistan with consanguineous marriages. Total no. of 5 cases; 3-females and 2-males. Clinical spectrum: mean age of diagnosis was 0.7 year. More in females (3:2) All(100 %) had umbilical bleeding at birth and subcutaneous/muscle bleeds. 4 (80 %) of them had CNS bleeds.PT/APTT/BT were normal in all cases. Clot was soluble in 5 M urea in all cases. 5 cases had factor XIII level assay, 2 of them had severe factor deficiency. 3 are doing well and 2 have expired. 1 died due to intracranial bleed and another due to intra-abdominal bleed following trauma. Mean age of death was around 4 years. During the bleeding episodes they received fresh frozen plasma as treatment Conclusion: Any child with umbilical bleeding at birth should be investigated for factor-XIII deficiency, especially if there is a history of consanguineous marriage. Intracranial bleed in these set of patients are very common and can be lethal. With timely diagnosis and treatment they can lead a long life.

Abstract No. 209 Molecular Analysis of Combined Factor VIII and IX Deficiency: A Case Report SV Raj Kumar, Neeraj Arora, Sankari Devi, Aby Abraham, SC Nair, GR Jayandharan, Alok Srivastava Department of Hematology, Christian Medical College, Vellore Introduction: Familial multiple coagulation factor deficiencies (FMCFDs) are a group of disorders characterized by the simultaneous deficiency of two or more coagulation factors. Combined factor (F) VIII

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Fig. 1 . and FIX deficiency is very rare (FMCFD type 2, 1:1,000,000) and known to arise as a result of coincidental inheritance. Only two patients with FMCFD type 2 have been reported in the literature. We report here the phenotypic and molecular analysis of a family from South India with combined factor VIII and IX deficiency. Case study: The index case was a 17 year old male who presented with a history of prolonged bleeding after minor trauma, gum bleeds, deep haematoma and haemarthrosis. There was a family history of mild haemophilia A in the mother who had base line FVIII: C of 24 % (Fig. 1). Coagulation tests in the patient revealed a prolonged aPTT (125 s, reference range 9.7–13.3 s) and reduced FVIII: C levels (16.2 %). As the aPTT was abnormally prolonged and since his clinical phenotype was severe, he was evaluated further that revealed severe FIX deficiency (FIX: C \ 1 %). This suggested that the proband had combined FVIII and FIX deficiency. To identify the molecular defect responsible for this condition, we performed mutation screening in F8 and F9 genes by CSGE and DNA sequencing strategy. The proband had a previously reported Glu162Lys mutation in FVIII. Glu162 is present in the coil structure between Leu160–Leu171 in the A1 domain. The replacement of this residue by Lys162 is predictive of a mild disruption to A1 domain in FVIII as a result of the charge switch (negative ? positive) and possibly resulting in mild haemophilia A. However, his F9 gene had a c.1322A[G transition predicting a p.Tyr441Cys missense substitution. This is predicted to alter the tertiary structure of the FIX serine protease domain and result in severe FIX deficiency. Analysis of F8 and F9 in the DNA of available family members (Fig. 1) revealed that both the F8 and F9 mutations were inherited in cis from the maternal grand parents. Conclusion: This report highlights the importance of recognizing and diagnosing FMCFDs in patients with discrepant phenotypic findings. Recognition of this phenomenon is also essential for accurate genetic testing in such families.

Abstract No. 210 Acquired Hemophilia A: A Rare Bleeding Disorder Nitinkumar N Rathod, V Kamath, V Ramya, S Singh, S Nair

349 FVIII:C which neutralize its procoagulant function and result in severe, often life-threatening bleeding. The antibodies arise in individuals with no prior history of hemophilia A. Materials and Methods: A retrospective analysis of the records of patients who were referred to our laboratory for evaluation of bleeding disorders between 2005–2013 revealed 5 patients with Acquired Hemophilia A. In the same period we diagnosed 1088 patients with inherited Hemophilia A. Results: The patient’s median age was 57 years. All patients had prolonged APTT which showed partial correction after the addition of normal plasma and F IX deficient plasma but not with F VIII deficient plasma, reduced FVIII:C (0.3–4.9 %), with evidence of FVIII:C inhibitor measured by the Bethesda assay. Range of inhibitors was 1.7–54 BU. All other screening tests were within normal limits. Discussion: Most common affected age group was elderly patients with spontaneous onset bleeding, no family history of or history of consanguinity. All patients had co-morbidities like diabetes, hypertension, Parkinsonism and in one case autoimmune disease. History of spontaneous hemorrhages present whereas haemarthrosis were uncommon. Conclusion: Bleeding pattern in acquired hemophilia A differs from the inherited form, mainly seen in elderly patients with underlying co-morbidities and show moderate to marked decreased in FVIII:C level and with quantifiable inhibitors.

Abstract No. 211 Hemostatic Abnormalities in Children (1–10 years) with Acute Infection: A Pilot Study Abhimanyu Sharma, Meera Sikka, Sunil Gomber, Satendra Sharma University College of Medical Sciences and GTB Hospital, Delhi Summary: Sepsis is a common cause of death in infants and children. Hemostatic abnormalities have been reported in patients with sepsis and contribute to the mortality. Early recognition of such abnormalities will help in reducing mortality. There is scant information on the nature of these abnormalities in children especially from India. Introduction: Coagulation abnormalities are common in sepsis. This study aimed to assess the nature of hemostatic abnormalities in children with acute infection and to predict the early development of DIC and organ dysfunction. Materials and Methods: The following tests were done on day 0, 3, 7 on 50 children with acute infection and on 50 controls on day 0: CBC, examination of stained PBF, PT, APTT, TT, plasma fibrinogen, factor VIII assay, D-dimer, CRP, LFT and serum creatinine. Results: Prolonged PT and APTT were seen in 18 and 46 % patients respectively. Plasma fibrinogen was decreased in 6 % and increased in 8 % patients. D-dimer was positive in 72 % patients. One or more hemostatic parameter was abnormal in 49 (98 %) patients. Hepatic and renal dysfunction were seen in 18 and 8 % patients respectively. Five (10 %) children died and 2 % developed organ dysfunction. Liver dysfunction was seen in all non survivors and renal dysfunction in 8 % patients. Coagulation parameters were abnormal in all non survivors. Many children who recovered continued to manifest hemostatic abnormalities even on day 7. Conclusion: Hemostatic parameters must be studied in children with infection preferably at admission. This may help in offering better treatment modalities and reduce mortality.

Christian Medical College Hospital, Vellore Summary: A retrospective study of rare life threatening bleeding disorder Acquired Hemophilia A in Christian Medical College Vellore. Introduction: Acquired hemophilia A is a rare disorder with an incidence of approximately 1 per million/year with a high mortality rate. The disease occurs due to auto antibodies against coagulation

Abstract No. 212 Evaluation of International Society for Thrombosis and Haemostasis Scoring System for DIC in Acute Leukemia Patients: A Retrospective Single Centre Study

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Shilpa Kusthe, Mansi Joshi, Khaliqur Rehman, Prashant Deshpande, Nilesh Deshpande, Sheetal Gaware, Deepti Karkhanis, Sona Duseja, Nikhil Patkar, PG Subramanian, Sumeet Gujral, Prashant Tembhare

Abstract No. 214

Hematopathology Laboratory, Tata Memorial Hospital, Mumbai

Rehan Ahmed, P Ganguli, Reena Bharadwaj, Ajay Sharma, V Nair, UD Gupta, Natwar Singh

Introduction: Disseminated intravascular coagulation (DIC) is one of the important complications to develop in patients with acute leukemia (AL). International Society for Thrombosis and Haemostasis (ISTH) has provided a laboratory-based objective scoring system for evaluation of DIC. In this study, we retrospectively evaluated ISTH scoring system in AL patients with suspicion of DIC and its predictive value for the requirement of fresh frozen plasma (FFP) and platelet transfusion. Materials and Methods: We retrospectively analyzed results of PT, PTT, INR, Fibrinogen, D-dimer assays from 145 cases of clinically suspected DIC in AL patients (58 B-ALL, 16 T-ALL, 40 APML & 33 AML) and ISTH DIC-scores were calculated. Cases with score C5 were defined as ‘‘Overt-DIC’’ and with \5 were defined as ‘‘Non-overt-DIC’’. In 48 cases, scores were correlated with history of FFP and platelet transfusion. Results: Out of 145 cases, 60 % cases had ‘‘Overt DIC’’ and 40 % had ‘‘Non-overt’’ DIC. Individually, in 62.5 % of B ALL, 50 % of T ALL, 52.5 % of APML and 70 % of AML had ‘‘Overt-DIC’’. For FFP transfusion, ISTH score C5 revealed positive predictive value (PPV) of 59.26 % & negative predictive value (NPV) of 42.86 %. For platelet transfusion, ISTH score C5 revealed PPV of 46.67 % & NPV of 22.22 %. Further details will be presented in meeting. Conclusion: ‘‘Overt-DIC’’ is most frequent in non-APML type of AMLs. It is followed by B ALL and then APML cases. Cut-off of ISTH score C5 is not a good predictor for requirement of FFP and platelet transfusion for treating DIC in AL.

Platelet Disorders Abstract No. 213 Platelet Function Studies on Various Days of Storage in Stored Blood Bank Platelets Nupur, Ashutosh Kumar, US Singh, Rashmi Kushwaha

Functionality of Transfused Platelets at Low Counts? A Novel Approach

Department of Pathology and Molecular Medicine, Army Hospital (R&R), Delhi Cantt-110010 Introduction: Single Donor Aphaeresis Platelet (SDP) is a precious tool in the transplant setting. It is used in patients with low counts (\50,000/cumm). This study determined the functionality of platelets as determined by platelet aggregometry (PA) in platelets donors, platelets concentrates and recipients. Aims & Objectives: (1) To evaluate platelet aggregation in low counts by platelet aggregometer. (2) To establish a method of estimating platelet aggregation by manual smear method. Materials & Methods: Part-I: Platelet function test was done using Chrono-Log (Model: 560VS, USA) aggregometer & agonists Adenosine Di Phosphate (10 lM), Arachidonic Acid (0.5 mM), Ristocetin (1.0 mg/mL) and Collagen (2 lg/ mL). (1) Donor: 30 volunteers for SDP were tested by Impedance method aggregometry prior to apheresis. (2) SDP: Single donor platelet was tested at collection, 2 and 24 h. (3) Recipients: Recipient were tested prior to and after SDP transfusion by impedance method of aggregometry. Part-II: (1) 20 volunteers with normal count were tested by Impedance & Optical method of aggregometry and by the manual examination of stained smear post aggregation. Minimum of 600 platelets/20 fields were examined. An aggregate was C3 platelets. (2) The novel method was used to evaluate in 20 recipients with varying low platelet counts. Observations & Results: Whole blood impedance method aggregometry was normal in all donors pre apheresis. The optical method of aggregation study on SDP at varying time revealed mild changes in aggregation. No aggregometry patterns were recorded for the recipients samples post transfusion. However the novel manual method demonstrated functionality of platelets in patients with low counts using impedance & optical methods. Conclusions: Platelet aggregation was estimated in patients with low counts.

Abstract No. 215

Department of Pathology, KGMU, Lucknow Summary: We did a study on functioning of stored platelets at KGMU in 2013 and showed normal platelet parameters and intact platelet procoagulant activity (PF3) on day 5, however, the aggregation activity of platelets was markedly diminished on day 1 and was totally absent after 3rd day of storage. Introduction: Platelet function studies measure the platelet’s ability to adhere and aggregate. During preparation and storage, platelets are exposed to a variety of mechanical and chemical influences that lead to their activation and progressive loss of platelet viability and hemostatic function. Materials and Methods: We prepared 50 units of PRP from whole blood within 8 h of collection and stored at 22 °C on platelet agitator. The platelet function studies on day 1, 3 and 5 included PF3 activity, platelet aggregation and platelet indices (platelet count, MPV and PDW). Results: All samples showed normal platelet indices on day 5 of storage. 42 (84 %) showed normal PF3 activity on day 5 of storage. out of 50 samples, only 1 showed above 60 % aggregation, 16 samples (32 %) showed 5–20 % aggregation and 33 samples (66 %) showed less than 5 % aggregation on day 1, and the aggregation ability of platelets was absent after 3rd day of storage. Conclusion: Stored platelets are essentially useful in improving hemostasis by taking part in coagulation, however, they don’t play a significant role in primary hemostasis which can be correlated to aggregation defect.

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New Diagnostic Modality: Platelet Mapping Assay to Show the Trend of Anti Platelet Drug Inhibition in a Tertiatry Care Hospital Swati Saxena, Vaishali Patil, Nita Munshi, Amit Kumar Singh, Jagdish Hiremath Department of Pathology, Grant Medical Foundation’s, Ruby Hall Clinic, Pune Summary: The platelet-mapping assay of the thromboelastograph (TEG) is used to measure platelet aggregation and to examine the effect of antiplatelet drugs on multiple platelet receptors in post PTCA and postoperative bleeding. It addresses the prothrombotic issue from both bleeding and ischemic perspectives. Introduction: Platelet Mapping can provide important information to help the clinician assess bleeding and ischemic risks in patients undergoing antiplatelet therapy. The Platelet Mapping assay specifically determines the MA (Maximum Amplitude) reduction present with anti-platelet therapy and reports the percent inhibition and aggregation. The assay uses arachidonic acid agonists to generate MAs that reflect the inhibiting effect of these antiplatelet agents: (a) Thromboxane A2 inhibitors such as aspirin. (b) ADP inhibitors such as clopidogrel, prasugrel, Ticagrelor. Materials and Methods: The percentage

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 platelet aggregation for adenosinediphosphate (ADP) and arachidonic acid was measured in 30 patients and with a reference of values of inhibition, a hospital data of varied trend of inhibition was correlated. Reference ranges for both clopidogrel/prasugrel and asprin are: 100–75 % indicates patient is responsive, 50–75 % semi-responsive and less than 50 % is non-responsive. Platelet mapping also helps the clinician to monitor the inhibition and alter the dose and drug according to the inhibition given by platelet assay and clinical correlation. machine used is TEG5000 hemoscope. Results: Out of 30 patients studied, 73.3 % showed inhibition to Clopidogrel/prasugrel between 75–100 %, 20 % between 50–75 and 6.66 % \50 %. 70 % patients showed inhibition to Aspirin between 75–100 %, 20 % between 50–75 and 10 % \ 50 %. Conclusion: The present study indicates that 30 % patients on aspirin and 26.6 % patients on clopidogrel/Prasugrel show platelet inhibition of B75 % which is significant clinically for patient management.

351 (*2/*2 or *2/*1) of CYP2C19 was found to be significantly higher in clopidogrel semi responders compared to responders (72 vs. 40 %, p = 0.0023 respectively). CYP2C19*3, CYP3A5*3 and PLA1/A2 did not showed any correlation between clopidogrel semi responders and responders. No significant correlation was found between clopidogrel semi responders and responders in relation to gender, duration of antiplatelet agents, coronary events, type of coronary intervention, hypertension, diabetes mellitus, statins and proton pump inhibitors. Conclusion: There was significantly higher frequency of mutant genotype of CYP2C19 in clopidogrel semi responders as compared to the responders associated with inadequate clopidogrel response. These findings have far reaching implications for appropriate therapy in patients with coronary artery disease.

Abstract No. 217 Abstract No. 216

Acquired Platelet Dysfunction with Eosinophilia: (APDE) A Case Report

Genetic Correlates of Aspirin and Clopidogrel Resistance in Patients with Coronary Artery Disease (CAD)

N Vijay, P Nayar, R Manchanda

Vandana Arya1, JPS Sawhney2, A Saraf1, M Bhargava1 Department of Hematology1 and Cardiology2, Sir Ganga Ram Hospital, New Delhi Summary: Platelet aggregation analysis in 50 Coronary Artery Disease (CAD) patients on dual anti platelet therapy showed 50 % patients semi responder to clopidogrel had association with mutant *2 allele of CYP2C19 gene. Also 4 % of the cases were semi responder to aspirin. Introduction: Combination therapy with clopidogrel and low dose aspirin is the current standard of care in the management of patients with Coronary Artery Disease (CAD) including acute coronary syndromes (ACS). Between 4–30 % of patients treated with conventional doses of clopidogrel show an inadequate platelet response. The present study was designed to determine the frequency of resistance to aspirin and clopidogrel in Indian patients with CAD and its correlation with four selected gene polymorphisms. Materials and Methods: Fifty patients with CAD who were stable on dual anti platelet therapy (Clopidogrel 75 mg OD and aspirin 150 mg OD), were investigated along with age and sex matched 50 controls. Demographic and clinical data was collected on a predesigned clinical proforma. Platelet function testing by light , transmission aggregometry was done with 4 agonists (ADP 10 , Collagen 2 /ml, Arachidonic Acid 0.75 mM) in Epinephrine 5 each patient/control. After meta analysis the criteria employed for the Aspirin resistance was mean platelet aggregation C70 % with ADP and C20 % with 0.75 mM of Arachidonic acid. Aspirin 10 semi responders were defined as those meeting only one of the above criteria. Clopidogrel resistance was defined as \10 % decrease from . Semi the baseline in platelet aggregation in response to ADP 10 responders were defined as 10–29 % (\30 %) decrease from the baseline. A baseline mean platelet aggregation was obtained from 50 controls. Polymorphisms CYP2C19*2, CYP2C*3, CYP3A5*3 and PLA1/A2 were genotyped by PCR-RFLP. Results: The 50 cases included 41 (82 %) males and 9 (18 %) females in the age range from 32 to 77 years (mean 56.7 ± 09). Employing resistance criteria we found 2 (4 %) patients were semi responders to aspirin and 25 (50 %) patients were semi responders to clopidogrel. Both the patients who were semi responder to aspirin were also semi responder to clopidogrel. However, none of the patient was completely resistant to either aspirin or clopidogrel. Genetic analysis showed significantly higher frequency of the mutant *2 Allele of CYP2C19 in clopidogrel semi responders than in responders (p = 0.037). The mutant homozygous and heterozygous genotypes

Department of Pathology, KEM Hospital, Pune Summary: We report a case of an acquired platelet dysfunction with eosinophilia. Introduction: Acquired platelet dysfunction with eosinophilia (APDE) is usually a self-limiting benign bleeding disorder characterized by an insidious onset of easy bruising in an otherwise well person. Hypereosinophilia is associated with a platelet storage pool disorder. APDE has been mainly reported from children in South-East Asia. We report a 4 years old male child who presented with large ecchymotic patches all over the body of recent onset. Materials and Methods: A detailed clinical history was noted and clinical examination done. Complete hemogram, peripheral blood smear study, complete coagulogram and platelet aggregation studies were evaluated. Results: O/E no organomegaly or lymphadenopathy was noted. His hemogram revealed mild leucocytosis with increased eosinophils and mild thrombocytopenia. Bleeding time was prolonged. Platelet count, Prothrombin time and Activated Partial Thromboplastin time were normal. Platelet aggregation studies showed normal platelet aggregation with Ristocetin and ADP. No aggregation was seen with collagen. Conclusion: Although rare, eosinophilia can be associated with an acquired bleeding disorder. It is essential to recognise this benign condition. Awareness of this would help to diagnose and treat early.

Abstract No. 218 Gray Platelet Syndrome: A Case Report K Deshpande, R Godbole, P Nayar, R Manchanda Department of Pathology, K.E.M. Hospital, Pune Summary: Gray Platelet Syndrome is a rare inherited platelet disorder, characterized by platelet specific Alpha granule deficiency. There is mild bleeding tendency, mild to moderate thrombocytopenia & large platelets with typical hypogranular, gray appearance by light microscopy. Our case was diagnosed on basis of clinical features, peripheral blood smear findings and platelet function studies and is being presented for its rarity. Introduction: The gray platelet syndrome is a rare, inherited and mild to moderate bleeding disorder characterized by marked decrease or absence of alpha-granules and of platelet-specific alpha granule proteins. Materials and Methods: A complete haemogram, peripheral blood smear study, complete coagulogram and Platelet Aggregation studies were evaluated. A detailed

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352 clinical history with previous laboratory investigations were recorded. Results: A 9 years old female, second product of third degree consanguineous marriage, brought by parents with complaints of easy bruisability after trivial trauma with a history of 1 episode of spontaneous gum bleeding 4 years before. The clinical diagnosis was? ITP? Inherited Coagulation defect? Inherited Platelet Disorder. Her haemogram revealed mild thrombocytopenia. Peripheral blood smear showed hypogranular, bluish gray colored platelets. Complete Coagulogram and platelet aggregation studies were suggestive of Gray Platelet Syndrome. Conclusion: Although, the gray platelet syndrome is a rare disorder, it can be diagnosed by history, clinical correlation, platelet morphology on peripheral blood smear and platelet function studies.

Abstract No. 219 Dynamics of Platelet Count in Critically Ill Medical Patients as a Prognostic Marker and Its Associated Risk Factors: Experience at a Tertiary Care Centre of North-West India Sandhya Gulati, Sudhir Mehta, Shubham Joshi, Nidhi Sharma SMS Medical College, Jaipur, Rajasthan Summary: Thrombocytopenia is highly prevalent in medical intensive care unit patients. Patients who develop thrombocytopenia have greater severity of illness, increased bleeding episodes, greater transfusion requirement and higher mortality. Therefore, monitoring platelet counts is not only a sensitive but readily available marker of patients’ clinical status and thus has a great prognostic value. Introduction: Thrombocytopenia is a major concern in medical intensive care units (MICU). Platelet counts below 50 9 109/L in critically ill patients have major impact on the morbidity & mortality as well as on the management strategies. In view of paucity of Indian data available on prevalence & dynamics of thrombocytopenia in MICU adult patients, we undertook the present study to find out proportion of MICU patients developing thrombocytopenia, follow dynamics of platelet count, and identify associated risk factors and to determine the outcomes. Materials and Methods: Critically ill patients with normal platelet count on admission to ICU & who stayed in ICU [24 h were recruited. Those with an underlying haematological disease affecting platelet counts were excluded. Data collected in performa included age, sex, illness, reason for ICU admission, co-morbidity etc. Daily recordings included pulse, BP, respiratory rate, temperature, drugs given, APACHE II score, mechanical ventilation, presence of bleeding/shock/DIC. Laboratory data included CBC, liver & renal function tests, X-ray chest, ECG, coagulation tests, FDP and D-dimer assay. Results: Thrombocytopenia is highly prevalent in MICU (49.26 %). ICU mortality was higher among thrombocytopenic patients as compared to non thrombocytopenic patients irrespective of illness and reason for admission in ICU. Patients who developed thrombocytopenia during their ICU stay had a lower mean platelet count at admission in ICU, higher mean APACHE II, severe organ insufficiency, higher INR values. Complications such as shock, bleeding, mechanical ventilation and DIC were significantly higher among thrombocytopenic group. Sepsis was an important risk factor for thrombocytopenia in ICU. Thrombocytopenic patients who expired subsequently had higher APACHE II score, lower level & longer duration of thrombocytopenia irrespective of cause. Thrombocytopenic patients had greater transfusion and vasopressors requirements. Mortality among thrombocytopenic patients was proportional to severity, duration & lowest level of thrombocytopenia. Conclusion: This study shows that nadir as well as dynamics of platelet count is an important marker of critical illness & is associated significantly with mortality & morbidity.

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Abstract No. 220 Polymorphism of TNF-a Gene as Potential Risk Factor for Primary Immune Thrombocytopenia (ITP) in Indian Patients Deependra Kumar Yadav1, Sanjay Mishra1, Divya Gupta2, Ashutosh Kumar1, KN Prasad2, Jyotsna Agarwal1, AK Tripathi1 1

King George’s Medical University, Chowk, Lucknow 226003, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow 226014

2

Summary: Primary Immune Thrombocytopenia (ITP) is an autoimmune hematologic disorder. It is associated with cytokine response and dysregulation. Tumor necrosis factor (TNF-a) is associated with susceptibility to ITP. Introduction: Primary Immune Thrombocytopenia (ITP) is an autoimmune hematologic disorder characterized by isolated thrombocytopenia (\100,000/cumm) in the absence of other causes or disorders that may be associated with thrombocytopenia. ITP has been reported to be associated with aberrant cytokine response and dysregulation. Several genes involved in immune system regulation including (HLA)-DPBI and tumor necrosis factor (TNF-a) are associated with susceptibility to ITP. The purpose of this study was to investigate whether the tumor necrosis factor (TNF-a, A-G) polymorphisms may be responsible for genetic susceptibility to ITP. Materials and Methods: Fifty patients and controls were genotyped for the A-to-G polymorphism of TNF-a gene by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) method. Results: The diagnosis of primary ITP was considered in patients with isolated thrombocytopenia (platelet count \100,000/cumm) in the absence of other causes or disorders that may be associated with thrombocytopenia. Genotypic analysis of TNF-a+252(rs909253) gene of 50 ITP patients showed 40 % homozygous (AA); 16 % mutant (GG) and 44 % samples had heterozygous (AG) genotype, whereas in controls it was 76 % (AA); 8 % (GG) and 16 % (AG). Conclusion: The odds ratio of TNF-a gene for development of (ITP) in carriers of mutant allele (G) vs. (A) wild allele is 4.794, homozygosity for the genotype (GG) increased likelihood for ITP 4.5 fold (p-value 0.025 with 95 % CI). TNF-a GG genotype is a potential risk factor for ITP. Heterozygosity for genotype of TNF-a gene (AG) increased likelihood 5.0 fold which indicate significant contribution of TNF-aˆ gene A/G polymorphisms. Identification of such would increase our understanding of the biology for the disease and allow a prediction of those ‘at risk’ in the general population.

Abstract No. 221 Clinicopathological Study of Thrombocytopenia Neha Ahuja, Jyotsana V Wader, Vijay Kumar S Wader Krishna Institute of Medical Sciences, Karad Summary: A Crosssectional Study was carried out in Krishna Institute of Medical Sciences Thrombocytopenia was observed in 1012 cases. In 90 % cases isolated etiology was responsible. In 8 % cases multiple etiologies were responsible. Introduction: Thrombocytopenia is defined as platelet count of \1.5 lakh/cumm, and it is one of the commonest cause of bleeding. Available comprehensive studies on this topic are few, study done with purpose of correlating it clinically and assessing the severity of thrombocytopenia. Materials and Methods: All samples with platelet count \1.50 lakh/cumm were screened and included in the study. Complete blood count, peripheral smear examination were done for the patients. Spurious thrombocytopenia was being ruled out. Investigations like ESR, Bone marrow examination, retic count were done wherever necessary. Haematological tests were correlated with other tests to decide for the etiology.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Results: Infection was the leading isolated cause of thrombocytopenia in (39 %) cases, Malaria being the commonest cause in infection in (17.1 %) cases. Anaemia was the next common cause in 26 % cases, other causes observed were hematological malignancies (1.4 %), use of myelosuppressants, alchoholism, obstetrics causes and others. Platelet count in most of cases was in between 50,000 to 1 lakh/cumm. Significant association was found between thrombocytopenia and hematological causes and significant association was found between decrease in platelet count and bleeding. Conclusion: The Preventable causes of thrombocytopenia like infections & anaemia are present in high percentage. Hence most of cases of thrombocytopenia have a favorable prognosis. Cases of hematological malignancies and obstetric causes should be followed for thrombocytopenia. Cases with severe thrombocytopenia should be treated on urgent basis to prevent disastrous effect.

Abstract No. 222 Idiopathic Thrombocytopenic Purpura: A Single Centre Experience Delon Dsouza, Sanjukta Rao, Ratnamala Chaudhary, Abdul Matheen, Cecil Ross St Johns Medical College, Bangalore Summary: A study on 250 patients with Idiopathic Thrombocytopenic Purpura (ITP) at our centre shows that most were female patients, and one third achieved remission with steroids. Two third of patients who underwent splenectomy achieved complete remission. Introduction: Idiopathic thrombocytopenic purpura is a common hematological disorder that is encountered in the medicine or haematology OPD. This study aims to understand the clinical response to treatment with steroids and splenectomy. Materials and Methods: We identified 250 consecutive patients with the discharge diagnosis of idiopathic thrombocytopenic purpura treated in the hematology outpatient department at SJMCH Bangalore. All patients had drug screening, HIV, hepatitis C, thyroid test and bone marrow study done to rule out secondary causes of ITP. Results: Mean age of patients was 30.6 years with 179 females and 79 males. The mean presenting platelet count was 15,000/mm3. On treatment with steroids, 33.6 % patients were in complete remission even after therapy was discontinued. 32.4 % patients required continued treatment after initial treatment (CRR) with a mean time to treatment failure of 110 days. 16 % patients attained stable partial response while 14 % patients had no response to steroids. 129 patients had splenectomy. After splenectomy, 63.5 % had CR till mean 238 days, 16.2 % had CRR in mean 304 days. Mean time to remission after splenectomy was 237 days. 5.4 % patients had partial response and 3.1 % patients had no response. 5.4 % patients who underwent surgery died, of bleeding infections or therapy complications, in an average of 171 days. Conclusion: We conclude that most patients with ITP attain stable remission, though on average this occurs slowly. However, a subpopulation with severe, resistant disease experiences significant morbidity and mortality.

Abstract No. 223 Rituximab Therapy for Chronic Refractory Immune Thrombocytopenic Purpura: A Single Centre Study Sanjeev, Nishad Dakate, Sneha Tandon, Pradeep Kumar, Rajesh Kashyap, Soniya Nityanand Department of Hematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh

353 Summary: Rituximab is emerging as a safe and effective alternative treatment of chronic refractory immune thrombocytopenic purpura (ITP). The aim of this study was to evaluate the response of this biological agent in patients of chronic refractory ITP presenting to SGPGI, Lucknow. Introduction: About 30 % of adult ITP and 10 % of childhood ITP patients fail to respond to conventional therapies and these patients develop a refractory disease. Rituximab may be considered for these patients who have failed first line therapy and have significant bleeding symptoms. It may also be considered as an alternative to splenectomy in children and adults with chronic ITP as well as in those who do not respond favourably to splenectomy. Materials and Methods: Rituximab was administered in a dose of 100–375 mg/m2, IV weekly for a total of four doses. Responses were recorded at 3 monthly intervals up to 1 year, as follows: (i) complete remission (CR)-platelet counts [100 9 109/L (CR); (ii) partial remission (PR)-platelet counts [50 9 109/L but \100 9 109/L (PR); (iii) minimal response (MR)-platelet counts [ but \50 9 109/ L-(MR); (iv) no response (NR)-no improvement in platelet count. Response was classified as sustained response (SR) when it was stable for a minimum of 6 months. Median time from first dose of Rituximab to response was recorded and classified in three patterns: early (before 4th dose of Rituximab), intermediate (7–10 weeks after Rituximab) and late ([10 weeks after Rituximab). All treatment related side effects were recorded. Results: Total 10 patients (8 male & 2 female) of chronic refractory ITP were treated with inj Rituximab in last 1 year. Median age was 23 years (range 3–49 years). Median platelet count was 12 9 109/L (range 3.0–21.0 9 109/L). CR was achieved in five patients (50 %), PR in three (30 %), MR in one (10 %), and NR in one (10 %). SR was achieved in 8 patients (80 %). The median period of follow-up after Rituximab therapy is 4 months (range 1–12 months). The median time for response was 14 weeks (range 4–18 weeks). Early response was found in 1 patient, intermediate response in 7 patients & late response in 1 patient. There were no adverse events with Rituximab therapy. Conclusion: Rituximab therapy in chronic refractory ITP, results in an overall response rate of 80 % (CR ? PR), which is sustained. It is safe with no adverse events. Thus it may be the treatment of choice in this subset of ITP patients.

Thrombophilia Abstract No. 224 Association of CYP2C9, VKORC1, CYP4F2 and EPHX1 Polymorphisms in Warfarin Treatment Related Adverse Events Tejasvita Gaikwad, Kanjaksha Ghosh, Bipin Kulkarni, Shrimati Shetty Department of Haemostasis and Thrombosis, National Institute of Immunohaematology (ICMR), KEM Hospital, Parel, Mumbai Summary: Warfarin is an anticoagulant, widely prescribed for prevention of thrombosis. Several polymorphisms have been found to be associated with dose requirement and other adverse effects. Four warfarin sensitive polymorphisms i.e. VKORC1, CYP2C9, EPHX1 and CYP4F2 were analysed in warfarin treated patients and these were correlated with warfarin dosage and other adverse events. Though VKORC1 and CYP2C9 are the strongest predictors of dosage and bleeding events, addition of EPHX1 and CYP4F2 showed a marginal increase in their predictive efficiency. Introduction: Warfarin, a widely prescribed anticoagulant for prevention of thrombosis, is associated with narrow therapeutic window where even small variations in dosing may result in hemorrhagic or thrombotic complications. Several polymorphisms in VKORC1, CYP2C9, CYP4F2,

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354 EPHX1, GGCX, factor IX, factor II and factor VII have been shown to be associated with warfarin dose requirement. Materials and Methods: (A) A total of 191 warfarin treated patients were genotyped for CYP2C9 and VKORC1 polymorphisms using PCR-RFLP method. (B) A total 128 warfarin treated patients were genotyped for EPHX1 T113C polymorphism using allele specific PCR method. (C) A total 107 warfarin treated patients were genotyped for CYP4F2 rs2108622:G[A polymorphism using allele specific PCR method. Results: VKORC1 variants found significantly associated with low warfarin dose requirement and risk of over anticoagulation. In CYP2C9 genotype, except CYP2C9*1/*2 all other variants were found significantly associated with low warfarin dose requirement and risk of over anticoagulation. EPHX1 and CYP4F2 polymorphisms showed moderate association with warfarin dosage but not with adverse events. Conclusion: Although the genotype of VKORC1 and CYP2C9 are clearly the most important genetic factors for warfarin response, yet inclusion of EPHX1 and CYP4F2 genotypes will be beneficial for accurate warfarin dosage in Indian patients.

Abstract No. 225 Current Guidelines Vis-a-Vis Unresolved Issues in the Diagnosis of Lupus Anticoagulant Vishal Mehrotra, Pradnya Chaudhari, Manisha Ramani, Amar Das Gupta

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Department of Clinical Hematology, Karturba Medical College, Manipal Summary: Lupus anticoagulants (LA), a Antiphospholipid antibody (APA), is commonly attributed cause of acquired thrombophilia. Among many recommended screening tests, Dilute Russel’s Viper Venom Time (dRVVT) is one of the most important. It is a 2 step process which involves determination of Screen time (ST) and Confirm time (CT). Objective was to see if a cut off value for DRVVT Screen time can be determined, below which confirmatory test is not necessary. Introduction: Commercially available dRVVT systems include a screening reagent with low phospholipid (PL) concentration and a confirmatory product with high PL concentration. Ratio (R) of ST to CT is calculated to obtain dRVVT ratio. LA is considered positive if the ratio is C1.2. However, it is observed that when the value of screen time is low, the ratio of 1.2 is not reached. Materials and Methods: A retrospective analysis of 111 dRVVT test values was done. Statistical analysis of Screen time values was done to determine a minimum cutoff. Results: Forty six cases were positive (R C 1.2), 46 were negative(R \ 1.15) and 16 were categorized as borderline (R = 1.15–1.19). Analysis by determining the ROC curve gave the best cutoff value of 49 for Screen time (Sensitivity-93.5 %, Specificity-89 %). A cutoff value of 45.5 s had the same sensitivity of 93.5 % but the specificity dropped to 80 %. Conclusion: If a cutoff value of 45 s is taken for the screen time, there is a 6.5 % chance of missing the positive cases. It is therefore mandatory to do the dRVVT Confirm test in all cases.

Sections of Hematology and Flow Cytometry, SRL Limited, Mumbai Summary: Application of 99th percentile cut off appears to increase specificity of tests for LA at the cost of their sensitivity when compared to 2SD cut off. This observation needs to be confirmed since there is a risk of missing weak LA positive cases using this approach. Introduction: The 99th percentile cut-off for detecting positive cases at each steps of Lupus Anticoagulant (LA) testing (ISTH/SSC guidelines 2009) is difficult to establish and implement in most of the labs due to the requirement of a large number of normal plasma samples. Therefore, most labs have been using 2 SD cut-offs for this purpose. This study compares the incidence of LA positivity in suspected cases using 99th percentile cut-off with that of 2 SD cut-off. Materials and Methods: APTT using silica as the activator, mixing studies using normal pooled plasma, dRVVT screening and confirmatory tests were performed in 120 healthy individuals (60 M, 60 F) to establishing reference ranges and 99th percentile cut-off values for the two sets of tests. Two thousand forty two cases were tested for LA and results were evaluated according to the ISTH/SSC guidelines. Results: Using 99th percentile cut-off values for dRVVT screening and normalized ratio, we observed a 16 and 7 % reduction in LA positive cases respectively when compared to application of 2SD cut off for LA positivity. Conclusion: Published data to evaluate the impact of introduction of 99th percentile cut-off in detecting ‘‘true’’ LA positive cases is unavailable. The specificity of the LA tests seems to increase at the cost of sensitivity with the use of 99th percentile cut off. However, there is no data to show if LA positive cases using 99th percentile cut off are ‘‘true positive’’ or are weak positive cases being missed when this approach is applied. There is need to verify this fact.

Abstract No. 226 Dilute Russel’s Viper Venom Time: Is Two Steps Testing a Necessity? Akriti, Chethan Manohar

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Abstract No. 227 Newer Anti Beta-2 Glycoprotein I & Anti Prothrombin Antiphospholipid Antibody Tests in Patients with Recurrent Pregnancy Loss: To Do or Not to Do? Angad Singh, Anita Nangia, Sunita Sharma, Manju Puri Lady Hardinge Medical College and Associated Sucheta Kriplani Hospital, New Delhi Summary: 50 women with recurrent pregnancy losses were tested for circulating antiphospholipid antibodies of whom 11/50 (22 %) women tested positive. Maximum positivity was seen for anti beta-2 glycoprotein I (12 %) followed by anti-prothrombin (6 %), lupus anticoagulant (4 %) and anticardiolipin antibodies (2 %). Introduction: Recurrent pregnancy losses are defined as three or more consecutive pregnancy losses. Antiphospholipid antibody syndrome is causal in 5–20 % of pregnancy losses. Materials and Methods: 50 women with recurrent pregnancy losses & 50 matched controls were evaluated for the presence of: Lupus anticoagulantscreened by LA sensitive aPTT & DRVV and confirmatory Staclot Assay. IgG & IgM anticardiolipin, anti beta-2 glycoprotein I & anti prothrombin antibodies—ELISA kits. Results: 11/50 (22 %) women in case group and none in control group had circulating antiphospholipid antibodies. 2 cases (4 %) had lupus anticoagulant. 1 case (2 %) had anticardiolipin antibody & 6 cases (12 %) were positive for anti beta-2 GP I antibody (p value = 0.027). 3 cases (6 %) had anti prothrombin antibody. All were mutually exclusive except for one. Conclusion: Women with recurrent pregnancy losses should be tested for anti beta-2 GP I antibodies & anti prothrombin antibodies in addition to conventional lupus anticoagulant and anticardiolipin antibodies. This approach can decrease the incidence of SNAP (seronegative antiphospholipid syndrome) cases while establishing the true prevalence of antiphospholipid syndrome in Indian women.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Abstract No. 228 Can Micro Particle be a Marker for Heparin Dose Adjustment During Pregnancy in Women with Recurrent Pregnancy Loss? Rucha Patil, Kanjaksha Ghosh, Purnima Satoskar, Shrimati Shetty Department of Haemostasis and Thrombosis, National Institute of Immunohaematology (ICMR), KEM Hospital, Parel, Mumbai Summary: Procoagulant microparticles (MPs) are found significantly increased in women with recurrent pregnancy loss (RPL). The effect of heparin therapy, both unfractionated (UFH) and low molecular weight (LMWH), was studied in seven pregnant patients. It was observed that as the heparin therapy progresses, the MP levels decrease and when normalized, led to a successful pregnancy. Perhaps if the dosage is adjusted so as to normalize the MP levels, successful pregnancy outcome may be achieved. Introduction: 15 % of reproducing couples suffer from PL and recurs in 2–3 %. A defective maternal hemostatic response leading thrombosis of the uteroplacental vasculature has been hypothesized to subsequently lead to PL in unexplained cases. Hereditary thrombophilia and antiphospholipid antibodies (aPLA) have been described as risk factors for RPL. Recently, procoagulant microparticles have been reported to have a major role in many thrombosis complicated diseases. However there is hardly any data on the effect of different anticoagulants on MP levels. Materials and Methods: Seven pregnant women on heparin therapy with unexplained RPL history were analyzed for procoagulant phosphatidylserine exposing MPs of different cell types. Results were compared with 18 healthy pregnant women. Methodology for analysis of MPs has been standardized on BD FACS Aria flow cytometer by participating in the ‘‘Vascular Biology SSC workshop’’. Results: MP levels were highly elevated at the onset of pregnancy. However, as heparin therapy continues MP levels decrease. Out of the 7 pregnancies, in 2 women, MPs normalized by 3rd trimester resulting in successful pregnancy; in one patient MPs did not normalize resulting in pregnancy loss, in 2 patients were highly elevated just before they miscarried and in 2 women were normal from the beginning of their pregnancy but still miscarried indicating some other unknown reason for the loss. Conclusion: MP levels decrease considerably as heparin therapy progresses during pregnancy and successful pregnancy outcome was achieved where MP levels normalized. If the dosage of heparin is monitored so as to normalize the MP levels, may be a successful pregnancy outcome may be achieved.

355 women with RSA together with 198 healthy fertile controls. Their demographic profile; clinical presentation and history were duly recorded. Protein C, Protein S, Antithrombin (AT), APC-R, Lupus Anticoagulant (LAC) and antiphospholipid antibody assays were performed. Genetic variants for inherited Thrombophilia such as factor V leiden (FVL), Prothrombin (G20210A), MTHFR (C677T), GPIIb/IIIa (PLA1/A2) and PAI-1(4G/5G) were analysed by PCRRFLP and ASO-PCR. Results: Of the evaluated 250 females and 198 controls, the mean age was 30 (range: 19–44) and 27 (18–36) years respectively. Amongst 250 cases, 128 had 2 and 122 had C3 consecutive pregnancy losses. Procoagulants protein C, protein S and antithrombin deficiency was present in 2.4 vs. 0 %, 8 vs. 2 % and 3.2 vs. 0.5 % of patients versus control respectively. Anti phospholipid antibodies: ACA IgG & IGM were positive in 1.6 vs. 1.5 % and 6.8 vs. 2.5 %; b2 IgG & IgM in 0.8 vs. 1.5 % & 1.6 vs. 0.5 % of patients versus controls. LAC was positive in 9.2 % patients vs. 6.6 % controls. The mean levels of Protein C, S, AT, ACAIgG, IgM, b2IgM & LAC in patients were 57.4 ± 19, 44.5 ± 8.1, 68.5 ± 20.5, 29.4 ± 10.2, 17.7 ± 5.5, 47.8 ± 35 & 1.4 ± 0.16 respectively. A strong association was observed with Protein C (p = 0.02), Protein S (OR 3.9, 95 % CI: 1.3–11.4, p = 0.005), Antithrombin (OR 6.3, 95 % CI: 0.79–50.2, p = 0.04) and ACA IgM (OR 2.7, 95 % CI: 1.0–7.17, p = 0.03). Allele frequency of mutant T allele of MTHFR, A2 of PLA1/A2 & 4G of PAI-1 were 20 vs. 11.3 %, 9.9 vs. 6 % & 53 vs. 40.1 % in patients vs. control. The mutant T allele of MTHFR, A2 allele of PLA1/A2 and 4G allele of PAI-1 polymorphism were significantly associated with the RSA (OR 19, 95 % CI: 1.3–2.8, p \ 0.005; OR 1.7, 95 % CI: 1.02–2.8, p \ 0.05 and OR 1.7, 95 % CI: 1.2–2.2, p \ 0.0005 respectively) when compared with the controls. Abnormal APCR and FVL mutation was found in 2.8 % (7/250) patients. No mutant allele of prothrombin gene was observed either in patients or controls. Conclusion: Thromophilia screening of pregnant women with unexplained recurrent spontaneous abortions revealed multiple abnormalities in procoagulant factors as well as in molecular markers such as MTHFR, PLA1/A2 and PAI-1 polymorphism.

Abstract No. 230 Profile of APLA Syndromes J Latha Fathima, P Parimala, A Vanamala, AM Shanthala, S Sitalakshmi St. John’s Medical College Hospital, Bangalore 560034

Abstract No. 229 Inherited/Acquired Thrombophilia in Unexplained Recurrent Spontaneous Abortions Vandana Arya, Abha Majumdar1, Manorama Bhargava Department of Haematology & 1Department of Obstetrics and Gynecology, Sir Ganga Ram Hospital, New Delhi Summary: A study of thrombophilic factors in 250 women with recurrent spontaneous abortions along with controls revealed multiple abnormalities in procoagulant factors such as protein C, S, Antithrombin, LAC, antiphospholipid antibodies. In addition, genetic defects in MTHFR, PLA1/A2 & PAI-1 were also found to be associated with RSA. Introduction: Pregnancy is a hypercoagulable state and thromboembolism is the leading cause of adverse pregnancy outcome includes recurrent spontaneous abortions (RSA). In the present study we have tried to evaluate the role of inherited/acquired thrombophilia factors in aetiology of RSA in Indian women. Materials and Methods: A retrospective analysis was done in 250

Summary: APLA syndromes are associated with development of antibody against the phospholipids. The present study showed that females are involved 5 times commonly than the males and the age group affected was in third and fourth decades. LA was positive in 61 %, ACL was positive in 16 % of cases and both LA and ACL was positive in 22 % of cases. Introduction: APLA (antiphospholipid antibody) are directed against certain phospholipids. APLA are associated with a syndrome that includes a hypercoagulable state, thrombocytopenia, fetal loss, stroke, and dementia and skin lesions. Materials and Methods: Aim: (1) To study the clinical presentations of APLA syndrome. (2) To study the laboratory profile in APLA. A retrospective study was done from the department of clinical Pathology for a period of one year (January 2012–December 2012). The laboratory profile and the clinical history were retrieved from the case records. Results: The total numbers of patients screened were 854. A total of 36 (4 %) patients were found to be positive. The predominant age group affected was between 20–40 years. One patient was a female aged 14 years. The male to female ratio was 1–5. Among these 36 patients, 10 (27 %) patients presented with repeated miscarriages and intrauterine death of the fetus. 8 (22 %) presented with deep vein thrombosis, 6 (16 %) with stroke. Of the 36 patients,

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356 LA alone was positive in 22 (61 %) patients and LA with ACL were positive in 8(22 %) cases ELISA for ACL (anticardiolipin) was positive in 6 (16 %) cases in which LA was negative. 7 (19 %) cases were found to have thrombocytopenia. Conclusion: The APLA syndrome is found 5 times commonly in females than in males. The predominant age group affected was between 20 and 40 years. 27 % of the female had miscarriages/abortions. 61 % patients were positive for LA.

Abstract No. 231 Evaluation of Antiphospholipid Antibodies in Women with Bad Obstetric History Narender Kumar, Jasmina Ahluwalia, Sunil Kumar Bose, Joseph, Varun Uppal, Neelam Varma Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh Summary: This was a retrospective study. Seven hundred (700) women with bad obstetric history were screened for antiphospholipid antibodies over a period of 3 years. Anti b2 GP1 antibody was most commonly detected in the screened cases. Introduction: The presence of antiphospholipid antibodies, such as anti cardiolipin antibodies (ACA), anti-beta 2 glycoprotein 1 (Anti b2 GP1) and the lupus anticoagulant (LA), are significantly associated with a history of thrombosis, thrombocytopenia, recurrent miscarriages and late foetal death. The prevalence of these antiphospholipid antibodies in women with bad obstetric history (BOH) is not very clear in north India. The aim of the study is to evaluate the prevalence of antiphospholipid antibodies, namely anti cardiolipin antibodies (ACA), anti-beta 2 glycoprotein 1 (Anti b2 GP1) antibodies and the lupus anticoagulant (LA) among women with BOH. Materials and Methods: Data was collected from last 3 years (2010–2012). Only Women with bad obstetric history were included in the study (BOH defined as one with poor obstetric outcome that can again adversely influence the future pregnancy and labour. Still birth, intrauterine foetal death and recurrent pregnancy loss are common varieties of BOH). ACA (IgM & IgG) and Anti b2 GP1 (IgM & IgG) antibodies were detected quantitatively by ELISA kit (Orgentec GmBH). LA was detected by clot based assay. Results: All the cases which showed presence of antibodies are divided in to two groups. Group 1 comprises of 23 case showed presence of either one or more antibody which was confirmed by repeating the test after 12 weeks and hence called as True positive. The positivity of individual tests was as follow—Anti b2 GP1: 16 cases; LA: 5 cases; and ACA: 13 cases. Group 2 contained 50 cases which showed presence of one or more antibodies. The repeat testing after 12 weeks was not performed (patients did not came back for repeat testing) to confirm the persistent positivity. In this group the individual test positivity were as follow: Anti b2 GP1: 34 cases; LA: 6 cases; and ACA: 1 case respectively. Six cases showed presence of both LA and Anti b2 GP1. One case was positive for Anti b2 GP1 and ACA while one case was triple positive. Conclusion: 3.2 % of total cases (23/700) showed presence of antiphospholipid antibodies which were confirmed by repeat testing after 12 weeks. Anti b2 GP1 is the most frequently detected antibody in both groups.

Abstract No. 232 A Case of ‘‘Catastrophic’’ Antiphospholipid Antibody Syndrome Arihant Jain, Raghavendraswami Amoghimath, Milind S Tullu, CT Deshmukh, Sunil Karande

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Department of Pediatrics, Seth GS Medical College & KEM Hospital, Parel, Mumbai Introduction: Catastrophic antiphospholipid antibody syndrome— Asherson syndrome is rare [Incidence \1 % of patients of Antiphospholipid antibody (APLA) syndrome]. It is characterised by diffuse thrombotic microvasculopathy resulting in multiorgan failure. The mortality is 50 % despite treatment. Case summary: A 5 month female child presented with fever & erythematous purpuric rash all over body (with gangrenous changes & ulceration over tips of fingers & toes) for 15 days. Subsequently she developed subacute hemorrhagic cerebral infarcts in left middle cerebral artery territory (deep white matter & basal ganglia) & pulmonary hemorrhage. Skin biopsy showed small vessel vasculitis with fibrin thrombi. APLA profile was positive (APLA IgM/ACLA IgM/Anti beta-2 glycoprotein-1 IgM—all positive); thus fulfilling all diagnostic criteria for catastrophic APS. The child had high total leucocyte counts— 50,100/mm3, high CRP-295 mg/lt (0–7) & blood culture showed MRCONS. ANA, dsDNA, ANCA & cryoglobulins levels were normal. Infection was precipitating factor in our patient and she was treated with systemic antibiotics (Meropenem, Linezolid), intravenous immunoglobulin, anticoagulants and steroids. Child showed dramatic response and was successfully discharged on oral anticoagulants & steroids. The child is asymptomatic for past 7 months. Conclusion: Our patient was diagnosed as catastrophic APS on the basis rapid involvement of three organ systems with small vessel vasculitis on skin biopsy and positive serology for antiphospholipid antibodies. High degree of clinical awareness is required in such cases for early diagnosis & treatment.

Abstract No. 233 A Study on Antithrombotic Factors and Platelet Functions in Epithelial Neoplasia Rashmi Kushwaha, Ashutosh Kumar, Ashima Jain, US Singh King Georges Medical College, Lucknow Introduction: The association between malignancy and thromboembolic disease is well established. It has been estimated that hyper coagulation accounts for a significant percentage of mortality and morbidity in cancer patients. Venous thromboembolism is the most common complication of cancer and second most common cause of death in cancer patient. The aim of this study was to assess the role of prothrombotic factors and platelets in cancer related thrombosis and cancer metastasis. Materials and Methods: A total of 70 Patients (28 with metastasis and 42 without metastasis) with histopathologic diagnosis of either cancers of gastrointestinal tract, lung or breast, were recruited. Protein C and Protein S estimation was done by Hemostar Coagulometer. PT, APTT, Fibrinogen estimation and FDP Assay was done. Platelet aggregation was done by OpticalAggregometer with AGGROLINK8 software. Results: We evaluated 98 individuals (28 with and 42 without metastasis and 28 as control). comparing mean level of Protein C and S factor between three groups, ANOVA revealed significantly different levels of Protein C (F = 55.53, p \ 0.001) and Protein S (F = 41.76, p \ 0.001) among the groups. Further, Tukey test revealed that the mean levels of Protein C and Protein S lowered significantly (p \ 0.001) in Group 2 as compared to both Control and Group 1. Furthermore, the mean levels of Protein C and Protein S also lowered significantly (p \ 0.001) in Group 1 as compared to Control. Our study revealed statistically significant difference and higher percentage of positive D-Dimer index in patients with metastasis (Group 2) as compared to both normal healthy (Control) and without metastasis patients (Group 1) (p \ 0.001). Comparing mean

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 level of fibrinogen between the three groups the study revealed that the mean level of Fibrinogen did not significantly differ among the three groups (p = 0.220). 15 patients in this study was assessed for platelet aggregation. 13 % patients had increased aggregation. The remaining 73 % patients had decreased platelet aggregation irrespective of their metastatic status. Though unexpected our result showed no difference in platelet aggregation patterns in the two groups. Conclusion: Epithelial malignancies have a subclinical prothrombotic state, progressing to thrombotic events that contribute significantly to morbidity and mortality in metastatic patients. Anti coagulant markers have a role in tumor metastasis. Protein C and S have a potential role in predicting thrombosis although larger studies are needed to establish its definite role.

Abstract No. 234 A Study of the Genetic Markers of Thrombophilia in Ischemic Stroke and Their Association with Stroke Severity Vandana Arya1, P Correia2, P Bhatt1, CS Agrawal2, M Bhargava1 Department of Haematology1 & Neurology2, Sir Ganga Ram Hospital, New Delhi Summary: Analysis of thrombophilic genetic markers in 120 adult stroke cases showed association of mutant A2 allele of PLA1/A2 polymorphism with moderately severe and severe group of patients. Also anterior circulation involvement was strongly associated with high severity. Introduction: Attempts to study the role of genetic variation in development of stroke in Indian population are few and far between. This study has looked at correlations, if any, between the thrombophilic genetic markers. Materials and Methods: 120 adult patients of ischemic stroke underwent gene mutation analysis for Factor V Leiden, MTHFR (C677T), Prothrombin(G20210A), GPIIb/IIIa(PLA1/A2) and PAI-1(4G/5G) polymorphisms by PCR RFLP. Severity of stroke was assessed on admission by the NIHSS stroke severity scale. Multiple risk factors for ischemic stroke were also included. Results: The mean age of the patients was 52 years (range 20–92 years) including 77 males and 53 females. As per NIHSS scoring the severity of stroke was mild in 24.1 % (29), moderate in 63.3 % (76), moderately severe in 5 % (6) and severe in 7.5 % (9). The T allele frequency of MTHFR among mild, moderate, moderately severe and severe groups were 17.4, 17.7, 33.3 & 33.3 % respectively showing an increasing trend in moderately severe and severe cases even though the difference was statistically not significant. A2 allele of PLA1/A2 was significantly associated with moderately severe and severe phenotype (p = 0.02). Frequencies of 4G allele of PAI-1 were 50 % in mild, 62.5 % in moderate, 91.6 % in moderately severe and 72 % in severe phenotype, the difference being borderline significant (p = 0.06). FVL mutation was present in only two patients one each from mild and moderate group. Prothrombin mutation was completely absent in our patients. Among all the risk factors chronic alcoholism, obesity and low vitamin B12 levels were significantly associated with stroke of high severity (p = 0.03, 0.04 & 0.03 respectively). Moderate, Moderately Severe and Severe sub group had Anterior circulation involvement in [50 % of cases and the association was also found to be statistically significant (p = 0.0001). Conclusion: A2 allele of PlA1/A2 polymorphism is associated with moderately severe and severe group of stroke patients. Chronic alcoholism, Obesity and Vitamin B12 levels are associated with the higher severity of the stroke. Anterior circulation involvement is significantly associated with moderate to severe stroke.

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Abstract No. 235 Correlation of Prothrombotic Coagulation Parameters with Genetic Markers of Thrombosis in Ischaemic Stroke Vandana Arya1, P Correia2, CS Agrawal2, M Bhargava1 Department of Haematology1 & Neurology2, Sir Ganga Ram Hospital, New Delhi Summary: In a study of 39 adult patients of stoke, multiple thrombophilic factors predisposed to large vessel stroke of moderate severity. There was a strong association with younger age and presence of lupus anticoagulant. Introduction: The role of prothrombotic coagulation parameters in acute phase of thrombosis is debatable. The present study is an attempt to study these parameters together with genetic markers of thrombosis in untreated cases of stroke to see if there is any contribution of these parameters in development of stroke. Materials and Methods: 39 adult patients of ischemic stroke were included. Their demographic profile clinical presentation and history were duly recorded. EDTA, Citrated and Plain blood samples were used for various investigations before institution of therapy. Protein C, Protein S, Antithrombin (AT), APCR, Lupus Anticoagulant (LAC) and antiphospholipid antibody (APA) assays were performed. Common genetic variants accounting for inherited Thrombophilia such as factor V leiden (FVL), Prothrombin (G20210A), MTHFR (C677T), were analysed by PCR-RFLP. Severity of stroke was assessed on admission by the NIHSS stroke severity scale. The important clinical and biochemical risk factors for ischemic stroke were also compiled. Results: In all the 39 patients thrombophilic factors were analysed within a period of 15 days of the onset of stroke. Detailed clinical findings with NIHSS severity score were recorded and the cases were sub divided as per TOAST criteria into various groups. Most of the strokes occurred in young to middle aged (mean age 40 year, range 18–57 years) healthy adults without any noteworthy presence of comorbidities (diabetes, hypertension, obesity, smoking, alcoholism, previous history of stroke and coronary artery disease). The strokes were mostly of moderate NIHSS severity (79.5 %, 31/39). Various inherited/acquired thrombophilic factors contribute to about 36 % of adult ishaemic stroke. Procoagulants protein C, protein S and antithrombin deficiency was present in 5.1, 12.8 and 2.5 % of patients respectively. Anti cardiolipin IgG antibody was positive in 2.5 % and IgM in 7.7 % of the stroke patients. One case each was positive for b2 glycoprotein IgG & IgM. Lupus anticoagulant was positive in 23 % (9/39) patients of which 5 were females. Factor V leiden was present in two cases whereas 17 cases were positive for MTHFR gene polymorphism. The presence of Lupus anticoagulant both alone and in combination with other thrombophilic risk factors is an important independent risk factor for stroke of moderate severity (p = 0.04). Majority of the patients with presence of thrombophilic risk factors had anterior circulation (71.4 %) and large vessel involvement (50 %). None of our patients had small vessel disease. Conclusion: The presence of multiple thrombophilic risk factors is an independent predisposing cause for Large vessel stroke of Moderate Severity. This showed strong association with younger age. Presence of lupus anticoagulant was an independent risk factor.

Abstract No. 236 Analysis of Thrombophilic Mutations in Young Patients of Thrombosis: A Pilot Study in a Tertiary Care Hematology Lab Monika Aggarwal, Jyoti Kotwal, Kshitij Srivastava, V Manu, Rajan Kapoor, SM Vibha Dutta, Velu Nair

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358 Armed Forces Medical College, Pune Summary: Evaluation of nine thrombophilic mutations and their polymorphisms for its role in unexplained thrombosis in young patients. Introduction: Nine thrombophilic mutations and their polymorphisms has come as a newer diagnostic modality for thromboembolic disorders. Mutations should be evaluated to know its utility in clinical presentation and preventive measures in patients of thrombophilia. Materials and Methods: We studied nine mutations in 20 cases of venous thrombosis in individuals \45 years and 20 age and sex matched controls over a period of 1 year by Strip based multiplex polymerase chain reaction reverse-hybridization method. Cases were of unexplained thrombosis in whom routine thrombophilia work up for protein C, protein S, Antithrombin III and APC-R was negative. All the controls selected were negative for any history of thrombosis. Results: Analysis of mutations by CVD strip assay has helped in interpreting mutations in all 20 cases and 20 controls. Presence of MTHFR C677T mutation (OD-2.25), MTHFR A1298C mutation (OD-4.88), PAI-1 4G/5G mutation (OD-1.50) showed positively statistically significant correlation with thrombosis. Prothrombin G20210A heterozygous was seen in one case only. However there was no increased association in cases with Factor V G1691A (OD-ZERO), Factor V H1299R(OD-1), Factor XIII V34L(OD-1), mutations. The EPCR A4600G, G4678C mutation (OD \ 1) was found positive in controls and protective for thrombosis. Conclusion: It is a valuable new test showing mutation association and prediction of occurrence of deep venous thrombosis in young patients.

Abstract No. 237 Co-Inheritance of Factor V Leiden in Cases with Inherited Hemophilia A in North India Namrata P Awasthi, Praveen Kumar, Shivbrat Upadhyay, Nuzhat Husain Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, UP Summary: The present study was undertaken to determine coinheritance of Factor V Leiden mutation in Cases with Hemophilia A (CWH) and evaluate the phenotypic effect on clinical severity. In hundred CWH studied, Factor V Leiden mutation was found to be 3 %. Factor V mutation did not appear to influence the clinical severity in our cases. Introduction: Hemophilia A is an X-linked recessive bleeding disorder resulting from mutations in FVIII gene including Inversion 1, Inversion 22 and small mutations in any of the 26 exonic regions. Clinical phenotype is dependant of FVIII bioactivity categorized into severe (\1 %), moderate (1–5 %) and mild (6–35 %) which in turn depends on the type of mutation in the family. Factor V Leiden mutation has been reported to reduce the severity of bleeding in CWH. The present study was designed to determine the prevalence of Factor V Leiden mutation in CWH and its effect on modulating the clinical phenotype. Materials and Methods: A total of 100 CWH were recruited for this study, including 56 severe, 35 moderate and 9 mild cases based on Factor VIII activity. Clinical assessment for age at onset, severity of bleeding and Factor VIII bioassay was performed. Factor V Leiden mutation was assessed by PCR-RFLP. Results: Factor V Leiden mutation was observed in 3 % CWH in this case series. All the three patients had Factor VIII levels \1 % with Intron 22 inversion, were heterozygous for Factor V Leiden and were clinically severe. Out of these three patients, two were real brothers and the third sibling who is also a severe hemophiliac, did not carry Factor V Leiden mutation. Conclusion: Overall

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 prevalence of Factor V Leiden mutation in CWH is similar to the nonhemophilic population and this mutation has no definite influence on the clinical severity in CWH.

Red Cell Disorders Abstract No. 238 Protective Effect of HFE Mutation Against Iron Deficiency Anemia? Sanjay Kumar Pandey, UK Chauhan, Arvind Tripathi, Vivek Yadav, Vineet Shah, Sweta Pandey Centre for Biotechnology Studies, Awadhesh Pratap Singh University, Rewa 486003 (MP) Summary: This study was aimed to optimize the phenotypic effect of HFE heterozygosity in iron deficiency anemia. Protection against iron deficiency and its morbid consequences may have selection advantage for heterozygous carriers of the mutation in IDA patients. Hence with the objective to study the effect of HFE genotype on phenotype of Iron deficiency anemia, we had explored the presence of HFE mutations in iron deficiency anemia patients. HFE heterozygosity was correlated an altered iron metabolism and ameliorate the phenotype of iron deficiency anemia. Introduction: HFE is a key regulator of iron hemostasis. In mutant condition it leads to iron overload, but in heterozygous condition it absorb more efficient iron than wild conditions. It may be protective effect on IDA Patients. The genetic forms of IDA are also associated with iron overload and a non responsive phenotype. Based on this background it was hypothesized that presence of any of these may influence the iron absorption and render protection from severity of iron deficiency. Materials and Methods: Study subject were iron deficiency anemia patients. Screening of the patients was done by clinical evaluation and iron profiling. Five ml venous blood was taken for DNA extraction and iron study. Hemogram was measured by automated cell analyzer. DNA extraction done by phenol-chloroform method. HFE gene mutations were determined by PCR-RFLP according to Aysen GunelOzcan et al. (2006). Serum ferritin, serum iron, TIBC nand % transferring saturation estimation was done by standard laboratory method. Results: Total 150 IDA patients sample were collected. Out of them 90 were male (mean age = 24.3 ± 3.5 years) and 60 were female (20 were in gestation and 30 were non gestation with mean age of 21.5 ± 2.4 and 17.3 ± 3.1 years respectively). Patient of IDA were presenting weakness, Easy fatiguability, breathlessness, tachycardia, systolic heart murmur, atrophic glossitis, angular stomatitis and pica. Total 200 healthy control samples were collected to compare the investigation value of patients. Heterozygotes for either C282Y or H63D was showing slightly higher Hb along with significantly raised serum iron, TS and serum ferritin compared with subjects and controls lacking these mutations. Also their iron supplementation frequency was fewer. Lower prevalence was noted among women carrying these mutations. Conclusion: The identification HFE heterozygotes influence on iron absorption modulating the phenotype of iron deficiency anemia may potentially present new therapeutic and diagnostic opportunities for the management of irondeficiency anemia.

Abstract No. 239 Role of Serum Hepcidin in Differentiating Anemia of Chronic Disease from Iron Deficiency Anemia in Children

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Gunjan Mahajan, Sunita Sharma, Jagdish Chandra1, Anita Nangia Department of Pathology and Paediatrics1, Lady Hardinge Medical College and Associated Hospitals, New Delhi Summary: Hepcidin, a recently discovered peptide, is a key regulator of iron homeostasis. It was significantly increased in Anemia of chronic disease (ACD) as compared to iron deficiency anemia (IDA) and ACD with coexistent ID. Introduction: ACD and IDA are two most common types of anemia, characterized by hypoferremia. The functional ID present in patients with ACD can be complicated by true ID. Differentiation between ACD and ACD/IDA is important clinically. However, in clinical practice it is difficult to differentiate between ACD, IDA and ACD/IDA by routine conventional laboratory tests. Hepcidin a key regulator of iron metabolism, might have promising role in differentiating the above conditions. Materials and Methods: Thirty children of ACD, IDA and controls each were studied. All were subjected to Complete blood count with peripheral smear, serum C-reactive protein, serum IL-6, Iron studies, serum soluble Transferrin receptor and serum Hepcidin. Patients of ACD with raised sTfR levels ([3’ g/ml) were classified as ACD with coexistent IDA. Results: Hepcidin levels were significantly increased in all ACD patients (mean = 143.85 ± 42.75 ng/ml), reduced in all IDA patients (mean = 6.0 ± 2.83 ng/ml) and ACD with coexistent IDA (mean = 10 ± 2.97 ng/ml) as compared to controls (p \ 0.001). Conclusion: Hepcidin is a new biomarker of iron homeostasis and its ability to differentiate ACD from IDA is yet to be established. This study indicated that hepcidin had a definitive role in identifying ID in ACD patients and differentiating ACD from IDA. Newer drugs available against hepcidin ferroportin axis or cytokines which stimulate hepcidin transcription might be useful in treating anemia of inflammation.

Abstract No. 240 Prevalence and Clinical Profile of Megaloblastic Anemia in Children Govind Kendre, Sujata Sharma, Vishal Jadhav, Nikita Shah, Smita Dsouza, Yashwant Gabhale, Mamta Manglani Division of Pediatric Hematology-Oncology, LTMM College & General Hospital, Sion, Mumbai Introduction: Megaloblastic anemia is an underestimated disease of nutritional origin in children. Contrary to earlier times, this entity has become much more frequent today. Veganism is one of the common reasons for an increase in the prevalence of nutritional megaloblastic anemia. Deficient mothers often have children with vitamin B12 deficiency with megaloblastic anemia and various neurologic manifestations. It presents with varied clinical features in childhood, sometimes mimicking a hematological malignancy like leukemia. Diagnosing this disease assumes great clinical importance since it responds exceedingly well to treatment. Objective of the Study: (1) To study the prevalence and clinical profile of megaloblastic anemia in children referred for anemia. (2) To determine the high risk factors for occurrence of megaloblastic anemia in children and (3) To find out the effectiveness of treatment in these children. Materials and Methods: This study was a retrospective analysis of children referred for anemia to the Pediatric Hematology-Oncology Division of LTMG Hospital over the last 3.5 years from January 2009 to July 2013. Clinical data was charted in a proforma and all patients underwent a complete hemogram, corrected reticulocyte count and peripheral blood smear examination, biochemical investigations including serum bilirubin levels, lactate dehydrogenase levels, serum vitamin B12 levels (NR: 200–950 pg/ml) and red cell folate levels (NR:

359 175–700 ng/ml). Bone marrow was done to confirm the diagnosis in those children who could not afford B12 and folate levels. The diagnosis of megaloblastic anemia was established on the basis of serum Vitamin B12 levels below 200 pg/ml and/or RBC folate levels below 175 ng/ml or bone marrow suggestive of megaloblastic changes along with the clinical findings and smear picture suggestive of megaloblastic anemia. Results: A total of 1,365 patients were referred for anemia during the study period. Of these 56 (4.1 %) children were diagnosed as per the predefined criteria as megaloblastic anemia. Among these children, the age and sex distribution was as follows: 25 (44.64 %) were males 31 (55.35 %) were females with male to female ratio of 0.8:1. The youngest was 3 month old and the oldest was 12 years old. 22 (39.28 %) of them were in age group of 0–12 months, 8 (14.28 %) in 1–3 years, 9 (16.07 %) in 3–6 years, 5 (8.92 %) in 6 to 9 years and 12 (21.42 %) in 9–12 years age group. When evaluating diet in these children, history of milk based diet was obtained in 24 (42.85 %) children, 15 (26.78 %) were lactovegetarians and 17 (30.35 %) consumed a mixed (non-vegetarian) diet. Clinical presentations varied from recurrent infections to various hematological manifestations including mucosal or skin bleeds. 23 (41.07 %) patients were incidentally found to have megaloblastic anemia on routine blood counts when they were seen for acute infections. 6 (10.71 %) children had listlessness and 12 (21.42 %) had pallor as presenting complaint. 5 (8.92 %) children presented with skin and mucosal bleeds. 10 (10.71 %) of them presented with neurological complications; 4 (7.14 %) of these had infantile tremor syndrome and the remaining 6 had gross developmental delay. On examination, pallor was present in all, hyperpigmented knuckles were seen in 47(83.927 %) children, where as sallow complexion was found in 30 (53.57 %) children. 38 (67.85 %) children had mild hepatosplenomegaly. On investigations, hemoglobin ranged from 1.6 to 7.5 g % (mean Hb 5.7 ± 1.02 g %). MCV ranged from 85 to 123 fL (Mean MCV was 102.43 ± 6.94 fL). 14 (25.00 %) children had pancytopenia and 18 (32.14 %) had bicytopenia (anemia with leucopenia). 6 (10.71 %) patients who presented with mucosal and skin bleeds had thrombocytopenia (platelet counts ranged from 30,000 to 50,000 per c.mm). Peripheral blood smear showed macrocytes, macroovalocytes and pear shaped poikilocytes in all of them. Hypersegmentation and large sized neutrophils were seen in 31 (55.35 %) cases. Serum bilirubin levels ranged from 0.4 to 4.7 mg/dl (with high indirect bilirubin in those with elevated levels). Serum LDH levels [1,000 IU/L was seen in as many as 34 (60.71 %) children. Serum vitamin B12 and red cell folate levels could be done in 45 (80.35 %) patients. Of these, 8 (17.77 %) patients had low levels of both serum vitamin B12 and RBC folate, 24 (53.33 %) had only low serum vitamin B12 levels, 6 (13.33 %) had low folate levels and 7 (15.5 %) patients had normal levels. 11 (19.64 %) children were diagnosed on the basis of bone marrow aspirate findings of megaloblastic anemia. Treatment was effective in a short duration with a mean rise in hemoglobin of 1–2 g % in the 1st week and by 4–4.3 g % in 2nd week. Thrombocytopenia and other morphological parameters also showed improvement on treatment within 4 weeks. Conclusion: We conclude that appropriate diet would prevent occurrence of megaloblastic anemia in children, which is almost always nutritional in etiology. Additionally, timely diagnosis and treatment with specific hematinics can avoid unnecessary transfusion of blood components.

Abstract No. 241 A Study of UGI Endoscopy Features and H Pylori Infection in Megaloblastic Anemia Vineet Behera, Velu Nair, Vineet Behera, Rajan Kapoor, Jyoti Kotwal, Pankaj Puri, M Dwivedi, P Chauhan

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Department of Hematology & Department of Internal Medicine, Armed Forces Medical College, Pune

Abstract No. 243

Summary: Objective To study the UGI endoscopy features and incidence of helicobacter pylori infection in MA. Introduction: H. pylori has been proposed as etiology of megaloblastic anemia (MA). H. pylori causes gastritis which result in destruction of the parietal cells leading to decreased production of intrinsic factor and MA. H pylori in MA has been studied in occasional studies but Indian s sparse. Materials and Methods: We designed a two year cross sectional study in a tertiary care hospital. The study population comprised of patient of MA (diagnosed by PBS/bone marrow/low vitamin B12 or folic acid). A detailed clinico pathological record and etiological workup was done. UGI endoscopy was done to look for macroscopic features and a gastric biopsy was done and H pylori testing was done on the biopsy tissue for all patients. Results: (1) A total of 42 patients of MA were included. UGI symptoms like dyspeptic symptoms were seen in 28.5 %, nausea/vomiting in 19 % and abdominal pain in 14.2 % cases. (2) UGI endoscopy showed evidence of gastritis in 14.2 %, atrophic gastritis in 23.8 %, gastric or duodenal ulcer in 9.5 % and a normal appearance in 52 % cases. Histology showed features of gastritis in 33.3 %, non specific inflammation in 23.8 % and a normal histology in 42.8 % cases. Direct evidence of H. pylori was seen in 31 cases (71.3 %). (3) H. pylori infection was seen in 58 % of MA with known etiology (n = 18) and about 41.9 % of MA with unknown etiology (n = 13). There was statistically significant correlation between presence of H pylori and MA and presence of dyspeptic symptoms in MA.

Automated High Speed Micro Fluidic Chip Based Capillary Electrophoresis Technique for the Identification of the Red Cell Membrane Protein Defect

Abstract No. 242 Clinico-Pathological Study of Geriatric Anemia in and Around Varanasi: A Hospital Based Study Deepa Rani, Vijai Tilak, IS Gambhir Institute of Medical Sciences, BHU, Varanasi Summary: We did a prospective study in 139 geriatric anemia patients and found that the incidence of anemia due to hematological malignancy is high in this age group and especially for that of multiple myeloma. Introduction: The number of elderly individuals is expected to reach unprecedented levels in the twenty-first century. Anemia represents an emerging global health problem that negatively impacts quality of life in a significant proportion of the elderly population and requires an ever-greater allocation of healthcare resources. Materials and Methods: In the geriatric patients ([60 years) presented with anemia (hemoglobin in male \13 g/dl, hemoglobin in female \12 g/dl), complete blood count, general blood picture, bone marrow aspiration and biopsy was done, as well as wherever needed serum iron, TIBC, vitamin B12, lactate dehydrogenase levels were evaluated. Results: Anemia is common in age group of 60–65 years in both the sexes. Anemia due to hematological malignancy formed the major group. Multiple myeloma (21.6 %) was the commonest cause of geriatric anemia followed by iron deficiency anemia (12.3 %), megaloblastic anemia (12.3 %), CML (10.1 %) and tuberculosis (7.2 %). The uncommon causes were AML (6.5 %), Aplastic anemia (4.3 %) and anemia due to diabetes (3.6 %). CLL (2.2 %), Myelofibrosis (2.2 %), Kala-azar (2.1 %) and MDS (1.4 %) were the rare causes of geriatric anemia. Conclusion: Haematological malignancy cases may present with severe anemia hence proper investigative workup is needed to reach up to the cause in each and every patient of geriatric anemia.

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Prabhakar Kedar, Prashant Warang, Kanjaksha Ghosh, Roshan Colah National Institute of Immunohaematology (Indian Council of Medical Research), K.E.M. Hospital Campus, Parel, Mumbai Summary: Qualitative or quantitative defects of red blood cell (RBC) membrane proteins in hereditary spherocytosis cases is identified by automated high speed micro fluidic chip based capillary electrophoresis technique. Introduction: Hereditary spherocytosis (HS) is a congenital hemolytic anemia. The disease is caused by defects in red cell membrane cytoskeleton resulting from mutations in genes encoding various red cell membrane and skeletal proteins. The screening tests for HS are the osmotic fragility test and flowcytometric eosin-50 -maleimide (EMA)-binding test. SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins is carried out to confirm the diagnosis. Capillary electrophoresis (CE) is also known as a separation technique for the separation of both charged and noncharged molecules. Materials and Methods: We investigated erythrocyte membrane protein defects in 30 Indian HS patients and 10 controls. The Agilent 2100 Bioanalyzer employs microfluidic capillary gel electrophoresis for protein analysis where fluorescence intensities of proteins are measured as a function of their migration times. Fluorescence intensity is related to the concentration of these proteins. Data analysis is performed by the Agilent 2100 Expert Software, automatically determining molecular weight and concentration of the proteins in the sample. Results: We used automated high speed micro fluidic chip based capillary electrophoresis technique to identify the protein defects causing HS. Protein deficiencies related to HS were demonstrated in 57 % of HS patients. Spectrin deficiency was detected in 12 % of cases (4/30), ankyrin deficiency in 16 % (5/30), Band-3 protein deficiency in 20 % (6/30), combined spectrin and ankyrin deficiency in 6 % (2/30). Forty three percent of cases (13/30) showed normal protein contents. Conclusion: Automated high speed micro fluidic chip based capillary electrophoresis (1DE) may reveal qualitative or quantitative defects in red blood cell (RBC) membrane proteins in hereditary spherocytosis cases. It may be substitute for SDS-PAGE technique.

Abstract No. 244 Effect of UGT1A1 and Hemoxygenase Gene Polymorphisms on Serum Bilirubin Levels of Patients with Hereditary Spherocytosis Prashant Warang, Rati Devendra, Selma D’Silva, Prabhakar Kedar, Kanjaksha Ghosh, Roshan Colah, Malay Mukherje National Institute of Immunohaematology, Mumbai Summary: The present study demonstrates that (TA)n repeat polymorphism in the UGT1A1 gene is strongly associated with high serum bilirubin levels as well as the risk of developing gall stones in the patients with hereditary spherocytosis. Introduction: Hereditary spherocytosis (HS) is one of the most common causes of inherited hemolytic anemia. The aim of this study was to investigate the effect of UGT1A1 and hemoxygenase gene (HMOX1) polymorphisms on serum bilirubin levels in the patients with hereditary spherocytosis.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Materials and Methods: 100 HS patients with variable serum bilirubin levels (0.9–30.0 mg/dl) were studied. Screening for red cell membrane protein defects was done by flow cytometric analysis using the Eosin 50 maleimide dye test. The UGT1A1 (TA)n and HMOX1 (GT)n promoter polymorphisms were determined by Gene Scan analysis. Results: The (TA)7 promoter polymorphism was found to be significantly higher among the HS patients who were either heterozygous or homozygous for this mutant allele as compared to the wild type (TA)6 allele [77.0 vs. 23.0 % (p \ 0.001)]. The mean serum bilirubin levels as well as the formation of gall stones were significantly higher among the cases who were homozygous for the (TA)7 allele associated with Gilbert’s syndrome as against the wild type (TA)6 allele (6.85 ? 5.87 vs. 2.65 ? 1.24 mg/dl and 52.0 vs. 0.0 % respectively). No significant difference were seen in the HMOX1 (GT)n promoter polymorphism. Conclusion: These finding indicate that Glibert’s syndrome could be the major contributing factor for hyperbilirubinemia and developing gall stones in HS patients.

Abstract No. 245 Autoimmune Hemolytic Anemia with Thrombocytopenia: A Case Series Rajan Kapoor, Mukesh Dhillon, Jyoti Kotwal, Devika Gupta, Satish Mendonca, Velu Nair Armed Forces Medical College Summary: Ten cases of Direct antiglobulin test (DAT) positive hemolytic anemia and thrombocytopenia were analysed in this study. Out of these 4 (40 %) had primary Evans syndrome (ES), SLE was seen in 3 (30 %) while 1 patient each had common variable immune deficiency (CVID), autoimmune lymphoproliferative syndrome (ALPS) and lymphoproliferative disorder. Introduction: Evans syndrome (ES), first described in 1951 by Evans and colleagues, is a rare autoimmune disorder characterized by the simultaneous or sequential development of autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP) and/or immune neutropenia in the absence of any underlying cause. These patients present with various combinations of cytopenias and warrant extensive evaluation to rule out secondary causes which are common in this setting. We report cases of AIHA and ITP reporting to a tertiary care centre of armed forces. Materials and Methods: This study was conducted in department of clinical hematology at a tertiary care centre of armed forces, and included 10 new patients of Evans syndrome diagnosed from January 2011 to April 2013. The diagnosis was based on detailed medical history, physical examination and laboratory investigations including specialized investigations when clinically indicated. Bone marrow aspiration and biopsy was carried out in all cases to rule out other causes of bicytopenias. Serology was carried out for hepatitis B, C and Epstein Barr virus infection, Cytomegalovirus and HIV. Connective tissue disorders and vasculitis were investigated for by antinuclear, anti-double stranded DNA antibodies, anti-cardiolipin, b-2 glycoprotein and lupus anticoagulant assays. Immunoglobulin profile for primary immune deficiencies and flow cytometry to look for lymphoproliferative disorders was done when clinically suspected. Results: Total of 10 patients, 9 females and 1 male were analyzed. Mean age was 28.9 years (2 patients were more than 60 years). Mean hemoglobin was 5.08 g %, mean platelets were 34,400/ll. Bone marrow of all patients showed erythroid hyperplasia with increased megakaryocytes. Out of 9 female patients, 3 were diagnosed as systemic lupus erythematosus (SLE), 1 was found to have common variable immunodeficiency (CVID), 1 was diagnosed as autoimmune lymphoproliferative (ALPS) disorder and rest 4 were diagnosed as primary ES. The only male patient was diagnosed as a case of lymphoproliferative

361 disorder. Conclusion: Evans syndrome is predominantly seen in females. The diagnosis of ES still implies the exclusion of secondary causes to avoid any detrimental misdiagnosis. Our study showed that 60 % patients have secondary causes, most commonly SLE which was seen in 30 %. The careful analysis of the peripheral smear, DAT with relevant specialized investigations based on clinical settings is of paramount significance. ES may well precede a variety of underlying disorders which may influence both the management and outcome. We believe that ES should no longer be considered only as an idiopathic condition but rather be classified as primary (idiopathic) or secondary (i.e., associated with an underlying disease) syndrome; a terminology well accepted and used in the setting of AIHA and ITP.

Abstract No. 246 Assessment of Therapeutic Response in Management of Anemia in CKD Vineet Behera, Vineet Behera, KV Baliga, RS Vasant Kumar, Velu Nair, N Naithani, Arun Kumar, Mayank Mishra Department of Internal Medicine, Armed Forces Medical College, Pune Summary: Title-To assess the therapeutic response in management of anemia of CKD and compare the response in different stages of CKD. Introduction: Anemia is a common cause of morbidity and mortality in CKD and is multifactorial. Despite major advances and newer therapies, it is often difficult to treat with variable response in different cases. But, there is no Indian literature on the therapeutic response of the conventional agents used in anemia of CKD in Indian population. Materials and Methods: This is an observational study which included patients of CKD with iron deficiency anemia(recently detected or on no therapy for anemia). All patients were treated with erythropoietin 150 U/kg/week SC (3 divided doses/week), injection iron sucrose 100 mg IV once a week for 10 weeks and oral hematinics. The patients were serially monitored to assess therapeutic response. Results: The study population (n = 111) was divided into three groups. Group A (CKD stages 1,2 and 3) (n = 33, 30 %), Group B (CKD stages 4 and 5 not on dialysis) (n = 29, 26 %); and Group C (ESRD patient on dialysis) (n = 49, 44 %). Anemia was seen in 83 (74 %) of total patients with 18 (54 %) of group A, 20 (69 %) of group B and 45(92 %) of group C. Anemia was normocytic normochromic in 56 %, microcytic hypochromic in 31.5 % and dimorphic in 11.7 %. The mean Hb (g %), serum ferritin(ng/ml) and transferrin saturation (%) measured at start of therapy, after 3 and 6 months. In group A the levels were 11.34, 198.50, 27.17; 11.39, 230.67, 30.00 and 11.9, 298.17, 33.15 respectively (p [ 0.01); in group B the levels were 10.73, 180.67, 23.33; 11.3, 204.0, 27.83 and 11.8, 271.83, 31.33 (p \ 0.001) and in group C the levels were 9.24, 127.48, 13.7; 10.1, 165.0, 15.97 and 11.4, 189.58, 18.03 respectively (p \ 0.01). Conclusion: Anemia was more prevalent and severe and showed better therapeutic response in advanced stages of CKD than early stages. Patients on dialysis showed less improvement than the cases not on dialysis.

Haemoglobinopathies Abstract No. 247 Sehgal Index: A New Index and Its Comparison with Other CBC-Based Indices for Screening of Beta—Thalassemia Trait in a Tertiary Care Hospital Dia Mansukhani, Kunal Sehgal, Meher Irani, Tina Dadu, Shanaz Khodaiji

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Hematology Laboratory, Department of Laboratory Medicine, P.D. Hinduja National Hospital and MRC, Mumbai Summary: Various existing CBC-based indices along with newly proposed index (SEHGAL Index) were evaluated for the best combination of sensitivity and specificity to predict Beta thalassemia trait (BTT). SEHGAL index and Mentzers-index (\14) were found to be the best for screening of BTT in a hospital setup. Introduction: BTT must be differentiated from iron deficiency anemia (IDA) to avoid unnecessary iron therapy and for prevention of thalassemia major by genetic counseling. In a tertiary care hospital, it is vital that the screening tool is not only sensitive but also specific so as to be cost effective and save time. Aim: The aim of this study was to evaluate the new SEHGAL index and compare it to existing CBC-based indices for the best combination of sensitivity and specificity to predict BTT. Materials and Methods: Study was done in 2 phases: (a) Phase 1: A retrospective analysis of 1022 consecutive HPLC cases from July 2008–June 2011. (b) Phase 2: A prospective analysis of 973 consecutive HPLC cases from July 1, 2011–June 10, 2013 was done to further evaluate the new SEHGAL index. Results: In phase 1, prevalence of BTT was 28.8 % (294 cases/1022 cases). ROC-AUC and Youden index was highest for new SEHGAL index, followed by MI \14 (Table 1). The prospective study shows results as we had expected from the trends in the retrospective study. Conclusion: SEHGAL index and Mentzers Index \14 showed the best combination of sensitivity and specificity in predicting BTT. The best indices or combination can be used as a ‘validated flag rule’ in the analyser middleware program in a hospital for identifying suspected cases of BTT.

Table 1 ROC-AUC of various CBC-based indices CBC-based index

Abstract No. 249

Phase 1 ROC-AUC

Phase 2 ROC-AUC

\12

0.71

0.784

\13

0.76

0.802

\14

0.80

0.808

0.77

0.755

Mentzers index (MI)

Green and King (G&K) Shine and Lal (S&L)

0.69

0.735

RDW index (RDWI)

0.79

0.783

Srivastva index (SI)

0.78

0.785

England and Fraser (E&F)

0.71

0.731

Ricera index (RI) RDW-CV

0.65 0.629

0.637 0.634

SEHGAL index

0.81

0.823

Abstract No. 248 Transfusion Associated Unknown Peak on Hb HPLC—A Diagnostic Dilemma: A Case Report Aruna Chhikara, Sunita Sharma, Preeti Rai, Richa Chauhan, Jagdish Chandra1 Department of Pathology & Paediatrics1, Lady Hardinge Medical College & Kalawati Saran Children’s Hospital Summary: Haemoglobin High Performance Liquid Chromatography (Hb-HPLC) is used to diagnose various hemoglobinopathies. Recent

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blood transfusions from asymptomatic donors with hemoglobinopathies can lead to spurious peaks. We report a case of transfusion associated hemoglobinopathy in infant with aˆ-thalassemia major. Introduction: Abnormal peaks on HPLC can lead to difficulty in diagnosis. Transfusion of blood from asymptomatic donors with hemoglobinopathy can sometimes lead to disastrous consequences. Materials and Methods: A 7 month infant presented with pallor & hepatosplenomegaly. The laboratory investigations revealed: Hb 7.9 g/dl, TLC 18.3 9 109/l, platelet count 130 9 109/l, RDW 44.7 %, MCV 65.9 fl, MCH 21.4 pg and MCHC 32.5 g/dl Peripheral smear revealed microcytic hypochromic red cells with marked anisopoikilocytosis and 20 nRBCs/100 WBCs. Corrected reticulocyte count was 4.8 %. A diagnosis of Beta Thalassemia Major was suggested and HPLC was done. Results: Hb-HPLC chromatogram revealed 47.6 % HbF, 35.9 % HbA0, 3.2 % HbA2 & an unknown peak of 9.1 % at retention time (RT) of 4.71 min. Possibility of aˆThalassemia major with HbQ India was kept. However, due to 35 % HbA0, possibility of transfusion associated hemoglobinopathy was also considered. Further workup revealed transfusion of one unit of blood a week prior to admission. Both parents were heterozygous for aˆ-thalassemia and none showed peak at RT-4.71 min. Final diagnosis of aˆ-Thalassemia major with transfusion associated hemoglobinopathy was made. Conclusion: Low percentage of Hb peaks and their sudden appearance/disappearance are clues to pick up unknown peaks in Hb-HPLC. Therefore, family study, history of blood transfusion and repeat HPLC should be done to rule out presence of spurious haemoglobin peaks.

Detection of Haemoglobinopathies by HPLC in North Indian Population: A Tertiary Care Hospital Based Study Richa Chauhan, Sunita Sharma, Preeti Rai Department of Pathology, Lady Hardinge Medical College, New Delhi Summary: Cation Exchange High Performance Liquid Chromatography (CE-HPLC) is an essential tool to screen various haemoglobinopathies and diagnose haemolytic anemias. A cross sectional study of 1,360 subjects was carried out to determine occurrence of various haemoglobinopathies in population catered by tertiary care hospital in Delhi. Introduction: HPLC is a useful method to promptly identify various haemoglobinopathies. Nowadays, it has emerged as an essential screening method for detecting haemoglobin variants. Premarital and prenatal screening plays an important role in reducing disease burden in the country. Materials and Methods: A total of 1,360 blood samples were run on the BioRad Variant II HPLC system by b-Thalassemia Short Programme. The retention time (RT), percentage of haemoglobin peaks and characteristics of chromatogram were analyzed. Family study was done wherever indicated. Results: Out of 1,360 cases, 383 (28.16 %) showed abnormal HPLC chromatogram. Most common haemoglobinopathy was b-thalassemia trait (18.82 %) followed by b-thalassemia major (3.24 %). HbE and HbS were seen in 3.16 and 1.02 % cases respectively (heterozygous/homozygous/doubly heterozygous state). Eleven cases (0.8 %) of HbD-Punjab as heterozygous/doubly heterozygous state were also observed. Single case of Hb D-Iran and Hb Q-India were seen. Early peaks were detected in 4 cases which were later confirmed as HbH disease by demonstration of golf ball inclusions on reticulocyte preparations. Conclusion: HPLC is a reliable method to detect haemoglobinopathies. Most cases can be accurately diagnosed by studying retention

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 times, characteristic and percentage of Hb peaks in chromatogram. Family study should be done for confirmation of haemoglobin variants.

Abstract No. 250 The Prevalence of Thalassaemia Carrier in West Bengal Basab Bagchi, Maitryee Bhattacharyya, Swarupa Bhattacharya, Soumita Chowdhury, Sanjay Misra, Shyamali Dutta, TK Dolai, Rajib De

363 antenatal mothers were screened. Only 1,737 (8.32 %) mother attended antenatal clinic in their 1st trimester, 12,096 (57.91 %) in 2nd and 7,050 (33.75 %) in 3rd trimester. 2,193 (10.5 %) were carriers. 1,065 (48.6 %) husbands agreed for screening, Both husband & wife detected to be carrier in 119 (11.2 %) cases and out of them 46 (39.13 %) underwent PND. Conclusion: Since most of the mothers attended antenatal clinic late and only a small percentage of husbands agreed for screening it suggests that more emphasis to be given on pre-marital rather than antenatal screening.

Abstract No. 252

Department of Haematology, NRSMCH Summary: Population screening for thalassaemia carrier showed that HbE trait was the most common hemoglobinopathy followed by b-thalassaemia trait. Among the symptomatic haemoglobinopathies HbEb was commonest. Rare Hb variants like Hb Lepore, HbJ Toronto, Hb New York and HbC trait were also detected in West Bengal. Introduction: This study was conducted to determine the prevalence of haemoglobinopathies in Bengal. The samples were collected from mothers attending the antenatal clinic and also from healthy people attending the outreach camps organized in different parts of Bengal. Materials and Methods: The blood samples from 16,818 peoples (12,343 antenatal mothers and 4,475 healthy people from outreach camps) collected during the period from June 2010 to Jan 2013 were analyzed by HPLC using Biorad variant 1 and 2. In selected cases molecular analysis were done. Results: The most prevalent haemoglobinopathy was HbE trait 643 (3.8 %) followed by btt (618) (3.67 %). 25 cases HbEb thalassaemia was detected and no case of bTM was detected. 45 cases of HbS trait (0.26 %), 23 cases of HbD trait (0.14 %) and 2 cases of HPFH trait were detected. Among the rare forms 3 cases each of HbDb and HbDE were detected. 2 cases were double heterozygous for HbS and HbD. One case each of Hb Lepore, HbJ Toronto, Hb New York, and HbC trait was detected. Conclusion: HbE trait was the most common carrier state followed by b-thalassaemia trait and HbS trait. Among the symptomatic haemoglobinopathies, HbE/b thalassaemia was most prevalent.

Abstract No. 251 Effectiveness of Antenatal Screening for Prevention of Thalassaemia in a Developing Country Basab Bagchi, Maitryee Bhattacharyya, Soumita Chowdhury, Sanjay Misra, Shyamali Dutta, TK Dolai, Rajib De Department of Haematology, NRSMCH Summary: Screening of antenatal mothers showed that a significant proportion (10.5 %) of antenatal mothers were carriers. However the majority of them attended antenatal clinic first in the 2nd (57.91 %) and 3rd trimester (33.75 %) thereby reducing the utility of antenatal screening and PND. Introduction: Thalassaemia is a major public health burden in India. Screening of pre-marital population & antenatal mothers considered an effective way of preventing birth of thalassaemia children. However screening of mother needs to be done early in pregnancy so that if both the partners are detected to be carrier, there is an option of PND and termination of pregnancy if required. Materials and Methods: The study was conducted in the haematology department of NRSMCH in collaboration with G&O department from Feb, 2009 to Nov, 2012. Antenatal mothers were screened for thalassaemia carrier. Husbands of carriers mothers were advised to undergo screening. The couples were counseled for PND if both of them were thalassaemia carriers. Results: Total 20,889

Thalassemia/Hemoglobinopathies Associated with Mutations in Terminal Exon of b Globin Gene KJ Sampath Kumar, K Neelagandan, Eunice Sindhuvi, Biju George, Vikram Mathews, Alok Srivastava, RV Shaji Christian Medical College, Vellore Multiple mutations including substitutions, deletions and frameshift mutations have been described within the exonic, intronic and promoter regions of b-globin gene causing b-thalassemia/ hemoglobinopathies. Mutations in the exon-3 of b-globin gene are uncommon, even though it represents 30 % of the coding region bglobin gene. Thalassaemic haemoglobinopathies arising from mutations in the exon 3 of b-globin gene exhibit a wide spectrum of phenotypes ranging from mild thalassaemia intermedia to severe thalassaemia major. The majority of the mutations in exon 3 present as dominant beta-thalassemia. In our center, molecular diagnosis of beta thalassemia was carried out for 3,240 suspected individuals from January 2005 to July 2013. Genomic DNA was extracted by a standard protocol and eight common mutations that cause b-thalassaemia were screened by reverse dot blot (RDB). Rare and novel mutations were identified by sequencing of the entire b-globin gene. Five different mutations in exon3 of b-globin gene were indentified in 7 individuals. These mutations include four previously reported mutations {Codon 110 (T-C){Hb Showa-Yakushiji}, Codon 114 (T-C){Hb Durham-N.C./Brescia}, Codon 106/107(+G), Codon 126–131(17 bp)} and a novel mutation{Codon 130 (T-A)}. These mutations were present in heterozygous (n = 2), homozygous (n = 1) and compound heterozygous states (n = 4). The clinical phenotype of these cases ranged from mild thalassemia intermedia to severe thalassemia major. Codon 110 and 114 mutations affect the interaction of a–b subunits results in unstable b-globin chain. Codon 106/107(+G) and Codon 126–131(-17 bp) results in a frameshift with premature termination codon at 139 and 133 positions respectively results in a thalassemia phenotype. The novel mutation at Codon 130(T-A) results in the replacement of threonine by stop codon, producing a shorter b-globin. This report describes the different mutations occurring in exon3 of b-globin gene observed in our center. To conclude, mutations in exon3 of b-globin gene are rare and results in a heterogenous phenotype, with some mutations resulting in dominant phenotype.

Abstract No. 253 Detection of Uncommon Beta Thalassemia Mutations Using Direct Sequencing and Detection of Alpha Gene Triplication for Suspected Thalassemia Cases with Diagnostic Confusion in the Population of West Bengal Krishnakoli Sen, Shila Chakrabarti, Uttam Nath, Siddhartha Sankar Ray, Prantar Chakrabarti, Utpal Chaudhuri

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364 Institute of Haematology and Transfusion Medicine, Medical College, Kolkata Summary: Sequencing studies done on patients with beta thalassemia trait revealed that several uncommon mutations exist in the population of West Bengal. Alpha triplication in combination with heterozygous beta mutation was also found to exist in this population and results in diagnostic confusion. Introduction: In some antenatal/ prenatal cases, where RBC indices suggested beta trait or there was history of thalassemia in offspring, the Hb HPLC was inconclusive, and the array of common beta mutations were absent. Such samples were subjected to sequencing to detect uncommon mutations and alpha triplication studies were performed. Alpha triplication was also studied in suspected thalassemia cases who may be requiring blood transfusion in spite of being heterozygous for a beta thalassemia mutation. Materials and Methods: The beta globin gene was investigated by amplifying the gene in two fragments—the first fragment being 916 bp and the second fragment being 454 bp in length. The amplified products were then sequenced and the data obtained was analyzed using available bioinformatics tools. Alpha triplication detection was performed using oligonucleotide primers specifically designed to amplify unequal crossover regions of alpha globin gene and the product was observed by agarose gel electrophoresis. Results: Some of the rare mutations observed in the study included substitution mutation at 50 UTR(–90C[T), frameshift mutation (-T) at codon 15 of exon 1, frameshift mutation (-C) at codon 16 of exon 1, substitution mutation in the capsite 1 (A[C), substitution mutation at IVSI-129(A[C). Rare haemoglobin variants observed were Hb Hofu (substitution at codon 126 of exon 3 GTG[GAG), Hb Grange-Blanche(substitution at codon 27 of exon 1 GCC[GTC). 42 patient samples were studied for alpha triplication detection out of which 11 patients (26.2 %) were found to be positive for triplication. Conclusion: Sequencing studies help in the detection of rare mutations in some unexplained cases of thalassemia. Also, alpha triplication studies can clinch the diagnosis in a significant percentage of cases including couples presenting for prenatal diagnosis.

Abstract No. 254 Phenotype Scoring System to Evaluate the Clinical Severity of Thalassemia Intermedia and Its Correlation with Genotype Krushna Chandra Pani, M Murari, S Sharma, SR Phadke, S Agrawal Department of Pathology, Department of Medical Genetics, SGPGIMS, Lucknow Summary: Thalassemia intermedia is a common hemoglobinopathy associated with variable clinical profile. An already published scoring system was applied to 41 cases Indian patients to evaluate the clinical severity. Introduction: Thalassemia intermedia patients show clinical severity varying between thalassemia major and asymptomatic carriers. The genotype of different thalassemia intermedia patients also vary, which includes the cases of b0 homozygotes, b? heterozygotes, E-b thalassemia, sickle-b thalassemia and delta-b Thalassemia etc. This study was conducted to evaluate the clinical severity of thalassemia intermedia patients based on phenotype scoring system. Materials and Methods: Forty one cases of thalassemia intermedia were included in this study. Based on scoring system developed by Sripichai et al., these patients were scored for clinical severity and grouped into mild, moderate and severe category. HPLC and mutation analysis were used to classify the genotype of the patients. The clinical and haematological parameters were compared in the three groups. Results: Genotypically the patients were

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 categorized as b0 homozygotes (n = 4), b+ heterozygotes (n = 5), E-b Thalassemia (n = 16), sickle b thalassemia (n = 15) and Hb D-b thalassemia (n = 1). Phenotypically 16 patients had mild disease whereas 8 had severe disease. E-b thalassemia patients were equally distributed into mild, moderate and severe category. Most of the sickle-b thalassemia cases showed mild to moderate clinical severity while majority of the b0 and b+ thalassemia intermedia cases had moderate clinical severity. Conclusion: The scoring system classifies the severity and is useful for semiquantification of the phenotypes of thalassemia intermedia patients. Thus it can be used in planning, management, follow up and adaptation to newer treatment strategies.

Abstract No. 255 Results of Prenatal Diagnosis of Thalassemia and Haemoglobinopathies in 190 Cases: A Single Centre Study from West Bengal Soujatya Dhar, Sila Chakrabarti, Uttam Nath, Siddhartha Sankar Roy, Sambit Samanta, Arnab Chattopadhyay, Prantar Chakrabarti, Utpal Chaudhuri Institute of Haematology & Transfusion Medicine, Medical College, Kolkata Summary: The goal of the study was to identify the presence of thalassemia mutations for prenatal diagnosis in pregnant women attending our centre in West Bengal, so that the birth of children with thalassemia and haemoglobinopathies could be prevented. Introduction: Hb E beta thalassemia is the most common thalassemia in West Bengal, followed by homozygous beta thalassemia and some cases of compound heterozygosity for beta thalassemia and other hemoglobin abnormalities like HbS, Hb D, Hb Lapore, Delta Beta/ HPFH and so on. The best form of management of these disorders is prevention by premarital screening and in some cases by prenatal diagnosis. Materials and Methods: Most of the couples had one or two affected children and mostly came from rural areas with poor economic status. Characterization of Beta thalassemia and hemoglobin E, S/D and delta beta/HPFH mutations were done by ARMS— PCR and RFLP technique after analyzing HPLC pattern. Prenatal diagnosis was done by chorionic villus biopsy at 9–12 weeks. VNTR analysis was done to rule out maternal contamination in isolated CVS sample. Results: In the present study 295 couples were offered prenatal diagnosis after screening for thalassemia and haemoglobinopathies by HPLC. CVS was performed in 190 of these cases after appropriate counseling of the carrier couples. Out of these cases, homozygosity/double heterozygosity was detected in 47 pregnancies, which were successfully terminated. In our study IVS 1-5(G-C) mutation was the most common among beta thalassemia carriers. Conclusion: Facilities for Prenatal diagnosis by genetic studies is an important tool for the prevention of thalassemias and haemoglobinopathies.

Abstract No. 256 To Evaluate the Association of HFE Gene Mutations in BetaThalassemia Major Patients with Iron Overload—A Pilot Study Amit Kumar Tiwari, Satendra Sharma, Sunil Gomber, Usha Rusia Depatment of Pathology & Pediatrics, UCMS and GTB Hospital, Delhi Summary: DNA analysis for HFE gene mutation in beta-thalassemia major patient to investigate their influence on iron overload.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Introduction: Thalassemic children are dependent on frequent blood transfusion and develop iron overload. HFE gene mutation is characterized by increase intestinal absorption of iron and progressive iron overload. HFE gene associated mutations are—C282Y, H63D, and S65C. Although iron overload is often attributed to frequent blood transfusion, coinheritance of HFE gene mutations may exaggerate iron overload. Most of the studies conducted earlier are related to b-thalassemia trait and intermedia. But, b-thalassemia major was virtually unexplored. The present study is designed to determine the association of HFE gene mutation in b-thalassemia major patients and investigate their influence on iron overload. Materials and Methods: A descriptive observational, pilot study was conducted. Which comprised of 50 diagnosed beta-thalassemia major cases with iron overload. Detailed clinical history including history of any recent infection was taken. Complete blood count, serum iron levels, TIBC, percent transferrin saturation and serum ferritin levels were performed. DNA analysis by PCR-RFLP method for HFE gene mutations was done. Results: Out of three HFE gene mutations only H63D mutation was detected in 8 out of 50 cases. There was no significant difference between serum ferritin values (a marker of iron overload) with and without mutation (mean ferritin level 4,641 ± 2,166 and 4,170 ± 2,461 ng/ml respectively). Conclusion: To conclude, observed frequency of HFE mutations of C282Y, H63D and S65C were 0, 16, 0 % respectively. The coinheritance of HFE gene mutations does not exaggerate iron overload in b-thalassemia major patients.

Abstract No. 257 Comparative Efficacy and Safety of Deferiprone, Deferasirox and Their Combination in Thalassemics Prachi Jain, Sunil Gomber, Manish Narang, Satender Sharma Department of Pediatrics, UCMS & GTB Hospital, Delhi-110095 Summary: Using combination of oral iron chelators in iron overloaded multi-transfused thalassemics to establish the efficacy and safety of oral chelation combination versus the monotherapy. Introduction: Iron overload causes significant complications in transfusion dependent thalassemia major patients. Three well known iron chelators (one parenteral: Desferrioxamine and two oral: Deferiprone and Deferasirox) have been used to combat some of these complications. Combination and effective use of parenteral iron chelator with either of the two oral chelators is practised, but without alleviating the pain of injections. Combining the two oral iron chelators would overcome this pain and may prove to be effective therapy in multi-transfused thalassemics. No such combination has been tried so far in the Indian population and in pediatric age group. Materials and Methods: A Prospective Comparative Interventional Trial was carried out over 15 months duration. A total of 49 multi-transfused thalassemic patients with mean age 11.6 ± 6.21 years were enrolled and divided in three groups. They were chelated with either Deferiprone alone (75 mg/kg/day in 3 divided doses) or Deferasirox alone (30 mg/kg/ day single dose) or their daily combination (same dose as monotherapy) for 12 months duration. Serum Ferritin, MRI T2* of Liver and Heart and 24-h urinary iron excretion were estimated at various intervals in all three groups. Adverse effects were monitored both clinically and biochemically. Results: We found that the combination therapy was most efficacious in causing fall in serum ferritin levels (P = 0.035) and produced maximum negative iron balance in the body by maximally increasing the iron excretion in urine (P = 0.002). Results of MRI T2* were equivocal. No increased incidence of adverse reactions was noticed in the monotherapy or the combination group. Conclusion: Oral combination therapy of Deferiprone and Deferasirox is the most efficacious followed by

365 Deferasirox monotherapy and then Deferiprone monotherapy. The combination therapy is safe with no serious adverse effects in multitransfused thalassemics.

Abstract No. 258 Combination Chelation Therapy for Transfusional Haemosiderosis in Thalassaemia Major G Vimal Kumar2, M Deenadayalan2, Hemalatha2, Karuna Sri2, Venkatadesikalu1, Mythili1, Saarulatha1, Revathi Raj1 VHS Thalassaemia Centre, Chennai1, Apollo Speciality Hospital, Chennai2 Aims: To assess efficacy of combination chelation therapy for transfusional haemosiderosis in thalassaemia major. Materials and Methods: We have studied iron chelation in patients with transfusion dependent thalassaemia major for 10 years at our centre. Lack of response to a single iron chelator has been a major challenge in 24 out of our 220 patients. We have collected comprehensive information since 2009 in these patients regarding the use of iron chelating drug combinations to reduce high levels of serum ferritin. The study consisted of 11 male and 23 female patients between the ages of 6–30 years who were initially started on the oral chelator deferasirox. A therapeutic dose of 40 mg/kg/day of deferasirox was given for at least 3 months. If serum ferritin levels continued to increase despite adequate compliance, a second chelator was offered. Subcutaneous administration of desferrioxamine (DFO) along with the oral chelator was commenced in 18 patients. All families belonged to poor socioeconomic background and could not afford an infusion pump. Desferral was administered at a dose of 500 mg in two divided doses as a subcutaneous injection 3 to 5 nights a week depending on the serum ferritin. Results: The average ferritin level was 4562 ng/ml at the start and reduced to 2,708 ng/ml after 6 months therapy. All our patients showed significant decrease, possibly due to a synergistic and additive effect. There were no adverse effects noted. Four families had initially refused injectable iron chelators and were offered deferasirox and deferiprone at 50 mg/kg/day as a combination. All of these patients showed a serum ferritin level drop by 1,000 ng/ml over a 4 month period. Deferiprone had to be withdrawn in 1 child due to arthralgia. Two children had intolerance to deferasirox with high ferritin. They have been treated with deferiprone with subcutaneous desferral schedule and have also shown a 1,000 ng/ml reduction in serum ferritin over a 6 month period. There were no major gastrointestinal, liver or renal toxicity with either of the combinations during our 3 year study period. Conclusion: We conclude that deferasirox can be safely combined with desferioxamine or deferiprone in patients who fail to respond to the single agent and subcutaneous desferrioxamine can be used as an adjuvant without an infusion pump to help reduce serum ferritin in developing countries.

Abstract No. 259 Use of Hydroxyurea and Wheatgrass Therapy in Reducing Transfusion Frequency in Beta-Thalassaemia Patients V Ramanan, K Kelkar Yashoda Hematology Clinic, Pune Summary: The use of hydroxyurea and wheatgrass have been associated with clinical and hematologic improvements in thalassaemia intermedia patients, but responses in patients with thalassemia major are controversial. The aim of this study was to find out the effect of hydroxyurea and wheatgrass tablets in reducing the blood transfusion

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366 frequency in Beta thalassaemia major and intermedia patients. Introduction: Hydroxyurea could benefit patients with B-thalassaemia, by increasing the synthesis of gamma chains which would neutralize the excess alpha chains and partially correct ineffective erythropoiesis. Wheatgrass juice has been known to reduce blood transfusion frequency in Beta-thalassaemia major patients. Materials and Methods: The study was carried out between January 2008 and June 2013 on 74 patients diagnosed as Beta-thalassaemia by HPLC. Results: The study had 34 males and 40 females with age group ranging from 1 to 35 years. The number of patients who received 2 blood transfusions/month, 1 transfusion per month and less than 1 transfusion per month were 16, 42 and 14 respectively. 2 of the 74 patients diagnosed as thalassaemia homozygous at the age of 6 and 7 months were started immediately on wheatgrass tablets and never required transfusion. Frequency of blood transfusion after wheatgrass and hydroxyurea decreased in a significant percentage of patients. Conclusion: Wheat grass and hyroxyurea have the potential to increase the Hb levels and decrease blood transfusions in BetaThalassaemia patients.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 with various complications arising out of iron overload and transfusion transmitted infections. With increasing lifespan of these patients due to advances in the medical management, quality of life has become an important parameter reflecting the efficacy of health care system. Materials and Methods: 60 thalassemia patients between 10–18 years of age regularly attending Thalassemia Day Care Centre were included in the study. Quality of life was assessed by using WHOQOL-BREF scoring. The QOL was assessed on four parameters. independent variables evaluated included clinical and sociodemographic characteristics. Results: The mean overall score was found to be 119.37 with scores in physical, psychological, social and environmental domains being 135.7, 101.6, 59.23 and 134.89 respectively. Higher socioeconomic status was related to higher scores. Presence of co-morbidities like TTI’S & endocrinopathy was associated with lower overall score. Conclusion: Adolescent thalassemics in our unit have reasonable quality of life. An association is found between co-morbidities and quality of life.

Abstract No. 262 Abstract No. 260 Safety and Efficacy of Interferon and Ribavirin Therapy in Thalassemia Patients with Chronic Hepatitis C Shruti Kakkar, PC Sobti, A Sood, A Markan Dayanand Medical College, Ludhiana Summary: Thalassemia patients with chronic hepatitis C were treated with Interferon and Ribavirin combination to assess the safety and efficacy of the combined regimen. Introduction: Transfusion transmitted infections are the most dreaded complications of regular blood transfusion. Chronic HCV infection can lead to long term morbidities like cirrhosis and HCC. Materials and Methods: Fourteen thalassemia major patients with chronic hepatitis C (genotype 3) who gave informed consent for the treatment and were negative for HBsAg and HIV were included in the study. Patients were treated with Pegylated interferon a 2a and ribavirin for 24 weeks. Results: The mean age of the patients was 15.14 (7–28) years. Ten (71.4 %) were male and 4(28.6 %) were female. Twelve (85 %) patients achieved rapid virological response. All patients achieved complete early and end of treatment response. SVR was attained in 7/13 (53.8 %) patients. Response rate in patients being treated for the first time was 60 % (3/ 5) as compared to 50 % (4/8) in non responders. Flu like symptoms is the most common adverse effect seen. Two patients required growth factor support for neutropenia. Eight (57 %) out of 14 patients developed thrombocytopenia (platelet count \100,000/mm3). Transfusion requirement increased by 35 % during the treatment. Conclusion: The efficacy of interferon and ribavirin combination in achieving SVR in thalassemia patients with chronic hepatitis C (genotype 3) is lower than that in general population.

Assessment of Fertility in Males with Thalassemia Major Shruti Kakkar, PC Sobti, P Dean Dayanand Medical College, Ludhiana Summary: To assess the fertility status of males above 18 years of age attending thalassemia clinic of a tertiary care hospital. Introduction: With an increasing number of thalassemia patients reaching adulthood due to advances in medical management, fertility issues have gained prime importance. Materials and Methods: Consenting males [18 years of age were included in the study. Weight, height and sexual maturation stage was recorded. Full blood count, LFT and RFT were done to rule out any underlying renal or hepatic disease. Baseline LH, FSH, testosterone and thyroid levels were obtained. Urine and semen samples were analyzed. Results: A total of 26 patients were included in the study. Eleven (42 %) had height \5th centile. Seventy seven percent (n = 20) patients were in SMR stage 4, 11 % (n = 3) were in stage 3 and 4 % (n = 1). Seven percent (n = 2) patients had hypothyroidism and diabetes mellitus each. Normal LH and FSH values were found in 90 and 95 % patients respectively. Low testosterone levels were detected in 11 % (n = 3) patients. Azoospermia was present in 19 % (n = 5) of our patients and oligozoospermia in 15 % (n = 4). Four patients (26 %) had sperm motility \50 % (asthenozoospermia). Transrectal ultrasound revealed low prostrate volume in 4 patients. Another patient who had azoospermia underwent a testicular biopsy showing spermatogenic cells in all stages of maturation including mature spermatozoa. Conclusion: Fifty percent patients with thalassemia major can preserve fertility if adequately managed.

Abstract No. 263 Abstract No. 261 Quality of Life Among Adolescent Thalassemics Shruti Kakkar, Praveen C Sobti, P Dewan Dayanand Medical College, Ludhiana Summary: this study is to assess the quality of life amongst 60 patients between 10–18 years being managed at Thalassemia Day Care Centre, Dayanand medical College and Hospital Ludhiana. Introduction: Thalassemia major is an inherited disorder requiring life-long blood transfusion and chelation therapy. It is also associated

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Assessment of Fertility in Females with Thalassemia Major Shruti Kakkar, PC Sobti, Raina Nagpal Dayanand Medical College, Ludhiana Summary: Hypogonadiam secondary to iron overload ia common in thalassemia. The study aims to assess the fertility status of females with thalassemia major. Introduction: Infertility can occur in females with thalassemia due to iron deposition in gonadotropin secreting cells or damage to ovaries. Materials and Methods: Ten patients [16 years old attending our thalassemia unit were included in the study. Age of onset of thelarche and/or menarche and menstrual

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 history was recorded. Weight, height and sexual maturity stage was documented. Baseline full blood counts, ESR, liver and renal function tests were done to rule out any secondary cause of infertility. Serum levels of LH, FSH, TSH and Prolactin were tested. Ultrasound pelvis was done to assess the status of female genital tract and ovulatory function. Results: Ten patients [16 years old attending our thalassemia unit were included in the study. Age of onset of thelarche and/ or menarche and menstrual history was recorded. Weight, height and sexual maturity stage was documented. Baseline full blood counts, ESR, liver and renal function tests were done to rule out any secondary cause of infertility. Serum levels of LH, FSH, TSH and Prolactin were tested. Ultrasound pelvis was done to assess the status of female genital tract and ovulatory function. Results: Mean age of patients was 21.8 years (16–32 years). Weight and height \3rd centile was present in 50 % patients. Thelarche occurred spontaneously in 7 patients and was induced in 3. Mean age of onset of thelarche was 14.5 years. Eight (80 %) patients had attained menarche at a mean age of 16.1 years. Menstrual irregularities and oligomenorrhea was present in 50 and 37 % patients respectively. Low LH and FSH values were present in one patient each with high serum prolactin in 2 patients. Ultrasound abdomen revealed hypoplastic uterus in a patient who did not attain menarche and polycystic ovarian disease in a patient with history of menstrual irregularity. Ovulation studies showed delayed ovulation in 2 patients. Conclusion: Females with thalassemia major attain puberty later than their normal counterparts and gonadal dysfunction is common.

Abstract No. 264 Autoimmune Hemolytic Anemia in Patients with Thalassemia Major Dipanjan Haldar, Suvro Dutta, SS Roy, S Samanta, UK Nath, P Chakrabarti Institute of Hematology and Transfusion Medicine, Medical College, Kolkata Summary: Hemolysis is a common feature in patients with thalassemia major. As a result, autoimmune hemolytic anemia complicating thalassemia is easily overlooked. We describe the clinical features and management of 5 patients with thalassemia major and autoimmune hemolytic anemia (AIHA). Introduction: Patients with Thalassemia major require long-term blood transfusions and chelation therapy. Therefore, these patients are at high risk of developing antired blood cell (RBC) antibodies due to multiple allogeneic blood transfusions. Autoimmune hemolytic anemia (AIHA) is characterized by production of autoantibodies against the patient’s own RBCs leading to increased hemolysis. Therefore, AIHA developing in addition to thalassemia, already a hemolytic disease, is easily overlooked. Materials and Methods: A total of 5 cases of AIHA complicating Thalassemia Major were recruited in our study. 4 patients each were of Hb Eb Thalassemia and 1 of b Thalassemia Major. The diagnosis of thalassemia major and AIHA was based on HPLC analysis (and mutation studies where HPLC was inconclusive) and Coombs’ tests, respectively. The clinical and transfusion records were evaluated for all patients. Results: All these patients had received inadequate transfusion and irregular iron chelation therapy. One of these patients was Anti HCV positive. All the patients had Ig G subtype of auto antibody. Anti C 3 was also positive in one of these patients. In addition, two of them showed positive in the indirect antiglobulin tests, and the alloantibodies were identified as anti-Rh c & E. All of them were started at Prednisone 1 mg/kg/day. Four patients responded to steroids while one required splenectomy for AIHA. Conclusion: AIHA should be considered as one of the possibilities in Thalassemic patients when they present with increased

367 transfusion requirements. A proper diagnostic algorithm and therapeutic approach should be followed in such cases.

Abstract No. 265 High Incidence of Non-deletional Alpha Thalassemia in the MidEast Poses Diagnostic Problem Even in Well Equipped Laboratories Amar Dasgupta, Sandip Dey, Hemraj Singh, Shailendra Rathod and Sam Daniel SRL Diagnostics, Dubai, United Arab Emirates Summary: Our study of suspected cases of hemoglobinopathies in the local UAE population reveled the possibility of a high incidence of non-deletional alpha thalassemia in addition to deletional alpha thalassemia posing greater challenges in the diagnosis of microcytic anemia in this population. Introduction: The high incidence of alpha thalassemia in the Arab population adds to the diagnostic challenges in microcytic anemia in this region. Here we describe the spectrum of lesions in suspected cases of hemoglobinopathies referred to our laboratory. Materials and Methods: One hundred nine suspected cases of thalassemia were examined using HPLC (Bio Rad Variant II) and molecular methods. A PCR amplification of the alpha globin genes was performed using specific primers for detection of 3.7 and 4.2 bp deletions and for SEA, FIL and THAI deletions. Results: Sixty-two (*57 %) cases had molecular lesions consistent with alpha thalassemia. Of these 36 and 23 cases respectively showed heterozygous and homozygous deletions for 3.7 bp; 1 case each had homozygous and heterozygous 4.2 bp deletions, and HbH disease on account of 3.7 kb and SEA deletions. Two cases each of HbS trait with 3.7 bp heterozygous and homozygous deletions and of HbS disease with 3.7 bp heterozygous deletion were seen. A small numbers of beta-thalassemia trait (3 cases), HbS trait (2 cases), and HbD trait, HbE trait and double heterozygous for HbD-beta thalassemia (1 case each) were also encountered. In the remaining 39 cases no thalassemia or structural Hb variant was detected although the red cell indices suggested a thalassemic phenotype-raising the possibility of non-deletional alpha thalassemia in these cases. Conclusion: Alpha thalassemia seems to be significantly more common than beta thalassemia in the UAE patients. The large number of cases with potential non-deletional alpha thalassemia in the Arab population as shown above calls for additional investigative measures for diagnosis.

Abstract No. 266 South East Asian (–SEA) Deletion as a Common a Thalassemia Mutation in Kerala S Eswari, K Neelagandan, Sankari Devi, Auro Viswabandya, Biju George, Vikram Mathews, Alok Srivastava, Eunice S Edison, RV Shaji Department of Haematology, Christian Medical College, Vellore a-Thalassemia refers to a group of hereditary anaemias caused by absence or decreased production of the a-chains of haemoglobin. Single and two gene deletions in a-globin gene cluster result in a relatively mild anemia whereas three gene deletions result in a moderate to severe disease (HbH disease) characterized by ineffective erythropoiesis and requirement for intermittent transfusions. Single gene deletions (-a3.7 and -a4.2) are the most common mutations for a-thalassemia in India. The two gene deletion, –SEA, is predominantly seen in South East Asia and the Middle East and the Mediterranean populations. A few cases with this mutation have been reported in

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Table 1 Hematological parameters of patients with alpha deletion (–SEA) UPN.NO Age/ Sex

Hb MCV MCH Ferritin HbH HbF HbA2 ALPHA (g/dL) (fl) (pg) (ng/ml) (%) (%)

G-254

4/F

8.10

55.50 15.50

81.70

*

0.60 1.20

-a3.7/–SEA

G-425

39/F

9.10

60.10 18.70

67.40

+

0.00 1.60

-a3.7/–SEA

G-427

39/F

61.60 18.70 112.50

+

0.00 1.40

-a3.7/–SEA

a-83

23/F

*

0.00 1.20

-a3.7/–SEA

H-112

33/M 12.50

56.90 17.70 192.40

*

0.00 1.80

-a3.7/–SEA

B-311

7/M

61.90

-

0.00 2.50

aa/–SEA

a-73

66 M 13.50

65.70 21.00 354.00

*

0.00 2.30

aa/–SEA

2012/22

39/F

67.80 21.60

0.00 2.30

aa/–SEA

9.20 *

10.60

11.00

*

*

*

*

*

*

*

* Not available

India. However, its clinical phenotype and ethnic origin in this population are not known. We describe the hematological and molecular characterization of a series of cases with the –SEA deletion in Indian population. Patients with suspected a-thalassemia based on the hematological parameters were included in the study. Haematological parameters (CBC) were measured on an automated cell counter (LH 750, BC, USA). Haemoglobin F and A2/E were quantified using an automated high performance liquid chromatography system (VARIANT, BIO-RAD, USA). Genomic DNA was isolated from peripheral blood leukocytes by standard protocols. The common alpha (a)-globin deletions (-a3.7, -a4.2, –SEA, –MED, –SA) were screened by multiplex PCR (Shaji et al. 2003). We screened 252 suspected cases of a-thalassemia from 1998 to 2012. The single gene deletion -a3.7 was the most common mutation in the patients included in the study. The other deletions identified in this group were a4.2, –SA and –MED and –SEA. The South East Asian deletion (–SEA) along with -a3.7 deletion was found in five cases of HbH disease. The compound heterozygous state of -a3.7/–SEA deletion resulted in moderate to severe anemia with microcytosis and very low HbA2 levels. This mutation was found in heterozygous state in three individuals. The presence of –SEA deletion was confirmed by sequencing of the PCR products produced by the primers that flank the breakpoints. The hematological parameters observed in all the cases with –SEA deletion are shown in Table 1. Interestingly, all the subjects were from Kerala. A haplotype was constructed to study the origin of this mutation in our population. Haplotype analysis revealed that this mutation was associated with haplotype A. This is the most common haplotype seen in many populations. Haplotype analysis is being carried out in controls and the data will be presented. The identification of this deletion (–SEA) in the Indian population and its high frequency in Kerala is an important finding that will aid in a-thalassemia screening programs in the country.

Abstract No. 267 Influence of Single Nucleotide Polymorphisms in the BCL11A Gene on HbF Levels and the Clinical Presentation of Sickle Cell Disease in Central India Dipti Upadhye, Dipty Jain, Anita Nadkarni, K Ghosh, Roshan Colah National Institute of Immunohematology (ICMR), KEM Hospital Campus, Parel, Mumbai 400012 Summary: Three SNPs in the BCL11A gene which may be responsible for variations in HbF levels and clinical severity in sickle

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cell disease were studied. Introduction: Sickle cell disease has an extremely variable clinical expression. High HbF levels are often associated with a milder clinical phenotype. Genetic variations at 3 loci, HBB cluster on chromosome 11, HBS1L-MYB on chromosome 6q and BCL11A on chromosome 2p influence HbF levels and disease severity in sickle cell disease (SCD). Xmn1 polymorphism (rs7482144) accounts for around 20 % of the variation in HbF levels. Previous studies in two independent SCD cohorts showed a strong association between variants in the BCL11A gene and HbF levels and the clinical severity. We undertook a preliminary study to evaluate the effect of three BCL11A polymorphisms on HbF levels and clinical severity in sickle cell disease. Materials and Methods: 43 SCD patients were studied. HbF levels were measured by CE-HPLC. Three polymorphisms in the BCL11A gene: rs4671393, rs11886868 and rs7557939 were genotyped by DNA sequencing. Results: Patients with the mutant allele GG and the AG allele in the rs 4671393 SNP showed lower HbF levels (22.0 ± 7.27 %) than the AA allele (28.8 %). The CC allele of the rs 11886868 SNP was associated with higher HbF levels (25.0 ± 7.69 %), than the CT allele (21.0 ± 7.98 %) and the TT allele (18.6 ± 5.93 %). Cases with the GG allele of the rs7557939 also showed higher HbF (24.0 ± 7.69 %) than the AG allele (21.0 ± 7.06 %) and the AA allele (19.0 ± 12.02 %). 60 % of the patients were classified as severe. The GG allele of the rs4671393 SNP was much higher in severe (84 %) vs. milder patients (41 %). Conclusion: Different alleles of the 3 SNPs in the BCL11A gene had variable HbF expression and the GG allele of the rs4671393 was more common in patients with severe disease.

Abstract No. 268 Sickle Cell Disease—An Unusual Presentation: A Case Report M Shah, R Godbole, P Nayar, R Manchanda Department of Hematology, K.E.M. Hospital, Pune Summary: Sickle hemoglobinopathies are hereditary disorders in which red cells contain HbS. Here is a case of sickle cell disease which presented for the first time at the age of 63 years. He presented with complaints of giddiness since two weeks with bluish discoloration of ring & little fingers, O/E there was no hepatosplenomegaly. Hemogram revealed leucocytosis with thrombocytosis. Myeloproliferative disease was suspected. Introduction: Sickle hemoglobinopathies are autosomal recessive hereditary diseases. The homozygous state (HbSS) is the most common form of sickle cell disease. Interaction of HbS with thalassaemia & certain variant hemoglobins like Hb C & Hb D potentiate sickling while, HbF has opposite effect & tend to diminish sickling. Materials and Methods: Detailed clinical history, Complete Hemogram, Peripheral blood smear study, USG Abdomen, FISH for bcr-abl, Bone marrow examination & HPLC was done. Results: Routine Hemogram revealed leucocytosis with shift to left & thrombocytosis. FISH for bcr-abl was negative. Bone marrow did not confirm myeloproliferation.PBS revealed many target cells, few Howelljolly bodies & occasional sickle shaped RBCs. So HPLC was done & it revealed HbS of 60 % & HbF of 40 %. Sickling test was positive. Diagnosis of Sickle cell disease with autosplenectomy causing Leucocytosis & thrombocytosis was offered. Conclusion: Sickle cell disease presenting at the age of 63 for the first time is rare. Because of its uniqueness regarding age at presentation, diagnostic dilemma with myeloproliferative disease & high levels of HbF and HbS.

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Abstract No. 269 A Study on Genotypic Characterization of Beta Thalassemia and the Causes of the Phenotypic Diversity of Hemoglobin E Beta Thalassemia in West Bengal Soujatya Dhar, Sila Chakrabarti, Uttam Nath, Siddhartha Sankar Roy, Sambit Samanta, Arnab Chattopadhyay, Prantar Chakrabarti, Utpal Chaudhuri Institute of Haematology & Transfusion Medicine, Medical College, Kolkata Summary: The goal of the study is to identify the Beta thalassemia mutations in West Bengal and to reduce the birth of thalassaemic child by prenatal diagnosis. And at the same time provide a hope to the unfortunate carrier couples with a previously affected thalassemic child to plan for another child free from the disease. Introduction: Beta thalassemia and Hb E Beta thalassemia is the most common haemoglobinopathy in West Bengal. Hb E beta thalassemia in compound heterozygous and Hb E and co-inheritance of beta thalassemia with HbS/D, delta beta/HPFH and in homozygous state results in severe anaemia and transfusion dependent survival in many cases. Materials and Methods: For characterization of Beta thalassemia and hemoglobin E, S/D and delta beta/HPFH mutations have done by CR and RFLP technique after analyzing HPLC pattern. ARMS Prenatal diagnosis is done by chorionic villous biopsy. VNTR analysis is done to rule out maternal contamination in isolated CVS sample. Results: In the present study of prenatal diagnosis 295 couples were counseled and 190 were offered CVS studies were carried out of these couples. Most of the couple have one or two affected child or death of affected child and mostly came from rural areas and have poor economic status. In our study IVS 1-5(G-C) mutation is most common among beta thalassemia carriers. We have prevented the birth of 47 affected child by CVS study. Conclusion: The birth of affected child with beta thalassemia major or E beta thalassemia may be prevented with proper awareness campaign and the facility to carry out prenatal diagnosis.

Abstract No. 270 Clinical, Hematological and Molecular Profiling of Homozygous HbE in Indian Population Divya Jayashree, Divya Jayasree, RV Shaji, Auro Viswabandya, Biju George, Vikram Mathews, Alok Srivastava, Eunice S Edison Department of Haematology, Christian Medical College, Vellore Introduction: Hemoglobin (HbE) is the most common abnormal haemoglobin (Hb) variant in the world. In India, HbE has been observed with a prevalence of 7–50 % in north eastern region and 1–2 % in West Bengal. Homozygous state of HbE shows a mild thalassemic feature as a result of reduced b-globin synthesis and present with mild anemia. Even though it is considered as a benign disease, the patients with this disease show significant heterogeneity in their phenotypes. There are no comprehensive reports describing the haematological, biochemical and genetic parameters in homozygous HbE from India. So we aimed to study the clinical features of patients with HbE syndromes in India and comprehensively analyze the molecular and genetic factors in this cohort. A total of 61 patients were included in this study. Haematological parameters were measured on an automated cell counter. Haemoglobin F and A2/E were quantitated using an automated HPLC system. Genomic DNA was isolated from peripheral blood leukocytes by standard protocols. The homozygosity of codon 26(G-A) (bE mutation) was confirmed by reverse dot blot (RDB). The common alpha (a)-globin deletions

369 (-a3.7,-a4.2, –SEA, –MED, –SA) were screened by multiplex PCR (Shaji et al.). The b globin gene cluster haplotype consisting of 50 eHincII, 50 GcXmnI, GcHindIII, AcHindIII, 50 wbHincII, 30 wbHincII, AvaII in b and 30 bHinfI sites and association of genetic variants in BCL11A (rs11886868) and HBSIL-MYB (rs4895441) were screened by PCRRFLP. Analysis of polymorphic sequence in (AT)X(T)Y motif and UGT1A1 gene promoter were analyzed by PCR-Genescan method. Data analysis was performed by SPSS software. Sixty one patients were confirmed to be homozygous HbE by phenotype and molecular analysis. Majority (63 %) of the patients were from north eastern parts (West Bengal, Assam & Meghalaya) of the country. The median age was 35 years (3–62 years). On physical examination 35 % patients presented with hepatosplenomegaly and 14 % with cholelithiasis. One of the common presenting symptoms among patients was jaundice. The mean Hb, Hb F and Hb A2/E levels in this group were 9.6 ± 2.57 g/dl (7–12 g/dl) 3.5 ± 2.4 % (1–14.3) and 87.0 ± 4.65 % (76–97) respectively. In the literature, homozygous HbE is presented as a clinically benign disorder with near normal Hb levels (11.4 ± 1.8 g/dl), and absence of hepato-splenomegaly (Fuchaeron et al. 2000). In India the severity of homozygous HbE ranges from mild to moderate anemia with hepato-splenomegaly and jaundice. Serum total bilirubin values in these patients ranged from 0.4–14.2 mg/dl (mean 2.35 mg/dl). Single a globin gene deletion (a3.7/aa) was found in one patient. We were able to construct a haplotype successfully in 84 chromosomes of HbE. Most of the bE genes in the Indian population were found to be associated with two haplotypes, -??-???- (haplotype A-62.8 %) and ??----?(haplotype B-31.8 %) which is a novel haplotype found in our study. Among these, haplotype A has already been reported to be associated with bE gene in Southeast Asian population. No significant difference in haematological parameters [Hb (p = 0.91) HbF (p = 0.717) HbA2/ E (p = 0.191)] was found between Haplotype A and B. The allelic frequencies of different SNPs modulating HbF levels are tabulated (Table 1). The genotype CC in BCL11A SNP rs11886868 was strongly associated with relative or absolute HbF when compared to TT genotype (p = 0.04) (Fig. 1). The median HbF % values of CC vs. TT genotype were 8.1 (5.5–13.6) and 3.5 (1.5–8.3). No significant association was observed between SNPs rs4895441 located within the HBS1LMYB intergenic region and rs7482144 in HBG with the level of HbF. Hyperbilirubinema was observed in 27 cases. Since hemolysis is not an expected feature of Homozygous HbE, we screened for the repeat polymorphism in the UGTA1 gene. In this cohort, 50 % were normal (TA 6/6), 15 % (TA 7/7) and 35 % heterozygous for (TA)6/7 of UGT1A1 polymorphism. This suggests that the variant TA7 is associated with hyperbilirubinemia in homozygous HbE patients. The polymorphic repeat sequence in the silencer region of the b globin, AT9T5 was found in a strong linkage with bE mutation.

Table 1 Allelic frequencies of SNPs at the BCL11A, HBS1L-MYB and HBG2 loci Gene

SNP

BCL11A

rs11886868 C/T

HBS1LMYB

Alleles Allele frequency (present study) homozygous HbE 0.30 (C)

Allele frequency HbE-BETA (unpublished data) 0.61 (C)

0.70 (T)

0.39 (T) 0.91 (A)

rs4895441

A/G

0.88 (A) 0.12 (G)

0.09 (G)

HBG2 rs7482144 (-158 XmnI)

G/A

0.13 (G)

0.55 (G)

0.87 (A)

0.45 (A)

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 15

HbF Levels

P=0.04 10

5

TT

C

C

0

BCL11A(rs11886868)

Fig. 1 Association between BCL11A (rs11886868) genotypes and proportion of HbF This is the first extensive study carried out in India to show the clinical, haematological and interaction of various genetic factors in homozygous HbE.

Abstract No. 271 Incidental Finding of Hb Q-India Trait During Thalassemia Screening

Summary: Thalassemia is a quantitative genetic disorder resulting due to mutations in the globin genes of hemoglobin. Prevalence of thalassemia and qualitative hemoglobinopathies like HbD-Punjab and HbQ-India together in a heterozygous state is very rare and hence clinically significant. Introduction: HbQ-India and HbD-Punjab are hemoglobinopathies which are inherited as an autosomal dominant trait. Co-inheritance of thalassemia trait along with HbQ and HbD can result in clinically significant anemia and their screening in certain regional groups and community is extremely important. Materials and Methods: During routine beta-thalassemia screening in our lab we detected a Sindhi family with HbD-Punjab as well as HbQ-India. The EDTA-anticoagulant blood samples of the family members were screened for thalassemia test using TOSHO G8 HPLC analyzer while complete blood count was performed using SYSMEX XN-1000 automated hematology analyzer. Results: All the four members of the family were analyzed for hemoglobin variant analysis. The father was detected with HbD-Punjab trait. The mother was found to be compound heterozygosity for HbQ-India and beta-thalassemia trait with microcytic anemia. Their elder daughter has been reported to be again having compound heterozygosity for HbQ-India and beta-thalassemia trait with microcytic indices while the younger daughter was detected with the HbD-Punjab trait. Conclusion: Our case report to the best of our knowledge is one of the first few to document and report a familial case of compound heterozygosity for beta-thalassemia with HbQ-India and HbD-Punjab trait in a single Sindhi family. Such data generated throws light on the significant prevalence of different hemoglobinopathies in regional groups and community especially in the central region of the country.

Suhas Sakhare, Sandhya Iyer, Caesar Sengupta, A Velumani Thyrocare Technologies Limited, Turbhe, Navi Mumbai-400703 Summary: Hemoglobinopathies are a group of disorders affecting the structure as well as function of hemoglobin. This can result in possible serious health complications and their timely clinical diagnosis is extremely important. Introduction: Hb Q-India is an extremely rare hemoglobinopathy and is an autosomal dominant disorder. Its clinical detection is extremely significant as this in conjunction with other hemoglobinopathy can cause serious health complications. Materials and Methods: Our lab conducted massive thalassemia screening camps in two places viz. Bhusawal and Wadsa in central India. A total of 647 samples were collected and screened for betathalassemia. Screening for thalassemia was done using HPLC technology using G8 Tosoh analyzer, while complete blood count data was generated using SYSMEX XN-1000 automated hematology analyzer. Results: Of the 647 samples screened for thalassemia, we detected ten samples with the Hb Q-India trait. From the complete blood count report, we detected two subjects to be suffering from microcytic anemia while the other eight were asymptomatic. Conclusion: The finding of Hb Q-India trait is very rare and significant clinically. Previous reports of finding of this trait had emphasized the distribution pattern of this trait to be specifically concentrated in the central and northern part of the country. The screening camps carried by our lab were also done in the central region of the country. Our finding of this trait in these camps conducted also reiterates the regional distribution fact.

Abstract No. 272 A Rare Case Report of Inheritance of HbD-Punjab and HbQIndia in a Sindhi Family During Routine Beta Thalassemia Screening Suhas Sakhare, Sandhya Iyer, Caesar Sengupta, A Velumani Thyrocare Technologies Limited, Turbhe, Navi Mumbai-400703

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Bone Marrow Morphology Abstract No. 273 Assessment of Iron Stores in Bone Marrow Using an Intensive Grading Method Purnima S Rao, Binit Khandelia Kasturba Medical College—Mangalore, Manipal University Summary: A total of 1,360 bone marrow aspirates were received in the KMC Hospital Laboratory during the study period, out of which 97 cases met the inclusion criteria defined for the study. 22 of these had to be excluded. Age, gender distribution, hemoglobin levels, RBC indices, peripheral blood smear, bone marrow findings and diagnosis, and marrow iron stores were studied in the remaining 75 cases. The marrow iron findings were correlated with serum ferritin levels, CRP and ESR wherever available. Introduction: This retrospective and prospective study was undertaken for a period of two and a half years between January 2010 to June 2012 at the Haematology Unit of the Department of Pathology, Kasturba Medical College—Mangalore. Causes of microcytic anemia include iron deficiency anemia (IDA), thalassemia, anemia of chronic disease (ACD) and rarely sideroblastic anemia. (2) Iron deficiency is the most common cause of anemia, and serum ferritin levels help us to confirm the diagnosis. (3) The gold standard for diagnosis for IDA is demonstration of absent iron stores on Perls’ Prussian blue stain in bone marrow aspirate (BMA) smears. (4) The bone marrow iron stores are graded according to Gale’s grading method as grade 0 to 6?. (7) A new intensive grading method analyzes the fragment iron, macrophage iron, erythroblast iron and grades the iron stores as normal, functional iron deficiency and/or storage iron deficiency. (8) The aim of the study is to assess the iron stores in the bone marrow using an intensive grading method in cases with microcytic anemia for which bone marrow aspiration was done. The objectives of the study are: (1) To compare the Gale’s grading

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 method with the intensive grading method in diagnosis of IDA and ACD. (2) To correlate the BMA iron stores with serum ferritin levels. (3) To correlate the C-reactive protein (CRP) levels and ESR with bone marrow findings. Materials and Methods: The present study has been undertaken to assess the iron stores in the marrow using the intensive grading method in cases of microcytic anemias for which marrow aspiration was done and compare it with Gale’s grading method, thereby providing a better assessment of marrow iron, both storage and functional. Bone marrow samples of those patients were taken for study who met the following criteria: Hemoglobin \13 g/dl for males and \12 g/dl for females, MCV \80 fL, Peripheral Blood Smear (PBS) picture of Microcytic Hypochromic Anemia Exclusion Criteria were: Patients presenting with leukemia, plasma cell dyscrasias, hemolytic anemias and patients on chemotherapy. The following parameters were studied on the blood samples: (1) Hemoglobin, (2) RBC indices (MCV, MCH, MCHC, RDW-CV). The blood samples were collected in EDTA and fed to the Sysmex XT 1800i automated cell counter (January 2010–October 2011) and Beckmann Coulter LH 780 automated cell counter (November 2011– June 2012) The bone marrow aspirates were collected in EDTA anticoagulant. Within 2 h of aspiration, the aspirate material was poured onto a watch glass and the marrow particles were picked up by a fine forceps and placed on a clean slide. A second slide was then placed perpendicular to the first and then slides were quickly pulled apart along the long axis. Bone marrow smears were air dried and few of them were stained with standard Leishman stain. The unstained smears were analyzed for presence of marrow particles. Only those smears were taken which had a minimum of seven marrow particles were stained with Perls Prussian Blue stain. In this study, Nuclear Fast Red stain was used as a counter stain. Results: A total of 1,360 bone marrow aspirates were received in the KMC Hospital Laboratory during the study period, out of which 97 cases met the inclusion criteria defined for the study. However 15 cases yielded aparticulate matter and hence were excluded from the study. Seven cases did not have enough particles to analyze iron stores and hence were excluded. Age, gender distribution, hemoglobin levels, RBC indices, peripheral blood smear, bone marrow findings and diagnosis, and marrow iron stores were studied in the remaining 75 cases. The marrow iron findings were correlated with serum ferritin levels, CRP and ESR wherever available. Microcytic anemia was encountered in a wide range of age groups. The fifth decade was the age group with the maximum number of cases of microcytic anemia. Minimum number of cases was seen in the eighth decade and above. Mean age of presentation for microcytic anemia in bone marrow aspirates was 38.7 years. Microcytic anemia was observed more in males than in females with a Male to female ratio of 1.27:1. Microcytic anemia was seen more commonly in males than in females in all age groups except for the second and third decade. Iron deficiency anemia was found to be the most common cause of microcytic anemia, followed by anemia of chronic disease. Iron deficiency anemia was observed in 46 cases, with mean age—35.8 years. The gender distribution M:F ratio of 1.7:1. Males predominated in each age group except the second decade. Among the females, most cases of IDA were found in the reproductive age group (11 cases, 15–45 years). Among the males, fifth decade was the most common age group with IDA. Overall, maximum cases of IDA were seen in the fifth decade. Anemia of chronic disease was observed in 29 cases, with mean age—43.38 years. The gender distribution was almost equal. Overall, maximum cases of ACD were seen in the sixth decade. The hemoglobin levels ranged from 1.5 to 11.5 g/dl (mean 7.7) in all cases of microcytic anemia. In cases of IDA, the Hb levels ranged from 1.5 to 11.5 g/dl (mean 7.3). For ACD, the Hb levels ranged from 3 to 11.3 g/dl (mean 8.5). Based on Hb cutoff values, there were 3 cases of mild anemia, and 36 cases each of moderate and severe anemia. Most of the cases of IDA presented with severe anemia (27/46—58.6 %). However, most cases of ACD presented with moderate degree of anemia (18/

371 29—62.1 %). All the bone marrow aspirates with adequate marrow particles were analyzed for iron stores after staining with Perls Prussian Blue stain. BMA iron stain was analyzed by Gale’s grading method and the newer intensive grading method. Gale’s Grading Method: Out of the 75 cases of bone marrow aspirates, 46 cases (61.3 %) presented with absent iron stores (grade 0), and were classified as IDA. The rest of the cases had adequate to increased iron stores and were diagnosed as ACD. None of the cases presented with depleted iron stores or grade 1. Intensive Grading method: Functional iron stores/sideroblasts were graded as reduced or absent, macrophage iron was graded as present or absent and fragments/extracellular iron was graded as present or absent. All the cases of iron deficiency anemia had absent functional and storage iron. Among the cases of ACD, 37.9 % had absent functional iron, and the same number of cases had absent extracellular iron/fragments. Based on this grading system, all cases of IDA had both functional and storage iron deficiency. However, all cases of ACD had only functional iron deficiency, but had adequate macrophage iron and/or extracellular iron. This was statistically significant (p value \0.001). Conclusion: Under intensive grading method, the iron was graded as fragments, macrophage iron/reticulum cells, and sideroblast iron. Functional iron stores/sideroblasts were graded as reduced or absent, macrophage iron was graded as present or absent and fragments/extracellular iron was graded as present or absent. There were significant differences in the detection and correlation of various criteria in the diagnoses of anaemias between the traditional Gale’s Grading System and the new Intensive Grading System, that were compared and contrasted in this study.

Abstract No. 274 A Study of Bone Marrow Aspiration, Trephine Touches & Trephine Biopsy in the Diagnosis of Haematological Disorders Vijai Tilak, Soobasschan Bundhun, Mohan Kumar, M Rai, Vineeta Gupta Department of Pathology, Institute of Medical Sciences, B.H.U., Varanasi Summary: This prospective study comprises 70 cases in which bone marrow aspiration(BMA), imprint & biopsy were performed. Biopsy is the gold standard for the assessment of cellularity & is extremely useful in the assessment of architecture, necrosis, granuloma & myelofibrosis. BMA & Imprint are valuable in morphological assessment. The three are complementary. Introduction: BM examination forms the cornerstone of diagnosis & management in myriad of clinical conditions both haematological & non-haematological. This is a prospective study carried out in both the paediatric & adult patients presenting with haematological disorders to compare the relative strengths and weakness of the three techniques. Materials and Methods: BMA, Imprint & biopsy were undertaken from the posterior superior iliac spine, under aseptic condition. The biopsy was formalin fixed, decalcified & processed to get H & E & reticulin sections. The smears & imprints were stained with Leishman & perls stain. Cell density was graded as good (3?), fair (2?), poor (1?) & acellular (0). Results: Seventy cases were analysed. Using biopsy as the gold standard for assessing cellularity, there was very good correlation with cell density on imprint & evaluation of cellularity on BMA. In aplastic anemia absence of increase in reticulin on biopsy helped in ruling out MDS. In acute leukemia there was very good correlation between blast count evaluation on imprint & aspiration. Lymphoglandular bodies on imprint in a case of ALL provided a valuable aid in diagnosis. Biopsy is misleading in megaloblastic anemia but of tremendous value in myelofibrosis, granuloma & MDS. Conclusion: The three are complementary to each other. Undertaking

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372 them together significantly augments the chances of reaching a correct diagnosis.

Abstract No. 275 Comparative Study of Bone Marrow Aspiration and Trephine Biopsy in Pancytopenia Vandana Kansal, Mridu Manjari, Daljit Singh, Tejinder S Bhasin SGRDIMSR, Sri Amritsar Summary: The prospective study was done in 61 cases presenting with pancytopenia from period June 2011–May 2013 to find out the cause and compare bone marrow aspiration with bone marrow biopsy. The various diagnosis made were Megaloblastic anaemia (67.2 %), Leukemia (21.3 %), Aplastic anaemia (8.2 %) and Myelodysplastic syndrome (3.2 %). Bone marrow aspiration was conclusive in 55 cases and in rest bone marrow biopsy made the diagnosis. Introduction: Pancytopenia is a striking feature of many serious illnesses ranging from megaloblastic anaemia to fatal bone marrow aplasia and leukemia. The severity and underlying pathology determines the treatment and predicts the prognosis. Materials and Methods: A prospective study was done on 61 cases of pancytopenia in the Department of Pathology, SGRDIMSR Amritsar. After clinical evaluation bone marrow aspiration and biopsy were performed for correlation. Results: Out of total 61 cases the most common cause was Megaloblastic anaemia (67.2 %), Leukemia (21.3 %), Aplastic anaemia (8.2 %) and Myelodysplastic syndrome (3.2 %). Age of patients varied from 3 to 80 years with mean age of 41.5 years and Male:Female ratio 1.4:1. The most common complaint was generalised weakness with fever. Bone marrow aspiration was inconclusive in 06 cases whereas biopsy was conclusive in all the cases. Special staining and immunohistochemistry was undertaken in few cases. Conclusion: It was concluded that primary haematological investigations with bone marrow aspiration are helpful in making the diagnosis in majority of the cases but in some bone marrow biopsy is conclusive and can also be subjected to immunohistochemistry. Thus showing that bone marrow biopsy should be performed along with aspiration for planning management of patient.

Abstract No. 276 To Study Spectrum of Bone Marrow Morphological Changes in Chronic Liver Disease Patients Referred for Evaluation of Haematological Derangements Anshu Palta, Vidhi Gupta, Sanjay D’cruz Government Medical College and Hospital, Sec-32, Chandigarh Summary: The present study was undertaken: To study spectrum of morphological changes on Bone Marrow examination in Chronic Liver Disease patients.—To study the diagnostic utility of bone marrow examination in these patients. Introduction: Liver diseases are frequently associated with hematological abnormalities and cytopenias of diverse nature occur in these patients. Materials and Methods: This is a retrospective analysis of bone marrow findings in 27 cases of chronic liver disease referred for haematological derangements in the haematology section of department of pathology, GMCH-32B, Chandigarh over 9 years (June 2004–June 2013). Results: 27 cases of chronic liver disease were in the age range of 18 years to 65 years with a mean age of 45.5 years. The male to female ratio was 17.4:1. 14 cases (51.8 %) were of alcoholic liver disease, one had chronic HCV and one had chronic HBV infection.

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 The main indications of bone marrow examination in the current study was pancytopenia in 13 patients (48.2 %) while 12 patients (44.4 %) had bicytopenia, one patient presented with anaemia and one had leucocytosis. The value of haemoglobin ranged from 2.8–14.5 g % with a mean value of 9.27 g %. The TLC ranged from 1,500 to 32,000/ll. Platelet count ranged from 25,000 to 3.5 lakh/ll. 15 cases (55.5 %) had moderate thrombocytopenia while 6 cases each (22.2 %) had severe and mild thrombocytopenia. Bone marrow smears were hypocellular in 2 cases (7.4 %), hypercellular in 20 cases (74 %) and normocellular in 5 (18.6 %) cases. Eosinophilia was found in 21 cases (77.8 %). Iron stores were adequate in 17 (63 %) cases while 5 (18.5 %) patients had increased iron stores and 5 (18.5 %) patients had absent iron stores. Hypersplenism was found in 15 (55.6 %) cases and was the most common cause of cytopenias. Also it was found to be associated with megaloblastic anemia in 13 cases (48 %). Megaloblastosis was found to be the sole cause of anemia in 7 cases (26 %). One patient (3.7 %) had dimorphic anemia and one patient (3.7 %) had storage disorder. Conclusion: Hypersplenism was found to be the most common cause of cytopenia in patients of chronic liver disease on bone marrow examination. Eosinophils with precursors were increased in majority of patients and need further work up. Bone marrow examination is an important test in work up of patients with chronic liver disease especially for those presenting with severe pancytopenia and sudden alteration in peripheral blood counts.

Abstract No. 277 Case Report of Metastasis of Small Cell Carcinoma of Lung in Bone Marrow Nainesh Menat, Biren Parikh, Dhaval Jetly, Manoj Shah Pathology Department, The GCRI, Civil Campus, Ahmedabad Summary: Bone marrow involvement by small cell carcinoma is a very rare phenomenon and it was documented in our institute and was diagnose with the aid of bone marrow aspirate and bone marrow trephine biopsy examination. Introduction: Small cell carcinoma also known as oat cell carcinoma which is highly malignant cancer arises in the lung. It involves mainly lung and pleura but rare involvement of cervix, prostate, liver,gut and bladder. Bone marrow involvement is very rare in cases of small cell carcinoma. Materials and Methods: a patient named Mavjibhai Korot, 70 years male came to the GCRI with complains of neck swelling, pain, breathlessness and cough with expectoration since three months. CBC, lymph node biopsy,bone marrow aspiration, bone biopsy, IHC were done for definite diagnosis. Results: Bone marrow aspirate and Bone marrow trephine biopsy shows high grade malignant infiltration. In IHC AE1, Chromogranin, Synaptophysin were positive. On lymph node biopsy it was diagnose as metastatic small cell carcinoma. In CT scan left peribronchial mass with collapsed left lung, left pleural effusion, mediastinal nodes, liver metastasis and lytic lesion in right iliac wing were noted. Conclusion: Bone marrow involvement by small cell carcinoma is a very rare phenomenon and can be diagnosed by bone marrow aspiration, bone marrow trephine biopsy and IHC.

Abstract No. 278 A Study of Bone Marrow Micrometastases in Cases of Epithelial Malignancies Akanksha Agarwal, Ashutosh Kumar, Rashmi Kushwaha King George’s Medical University, Lucknow

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Summary: This is a prospective study to analyse the percentages of various epithelial malignancies presenting with bone marrow metastases or micrometatstases. Introduction: The most common malignancies are derived from simple epithelia of breast, lung, colon, prostate, ovaries, and endometrium. Carcinomas from these sites usually metastasize to regional lymph nodes and other organs, such as lung, brain, liver and bones. Among various epithelial malignancies breast (28.8 %), lung (23.3 %) and prostate (6.8 %) are common to metastasize to bone marrow. Materials and Methods: Over 2 years 45 patients of various epithelial malignancies were evaluated. Their bone marrow aspiration, clot section, imprint smears and bone marrow biopsy were done and analysed. CK 7, CK 20 and PSA immunohistochemistry were used to confirm the bone mets. Results: Out of all the cases 4 cases of prostate malignancies showed positive bone mets and were confirmed on IHC and histopathology. Conclusion: Bone marrow metastases analysis will help to stage and grade the malignancy. A positive case indicates a relatively poor prognosis and continuos follow up, even after the cure of primary lesion is required in these cases to avoid the episodes of recurrences.

Cell Counters Abstract No. 279 Automated Red Cell Indices in Evaluation of Anemia

373 Summary: The existing diagnostic parameters for megaloblastic anemia such as high mean corpuscular volume (MCV) within a normal range could mask an existing or progressive cobalamin deficiency. New parameters like mean neutrophil volume (MNV) and mean monocyte volume (MMV) were found to be valuable in supplementing the conventional methods. Introduction: Conventionally, the tools for laboratory diagnosis of megaloblastic anemia, namely, serum vitamin B12 and folate levels have proven to be time tested and reliable. Automated haematology parameter such as a raised MCV has further enhanced the diagnostic acumen. New parameters which could possibly facilitate early detection of cobalamin deficiency include MNV and MMV. Hence, we decided to analyze the significance of MCV, MNV and MMV in megaloblastic anemias. Materials and Methods: A retrospective analysis of 65 blood samples, all having serum vitamin B12 level \200 pg/ml (normal: 220–914) and/ or folate level \4 ng/ml (normal 4.1–12) was conducted in the Hematology division of Kasturba Hospital, Manipal for 6 months. Using Beckman Coulter LH 750 and LH755 analyzers, we extracted the MCV, MNV, MMV parameters. Corresponding ESR and CRP values were corroborated to rule out a false positive increase in these parameters. Results: Both MNV and MMV were increased in almost all cases of cobalamin deficiency. Furthermore, there was a good correlation between MCV and these parameters. Also, an inverse relation was noted between serum levels and both MNV and MMV. Conclusion: Both MNV and MMV are reliable predictors of cobalamin deficiency. The machines can be calibrated to flag the cases of cobalamin deficiency.

SG Joshi, NV Kadgi, MV Jadhav Department of Pathology, BJGMC, Pune Summary: Present study of automated RBC indices in anemia was rewarding in underlining their importance in evaluation of anemia. Introduction: Given the multitude of clinical presentations and pathogenetic mechanisms, evaluation of anemia is ever challenging. Present study is a humble attempt to elucidate role of automated RBC indices in evaluating anemia. Materials and Methods: 115 prospectively registered patients with primary complaints of anemia admitted in SGH, Pune & referred to hematology department for bone marrow examination were enrolled in the study. After obtaining informed consent, they were evaluated clinically and laboratory investigations namely complete blood count (CBC) on 18 parameter, 3 part differential automated hematology analyzer (Sysmex-K4500), Peripheral blood smear, reticulocyte counts, iron studies and other pertinent investigations. Results: Diagnostic accuracy of MCV in classifying anemias was 77.1 %, that of MCHC was 61.7 %, that for MCH was 82.8 % and that of RDW was 58.5 %. Diagnostic accuracy of conventional morphological classification was 65.2 %, that of Bessman’s classification was 54.9 % & that for newer classification invented in present study & based on MCV & MCH was 80.9 %. MCV correlated maximally with MCH followed by MCHC, RDW, Se. iron & transferrin saturation. The RDW showed negative correlation with WBC count. Conclusion: Of the automated RBC indices, MCH emerged as a very useful index with highest diagnostic accuracy in classifying anemias. A newly invented classification based on this index along with MCV offers highest diagnostic accuracy in morphologically classifying anemias.

Abstract No. 280 Megaloblastic Anemia and Automated Parameters: Old Friends with New Discoveries Ayushi Agarwal, Deepak Nayak M Kasturba Medical College, Manipal, Manipal University

Abstract No. 281 Reference Range Evaluation of CBC Parameters with Emphasis on Newer Research Parameters on the CBC Analyser Sysmex XE-2100 Kunal Sehgal1, Tina Dadu1, Urmi Choksey2, Reeta Dalal2, Shanaz Khodaiji1 1

Hematology Laboratory, Department of Laboratory Medicine, Department of General Medicine, P.D. Hinduja National Hospital and MRC, Mumbai

2

Summary: Reference ranges were evaluated for 122 normal CBCs on the Sysmex XE-2100, with emphasis on the novel research parameters. Introduction: Since the advent of automation in the field of hematological cell counters with constant refinement of the technology, many newer parameters are available. Knowledge of laboratory reference ranges is essential for their use in clinical practice. The aim of this study was to evaluate the reference interval for CBC samples on the Sysmex XE-2100 autoanalyser, with emphasis on the novel or newer research parameters. Materials and Methods: Blood samples from a total of 122 clinically asymptomatic subjects were evaluated and following blood examinations were performed for all samples: CBC, Reticulocyte Count, ESR, Iron Studies, Vitamin-B12 levels and Folate levels. Only patients with completely normal CBC with no flags and normal values for all above tests were further evaluated. A final of 100 patients (54-M, 46-F) were included in the study after excluding patients with high ESR levels (n = 6), iron deficiency (n = 10), Vitamin B12/Folate deficiency (n = 6). Appropriate statistical evaluation was done. Results: The normal reference ranges for all parameters are shown in Table 1 and will be discussed in detail on the day of the conference. Conclusion: The reference intervals we report here for the various parameters match quite well with few minor variations as compared to published literature. Such probably population-based shifts stress the importance of determining reference limits in every laboratory or verifying the applicability of a published

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Table 1 Reference ranges for parameters reported on Sysmex XE2100 Parameter

Mean

Range Mean ± 2 SD

Hb (g/dl) RBC M-5.4 (1012/L) F-4.8

Median

Range 2.5–97.5 percentile

M-15.4

13.18–17.22

F-13.4

12.1–14.6

4.56–6.16 M-47.6

40.24–53.48

F-41.7

37.33–46.05

MCHC (g/dl)

M-32.4

30.88–34.96

F-32.1

30.47–33.95

RDW (%)

13.400

12.3–15.14

Retic count (%)

.920

0.42–1.82

MCV (fl)

87.287

MCH (pg)

M-28.39

26.12–30.67

F-27.87

25.5–30.2

81.125–93.448

RPI

M-1.1

0.37–2.26

F-0.8

0.4–1.29

DHe (pg)

3.200

1.42–4.35

IRF (%)

6.700

2.00–16.52

LFR (%)

93.350

82.47–97.99

MFR (%)

6.300

2.0–14.03

.400

0–2.25

Ret He (pg)

M-31.4 F-30.59

27.7–33.4

RBC He (pg)

M-28.25

26.09–30.42

F-27.63

25.45–29.81

28.7–34.1

HFR (%) Ret Y

159.76–178.24

M-160.83 152.89–168.77 F-158.46 150.42–166.50

TLC (109/L)

6270.000

PLT (109/L)

M-259

153–366

F-295

182–409

PLT I (109/L)

M-254

132–376

F-295

182–409

PLT O (109/L)

M-276

161–391

F-302

178–427

PLCR

30.510

14.86–46.16

PCT (%)

0.27 0.31

0.17–0.37 0.21–0.41

PDW (%)

12.230

9.44–15.02

MPV(fl)

10.660

9.32–11.99

IPF (%)

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Utilization of Histograms in Routine Practice Ravikiran N Pawar, Vijay H Juvekar, AG Valand, Shubhangi V Agale

Summary: Blood cell histograms supplied by the automated hematology analyzers are of great diagnostic and morphologic importance. Reviewing the histograms is useful for evaluation of peripheral blood films. Introduction: Histograms of the samples received in hematological laboratory were analysed and correlated with the blood indices to detect the diseases which were not diagnosed in normal peripheral blood smear examination. Materials and Methods: The study comprised of histograms of over a period of 2 years in the hematology section of pathology department. Detailed clinical history, examination findings helped to arrive at the diagnosis. Smears of the fresh samples were made and stained with Field’s and Leishman’s stain for microscopy. All data was analysed and tabulated. Results: Comparison of abnormal red blood cell histogram with red blood cell indices and RDW-CV showed highest sensitivity (59.5 %) and specificity (98.33 %) than indices alone. Sensitivity of histograms in detecting neutrophilia (87 %) was better than that of peripheral blood smear (80.4 %). Sensitivity of peripheral smears was found to be 89.78 % in abnormal white blood cell histograms and highest sensitivity (100 %) in cases of Chronic myeloid leukemia. Conclusion: Blood cell histograms supplied by the automated hematology analyzers are of great diagnostic importance.

Abstract No. 283 Can the Sysmex Xn, 6-Part Cell Counter be an Effective Tool to Diagnose Malaria? R. Rani, Leela Anthony, M Murli Mohan, Emmimal, Mohanapriya Hitech Diagnostic Centre, Chennai

M-171.71 162.81–180.61 F-169

RBC Y

Abstract No. 282

Grant Government Medical College, Byculla, Mumbai

4.20–5.39

HCT (%)

reference interval for a given hematology analyser before its use as suggested by CLSI.

1.600

4,240–10,075

0.70–4.32

Summary: A prospective study conducted to improve the efficiency of the diagnosis of malaria by using the data obtained from the Sysmex XN, automated 6-part haematology cell counter. The data obtained from 105 positive cases were analysed for IP messages/flags and scattergram. The data revealed that the shape of the scattergram and the abnormal scatter, flags like blasts/abnormal lymphocyte, left shift were highly predictive of Plasmodium Vivax infection. Introduction: The Indian sub-continent is endemic for malarial infections. The presence of the malarial parasite interferes with the counting of cells and the shape and manner of cell scatter by the laser in the 5-part automated cell counter This study attempts to use that deviation to predict the presence of malarial parasite in a sample of blood. Materials and Methods: This is a prospective study conducted between April 2013 and July 2013 in the Department of Haematology, Hitech Diagnostic Centre, Chennai. 350,000 samples were analysed on the Sysmex XN 3000, 6-part automated haematology cell counter and 105 cases were found positive for malaria. The samples were tested on the Malaria Antigen P.f/Pan SD BioLine (Alere Diagnostics) kit. It is a one step Malaria HRP-II (P.f) and pLDH (Pan) Antigen Rapid Test. All cases were taken for confirmation by the microscopic examination of thin smears stained by Leishman stain. The species and stages of malarial parasite were

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 determined by 2 independent staff. The data from the WNR (the channel to count white blood cells and nucleated red cells) and WDF (the channel used to classify white cells) channels, IP messages and the scattergram obtained from the cell counter were analysed. Results: 105 cases of were positive for malaria, 100 for Plasmodium Vivax and 5 for Plasmodium Falciparum. The cell counter showed several features that were suspicious for the presence of malaria Difference between WNR and WDF [1.3 in 58 % of cases of Plasmodium vivax Narrowing of space between neutrophil and eosinophil cluster in the WDF scattergram in 78 % of all cases of Plasmodium vivax and 100 % of cases where the difference is [1.3. Presence of an abnormal cluster distinct but adjacent to the eosinophil/neutrophil cluster in 42 % of cases The most commonly occurring IP messages were Blast/Abnormal lymphocyte (57 %), Left shift (56 %), Atypical lymphocyte (37 %), and abnormal scatter (31 %). There were 42 % cases where the difference between WNR and WDF was \1.3, but 17 cases (41 %) showed narrowing of space between neutrophil and eosinophil cluster, 5 (12 %) abnormal cluster and IP messages like Blast/Abnormal lymphocyte (57 %), Left shift (45 %), Atypical lymphocyte (45 %), and abnormal scatter (19 %). There were no flags raised for samples with Plasmodium Falciparum infection. Conclusion: All samples that are run on the Sysmex XN cell counter that show a difference greater than 1.3 between WDF and WNR, with narrowing of space in the scattergram are highly suspicious for Plasmodium vivax infection. Can a flag for malaria be incorporated? Samples where the difference is less than 1.3 show narrowing along with IP messages that raise a suspicion for the presence of malaria.

Abstract No. 284 Diagnosis of Malaria by an Automated Haematology Analyser: Looking Beyond Conventional Methods K Indira Shastry, M Deepak Nayak, Sushma V Belurkar, Saroja Kasturba Medical College-Manipal, Manipal University Summary: The diagnosis of malaria has taken rapid strides. The application of VCS technology is one such progressive step in this regard. When used effectively; the new methodology is a useful supplement and possibly, a substitute to the conventional microscopy. Introduction: The diagnosis of malaria has always been a diagnostic challenge. The screening of peripheral blood films confers an additional load on pathologists. Thus, a need for a more sensitive and cost effective method has interested researchers in recent past. Automated haematology analysers provide scatter grams for leucocytes. Additionally, the research population data (RPD) may provide useful information regarding the presence of parasitemia. The objective was to achieve a working model for diagnosis of malaria using VCS technology. Materials and Methods: A retrospective case control study was conducted for period of three months in the Haematology division of Kasturba Hospital, Manipal. 90 cases of malaria and 220 controls were scrutinized using following parameters: (a) malaria factor = (SD volume of lymphocytes 9 SD volume of monocytes)/100, (b) thrombocytopenia, (c) monocytosis, (d) Mean volume of monocytes and (e) pseudoeosinophilia. Results: At cut-off value for malaria factor of 3.4, a 97 % sensitivity and 89 % specificity was achieved. Additionally, Monocyte count (11.6 %), Mean monocyte volume (180 fl), Platelet count (93 9 103/cu mm), Eosinophils (0.99 %) when added to the malaria factor; further increased the sensitivity and specificity. Conclusion: The automated detection of malaria by VCS technology is a useful tool. If calibrated, the machines have the potential to highlight low parasitemia cases due to its high sensitivity and also to exclude false positives.

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Abstract No. 285 Correlation of Eosinophilia in Automated Hematological Cell Counter Sysmex XS800i with Plasmodium Infection Diagnosis Ram Mohan Jaiswal, Shashikant Adlekha, Tandra Chadha, A Shina Singla Mahatma Gandhi Medical College, Sitapura, Jaipur Summary: Automated analysers based on flow cytometry have evolved as an effective adjuvant diagnostic tool in malaria diagnosis. We studied the correlation of interpretive message from automated hematology analyser XS800i and Plasmodium infection diagnosed in peripheral smear. Introduction: Automated analysers are widely used in laboratories across the world for routine hematological analysis. Automated hematology analysers though are not specifically designed for detection of malaria related abnormalities, they have sensitivity for detection of malaria parasite. Malaria diagnosis with flow cytometry based hematology analysers can become an important adjuvant diagnostic tool in the routine laboratory work up of patients, who have even not been referred by clinicians for light microscopic examination. This study have found that the autoanalysers give vital clues to the diagnosis of malaria infection. Materials and Methods: All cases with interpretation (IP) message of eosinophilia in Sysmex XS 800i analyser (Five part differential) were screened for microscopic correlation of eosionophilia and other peripheral smear findings. Results: In 22 out of 5,012 patients with eosinophilia, microscopic eosinophil counts were normal. In these 22 cases, IP message in hematology analysers were eosinophilia and WBC scatterogram showed narrowed space between the eosinophil (EO) and neutrophil (Neu) population. On careful examination of the peripheral smears of all 5012 cases, 39 cases revealed trophozoites, schizonts or gametocytes of Plasmodium vivax in erythrocytes. Conclusion: Peripheral smears of the cases having inconsistent eosinophilia result with that of Sysmex XS800i analyzer should be examined carefully for the presence of malaria parasites in the red blood cells. Sysmex XS800i analysers have moderate range of sensitivity and high degree of specificity in diagnosing malaria as spurious eosinophilia.

Abstract No. 286 VCS Parameters as an Indicator of Acute Bacterial Infections by the Automated Hematology Analyzer KS Pooja, Jessica Minal, Purnima S Rao, Kirthinath Ballal Kasturba Medical College, Mangalore, Manipal University Summary: In the present study, an elevated mean channel of neutrophil volume (MNV) & mean channel of monocyte volume (MMV) were seen to be an early predictors of acute bacterial infections. Introduction: The correct and timely diagnosis of severe acute infectious processes is extremely important for proper patient management. Complete blood picture is the most common investigation ordered in these patients. With the advent of automated Coulter haematology analysers, the VCS (Volume, Conductivity & Scatter) parameters of the leucocytes could be analysed for the early detection of the infections. Materials and Methods: Peripheral blood samples from 94 patients with infection (systemic infections, positive blood cultures for bacteria n = 36, localised infections m = 58) and 46 control subjects were studied using the VCS parameters by the Coulter LH 780 haematology analyser. Results: We observed a significant increase in the mean channel of neutrophil volume (MNV) & mean channel of monocyte volume (MMV) from patients with infections (both systemic & localised) compared with control subjects (MNV: 158.3 ± 13.7 vs. 137.2 ± 4.3; p \ 0.001) & (MMV:

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376 177.8 ± 16 vs. 161.7 ± 6.04; p \ 0.001). The mean channel of neutrophil light scatter (MNS) was decreased significantly in patients with infection (138.5 ± 9.2 vs. 147.2 ± 3.8; p \ 0.001). However, there was no significant difference in the MNV of patients with systemic infection & localised infections (160.5 ± 17.5 vs. 156.8 ± 10.5, p [ 0.05). Conclusion: Assessing the peripheral blood smears for the features of infection is tedious and labourintensive. Therefore, the diagnostic value of the manual differential count as an indicator of infection remains the subject of on-going debate. The VCS parameters like MNV & MMV are more sensitive parameters and could prove to be a quick diagnostic indicator of acute bacterial infections.

Abstract No. 287 VCS Parameters: Indicator for Predicting Diabetes Mellitus, Impaired Fasting Glucose and Diabetes with Complications Parul Arora, Praveen Kumar, Aashish Gupta1, Subhadra Sharma, Ashok Mukhopadhyay Departments of Laboratory Medicine and Pathology1, All India Institute of Medical Sciences, New Delhi Summary: Volume Conductivity Scatter (VCS) parameters mean platelet volume (MPV) and mean Neutrophil volume (MNV) were studied in patients with impaired fasting glucose (IFG), diabetes mellitus (DM) and diabetes with complications to find out the correlation of these parameters with the presence of the disease and its severity. Introduction: Diabetes Mellitus has been associated with increased risk of both micro and macrovascular complications in which platelet plays a pivotal role. MPV is an indicator of its function. MPV and MNV, another useful parameter can be easily determined on routine automated hemograms. The objective of this study is to compare the MPV and MNV in patients with DM, Pre-diabetes or IFG and non-diabetic controls. Materials and Methods: This is a retrospective observational study done in the Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi between September-2012 to April-2013. A total of 40 healthy controls and 153 patients were selected and sub-grouped as IFG (Gr A), DM (Gr B) and DM with complications(Gr C). Hemogram from both cases and controls were run on Beckman Coulter LH-750 and VCS parameters were noted. Fasting and post-prandial glucose were estimated in blood chemistry analyser. Results: MPV was significantly different between cases and control (10.6 ± 1.8 vs. 7.01 ± 1.4, p \ 0.05) as well as between groups Gr A vs. B (p \ 0.05), A vs. C (p \ 0.05) and B vs. C (p \ 0.05). Similarly, MNV was statistically significant between cases and controls (146.7 ± 14.6 vs. 140.57 ± 7.7, p \ 0.05) and also among the DM groups Gr A vs. C (p \ 0.05) and B vs. C (p \ 0.05). Discussion: MPV was significantly raised in the cases when compared to control group and lower in the IFG when compared to DM as well as with DM with complications. MNV was significantly raised in the overall DM group as well as in the DM with complications when compared to IFG and DM. Conclusion: Our study suggests a potential role of platelet size and activity in subjects with diabetes. MPV and MNV are strongly and independently associated and can predict the presence and severity of diabetes.

Abstract No. 288 Correlation of Mononuclear Cell Counts: Cell Counters Versus Manual Estimation Deepika Wali, Rehan Ahmed, Prosenjit Ganguli, Reena Bharadhwaj, Jasjit Singh, UD Gupta, Velu Nair, Natwar Singh

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Department of Pathology and Molecular Medicine, Army Hospital, (R&R), Delhi Cantt-110010 Introduction: Our studies suggest that mononuclear cell are the cells with one lobed nucleus, not the multilobed nucleus, which sometimes are wrongly interpreted by machine. We took 80 Patient for Peripheral Blood Sample and 20 Patient for Bone Marrow to evaluate the MNCs by Sysmex KX21 (3 part) and Coulter HMX (5 part) Versus Manual counting. Aims & Objectives: (1) To evaluate MNCs and correlating them with 3 Part, 5 Part and Manual counting under 4 different ranges of TLCs. (2) To evaluate amongst the 3 part and 5 Part counter, which one is supporting the Manual count more accurately. Materials and Methods: (1) 80 samples of Peripheral blood were included in the study. 20 samples of Bone marrow were too taken into the study. (2) 3 ml EDTA-K2 (BD) tubes were used for collecting peripheral blood, as well as Bone Marrow Sample. (3) Sysmex KX-21 (3 part) & HMX(5 part). (4) Slides, pipette, tips (Eppendroff/Axygen/Tarson). (5) Leishman Blue Stain (Eosin-methylene blue solution, Merck). (6) May-Gruenwald Solution modified (Eosin-methylene blue solution). (7) Microscope (Labomed) Vision 2000, 230 V and Motic Penta Head (for interobserver Correlation Study). Procedures: (1) We took 4 Different Ranges of TLC of Peripheral Blood, 20 Volunteers for each range and 20 volunteers for Bone Marrow Blood. (a) Less than 4,000/cu mm, (b) 4,000–12,000/cu mm, (c) 12,000–20,000/cu mm, (d) above 20,000/cu mm. (2) Blood Count done by Fully Automated Beckman Coulter Sysmex KX 21(3 part) & HMX (Beckman Coulter 5 Part). (3) Manual Counting by Smear preparation & visualizing its Morphology. (4) Viability done with Tryphan Blue. (5) Comparing the MNC (by both Coulter and Manually) & interpreting which one is giving the best result of mononuclear counts. Observations & Results: 5 Part shows more similarity with the manual counting, while a wide range of fluctuation was seen in 3 Part v/s Manual counting which implies that 5 Part Shows an accurate results than 3 Part and MNC which actually are monocytes and lymphocytes are only 25–40 % of the Total leukocyte. Conclusion: Our study, shows that there are differences in the PB-MNC and BM-MNC counted by 3 part (sysmex KX 21), 5 Part (Beckman Coulter HMX), and Manual counting (3 different individuals). Result of 3 part and 5 part were somewhat different, 5 Part was showing similarity with manual counting. Manually counted mixed population supports to 3 Part which is performed by 3 different observers. Mean of manual mixed population of different individual shows similarity with 3 Part. We cannot predict MNCs in 3 Part as it doesn’t gave the exact number of monocytes which actually constitute mononuclear cell.

Abstract No. 289 Performance Characteristics of BLAST Flags on Beckman Coulter LH-750 Automated Hematology Analyzers Ishwar Bihana, Prashant Sharma, Parveen Bose, Sudershan Kumar Sharma, Neelam Varma Department of Hematology, Level 5, Research Block A, Postgraduate Institute of Medical Education and Research, Sector, 12, Chandigarh 160012, India Introduction: Automated hematology analyzers can indicate atypical/immature leukocytes including blasts. Primarily designed to trigger smear review, these ‘‘flags’’ are currently considered insufficiently accurate/precise for use as reliable first-line diagnostic aids. Materials and Methods: We evaluated the diagnostic efficiencies of software-generated flags for suspicious cells from Beckman Coulter LH750 analyzers. Flow cytometrically characterized acute leukemia cases (n = 28, C20 % blasts) were studied along with 30 controls without circulating blasts. The analyzer’s blast lineage sub-

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 classification (granulocytic/lymphoid/monocytic lineages indicated as NE/LY/MO blasts respectively) was also assessed. Results: Blast flags were generated in 18 of 28 leukemia cases (64.3 %). Four did not show blast flags, but flagged for immature neutrophils (Imm NE1/ Imm NE2), variant lymphocytes (VarLymph) or manual differential count verification (VerifyDiff). The remaining six cases showed no abnormal cell flags although variable other abnormalities (cytopenias/ leukocytoses) triggered smear review in all but one case. Within blast subtypes, NEblasts flags were generated in 4 cases [3 (75 %) correctly identified], LYblasts in 12 cases [9 (75 %) correctly identified], and MOblasts in 6 cases [2 (33 %) correctly identified]. Of the non-leukemia cases, only 1/30 (3.3 %) showed a MOblast flag: smear review confirmed the presence of transformed lymphocytes without any blasts. Conclusion: Automated blood cell counter generated blast flags are highly specific (96.7 %) for the presence of blasts and, together with other abnormal WBC flags, are sufficient to trigger smear review. However, their relatively low sensitivity (64.3 %) for these hematologically critical cells indicates a need for continuing technological improvements in flagging software as well as 5-part differential calibrators and controls.

Abstract No. 290 Comparison of CBC Parameters in Venous and Capillary Blood in Oncology Patients Manikchandra R Tiwari, Umakant A Gavhane, Sanjay Kumar, Ulka M Gosavi, Preeti Chavan, Vivek Bhat Advanced Center for Treatment Research and Education in Cancer, Tata Memorial Center Summary: Venous and capillary blood samples collected from 200 cancer patients were analyzed. WBC, hemoglobin and MCV showed good correlation with either technique of blood collection whereas RBC and platelet showed good correlation only for low counts. Introduction: Venous blood is normally collected for CBC test. In chemotherapy patients collection of finger-prick blood may be easier. The comparison between CBC from capillary and venous blood would enable us to explore the possibility of using the two methods interchangeably. Materials and Methods: Venous and capillary blood collected from 200 cancer patients were analyzed in 5-part differential HMX-BC hematology analyzer and smears were screened. The results were compared for WBC, RBC & platelets. Results: WBC—Consistence correlation with R2 = 0.9066, 0.8983, 0.9861 was observed for WBC counts \4, 4-11, [11 9 109L respectively. RBC—Correlation with R2 = 0.9646 was observed for RBC count \4.5 9 1012/L whereas, for RBC [4.5 9 1012/L, R2 = 0.7731. Hemoglobin—Consistent correlation with R2 = 0.9567 and 0.8731 was observed for hemoglobin \12.5 and [12.5 g/dL. MCV—Consistence correlation with R2 = 0.9621, 0.9766, 0.9787 was observed for MCV \80, 80–100 and [100 fL. Platelet—Correlation with R2 = 0.9107 was observed for platelet count 0 to 150x109Cells/L whereas high platelets [150 showed R2 = 0.7575. Conclusions: It can be concluded that finger prick technique can be used as a suitable method for blood collection for CBC test in patients with RBC count \4.5 and platelet count \150.

377 Summary: The platelet dynamics and immature platelet fraction (IPF) were monitored in 286 serologically confirmed dengue patients. 76.6 % were thrombocytopenic and 79 % had platelet recovery within 48 h of high IPF. Introduction: The commonest hematologic abnormality in dengue is thrombocytopenia. Platelet recovery is expected within 24–72 h of rise in IPF as this reflects thrombopoiesis. The study involves platelet dynamics with particular reference to immature platelet fraction. Materials and Methods: This retrospective study (Jan 2012–Dec 2012) was conducted in the Department of Pathology, Sri Ramachandra University on 286 dengue patients (NS1antigen or IgM or both positive and platelet count done up to 7 days of admission). The platelet count and IPF run on SYSMEXXE2100 analyzer were retrieved for day 1,3, 5 and 7 of admission. The rising and falling trend of platelet count and the IPF were analyzed. Results: Dengue was more common in males (61.2 %) less than 5 years. 76.6 % of patients had thrombocytopenia. 29 % had values below 60,000/cumm. The lowest platelet count were observed on day 1 in 71 % patients. 2.4 % patients with normal count on day 1 had thrombocytopenia on day 3. Platelet recovery of more than 10 % was observed in 79 % patients within 48 h of high IPF. Conclusion: Platelet count should be monitored every day for a minimum of 7 days. As platelet recovery is expected within 48–72 h of peak IPF, platelet transfusion may be deferred if there is rise in IPF.

Abstract No. 292 Role of Mean Platelet Volume in Evaluating the Cause of Thrombocytopenia in Neonatal Intensive Care Unit Neeti Goyal, J Kotwal, Ganesh Kumar Parajuli, SM Vibha Dutta, Amit Devgan Armed Forces Medical College, Pune Summary: Mean platelet volume (MPV) is a rapid, non invasive, automated cell counter index to diagnose the cause of thrombocytopenia in neonates as bone marrow biopsy is generally not done at this age. Introduction: Neonatal thrombocytopenia can be due to increased platelet destruction or due to decrease platelet production. If bone marrow biopsy shows few megakaryocytes it confirms low platelet production and many megakaryocytes provides clue to increase platelet utilization. Bone marrow biopsy is invasive procedure so generally postponed in neonatal period. Therefore in this situation the MPV may be a good non-invasive alternative. Patients with decreased production may require less transfusion viz. those with increase platelet destruction. Materials and Methods: We measured MPV along with haemogram in 100 suspected cases of thrombocytopenia by automated cell counter. PBS was examined to access the cause of thrombocytopenia and other test like PT, PTT, D-dimer, Fibrinogen were done if indicated. Results: We studied 100 neonates of thrombocytopenia in whom 62 had increased platelet destruction (Sepsis, DIC, etc.) and 38 had decrease platelet production (IUGR, genetic, etc.) as the cause of thrombocytopenia. We found that MPV was \8.5 fl in all cases with decrease production and [9.2 fl in all cases with increase platelet destruction. Conclusion: A simple index like MPV which is available on cell counter can provide information for cause of thrombocytopenia and helps in deciding the frequency of blood transfusion required.

Abstract No. 291 Dengue: Platelet Dynamics and Immature Platelet Fraction D Febe Renjitha Suman, Lawrence D’ Cruze, Suresh Varadarajan Sri Ramachandra University, Chennai

Abstract No. 293 A Study of Platelet Volume Indices (PVI) in Patients of Coronary Artery Disease and Acute Myocardial Infarction in Tertiary Care Hospital

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

Killol Nathubhai Desai, Killol Nathubhai Desai

Flow Cytometry

Pramukhswami Medical College, Shree Krishna Hospital, Karamsad, Anand, Gujarat

Abstract No. 295

Summary: The study was performed to analyse PVI that are useful for identifying large platelets, which are hemostatically more active and risk factor for developing coronary thrombosis and myocardial infarction. Introduction: Ischemic heart disease is the leading cause of death worldwide. Platelets have definite role in its pathogenesis and complications. Platelet size reflects its activity. Large platelets are metabolically and enzymatically more active and produce more thromboxane A2. Materials and Methods: In this retrospective study, 200 cases were studied, 100 patients had unstable angina (UA) or acute myocardial infarction (AMI), 70 patients had stable coronary artery disease (stable CAD). The third group comprised of 30 healthy control with no history of heart disease and normal electrocardiogram. The anticoagulated peripheral blood sample was collected from each patient and analysed in a 3-part automated haematology analyser. Results: All three Platelet indices i.e. Mean Platelet Volume (MPV), Platelet Distribution Width (PDW) and Platelet to Large Cell Ratio (P-LCR) were increased in patients of UA and AMI. Mean MPV was 11.2 fl, mean PDW was 14.3 fl and mean P-LRC was 28.6 % in unstable coronary artery disease. In stable coronary artery disease, mean MPV was 10.7 fl, mean PDW was 13.2 fl and mean P-LRC was 23.0 %. In the control group mean MPV was 9.3 fl, mean PDW was 11.3 fl and mean P-LRC was 19.86 %. Conclusion: Patients with large platelets can be identified during routine work up. PVI is simple and cost effective tool for predicting possibility of impending acute coronary events and further patients can be refereed for preventive measures.

Assessment of Blast Flag in Advia 2120i by Peripheral Blood Smear Examination Sanjay Kumar, Manikchandra Tiwari, Ashwadeep Karmore, Umakant Gavhane, Ulka Gosavi, Preeti Chavan, Vivek Bhat ACTREC, TMC, Kharghar, Navi Mumbai Summary: The fully automated haematology analyzer—Advia 2120i provides additional parameters in the form of % LUCs and blasts. We evaluated 100 samples which generated blasts flag. Blast % of [4 was seen to correlate with presence of blasts in the peripheral smear. There was no correlation between LUC % & presence of blasts Introduction: Advia 2120i, a fully automated hematology analyzer by Siemens provides, in addition to routine parameters, flags for blasts and LUC %. The criteria for generating blasts flag are %blasts [1.5 & %LUC (Large unstained cells) C4.5 or % blasts [5 of the total WBC. It is recommended that peripheral blood smear examination should be done for such samples. The aim of this exercise was to correlate the blast flags with the presence of blasts. Materials and Methods: We evaluated 100 samples which generated the blasts flag. Peripheral blood smears were prepared and manual differential count (MDC) was done. The count was correlated with the LUCs & blast %. Results: Seven samples showed presence of blasts. There was no correlation between LUC % & presence of blasts. Blast % of [4 was seen to correlate with presence of blasts in the peripheral smear. Conclusion: From our study we found that LUC % does not have any bearing on the presence of blasts in the sample. However, samples with blasts % of more than 4 should be reviewed for presence of blasts on peripheral smear.

Abstract No. 294 Mean Platelet Volume in Diabetes Mellitus Abha Thakur, S Marwah, B Kulshreshtha, V Kumar, AS Nigam, G Buxi PGIMER, Dr. RML Hospital, New Delhi Summary: 150 diabetic patients and 150 age and sex matched healthy controls were taken as study group. Complete haemogram, HbA1C and FBS along with clinical history and previous medical records were analyzed. The differences in Platelet Indices i.e., MPV, PDW and LPCR were found to be statistically significant among cases and controls. Micro and Macrovascular complications were also found to be associated with various platelet indices. Introduction: The prevalence of diabetes is increasing all over the globe at an alarming rate. Platelets play an important role in the integrity of normal haemostasis and MPV is an indicator of its function. Materials and Methods: A total of 150 diabetic patients and an equal number of age and sex matched healthy subjects were taken. History, examination, previous medical records, routine haemogram, HbA1C and FBS were evaluated. Data was collected and statistical analysis was done with p value of 0.05 or less taken as statistically significant. Results: MPV, PDW and LPCR were found to be higher and statistically significant among cases than controls. In Logistic regression analysis, micro and macro vascular complications as a dependent variable was associated with MPV, PDW and LPCR. Conclusion: MPV is an important, simple, effortless and cost effective tool that should be used in developing countries for predicting the possible impending acute events.

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Abstract No. 296 Flow Cytometric Immunophenotypic Analysis of Blastic Plasmacytoid Dendritic Cell Neoplasm: Report of 2 Pediatric Cases Preeti Dharmani, PG Subramanian1, Mittal Neha1, Galani Komal 1 , Badrinath Yajamanam1, Sridhar Epari2, V Seethalakshmi2, Nair Reena3, Banavali SD3, Gujral Sumeet1 Hematopathology Laboratory, 2Department of Surgical Pathology, Department of Medical Oncology, Tata Memorial Hospital, Parel, Mumbai 1

3

Summary: Two cases of Blastic plasmacytoid dendritic cell neoplasm (BPDCN) in pediatric patients. Introduction: BPDCN is a rare haematological malignancy. Most affected are older adults, however, pediatric experience is limited. 2 cases in pediatric patients: Case history: Case I: A 12 year-old girl with arm swelling, reported elsewhere as NHL T-lymphoblastic type. Bone marrow showed replacement by large cells with monocytoid morphology. On flow cytometry, gated cells expressed CD33, HLA-DR, CD4, CD56, and were negative for myeloid, B, T lineage markers. On additional panel, tumour cells expressed specific markers like CD303/BDCA-2 and CD123, confirming BPDCN diagnosis. Patient was started on BFM90 protocol. Post induction, marrow was in remission. 15 months later, she returned with marrow, CNS relapse and succumbed. Case II: A 17-year-old girl presented with pre-auricular swelling and incisional biopsy was reported as high grade malignancy, possibly of histiocytic lineage. Staging marrow showed involvement by

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 monocytoid blast-like cells. On flow cytometry, cells expressed D4, CD56 and negative for T, B, myeloid markers. CD303 and CD123 were positive on additional panel, proving involvement by BPDCN. BM did not achieve remission—expired 7 months later. Conclusion: Morphology can be variable although, in most cases it is monocytoid blast-like cells involving skin, marrow. A high index of suspicion is needed in cases of acute leukemia which co-express CD4 and CD56. Newer and more specific markers like CD303, TCL1, and CD123 must be done so as to not misdiagnose BPDCN as T-ALL or undifferentiated leukemia. These specific markers can be easily standardized on flow cytometry. Early detection followed by SCT is the only hope for a long term survival in these patients.

379 T-lymphocytes have been described in peripheral blood almost twenty years ago. CD20 positive T-cell lymphoma is rare with few case studies. Two theories to explain expression of CD20 by neoplastic T-cells. The first possibility, lymphomas develop from CD20positive subset of normal T-cells. The second theory regards CD20, an activation marker. Prognosis and treatment of these lymphomas remain to be elucidated. The need of the hour is to quantify these lymphocytes and gain an insight to their role in immunity. Aim: To quantify and determine detailed immunophenotype by flow cytometric analysis of CD20-positive T-lymphocytes in peripheral blood, BM and body fluids. Materials and Methods: Samples studied will be 20 normal peripheral blood, 10 bone marrows, 5 uninvolved lymphoma marrow, 5 ITP, 5 Body fluids, 5 multiple myeloma, 5 MDS BM. The antibody panel used in this assay is as follows-

Abstract No. 297 Evaluation of Platelet Activation Markers in Sepsis Patients and Correlation with SAPS (Simplified Acute Physiology Score) III Admission Scores Using Flowcytometry: Our Experience in a Tertiary Care Centre in India

FITC

PE

Percp cy5.5

Pecy7

APC

APCH7

BV421

V500

Tube 1

CD20

CDTCR ab

CD3

CD2

CDTCR gd

CD19

CD5

CD45

Tube 2

CD20

CD16 + 56

CD3

CD4

NKG2D

CD8

CD7

CD45

BK Chakrabarty, V Manu, S Shivinder, V Dutta Armed Forces Medical College, Pune Summary: Platelet hyper-reactivity and or circulating activated platelets have been reported to be associated with many common clinical settings, including sepsis. Whole blood flowcytometry is a powerful new technique for the assessment of platelets using specific monoclonal antibodies against various receptors. Introduction: Activation of platelets play an important role in the pathophysiology of septic shock and multiple organ failure. Scanty information is available to understand the patho-physiology and risk indicators associated with the development thrombocytopenia and platelet dysfunction in adult sepsis patients. Materials and Methods: In the present study we used flow cytometry to examine markers of activation on platelet in sepsis patients, and look for correlations between these markers and the severity of the illness based on current standardized risk scoring systems for sepsis. Results: Fifteen sepsis patients admitted to the Intensive care unit (ICU) in a tertiary care centre and twenty control patients (ten normal individuals and ten non sepsis ICU patients) were included in this study. Patients were evaluated on admission, for sepsis criteria and SAPS (Simplified Acute Physiology Score) III score. Indicators for platelet activation were analysed by flow cytometry. The obtained results are compared with patients and controls. Results and statistical analyses will be discussed. Conclusion: The use of flowcytometric analysis of the platelet markers may provide accurate, sensitive and reproducible parameters for their evaluation in critically ill patients so as to improve clinical decision-making when addressing this common problem in sepsis.

Abstract No. 298 CD20 Positive T Cells: Quantification and Immunophenotype in Normal Body Tissues Dia Mansukhani, Kunal Sehgal, Shanaz Khodaiji Department of Laboratory Medicine, Hematology Laboratory, P.D. Hinduja National Hospital and MRC, Mumbai Summary: To quantify and evaluate detailed immunophenotype of CD 20 positive T-cells. Introduction: CD20 is expressed by all B-lymphocytes and some T-lymphocytes. CD20-positive

Results: Detailed results will be discussed in poster to be presented during conference. Conclusion: CD20 positive cells will be quantified as a percentage of all cells, of total lymphocytes and evaluation of other T cell markers on these cells will be done. Quantification of Normal CD5 positive B cells and normal TBNK subset analysis will be done.

Abstract No. 299 Lymphocyte Subset Analysis in Multiple Myeloma Lata Rani, Shuchi Smita, Nitin Mathur, Atul Sharma, Lalit Kumar, Ajay Gogia, Ritu Gupta All India Institute of Medical Sciences, New Delhi Summary: Multiple myeloma (MM) is associated with immunological abnormalities of cellular and humoral system. Enumeration of lymphocyte subsets in peripheral blood (PB) and bone marrow (BM) compartments of MM patients suggested no significant compartmental differences; however, regulatory T cells’ number was higher than Th17 cells. Introduction: Altered frequency of lymphocytes has been demonstrated in many cancers but conflicting results for preferential accumulation in PB and BM compartment have been reported in MM. The objective of this study was assessment of T-cell subsets in PB and BM of myeloma patients for any evidence of their preferential localization. Materials and Methods: Patients with diagnosis of MM established as per the international myeloma working group criteria were enrolled. NK (3-16+56+), Treg (4+25+FoxP3+) and Th17 (3+8-IL-17+IFN-c-) cells were assessed using multi-parametric flow cytometry. Unpaired student’s t-test was used to calculate the significance. Results: A total of 30 patients of MM including 22 males and 8 females with median age of 57 years were evaluated. The mean frequency of NK cells was significantly higher in PB (12.24 %) than in BM (8.45 %; p = 0.03). T cell profile showed no significant difference of CD4+:CD8+ ratio in BM (0.78 ± 0.53) and PB (1.12 ± 0.74) compartment. The difference in the mean percentage of Treg and Th17 cells in BM and PB was also not statistically significant. Conclusion: Treg and Th17 cells in MM patients do not show preferential localization in PB or BM compartment, the data, however, needs to be validated on large cohort of MM patients.

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380

Abstract No. 300 Flowcytometric Characterization of Mantle Cell Lymphoma on Lymph Node Aspirates Aneesha Mohanpuria, Poojan, Snigdha Goyal, Vijay Kumar, Sadhna Marwah, AS Nigam, G Buxi PGIMER, Dr. RML Hospital, New Delhi Summary: Mantle cell is a subtype of B-cell lymphoma. MCL cells generally over-express cyclin D1 due to a t(11:14). At diagnosis, typical patients are elderly and usually present to the oncologist with advanced disease. Thus, lies the importance of early diagnosis in the treatment of patients. Introduction: Three cases of mantle cell lymphoma were analyzed by flowcytometry on lymph node aspirates and showed expression of CD5, FMC7, CD79b, CD22 and Kappa/ lambda light chain restriction. Correlating the clinical picture and cytomorphology with flow cytometric immunotyping, a diagnosis of mantle cell lymphoma was offered. Materials and Methods: Flow cytometric analysis was carried out on lymph node aspirates which were suspicious of small cell lymphoma on cytological examination. The gating strategy employed was CD19 vs. SSC. A panel of antibodies comprising of CD 45, CD5, CD23, CD7, CD3, FMC7, CD79b, CD10, CD19, CD22, Lambda and Kappa was used for immunophenotyping. Results: Three of the analyzed aspirates showed expression of CD5, FMC7, CD79b, CD22 and Kappa/lambda light chain restriction. These cells were negative for CD23, CD10 and CD7. Correlating the clinical picture and cytomorphology with flow cytometric immunotyping, a diagnosis of mantle cell lymphoma was offered. Conclusion: These cases emphasize the role of flowcytometric immunophenotyping on lymph node aspirates as a rapid diagnostic modality in mantle cell lymphoma.

Abstract No. 301 Challenges in Immunophenotypic Diagnosis and Classification of Monocytic Leukemia Manisha Ramani, Santosh Parab, Vishal Mehrotra, Pradnya Chaudhary, Amar Dasgupta SRL Limited, Mumbai Summary: Non-representative nature of the sample poses difficulties in subclassification of acute leukemia (AL) with monocytic component. A proper multiparametric approach allows diagnosis in most of these cases. Introduction: Subclassification of AL with monocytic component requires adherence to strict cut offs for myeloblasts, monoblasts, promonocytes, monocytes and granulocytes in bone marrow (BM) and blood. Therefore, non-representative samples, especially of hemodiluted BM, pose a major challenge in diagnosis. Here we share our experience of immunophenotyping of these cases. Materials and Methods: AML cases (685) were classified according to WHO (2008) criteria. Immunophenotypic features used for identification of leukemic cells of granulocytic and monocytic lineages were, myeloblasts: CD45 dim, SS low, CD4-, CD13+, CD14-, CD15?/-, CD33?, CD117?, CD34?, HLA DR?; monoblasts: CD45?, SS mod, CD4-/?, CD13 dim/-, CD14-, CD15?, CD33 strong, CD117-/?, CD34-/?, HLA DR strong; promonocytes and monocytes: CD45 strong, SS high, CD4?, CD13?/-, CD14?, CD15 ? hetero, CD33 strong, CD117-/?, CD34-, HLA DR? hetero. Results: AL with monocytic component constituted 18 % (127/685) of all AML cases. In 49 (40 %) cases only BM samples and in 76 (60 %) cases only blood samples were available. Diagnosis and subclassification could be made in 48/49 BM samples and 70/76 blood samples. Blood showed higher percentage of blast cells where both BM

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 and blood samples were examined—due to the presence of accompanying hemopoietic cells in BM compared to blood. Overlapping morphological and phenotypic features of promonocytes and leukemic monocytes, and those between dysplastic granulocytes and monocytes posed diagnostic challenges. Conclusion: In spite of the non-availability of representative BM samples in a large number of cases, our data show that it is possible to subclassify AL with monocytic component from blood samples using a multiparametric approach.

Abstract No. 302 A Short Primary Immunophenotyping Panel has a Role in the Diagnosis of Pediatric Acute Leukemia (PAL) Pradnya Chaudhary, Amar Das Gupta, Manisha Ramani, Vishal Mehrotra, Santosh Parab, Janmejay Yadav, Pallavi Gujrathi SRL Limited, Mumbai Summary: We have shown the usefulness of an abridged, cost-effective immunophenotyping panel in PAL. Introduction: There is need for a primary screening panel for AL that is both cost-effective and informative. We describe here our experience with such a panel for PAL. Materials and Methods: Cases (114) of PAL were immunophenotyped using a multi-colour antibody panel against CD3, 5, 10, 13, 19, 20, 45, and HLA-DR. The composition of the panel was influenced by the fact that B-ALL is the commonest PAL. Cases undiagnosed by the abridged panel were diagnosed in the next step using cytochemical MPO and additional markers (CD2, 4, 7, 8, 22, 33, 34, 41, 61, 99, 117, glycophorin, cMPO, cCD79a and cCD3). Results: In 74/76 (97 %) cases of B-ALL the diagnosis could be made in the first instance with the help of the abridged panel; and in 19/21 cases of AML (CD13+ and HLA-DR+) and in all 14 cases of T-ALL (CD3- and HLA-DR-; CD5+) a presumptive diagnosis could be made. Additional CD markers (2-9) were required in 40/114 (35 %) cases (T-ALL = 14; AML = 21; B-ALL = 2; others = 3). On further workup CD13?, CD19? and HLA-DR? phenotype by abridged panel represented AML and not B-ALL. Conclusion: We were able to correctly diagnose twothirds of PAL cases by applying an abridged, low cost primary antibody panel consisting of only 8 CD markers. In the remaining 35 % of cases also, a presumptive diagnosis of the type of leukemia could be made which was confirmed by examination of additional markers, thereby proving the usefulness of the abridged panel.

Abstract No. 303 A Novel Flow Cytometric Method for Simultaneous Assessment of DNA Ploidy, Cell Cycle Analysis and Multicolor Surface Immuno-Phenotyping in Hematolymphoid Neoplasms Vandana Baloda, Y Badrinath, PG Subramanian, Sitaram Gogale, Ashok Kumar, Nihkil Patkar, Hari Menon, Manju Senger, Navin Khatri, Sumeet Gujral, Bhausaheb Bagal, Prashant Tembhare Hematopathology Laboratory, Department of Pathology, Tata Memorial Center, Mumbai Introduction: Flow cytometric (FC) analysis of DNA content is a powerful tool for assessing prognostic variables in different malignancies. In both, B cell acute lymphoblastic leukemia (ALL) and multiple myeloma (MM), hyperdiploidy is associated with good prognosis and hypodiploidy with worse prognosis. Many methods are described for FC DNA ploidy; however, no method can be satisfactorily used for simultaneous evaluation of both surface

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 immunophenotype and DNA ploidy which limits ploidy evaluation of selective tumor population in the background of other hematopoietic cells. In this study, we described a novel method of simultaneous evaluation of immunophenotyping and DNA ploidy in cases of acute leukemia and multiple myeloma using a new dye—FxCycle Violet stain. Materials and Methods: Peripheral blood & bone marrow samples from 44 acute leukemia (39 B-ALL, 5 T-ALL) patients and 13 MM patients were studied using 6–8 color FC. DNA ploidy was evaluated using first tracking surface markers like CD45, CD34, CD19 & CD38 and subsequently followed by staining with FxCycle Violet. Minimum 1,000 events of tumor cells were analyzed. Results: CV of diploid peak ranged between 3–4 %. Out of 39 B-ALL cases, 2.6 % cases were hypodiploid and 12.8 % were hyperdiploid. Out of 13 MM cases, 7.7 % cases were hyperdiploid. Remaining cases were diploid. The results will be correlated with cytogenetics findings and other prognostic factors. Conclusion: This novel method using FxCycle Violet is objective & sensitive technique for simultaneous evaluation immunophenotyping & DNA analysis. It can be easily incorpo-rated in routine immunophenotyping for diagnosing aneuploidy even in a minute tumor population.

Abstract No. 304 CD99 Expression is not Useful as a Marker of Immaturity in T-Lymphoid Neoplasms Vishal Mehrotra, Amar Das Gupta, Manisha Ramani, Pradnya Chaudhary, Santosh Parab, Janmejay Yadav, Pallavi Gujrathi SRL Limited, Mumbai Summary: Our study shows limited usefulness of CD99 as a marker of immaturity in T-cell leukemia contrary to the commonly held view. Introduction: Cases of T-Acute Lymphoblastic Leukemia (T-ALL) are morphologically highly heterogeneous and blast cells in T-ALL can be indistinguishable from the neoplastic cells in T-Chronic Lymphoproliferative Disorders (T-CLPD). CD99 has been reported to be useful in distinguishing T-lymphoblasts from T-lymphoma cells by virtue of being a reliable marker of immaturity—even better than CD34 and TdT in this regard. Our experience with CD99 as a marker of immaturity in T-lymphoid neoplasms described here, however, contradicts the above view. Materials and Methods: CD99 expression was examined as a part of immunophenotyping of 17 cases of T-ALL and in 5 cases of T-CLPD (PTCL-1; T-PLL-2; Cutaneous T-cell lymphoma-2) using a multi-colour antibody panel against CD2, CD3, CD4, CD5, CD7, CD8, CD10, HLA-DR, CD34, CD99 and TdT (in some cases) in addition to B-lymphoid and myeloid antigens. Results: While all T-ALL cases expressed variable degrees of CD99, it was strongly expressed by blast cells in only 3 of the 17 cases. In the majority of these cases CD99 was dimly expressed. CD34 was positive in only 4 cases. TdT was dimly positive in all the 4 cases tested for this nuclear antigen. Interestingly, 4 out of 5 cases of T-CLPD were positive for CD99, albeit dimly. There was no correlation between CD99 expression and that of any of the other markers (T, B, myeloid or non-lineage specific) by the neoplastic T cells or their maturity level. Conclusion: Our observation raises a question about the reliability of CD99 as a marker of immaturity for distinguishing T-lymphoblasts from T-lymphoma cells.

Abstract No. 305 Flow Cytometric Evaluation of Severe Combined Immunodeficiency

381 Harikrishnan Babu, Kotteeswari Kathirvel, Ansu Abu Alex, Faranaz Khamrudin, Biju George, Auro Viswabandya, Aby Abraham, Alok Srivastava, Vikram Mathews Department of Haematology, Christian Medical College, Vellore Summary: Severe combined immunodeficiency (SCID) is a group of potentially fatal inherited disorder characterized by defective T, B and natural killer (NK) cell function. There is limited data on the common phenotypes in India. Introduction: We undertook a prospective study to identify SCID by using simple lymphocyte subset panel by multicolor flowcytometry. Materials and Methods: Peripheral blood samples with suspected clinical and hematological parameters for immunodeficiency disorders were received for lymphocyte subset analysis from 2008 to 2013. Briefly cells were labeled using a panel of monoclonal antibodies to CD3, CD4, CD8, CD19, CD56 and CD45 directly conjugated with FITC, PE, PerCP or APC followed by red cell lysis and were then washed and 20,000 events were acquired using FACS Calibur and analyzed by Cell Quest pro software. Cells were gated using CD45 versus side scatter dot-plots. Absolute lymphocyte subset counts were calculated for the analysis and were compared with the normal reference ranges. Results: Among the 210 samples received for lymphocyte subset analysis, 13 patients were detected to have SCID based on flow cytometric evaluation. 8 cases were found to be T -B-NK+ (61.5 %) and 5 cases were T- B+ NKSCID (38.4 %). The median total lymphocyte count was 684 cells/ mm3. Table 1 summarizes the subset and immunoglobulin profile. In the T- B+ NK- SCID cases, median CD19 percentage was 88 % (71.6–96.7) with a normal absolute B cell counts of 489 cells/ll (range: 348.12–994.5) though the immunoglobulin level were still low or in the low normal range (Table 1). The median age of this cohort at diagnosis was 6 months (range: 3–10) with male predominance of 61.5 %. All infants were symptomatic at evaluation with fever, pneumonia, persistent diarrhea or a combination of these. Among the 11 patients where clinical history was available in 3 there was a history of consanguinity and in 3 a history of sibling death. One patient underwent allogenic stem cell transplantation and is well with a follow up of 60 months. Two of the patients died while in hospital and there was no follow up data available in the remaining. Conclusion: This is a descriptive single centre experience of a cohort of patients with SCID from southern India. T -B-NK+ and T – + B NK- are the most common phenotypes seen in our center. A high index of suspicion is required to diagnose these cases before they become symptomatic.

Table 1 Immunophenotype and immunoglobulin levels in cases diagnosed as SCID T- B- NK+ (n = 8)

T

-

B+ NK

–

Median

Range

Median

Range 3–10

(n = 5)

Reference ranges

Age (months)

6

4–10

7

TLC (cells/mm3)

691

324–3348

684

360–1170

3400–7600

CD3 (cells/ll)

13.9

1.8–81.62

39.78

4.32–64.15

2500–5500

IgM (mg/dl)

\10

–

27

10–44

IgA (mg/dl)

\10

–

\10

–

50–190 140–420

IgG (mg/dl)

238

124–866

138

100–354

800–1700

CD4 (cells/ll)

17.01

0.4–73.92

11.7

4.68–34.2

2700–3900

CD8 (cells/ll)

1.5

0.4–30.2

10.53

0.89–23.94

350–2500

CD19 (cells/ll)

7.039

0.34–26.78

489.7

348.12–994.5

300–2000

CD56 (cells/ll)

568.149

307.8–2356.9

6.12

0.5–25.74

170–1100

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382

Abstract No. 306

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Table 1 . Diagnosis

Flow Cytometry Based Prenatal Diagnosis of Primary Immunodeficiency Disorders Maya Gupta, Manisha Madkaikar, Anju Mishra, Aparna Dalvi, and Kanjaksha Ghosh National Institute of Immunohaematology, Mumbai Introduction: Primary immunodeficiency (PID) encompasses a heterogeneous group of genetic disorders causing defects in different components of immune system. Patients affected with severe form of PID are highly susceptible to severe and recurrent infections and do not survive unless treated with immune reconstituting treatments. Thus in families who already have one severely affected child genetic counselling and prenatal diagnosis becomes important part of the management. Methods and Materials: The aim of the study was to establish phenotypic prenatal diagnosis on cordocentesis sample by flow cytometry for severe Primary immunodeficiency diseases (PID) in families having affected child with PID. Normal reference ranges of lymphocyte subsets, CD 18/CD11 integrins on leukocytes, MHC class II expression, BTK expression on monocytes and oxidative burst activity of fetal neutrophils at 18 weeks of gestation were previously established on 30 cord blood samples. Prenatal testing was performed in 9 families with PIDs. Maternal contamination was ruled out by VNTR analysis. Results: Out of 13 fetuses, 9 were found to be unaffected (3 cases with leukocyte adhesion deficiency (LAD-I), 4 cases with severe combined immunodeficiency diseases (SCID), 1 with X-linked agammaglobulinemia (XLA), and 1 with chronic granulomatous disease (CGD)] and 3 were found to be affected (1 with T-B+NK-SCID, 1 with MHC class II deficiency and 1 with LAD-I). Diagnosis was confirmed a by testing the cord blood samples after delivery and further follow-up of the children. In one family diagnosis could not be offered due to maternal contamination. No procedure related complications were observed. Conclusion: Flowcytometry offers rapid and sensitive method for prenatal diagnosis and genetic counseling for PID especially where facilities for molecular diagnosis are not available.

Laboratory Haematology Abstract No. 307 Comparative Analysis of Molecular Genetic and Conventional Cytogenetic Detection of Recurrent Genetic Translocations in Leukaemia J Priyadarshini, Neeraj Arora, Vivi M Srivastava, A Senthamizhselvi, M Sathya, Auro Viswabandya, Biju George, Alok Srivastava, Vikram Mathews, Poonkuzhali Balasubramanian Department of Haematology, Christian Medical College, Vellore Summary: The contemporary diagnosis of leukemia has moved on from the purely morphologic approach in FAB classification system to a multifaceted WHO 2008 classification system which utilizes morphologic, cytochemical, immunophenotypic, cytogenetic and molecular findings into consideration for proper classification. The techniques employed include karyotyping, fluorescence in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR). For a cost effective and rapid diagnosis of leukaemia, knowledge of the accuracy and efficiency of these techniques is essential in one particular setup. The aim of the study was to compare these three techniques for detection of common recurrent chromosomal translocations in leukemia. Materials and Methods:

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CML

APML

Ph+ ALL

t(12;21)

t(1;19)

t(4;11)

Total cases

307

301

176

116

62

16

Concordance RT-PCR/ Cytogenetics (%)

98.9

95

93.8

59.5

93.8

85.7

(186/ 188)

(192/ 202)

(136/145)

(50/84)

(45/48)

(12/14)

Discordance RT-PCR/ Cytogenetics (%)

1.1

5

6.2

40.5

6.2

16.7

(2/188)

(10/202)

(9/145)

(34/84)

(3/48)

(2/12)

Concordance RT-PCR/FISH (%)

99.3

97.3

98.6

83.3

0

100

(290/ 292)

(214/ 220)

(68/69)

(10/12)

Discordance RT-PCR/FISH (%)

0.7

2.7

1.4

16.7

(2/292)

(6/220)

(1/69)

(2/12)

(1/1) 0

0

This is a retrospective study carried out in the department of Haematology, Christian Medical College Vellore and included all newly diagnosed cases of leukaemia between January 1997 and June 2012 referred to the molecular laboratory where information about either karyotyping, and/or FISH were also available. Patients were diagnosed and sub classified according to the WHO classification 2008 based on morphology, immunophenotyping and cytogenetic analysis. Samples were processed using standard cytogenetic protocols. FISH was carried out by standard protocols and hybridization procedure. The type of aberration was determined in patients’ samples by nested qualitative RT-PCR based on the previously published Biomed Concerted Action Protocol. Results: During the study, a total of 978 newly diagnosed patients with leukemia who were positive for any of the fusion transcripts by RT-PCR, were included in this study; chronic myeloid leukaemia (CML) (n = 307), acute promyelocytic leukaemia (APML) (n = 301) and acute lymphoblastic leukaemia (ALL) (n = 370). Among the 370 ALL, the majority were BCR-ABL positive (n = 176), followed by TEL-AML1 (n = 116), E2A-PBX (n = 62) and MLL-AF4 (n = 16). Cytogenetic analysis was performed in 681 of the 978 (69.6 %) cases of which the translocations were not seen in 8.8 % (60/681) cases. FISH analysis was performed in 589 (60.2 %) cases of which the translocations were not seen in 1.9 % (11/593) of cases. Table 1 shows overview of the concordant and discordant results of karyotyping, FISH and RT-PCR in various tested aberrations. There was a single case of t(1;19) which was not detected by RT-PCR but was positive on karyotype analysis, probably due to the fact that peripheral blood sample used for RT-PCR while bone marrow is used for cytogenetic analysis. Conclusion: Karyotyping, FISH and RT-PCR are powerful tools for the detection of the major chromosomal abnormalities in leukemia. Although each method has its own limitations, the judicious use of these techniques will deliver the best cost effective treatment in our country.

Abstract No. 308 Comparison of Turbidimetric and Nephalometric Methods for Immunoglobulin Estimation Babu Pillai, Pratik Poladia, Hemali Dethe, Preeti Chavan, Vivek Bhat, Meera, Ghadge1, Nitin Inamdar1

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

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ACTREC, TMC, Kharghar, Navi Mumbai1, Tata Memorial Hospital, TMC, Parel, Mumbai

Department of Pathology, Lady Hardinge Medical College, New Delhi-1

Summary: This bicentric study was conducted at the biochemistry services, ACTREC and TMH. We analysed 50 patient samples for Immunoglobulin (IgG, IgM, IgA) estimation that had been assayed by nephelometry and turbidimetry. The results were analysed for correlation and bias. Introduction: Nephelometric and turbidimetric methods are used commonly for estimation of immunoglobulins & measure amount of ag-ab complexes formed. Nephelometry measures scattered light, turbidimetry measures transmitted light. Aim of this study was to determine the correlation between the results of the two tests and assess the suitability of using them interchangeably. Materials and Methods: We used Siemens Dimension RxL analyser for turbidimetric and Beckman Coulter Image for nephelometric studies. Results were analysed for correlation using Regression analysis and for bias using Bland Altman Plots. Results: ‘R’ value between nephelometry & turbidimetry for IgG, IgM and IgA were 0.985, 0.994 & 0.994 respectively. Bias was observed between the two methods as 9.5, -23 and 9 units for IgM, IgG and IgA respectively. Conclusion: The results of this investigation reinforce the findings that both nephelometry and turbidimetry are suitable techniques for the assay of immunoglobulins. The bias showed in the results of two methods should be evaluated and correlated clinically.

Summary: Coagulation testing is central to diagnosis of hemostasis disorders and is critical for monitoring of antithrombotic therapies. Usually hemolysed samples for coagulation studies are rejected. We have analysed the influence of blood cell lysis on routine coagulation testing. Introduction: The complexity of coagulation testing is most prone to pre analytical errors in the clinical laboratory. Hemolysis is one of the common reasons for rejecting specimens as it influence the reliability of coagulation tests. So if specimen is hemolyzed, a comment to the report can be added that result may be mildly affected and re-collection is recommended if this difference is affecting diagnostic or therapeutic decisions. With this approach, repeat sampling, especially in pediatric patients can be reduced. Materials and Methods: Blood samples from 50 patients for PT, APTT were collected in citrate vaccutainer and tested on CA-50 semiautomated coagulation analyzer. Blood samples were then mechanically lysed. Hemolysed plasma was analysed for hemoglobin and re-tested for PT, APTT. Results: Statistical analyses were performed using SPSS version 16, Chicago, Illinois, USA; p value of \0.05 was considered significant. Paired ‘t’ test was applied for comparison. The values of PT, APTT in pre and post hemolysis sample revealed no statistically significant difference (p = 0.3, 0.31 respectively). Conclusion: Certain hemolysed samples received for coagulation profile in emergency may not be always refused. Though ideally a non hemolysed sample is preferred, an attempt may be made to give a clue to the treating physicians, especially in pediatric patients and those having underlying cause of hemolysis.

Abstract No. 309 Comparison of Erythrocyte Sedimentation Rate Measurement by the Automated Vesmatic Cube 80 System and Conventional Westergren Method Seema Sharma, Preeti Agarwal Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow Summary: Although the conventional Westergren method for ESR remains the gold standard for measuring ESR, it is laborious, time consuming and involves high biohazard risks. Several automated closed systems like Vesmatic cube systems are now available that would overcome these drawbacks. Introduction: This study was done to compare the performance of Vesmatic cube 80 system (DIESSE Diagnostica),a fully automated analyzer for the measurement of the erythrocyte sedimentation rate (ESR), with the manual Westergren method. Materials and Methods: Both methods were applied to 104 randomly selected subjects. The linear regression and Bland and Altman data analysis methods were used to measure the agreement between the automated and manual methods. Results: The ESR measurement using Ves-Matic Cube 80 and Westergren methods from 104 patients were compared. Mean ESR value measured with Ves-Matic Cube 80 method was 60.8 and 63.5 mm/h by Westergren method. Pearson regression analysis showed a good correlation (r = 0.87). A greater scatter of data was observed with abnormally high ESR compared with normal readings on Bland and Altman data analysis. Conclusion: The Ves-Matic Cube 80 analyzer offers a fast determination of ESR and good correlation with reference Westergren method. This procedure allows the use of the same standard EDTA tube for multiple hematological analyses, thus avoiding biological hazard and reducing blood sample volume taken from the patient.

Abstract No. 310 The Effect of In Vitro Blood Cell Lysis on Routine Coagulation Tests Preeti Rai, Sunita Sharma, Kaushik Majumdar, Deepa

Abstract No. 311 Cytochemical Myeloperoxidase Staining Using 4-Chloro-1napthol is a Safe, Sensitive and Reproducible Alternative to Benzidine Di-hydrochloride Sarika Narkhede, Shashikant Mahadik, Rashida Ansari, Prashant Deshpande, Shaila Shinde, PG Subramanian, Nikhil Patkar, Sumeet Gujral, Prashant Tembhare Hematopathology Laboratory, Tata Memorial Hospital, Mumbai Introduction: Cytochemical detection of myeloperoxidase (MPO) is a strong indicator of myeloid differentiation. It is routinely used to differentiate between myeloid and lymphoid blasts. Benzidine-dihydrochloride is the main reagent used for cytochemical MPO staining; however, it is known to have potential toxic and carcinogenic effects and has been banned in many countries. 4-chloro 1-Naphthol (4C1N) is a known but poorly studied alternative for benzidine. In this study, we standardized MPO staining using 4C1N and compared the results with benzidine-based MPO staining. Materials and Methods: Air dried bone marrow and peripheral blood smears from 20 cases of acute leukemia [10 lymphoblastic, 10 AMLs (FAB—3 M1, 3 M2, 2 M3 & 2 M4)] and 20 normal individuals were evaluated for MPO activity using benzidine-based and 4C1N-based methods. Minimum 200 cells (granulocytes in normal individuals & or blasts in leukemia cases) were examined. Cut off of C3 % positivity is used. Results were validated with flow cytometric immunophenotyping in leukemia cases. Additional cases will be done. Results: In 20 normal individuals, 100 % neutrophils showed myeloperoxidase activity by both methods. Blasts from lymphoblastic leukemia showed no myeloperoxidase activity. Of the 10 cases, abnormal-promyelocytes showed extremely strong MPO-reactivity and blasts from remaining AMLs showed weak to moderate MPO-reactivity. Results of both methods revealed a very good correlation. Conclusion: 4-Chloro-1-Naphthol based MPO staining method is a safe, sensitive and reproducible

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384 alternative for determining cytochemical myeloperoxidase activity. It can easily replace the benzidine-based method.

Abstract No. 312 Review of External Quality Assessment Scheme (EQAS) of Clinical Biochemistry in an Oncology Setup

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 (59.29 %) were limited to the first 30 min. Most common delay was noted during lunch breaks and early mornings. Common source of delay was the pre-analytic phase (sample transport and pre-analytical processing).Significant improvement in TAT from year 2011 (8.02 %) to 2012 (5.4 %) was due increasing staff, regular audit and corrective actions. Conclusion: Regular audit of data helps in evaluation of the efficiency of laboratory which is then followed by appropriate measures that lead to improvement in service to patients and clinicians.

Pratik P Poladia, Babu G Pillai, Umakant Gavhane, Preeti Chavan, Vivek Bhatt TATA Memorial Centre ACTREC, Kharghar, Navi Mumbai Summary: The Biochemistry lab at ACTREC, TMC participates in the External Quality Assurance Scheme (EQAS) conducted by BIORAD. We perform EQAS for 18 parameters in our laboratory and is one of the essential requirements for accreditation. The results of two cycles were evaluated; twelve lyophilized serum samples were received in each cycle, one every month. Corrective action was taken against the parameters that have gone out of ±2SDI (Standard Deviation Index), The samples were also observed for trend/bias if any. Introduction: Proficiency Testing is one of the important tools to determine the technical competence of the testing laboratories. The Biochemistry EQAS program that we participate in is conducted by BIO-RAD and is used to evaluate quality and reliability of tests performed by clinical labs. More than 250 labs participate in our peer groups depending on the equipment infrastructure and methodologies used in the labs. Materials and Methods: We evaluated 24 EQAS samples for Siemens Dimension-Xpand analyser and 12 samples for Siemens RXL analyser for past 2 cycles (June 2011–May 2013). Test results of 18 routine parameters were analysed and documented. Results: Discordant results were observed for some parameters having Standard Deviation Index (SDI) more than ±2 SDI and corrective actions were taken. Conclusion: Regular participation in EQAS has helped us improve our clinical services in terms of performance, patient care and overall lab quality. We recommend active participation in the EQAS program to ensure greater accuracy & better quality of laboratory results.

Abstract No. 313 Determination of Turn Around Time (TAT) in NABL Accredited Hematology and Clinical Laboratory

Abstract No. 314 Analysis of External Quality Assessment Scheme (EQAS) Results of Clinical Hematology in an Oncology Setup Umakant Gavhane, Sanjay Kumar, Manikchandra Tiwari, Ulka Gosavi, Preeti Chavan, Vivek Bhat, Chital Naresh TATA Memorial Centre, ACTREC, Kharghar, Navi Mumbai Summary: The composite laboratory (Haematology) at ACTREC, TMC participates in ISHTM-AIIMS EQAP Programme. Corrective actions were taken on outliers. For assessment results 2010–2013 trend analysis was done. Overall this study gives an account of participation in EQAS being a very important tool in monitoring the quality of the laboratory practices. Introduction: Proficiency Testing is one of the important tools to determine the technical competence of the testing laboratories. Participation in EQAS provides laboratories with an objective demonstration of reliability of the data they produce. In May 2013 out of 200 participants, 86 % responded with their results. Z-score is given as assessment score for the results among the laboratories (EQA) and within the laboratory (IQA). Materials and Methods: In four years, fourteen EQAS samples-blood, peripheral and reticulocyte smears were analyzed. Corrective actions taken for Z-score [3. Result: Result of May-2012 showed Z-score for WBC-4.24, MCV-3.19. This sample had deteriorated hence no corrective action could be taken. In March-2010, Z-score for MCV-3.04 and Reticulocyte count-4.27 and corrective action comprised calibrating analyzer for MCV and training staff for reticulocytes screening. All other results of EQAS showed satisfactory Z-score. Conclusion: Regular participation in EQAS has helped us improve our clinical services in terms of performance, patient care and overall lab quality practice. Therefore we recommend active participation in the EQAS program to ensure greater accuracy & better quality of laboratory results.

Killol Nathubhai Desai, Krishna Patel, Megha Shah, Mustafa Ranapurwala, Menka Shah Pramukhswami Medical College, Shree Krishna Hospital, Karamsad388325 Summary: The study was performed to analyze Turn Around Time (TAT) in NABL accredited hematology and clinical laboratory. Number of samples and the reasons for delay were analyzed. Increasing staff, regular audit and corrective actions lead to improvement in TAT. Introduction: Along with accuracy and reliability, timely reporting of laboratory test result is now considered an important aspect of service provided by a laboratory. The aim of this study is to evaluate the cause of delay in TAT and scope for improvement. Materials and Methods: This was a retrospective study from January 2011 to December 2012, where the TAT of stat tests were analyzed. TAT was specified as the time from sample collection to final dispatch of report. The delay was categorized in to 0–30 min, 30 min–1 h, 1–2 h, 2–5 h, 5–10 h and [10 h. Reasons and time of the day in which delay were looked into. Results: Total 102,331 samples were received, out of which 6,989 (6.8 %) samples were reported out of range for acceptable TAT. Most delays

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Immunodeficiency Syndromes Abstract No. 315 A Case Report of Chediak-Higashi Syndrome Vikram Raj Gopinathan, Garima Rakheja, Richa Gupta, Tejinder Singh, Seema Kapoor Department of Pathology and Paediatrics, Maulana Azad Medical College, New Delhi Summary: Chediak-Higashi syndrome (CHS), an autosomal recessive disorder, is characterized by immunodeficiency, oculocutaneous albinism, bleeding disorder and progressive neurological disease. A characteristic feature is the presence of large granules in lysosomes of many cell types. A case of 5 months female presenting with ChediakHigashi syndrome is presented. Introduction: Chediak-Higashi syndrome is a rare disease with diverse clinical manifestations. Patients have decreased pigmentation, giant intracellular granules which are

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 pathognomonic, a bleeding disorder related to platelet dense bodies which are reduced or absent in number and progressive neurodegeneration. Granules are azurophilic and positive for myeloperoxidase and acid hydrolases. They are primarily seen in neutrophils and also in eosinophils, basophils and monocytes. There is neutropenia and platelet count is normal. Materials and Methods: A 5 month female presented with albinism, recurrent respiratory tract infections and silvery hair. Initial blood counts suggested neutropenia. On peripheral smear examination, intracellular granules were seen in neutrophils, monocytes, eosinophils and also in lymphocytes. These granules were MPO positive. Bone marrow examination also revealed coarse granulation of cells of myeloid series, which was MPO positive. Results: Based on the above clinical features and the presence of MPO positive large intracellular granules in leucocytes, a diagnosis of Chediak Higashi syndrome has been made. Conclusion: Though the disorders of lysosome related organelle biogenesis and trafficking like Chediak-Higashi sydrome are rare, they should be kept as part of differential diagnosis whenever a patient presents with defective pigmentation, bleeding tendency and immunodeficiency.

Abstract No. 316 Accelerated Phase of Chediak—Higashi Syndrome: A Case Report A Deshmukh, K Ramanan, P Nayar, R Manchanda, A Bawdekar Department of Hematology, K.E.M. Hospital, Pune 411011 Summary: 11 year male child, presented with oculocutaneous albinism and cervical lymphadenopathy. Hemogram showed lymphocytosis. PBS and bone marrow aspirate smears showed giant inclusion bodies in lymphocytes and cytoplasmic vacuolations in neutrophils. Trephine biopsy showed lymphohistiocytic collections with few plasma cells and eosinophils. Hair study, Histopathological examination and immunohistochemistry of cervical lymph node will be discussed. Introduction: Che´diak-Higashi syndrome (CHS), a rare childhood autosomal recessive disorder, results from defective regulation of fusion of primary lysosomal granules and is characterized by oculocutaneous albinism, repeated infections and peripheral neuropathy. 50–80 % of patients with CHS enter into ‘‘accelerated phase,’’ manifested by fever, jaundice, hepatoslenomegaly and lymphohistiocytic organ infiltrate. Materials and Methods: Detail clinical history and examination. Hemogram, thorough PBS study, bone marrow aspirate and biopsy examination, cervical lymph node biopsy immunohistochemistry and hair study. Results: 11 years male child presented with oculocutaneous albinism and bilateral cervical lymphadenopathy. Hemogram revealed mild lymphocytosis. Giant inclusion bodies in lymphocytes and cytoplasmic vacuolations in neutrophils were noted on PBS and marrow smears. Bone marrow trephine biopsy showed lymphohistiocytic collections comprising of lymphocytes, plasma cells, and few eosinophils. Hair study, Histology and immunohistochemistry of lymph node will be discussed. Conclusion: This case emphasizes the importance of careful examination of well prepared and stained blood film by an experienced morphologist and also recognizes the potential of CHS to enter into an ‘‘accelerated phase’’.

Abstract No. 317 Accelerated Phase of Chediak Higashi Syndrome Mimicking Lymphoma: A Case Report Divesh Goyal, K Parikh Sonia, N Shukla Shilin, S Talati Shailesh, S Anand Asha, Shah, A Sandip, P Panchal Harsha, A Patel

385 Apurva, B Parikh Bhavesh, Maniar, Vashistha, Gohel Vandana, Raut Shreeniwas Department of Medical and Pediatric Oncology, Gujarat Cancer & Research Institute, Ahmedabad Introduction: Chediak Higashi Syndrome (CHS) is a rare autosomal recessive disease, characterized by partial oculocutaneous albinism, frequent pyogenic infections, and abnormally large granules in the leukocytes. About 50–85 % of patients with CHS enter into an ‘‘accelerated phase,’’ manifested by fever, jaundice, hepatosplenomegaly, lymphadenopathy and widespread lymphohistiocytic organ infiltrates with hemophagocytosis, leading to pancytopenia, hypertriglyceridemia and bleeding disorders. The first accelerated phase of CHS may occur shortly after birth or several years later. Most patients undergo a variable period of recurrent infections before going into the accelerated phase. Therefore, primary presentation in the accelerated phase is unusual. This case was referred to our institution with a suspected diagnosis of lymphoma/leukemia. Case Report: In our case a 7 years old boy presented with complaint of right iliac fossa pain & fever & was diagnosed as appendicular perforation & underwent appendicectomy. On third postoperative day patient developed high-grade fever and pus discharge from operative site. Peripheral smear was suggestive of pancytopenia & giant granules in neutrophils & referred. Past history revealed recurrent fever & respiratory tract infection for 6 years. Examination revealed silvery hair, pallor, anasarca, subcentimeter size submandibular, axillary lymphadenopathy, hepatosplenomegaly, bilateral iris hypopigmentation and pale retina. On investigation peripheral smear revealed pancytopenia, giant granules in neutrophils with reduced TLC and platelets, deranged LFT, hypertriglyceridemia. USG abdomen and pelvis revealed hepatosplenomegaly with no infiltration & free fluid in peritoneal cavity. Lymphnode biopsy was not possible. The bone marrow aspirate & biopsy revealed giant pink cytoplasmic granules. Histiocytes arranged in sheets with pale foamy cytoplasm and engulfing intact red blood cells and fragmented erythrocytes, both features consistent with CHS and Haemophagocytic Lymphohistiocytosis (HLH) were seen. On the basis of above finding, a diagnosis of accelerated phase of CHS was made. The patient was treated with antipyretics, antibiotics and stem cell transplantation was suggested and put on close surveillance for lymphoma. Conclusion: A patient presenting with oculocutaneous albinism and recurrent infections, careful examination of the peripheral blood smear cannot be overemphasized. A high degree of awareness and early recognition of the syndrome, could lead to the institution of the only possible curative treatment, bone marrow transplant, before the accelerated phase supervene.

Abstract No. 318 Mutation Analysis of ITGb2 Gene in Patients with Leukocyte Adhesion Deficiency Anju Mishra, Sushant Chavan1, Maya Gupta1, Mukesh Desai2, Kanjaksha Ghosh1, Manisha Madkaikar1 National Institute of Immunohematology, 2Bai Jerbai Wadia Hospital, Mumbai

1

Introduction: Leukocyte adhesion deficiency-I (LAD-I) is an autosomal recessive disorder caused by mutations in the ITGb2 gene, encoding the beta2 integrin family, which leads to markedly reduced expression of CD18 & CD11 on leukocytes. This study was undertaken with aim to identify the molecular defects underlying LAD-I in Indian patients with and to develop a strategy for mutation detection in these patients and utilize it for prenatal diagnosis. Materials and Methods: The entire coding region and intronic splice sites of the

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386 ITGb2 gene were sequenced from the genomic DNA of 20 patients diagnosed with LAD-I on the basis of clinical manifestations and markedly reduced expression of CD18 and CD11 on peripheral blood leukocytes. Results: In 20 LAD-I cases genetic screening of entire coding region and intronic splice sites of the ITGb2 gene revealed 6 novel mutations in 10 cases and 7 known mutations in 10 cases encompassing exon 5–13. Seventeen patients carried homozygous, one patient carried compound heterozygous mutation and two were partially characterized with heterozygous mutation. Several types of mutations were detected viz. missense, deletion/insertion, nonsense and splice site defects. Conclusion: High heterogeneity in mutational profile has been observed in the present study with no association with ethnicity of the patient.

Abstract No. 319 Characterization of CD40LG Gene in Patients with X-Linked Hyper IgM Syndrome Aparna Dalvi1, Sushant Chavan1, Anju Mishra1, Maya Gupta1, Mukesh Desai2, Kanjaksha Ghosh1, Manisha Madkaikar1 1 National Institute of Immunohematology, 2Bai Jerbai Wadia Hospital, Mumbai

Introduction: Hyper-Immunoglobulin M (HIGM) syndrome is a heterogeneous group of primary immunodeficiency disorders, characterized by recurrent infections associated with decreased serum levels of IgG, IgA and IgE and normal to increased levels of IgM. Mutations in five genes have so far been associated to the disease; of these X-linked HIGM caused due to CD40 ligand (CD40LG) mutations is the most common. The aim of this study was to analyze the nature of mutations in patients diagnosed with X-linked HIGM. Materials and Methods: The entire coding region and intronic splice sites of the CD40LG were sequenced from the genomic DNA of 4 patients diagnosed with X-linked HIGM. Results: We identified four distinct mutations of which three were novel and one was previously described. Patient P1, P2 & P3 harbored three novel non sense mutations viz. p.Asn58IleX8, p.Arg77X in exon 2 & p.Gln160X in exon 5 respectively. Patient P4 was detected with known splice site mutation c.346 ? 2T [ C in exon 3. All of these mutations abruptly affected expression of CD40LG. Carrier status was confirmed in 2 out of 3 mothers. In one family mutation was not detected in the mother thereby suggesting either a spontaneous mutation or germline mosaicism. Conclusion: Pattern of mutations detected was different from that reported in the western literature. Molecular characterization of HIGM is important for detection of carrier status and further genetic counseling and prenatal diagnosis in affected families.

Infections in Haematology/Oncology Abstract No. 320 Tuberculosis in Pediatric Oncology—A Single Centre Experience K Anand Kumar, Vasant Chinnabhandar, Nita Radhakrishnan, Anupam Sachdeva Pediatric Hematology Oncology-Sir Ganaga Ram Hospital Summary: The etiology of fever in pediatric oncology patients are multiple. Our experience shows that after evaluating for commoner causes, one should possibly explore the possibility of tuberculosis. Introduction: Tuberculosis (TB) is an important differential diagnosis of children with malignancies who have uncontrolled persistent

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 fever, especially in the setting of high prevalence as in developing countries. Materials and Methods: Retrospective analysis of data of TB in pediatric-oncology patients admitted at our hospital between 2005–2012. The diagnosis was made on clinical and standard laboratory tools {Biopsy,Quantiferon-gold test, Adenosine-deaminase levels (ADA)}. Results: Eight cases of TB were diagnosed among 800 newly diagnosed cases of childhood cancer. The median age was 5.5 (2.9–13.5) years. The underlying diseases were five acute lymphoblastic leukemia, two acute myeloid leukemia and one Hodgkins lymphoma. The disease categories were two pleuro-pulmonary-TB, one cutaneous-TB, one hepatic-TB and one disseminated-TB. None had a positive family history of TB. The clue of TB was persistent fever in all except for one who was incidentally diagnosed during the pre-stem cell transplant investigations, one had skin eruptions and three had chronic cough with chest pain. We diagnosed one case by AFB positive on liver biopsy, another by positive quantiferon gold with elevated pleura-fluid-ADA, skin biopsy of another showed erythema nodosum and the rest clinically. We treated all cases with 4 drug anti-tubercular treatment (ATT). There were 3 therapy-related mortalities, not attributable to TB. One child has completed treatment and is off ATT for 6 months, other two are on ATT, one had a very early CNS relapse and the other is doing well. Conclusion: Tuberculosis is an uncommon but significant cause of PUO in the oncology setting, especially hemato-lymphoid malignancies.

Abstract No. 321 Detection of Oral Herpes Simplex Virus 1 and 2 in Children on Chemotherapy: A Comparison of Serology, Conventional PCR and Quantitative Real-Time PCR Deepak Bansal, Ritu Aggarwal, Deepak Bansal, Jasmine Naru, Manila Salaria, Ranjana W Minz, Amita Trehan1, RK Marwaha1 Department of Immunopathology and Pediatric HematologyOncology Unit, Advanced Pediatrics Centre, Post Graduate Institute of Medical Education and Research, Chandigarh Summary: Prevalence data of herpes simplex virus (HSV) in oral mucositis in children on treatment for cancer is limited. QuantitativePCR has been seldom utilized for detection of HSV-1/2 in oral mucosa. Introduction: The aim was to study the prevalence and compare diagnosis of HSV 1 and 2 in oral mucosal lesions of children receiving chemotherapy by serology (ELISA), conventional and realtime PCR. Materials and Methods: Children on treatment for cancer with oral mucositis were enrolled as cases and healthy children as controls. An oral swab from the lesion in cases and scape material in controls was obtained. Both qualitative and real-time quantitative PCR for HSV-1/2 were performed. Serum ELISA-IgG/IgM for HSV1/2 antibodies (NovaLisaTM-Dietzenbach-Germany) were measured. Study was funded by Indian Council of Medical Research, N Delhi. Results: 32 cases (age: 6.3 ± 3.4 years) and 30 controls were enrolled. Majority (69 %) of cases had ALL. All patients had febrile neutropenia, except two. ELISA-IgM-HSV-1/2 was not positive in any case or control. ELISA-IgG-HSV-1/2 was positive in 11 (34 %) cases and 9 (30 %) controls (p = 1.0). Qualitative-PCR for HSV-1 detected the virus in 8 (25 %) cases and nil controls (p = 0.009). HSV-2 was not detected in any case/control by qualitative-PCR. Quantitative-PCR detected HSV-1 in 21 (66 %) and HSV-2 in 22 (69 %) cases. In controls, quantitative-PCR detected HSV-1 in 3 (10 %) and HSV-2 in none. In patients, the mean viral load of HSV-1 (5,500 ± 15,987 9 104 copies/nano-gram DNA) was more than HSV-2 (4.03 ± 8.5 9 104) (p = 0.11). There was no correlation of HSV-1/2 with grading of mucositis. Conclusion: Both HSV-1 and 2 are commonly shed from oral mucosal lesions in children receiving chemotherapy. In a novel finding, real-time PCR detected copies of

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 HSV-2 in 69 % cases, all missed by conventional PCR. Implication for morbidity, if any, or treatment, needs to be determined.

Abstract No. 322 ‘‘Uro’’philic Virus Troubling Bone Marrow: An Unusual Presentation & a Successful Diagnosis Prachi S Kate, Nita Munshi, Vaishali Patil, Sohini Arora, A Sadre Grant Medical Foundation, Ruby Hall Clinic, Pune Summary: In post renal transplant patients, B K polyoma virus infection is known to present as pyuria, renal dysfunction, haemorrhagic cystitis or it may be asymptomatic. Here, we had a case which presented only with unexplained severe anaemia. Etiology was diagnosed on bone marrow aspiration & confirmed with RT PCR. The patient was treated for the same & anemia got treated successfully. Introduction: We received bone marrow aspiration of 29 years old female with history of severe anaemia, a month after renal transplant. The renal function tests were normal throughout the follow up. No h/o oliguria, edema. No h/o hematuria, fever, malaise. Materials and Methods: This is a case report. The case was referred to hematologist by nephrologist for post transplant unexplained anemia without any other symptoms & with perfectly normal renal function tests, electrolytes etc. The etiology was diagnosed in hematology lab. The instruments & methods used were as follows: The bone marrow aspiration (with Leishmann & geimsa stain); CBC (COULTER LH 750); Peripheral blood smear (Leishmann & Geimsa stain); Reticulacytes count (Supravital stain); Viral load (Rosch Taqman by RT PCR). Results: The bone marrow aspiration showed erythroid hyperplasia with dyserythropoesis, increased number of erythroblasts & promyelocytes showing prominent intranuclear inclusions. In view of intranuclear inclusions, viral load for parvo virus, BK polyoma virus & CMV was done. Viral load came positive for B K polyoma virus. Other investigations were: CBC-Hb: 6.7 g/dl; Hematocrit-20.2; RBC count-2.4 9 106/mm3; RDW, WBC count, differential count, platelet count were within normal range. Peripheral blood smearNormocytic normochomic anaemia; No anisocytosis; No schistocytes. Reticulacytes count: 0.3 %. Conclusion: B K polyoma virus may present as severe anemia without any urinary symptoms. Bone marrow aspiration is a simple & cheap method to get a clue of bone marrow infestation B K polyoma or related virus.

Abstract No. 323 Detection of Bacteria by Using Pan-Bacterial Primers in Patients with Bone Marrow Failure of Various Disease Conditions in Adults Lingaraj Nayak, KK Gupta, G Nath, Arun Sarvanan Institute of Medical Science, BHU, Varanasi Summary: In our study we found M. tuberculosis DNA in significant number of cases of bone marrow failure syndrome(aplastic anemia and MDS) cases. Introduction: A very high incidence rates of Bone marrow failure/aplastic anaemia (AA) are reported from Uttar Pradesh, Bihar and Delhi/NCR states. The exact agent from environment which is responsible for this is still a mystery as 70 % cases are idiopathic. This study was conducted to find out the bacterial association with marrow failures. Materials and Methods: 32 cases of bone marrow failure [20 years including aplastic anemia and MDS were enrolled in the study. Vis-a-vis 20 healthy age & sex matched control were taken from orthopaedic department. Pan bacteria primer was used for nested PCR in 32 samples of bone marrow failure.

387 Simultaneously nested PCR for S. typhi & M. tuberculosis was also done in all samples. For statistical analysis, Z-test and Fisher exact test was used. Results: 44 % patients with aplastic anemia were positive for M. tuberculosis whereas none of the controls were positive for M. tuberculosis which is quite significant (p \ 0.001). 85.7 % patients with MDS were positive for M. tuberculosis whereas none of the controls showed M. tuberculosis positivity and was quite significant (p \ 0.001). Panbacteria DNA was positive in all samples. Conclusion: In our study we found M. tuberculosis DNA was found in significant number of cases (p \ 0.001). we can make a hypothesis that tuberculosis could be the main infectious trigger for the development autoimmunity in this part of our country against bone marrow in patients of bone marrow failure in India.

Abstract No. 324 Review of Clinical Profile and Bacterial Spectrum and Sensitivity Patterns of Pathogens in Febrile Neutropenic Patients in Hematological Malignancies: A Retrospective Analysis from Single Centre Kaushal Patel, Arun Karanwal G C R I, Ahmedabad Summary: Febrile neutropenia (FN) is a common complication of cancer treatment. With the development of more aggressive chemotherapy regimens for haematological malignancies, the survival of these patients have improved. At the same time these patients frequently succumb to febrile neutropenia, which is the major cause of morbidity & mortality. Studies have reported that 48–60 % of patients admitted with febrile neutropenia have an infection, which can be life threatening as well. Introduction: The aim of this study was to study clinical profile with bacterial spectrum & susceptibility patterns of pathogens in culture positive febrile neutropenic (FN) patients of haematological malignancies. Materials and Methods: We retrospectively reviewed the medical records of 23 hematological malignancy patients admitted with chemotherapy induced febrile neutropenia with culture positive results, at our institute between June 2011 to December 2011. Results: A total of 23 patients were studied, 12 males & 11 females, with median age of 35 years. Most common diagnosis was acute leukemia (78 %) with majority of patients developing febrile neutropenia during induction phase of treatment. Most common presenting symptoms were fever, cough, dyspnea & diarrhoea. Primary site of infection was not found in 47 % of patients, while rest had lung, gastro-intestinal & skin/soft tissue infection. Overall 23 organisms were isolated during the study period, from blood (56 %), sputum (46 %), stool (23 %), & nasal swab from 1 patient. Gram negative bacteria accounted for 78 % of organisms, while gram positive organisms accounted for 22 % of the total isolates. The most common organisms were: Escherichia coli (43 %), Staphylococcus aureus (22 %), Pseudomonas aeruginosa (17.4 %) and klebsiella pneumonia (17.4 %). Antibiotic sensitivity patterns of these bacteria were studied. Gram negative bacterial infections were associated with higher mortality (89 %). Conclusion: Induction phase of treatment in acute leukemias is the major cause of FN in haematological malignancies at our institute & gram negative organisms are the predominant organisms with E coli as major isolate while staphylococcus aureus represents most common gram positive organism. Amikacin & Cefoperazone/Sulbactum (C/ Su) appears to be initial antibiotic appropriate to cover most gram negative pathogens while vancomycin to be added for suspected gram positive infections. FN represents a major cause of morbidity & mortality in haematological malignancy patients, high index of suspicion & early empirical antibiotics with supportive care are main interventions to reduce high mortality for these patients. Antibiotics

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388 should be modified according to culture sensitive report as soon as possible.

Abstract No. 325 Antibiotic Resistance: High Prevalence of Resistant Bacteria in Fecal Surveillance Cultures of Patients from a New Tertiary Care Cancer Centre Vivek Sulekha Radhakrishnan, Gaurav Goel, Dipmala Das, Sriram Ravichandran, Vishvdeep Khushoo, Saurabh Jayant Bhave, Anupam Chakrapani, Reena Nair, Sanjay Bhattacharya, Mammen Chandy Department of Clinical Hematology, Tata Medical Center, Kolkata Introduction: In India, infections in the critical care setting are more often due to drug resistant gram negative organisms. Surveillance Cultures on admission, is part of a multi-factorial intervention to control MDRO (Multi-Drug Resistant Organisms). Colonization surveillance may have the potential to improve empiric antimicrobial treatment in a critical care setting. Materials and Methods: Design: Retrospective. Setting: Newly started Comprehensive Cancer Center in Eastern part of India. Period: Nov 2011–Jan 2013. Review of fecal surveillance cultures of patients undergoing intensive chemotherapy for hematological cancers or haemopoietic stem cell transplant. Fecal Surveillance cultures were done prior to Chemotherapy using a method based on Landman et al. (J Clin Microbiol 2005). Results: 49 patients (36 male and 13 female) admitted with diagnosis of AML (22), ALL (6), APL (1), Acute Mixed Lineage Leukemia (1), MDS (2), Hodgkin Lymphoma (1), DLBCL (1), Fanconi Anemia (1), Multiple Myeloma (4), and Myeloproliferative Disorder (1) had information on Fecal Surveillance Cultures taken prior to intensive chemotherapy. Mean age: 35 years (range; Male: 6–67 years, Female: 10–59 years). Total Samples collected: 67. Median sample per patient: 1 (range 1–4). All samples excepting one, grew MDRO isolates (98.5 %). No. of Isolates: 136 [95 gram negative bacilli (GNB), 41 gram positive cocci (GPC)]. Most common GNB: Escherichia coli (57.9 %). Most common GPC: Enterococcus family. Among GPC isolates, resistance to Ampicillin, Vancomycin, High level Gentamycin, and Linezolid were 86.5 % (32/37), 7.3 % (3/41), 60.9 % (25/ 41) and nil (0/41) respectively. Among GNB isolates, resistance to Cefotaxime, Ciprofloxacin, Co-Amoxiclav, Gentamycin, Amikacin, Piperacillin-Tazobactum, Meropenem and Colistin were respectively 90.8 % (79/87), 67.4 % (60/89), 67.4 % (60/89), 45.2 % (43/95), 23.4 % (22/94), 39.7 % (37/93), 25.8 % (24/93) and 6.4 % (5/78). Conclusion: Background antimicrobial resistance in the enteric flora is highly prevalent in patients receiving intensive chemotherapy at our centre. The causes of widespread MDRO in Indian settings need further studies.

Abstract No. 326 A Randomized, Multi Centre, Open-Label Study to Evaluate the Efficacy and Safety of Peg G-CSF as Compared to GrafeelÒ in the Prophylaxis of Severe Neutropenia in Cancer Patients Receiving Cytotoxic Chemotherapy Anupama Ramkumar, Ramesh Nimmagadda, SS Nirni, Tazeen AIdris, Arun Anand Dr. Reddy’s Labs, Biologics, Hyderabad Introduction: Pegfilgrastim is a pegylated form of filgrastim and is a colony-stimulating factor (CSFs) for the prophylactic use during chemotherapy to reduce the risk and severity of severe and febrile

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 neutropenia. Pegfilgrastim has a sustained effect on granulopoiesis compared to filgrastim because of a longer half life. Materials and Methods: This multicenter, open label, randomized study sought to establish the safety and efficacy Peg G-CSF compared with GrafeelÒ over six cycles in 162 adult chemo-naı¨ve patients with advanced (stage III B and IV) unresectable non small lung cancer (NSCLC) or breast cancer eligible to receive suitable cytotoxic chemotherapy as a first line treatment. Efficacy of Peg G-CSF or GrafeelÒ prophylaxis was measured by duration of severe neutropenia (DSN), neutrophil nadir and recovery in cycle 1, and incidence of severe and febrile neutropenia over 6 cycles. Pharmacokinetic parameters and immunogenicity was assessed. Results: The mean DSN at cycle 1 was comparable between peg-G-CSF and GrafeelÒ (1.9 and 2.6 days respectively) and comparable to data published previously for pegfilgrastim (1.8 days). In cycle 1, the incidence of severe neutropenia was 64.4 % (grade 3 and 4) and 41.4 % (grade 4) in the Peg G-CSF arm which recovered in 0–3 days as compared to 65.9 % (grade 3 and 4) and 56.8 % (grade 4) in the GrafeelÒ arm. Median time to ANC recovery in days was found to be 9, 10 and 16 days for patients enrolled in Peg G-CSF, GrafeelÒ and no prophylaxis arm in cycle 1. ANC nadir was higher for Peg G-CSF arm and median day of ANC nadir and median time to ANC recovery were comparable for Peg G-CSF and GrafeelÒ arms. Plasma concentrations of the study medication declined when neutrophil recovery commenced. Treatment emergent related adverse events were similar to as reported in literature. Conclusions: The safety and efficacy of PegG-CSF was comparable to GrafeelÒ in this study. The advantage of administering this drug as a single dose in each cycle would have significant advantages in patient compliance and cost effectiveness of therapy.

Transfusion Medicines Abstract No. 327 Knowledge, Attitude and Practice of Transfusion Medicine Amongst Resident Doctors: A Survey Based Study Sudeep Kumar, Joseph Philip, T Chatterjee, RS Mallhi Department of Immunohematology and Blood Transfusion, AFMC, Pune-40 Summary: Clinicians have active role in decision making for blood transfusion. To assess awareness and transfusion medicine knowledge for resident doctors, a self administered anonymous survey was conducted on resident doctors from different clinical specialities. The overall mean score for correct response was found to be 46.6 %. A mandatory two weeks training for residents doctors of clinical speciality, is recommended in transfusion medicine. Introduction: Clinician’s knowledge of transfusion medicine (TM) may have profound impact on transfusion outcomes. Variations from the standards in practices in blood transfusion may jeopardize patient care. We assessed the essential knowledge of transfusion medicine in resident doctors and explored methods to improve their knowledge of the subject in order to have a better connect between clinician and TM. Materials and Methods: A descriptive cross sectional study using a self administered anonymous survey was conducted. A questionnaire comprising of 45 items and a feedback was developed to assess the essential knowledge of transfusion medicine for 80 resident doctors from various clinical specialties. Results: Survey revealed an overall mean score of 46.6 % for correct responses. None of the residents groups showed significant difference over the other. Lowest knowledge was found for tests and procedures carried out in the blood bank. Conclusion: Our study shows that majority of resident doctors have bare essential knowledge of transfusion medicine. A mandatory two

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 weeks training for all residents from clinical specialties, is recommended in transfusion medicine which will definitely connect our clinician to transfusion medicine better, and ultimately result in improved patient care.

Abstract No. 328 Appropriate & Inappropriate Use of Blood Product in a Tertiary Care Hospital: A Retrospective Study Ansuman Sahu, Smita Mahapatra, Dilleswari Pradhan, Sushreeta Patra Department of Transfusion Medicine, SCB Medical College, Cuttack Summary: A total 9,380 number of blood and blood product unit request forms are analyzed during three months showed high rate of inappropriate use in whole blood and components in various wards. This can be minimized by prospective controlled clinical trials. Introduction: To determine the retrospective monitoring of inappropriate use of whole blood & blood components (Packed red cell, Random donor platelets, Fresh frozen Plasma) for a period of 3 months. Materials and Methods: This study was conducted in SCB Medical College & Hospital Blood Bank, Cuttack in 9,380 whole blood & component units from March to May, 2013 with blood product request forms specifying the whole blood & component with their clinical diagnosis in various specialties and superspecialties wards. 4,425 units of whole blood, 1,356 units of packed cells, 1,849 units of Random donor platelet (RDP) & 1,750 units of Fresh Frozen Plasma (FFP) were analyzed in total. Results: Maximum percentage in use of packed cell (Out of whole blood and packed cell) was observed in Haematology ward followed by Paediatrics ward. 77.4 % of FFP was inappropriately used in Surgery. FFP most appropriately used in Obstetrics and Gynaecology ward (40.7 %). For RDP maximum inappropriate use was accounted in Surgery and most appropriately used in Cancer ward. Conclusion: Retrospective study of the blood products units shows high rates of inappropriate use of whole blood, FFP & RDP in various surgical wards possibly reflect uncertainty of appropriate laboratory criteria for component transfusion which could have been reduced by prospective controlled clinical trials.

Abstract No. 329 Iron Status in Regular Blood Donors: An Emerging Problem Ajit C Gorakshakar, Pradnya Kamble, Vandana Pradhan, Jayashree Sharma, Kanjaksha Ghosh National Institute of Immunohematology, Department of Transfusion Medicine, KEM Hospital, Parel, Mumbai 400012 Summary: In the present study iron status of the voluntary blood donors was evaluated by estimating their serum ferritin level. Genderwise comparison of serum ferritin levels showed that females are more prone of getting anemia than male donors. With increase in number of donations body’s iron stores decreases although hemoglobin level remains acceptable. This study strongly recommends that regular voluntary donors especially females should receive iron supplementation to prevent iron deficiency and depletion of iron stores. Introduction: India with a huge population of over one billion is lagging behind in blood collection with a shortage of about 2 million bags per year. Therefore there is an urgent need to fill up this gap. However regular blood donations by the donors can lead to

389 preclinical iron deficiency as well as iron deficiency anemia. Therefore donors should be evaluated to see the impact of regular blood donations on their iron status. Materials and Methods: 500 voluntary blood donors (399 males and 101 females in the age group of 18–55 years) from Mumbai were included in this study. Prior to donation proforma including age, sex, number of donations in the previous year, past and present illness, whether ready to take iron supplement was collected from each donor. Their hemoglobin estimation and other RBC indices were done on automated cell counter. Serum ferritin was estimated by indirect ELISA. The donors were divided into four groups depending on the number of donations in the previous year. Results: 8.4 % of males and 43.7 % of females were having hemoglobin less than the cut off value for donation. First time donors had higher mean serum ferritin levels than that in repeat donors. First time female donors had lower mean serum ferritin level than that in first time male donors. Increase in number of donations was associated with significant decrease in serum ferritin level. 5 % of the donors were already having anemia while 28 % of the donors showed iron deficiency at the time of donation. 23 % of the donors were ready to take dietary advice for replenishing their iron stores while only 5 % were ready to take oral iron supplements. Conclusion: Copper Sulphate test for hemoglobin estimation has poor sensitivity. Evaluation of serum ferritin in both first time and repeat female donors must be done before their phlebotomy. The use of parameters that reflect iron status more accurately like serum ferritin level must be included as a part of donor assessment programme.

Abstract No. 330 The External Quality Assessment Scheme: Five Years of Experience as a Participating NABH Accredited Blood Bank Krishna Patel, Killol Nathubhai Desai, Megha Shah, Kirti Rathod, Monica Gupta, Menka Shah A.D. Gorwala Blood Bank, Shree Krishna Hospital, Karamsad, Anand, Gujarat Summary: We participated in External Quality Assessment Scheme (EQAS) during the year 2008–2012 as per recommendation by National Accreditation Board for Hospitals & Healthcare Providers (NABH). We present our five years’ experience. Analyses of results have helped us to significantly improve our transfusion service. Introduction: Quality assurance in blood bank includes active participation in EQAS. Such programme offers valuable benefits in terms of performance evaluation, improvement in patient care and safety and overall quality of blood bank practices. Materials and Methods: For EQAS, we receive serum samples, from Baroda for HIV1/2 six monthly, from Bombay for all transfusion transmitted disease (TTI) six monthly and from Jaipur for ABO grouping, Du typing, direct and indirect Coomb’s test and TTI six monthly. We analysed the results of EQAS from 2008 to 2012. Results: We observed 100 % correlation in ABO grouping, Du typing, direct and indirect Coomb’s test and HBsAg. Discordant results were observed in HIV 1/2 two times and in anti-HCV and VDRL once. In root cause analysis we found that false positive results in HIV 1/2 were due to random error and machine break down. The false negative result in anti-HCV was due to sample integrity. VDRL was done by RPR card test method, which has low sensitivity than treponema pallidum hemagglutination method. Conclusion: We believe that participation in EQAS program will definitely improve quality of hospital service because no health care facility can be totally self-sufficient and there is always an inclination for improvement and development in a system.

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Abstract No. 331 RBC Alloantibodies in Patients: Experience of Detecting and Providing Compatible Antigen Negative Red Cell Units in a Tertiary Care Centre in India Siddhi Shah, Sangeeta Kalgutkar, Rajesh Sawant, Anand Deshpande Transfusion Medicine, Department of Laboratory Medicine, PD Hinduja National Hospital & Medical Research Centre Introduction: The development of red cell alloantibodies and/or autoantibodies in patients can significantly complicate the transfusion therapy as it becomes difficult in providing compatible blood units. RBC alloimmunization can cause destruction of transfused red blood cells i.e. Hemolytic Transfusion Reactions (HTRs). Alloimmunization to red cell antigens may result from pregnancy, transfusion, injection with any immunogenic material or recent drug therapy. Thus, identification of such clinically significant unexpected antibodies (other than ABO) in patients is essential for providing appropriate blood units for transfusion. Aim: To detect the alloantibodies in patients showing positive antibody screen and to provide compatible red cell units. Materials and Methods: 11,824 patients were screened using 3 cell screening panel cells (Bio-Rad, Cressier, Switzerland). In case of positive antibody screen, antibody identification was carried out using 11 cell identification panel cells (BioRad, Cressier, Switzerland). When alloantibodies to the Rh-Kell blood group system were identified, Rh-Kell phenotyping (Bio-Rad, Cressier, Switzerland) of the patient and the donors was done. All tests were carried out using Column Agglutination Technology. Results: During the study period of 2 years, 11,824 patients were screened for the presence of any unexpected red cell antibodies. 148 (1.24 %) showed a positive antibody screen, of which, 109 (73.65 %) alloantibodies, 13 (8.78 %) autoantibodies, 3 (2.03 %) cold agglutinins, 1 (0.68 %) drug induced antibody, 1 (0.68 %) against the preservative used in the panel cells and 17(11.49 %) inconclusive antibodies were identified. Amongst 52 male patients having alloantibodies, 34(31.19 %) had history of transfusion and showed the presence of anti-E(7), anti-Lea(5), anti-C(2), anti-c(2), anti-Fyb(2), anti-M(2), anti-K(1), anti-Kpa(1), anti-N(1), anti-S(1), anti-Xga(1) and multiple alloantibodies developed in 9 patients. 18 (16.51 %) had no history of transfusion and showed the presence of anti-M(8), antiLea(3), anti-Lua(3), anti-E(1), anti-Cw(1), anti-D(1) and multiple alloantibodies developed in 1 patient. 56 female patients developed alloantibodies, of which, 30(27.52 %) had history of transfusion and showed the presence of anti-E(6), anti-K(3), anti-Lea(3), anti-c(2), anti-e(1), anti-Kpa(1), anti-Jka(1), anti-Jkb(1), anti-P1(1), anti-Lua(1) and multiple antibodies developed in 10 patients. 27 (24.77 %) had no history of transfusion and showed the presence of anti-D(9), anti-E(4), anti-M(3), anti-Leb(2), anti-P1(2), anti-Cw(1), anti-Fyb(1), anti-Lua(1) and multiple antibodies developed in 4 patients. All these female patients had history of pregnancy. A total of 1,218 units were crossmatched for 148 patients showing a positive antibody screen of which, 621(50.98 %) units were found to be compatible and 42 (3.45 %) units were least incompatible. We could provide 304 antigen negative compatible blood units to the patients. 27 patients were available for follow-up and did not develop any new antibodies except one patient who developed another alloantibody. Conclusion: Unexpected red cell antibody screening was found to be positive in 1.24 % patients screened and alloantibodies were detected in 0.92 % patients screened. Most commonly encountered antibodies in patients with and without transfusion history were from Rh-Kell blood group system(43/109) followed by anti-M(13) and anti-Lea(11). Multiple antibodies were identified in 24 patients. From this study, we can safely conclude that adverse transfusion reactions can be prevented by

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 transfusing antigen negative blood. Lastly, it also prevented further alloimmunization in patients requiring repeated transfusions.

Abstract No. 332 Multiple RH Alloantibodies in a Patient of Hereditary Spherocytosis: A Case Report with Review of Literature P Lalita Ajyotsna, Sangeeta Pahuja, Sunita Sharma Lady Hardinge Medical College Summary: We report the case of a 23 year old primigravida female, patient of hereditary spherocytosis, who presented with severe anaemia and required transfusion. Antibody screening followed by identification testing revealed multiple alloantibodies which developed as a result of a previous transfusion. Introduction: Alloimmunisation is a known complication of transfusion. It can complicate the course of future transfusions in the patients and can cause problems in cross-matching and identifying compatible blood units. Multi-transfused patients are at further increased risk of developing allo-antibodies, with an overall reported frequency of 2.6–47 %.However, in hereditary spherocytosis (HS), there is paucity of literature regarding alloimmunisation risk. Transfusion requirement depends on severity of disease. Patients with severe disease need frequent blood transfusions while in cases of moderate HS, anaemia is mild-moderate and patients may require transfusion during intercurrent illness or pregnancy. Materials and Methods: A 23-year old female, primigravida with 31 weeks pregnancy, known case of hereditary spherocytosis, presented with complaints of swelling of feet, easy fatiguability and breathlessness on exertion for 1 week. Routine hematological investigations were done. A requisition for transfusion was received in the blood bank. Antibody screening using 3-cell panel followed by antibody identification and adsorption studies using 11-cell panel was carried out. Results: The hemoglobin concentration was 3.2 g %. The antibody screen using a 3-cell panel revealed a 4+ positive reaction with two of the cells. The 11-cell identification panel on the serum after adsorption studies was suggestive of presence of anti-c and anti-E antibodies. Based on our results, she was transfused with blood according to her antigen profile, which was c and E antigen-negative. Conclusion: Our case highlights the need for extended antigen-typing before first transfusion with transfusion of at least Rh and Kell-matched blood in patients of hereditary spherocytosis (and other chronic haemolytic anaemias) to prevent allo-immunisation.

Abstract No. 333 Blood Group Discrepancy Leads to Diagnosis of Secondary Cold Auto Immune Haemolytic Anemia due to Chronic Lympho Proliferative Disorder Ritam Chakrabarty, Suvro Sankha Datta, Biplabendu Talukder, Prasun Bhattacharya, Krishnendu Mukherjee, Oona Mandal Medical College and Hospital Summary: Blood group discrepancy in a 64 year old male patient of chronic anemia was found to be due to a low titre, increased thermal amplitude cold antibody and on further investigation revealed Chronic Lympho Proliferative Disorder. Introduction: Immune haemolytic anemia is defined by in vivo shortened RBC survival mediated through the immune response. Cold reactive auto-antibodies usually associated with blood group discrepancies. Here we present a

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 case of secondary cold auto immune haemolytic anemia in a 64 years old male with features of chronic anemia and Hb level around 5 g % for last 1 year. History of 2 units of blood transfusion 2 years ago. Materials and Methods: We received requisition of blood transfusion for this patient. Conventional tube groupings were done initially at room temperature, and then at 2–4 and 37 °C, to resolve the blood group discrepancy. Subsequently Direct Antiglobulin test (both at polyspecific coombs’ and mono-specific card), Autocontrol, Alloantibody screening, Titration and Thermal Amplitude tests were performed to identify the type of antibody and its clinical significance. Results:

Conclusion: So, a low titre and increased thermal amplitude type of cold auto antibody is present in this patient. As most of the cold reactive auto antibodies are associated with lympho proliferative disorders we suggested a detailed investigation which revealed CLPD. Hence a mere blood group discrepancy led to the diagnosis of a major disease.

Abstract No. 334 Blood Group Discrepancy in Newborn: A Real Life Nightmare Nidhi Mehta Kokilaben Dhirubhai Ambani Hospital, Mumbai Introduction: ABO discrepancies happen when there is no match in results between forward and reverse grouping. These discrepancies are usually technical in nature and can be simply resolved by correctly reporting the testing and carefully checking reagents with meticulous reading and recording of results. Group I discrepancies These are most common discrepancies, between forward and reverse grouping due to weak reaction or missing antibodies. The reason for the missing antibody or weak reaction is that the patient has depressed antibody production or cannot produce the ABO antibodies. (1) This type of discrepancy can be seen in new born infants, elderly patients. (2) Patients with lymphoma. (3) Patients using immunosuppressive drugs. (4) Patients with immunodeficiency disease, BM transplant. Group II discrepancies: These are least one discrepancies, between forward and reverse grouping due to weak reaction or missing antigens. Can be caused by some subgroups of A or subgroups of B or both. Also it can be present in patients with leukaemia and Hodgkin’s disease. Group III discrepancies: These discrepancies are between forward and reverse grouping due to protein or plasma abnormalities. These can be caused by elevated levels of globulin from certain diseases such as multiple myloma, Hodgekin’s lymphoma. Some are caused by (Rouleaux formation). Group IV discrepancies: These kind of discrepancies are between forward and reverse groping due to miscellaneous problems. Polyagglutination can occur due to exposure of hidden erythrocyte Ag. (T antigen) in patients with bacterial or viral infection. Bacterial contamination in vitro or vivo produces an enzyme that alters and exposes the hidden Ag. on red cell leading to T activation. Case study: 40 days old baby admitted with Clinical condition—Congenital ‘Situs inversus at Kokilaben Dhirubhai Ambani Hospital. Situs Inversus is an autosomal recessive genetic condition. On 14th November, the request for the blood along with

391 patient’s blood sample was received in the Department of Transfusion medicine. The blood grouping was done using column agglutination technology using Ortho AutoVue Innova system and reported as ‘AB positive’. The baby was transfused with O positive packed RBC units on 14th Nov Feb followed by AB plasma on 19th Nov and O positive packed RBC on 22nd Nov 2011 and got discharged. On 6th Dec 2011, the baby blood sample was retested for blood group and the blood group was reported as A positive. On cross verifying the earlier record, the grouping discrepancy was observed. Retrospective analysis of the reaction pattern in the blood group report in the month of November 2011 in Ortho AutoVue Innova system showed strong reaction with Anti A column (4+), weak reaction in Anti B column (2+) and strong reaction with Anti D(4+) and the system reported the result as AB+ve. The result was withheld by the system with a flag for ‘trigger the threshold notification’ as the threshold limit for Anti A, Anti B and Anti D reaction was 2+ and so the result was manually accepted. Since the sample was from one month old baby, the reverse grouping was not carried out. Due to the weak reaction reported in CAT technology with Anti B and the result was withheld by the automation system, the sample was retested in tube method during that time and observed the similar trend of reaction pattern. The case history of the baby was collected from the hospital record and analyzed. The microbiology report of the patient revealed that the baby was having the E. coli and Staphylococcus infection and reported as the case of ‘Septicemia’. It was concluded that the bacterial infection is the root cause for the acquired B antigen which leads to the Blood group discrepancy in the baby. Discussion: The acquired B phenotype arises when microbial deacetylating enzymes modify the A antigen by altering the A-determining sugar (N-acetylgalactosamine) and it resembles the B-determining sugar (D-galactose). Acquired B antigens are usually associated with tissue conditions that allow colonic bacteria to enter the circulation. Campbell and Palmer (1980) reported two elderly patients with acquired B antigen, one secondary to Escherichia coli and the other to Pseudomonas septicemia. Acquired B phenomena can be usually identified by the discrepancy between forward and reverse group. In case of new born babies, since reverse grouping is not warranted before 6 months of the baby, it becomes difficult to identify the acquired B syndrome. In this case study, we reported the variation in blood groups of the new born baby within the first 3 months of the baby due to acquired B phenotype. Conclusion: This study warranted that reporting Blood group of babies in the first 3 months has to be done with caution as it may lead to discrepancy due to various clinical conditions similar like the above reported case of septicemia. Also, this study proved the safety and security of the Ortho AutoVue Innova system by flagging the results which needs further analysis before reporting.

Abstract No. 335 Seroprevalence of Infectious Markers Among Blood Donors from Blood Bank of a Tertiary Care Hospital Tasneem V Bisht, Sujata R Kanetkar, Sujata S Kumbhar, Surekha Chavan Krishna Institute of Medical Sciences, Karad Summary: Blood transfusion services form an integral part of health care; but simultaneously carries the risk of transmission of transfusion transmissible infections. We conducted a 5 year retrospective cross sectional study to estimate the prevalence of various infectious markers in the blood donors which was found to be 2.01 %. Introduction: Blood safety is a major concern among health care personnel. We undertake this study to assesss the seroprevalence of

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392 transfusion transmissible infections (TTIs) namely HIV, Hepatitis C, Syphilis and Malaria among blood donors from blood bank of a tertiary care hospital, in Western Maharashtra, India. Materials and Methods: A total of 21,293 blood units were collected from donors. All blood units were screened for HIV I &II, HBsAg and HCV using ELISA. Test for syphilis was done by Rapid Plasma Reagin card test and peripheral smear examination was done to detect malarial parasite. Results: A total of 21,293 blood donors were tested, of which 19,940 (93.65 %) were voluntary donors and 1,353 (6.35 %) were replacement donors. The highest seroprevalence observed was for HBsAg (1.56 %) followed by HIV (0.39 %), HCV (0.08 %) and Syphilis (0.02 %). No donor was found to be positive for malaria parasite. Of the 82 donors who were positive for HIV, 6 also showed positivity for a second TTI (4 for HBsAg, 1 for HCV and 1 for Syphilis). Maximum seroprevelance was noted in the males between age group of 26–35 years. Conclusion: Strategies need to be implemented to improve donor selection, using highly sensitive and specific screening tests and a better structured voluntary donation system. Nucleic acid amplification test would help to detect donors in window period for HIV infection. In view of high prevalence, effective community based programs and health education with emphasis on sexually transmitted diseases may prove helpful to decrease the seroprevalence.

Abstract No. 336 RDP Prepared by Buffy Coat Method from Buffy Coat Stored Overnight: Advantages and Benefits Samantha Kumarage, Joseph Philip, T Chatterjee, RS Mallhi Department of Immunohematology and Blood Transfusion, Armed Forces Medical College, Pune 40 Summary: In our study we have shown that Random donor platelet (RDP) prepared at 24 h of collection is superior to that prepared within 8 h. It provides better quality due to the advantage of preparation in business hrs of the day, and has better platelet counts because of which the patient requires fewer RDPs, resulting in fewer donor exposure with its resultant benefits. Introduction: When RDP is prepared in India by the buffy coat method, it is from whole blood stored at room temperature, within 8 h of collection. Our aim was to evaluate the platelet count, WBC contamination, platelet activation, and biochemical parameters of RDPs prepared from buffy coat within 8 h and within 24 h of collection. Materials and Methods: We prepared 40 units of RDP by the buffy coat method from whole blood stored at room temperature within 8 h of collection (Fresh BC), & another 40 units from buffy coat stored at 22 °C for less than 24 h Stored BC). We analyzed the platelet counts, CD62P expression, WBC counts, glucose levels, pH, PO2, PCO2 in both the groups of RDPs, 24 h after respective preparation. Results: The platelet counts from stored BC was higher in fresh BC. CD 62P expression was low in stored BC compared to fresh BC. There were no differences of pH, pO2, pCO2 and glucose levels in fresh BC and stored BC. WBC contamination was more in fresh BC. Conclusion: RDP prepared from stored BC is the better method for RDP production.

Abstract No. 337 Review of Therapeutic Apheresis Procedures in a Tertiary Care Hospital in India Sangeeta Kalgutkar, Rajesh Sawant, Anand Deshpande

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 Transfusion Medicine, Department of Laboratory Medicine, P D Hinduja National Hospital & Medical Research Centre Introduction: Therapeutic apheresis has a role in various acute & chronic disease conditions. Therapeutic plasma exchange is beneficial in myasthenia gravis (MG) crisis or Guillain Barre syndrome (GBS) and hemolytic uremic syndrome (HUS) in children. In patients with severe malaria, sickle cell disease, therapeutic red cell exchange is beneficial. Aims: To review therapeutic plasma exchange (TPE) & red cell exchange (TREX) procedures. To compare use of continuous flow (CFCS) & intermittent flow cell separators (IFCS) in TPE. Materials and Methods: A retrospective analysis of 438 TPE, 07 TREX procedures carried out in both adult and pediatric patients during a 13 year period was done. 92 procedures were carried out using IFCS & CFCS was used in 346 procedures for TPE. For TREX CFCS was used. Results: 438 TPE procedures in 77 patients (18pediatric, 59-adult). Myasthenia gravis, GBS & HUS were the commonest indications. An average of 5 procedures per patient were required for clinical benefit. 1.5 volume plasma was exchanged with FFP or colloids & crystalloids as replacement fluids. Procedure time with IFCS was from 130–200 and 33–130 min with CFCS. Citrate toxicity,low access pressure, reversible hypotension and allergic reactions was the commonest complications. Clinical outcome was satisfactory. 07 TREX procedures, in 2 pediatric patients with malaria, 2 with sickle cell disease (SCD) & one adult patient with SCD. The procedure time ranged from 52–90 and 97–117 min in adult patient. Group specific cross-match compatible packed red blood cells were used as replacement fluid. In cases of malaria the infestation rate dropped from 75–18 and 67–08 % after the TREX. In the adult patient with SCD, Hb S levels dropped to \20 % after each procedure and in pediatric patients HbS levels dropped five fold in both cases post procedure. Conclusion: Therapeutic apheresis procedures (TPE, TREX) are safe & effective in various clinical disorders. CFCS are well tolerated by patient and require less procedure time. Therapeutic apheresis can be safely performed in patients of pediatric age group maintaining their hemodynamic stability.

Abstract No. 338 ABO-Identical Versus Non-identical Platelet Transfusion: Comparison of Therapeutic Efficacy in a Mixed Patient Population Clara Thomas, Atul Shringare, Vidya Patne, Maithili Gangurde, Rajesh Sawant, Anand Deshpande Transfusion Medicine, Department of Laboratory Medicine, P D Hinduja National Hospital & Medical Research Centre Introduction: The significance of ABO matching for platelet transfusion has not been clearly defined. Providing ABO-identical platelets can be difficult because only limited inventories are available. Therefore determining the clinical value of transfusing ABO-identical platelet is essential. Aim: To compare the therapeutic efficacy of ABO-identical versus non-identical platelet transfusion in a mixed patient population. Materials and Methods: In a prospective, observational study, corrected count increment (CCI) and percentage platelet recovery (PPR) were calculated at 24 h post transfusion. Platelet count, WBC count and volume of all platelet units were noted. Choice of the platelet preparation i.e.; random donor platelet (RDP) or apheresis platelet (SDP) was made by the treating clinician. All patients were monitored for presence of clinical bleeding, fever, hypertension, DIC, any adverse transfusion reaction and time to next transfusion. The co-relation between ABO compatibility and platelet dose with CCI and PPR was studied. Results: 79 transfusion episodes (36 RDP and 43 SDP) in 37 patients were studied. Mean patient age

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 was 45 (5–87) years, weight 58 (9–103) kg and height 156 (67–185) cm. Dose of platelet was higher in SDP (p = 0.004) than RDP. 52 % RDP and 55 % of SDP transfusion were ABO compatible. All transfusions were prophylactic in nature. The mean transfusion threshold for platelets was 20,812. Mean CCI and PPR in the SDP group were 15,206 and 37 % than in the RDP group were 12,446 and 22 % respectively. Mean CCI and PPR with ABO identical RDP transfusion were 13,358 and 27 % and in the ABO non identical RDP transfusion were 9,491 and 14 % respectively. CCI and PPR with ABO identical SDP transfusion were 16,670 and 44 % and with ABO non identical SDP transfusion were 15,091 and 29 % respectively. ABO non identical transfusions lead to significantly reduced (p = 0.005) PPR with both, RDP and SDP preparations. Leukocyte content of RDP’s was greater than SDP’s. Allergic reactions and FNHTR’s occurred in 10 % of the cases and were associated more (p = 0.003) with RDP transfusions. The CCI and PPR showed on significant correlation with the platelet preparation or platelet dose transfused and time to next transfusion. Conclusion: ABO non identical platelet Transfusion lead to a statistically significant decrease in the PPR at 24 h post Transfusion. This is independent of the type of platelet preparation and the platelet dose transfused. This study shall continue in future to further analyse results in a longer patient cohort.

Abstract No. 339 Fenwal Amicus or Fresenius Com.Tec? A Comparative Analysis of Various Aspects of Plateletpheresis on Two Cell Separators AK Biswas, Joseph Philip, T Chatterjee, RS Mallhi Department of Immunohaematology and Blood Transfusion, Armed Forces Medical College, Pune Summary: Comparison of two apheresis machines for platelet collection was done. Donor & instrument variables along with product quality were estimated. Both machines were comparable, however Amicus reached the PLT target yield more quickly. Introduction: In our study, we compared two apheresis instruments (Fenwal Amicus and Fresenius COM.TEC) at our centre with regard to the donor peripheral blood parameters, operational variables of the instruments and the quality control parameters of the products obtained. Additionally, the collection rates, collection efficiencies, platelet yield & product activation levels (CD62P) from both the instruments were also estimated & compared. Materials and Methods: Donors undergoing plateletpheresis were randomly separated into two groups (Amicus and COM.TEC). One hundred platelet collections were carried out. Platelets were measured by an automated analyzer and activation statuses of platelets were analyzed by a flowcytometer. Results: The blood volume processed to reach a target PLT yield of C3.0 9 1011/bag was higher in Amicus compared to the COM.TEC (2,972 vs. 2,853 ml; but it was not significant, p [ 0.05). No major adverse events were observed during the procedures, in both the machines. The median time needed for the procedures was significantly longer with the COM.TEC (74 vs. 68 min). All products obtained with both instruments had WBC counts lower than 5 9 106/bag. There was no statistical difference with regard to collection efficiency and collection rates between the devices. Conclusion: Both instruments collected platelets efficiently with minimal donor discomfort. Consistent leuco-reduction was achieved with both the instruments. However, Amicus reached the PLT target yield more quickly.

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Abstract No. 340 Development of Anti-platelet Antibodies & Refractoriness in Multi Platelet Concentrate Transfused Patients, Their Detection and Identification by a Solid-Phase Modified Antigen Capture Elisa (MACE) Technique Neelesh Jain, RS Sarkar, J Philip Armed Forces Medical College, Pune-40 Summary: Detection & identification of platelet antibodies and its correlation to platelet refractoriness by measuring the corrected count increment (CCI) at 1 & 24 h after transfusion. Introduction: Platelets express polymorphic glycoproteins (IIb/IIIa, Ib/IX, Ia/IIa, IV, and HLA-1) which can become targets for antibody responses. Patients at threat are those given multi-platelet transfusions, such as those suffering from hemato-oncological diseases and Bone marrow failure syndromes. MACE is a qualitative solid phase ELISA designed to detect IgG antibodies against platelet specific antigens. Materials and Methods: This is a prospective analytical study, carried out in our department. A total of 100 patients were selected, who had been transfused with at least 15 units of platelet concentrate. Platelet antibodies were identified using MACE technique. Results: 39 % of the patients were found to be alloimmunized against platelet antigens. Twenty patients showed corrected count increment (CCI) \5,000 of which thirteen showed refractoriness. Production of HLA-1 antibody and the development of refractoriness was found to be significant with OR 14.05 and p value 0.0025. Conclusion: MACE enabled specific detection and identification of platelet antibodies, which in turn correlated well with the development of refractoriness. This technique should specifically be utilised for patients who are at an increased risk of developing alloimmunisation due to repeated platelet transfusions, thereby permitting us to provide matched platelet units & help in reducing the risk of refractoriness. An important observation of this study is that, in our patient population the most common platelet specific antibody detected was GP Ib/IX (HPA-2A/2b) unlike in the west, where it is GPIa/IIIa (HPA-1a/5b).

Abstract No. 341 Evolving Testing Protocols for HBV-Serology Yield Cases: Our Experience P Satawase, S Kalgutkar, A Athare, R Sawant, A Deshpande Transfusion Medicine, Department of Laboratory Medicine, P D Hinduja National Hospital & Medical Research Centre Introduction: Nucleic acid Amplification Testing (NAT) is a molecular technique that provides simultaneous detection of HIV RNA, HCV RNA and HBV DNA in a single reaction. In March 2009, Individual ULTRIO NAT (ID-NAT) was implemented in conjunction with EIA at a tertiary care multispecialty hospital in Mumbai. However, there is a need to have a uniform testing algorithm for evaluation of discrepant results of EIA and NAT especially with reference to HBV, which is most prevalent Transfusion Transmitted Infection (TTI) in India. Aims: To study the HBV discrepant samples and evaluate the effectiveness of algorithm and ULTRIO Plus. Materials and Methods: Routinely, all blood donor samples were tested by EIA (Axsym and Architect) for the detection of anti-HIV and anti-HCV antibodies, HBsAg and also by ID-NAT assay using Transcription Mediated Amplification (TMA) (ProcleixÒ UltrioÒ Assay, Novartis). All reactive donors by ID-NAT were subjected to discriminatory assay for the detection of specific virus. December

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394 2011 onwards, an algorithm was implemented in case of discrepancy in EIA and NAT results. According to algorithm, NAT assay was repeated 3 times each with EDTA sample, plasma bag sample and discriminatory assay to detect NAT-yield. To detect sero-yield, NAT assay was repeated 3 times with EDTA sample and confirmed when at least one NAT assay was reactive. Additional biochemical, serological and molecular tests were also performed in such cases. Discrepant results were evaluated using Real-time PCR, algorithm and ULTRIO plus assay. Results: Out of 22,656 donors 245 were HBV reactive by EIA however only 238 were reactive by both EIA and NAT resulting in 7 discordant cases. All the cases had normal liver function (SGPT, SGOT, Alkaline phosphatase, Bilirubin). HBV serology profile of all seven donors showed positive results for anti-HBc(total), negative anti-HBc (IgM) and negative anti-HBsAg. Out of these 7 cases, 5 had detectable HBV viral load by real-time PCR (sample was not sufficient in 2 cases). Out of these 5 cases, 4 were reactive by ULTRIO plus, resulting in one sero-yield case. After the implementation of algorithm, 3 more HBV cases were observed to be reactive by EIA (titer [250 IU/ml) and non-reactive by initial NAT assay. However all these cases were detected to be NAT reactive using the algorithm. Moreover, all the 3 cases were reactive by ULTRIO Plus. Out of 22656 blood donors one was observed to be NAT-yield case when tested using algorithm (2/3 times NAT reactive with EDTA, 1/3 reactive with plasma bag and 1/3 with discriminatory assay). Conclusions: Implementation of NAT algorithm and ULTRIO plus enhanced the analytical sensitivity of NAT assay. This has helped us in resolving discordant HBV sero-yield cases.

Abstract No. 342 Major Blood Group Antigens and Growth of Plasmodium Falciparum in In-Vitro Culture Vrushali Pathak, Kanjaksha Ghosh, Roshan Colah National Institute of Immunohaematology, Mumbai Summary: ABO blood group system is the most important blood group system in clinical practice and found to be associated with various infections. This study was undertaken to correlate the P. falciparum parasitemia with the ABO blood groups in an in-vitro culture system. Introduction: During the period of parallel co-evolution of Plasmodium falciparum and its human host, multiple erythrocyte related polymorphisms associated with survival advantage in the infection are thought to have developed. These include blood group antigens, hemoglobinopathies, red cell enzymopathies and membranopathies. ABO blood group antigens are the most profound antigens present on the erythrocyte membrane. The clinical manifestations of malaria result from the intra-erythrocytic growth cycles of plasmodium suggesting hyperparasitemia as an important indicator of disease severity. Materials and Methods: We used an in-vitro culture system that involved co-culturing of P. falciparum (strain 3D7) infected erythrocytes with uninfected erythrocytes of different blood groups. Parasites were allowed to grow in fixed amount of erythrocytes for up to 5 days. Mean percent parasitemias were calculated after every 24 h. Results: Percent parasitemia (Mean ± SD) in A,B and O group cultures (Ten of each) on fourth day i.e. after two growth cycles were 15.4 ± 0.7, 13.3 ± 1.3 and 19.9 ± 3.2 respectively. Parasitemia was found to be significantly higher in O blood group culture when compared to A and B group cultures (p \ 0.01). Conclusion: This work demonstrated that blood groups differentially allow P. falciparum growth. Thus indicating blood group specific determinants might be involved in the invasion process of parasite.

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Miscellaneous Abstract No. 343 Clinico-Haematological Evaluation of Splenomegaly KR Kabra, MM Kulkarni, MV Jadhav Department of Pathology, BJGMC, Pune Summary: Present study on clinico-haematological evaluation of splenomegaly was rewarding in underlining the correlation between etiology, grading of splenomegaly & its associated haematological manifestations. Introduction: Palpable spleen in a symptomatic person is always significant. Splenomegaly is more significant when associated with lymphadenopathy & hepatomegaly. Sometimes its the first & only sign of serious disease. Questions concerning the frequency, etiology and significance of finding a palpable spleen are raised from time to time. Present study is a humble attempt to elucidate etiopathogenesis of splenomegaly and its associated hematological manifestations. Materials and Methods: It was a prospective study which included 90 cases of splenomegaly referred by clinicians. After obtaining informed consent all patients were evaluated clinically & ultrasonographically for grading & etiology of splenomegaly & investigations namely Peripheral Blood Smear, Haemoglobin, automated cell count on Sysmex 18 parameters, 3 part cell counter-K4500, pertinent haematological investigations like Sickling test, Osmotic fragility etc & bone marrow examination were carried out. Splenectomy was done in 7 cases & after 7 days investigations were repeated. Data was analysed using Chi Square test. Results: 88.57 % cases of anemia, 61.5 % cases of congestive splenomegaly and 50 % cases of undetermined splenomegaly had Grade I to II enlargement. 63.3 % cases of myeloproliferative disorders and lymphoproliferative disorders had Grade III to IV splenomegaly. Hypersplenism was present in 31 % of cases. The most frequent cause of Hypersplenism was congestive splenomegaly either Grade II or III, secondary to liver cirrhosis and portal and splenic vein thrombosis (35.71 %). Conclusion: Most frequent cause of splenomegaly was anemia followed by myeloproliferative and lymphoproliferative disorders followed by congestive splenomegaly. There was statistically significant association between increasing size of spleen & hypersplenism. There was no correlation between splenic size & degree of anemia & cytopenia. Splenectomy resulted in normalization or improvement of hematological parameters in all the cases.

Abstract No. 344 Multiple Splenic Nodules due to Sarcoidosis Priya Wadhwa, Pradeep Vaideeswar, Daksha Prabhat Department of Pathology, Seth GS Medical College, Mumbai Summary: We present a case of splenic sarcoidosis, manifesting as multiple nodules in a 62 years woman with systemic disease in a medico-legal autopsy. Introduction: Sarcoidosis is an idiopathic chronic systemic granulomatous disease that commonly presents with pulmonary disease. Extrapulmonary manifestations also occur with varying frequency and splenic involvement is seen about 15 % patients. Materials and Methods: A medico-legal autopsy was performed in a 62 years woman who suddenly collapsed at home. Results: The cause of death was due to critical stenosis of the proximal segment of the left anterior descending artery. However,

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 there were other interesting findings. The spleen (210 g) showed mild enlargement with the presence of multiple (about 15) firm yellowish well circumscribed nodules ranging in size from 1 to 2 cm. Smaller glistening nodules were also found in the liver. The hepato-splenic lesions were accompanied by enlarged mesenteric, perigastric, portahepatis lymph nodes leading to a provisional diagnosis of a lymphoma or metastases. The nodules on histology were represented by discrete or confluent non caseating granulomas. Similar but miliary lesions were scattered in both lungs. Special stains for organisms were negative. A diagnosis of sarcoidosis was made. Conclusion: Sarcoidosis can manifest as multiple nodules in the spleen leading to a diagnostic dilemma. The problems can be accentuated when spleen is the sole organ involved.

Abstract No. 345 Hemophagocytic Lymphohistiocytosis in Adults and Adolescents: Experience from a Tertiary Care Centre in South India

395 and thrombocytopenia. Raised ferritin levels were found in 100 % patients with mean of 13,500 ng/ml (range 680–95,000). Hepatic dysfunction in the form of bilirubin elevation and deranged hepatic enzymes was found in 85 % of the cases. Bone marrow evidence of haemophagocytosis was found in 75 % of cases. No underlying etiology could be found in 3 patients (38 %), and diagnosis of disseminated tuberculosis, NK/T cell lymphoma, EBV infection, Dengue and Scrub typhus was made in one patient each after extensive investigations. Patients with diagnosis of disseminated tuberculosis, lymphoma and scrub typhus expired during or just after diagnostic workup. Among rest of the surviving 5 patients, one received only IV immunoglobulin and others dexamethasone with cyclosporine. All these 5 patients (62 %) survived and till now with a median follow up of 11 months none of the patients relapsed. Conclusion: HLH is uncommon but not rare and high suspicion is required especially in patients with fever and cytopenias. Hepatic dysfunction is a prominent feature at presentation. Underlying etiology is not clear in more than 1/3 rd of the patients. Immediate immunosuppression with steroids and cyclosporine and treatment of underlying cause is of utmost importance to avoid mortality.

Shailendra Prasad Verma, V Rakesh Nayak, TK Dutta, Debdatta Basu, BS Suryanarayana, KV Vinod, Rakhee Kar, Sajini Elizabeth Jacob

Abstract No. 346

Jawaharlal Institute of Post Graduate Medical Education and Research (JIPMER)

A Common Tropical Infection Presenting as Secondary Hemophagocytosis

Summary: HLH is a condition of immune dysregulation having various underlying causes. HLH is uncommon but not rare entity and high suspicion is required especially in patients with fever and cytopenias. We studied case records and followup of 8 patients with HLH. Along with fever, cytopenias, splenomegaly and raised ferritin, hepatic dysfunction is a prominent feature at presentation. Underlying etiology is not clear in more than 1/3rd of the patients. Immediate immunosuppression with steroids and cyclosporine and treatment of underlying cause is of utmost importance to avoid mortality. Introduction: Haemophagocytic Lymphohistiocytosis (HLH) is a rare disorder due to immune dysregulation of varied origin which leads to cytokine storm, unregulated macrophage and NK/T cell activity leading to progressive cytopenias and death if untreated. Familial HLH is usually restricted to young children and usually requires transplant as the treatment modality. Most of these events are triggered by infections. Secondary HLH is mainly due to underlying infections, malignancies or autoimmune disorders. We retrospectively studied clinical presentation, underlying cause, laboratory investigations, management and follow up outcome of eight cases of HLH, diagnosed during January 2011 to January 2013 under division of haematology and Department of medicine JIPMER a tertiary care centre in South India. Materials and Methods: We reviewed case records of patients diagnosed with HLH (as per HLH 2004 criteria) during the period of January 2011 to January 13 in Department of Medicine, Division of Haematology JIPMER. Eight cases were included in the study. All the patients were either adolescents or adults. We collected and analyzed details of age, sex, clinical history, underlying etiology, laboratory investigations, management and follow up outcomes of these 8 patients. Only one patient could be investigated for underlying perforin mutation by flowcytometry. Results: Mean age of our patients was 27 years (Median 20.5 years, range 13–57 years). Three patients were of adolescent age group. Most common presentation was prolonged undiagnosed fever (100 %) with median of 10 days fever (range-10 days to 8 week). Fever was commonly associated with jaundice (70 %) and rash (37 %). Most common physical findings were rash, pallor, splenomegaly (75 %), hepatomegaly (62 %), lymphadenopathy and pedal edema. All patients had anemia and 75 % of patients had leucopenia

Anusree, Pankaj Malhotra, Ram Nampoothiri, Navneet Sharma PGIMER, Chandigarh Summary: Hemophagocytic syndrome is characterised by presence of fever, pancytopenia, hepato splenomegaly, elevated serum ferritin ([500 ng/ml), triglycerides, LDH, sCD25, decreased NK cell activity and evidence of hemophagocytosis in the bone marrow, lymph nodes or spleen. It can be primary or secondary. This young girl was diagnosed with secondary HLH based on the presence of above mentioned features and etiology turned out to be enteric fever. Introduction: Secondary hemophagocytosis is generally associated with autoimmune disorders, infections, drugs and hematological malignancies. This condition can be fatal if untreated or remains uncontrolled. We present a case of young girl who presented with frank hemophagocytic syndrome with multiorgan dysfunction. She was initially managed with IVIG. Later bone marrow culture grew Salmonella typhi and she improved with specific management. There are 4 cases of enteric fever with hemophagocytosis reported in 1992 from Iran; one patient was diagnosed similar to index case with positive bone marrow culture. Materials and Methods: A-12 years girl presented with 10 days fever and 3 days bleeding manifestations—epistaxis, gum bleeds and hematochezia. On examination PR-140/min, BP-100/70, RR-40/ min, temp-39–40 °C, bony tenderness + hepatosplenomegaly + Results: Investigations: pancytopenia (Hb 7 g/dl, TLC 3,300/ c.mm, Platelet 12,000/c.mm), elevated liver enzymes (SGOT105/ SGPI81/ALP364), LDH (807U/L), triglycerides (272 mg/dl) and serum ferritin (7,265 ng/ml). Possibilities were acute leukemia and viral fever with secondary hemophagocytosis. BM examination was suggestive of hemophagocytosis. She was managed with IVIg 2 g/ kg over 2 days. Blood cultures were sterile, BM culture grew salmonella typhi. She was managed with ceftriaxone for 14 days. She became afebrile in a week and counts normalized. Conclusion: Hemophagocytosis is a clinical syndrome manifesting with pancytopenia and multiorgan dysfunction with very high mortality. Tropical infections should be kept in the differential diagnosis of HLH patients in developing countries. With proper diagnosis and treatment, results can be gratifying in an otherwise fatal condition.

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Abstract No. 347 Apoptotic Activity of BNW-8 Isolated from Neanotis Wightiana on Human Leukemic Cell Lines Prashanta Kumar Deb1, Niranjan Das2, Rajat Ghosh3, Nilanjana Deb1, Sudipta Chanda1, Shila Elizabeth Besra1 1 Drug Development/Diagnostic & Biotechnology Division, CSIR, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata-700032, West Bengal; 2Department of Chemistry/Pharmacy, Tripura University (A Central University), Suryamaninagar-799 130, Tripura (W)

Summary: The current study was aimed to investigate the anti-leukemic activity of isolated b-carbolene alkaloid (BNW-8) from Neanotis wightiana in two different human leukemic cell lines (MOLT-3 and K562). Introduction: Cancer, the second most dreadful disease is still incurable. Of therapies employed chemotherapy is of vital importance. Tyrosine kinase inhibitors that are used often have side-effects. Hence, we have opted for natural products for cure of cancer. BNW-8 isolated from Neanotis wightiana (Family: Rubiaceae), a perennial herb with sessile or sub-sessile leaves was evaluated for anti-leukemic activity. Materials and Methods: Cell viability study was done by Trypan blue exclusion method and cytotoxicity study performed by MTT assay, morphological study was analyzed by Fluorescence and Confocal microscopy. Agarose gel electrophoresis study was performed to mark the DNA fragments. Results: The isolated compound BNW-8 from the Neanotis wightiana showed significant anti-proliferative and cytotoxic activity in both the human leukemic cell lines MOLT-3 and K562 with IC50 of 2.1 lg/ml and IC50 of 3.6 lg/ml respectively. Morphologically, BNW-8 treated leukemic cells (MOLT-3 and K562) showed membrane blabbing, nuclear fragmentation, chromatin condensation and formation of apoptotic bodies than the control cells. Agarose gel electrophoresis study showed apoptosis by the formation of DNA ladder. Conclusion: The present study reveals that, the isolated b-carbolene alkaloid (BNW-8) from Neanotis wightiana has potent anti-leukemic activity against MOLT-3 (Lymphoid) and K562 (Myeloid) cell lines.

Abstract No. 348 Anti-leukemic Activity of Cirsimarin: An Active Constituent of Ruellia Tuberose Leaves Shila Elizabeth Besra1, Sayantan Dey1, Subhadeep Roy1, Nilanjana Deb2, Kalyan Kumar Sen2, Santanu Basu3 1

Drug Development/Diagnostic & Biotechnology Division, CSIR, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata-700032, 2Gupta College of Technological Sciences, Ashram More, G.T. Road, Asansol, West Bengal-713301. 3Employee State Insurance Hospital Sealdah, 301/3, A.P.C. Road, Kolkata-700009 Summary: Cirsimarin, a flavonoid has been isolated from the ethyl acetate fraction of methanolic extract of Ruellia tuberosa L. (Acanthaceae) leaf. We have studied the anti-leukemic activity of Cirsimarin on ALL and AML patient’s cells and two human leukemic cell lines (U937, K562). Introduction: Elimination of Cancer cells through apoptosis is the main goal of cancer therapy. The safest way to initiate cellular apoptosis is by using natural compounds than that of Tysrosine Kinase inhibitors specially in case of treating leukemia. Materials and Methods: Cytotoxicity study by MTT assay, morphological study was determined by Fluorescence and Confocal microscopy. Agarose gel electrophoresis study was performed. Detection of Apoptosis and cell cycle arrest study by flow cytometric analysis was performed. Cell signaling study by Caspase 9 & 3 assay

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 was also done. Results: The Cirsimarin isolated from the leaves of Ruellia tuberosa showed significant cytotoxic activity in human leukemic cell lines U937 and K562 with IC50 of 5.03 lg/ml & IC50 of 4.25 lg/ml as well as on PBMNCs of ALL IC50 of 7.47 lg/ml and AML IC50 of 21.86 lg/ml doses respectively. Cirsimarin treated leukemic cells showed formation of DNA ladder, chromatin condensation, externalization of phosphatidylserine apoptotic body formation and increased caspase 9 & 3 production indicating apoptosis. Cellular apoptosis has been confirmed further by flow cytometric analysis. Cell cycle study revealed that the treated leukemic cells get arrested in G0 & mostly in the G1 phase. Conclusion: The present study reveals that Cirsimarin, a bioactive flavonoid isolated from Ruellia tuberosa leaf have potent anti-leukemic activity.

Abstract No. 349 Evaluation of Monocyte CD36 Expression as Potential Marker of Macrovascular Complications in Patients of Diabetes Mellitus C Jayanthi, Neelam Wadhwa1, Rajive Kumar3, SV Madhu2, Satendra Sharma1, Anita Chopra3, Saroj Singh3 1

Department of Pathology, UCMS and GTB Hospital, Delhi, Department of Medicine UCMS and GTB Hospital, Delhi, 3 Laboratory Oncology Unit, BRA-IRCH, AIIMS, Delhi 2

Introduction: Predisposition of diabetes mellitus (DM) patients to macrovascular complications including atherosclerosis is well documented. Till date, there is no definitive investigation for atherosclerosis. CD36, a class 2B scavenger receptor expressed on monocyte plays an important role in uptake of oxidized LDL, a key event in atherosclerosis. Its increased expression on monocytes is proposed to be a potential diagnostic tool, besides being amenable to therapeutic targeting. Materials and Methods: 21 patients of DM and 20 normoglycemic controls (both HbA1C \6.5 and fasting plasma glucose \126 mg/dl) were recruited prospectively. Monocytes were identified flowcytometrically using PC5 labelled CD14 and side scatter characteristics. Monocyte CD36 (mCD36) expression was compared between cases and controls. Lipid profile was also performed in all subjects. Results: Monocyte CD36 expression in diabetics was significantly higher (mean ± SD: 101.59 ± 31.535, median ± SD: 107 ± 31.535) than normoglycemic controls (mean ± SD: 72.76 ± 31.973, median ± SD: 67.25 ± 31.973) (p B 0.006 and p B 0.016 respectively). Monocytes of DM patients were more likely to express CD36 than normoglycemic controls (83.86 vs. 55.67 %). The area under curve of ROC curves constructed for all the above mentioned parameters (namely mean mCD36, median mCD36 and % of monocytes expressing CD36) showed the highest value for % of monocytes expressing CD36 with sensitivity and specificity of 90.5 and 75.0 % respectively. The frequency of dyslipidemia was 1.5 folds higher in patients of DM in comparison to controls. The mean mCD36 expression increased in presence of dyslipidemia both in diabetic patients and normoglycemic controls (106.3 and 73.6 respectively) than their counterparts with lipid values in healthy range (72.5 and 67.7 respectively). Conclusion: Monocyte CD36 expression increases in patients with DM and those with dyslipidemia, conditions associated with increased risk of atherosclerosis. Being a novel marker, more studies are warranted to establish clinical utility of mCD36 as a potential marker of macrovascular

Abstract No. 350 Study of Hematological Changes in Wilson’s Disease K Kelkar1, P Patil1, R Manchanda1, A Bavdekar2, A Pandit2

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 1

Department of Pathology, K.E.M. Hospital, Pune; 2Department of Paediatrics, K.E.M. Hospital, Pune

Summary: Wilson’s disease is characterised by excessive copper deposition in liver, brain and other tissues due to genetic defect affecting the copper transporting ATPase gene leading to progressive liver failure, encephalopathy, coagulopathy and eventually death. Hematological changes in Wilson’s disease have been discussed in our study. Introduction: Hematological changes in liver disease are known. But there are few studies on large number of cases. The aim of this study was to study RBC, WBC and platelet parameters and to classify the type of anemia. Materials and Methods: The study was carried out between August 2005 and August 2007 on 50 patients who were clinically and biochemically proven to be Wilson’s disease. Hemogram and reticulocyte count and special tests like Bone marrow, PT,PTTK were studied. Results: Anemia was a common finding in all age groups. It was normocytic normochromic in 40 % new cases and 71 % on patients on treatment. Leucopenia was seen in 20 % new cases and 28.6 % in patients on treatment. 30 % new patients and 36.8 % patients on treatment showed thrombocytopenia. Pancytopenia was seen in only 15.7 % patients on treatment. Schistocytes, target cells and polychromatic cells were seen in new cases as compared to patients on treatment. Conclusion: Hematological changes in Wilsons disease are not infrequent. Patients on treatment need a close follow up of their hematological parameters. Neurological Wilsons is more often seen in adults, thus the diagnosis of Wilsons disease should be considered in an adult with liver failure, neurological and psychological symptoms.

Abstract No. 351 Haematological Profiles of Patients with Antinuclear Antibodies at a South Indian Tertiary Care Centre Mridula Laxman Bengre, B Prashantha Kasturba Medical College, Mangalore, Manipal University Summary: A retrospective analysis of haematological profiles of 82 patients with antinuclear antibodies was conducted at a South Indian tertiary care centre. Haematological abnormalities in these patients are multifactorial. Introduction: Antinuclear antibodies are autoantibodies that target normal proteins within the nucleus of the cell. The mere presence of ANA doesn’t indicate autoimmune disease. These ANAs could signal the body to begin attacking itself resulting in autoimmune diseases. Abnormal immune regulation patterns and persistent inflammation are hallmarks of autoimmune diseases. In such a context, autoimmunity/inflammation-associated cellular and molecular networks drive a complex of reactions that may involve haemopoietic tissue and peripheral blood cells. The purpose of this study is to evaluate the hematological abnormalities in patients with antinuclear antibodies. Materials and Methods: This retrospective study was conducted at a South Indian Tertiary care centre and included 82 patients with antinuclear antibodies detected by indirect IFT, irrespective of age, sex and clinical profiles. Haematological parameters were reviewed and evaluated. Results: 66/82 were females. Commonest ANA staining pattern was suggestive of dsDNA (32/82). Anemia was the commonest haematological abnormality (32/ 82) with normocytic normochromic anemia (13/32) as a majority, microcytic anemia (10/32), hemolytic anemia (3/32), pancytopenia (4/32) & others(2/32). Thrombocytopenia was the second most common abnormality (25/82). 15/25 patients had secondary Immune thrombocytopenic purpura. Leucopenia was least common (6/82). Conclusion: Anemia & thrombocytopenia are the common abnormalities seen in patients with antinuclear antibodies. Anemia of chronic disease and iron deficiency are more prevalent compared to

397 autoimmune hemolytic anemias. Secondary ITP is an underdiagnosed entity.

Abstract No. 352 Plumbism due to an Unusual Cause—A Case Report Ankita Shende, Daksha Prabhat, Anil Tambe1, Milind Tullu1, Mukesh Agarwal1 Department of Pathology, Seth G. S. Medical College and K.E.M. Hospital; 2Department of Pediatrics, Seth G.S. Medical College & K.E.M. Hospital Summary: We present a case of Plumbism in a child due to application of an indigenous paste made from a stone applied on the wound due to burns. Introduction: Lead poisoning (Plumbism) in children is usually due to ingestion of lead containing paint. It clinically manifests as colic, encephalopathy and anemia which is characterized by basophilic stippling of RBCs. Materials and Methods: Complete blood count (CBC) along with detailed study of Peripheral smear & reticulocyte count performed. Correlation with history and clinical presentation done. Serum lead levels were performed. Results: 11 year old female child came with pain in abdomen, constipation, vomiting since 4 days. There is past history of 30 % burns, 2 months back for which indigenous paste made from a stone was applied for 15 days.CBC revealed hemoglobin of 8.5 g/dl, extensive coarse basophilic stippling of RBCs. Reticulocyte count was increased12 %. Serum lead levels increased-89 lg/dl (Normal range in children upto 10 lg/dl). Lead levels of indigenous paste from the stone— 370 ppm. (Normal Limit—10 ppm). X-ray of Chest & abdomen revealed multiple superficial opacities corresponding to the site where the paste was applied and which cleared on stoppage of the application. A diagnosis of Plumbism due to application of an indigenous paste on the burns wound was made. Patient clinically improved after stoppage of the application. Hence no chelating agent was administered. Conclusion: Coarse basophilic stippling of RBCs should arouse a suspicion of lead poisoning & eliciting the cause by detailed history is important.

Abstract No. 353 Assessment of Blood Lead Levels and Haematological Changes in Employees of a Lead Battery Manufacturing Industry with Recommendations Suresh Venugopal, Adithya Suresh1, Rajesh Khan, Khaja K Khan Department of Laboratory, Nova Medical Centers Middle East, Muscat, Oman, Amrita Institute of Medical Sciences, Kochi Summary: A prospective study done in 2012 on 329 lead battery industry employees showed high blood lead levels in a vast majority. 71 % of those with high blood levels revealed hematological changes though basophilic stippling was seen in only 0.7 %. Recommendations were formulated based on blood level levels and hematological abnormalities. Introduction: Lead battery industry employees are exposed to lead causing adverse health effects. A study was undertaken to assess the blood lead levels among the employees, various hematological changes and formulate appropriate recommendations. Materials and Methods: Blood samples from 329 employees were tested for blood lead levels by atomic absorption method, Leishman stained peripheral smears were examined by a senior hematopathologist and complete blood counts performed on Cell Dyn emerald automated hematology analyser. Results: 283 (86 %) employees showed high blood lead levels. 191 out of 283 (58 %) employees

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398 showed blood lead levels exceeding the acceptable range for industrial exposure. Of these 283 employees, 71 % showed haematological changes. Erythrocytic changes (43 %) were the commonest followed by leucocyte changes (26.5 %). Basophilic stippling was seen in only 0.7 %. Apart from comprehensive annual medical checkups and strict compliance to preventive measures for all employees, specific recommendations were formulated for 3 categories based on blood lead levels and 5 categories based on hematological findings. Conclusion: A very high percentage of the target group showed high blood lead levels with the prominent hematological feature being erythrocytic changes (43 %) and the classical basophilic stippling seen in only 0.7 %. Specific recommendations were formulated for 3 categories based on blood lead levels and 5 categories based on hematological findings.

Abstract No. 354 Leucoagglutination In-Vitro—An Elusive Phenomenon: A Study of 11 Cases Kavita Gupta, M Deepak Nayak, Sushma V Belurkar Department of Pathology, Kasturba Medical College, Manipal University, Manipal Summary: Leucoagglutination in-vitro is an underrated event in laboratory practice. A morphological evaluation along with new automated analyzer parameters could possibly predict this phenomenon. Introduction: In-vitro aggregation is a well-known laboratory phenomenon for platelets and erythrocytes due to EDTA and cold agglutinins respectively. But leucoagglutination leading to spurious leukopenia has been reported predominantly as single case reports. These false results can be clinically perplexing; thus necessitating the recognition of this entity. We present a case series of 11 patients with spurious leukopenia due to leucoagglutination in-vitro. Materials and Methods: A retrospective analysis of 11 cases in the last two years which showed WBC clumping on the peripheral smear were retrieved using the LIS software. The machine data (including RPD data) for these cases were analysed. The corresponding peripheral smears before and after incubating the sample were also studied. A correlation with the clinical profile was done using the patient case files. Results: The WBC counts in all the 11 cases increased after incubating blood samples at 37 °C for 30 min suggesting a role of a temperature dependent antibody. The etiologies ranged from immune mediated (4 cases), liver diseases (2) and infections/drug induced (5). The mean neutrophil volume (MNV) was also noticeably high in 9 of 11 cases. Conclusion: Leucoagglutination in-vitro is possibly linked to an immunological phenomenon, may be due to a temperature dependent antibody. An increased MNV is a useful predictor in this regard. Hematopathologists should be aware of this spurious leukopenia to avoid unnecessary diagnostic tests and inappropriate treatment.

Abstract No. 355 A Rare Case of Pearson’s Syndrome with Predominant Hematological Manifestations and Novel Mitochondrial Deletion Parth Desai, Prasad Dange, Suniti Pahwa, Richa Gupta, Tejinder Singh, Seema Kapoor Department of Pathology and Paediatrics, Maulana Azad Medical College, New Delhi Summary: Pearson’s syndrome is a very rare cause of transfusion dependent anemia in infants with presence of pancreatic exocrine

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Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 deficiency and other systemic abnormalities. We present a case of Pearson syndrome in an infant with primary hematological manifestations and a novel mitochondrial deletion. Introduction: Pearson syndrome is a very rare cause of refractory transfusion dependent childhood anemia caused by mitochondrial DNA defects resulting in refractory sideroblastic anaemia, vacuolization of bone marrow precursors, exocrine pancreatic dysfunction and other metabolic defects Materials and Methods: A 6 month old infant presented with complaints of progressive pallor since one month, failure to thrive and fast breathing for 2 days. On examination he had severe anaemia with signs of congestive cardiac failure and hepatomegaly. Results: Peripheral smear findings revealed macrocytic anaemia with mild thrombocytopenia. Bone marrow aspirate revealed a cellular marrow with mild erythroid hyperplasia and characteristic cytoplasmic vacuolations in myeloid and occasional erythroid precursors. There were about 30 % ring sideroblasts observed on perl’s stain. In view of suspicion of Pearson’s syndrome, baby was further investigated which revealed high serum lactate levels (35 mg/dl). However, fecal fat excretion was normal. Mitochondrial DNA analysis revealed a point mutation in NADH dehydrogenase 3 (ND3 region) of mt DNA as opposed to large mtDNA deletions observed in previously reported cases of Pearson’s syndrome. Conclusion: Sideroblastic anemia presenting in infancy is an uncommon cause of anemia often requiring multiple transfusions. Pearson’s syndrome is a rare cause of sideroblastic anaemia, it presents in infancy, carrying a grave prognosis and requires high index of suspicion. Clinical picture is highly variable due to Mitochondrial DNA heteroplasmia. Mitochondrial DNA deletion/duplication studies carry a confirmative value. Pyridoxine is not very effective and child requires repeated transfusions. This case is presented for its rarity, hematological features and associated novel mitochondrial mutation.

Abstract No. 356 A Cross-Sectional Study of Nutritional Markers in Pregnancy Samanyoya Gochhait, Aashish Gupta, Sudhanshu Shekhar, Subhadra Sharma, Asok K Mukhopadhyay Department Of Lab Medicine, All India Institute of Medical Sciences, New Delhi Summary: Serum levels of nutritional markers (B12/Folate/Ferritin) in pregnancy and their association with high-risk factors like GDM and Hematological indices. Introduction: High prevalence of nutritional anemia has been reported in pregnant women in India. This cross-sectional study was undertaken to find out the correlation if any, of nutritional markers in different trimester of pregnancy and specially in association with various risk factors like GDM and hypertension. Materials and Methods: From the routine Antenatal Clinic (AIIMS, New Delhi) 251 pregnant women were subjected to assay of levels of Vitamin B12, Folate and Ferritin in the serum using Beckman Coulter’s Access-2 analyser. Serum from 31 non-pregnant healthy women of comparable age were used as controls. Complete Blood Count including indices were obtained by using EDTA sample from Beckmann Coulter’s Analyser. The data obtained from serum levels of the markers were analysed against the gestational age, gravida and with special relation to various high-risk factors in pregnancy and also correlated with CBC indices. The data being nonparametric Wilcoxon Rank-Sum Test was used. Results: Folate deficiency was found more commonly than VitB12 deficiency (p = 0.05). Folate deficiency was also shown to be associated more with high risk factors (single/multiple) (p = 0.05/0.02). Serum Ferritin was significantly (p = 0.02) raised in multiple high risk pregnancy. GDM was found to be significantly (p = 0.04/0.04) associated with decreased levels of VitB12 and Folate. Vit B12 was

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400 significantly reduced in second and third trimester (p = 0.02/0.02) while Ferritin was raised significantly in the third as compared to first and second trimester (p = 0.01/0.02). These nutritional markers were also found to be significantly associated with CBC indices in expected manner. Conclusion: Levels of nutritional markers merit monitoring in pregnancy which might help prevent high-risk pregnancy to deteriorate. There is association of Folate deficiency and high-risk factors. Gestational DM is associated with Folate and Vit B12 deficiencies.

Abstract No. 357 Prenatal and Intranatal Hypoxia: Important Cause of Increased Nucleated RBC in New Born Sonal Dhingra, SR Mehdi Era’s Lucknow Medical College Summary: Prenatal and Intranatal hypoxia are ignored but they are important causes of increased nRBC in the term new born. Introduction: Prenatal and intranatal history of acute or chronic hypoxia is significant in cases of increased nRBC in term neonate. Materials and Methods: Term baby’s APGAR at 1 and 5 min was taken. Maternal history, haematological and biochemical investigations, SPO2 monitoring and maternal and fetal blood grouping (mbg, fbg respectively) were performed. Results: APGAR at 1 and 5 min was 1 and 4 respectively. Increased nRBC = 125/100 wbc mbg = B+, fbg = B+, Patient Hb = 16,TLC = 67,700 (uncorrected), DLC = P35L60E3M2, plt = 70,000, PT = 17 s, APtt = 34 s, DCT = negative. PO2 decreased at six hrs postbirth to 73 % and patient expired ten hrs post birth. Maternal history ignored and other causes considered. No cause was found for increased nRBC. Mother was an unbooked case with Pregnancy induced Hypertension with Antepartum hemorrhage with active bleed at presentation with fetal distress, meconium stained liquor, cord around neck, labour duration = 28 h (increased) and Fetal Heart Rate = 80/min (reduced), maternal urea = 53.17 mg/dl, creatinine = 1.34 mg/dl, Conclusion: Prenatal and intranatal hypoxia, which are generally ignored should be considered as causes of increased nRBC along with considering other causes like Hemolytic disease of new born, Leukemia, Downs’ syndrome, Cyanotic heart disease or Pulmonary failure.

Abstract No. 358 CD4 and CD8 T Lymphocytes Counts and Their Ratio in Healthy Pregnant Females—Study at Tertiary Care Hospital Tarun Sharma, Sadhna Marwah, Indu Chawla, Gurdeep Buxi, AK Sen PGIMER and DR Ram Manohar Lohia Hospital, New Delhi Summary: The present study was undertaken to evaluate the levels of CD4 and CD8 T Lymphocytes count in normal pregnancy and to compare the results with controls. The CD4 and CD8 T Lymphocyte counts were also evaluated in each of the three trimesters. The study included 150 pregnant women (50 from each trimester) and 50 healthy women in reproductive age group. A detailed history were taken and any women (case or control) who had history of chronic disease, HIV positivity, derranged liver function test, kidney function test, raised blood sugar, or abnormal blood count was excluded from study. The levels of CD4 and CD8 T lymphocytes and their ratio were assessed by the four color flowcytometric machine and multitest kit. The results of this study showed that there was fall in the CD4 T Lymphocyte count, percentages and CD4:CD8 T lymphocyte ratio in pregnancy as compared to controls. Introduction: Pregnancy is

399 considered to be a physiological immunocompromised state. Any alteration in any parameter of immune system can affect health of pregnant women as well as outcome of pregnancy. The levels of CD4 and CD8 T Lymphocyte counts and their ratio serve as a guide for prophylactic and therapeutic intervention during the course of HIV infection. So it is important to know the baseline levels of CD4 and CD8 T Lymphocyte count in normal healthy pregnant women. Materials and Methods: The study included 150 pregnant women (50 from each trimester) and 50 healthy women in reproductive age group. A detailed history were taken and any women (case or control) who had history of chronic disease, HIV positivity, derranged liver function test, kidney function test, raised blood sugar, or abnormal blood count was excluded from study. The levels of CD4 and CD8 T lymphocytes and their ratio were assessed by the four color flowcytometric machine and multitest kit. Results: The results of this study showed that there was fall in the CD4 T Lymphocyte count, percentages and CD4:CD8 T lymphocyte ratio in pregnancy as compared to controls. Conclusion: This study showed that there was fall in the CD4 T Lymphocyte count, percentages and CD4:CD8 T lymphocyte ratio in pregnancy as compared to controls. Studies on large number of pregnant women are required to substantiate these findings.

Abstract No. 359 Improved Adeno-Associated Virus (AAV) Vectors for Hepatic Gene Therapy Dwaipayan Sen1, Dwaipayan Sen1,Akshaya Krishnagopal2, Rupali A Gadkari3, Govindarajan Sudha3, Vaani Roshini1, Ijaz Mahamadulla2, Narayanaswamy Srinivasan3, Alok Srivastava1,2, Giridhara R Jayandharan1,2 Department of Hematology, Christian Medical College, Vellore Introduction: Recombinant adeno-associated virus (AAV) vectors based on serotype-8 have shown significant promise for hepatic gene therapy of hemophilia B. However, the theme of vector dose dependent immunotoxicity seen earlier with AAV2 vectors persists. We had previously reported that novel AAV2 vectors bioengineered at specific serine (S)/threonine (T) ? alanine (A) or lysine (K) ? arginine (R) on its capsid have enhanced potential for liverdirected gene transfer (Gabriel et al., Hum Gene Ther Methods, 2013). Similar, proof-of-concept mutagenesis of equivalent residues in the homologous AAV8 serotype vectors improved coagulation factor IX expression with concomitant reduction in inflammatory and neutralizing antibody response in vivo (Sen et al., Hum Gene Ther Methods, 2013). In the present studies, we investigated if further modifications of capsid motifs identified by structural analysis on AAV8 can improve the efficiency of hepatic gene therapy. Results: Detailed structural analysis was carried out to scrutinize other potential sites on the AAV8 capsid (PDB: 2qa0), which would generate efficient novel vectors. We identified motifs such as phosphodegrons (phosphorylation sites recognized as degradation initiation signals by ubiquitin ligases) which contains phosphorylation and ubiquitination sites in AAV8 capsid structure. These sites being antigenic, away from interaction interface and receptor binding region served as potential target sites for mutagenesis to enhance gene transduction. Based on this analysis, we strategically mutated the following sites, K143, K652, S149, S156, T138 and T654 on AAV8 capsid. To test the efficiency of these novel vectors, we delivered luciferase gene under the control of chicken b-actin promoter (5 9 1010 vgs per mouse, i.v) into 8–12 weeks old male C57BL/6 mice. Hepatic gene expression was analyzed 2 and 5 weeks after gene transfer in a small-animal imaging system. As seen in Fig. 1a–c, a systemic distribution of luciferasegene expression was noted for K143R, K652R, S149A and T138A vectors, when compared to WT-

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400

2 weeks

WT-AAV8

K143R

5 weeks

Addendum

B Total Flux X 10^6 (photons/sec)

A

Indian J Hematol Blood Transfus (Oct-Dec 2013) 29(4):278–400

2 weeks 14

*

12 10

*

Clinical Profile of Children with Anaplastic Large Cell Lymphoma

6 4 2 0

Deepthi Boddu, Rikki R John, Narendra Chaudhary, Leni G Mathew

K652R

Hematology-Oncology, Department of Child Health, Christian Medical College, Vellore

S149A

T138A

T654A

Total Flux X 10^6 (photons/sec)

C S156A

5 weeks

30 25 20

* *

15 10 5 0 -5 -10

K137R +S671A

Fig. 1 Transduction efficiency of various novel AAV8 vectors 2 and 5 weeks post vector administration (a). Quantitation of data from A is presented in photons/s, 2 weeks (b) and 5 weeks (c) post vector administration. *p \ 0.05 AAV8 vectors 5-weeks post gene transfer. This was further corroborated by real time PCR analysis of vector copy numbers in organs like kidney (K143R—4, K652R—8, S149A—3, T138A—1.6 Vs WTAAV8—0.67 vector copies/diploid genome) and spleen (K143R— 1.4, K652R—14, S149A—6, T138A—2.7 vs. WT-AAV8—0.3 vector genome copies/diploid genome). Conclusions: Our data suggests that alteration of phosphorylation and ubiquitination sites in AAV8 capsid structure based on structural analysis highly improves the transduction efficiency of this promising gene therapy vector in vivo. Further on-going studies with these vectors expressing human coagulation factor IX in murine models, will demonstrate the feasibility of the use of these novel vectors for potential gene therapy of hemophilia B.

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Abstract No. 360

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Aim: Anaplastic large cell lymphoma (ALCL) is a distinct form of NHL, which accounts for 10–15 % of childhood lymphomas. In this retrospective analysis, we looked at clinical profile, staging, and outcome of a small cohort of children treated at our center. Methodology: Retrospective chart review of children with ALCL treated at the pediatric Hematology-Oncology unit of Christian Medical College, Vellore, India from January 2004–June 2013. Results: 19 children, 14 boys and five girls with ALCL were included, their age at diagnosis ranged from 1.5 to 14 years. Lymphadenopathy (16/19) and fever (14/19) were the most common presenting symptoms. One child had primary cutaneous ALCL; other symptoms included mediastinal disease, pericardial tumor, multiple bone involvement, fungating skin lesion, parameningeal disease with multiple cranial nerve deficits, mandibular tumor and maxillary tumor. Duration of symptoms ranged from 0.5 to 8 months. Two children had stage I disease, seven had stage II and five each had stage III and IV disease. Immunohistochemistry showed ALK positivity in all except three children, CD30 positivity in all, CD20 negativity in 15/19, and CD15 was negative in all 10 tested. All the children were treated with BFM-NHL 90 protocol. Twelve children achieved remission. Three died during treatment all of whom were high risk disease. Four relapsed all of whom were high risk of whom three were ALK-ve. Three of four children who relapsed were treated with Relapse protocol (Lomustine/VBL/ARAC) of whom two achieved remission. Conclusion: 63 % of children had high risk disease. Mortality was 16 % and the relapse rate was 21 % in this cohort. All the three children who had ALK negative ALCL relapsed.