Indian J Hematol Blood Transfus (Jan-Mar 2013) 29(1):61–64 DOI 10.1007/s12288-012-0225-0

ABSTRACTS

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

Ó Indian Society of Haematology & Transfusion Medicine 2013

Abstract P 211 To Evaluate Role of Annexin A1 by Immunohistochemistry in Diagnosis of Hairy Cell Leukemia (HCL) Dr. Gurmeet Singh, Dr. Mrinalini Kotru, Dr. Tulika Seth, Dr. Seema Tyagi, Dr. Renu Saxena, Dr. H.P. Pati All India Institute of Medical Sciences, New Delhi Objective: To evaluate role of Annexin A1 by immunohistochemistry in diagnosis of Hairy Cell Leukemia (HCL) as compared to immunophenotyping. Methods: A total of 19 cases of HCL diagnosed from a period of January 2006 to June 2012 were included in the study. 15 cases of NonHodgkins lymphoma were taken as controls. Annexin A1 was detected by immunohistochemistry on formalin fixed paraffin sections of bone marrow biopsy using Rabbit polyclonal antibody to Annexin A1. Results: A total of 19 cases of HCL were evaluated. Median age of HCL group was 50.5 years (35–65 years), with male preponderance (M:F = 18:1). Physical findings revealed splenomegaly in 100 % of cases with hepatomegaly and lymphadenopathy in 14/19 (73.7 %) and 8/19 (42.1 %) cases, respectively. Anemia (84.21 %) and thrombocytopenia (78.94 %) were prominent findings. Monocytopenia was a significantly different in HCL and controls (p \ 0.00001). Flowcytometry showed positivity for CD103 in 17 cases and CD25 and CD11c in 19/19 & 17/19 cases. Annexin A1 was positive in 17/19 (89.47 %) cases of HCL and more importantly was negative in all the 15 cases of controls. Annexin A1 has sensitivity of 89.45 % and specificity of 100 %. Diagnostic accuracy using Annexin A1 for diagnosis was 94.11 % (CI 80.3–99.3). Conclusion: Annexin A1 positivity is a very sensitive and specific for diagnosis of HCL.

Abstract P 212 Efficacy and Outcome of Dose Intensification of Daunorubicin from 45 to 60 mg/m2/day in Induction Chemotherapy for Acute Myeloid Leukemia: Experience from a Tertiary Care Center Suman Kumar, Avinash Singh, Sanjeev Sharma, Manoranjan Mahapatra, Nitin Gupta, Tulika Seth, Pravas Mishra, Renu Saxena Department of Hematology, AIIMS, New Delhi

Background: Recent trials from western world for AML patients showed that, escalating dose of daunorubicin to 90 mg/m2/day in standard ‘‘3 + 7’’ induction regimen improves relapse free survival and overall survival. There is scarce data from India on AML induction outcomes. Method: We compared outcomes by increasing daunorubicin to 60 mg/m2/day (ArmB: March 2010 to February 2012) to standard ‘‘3 + 7’’ regimen with daunorubicin 45 mg/m2/day (ArmA: August 2008 to Jun 2010). Second induction was given with high dose cytarabine and mitoxantrone (HAM) after first induction failure. Consolidation was given with 2–3 cycles of high dose AraC or allogenic SCT, if feasible. Results: There were 183 patients recruited (ArmA:94 and armB:89). Median age of patients was 30 years, with 64.5 % being males. Median duration of symptoms was 8 weeks (range 1–34), median time to start induction therapy from diagnosis was 22 days (range 1–106) with 11.4 % patients having delay of more than 1 month. 21 % patients were infected at baseline and 75 % patients had infections during induction therapy. 37.5 % of baseline infected patients (vs 16.8 % of those who were not infected, p = 0.009) died during first induction. 32.6 % of patients who developed pneumonia (vs 9.9 % of those who did not develop pneumonia, p \ 0.001) during induction died. Overall, 50.8 % patients achieved remission and 16.9 % died after first induction therapy, 65.6 % patients achieved remission and 32.2 % patients died after both inductions. 36.7 % of patients who achieved remission relapsed with median follow up of 176 days after last therapy. ArmB had remission rate of 46.1 %, death of 27 %, armA had remission rate of 55.3 % (p = 0.21) and death of 16 % (p = 0.12) after first induction. ArmB had 60.6 % remission, compared with 71.1 % in armA (p = 0.13) after both inductions. After excluding induction deaths, armA and armB had 96.4 & 96.9 % chances of achieving remission (p = 0.94) after both inductions. Estimated cumulative incidence of relapse in armA and armB were 61.4 and 51.9 % at 666 days (p = 0.43). Estimated OS at 1 year was 42.4 and 34.2 % for armA and armB (p = 0.28). Conclusion: Infections was the major contributor of induction deaths. A good number of patients presented with baseline infection. Increasing dose of daunorubicin was not sufficient enough to have increased induction remission, or reduced relapse rate or improved overall survival. We should reduce high induction mortality before attempting to further intensifying induction regimen in our patients.

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Abstract P 213 Central Venous Access Practices and Complications: An Observational Study from a Hematology Centre Suman Kumar, Avinash Kumar Singh, Tulika Seth, Manoranjan Mahapatra, Pravas Mishra, H. Pati Department of Hematology, AIIMS Background: Central venous access (CVA) forms an integral and an inescapable part of managing patients in hematology. These catheters are an important source of morbidity, and sometimes mortality in these patients. There is a scarcity of data on their usage and complications in India. Aims: This study was planned to observe and analyse central venous catheters (CVC), and their complications. Method and materials: The study was planned to be a prospective observational one over a period of 1 year, where catheters inserted to patients admitted under Hematology department were studied. Results: A total of 222 patients with 255 catheters were studied with a cumulative follow up of 9,636 catheter days (mean follow up of 37.8 days per catheter). 82.4 % of the catheters were inserted bedside, rest in OT/radiology room. 75.7 % catheters were PICC (usually Cavafix Certo, Braun), 7.1 % were IJV catheters and 17.3 % were tunneled CVC (Hickman, Bard). 80.4 % of the catheters were made of polyurethane and rest of silicone. Initial placement was wrong in 12.9 % of the cases (59.8 % were placed distally). Presenting diagnosis was aplastic anemia in 6.8 % and acute leukemia in 74 % of cases. CRBSI occurred at a rate of 0.93 per 1,000 catheter days. For PICC, IJV and Hickman catheters, rates were 1.19, 1.70 and 0.32 per 1,000 catheter days, respectively (p = 0.34). Local infective complications occurred at a rate of 5.89 per 1,000 catheter days. For PICC, IJV and Hickman catheters, rates were 6.68, 10.72 and 3.45 per 1,000 catheter days, respectively (p = 0.21). SVT occurred at a rate of 1.56 per 1,000 catheter days. For PICC, IJV and Hickman catheters, rates were 1.36, 1.73 and 0 per 1,000 catheter days, respectively. DVT occurred at a rate of 2.71 per 1,000 catheter days. For PICC, IJV and Hickman catheters, rates were 2.72, 6.81 and 0.32 per 1,000 catheter days, respectively. Conclusions: CRBSI occurred almost equally among PICC, IJV catheters and Hickman catheters. Local infective complications occurred almost equally among three types of catheters. DVT occurred more in IJV catheters, then in PICC and Hickman catheter. Our findings correlate well with data from other studies.

Abstract P 214 Anti-proliferative, Cytotoxic & Apoptogenic Activity of the Secretion of Bellamya bengalensis f. annandalei Moumita Ray1, Nilanjana Deb1, Subhadeep Roy1, Sayantan Dey1, Aparna Gomes1, Krishna Das Saha2, Prithvish Banerjee3, Santanu Bose3, Shila Elizabeth Besra1 1 Drug Development/Diagnostic & Biotechnology Division, Indian Institute of Chemical Biology (CSIR), 4 Raja S.C. Mullick Road, Kolkata-700032, West Bengal, India. Tel: 91-33-2413-3223, Fax-9133-2473-5197. Email- [email protected]; 2Immunology Division, Indian Institute of Chemical Biology (CSIR), 4 Raja S.C. Mullick Road, Kolkata-700032, West Bengal, India. Tel: 91-33-24133223, Fax-91-33-2473-5197; 3Employee State Insurance Hospital Sealdah, 301/3, A.P.C. Road, Kolkata-700009

Objective: Advances in the prevention and treatment of cancer will require the continued development of novel and improved chemotherapeutic and chemopreventive agents. Natural Products have been

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Indian J Hematol Blood Transfus (Jan-Mar 2013) 29(1):61–64 the most significant source of drugs and drug leads in history. Their dominant role in cancer chemotherapeutics is clear with about 74 % of anticancer compounds being either natural products, or natural product-derived. We have studied the anti-leukemic effect of the secretion of Bellamya bengalensis f. annandalei in K562 human immortalised myelogenous leukemia cell line and peripheral blood mononuclear cells of AML patients. Methods: Extraction, partial purification, SDS-PAGE and protein estimation by Lowry assay. Cell viability study by Trypan blue exclusion, cytotoxicity study by MTT assay, morphological studies were done by Confocal and Fluorescence microscopy, DNA ladder by agarose gel electrophoresis. Toxicity was studied on normal lymphocytes. Results: The secretion of Bellamya bengalensis (SBB) has potent anti-cancer activity against human leukemic cell line K562 & PBMNC of AML patients by anti-proliferative & cytotoxic activity. But in normal lymphocytes, have no toxicity. Morphologically it was found that externalization of PS in all treated leukemic cells than controls & fluorescence microscopic images also depicting the membrane blabbing, condensed chromatin & fragmented nuclei. Agarose gel electrophoresis showed DNA ladder formation, indicating the process of apoptosis. Conclusion: The active ingredient(s) present in the extrapallial fluid/ secretion of Bellamya bengalensis f. annandalei is found to be nontoxic therefore we can expect it to be a novel anti-cancer compound for the treatment of human from animal source.

Abstract P 215 Coagulation Profile in Diabetes Mellitus and Its Association with Microvascular Complications Dr. Tity. P, Prof. Dr. L.K. Meher, Dr. Uma Shankar Mishra, Dr. Jagannath Sarangi, Dr. Krishna Kumar K., Dr. Tapas Ranjan Behera, Dr. Karthik Sasalu, Dr. Sujal Shetty M.K.C.G. Medical College, Berhampur, Odisha Objective: This study intends to assess the changes in the simple routine Coagulation Parameters in diabetes mellitus and to investigate whether any relationship exists among changes in these coagulation parameters and development of microvascular complication in diabetes mellitus. Material and Method: Period of study was from 2010 to 2012. It was done in M.K.C.G Medical College with the approval from Berhampur University. It is a case control study. 50 diabetic patients and 50 age and sex matched non-diabetic patients were randomly selected. Simple coagulation parameters like activated Partial Thromboplastin time (aPTT), Prothrombin time (PT), Serum fibrinogen, platelet count, plasminogen activator inhibitor 1 (PAI-1) were measured. Statistical Study was done using unpaired T test and analysis and calculations were done using Graphpad software. Result: Serum fibrinogen was found to be increased in diabetic patients when compared to non-diabetic patients [Mean 278 ± 26.9 vs 232.52 ± 16.5, P value—0.009 significant]. PAI-1 levels was found to be higher among the diabetics when compared to non-diabetics (47.64 ± 8.82 vs 31.06 ± 7.12, the two-tailed P value is \0.0001, considered extremely significant). Platelet count through within normal limits it was found to be decreased in diabetic patient when compared to non-diabetic. [2.25 ± 0.18 vs 2.33 ± 0.03, P value—0.022]. Prothrombin time PT [13.15 ± 0.52 vs 13.04 ± 0.49, P value—0.28] and PTT [33.04 ± 1.31 vs 32.99 ± 1.29, P value 0.85 found to be statistically insignificant]. Among 50 diabetic patients 24 had neuropathy, 20 had nephropathy and 10 had retinopathy, 21 had none of these complications. On

Indian J Hematol Blood Transfus (Jan-Mar 2013) 29(1):61–64 comparing diabetic patients with microvascular complications and without microvascular complications, significant age difference was observed (59.55 ± 5.06 vs 51.00 ± 3.31, P = 0.003). This probably was a reflection of increase in microvascular complications with increasing duration of diabetes. Serum fibrinogen was found to be increased among diabetic patients with microvascular complications when compared to diabetic patient without microvascular complications (285.28 ± 32.36 vs 269.86 ± 13.08, P value 0.0449 statistically significant). PAI-1 levels was found to be higher among the diabetics with microvascular complications when compared to diabetics without microvascular complications (52.34 ± 7.40 vs 41.12 ± 6.31, the two-tailed P value is \0.0001, considered extremely significant) Comparison of diabetic patients with nephropathy and without nephropathy showed significant difference in fibrinogen and PAI-1 levels (292.39 ± 20.19 vs 269.80 ± 24.43, P value 0.002; 53.67 ± 7.59 vs 43.62 ± 7.31, P value \0.001). Serum fibrinogen and PAI-1 levels had significant difference on comparison among diabetic patients with neuropathy and without neuropathy(288.92 ± 26.42 vs 269.46 ± 24.38, P value 0.009; 52.86 ± 7.87 vs 42.83 ± 6.85, P value \ 0.001). Comparison of diabetic patients with retinopathy and without retinopathy showed significant difference in fibrinogen levels (249.50 ± 27.19 vs 286.13 ± 21.64, P value 0.0001). No significant difference in the PAI-1 levels was found among the diabetics with retinopathy when compared to diabetics without retinopathy (48.01 ± 6.95 vs 47.55 ± 9.30, the two-tailed P value is 0.8846, considered not significant). aPPT and, PT showed no significant difference in diabetic patients with and without microvascular complications. Conclusion: From this simple study it has been show that diabetes mellitus is a procoagulant state. Hypercoagulability as evidenced by increased fibrinogen and hypofibrinolysis as evidenced by in increased PAI-1 levels. Their levels are elevated in diabetic patients with microvascular complications when compared to those without. Though pathophysiology of microvascular complications not fully understood, it has been shown that there is significant coagulation abnormalities in diabetic patients with microvascular complications.

Abstract P 216 Comparative Assessment of Various Markers and Use of a Novel Limited Antibody Panel with CD127 as an Alternative to the Transcription Factor FoxP3 for Enumeration of Regulatory T Cells in Chronic Lymphocytic Leukemia Dr. Alakananda Dasgupta, Dr. Manoranjan Mahapatra, Dr. Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi Objective: This study compares the expression of CD127 with respect to FoxP3 and CD25 and explores the use of low CD127 expression as an alternative marker for enumerating of FoxP3+ Regulatory T cells (Tregs) in chronic lymphocytic leukemia (CLL). Methods: Treg enumeration was done using whole blood lysis and cell separation by I Magnet and T lymphocyte enrichment set in a series of 70 CLL patients and 30 age- and sex-matched controls by flow cytometric immunophenotyping. Antibodies used were CD4PerCPCy5.5, CD25FITC, CD127PE, and FoxP3AlexaFluor647. Results: The sensitivity, specificity, positive predictive value and negative predictive value of various combinations of markers were determined. CD4+FoxP3+ had the highest sensitivity and specificity. The other combinations with comparable sensitivity and specificity were CD4+CD127low/-, CD4+CD127low/-FoxP3+ and CD4+CD127low/-CD25+FoxP3+. There was significant correlation

63 between CD4+FoxP3+ expression (which is used as the reference population) and the various combination of markers. However, for the combination CD4+CD25+, positive correlation with CD4+FoxP3+ though present was far less (Spearman’s rho = 0.6443, p = 0.001) as compared to the other combinations where a much higher correlation coefficient was obtained. Thus, CD127 appears to be a better marker than CD25 for the identification of CD4?FoxP3?T cells. Our results also suggest that the specificity and sensitivity of CD4?CD127low/- cells is comparable to that of CD4?FoxP3? which is the gold standard and can be used as an alternative to it. Conclusion: This novel limited antibody panel comprising of CD4 and CD127 is cost-effective, can be used for Treg cell analysis and may have relevance in the context of resource limitation.

Abstract P 217 Determination of an Optimal Threshold Level for Regulatory T cell Expression in Chronic Lymphocytic Leukemia Dr. Alakananda Dasgupta, Dr. Manoranjan Mahapatra, Dr. Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi Objective: There is no uniform guideline or consensus regarding the optimal cut-off levels of frequencies of Regulatory T cells (Tregs) in chronic lymphocytic leukemia (CLL). Standardization and validation are warranted before Treg numbers in CLL can be generally adopted in routine laboratories as a predictive prognostic marker. This study explores the optimal threshold level for Tregs in patients with CLL. Methods: Treg enumeration was done in a series of 70 CLL patients and 30 age- and sex-matched controls by flow cytometric immunophenotyping. Antibodies used were CD4PerCPCy5.5, CD25FITC, CD127PE, and FoxP3AlexaFluor647. The cut-offs for Treg positivity (both percentage and absolute Treg cell count) that best discriminated between controls and cases were assessed by receiver operating characteristic (ROC) analysis. Results: For the ROC analysis, the sensitivity was plotted against the specificity, where the cutoff between negative and positive tests takes successively each value of a measure from lowest to highest. The area under the ROC curve (AUC) quantifies the degree of discrimination. A cut-off of 5.7 % for Treg cell percentage had a sensitivity of 87.14 %, a specificity of 86.67 %, positive likelihood ratio (LR?) of 6.535 and negative likelihood ratio (LR-) of 0.148 and AUC was 0.9124. For absolute Treg cell count, a cut-off of 35 cells/ll had a sensitivity of 84.29 %, specificity of 83.33 %, LR? of 5.057 and LR- of 0.188 and AUC was 0.9145. These cut-off levels therefore seem optimal in patients with CLL . Conclusion: The experiments presented here will aid in the optimization of quantification of Tregs in CLL patients with the optimal balance of specificity and sensitivity. However, further experiments are needed to validate our results and enhance the analytic certitude.

Abstract P 218 Homozygous b-Thalassemia with glucose6 Phosphate Dehydrogenase Deficiency: A Case Report J M Khungar, Rekha Singh, Monika Gupta, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi, India

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Indian J Hematol Blood Transfus (Jan-Mar 2013) 29(1):61–64

The thalassemias and glucose6 phosphate dehydrogenase deficiency are among the most common disorders worldwide. The marked oxidative stress caused by b-thalassemia, that is apparently incompatible with glucose6 phosphate dehydrogenase deficiency was seen in a rare case report in which these two disorders co-existed. A 6 month old baby boy presented in Hematology OPD with complaints of failure to thrive and required frequent blood transfusions. He had received 3 blood transfusions till date and had the following hematologic data; Hb 3.8 g/dL, Hct 14.1 %, MCV 75.0 fL, MCH 20.2 and MCHC 27.0 g/dL. Hemoglobin analysis revealed 96 % HbF and HbA2 3.1 %, his screening for glucose6 phosphate dehydrogenase enzyme showed a deficient enzyme activity. His parental study was done which revealed heterozygous b-thalassemia in both father and mother, while glucose 6 phosphate dehydrogenase screening showed a normal activity in father and a deficient activity in mother.

Chromatography (HPLC) was done which showed 100 % HbF. Provisional diagnosis of hereditary persistence of fetal hemoglobin was considered. Parental screening by HPLC for thalassemia showed normal results. Gene mutation studies for delta beta thalassemia were performed and patient was finally diagnosed as homozygous db-thalassemia. Both his parents showed compound heterozygosity for db-thalassemia in gene mutation analysis. There is a thin line of difference between hereditary persistence of fetal hemoglobin and db-thalassemia. The difference is subtle and is made on clinical and hematological basis. The characterization methods used in this study will prove useful in complementation of routine hemoglobin analysis to determine the genotype properly and will facilitate a prevention and control program of thalassemia and hemoglobinopathies in the region. Results of the patient and parents are listed in the table below:

Abstract P 219

Parameter

Patient

Mother

Father

Delta Beta Thalassemia: A Case Report

Sex/age (years)

M/10

F/35

M/38

J.M. Khungar, Rekha Singh, Monika Gupta, Ravi Ranjan, H. R. Pandey, Renu Saxena

Hb (g/dL)

9.0

11.6

13.0

Hct (%)

35.0

41.4

45.5

MCV (fL)

78.8

83.8

87.5

MCH (pg)

20.3

23.5

25.0

MCHC (g/dL)

25.7

28.0

28.6

HbA2 %

0.0

2.5

2.5

HbF %

100

16.6

18.0

Department of Hematology, All India Institute of Medical Sciences, New Delhi, India The hereditary persistence of fetal hemoglobin and delta beta-thalassemia are heterogeneous disorders characterized by increased levels of fetal hemoglobin in adult life. The distinction between these two conditions is not always possible with routine hematological analysis. We encountered such a rare case of db thalassemia. A 10 year old male child presented in Hematology OPD, with history of pallor, weakness and jaundice (Off & on) since 5–6 years. There was no history of blood transfusions or drug intake. There was no family history of similar disease. On examination, Liver and spleen were mildly enlarged. Laboratory investigations showed hemoglobin of 9.0 g%, MCV 78.8 fl, MCH 20.3 pg, MCHC 25.7 g/dl. High Performance Liquid

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Hb typing/HPLC

Mutation Homozygous Compound heterozygous Compound study for for both for breakpoint heterozygous db-thalassemia breakpoint A and B of for breakpoint A and B the Asian–Indian A and B of the of the Inv deletion Asian–Indian Asian–Indian Inv deletion Inv deletion