86 Indian J. Hematol. Blood Transfus 24(3):86–145

Indian J. Hematol. Blood Transfus 24(3):86–145

ABSTRACTS

49th Annual Conference of Indian Society of Hematology & Transfusion Medicine

ISHTM 2008, September 19–21, 2008, Location Jawaharlal Institute of Postgraduate Medicine and Research (JIPMER), Pondicherry, India Organizers Departments of Medicine, Pathology and Pediatrics, JIPMER, India

OP 1 Prevalence of genetic modulators and their effect on the phenotype of HbE/β thalassemia patients

Vineeta Sharma · Bijender Kumar · Sanjay Kumar Pandey · Ved Prakash Choudhry · Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi, India e mail: [email protected]

Background HbE, a β-globin chain variant occurs due to GAG to AAG i.e. Glu-Lys substitution at codon 26. HbE/β thalassemia results in a variable and often severe anemia, with the phenotype ranging from transfusion dependence to a complete lack of symptoms. The basis of the interaction and the cause of the variability remain unexplained. Some of the possible explanations for the observed variable clinical severity are coinheritance of mild β mutations, αdeletions and triplication, XmnI polymorphism HPFH and δβ deletions. Aim To determine the frequency of β mutations, α deletion and α triplication, XmnI polymorphism, HPFH3 and Asian-Indian Inv/DelGγ(Aγδβ)0 deletion in HbE/β thalassemia patients and to study their modulating effect on the phenotype of the Patients. Methods Subjects were 150 HbE/ßthalassemia patients diagnosed by HPLC and confirmed by PCR-RFLP. Patients were divided into three subgroups according to a scoring system based on seven clinical criteria as mild (score 0–3.5), moderate (score 4–7) and severe (score7.5–10).β-mutations were studied by ARMS PCR, XmnI polymorphism was

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Pondicherry, India

studied by PCR-RFLP while α deletions, HPFH-3 and Asian-Indian Inv/DelGγ(Aγδβ)0 deletions were studied by GAP-PCR and alpha triplication by multiplex PCR. Results The predominant β mutation was IVS 1-5 G→C (81.3%).α deletion was found in 19 (12.6%) out of which 3.7 3.7 13(12 αα/-α and 1 αα/-SA) were from Gp1 and 6(αα/α ) were from Gp2.α triplication was found in 11(7.3%) out of anti3.7 ) from Gp3 and 3 were these 11 patients 8 were (αα/ααα anti3.7 (αα/ααα ) from Gp2. XmnI was found in 94(62.6%), out of which 82 were heterozygous (+/–) and 12 were homozygous (+/+).XmnI +/+ was present in 9 Gp1 and 3 Gp2 patients, while XmnI +/– was present in 29 Gp1, 33 Gp2 and 20 Gp3 patients.HPFH-3 deletion was present in 8 (5.3%) out of which 5 were from Gp1 and 3 were from Gp2. Asian-Indian Inv/DelGγ(Aγδβ)0 deletion was present in only 1(0.7%) patient from GpII. Conclusion Patients with coexisting α deletion required lesser transfusions and had less severe phenotype while patients with α triplication were on frequent transfusions and had severe phenotype. XmnI polymorphism in homozygous state was observed to alleviate the severity while in heterozygous state it had no effect on the disease severity. Patients having HPFH3 deletions and AsianG A 0 Indian Inv/Del γ( γδβ) deletion showed mild to moderate disease severity. OP 2 Rapid screening of (δβ) thalassaemia deletions using gene dosage analysis of β globin cluster genes

Anu Moses · Eunice Sindhuvi Edison · Shaji R Velayudhan · Alok Srivastava · Mammen Chandy Department of Haematology, Christian Medical College, Vellore, India e-mail: [email protected]

Deletions that remove δ and β globin genes in the β globin cluster can cause (δβ)0 thalassaemia, characterized by elevated fetal hemoglobin with microcytic hypochromic anaemia. Homozygotes and compound heterozygotes

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exhibit the phenotypes of thalassaemia major or thalassaemia intermedia. In our center, 23 individuals from 12 families with (δβ)0 thalassaemia have been seen and a previously reported mutation causing this phenotype was found in 17(73.9%) individuals. For the remaining cases we developed a rapid and sensitive method that can detect all the deletions of the functional genes in the β globin cluster. Genomic regions from γ, δ and β globin genes and albumin (alb) gene were amplified by a multiplex PCR using fluorescently labeled primers using 200ng of DNA for 20 cycles. The PCR products were analyzed in ABI 310 Genetic Analyzer and peak heights were measured using Gene scan software. The values obtained from an individual with δβ thalassaemia were normalized by dividing them with those obtained from a normal individual. The copy numbers of γ, δ and β globin genes were calculated by assigning a value of 1 for albumin (alb) gene and calculating the relative values for the globin genes. Positive control samples with known (δβ)0 inversion-deletion mutation were used to validate this protocol. We used this protocol for successful molecular diagnosis in 6 individuals in whom the common inversion-deletion mutation was absent. In four of them, the mean gene dosage ratio for alb: γ: δ: β was 1: 1+0: 0.57+0.19: 0.50 +0.01, which is suggestive of heterozygous state of GγAγ(δβ)0 thalassaemia. In the two cases for which we performed prenatal diagnosis, the mean ratio was 1: 0.76+0.02: 0.52+0.01: 0.53+0, suggestive of Gγ(Aγδβ)0 thalassaemia. We, therefore, conclude that this gene dosage method can be used widely in all the populations to detect gross deletions affecting γ, δ and β globin genes avoiding cumbersome multiple southern blotting procedures. OP 3 Molecular characterization of 5 novel / rare β thalassemia mutations in India

Anita Nadkarni · A. Gorakshakar · R. Surve · P. Sawant · S. Phanasgaonkar · S. Nair · K. Ghosh · R. Colah National Institute of Immunohematology, 13th floor KEM hospital campus, Parel, Mumbai, India e-mail: [email protected]

Background β thalassemia is seen in every region and in majority of the caste groups in Indians. Molecular characterization of β-thalassemia continues to reveal an ever expanding profile of mutations in our diverse population. Objective To characterize novel and rare β-thalassemia mutations using a combination of mutation detection approaches and to correlate the genotype with phenotype. Methods As a strategy, we first screened for 6 common Indian mutations along with two abnormal hemoglobins (HbE and HbS) using reverse dot blot (RDB) hybridization.

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In the next step, we screened for 29 other known but rare mutations reported in Indians using amplification refractory mutation system (ARMS). The uncharacterized samples were then directly sequenced on the ABI – 310 automated DNA sequencer. Results Among 375 carriers of β-thalassemia analyzed during a period of 2 years, the mutations in 8 individuals remained uncharacterized after screening for 35 mutations by RDB hybridization and ARMS. Direct DNA sequencing reveled two novel [CD 16 C→T and IVS II 613 (C→ T)] and three rare mutations [CD15 (–T), CD 22/24 –7bp deletion and CD 8 (–AA)]. As the CD 15 (–T) mutation was seen in four families, we tried to look for a simple approach like restriction enzyme analysis for detection of this mutation. The deletion of T creates a restriction recognition site for the enzyme Bme1390I (SCrFI). The results of the restriction enzyme digestion were in concordance with the results of DNA sequencing. Conclusion Identification of such mutations shows the increasing repertoire of molecular defects causing β-thalassemia among Indians. This information is valuable for genetic counseling and a prenatal diagnosis programme. OP 4 Flowcytometric evaluation of CD59 expression for rapid diagnosis of patients with Paroxysmal nocturnal haemoglobinuria (PNH)

Bargavi Balakrishnan · Reena Rajasekar · K. Kotteeswari · Vikram Mathews · Auro Viswabandya · Biju George · Mammen Chandy · Alok Srivastava Department of Haematology, Christian Medical College, Vellore, India e-mail: [email protected]

Background PNH is an acquired stem cell disorder characterized by increased sensitivity of erythrocytes to complement-mediated cell lysis due to deficiency of membrane-bound GPI (glycosylphosphatidylinositol)anchored proteins such as CD59 and CD55. Objectives Evaluate flowcytometric expression of CD59 (MIRL - Membrane inhibitor of reactive lysis) on granulocytes and erythrocytes for diagnosis of PNH. Materials and methods Peripheral blood samples were collected between Jan 2006 and April 2008 from 324 patients with pancytopenia or clinical / laboratory features suggestive of PNH. Each sample was analyzed simultaneously with 324 healthy control samples. Expression of CD59 (antiCD59-PE, BD Biosciences) was analyzed both on erythrocytes and on granulocytes (confirmed by expression of CD11b). For each subject, both

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mean fluorescence intensity (MFI) and percentage of cells positive for CD59 was evaluated using FACSCalibur. Result The median age of the cohort was 32 years (range: 1–79 years). There were 118 (36%) females and 206 (64%) males. The normal healthy control reference range for CD59 expression on erythrocytes and granulocytes was 98.9+1.8% and 98.7+ 1.7% with MFI of 457.8+ 63.4 and of 480.3+65.9 channels respectively. In this group of patients with either pancytopenia or clinical / laboratory suspicion of PNH, reduced expression of CD59 was found in 55 (17%) patients. Among these, all showed reduced expression of CD59 on granulocytes with MFI (405.4+118.1) while only 17% of patients had reduced CD59 expression on erythrocytes with MFI (419.5+60.2). Based on CD59 expression patients were classified into three types, namely type I (Variant/normal, reduced MFI only) (n=7), type II (partial deficient) (n=18) and type III (complete deficient) (n=30). The mean MFI difference between healthy control and patient was 75 channels on granulocytes and 38 channels on erythrocytes. Conclusion The proportion of cells with reduced CD59 expression on granulocytes was usually higher than the proportion of abnormal erythrocytes, probably related to blood transfusion. Flowcytometric evaluation of CD59 as reported in our study is a simple, fast, sensitive and reproducible method for rapid diagnosis of patients with paroxysmal nocturnal haemoglobinuria. OP 5 A study of 32 patients of paroxysmal nocturnal hemoglobinuria from a tertiary center in western India

Indian J. Hematol. Blood Transfus 24(3):86–145

They were classified into Classical PNH, PNH with marrow hypoplasia and MDS-PNH. Physical examination, CBC, bone marrow aspiration biopsy and cytogenetics, biochemical tests for hemolysis, serum iron studies and urine hemosiderin were done. Patients were treated with prednisolone, danazol and immunosuppressive therapy. Results Of 32 patients, 13, 7 and 12 had PNH with marrow hypoplasia, PNH-MDS and classical PNH respectively. All had anemia, 50% had bleeding, jaundice, and hemoglobinuria. Pancytopenia was present in 100%, 92% and 33% in PNH-MDS, PNH with marrow hypoplasia and classical PNH respectively. All patients had raised LDH, two third had indirect-hyperbilirubemia and reticulocytosis and 25% had iron deficiency. Complement based test missed 10% of the cases diagnosed by flow cytometry. Thrombosis, hemoglobinuria and death were more common in patients with a larger neutrophil PNH clone. There were nine episodes of thrombosis, four cerebral venous sinus, one superior mesenteric vein, retinal vein, intracardiac thrombi and two strokes. There were five deaths; two due to thrombosis and bleeding each and one due to infection and thrombosis. About 75% of all the patients had some response to therapy. Conclusion Thrombosis was present in 30% patients. Thrombosis, hemoglobinuria and death were more common in patients with clone > 50%, but were almost equally distributed in the three groups. Three fourth patients had response to cheap and easily available therapy.

OP 6 Utility of an exon 14 BslI polymorphism for improved genetic diagnosis of hemophilia A in Indian population

Sonali Sadawarte1 · Farah Jijina1 · K Ghosh2 · Manisha Madkaikar2 1 Dept. of Hematology, Seth GS Medical College and KEM Hospital, Mumbai, India 2 National Institute of Immunohematology (ICMR), KEM Hospital Campus, Mumbai, India e-mail: [email protected]

Preethi Mukundan · Shrimati Shetty · Bipin Kulkarni · Kanjaksha Ghosh National Institute of Immunohaematology (ICMR), 13th floor, KEM hospital, Parel, Mumbai - 400 012, India e-mail: [email protected]

Background PNH is a rare disease with varied manifestations diagnosed previously by complement based tests. There is need to subclassify PNH patients and study the natural history on the basis of flowcytometric clone size. Objectives To study clinico-hematological profile of PNH patients, classify them in subgroups, study complications in correlation with clone size and assess response to therapy. Materials and methods Thirty two patients with clinicohematological features, diagnosed as PNH on flow cytometry (clone size >3%) were included in this study.

Haemophilia A is a common X linked recessive bleeding disorder caused by deleterious mutations in the gene encoding factor VIII. Though direct mutation analysis is the common practice in most of the developed countries, restriction fragment length polymorphism (RFLP) analysis using common polymorphic markers of factor VIII gene is still the most practical and feasible method in developing countries like India. We investigated the utility of a BslI polymorphism (A3864C) in exon 14 of F VIII gene for the genetic diagnosis of haemophilia A families by PCR followed by digestion with enzyme BslI. Out of 213 unrelated women

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examined for heterozygosity of this marker, 69 were found to be informative (32.4%), with an allele frequency of 0.32 and 0.68 for the ‘+’ and ‘–’ allele respectively. Subsequently 13 haemophilia A families, which were not informative with any of the common markers routinely used in genetic diagnosis of haemophilia and which were also negative for intron 1 and 22 inversions were analyzed. Three were found to be informative exclusively with this marker (23%) making it a highly useful marker in the genetic diagnosis of haemophilia A families in India. OP 7 Molecular characterization of factor (F) VII and X deficiency cases from western India and a first term prenatal diagnosis in a FVII family

Leenam Mota · Shrimati Shetty · Kanjaksha Ghosh National institute of Immunohaematology, ICMR, 13th floor of KEM hospital campus, Parel, Mumbai - 400 012 India

Factor (F) VII and X deficiencies are the commonest coagulation factor deficiencies amongst the rare coagulation disorders with a prevalence of 1 in 500,000–1,000,000 persons worldwide and inherited as autosomal recessive traits. Although these deficiencies are rare, there are still many families who require prenatal and carrier diagnosis, which can be provided with this study. We characterized 14 FVII and 15 FX deficiency patients for phenotype and genotype analysis. An endogenous thrombin potential (ETP) test was also performed in these samples. Conformation sensitive gel electrophoresis (CSGE) technique was used followed by DNA sequencing to detect mutations. FVII antigen levels varied between 5.5–130% (92.8% CRM positive), where as, in FX deficient patients 4/9 (44.44%) were CRM negative. FVII deficient patients show an increased lag time with decreased thrombin generation, whereas all the parameters of thrombin generation are affected by severe FX deficiency. Eleven different mutations were detected in FVII including 6 previously reported mutations and 5 novel mutations i.e. Ala191Pro, Asp338Gly, Ile138Thr, Trp284Argand Leu263Arg. A total of 7 mutations were detected in 11 FX patients of these 3 were previously reported and 4 were novel i.e. Ala-26Asp, Gly59Arg, Lys246Met and Val248Ala. Mother of severe FVII deficient boy reported to us in 10th week of pregnancy. The index case had an Arg152Gln homozygous mutation in exon 6 of FVII. The CVS DNA was extracted and a direct sequencing of the factor VII exon 6 was performed. The sequencing results confirmed that the

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fetus was a heterozygous carrier of the Arg152Gln mutation and thus a normal report was issued. From this study we have characterized about 25 families of these rare FVII and X deficient patients and have the registry for the same. We are now also equipped for prenatal and carrier diagnosis by molecular biology techniques at our center. OP 8 Antiphospholipid antibodies in haemophilia patients with severe bleeding tendency: cause, consequence or a consequential cause?

S. Shetty · S. Vora · L. Mota · B. Kulkarni · K. Ghosh National Institute of Immunohaematology (ICMR), 13 th Floor, KEM Hospital, Parel, Mumbai - 400 012, India e-mail [email protected]

The prevalence, cause and the impact of antiphospholipid antibodies (APA) on the clinical severity in haemophilia patients is poorly studied. We studied 72 severe seronegative haemophilia patients for the presence of four common antiphospholipid antibodies. Twenty six (36.1%) were positive for any one of the APA studied of which eight were positive only for anti cardiolipin (ACA), 3 for β 2 glycoprotein (β 2 GP), 4 for prothrombin (PT) and 6 for anti annexin antibodies. Remaining 6 patients showed multispecific antibodies. The study of in vitro thrombin generation showed a higher endogenous thrombin potential (ETP) in case of β 2 GP1 positive patients as compared to the remaining APA. Further, clinically severe hemophilia patients showed higher prevalence of APA as compared to the clinically milder group suggesting that these antibodies do not contribute in alleviating the clinical severity in haemophilia patients as has been observed with other inherited thrombophilia markers. High prevalence of APA in clinically severe haemophilia patients may be a consequence of continuing tissue damage in the clinically severe group; as in India, clotting factor concentrates cannot be used ad lib due to financial constraints. OP 9 A study of inherited and acquired thrombophilic factors in patients with cerebral venous thrombosis

J. Ahluwalia1 · R. Das1 · M. Modi2 · MUS Sachdeva1 · S. Prabhakar2 · P. Malhotra3 · S. Varma3 · N. Varma1 1 Departments of Hematology, Neurology2, and Internal

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Medicine3 PGIMER, Chandigarh. e-mail: [email protected]

Background Cerebral venous thrombosis (CVT) is an important neurological emergency, with considerable morbidity and mortality. Clotting in the cerebral venous sinus may be seen in predisposing circumstances such as infections, especially of the ear and sinuses and septicemia, in puerperium and in women on oral contraceptives. In some cases no underlying cause is readily identified. Aim To examine the role of acquired and inherited procoagulant states in patients with CVT. Methodology This retrospective analysis was performed on patients with the diagnosis of CVT referred for workup for the hypercoagulable state in the department of Hematology. Inclusion criteria were: confirmed diagnosis of CVT on neuroradiological examination, an interval of at least 6 weeks after the episode and at least 4 weeks after cessation of oral anticoagulants. Testing for functional Protein C, S and antithrombin was performed on the STA Compact coagulation analyzer. Anticardiolipin antibodies were tested with a commercial ELISA (Orgentec GmBH) and lupus anticoagulant was tested with the dRVVT kit. (Dade Behring) Results 102 patients were referred with the diagnosis of CVT in the 10 year period (1998–2007). The median age was 26 years (range 5 months–50 years). 20 children were included in the study. Male:Female ratio was 1.49:1. In 23.5% cases circumstantial factors could be identified, especially the postpartum state in females. 11.4% were heterozygous for the Factor V Leiden mutation, 4.4% had lupus anticoagulant and 11.8% had anticardiolipin antibodies. 7.1%, 14.6% and 18% were deficient for protein C, S and antithrombin respectively Conclusion CVT formed 4.2% of the referral for workup for a hypercoagulable state. Younger patients were commonly affected with a female preponderance. Predisposing factors were frequently present. The genetic causes for hypercoagulability were more common as compared to the acquired. Testing for these parameters is useful as they may predispose to recurrence / thrombosis at a different site and may require prolonged therapy. OP 10 A record based study on malaria in relation to anaemia and sickle cell disorders

Anindita Sinha Babu · Samira Kumar Behera · K. L. Dei · M. K. Patro · A. K. Bal Department of Pathology, M.K.C.G Medical College, Berhampur, Orissa, India

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Background Malaria is a major contributor of anaemia in places where malaria incidence is high. Orissa is a known hyperendemic zone for falciparum malaria and, therefore, asymptomatic parasitaemia, particularly in adults, is also likely to be high in this area. This is also a known sickle belt and sickle cell disorders are thought to be resistant to malaria. MKCG Medical College, being situated in Ganjam district of southern Orissa, gets sufficient number of cases to examine the association between sickle cell disorders and malaria. Objectives To find out 1) relative contribution of malaria to overall anaemia, 2) Extent of asymptomatic parasitaemia among malaria cases 3) extent of vulnerability of sickle cell cases to malaria. Materials and methods Records of all hematological cases referred to pathology department of MKCG Medical College for a period of 5 years (2003–2007) were analyzed to find out the relevant information relating to all anaemia, malaria and sickle cell cases. Result A total of 98,195 hematological cases were reviewed, of which 81.97% were anaemia. Malaria was second most common cause of anaemia (24.76%), next only to nutritional (62.24%). Among malaria cases 94.5% were falciparum, 4.8% mixed and 0.71% were vivax. Asymptomatic parasitaemia constituted 17.2% of total malaria cases. The bulk of asymptomatic cases belonged to the age group of 20–39 years (79.96%) with no asymptomatic case below 10 years. Percent prevalence of malaria among patients with sickle cell disorders was 12.7% Conclusion This area is hyperendemic zone for falciparum malaria with so many asymptomatic cases. In order to break the transmission chain through these cases, mass drug administration option should be seriously considered. Sickle cell disorders are not totally resistant to malaria. This issue of vulnerability needs further exploration. OP 11 Complications associated with silicon central venous access device in hematology practice Grace Angeline · Seema Thomas · Margaret Devadanam, Beulah Augustin · Sasi kumari · Jessa Varghese · Ranjitha Chacko · Selva Titus Chacko · Auro Viswabandya · Biju George · Vikram Mathews · Mammen Chandy · Alok Srivastava, Department of Haematology, CMC, Vellore, India Background Use of a Central Venous Access Device (CVAD) is critical in the management of patients undergoing stem cell transplantation, since they facilitate care of these patients for blood sampling, administration of medications, transfusion of blood products and total parental nutrition. Silicon CVAD’s (sCVAD) are most commonly used for this purpose.

Indian J. Hematol. Blood Transfus 23(6–9):86–145

Objectives The objectives of this study was to assess the sCVAD related complications and to focus on the common infections recorded in practice with sCVAD. Materials and methods An analysis was done on the outcome (post-insertion) of sCVAD catheters used in our department between March, 2006 to March, 2008. All catheters were dressed twice in a week as per standard protocol. Data was collected prospectively till discharge, using a checklist to audit their status and complications, if any. Results During this period, a total of 218 CVAD were inserted for stem cell transplantation. These included 183 sCVAD catheters and the remaining were peripherally inserted PVC central venous and subclavian catheters. Of the 183 sCVAD 134 (73%) were for patients who underwent allogeneic stem cell transplantation (alloSCT) and 49 (27%) were for those having autologous stem cell transplantation (autoSCT). In patients who had alloSCT, sCVAD remained insitu for an average of 57 days (range: 4-197) and in those having autologous sCVAD catheters remained insitu for an average of 29 days (range:4-55) accounting a total of 9743 catheter days. 39/183 (21.3%) sCVAD related complications were documented. These included 27/39(69.2%) bacterial infections of which 2/27 (7%) were entry site infection, 7/27 (26%) were luminal infections(positive blood culture taken during chills soon after line dressing) and 18/27 (67%) were positive blood cultures. The other complications were 3/39 (7.7%) catheter blocks and 9/39 (23%) skin allergic reactions to the adhesive. The most frequently isolated bacterial organism was coagulase negative staphylococcus. This was identified in alloSCT and also in autoSCT. Two third of these infections were reported during the neutropenic phase of SCT. No fungal infections related to sCVAD were identified. The overall sCVAD infection rate was 2.8/1000 catheter days. Conclusion We conclude that complications associated with the sCVAD in our centre are minimal and similar to that reported in literature. OP 12 Response to rituximab for immune cytopenia in adults and children: experience from a single Indian centre

Shailesh R. Singi · P. Srikant Care Hospital, Road no 1, Banjara Hills, Hyderabad 500038, India e-mail: [email protected]

Background The Incidence of immune Cytopenia, including idiopathic thrombocytopenic purpura (ITP) and

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Autoimmune hemolytic anemia (AIHA) is not known in Indian population. It is estimated that the incidence should be around 10 persons per 100,000 and 1 to 3 persons per 100,000, for ITP and AIHA respectively in western countries. Although ITP and AIHA respond to treatment with corticosteroids in most patients, the majority will relapse during steroid tapering. Splenectomy salvages approximately 60% to70% of these patients. Individuals who do not respond or who relapse after Splenectomy, various immunosuppressive treatments have been found to be of very limited effect and are temporary. Rituximab is a chimeric murine/human antiCD20 anti-body primarily developed to treat clonal Bcell malignancies. Antibody-producing cells are CD20 +B cells, rituximab have also been studied as treatment of various antibody-mediated autoimmune disorders, including ITP and AIHA. In this retrospective review from a single institution, we report on the efficacy of rituximab for refractory immune Cytopenia in children and adult patients. Objectives Evaluate the efficacy of rituximab in patients with ITP, AIHA and Evans syndrome. Materials and methods We retrospectively reviewed the medical charts of all patients treated with rituximab for immune Cytopenia at the CARE Hospital, Hyderabad, INDIA, through March 2007 till date. 26 patients (median age at first diagnosis, 23 years; range, 2–59 years) were identified who received 1 treatment course of rituximab for treatment of refractory ITP (17 patients), AIHA (7 patients), and Evans syndrome (2 patients). Data regarding age, diagnosis, date of diagnosis, previous treatments, co morbid conditions, and blood cell counts before taking rituximab, number of rituximab treatments, and response to treatment were extracted and analyzed. Results Of 17 patients treated for ITP, 4 were receiving corticosteroid-based treatment either alone or combined with other immunosuppressive therapy at the time they received rituximab. Complete remission occurred in 8(47%) of 17 patients with ITP and in 4 (57%) of 7 patients with AIHA. Response to rituximab in patients with Evans syndrome was seen in either ITP or AIHA, but not both. Complete response was durable in both ITP and AIHA. Response in pediatric (age less than 15 years) was much inferior to adult population. There was no difference in response in splenectomized and non-splenectomized patients. Conclusion Our findings, considered with the results of other studies, suggest that rituximab deserves early consideration as salvage therapy for immune Cytopenia that are refractory to both corticosteroid treatment and Splenectomy. This series represents the largest series of patients with ITP, AIHA and Evans syndrome from single centre in India.

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OP 13

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OP 14

Spectrum and antimicrobial susceptibility pattern of bacterial and fungal isolates in febrile neutropenia

Automated malaria detection by Beckman Coulter LH755 using VCS technology

Raghuveer Prabhu · Farah Jijina · K. Ghosh SGS Medical College and KEM Hospital, National Institute of Immunohematology (ICMR), KEM Hospital Campus, Mumbai, India

N. Arora · D. Inbakumar · U. Sitaram · S. C. Nair Department of Clinical Pathology, Christian Medical College, Vellore, India e-mail: [email protected]

Background Febrile neutropenia is a medical emergency which requires prompt administration of empiric broad spectrum antimicrobial therapy. It is essential to have a good knowledge of locally prevalent pathogens and their antimicrobial susceptibility patterns before starting empiric therapy. Aims To study the prevalence, risk factors, etiological agents, and antimicrobial susceptibility pattern and response to empiric antimicrobial therapy in febrile neutropenia. Materials and methods In this prospective study conducted in the Departments of Hematology and Microbiology of a major referral hospital in western India, from March 2006 to September 2007, blood for culture and susceptibility were collected during 156 febrile neutropenia episodes from 97 patients. The most common empiric antibiotic regimens used were piperacillintazobactam plus amikacin and cefoperazone-sulbactam plus amikacin. Results 63 (40.38 %) positive isolates were obtained, 55 bacterial and 8 fungal. Of the bacterial isolates, 72.73 % were gram negative and 27.27 % gram positive. Staphylococcus aureus among the gram positive, and Klebsiella pneumoniae, followed by Escherichia coli, Pseudomonas aeruginosa and Acinetobacter species were the predominant isolates. 72.73% of the K. pneumoniae and 60 % of E. coli isolates were ESBL producers. The sensitivity of the gram negative isolates to imipenem, meropenem, piperacillin-tazobactam and cefoperazone-sulbactam was 96.88%, 87.5%, 87.88% and 81.82% respectively. Among Candida, both albicans as well as non albicans species were prevalent. Conclusion Gram negative bacteria are the major isolates in febrile neutropenia in our hospital. Carbapenems should be judiciously used to prevent development of resistance. As MRSA is not a problem in our hospital, vancomycin need not be included in the initial empiric regimen. As bacteria develop resistance to antimicrobials, it is important to generate periodic data regarding the etiological agents and their susceptibility pattern.

Background The coulter LH 755 in addition to CBC parameters gives a set of investigative parameters called Positional Parameters (Research Population Data-RPD) which defines each WBC population and RBC’s in terms of mean and standard deviation (SD) of Volume (V), Conductivity (C) and Scatter (S). These parameters along with the scatterplot andWBC histogram were analysed in cases of suspected malaria. Objectives To evaluate VCS RPD in detecting the presence of malarial parasite in blood. Materials and methods Samples from 40 positive malaria cases confirmed by smear examination and Quantative Buffy Coat (QBC) and 33 negative (suspected malaria) cases were processed on LH 755 in both the Differential and in Reticulocyte mode. The RPD generated by these cases were compared with the RPD from 100 normal healthy donors. Results Comparison of the RPD of malaria positive cases and normal RPD are shown in table1. In addition, the WBC scatter plot in the malaria positive cases showed the presence of the abnormal population at the bottom of the scatterplot and the WBC distribution curve showed the presence of the abnormal population below 35fl.

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Neutrophil

Vmean

Cmean

SCmean

VSD

CSD

SCSD

P=0.000

NS

NS

P=0.000

NS

NS

Lymphocyte

NS

P=0.000

NS

P=0.000

P=0.000

NS

Monocyte

P=0.000

P=0.000

NS

P=0.000

P=0.000

NS

Lymphocyte volume SD was the only significant parameter on comparing positive malaria cases with suspected malaria cases. There was no abnormal population in scatter plot and the WBC distribution curve in the suspected malaria cases. Discussion and conclusion RPD along with an abnormal population at the bottom of the scatter plot and the abnormal population below the 35fl on the WBC distribution curve can form an effective screening and diagnostic tool for rapid detection of malaria.

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OP 15 Flowcytometric evaluation of CD38 and Zap-70 expression in chronic lymphocytic leukemia

M.U.S. Sachdeva1 · N. Varma1 · J. Ahluwalia1 · R. Das1 · P. Malhotra1 · S. Varma2 1 Department of Hematology 2 Department of Internal Medicine, PGIMER, Chandigarh, India e-mail: [email protected]

Backgroud Recent years have seen a shift in prognostic markers of CLL from the traditional Rai / Binet scoring and pattern of bone marrow infiltration on trephine sections, to CD38 and Zap-70 expression on flowcytometry. The positivity for CD38 and Zap-70 is indicative of a poorer outcome. Hence, this study was designed to have a background data on expression of these two markers in CLL patients treated at a large tertiary care centre in North India. Aim To evaluate the expression of CD38 and Zap-70 on flow cytometry, in patients diagnosed as CLL. Methodology All cases with clinical diagnosis of CLL, from January 2007 to April 2008, were subjected to bone marrow examination in the department of Hematology, PGIMER, Chandigarh. A panel of monoclonal antibodies was used for immunophenotyping and the diagnosis of CLL was confirmed on positivity of CD19, CD5 and CD23, complemented with other relevant markers. In addition, CD38 and Zap-70 expression was noted in each case. Results Twenty nine patients were diagnosed as CLL during the study period. Mean age was 64.6 years (45–90 years) with a male to female ratio of 4.8:1. All patients had peripheral blood lymphocytosis and bone marrow infiltration. CD38 expression was analyzed in all the 29 patients. Ten out of 29 (34.5%) cases were positive for CD38 and 19/29 (65.5%) were negative for the marker. Seventeen patients were analyzed for expression of Zap-70. Only one patient showed positivity, and all the rest 16 patients were negative for Zap-70 expression. None of the patients had a dual CD38 and Zap70 postivity. Conclusion This study documents CD38 positivity in 34.5% of our CLL patients which is slightly higher than noted in literature. On the other hand, our cohort had much less cases with Zap-70 expression, in contrast to positivity in about one-third of CLL patients, reported from western countries.

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OP 16 Frequency of commonly prevalent polymorphism present in Gilbert Syndrome among haemoglobinopathies in West Bengal

Sanjoy Misra1 · Maitreyee Bhattachryya1 · Santu Mondal1 · Abhijit chowdhury2 · Partha P. Majumder3 · Shalini Datta3 · Sarnava Roy1 · Malay K. Ghosh1 1 Department of Haematology, N.R.S. Medical College and Hospital, Kolkata, India 2 Department of Gastroenterology, I.P.G.M.E. and R., Kolkata, India 3 Human Genetics Unit, Indian Statistical Institute, Kolkata, India e-mail: [email protected] [email protected]

Introduction Hyperbilirubinaemia especially elevated level of unconjugated bilirubin (UCB) is often associated with haemoglobinopathies. Elevated level of UCB is also found in Gilbert Syndrome (GC) frequently due to “TA” insertion in the TATA box of the promoter region of the gene for UDP-glucuronosyl transferase synthesis (UGT1A1). It is reported that allele frequency of “TA” insertion in TATA box [A (TA)7 TAA instead of A (TA)6 TAA] in Indian population is 35.1%. This polymorphism is capable of modifying the phenotype of thalassaemia. Haemoglobin Ebeta thalassaemia is the most prevalent type of symptomatic double heterogygous state of thalassaemia in West Bengal and surroundings. An attempt was made by us to elucidate whether this particular polymorphism has any significant contribution towards elevated level of UCB among βTrait, E-Trait and E- β thalassaemia patient attending our department. Methods 200 subjects above 12 yrs of age (non-smoker) were included in the study extending over one year (May 07–May 08). Haemoglobinopathies diagnosis was confirmed by HPLC analysis. Patients with impaired hepatic function, associated haemolytic conditions, and subject with normal haemoglobin analysis were excluded by appropriate investigations. Only samples with haemoglobinopathy and UCB level > 1mg/dl were analyzed by P.C.R followed by resequencing to estimate the allele frequency for the particular polymorphism. Pearson Chi-square was used and p value < 0.05 was taken as significant. Results Frequency of ‘TA’ homozygous insertion in TATA box of UGT1A1 promoter was higher in β trait (59.3%) and E-trait (56.3%) then E-Beta Thalassaemia(22.4%) in our study. Although the proportion of heterozygous insertion was approximately equal in all three groups. Mean UCB level in E-Beta Thalassaemia was 2.900.But when we considered only “TA7/TA7”(homozygous) insertion in

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E-Beta thalassaemia then the finding was 2 fold increases in UCB level. The impact of “TA7/TA7” insertion was much less pronounced in β-trait and E- trait. Conclusion Internal genetic condition such as this particular polymorphism can be considered for the elevation of UCB levels in Hb E- Beta thalassaemia.

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valve was found to be the underlying cause in 2 patients each. Refractory iron deficiency state encountered in 10 PNH patients without any other recognized clinical association. Conclusion In the present day the etiology of the iron deficiency state has shifted from conventional worm infestation, nutritional deficiency to chronic GI blood loss and others.

OP 17 Profile of iron deficiency anemia in 21st century in tertiary health care centers

OP 18 Haematological spectrum of glanzmann thrombasthenia from a tertiary centre in India: a study of 189 cases

Partha Sardar1 · Pradipta Guha1 · Sarmistha De3 · Maitreyee Bhattacharyya2 · Malay Ghosh3 1 Department of Medicine, Medical College, Kolkata, India 2 IHTM, Kolkata, India 3 Department of Hematology, N.R.S Medical College, Kolkata, India e-mail: [email protected] [email protected]

N.V. Patkar1 · S. C. Nair1 · S. Baidya1 · R. Merlin2 · A. Viswabandya2 · V. Mathews2 · M. Chandy2 · A. Srivastava2 1 Department of Clinical Pathology and 2Department of Clinical Hematology. Christian Medical College, Vellore, Tamil Nadu. e-mail: [email protected]

Introduction Treatment of iron deficiency is incomplete without identification and correction of the underlying cause of iron imbalance. Hookworm infestation and nutritional deficiency were the major cause of iron deficiency anemia in the past. Health consciousness, social habits has changed dramatically the scenario. In this background the etiology of iron deficiency anemia needs re-evaluation. Materials and methods One hundred and seventy five patients attending Medicine and Hematology OPD of Medical College and N..R. S. Medical College, Kolkata with iron deficiency were evaluated for underlying cause. Requisite haematological and biochemical parameters were investigated to confirm the diagnosis. Stool for parasite, occult blood test, upper GI endoscopy, Colonoscopy and barium meal follow through, USG abdomen was done in all cases.. Few of the cases were also investigated fo rwell recognized but less common causes like PNH, idiopathic pulmonary hemosiderosis, prosthetic valve induced haemolytic anemia. Results Total no of patients evaluated were 175. In 44% cases a source of blood loss could be detected. GI tract (both small and large bowel ) was the site of blood loss in most of the cases. Stool occult blood was positive in 63% amongst the GI blood looser, hook-worm infestation detected in none. In 20% it was due hemorrhoids, other causes were oesophageal varices (6%), intestinal polyp 3%, peptic ulcer 0.8%, GI malignancies 7%,, inflammatory bowel diseases, resection anastomosis, angiodysplasia 6%. It is interesting to note that even amongst the woemen of reproductive age group chronic GI blood loss was detected in significant number. Pulmonary hemosiderosis and prosthetic cardiac

Background Glanzmann Thrombasthenia (GT) is an autosomal recessive inherited platelet function disorder resulting from qualitative or quantitative defect of the integrin αIIbβ3 characterised by normal platelet counts and morphology, prolonged bleeding time, varying clot retraction, absent or diminished platelet aggregation. Objectives To retrospectively analyze the clinical and haematological profile of Glanzmann thrombasthenia diagnosed in our institute. Materials and methods Clinical history of patients referred for evaluation between January 2001 to May 2008 was accrued, (demographic details, bleeding history, family history and consanguinity.) Laboratory data (haemogram, bleeding time [BT], plasma clotting tests, clot retraction test and platelet aggregometry) was also analysed. Where variant/type II disease suspected, flow cytometric quantification of GP IIb/IIIa was done. Results 189 patients (Age range: 20 days to 54 years; M: F = 0.93:1) presenting with gum bleeds (61%), epistaxis (53%), post traumatic bleeding (48.1%), easy bruisability (28.9%), ecchymoses (15.5%), tooth socket bleeds (13.9%) and menorrhagia (8%) were analyzed. Consanguinity was elicited in 56.5% and a positive family history in 30.5%. Normal platelet counts with prolonged BT and normal plasma clotting tests and fibrinogen levels were seen. Platelet aggregation studies showed an absent or markedly reduced response to ADP, collagen and epinephrine. Ristocetin showed normal (52.1%) and abnormal responses (47.9%) comprising of disagglutination and cyclical agglutination-disagglutination patterns. Flow cytometric quantification was available in 20 cases, fourteen of these

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showed good / residual clot retraction. Finally, cases were classified as Type I (86.8%), Type II (0.5%) and variant GT (6.3%). In 12 (6.3%) cases distinction between type 2 / variant GT not possible as flow cytometry was not done. Conclusion This series on GT is the largest from India. Cultural practices in this part of the world have resulted in a large number of cases with history of consanguinity. A significant number of cases with an abnormal response to ristocetin were noted. OP 19 Study of I.V.I.G plus high dose methylprednisolone vs methylprednisolone alone in ITP patients

P. K. Gogoi1 · S. Ali2 · J. Bhattacharya3 · P. Singh4 · D. Das5 1 Dept of Haematology 2 Dept of Medicine 3 Dept of Haematology 4 Dept of Medicine 5 Dept of Haematology, Guwahati Medical College, Guwahati, Assam, India

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at high risk for ICH) Till now 36 pts have been enrolled in the study of which 18 received combination IvIg and methylprednisolone and 18 pt received methylprednisolone alone. The administration of drugs was not blinded. Platelet count were evaluated at the presented, 24 hrs, 48 hrs, 1 week, and 4 week Results The median time to reach platelet count above 50000 was 2 days for 80% patients in IvIg plus methylprednisolone group and 60% in methylprednisolone group alone Median time required to raise the platelet count to a safe level was 1 day for IvIg plus methyprednisolone and 2 days for methylprednisolone alone. Sustained levels of platelet count > 50000 at 4 wk was found in 83% pts in IvIg plus methylprednisolone and 70% in methylprednisolone alone. Conclusion Thus finally to conclude that combination of IvIg and methylprednisolone does results in a quicker rise and more sustained rise in platelet count than when methylprednisolone used alone hence it is suggested to go for combination therapy specially in childrens with very low platelet count (<10,000) or if the child is actively bleeding irrespective of platelet count. OP 20

Background Immune thrombocytopenic purpura(ITP) is a disorder characterized by immune mediated platelet destruction. Majority of patients with ITP presents with skin and mucosal bleed it is therefore very important to raise the platelet count as quickly as possible.Role of corticosteroids in raising platelet count is well established, however only a few studies have determined the advantage of combining IVIG with Methylprednisolone in raising platelet counts immediately and for a sustained period especially in high risk patients. Aim and Objective The primary aim was to compare the effectiveness of combining IVIG with Methylprednisolone alone in raising platelet count ie to evaluate• The rapidity of rise in platelet as reflected by platelet count at 24 hrs. • Time taken to reach a platelet count >50000. Study proposal and methods This a ongoing study started in department of Haematology, GMCH to prospectively evaluate two treatment regimen in pts with ITP Regimen A- Combination of IVIG (1g/kg/dose × 2days) and Methylprednisolone (30 mg/kg/dose × 3 days) followed by oral prednisolone 1 mg/kg/day tapered over 4–6 weeks Regimen B- Methylprednisolone (30 mg/kg/dose × 3 days) followed by oral prednisolone 1 mg/kg/day tapered over 4–6 weeks. Estimated Enrollment – 50 Study started – March 2008 Estimated completion date – August 2008 Here we plan to study patients with ITP with special emphasis to pts with platelet counts < 1000/μL (those

Efficacy and safety of hydroxyurea in children with thalassemia intermedia

K. Naranje · I. Panigrahi · R. Das · R. K. Marwaha Advanced Pediatric Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India e-mail: [email protected]

Background Hydroxyurea(HU) is found to be effective in some chronic hemolytic anemias namely sickle cell anemia. Its usefulness in thalassemia syndromes is not clearly defined. Objectives The aim was to analyse the effectiveness and side effects of HU in children with thalassemia intermedia (TI). Materials and methods 73 children with TI were retrospectively evaluated. The relevant data was recorded on predesigned proforma. Results 46 (63.0%) out of 73 children showed response to HU therapy when change in hemoglobin (Hb) level was taken as response criteria. Out of this, 33 children (45.2%) showed good response and 13(17.8%) showed partial response. 27(37.0%) children showed poor or no response to HU. 28 (66.6% of transfused) children showed fall in transfusion requirements by more than 50%. No significant side effects were noted. Transient neutropenia and some cutaneous changes were observed. No dose adjustment was

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required. Only one child had severe myelotoxicity requiring HU to be stopped. There was significant rise in Hb after HU therapy (p–0.001). Response was observed to be better in those who were diagnosed after 3 year age (p–0.001). Conclusion HU therapy is a viable and quite safe option in children with TI especially those above 3 years of age with good response observed within 1–3 months after start of HU. OP 21 BCR-ABL, TEL-AML1, E2A-PBX and MLL-AF4 translocations in adult and childhood precursor-B acute lymphoblastic leukemia

The frequency of TEL-AML1 reported from most Caucasian studies in children with precursor B ALL ranges from 19-25%. Earlier reports from India suggested that TEL-AML1 is rare in Indian patients with ALL. The present figure of 17% is the highest reported so far suggesting that our population is not significantly different with regard to the frequency of this translocation compared to Western data. Low frequency of TEL-AML1 therefore cannot be used as an explanation for poorer outcome of ALL in Indian patients. Detailed analysis of the role of these fusion transcripts on the outcome of treatment in this cohort of patients has to be carried out. OP 22

B. Poonkuzhali1 · C. Ezhilarasi1 · V. Andrew1 · R. Reena 1 · D. Daniel2 · V. Mathews1 · B. George1 · A. Viswabandya1 · A. Srivastava1 · M. Chandy1 1 Department of Haematology, and 2Clinical Pathology, Christian Medical College, Vellore, India Analysis of the genetic abnormalities that occur in acute leukemia is important for prognostication and for the design of risk-adapted therapeutic strategies. Common translocations with fusion transcripts of prognostic significance that are seen in Acute lymphoblastic leukemia (ALL) are TEL-AML1, associated with a good prognosis and E2A-PBX, MLL-AF4, and BCR-ABL associated with poor prognosis. Ethnic differences in the frequency of these transcripts in patients with ALL have also been reported. The present study was conducted to determine the frequency of the most common fusion gene transcripts in a large group of adults and children with pre-B ALL diagnosed and treated at our center from 1999 to May 2008. The diagnosis of preB ALL was based on morphology and the presence of CD10 or CD19 B-cell markers by immunophenotyping. Molecular analysis of leukemia-specific fusion transcripts was carried out in 260 children (0–15 years) and 286 adults (>15 years) using RT-PCR. The frequency of the fusion transcripts in adults and children in the present study are similar to the reports from the West except for TEL-AML1. The table below gives the frequency of the different translocations: Adults (n=286)

BCR-ABL

TEL-AML1 E2A-PBX

MLL-AF4

75/286; 26%

8/282; 2.84%

10/282; 3.55%

5/282; 1.77%

45/259; 17.37%

12/259; 4.6%

1/259; 0.4%

e1a2:45 b3a2:11 b2a2:22 Pediatric 16/260; (n=260) 6.15% e1a2: 8/260 b3a2: 7 b2a2: 2

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Reduced intensity regimen of fludarabine and melphalan in young adult patients with acute myeloid leukemia (AML) in CR1 undergoing a matched related allogeneic stem cell transplant: a single centre experience

Abhijeet P. Ganapule1 · Auro Viswabandya1 · Biju George1 · Usha Sitaram2 · Dolly Daniel2 · Kavitha M. Lakshmi1 · Mammen Chandy1 · Alok Srivastava1 · Vikram Mathews1 1 Department of Haematology, Christian Medical College, Vellore, India 2 Department of Clinical Pathology, Christian Medical College, Vellore, India e-mail: [email protected]

Background Conventional myeloablative allogeneic stem cell transplant (SCT) for AML in CR1 has been associated with the lowest relapse rate. However, due to high transplant related mortality (TRM), the anti-leukemic effect has not translated into a significant improvement in event free survival (EFS) or overall survival (OS). Objective Assess clinical outcomes of a recently introduced RIC regimen fludarabine-melphalan (FluMel). Material and methods: RIC regimen consisting of fludarabine 30 mg/m2 × 5 days followed by one dose of melphalan 140 mg/m2 for young adults with AML in CR1 after induction chemotherapy. The clinical outcomes were compared with a historical control who received a conventional regimen Bu/Cy regimen. The graft source was either a PBSC or bone marrow. Results Sixteen cases received a Flu/Mel conditioning regimen while 41 received a conventional Bu/Cy. The baseline characteristics of these groups were comparable for sex, patient age, donor age, female to male transplants, AML subtypes and the number of courses of chemotherapy required to achieve CR1. All cases in the Flu/Mel group received a PBSC graft vs. 29(65.4%) in the Bu/Cy group (p=0.0006). The 100 day TRM in the Flu/Mel group was

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0(0%) vs. 8(19.5%) in the Bu/Cy group (p=0.06). Three patients died in the Flu/Mel group (1-relapse, day 399; 2 – chronic GVHD, day 299 and 127). Twenty six deaths were noted in the Bu/Cy group (11-infections, 10 relapses, 4 GVHD and 1-graft rejection). Acute GVHD grade 2-4 occurred in 5(31.3%) and in 25(56.8%) and chronic GVHD occurred in 7(43.8%) and 11(30.6%) among patients conditioned with Flu/Mel and Bu/Cy respectively (p=NS). The mean follow up in the Flu/Mel and the Bu/Cy group was 22 and 66 months respectively. One patient (6.3%) in the Flu/Mel group relapsed vs. 13 (29.5%) in the Bu/Cy group (p=0.086). The 5 year Kaplan-Meier estimate of EFS and OS for the Flu/Mel and Bu/Cy group was 72±14.34% vs. 34.73±7.4% and 72±14.34% vs. 40.0±7.4% respectively (p=0.019 and 0.034). Conclusion A conditioning regimen of Fludarabine and melphalan in young adults with AML in CR1 is associated with low TRM with a potential to translate into improved EFS and OS. OP 23 2-Chloro deoxyadenosine in hairy cell leukemia: series of 28 cases from a single centre

Rajiv Subramanian · Auro Viswabandya · Vikram Mathews · Mammen Chandy · Alok Srivastava Department of Hematology, Christian Medical College, Vellore, India

Background Management of Hairy Cell Leukemia (HCL) has significantly improved over the past two decades with the introduction of 2-Chloro Deoxyadenosine (2-CdA). Objectives: To evaluate the efficacy of 2-CdA in the management of newly diagnosed and relapsed Hairy Cell Leukemia. Materials and methods All newly diagnosed patients of HCL between January 2002 & December 2007, based on bone marrow morphology and immunophenotyping and treated with 2-CDA were included in the present study. Their follow up data was systematically retrieved and reviewed. Results Of the 28 patients diagnosed to have HCL, 23 underwent treatment, of which 18 patients had long term follow up. Five patients died due to fungal pneumonia during chemotherapy. Most common presenting symptom was fatigue (96.5%) followed by recurrent infections (79.3%). Two of the 28 patients were Variant HCL. Bone marrow IPT was consistent with HCL in 26 patients. All patients were positive for DBA44 and TRAP on bone marrow biopsy and aspirate respectively (TRAP done on buffy coat preparation). At diagnosis, anemia was seen in 58.6%, ANC <1000 in 68.9%, Platelets <1L/mm3 in 75.8%,

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pancytopenia in 34.4%, Splenomegaly in 93%. Of the 18 patients with follow up, 12 patients achieved a CR (66.7%), 7 achieved PR (33.3%) with an overall response rate of 100%. The mean Overall Survival is 59 months with a 75% survival at 78 months. Three patients relapsed (16.6%) at a mean follow up of 39.6 months, who had achieved PR after initial treatment. They were treated again with 2-CdA. All three achieved a CR with re-treatment and at a mean follow up of 18 months all were alive. Conclusion 2-CdA induces a 100% response in newly diagnosed and relapsed cases of HCL. The mean overall survival is 59 months with a 75% percent survival at 78 months. OP 24 Cytogenetic profile of patients with acute lymphoblastic leukemia V. M. Srivastava · B. J. Nancy · A. Viswabandya · V. Mathews · A. Srivastava · M. Chandy Department of Haematology and Cytogenetics Unit, Christian Medical College, Vellore, India Background Acute lymphoblastic leukemia is characterized by the accumulation of malignant, immature lymphoid cells in the bone marrow and peripheral blood. Characterization of the cytogenetic changes associated with acute lymphoblastic leukemia provides both diagnostic and prognostic information, thus facilitating patient management. Objective To define and classify the frequency and type of chromosomal abnormalities among patients diagnosed with acute lymphoblastic leukemia at Christian Medical College and Hospital between 2003 and 2007 Methods Chromosomal analysis of bone marrow samples was done at the institutional laboratory. Unstimulated overnight cultures were harvested and GTG banding was done. 20 metaphases were analyzed for each patient and karyotypes were designated according to the International System of Human Cytogenetic Nomenclature. Results Chromosome analysis was performed on 650 patients who were diagnosed to have acute lymphoblastic leukemia. This population consisted of 432(62%) children and 218(33%) adults. Successful cultures were obtained in 565 (87%). Abnormal karyotypes were present in 382 (59%). Among these patients, numerical abnormalities were present in 151 (24%) and structural abnormalities were present in 164 (25%). In 67 (10%) patients both numerical and structural abnormalities were present. The most common karyotypic abnormality in our series overall was hyperdiploidy (13%). The most common structural abnormality was t(9;22) (5%). Complex karyotypes with three or more abnormalities were seen in 55 (8%). Some of the other commonly reported

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abnormalities such as t(1;19), t(4;11), del6q and +21 were also present. Conclusion This is the largest series to be reported on the spectrum of cytogenetic abnormalities in acute lymphoblastic leukemia from India. Our findings are similar to that of previous reports published with respect to the success rate and proportion of abnormal karyotypes. However, there are differences with regard to the frequency of abnormalities detected. This could be attributed to differences in the study design. Molecular cytogenetic methods could help in the detection of cryptic cytogenetic abnormalities. OP 25 Spectrum of Bcr-Abl kinase domain mutations in patients with chronic myeloid leukemia receiving Imatinib mesylate therapy C. Ezhilarasi1 · M. Preetha1 · B. Poonkuzhali1 · V. M. Srivastava2 · V. Mathews1 · B. George1 · A. Viswabandya1 · A. Srivastava1 · M. Chandy1 1 Department of Haematology 2 Cytogenetics Unit, Christian Medical College Vellore, India Imatinib was the first targeted therapy developed to specifically inhibit the BCR-ABL kinase in Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML). Emergence of resistance to Imatinib remains a major problem in the treatment of CML. Several mechanisms of Imatinib resistance have been identified, including BCR-ABL gene amplification, point mutations in the BCR-ABL kinase domain, increased drug efflux and plasma sequestration. The aim of the present study was to analyze the frequency and the spectrum of bcr-abl kinase domain mutations in patients with clinical resistance to Imatinib. A total of 60 patients diagnosed in different phases of CML (chronic phase, accelerated phase and blast crisis) from 2004 to 2008 were included in the study for which mutation analysis was carried out. The median period from time of diagnosis to the time of suspected resistance was 16 months (range 4-48 months). Cytogenetic or molecular response to Imatinib was evaluated using FISH or RQ-PCR. The spectrum of mutations in BCR-ABL was evaluated by direct sequencing of the kinase domain. Mutations were identified in 18 of 60 cases (30%) whereas there are reports in the literature where the frequency of mutations is as high as 90% in patients with imatinib resistance. T315I mutation, which is shown to be associated with poor prognosis, was seen in only 2/18 cases, however the Ploop mutation M244V was more frequent (3/18). Among the other reported mutations, p-loop mutations (Y272H, G250E and Y253H), activation loop mutation (L403M) and

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mutations in STI 571 contact site (F336L) were observed. In addition, three mutations not previously reported occurred (F330I, D295G and Insertion) and their functional consequence remains to be characterized. RQPCR results (expressed as ratio of BCR-ABL/ABL copy numbers) were available for 47/60 cases using the same sample obtained for mutation detection. There was no significant difference in the RQPCR values of patients with mutations compared to that of patients without mutation (bcr-abl/abl ratio: 65+60 vs. 49+36). Among the 18 patients in whom mutation was detected, 6 cases received increased Imatinib dose up to 800mg, 6 cases were switched to other therapy including SKI606 or hydroxyurea, and 6 cases had disease progression. The very low frequency of ABL kinase domain mutations seen in this study suggests that the emergence of a mutation may not be the major mechanism of Imatinib resistance in this cohort of patients. Other mechanisms of resistance including pharmacogenetic variations, plasma sequestration of the drug or over expression of BCR-ABL need to be evaluated in these patients. OP 26 Extranodal non-Hodgkin lymphomas-an appraisal (3 years)

J. Sheba · K. Ramesh · M. Rama Apollo Speciality hospitals, Chennai, India e-mail: [email protected]

Background Extranodal non Hodgkin lymphomas have been a challenge to the clinician and pathologist alike. They are tricky to diagnose and classify.A frequently misdiagnosed entity in the past the extranodal lymphomas are now metamorphosing into well defined entities which are more meaningfully diagnosed, more accurately classified and more appropriately treated thanks to the light shed by newer modalities of research and practice. Objectives To analyse the distribution of different types of extranodal lymphomas at various sites and compare the findings with those of literature. Materials and methods Study design Two years Retrospective and one year Prospective study. (2004 to 2006) Study population Cases selected on the basis of inclusion criteria. Data management Data was entered into Microsoft office Excel 2003 and analyzed. Descriptive statistics (Mean, Median, Standard deviation) were used to describe the study variables. Results 222 cases of primary extranodal non-Hodgkin’s lymphoma, which constituted 34% of all lymphomas, had an age range of 1–95 years, mean age of 50.46 years and a

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male preponderance. Diffuse Large B-cell lymphoma was the commonest type followed by MALT lymphoma.The percentage of incidence of gastrointestinal tract lymphomas was 24.8%, like that of Middle East (25%) unlike western countries where the incidence is 4–18%.Waldeyer’s ring was the commonest site in the Head and neck region. Only NK/T-cell lymphoma of nasal type or T-cell lymphomas were found in the nasal region. 96% of the primary CNS lymphomas were Diffuse Large B-cell lymphoma.Mycosis fungoides was the most common subtype of cutaneous T-cell lymphomas.Orbit was the most common site among ocular adnexal lymphomas. All except one of the ocular adnexal lymphomas were low grade lymphomas and 82% were MALT lymphomas.

patients with fever, constitutional symptoms (p<0.02) and thrombosis (p<0.02). Conclusion This study has shown associations between certain clinical, hematologic, and immunologic features of SLE, reflecting specific patterns of disease expression. The evaluation of clinical and laboratory markers at diagnosis and during evolution may improve the clinical management of SLE patients.

OP 27

David Inbakumar · Kamal · Josphine · U. Sitaram · S.C. Nair Clinical Pathology, Christian Medical College Vellore, India. e-mail: [email protected]

Hematological manifestations of systemic lupus erythematosus

M. Murari · R. Dinkar · R. N. Misra Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow, India e-mail: [email protected]

Aim and objective To study the prevalence of the hematologic manifestations and to determine their relationship with clinical features and disease activity in systemic lupus erythematosus. Methods The study was done in 84 patients of SLE diagnosed on the basis of American College of Rheumatology (ACR) criteria, consecutively seen in Clinical Immunology department between January 2006 and January 2007. Clinical, hematologic and immunologic characteristics were studied according to the protocol. Results Of the 84 patients 76 were female and 8 were males, sex ratio (F:M ) was 9.5:1. Age ranged from 11–55 years (mean 29.7 yr and median 28 yrs). Clinical findings suggestive of antiphospholipid syndrome were present in 21 (25%) patients. Hematological abnormalities were seen in 80% cases. Main hematological abnormalities included anemia (79.7%), lymphopenia (63%), thrombocytopenia (22.7%), positive Coombs test (54.7%), and lupus anticoagulant (20.2%). Anticardiolipin antibodies were present in higher number of patients (IgG 32.7% and IgM 51.1%) compared to lupus anticoagulant. Anemic group showed higher renal involvement (p<0.03), neuropsychiatric manifestations (p<0.02) and lymphopenia (p<.001). Anemic and non-anemic groups had significant differences in ds-DNA (p <0.05) and SLEDAI score (p<0.01). Patients with thrombocytopenia showed higher incidence of arthritis (p<0.01), serositis and thrombosis / foetal loss. The patients in the LA positive group showed higher number of

OP 28 Reticulocyte research population data for the detection of ovalocytes and elliptocytes on Beckman Coulter LH 750

Introduction The Coulter LH 750 system uses VCS technology which measures volume, conductivity and light scatter on each cell in a hydrodynamically focused stream. This system also includes an investigation screen that gives statistical information (Mean and SD) of VCS about the main WBC population and also describes about the red cell VCS during reticulocyte analysis. The irregular shape of mature red cells and reticulocytes produce unpredictable light scatter information when subjected to a laser beam at angle 0 deg to 90 deg. This was used as basis to study the mature red cells in the non – retic area and to correlate with the presence of poikilocytes in smears and which shows increase in scatter mean and SD of red cells in non-retic area. Methods The total of 232 samples (K2 EDTA) were processed in the reticulocyte mode on the Coulter LH750 analyser 159 of these samples were having normal blood picture (No poikilocytosis) and 73 samples were having poikilocytes on smear review. We compared the non-retic Scatter Mean and SD of samples with no poikilocytes to the samples having ovalocytes/elliptocytosis and found that the non-retic mean scatter and SD of the sample having ovalocytes/elliptocytes is higher than the sample with no poikilocytes. Results For the detection of ovalocytes we found that the best parameters were the mean reticulocyte mean scatter (nretscme) and RDW when the presence of ovalocytes were not so high (Ovalocytes +) (Table 1). When the presence of ovallocytes was very high (++++) also MCHC and the Reticulocyte Scatter SD (nretscsd) were useful for its detection (Table 2). For the detection of elliptocytes we found that the best parameters were also Mean reticulocyte Mean Scatter (nretscme) and RDW when the presence of elliptocytes

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Table 1 Ovalocytes + ROC AUC Sens

spec

Cut-off

nretscme

0.819

86.11

64.62

>56

RDW

0.8425

88.89

63.59

>17.1

Table 2 Ovalocytes ++++ ROC AUC Sens

spec

Cut-off

nretscme

0.8877

100

81.94

>62

RDW

0.9681

100

88.55

>23.1

nretscsd

0.9824

100

96.48

>18

MCHC

0.8265

100

62.56

>33.2

Table 3 Ellitocytes + ROC AUC Sens

spec

Cut-off

nretscme

0.7847

78.26

67.31

>57

RDW

0.8695

91.3

70.19

>18.1

Table 4 Ellitocytes ++++ ROC AUC Sens

spec

Cut-off

nretscme

0.963

100

96.09

>74

RDW

0.9587

100

94.35

>25

nretscsd

0.987

100

98.7

>21

MCHC

0.7413

100

72.17

>33.5

were not so high (Elliptocytes +) (Table 3). When the presence of Elliptocytes was very high (++++) also MCHC and the Reticulocyte Scatter SD (nretscsd) were useful for its detection. (Table 4). Conclusion The mean reticulocyte Scatter was also useful and we have to investigate is also specific of these abnormalities. It is possible today with the instruments of the LH700 series from Beckman Coulter to create rules with the classical parameters MCHC, RDW, etc. but also with the reticulocyte Research Population Data and produce flags that may be will increase the detection of cases with these red cell abnormalities. OP 29 Performance evaluation of Beckman coulter LH 755 series in screening of hereditary spherocytosis

S. Jain · D. Inbakumar · U. Sitaram · S. C. Nair Department of Clinical Pathology, Christian Medical College, Vellore, Tamil Nadu, India e-mail: [email protected]

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Background While performing evaluation of blood samples on the LH 755 on the reticulocyte mode and using the Volume, Conductivity and Scatter (VCS) technology, reticulocyte indices showed a volumetric research parameter Mean Sphered Cell Volume (MSCV). A compound derived from methylene blue first precipitates the ribosomal RNA of reticulocytes, then red cells are sphered in acidic hypo osmotic condition and the mean volume of the whole RBC population (MSCV) is calculated. In normal people MSCV is more than Mean Corpuscular Volume (MCV). In patients of Hereditary Spherocytosis (HS), spherocytes reach a critical osmotic volume leading to cell fragmentation and show decrease in MSCV. On observation the difference between MCV and MSCV is higher in cases of HS. This may be a good screening tool for HS. Aim Utility of MCV and MSCV during reticulocyte analysis on the LH 755 in screening for HS. Materials and methods EDTA samples were collected from 30 cases diagnosed to have HS on the basis of clinical history, peripheral blood findings, reticulocyte count, biochemical markers and osmotic fragility. These patients had not received any transfusions over a 6 week period. The analysis was done on the LH 755 for both complete blood count and reticulocytes indices using VCS technology. Results Of the 30 cases of HS, 25 (83.3%) showed the difference between MCV and MSCV to be more than 10, the peripheral blood smear showed significant spherocytosis and there was marked increase in osmotic fragility. 2 (6.7%) cases had difference between 5–10 and 3 (10%) cases had a difference between 0–5. Conclusion Using the reticulocyte analysis on the LH 755, the difference between MCV and MSCV allowed us to analyse the particular behaviour of HS red cells. MCVMSCV > 10 is highly indicative of HS which can be used as a useful screening test for hereditary spherocytosis by automated haematology analyzers OP 30 Hematological manifestations of HIV infection

Pallavi Agrawal · Tejinder Singh · Nita Khurana · Vikas Sharma · Naresh Gupta Department of Pathology and Medicine, Maulana Azad Medical College, New Delhi, India

Background Hematological abnormalities like cytopenias are common among patients with HIV infections. Bone marrow examination in these patients reveals a spectrum of morphologic characteristics, ranging from normal hematopoiesis to marrow dysplasia, lymphocytic infiltration, plasmacytosis, fibrosis, granulomatous myelitis and other conditions.

Indian J. Hematol. Blood Transfus 23(6–9):86–145

Objective To study hematological profile in HIV positive patients Materials and methods 60 HIV positive patients were studied. Peripheral blood and bone marrow examinations were carried out in each case. Results Almost all the cases showed anemia of which 40 cases were normocytic normochromic, 10 cases were microcytic hypochromic, 3 cases were dimorphic and 7 cases were of macrocytic anemia. Bone marrow was normocellular in 20 cases and hypercellular in 40 cases. 7 cases revealed granulomas, 3 cases marrow necrosis, 4 cases gelatinous marrow transformation, 2 cases fungal infection, 14 cases marrow plasmacytosis, and 20 cases demonstrated dyspoiesis. A detailed pathological profile will be presented. Conclusion It is important for the hematologist to be aware of the features of HIV infection since diagnostic confusion may occur. All cases must be searched for tubercular and fungal infection.

OP 31 Autologous stem cell transplantation in young adult patients with acute myeloid leukemia (AML) in CR1: a single centre experience

Neeraj Sidharthan1 · Auro Viswabandya1 · Biju George1 · Vivi M. Srivastava2 · Usha Sitaram3 · Dolly Daniel3 · Kavitha M. Lakshmi1 · Mammen Chandy1 · Alok Srivastava1 · Vikram Mathews1 1 Department of Haematology, Christian Medical College, Vellore, India 2 Cytogenetic Unit. Christian Medical College, Vellore, India 3 Department of Clinical Pathology, Christian Medical College, Vellore, India e-mail: [email protected]

Background Randomized trials have established the lower risk of relapse when an autologous stem cell transplant (ASCT) was used as consolidation therapy in comparison with chemotherapy alone. Failure of an ASCT to translate to an improved EFS and OS has resulted in its role remaining controversial. Objectives Assess clinical outcomes of young adults with AML in CR1 who underwent an ASCT at our center. Material and methods: Newly diagnosed AML in CR1 under went an ASCT after receiving 2–3 high dose consolidation regimens. Conditioning regimen consisted of a standard Bu/Cy regimen. Cytokine mobilized PBSC were harvested after 2–3 cycles of consolidation chemotherapy and cryopreserved.

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Results Thirty four patients underwent an ASCT between 1993 and 2008. Majority, 28 (82%) were done after the year 2000. Mean age of the patients was 30 years (range: 11–62). There were 21(61.8%) males. In 10(33%) cases a cytogenetic report were not available, 17(50%) had normal cytogenetics and 7(20.5 %) had a complex karyotype. 28 (82.35%) attained CR1 after single induction whereas 6(17.65%) patients required two inductions. 22(64.70%) received 3 cycles of consolidation therapy with HiDAC. The mean MNC dose was 6.0 × 108cells/kg (range: 1.3–14.5) and the mean CD 34 dose was 8.2 × 106 cells /kg (range: 1.4–24.6). The median time to ANC>500/mm3 and Platelet count >20,000/mm3 was 10 (range: 8–31) and 14 days (range: 8–86). Four patients (11.7%) died in the peri-transplant period (by day 100) (only one after the year 2000). Three patients developed hemorrhagic cystitis and two developed acute hepatitis during the peritransplant period. Seven patients (20.5%) had documented infections. One patient each developed cerebral abscess and cerebral hemorrhage. Overall 9 patients (26.5%) relapsed and 12 (35.3%) died. The 5 year Kaplan-Meir estimate of overall survival and relapse free survival was 50.60±10.93% and 56.73±11.67%. Conclusion ASCT appears to be a reasonable option for patients with AML with a low TRM and acceptable relapse risk.

OP 32 Autologous peripheral blood stem cell transplantation (auto PBSCT) in multiple myeloma (MM) – results from a tertiary center in India

Rayaz Ahamed · Auro Viswabandya · Vikram Mathews · Mammen Chandy · Alok Srivastava. Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India

Background MM is an incurable malignant plasma cell disorder. Auto PBSCT has been shown to improve both disease free and overall survival in patients with MM in comparison to conventional chemotherapy. Aim of the study To analyze data of MM patients who have undergone autoPBSCT between January 1996 and December 2006 in our center. Methodology All cases of MM who underwent auto PBSCT during the chosen period, whose data could be retrieved for analysis were included for this analysis. All patients were mobilized with G-CSF at a dose of 10 microgm/kg and stem cell harvest was done on days 5 and 6. All of them were given Melphalan based conditioning regimen. G-CSF mobilized un-cryopreserved peripheral blood stem cells were infused within 24 hours of melphalan administration.

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Results Of the 73 patients who underwent auto PBSCT data could be retrieved for 70 patients. Fifty two (74.3%) were males, 18 (25.7%) were females. Median age at transplant was 50.5 years (range: 28–67). Thirty four (48.6%) cases were in complete/near complete remission (CR/nCR) and 36 (51.4%) were in partial remission (PR) prior to transplantation. Average number of chemotherapy regimens received prior to transplant was one in 43 (61.4%) patients and more than one in 27 (38.6%). The mean mononuclear cell (MNC) dose was 3.6 × 108/Kg (n = 70; range: 0.96–8.0) and mean CD 34+ cell dose was 5.5 × 106/Kg (n = 37; range: 1.4–20). The median time to achieve ANC >500/mm3 was 11 days (range: 9–19), and median time to achieve platelets >20,000/mm3 was 14 days (range:9–42). With a mean follow up period of 108 months; 51 (72.9%) are alive and well (CR/nCR/PR), 12 (17.1%) are dead and 7 (10%) were lost for follow up. Conclusion Autologous PBSCT is feasible in patients with MM in India, with a progression free survival of around 60months. OP 33 Predictive value of peripheral blood CD34 counts for the yield of stem cells collected by leukaphaeresis after mobilization by granulocyte colony-stimulating factor (G-CSF) for autologous stem cell transplant

K. Kotteeswari · Reena Rajasekar · Bargavi Balakrishnan · Vikram Mathews · Auro Viswabandya · Biju George · Mammen Chandy · Alok Srivastava. Department of Haematology, Christian Medical College, Vellore, India e-mail: [email protected]

Background Mobilized peripheral blood stem cells (PBSC) are the preferred stem cell source for autologous haematopoietic stem cell transplantation. The ability to collect adequate stem cells (CD34+) is a prerequisite for this procedure. Peripheral blood (PB) CD34 prior to aphaeresis could potentially predict adequacy of the harvested graft. Objectives To evaluate the ability of PB CD34 cell count to predict adequacy of CD34 cell dose of the PBSC harvest. Materials and methods: Patients were mobilized with GCSF 10ug/Kg/day for 5 days before leukaphaeresis using Cobe Spectra system. On the day of the aphaeresis pre and post aphaeresis samples were collected for stem cell enumeration using FACSCalibur by employing ISHAGE gating strategy. Results Eighty six patients underwent 152 aphaeresis’, for an autologous PBSC transplantation, at our centre between April 2005 and May 2008. The median age of the cohort was 45 years (range: 7–67) and there were 96

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(63%) males. The median WBC and CD34 cell count on the day of aphaeresis was 39600 cells/ul (range: 2900–95300) and 40 cells/ul (range: 1–633) respectively. The median mononucleated (MNC/Kg) and CD34 cell dose collected was 2.7x108 /kg/ aphaeresis (range: 0.5–8.4) and 3.5 x106 /kg (range: 0.2–79.1) respectively. The PB CD34 cell count post G-CSF (pre-aphaeresis) correlated well with CD34 cell dose (r=0.8, P-0.000) while it was weak, though significantly associated with the MNC cell dose (r=0.2, P value = 0.004). 103 (68%) achieved the target dose of CD34 > 2 x106 /kg / aphaeresis. A threshold of post G-CSF PB CD34 cell count of >18 cells /ul was associated with adequate mobilization (P=0.000). Only 14% of patients with post G-CSF PB CD34 cell count of >18 cells/ul did not achieve the minimum CD34 cell dose whereas 87% below threshold value had inadequate stem cell dose. The post GCSF CD34 value of >18 cells/ul gave a sensitivity of 86% and a positive predictive value of 95% to achieve the target CD34 cell dose of > 2.0 × 106/kg/aphaeresis. Conclusion PB CD34 cell count prior to aphaeresis correlates well with the stem cell content of the product. A pre-aphaeresis PB CD34 value of >18 cells/ul has a high predictive value of adequacy of the harvested graft. OP 34 Peripheral blood stem cell harvest using a CobeSpectra Apheresis System: a single center experience

Chidambaram Sahadevan · Bharatidasan Subramani · Reena Rajasekar · Kavitha M. Lakshmi · Vikram Mathews · Auro Viswabandya · Biju George · Mammen Chandy · Alok Srivastava Department of Haematology, Christian Medical College, Vellore. India e-mail: [email protected]

Background Mobilized peripheral blood stem cells (PBSC) are increasingly used in both autologous and allogeneic hematopoietic stem cell transplantation. Objectives Descriptive study of peripheral blood stem cell harvests done at our center. Materials and methods PBSC was done after G-CSF mobilization of stem cells. G-CSF was administered at a dose of 10ug/Kg/day × 5 days. PBSC harvest was done using the Cobe-Spectra Apheresis system (manual method with colourgram) on the fourth and if required the fifth day as well. Result Between Jan 2006 and May 2008 a total of 426 PBSC harvests were done on 251 patients. This included 192 (45%) autologous and 234 (55%) allogeneic PBSC collections. The baseline characteristics of the autologous and allogeneic donors are summarized in table 1.

Indian J. Hematol. Blood Transfus 23(6–9):86–145 Table 1 Baseline characteristics of donors Characteristics n (%) / median (range)

Autologous PBSCT

Allogeneic PBSCT

Patient /Donor Males

119 (62%)

129 (55%)

Age (years)

46 (7–67)

33(4–60)

Hemoglobin (g%)

11.4 + 1.6

13.4 + 1.4

Total WBC -Pre harvest (cell/mm3)

36,400 (6800–94800)

45350 (15700–84200)

Platelets (cell/mm3)

1,45,000 (25000–581000)

2,09,000 (20300–486000)

The median blood processed volume was 12420ml (range: 1056-21970) with a processing time of 206 min (range: 87-313) to yield a final PBSC product of 196ml (range: 81-257). For allogeneic transplants, 64 (44%) harvests achieved the target dose on the first day and 84 (55.6%) patient required a second day harvest. For autologous PBSC collection, 17 (16%) achieved the target dose with a single apheresis, while 80 (79.5%) had required a second day procedure and 5 (4.5%) did not achieve the target cell dose. The median MNC and CD34 cell dose for allogeneic and autologous harvests was 3.9 × 108/Kg/apheresis (range: 0.4–25.3) vs. 2.6 × 108/Kg/apheresis (range: 0.3–10.7) and 8.75 × 106/Kg/apheresis (range: 0.7–74.6) vs. 3 × 106/Kg/ apheresis (range: 0.2–79.1) respectively. The procedure was well tolerated with minor complications seen in 23 (15.4%) of allogeneic donors and in 6 (5.8%) of autologous patients. Complications were mild and self limiting in all cases and included symptoms of hypocalcemia, pain at venipuncture sites, chills, fever and nausea and vomiting. No long term complications were reported in any donor. Conclusion PBSC harvest is a safe and well tolerated procedure and provides adequate stem cell dose in the majority of cases. OP 35 Peripheral blood stem cell pheresis – an experience of a tertiary care hospital in South India i.e., S.V.I.M.S., Tirupathi, Andhra Pradesh K. V. Sreedhar Babu · Anju Verma · A. Gururaj Kumar Sri Venkateswara Institute of Medical Sciences, Tirupathi, India Background Stem cells are “Master Cells” found in all vertebrate animals including humans which can divide to give cells either identical to themselves or differentiate into a specific cell type for indefinite periods often throughout the life of an organism. With these properties, they play important roles in the process of normal development, regeneration and repair of damaged tissues-which can be genetic, developmental, traumatic or age related.

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These stem cells can be retrieved either from an embryo (unethical), umbilical cord blood, bone marrow, skeletal muscle or peripheral blood. The utilization of peripheral blood stem cells is gaining wide importance and acceptance because of so many advantages of them over others. Stem cells therapy is an autologous stem cell treatment derived from a patient’s own blood to treat various diseases such as Myocardial Infarction, Cardiomyopathy, Leukemias, Myeloma, Hepatic failure, etc., Stem cell therapy gives desperate ailing people confidence, strength and hope by adding years to life. Aim To evaluate utility of cell separator for Autologous peripheral Blood Stem cell pheresis in cardiac patients. Material and methods After identifying the suitable cardiac patients by the cardiologist for Autologous Peripheral Blood Stem Cell Transplantation, the patient was given a long acting Granulocyte Colony Stimulating Factor (Pegfilgrastim), in the dose of 10μg/Kg/day, subcutaneously for 4 to 5 days to mobilize peripheral Blood Stem Cells from the Bone marrow into the peripheral blood. From 2nd day onwards daily total leukocyte count was done. When the count reaches over 30,000/μl, the patient was subjected to Apheresis by continuous flow centrifugation to collect 30–40 ml of peripheral blood stem cells concentrate using either RVY or C4Y disposable stem cell pheresis kits. After identifying the presence of CD34 + cells by Immunohistochemistry, these stem cells were transplanted at the site of damaged myocardium via coronaries through Angio by the Cardiologist. Results During the period from September, 2007 to August, 2007, a total of 19 peripheral Blood Stem Cell pheresis were done. The commonest age group was 6th decade (31.58%) followed by 1st decade (21.05%). The youngest patient was 3 years old male child and the oldest was 69 years male. For most of the patients the Ejection Fraction is around 30%. Majority of the patients belong to Blood Group ‘O’ Positive (47.36%) followed by ‘B’ Positive (26.32%). Most of the patients are showing improvement in their functional status. Conclusion Adult stem cells collected, harvested and re-infused showed benefits especially to the greater satisfaction of the people who underwent these procedures which s associated with minimal risks. But as it is a small study of short duration, it needs further work-up and review for definitive conclusion. OP 36 Serological characterization of partial D variants

Swati Kulkarni · K. Vasantha · Kanjaksha Ghosh National Institute of Immunohaematology, 13th floor, NMS building, KEM hospital campus, Parel, Mumbai, India e-mail: [email protected]

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Background The variants of D antigen can be divided into two groups: quantitative variants characterized by lower or higher number of D antigenic sites and qualitative variants characterized by the absence of one or more number of D epitopes (partial D). It has been established that D antigen has more than 37 well defined epitopes. If partial D variant subjects are exposed to normal D antigen they can produce anti–D antibody against missing epitopes. Hence the identification of partial D variants is clinically important. Polyclonal anti-D have been replaced by monoclonal antiD reagents for RhD typing. Partial D variant red cells are known to give discrepant results with different monoclonal anti-Ds Objectives To identify and classify partial D variants serologically with panel of monoclonal anti-Ds Material and Methods: Twenty samples referred to the Institute for discrepancy in RhD grouping were tested with ALBAclone Advanced Partial RhD typing kit by Indirect antiglobulin test (a panel of twelve monoclonal anti-D). The kit can identify and classify weak D type 1 and 2 and partial D variants (DII and DNU, DIII, DIV, DVa, DCS, DVI, DVII, DOL, DFR, DMH, DAR, DHK and DAU-4, DBT and Rohar). Results Out of the twenty RhD discrepant samples tested with ALBAclone Advanced Partial RhD typing kit, seven were found to be DOL, four DFR, two DAR, three DVa, one DVI and three were of weak D type. Conclusion In the present study the ALBAclone Advanced Partial RhD typing kit could classify all cases of RhD discrepancies into weak D and different partial D variants. OP 37 Retesting the deferred donors hemoglobin: does it help?

R. Sawant · K. Arun · A. Marathe Department of Transfusion Medicine, ACTREC – Tata Memorial Centre, NaviMumbai, Mumbai, India

Background Low donor Hb is the most common reason for deferral of blood donors. Preventing unnecessary donor deferrals due to inappropriate Hemoglobin (Hb) testing is essential for recruitment and retention of eligible donors. Objective To compare the performance of the CusO4 method with the Hemocue (Hb 201) method and evaluate the impact of a new strategy to retest the Hb of donors (deferred by CuSO4 method) on donor recruitment. Material and methods The hemoglobin of deferred blood donors was retested using a capillary blood sample with Hemocue and the blood donation was accepted when the Hb was greater than 12.5 g/dl. Hb results with calibrated

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automated cell counter using venous blood sample were considered as standard reference value. Results Hb of 200 deferred blood donors was re-evaluated. The sensitivity, specificity, PPV and NPV of Hemocue method was better than the CuSO4 method with both capillary and venous samples. Bland and Altman plots showed that there was a good agreement between the Hemocue and reference method using the capillary (Avg. difference = 0.25 and r = 0.90) and venous blood samples (Avg. difference = 0.15 and 0.89). With the new strategy, 18% of the deferred donors could be accepted back for blood donation. Moreover, Hb values of 18% of the deferred donors were between 12–12.4 g/dl with the reference method. Conclusion The Hemocue (Hb 201) can be used reliably with capillary samples for donor hemoglobin estimation. The Hb retesting strategy helps in recruiting additional eligible donors for blood donation. A lower threshold of 12g/dl would result in increased acceptance of eligible donors for blood donation and therefore needs to be seriously considered in our country. OP 38 Influence of pre-donation counseling on adverse donor reaction

J. Jamukiya · A. Agrawal · N. M. Bhatnagar · M. Mittal · M. D. Gajjar · B. H. Parmar · Ashok Agnihotri · Sapre Jyoti Dept. of IHBT, BJ Medical College, Ahmedabad, India e-mail:[email protected]

Background Blood donation is a safe procedure and usually causes no harm to the donor.Most of the donor reactions are psychological occurring due to vasovagal reaction and are thus preventable. Though most of the adverse donor reactions are reported but there are not many studies which take into account the effects of Pre Donation Counseling (PDC) on Adverse Donor Reactions (ADR). So we had taken up this study in our tertiary health care set up with attached medical college having superspeciality in cardiosurgery, neurosurgery, urology, plastic surgery. We performed a pilot study on PDC of donors to reduce the incidence of ADR. ADR reported are mainly mild, moderate, severe. Aims and objectives To study the effect of PDC on ADR mainly Pain at the phlebotomy site(P), giddiness, nausea (G/N), tingling/twitching (T) with reference to age, gender, type of donor-(replacement(R), voluntary(V), directed (D). To study the effect of PDC on whether donor is willing to donate again and recommend others for donation. Whether Incidence of ADR varies according to age, gender(M/F) and type of donor-R, V, D.

Indian J. Hematol. Blood Transfus 23(6–9):86–145

Material and methods In our set up majority of collection, around 60% is through replacement donors. Randomly selected donors are given PDC explaining that donation is a safe procedure and majority of ADR are psychological. The study period is from Jan. 2007 to December, 2007. 10,000 random donors are selected according to standard criterion recommended by National Aids Control Organization. Among these donors 62% (6200) PDC was given explaining the phlebotomy procedure. Informed written consent was taken by every donor participating in the study and a questionnaire was given to fill up which was analysed. Results Pre-donation Counseling was done on 6200 random donors out of which 5540 were male and 660 werefemale. Most common ADR was Pain at the phlebotomy site (8.0%) followed by Giddiness/Nausea (3.2%) and Tingling/Twitching (0.4%). There was a drastic difference in ADR in donors who were pre-counseled particularly Voluntary (5.0%), male (5.05%), 1st time donors (2.03%) of age group 26–35 years (7.85%) as compared to non-counselled voluntary (6.08%) male (10.43%) 1st time donors (6.35%) of age group 26–35 years (11.39%). Conclusion As there is a big gap in demand and supply of blood, in our country, there should be judicious and proper use of blood and blood products along with serious donor motivation and recruitment. Donor if having unfavorable experience during donation is less likely to be available again for donation. So in these cases pre-donation counseling will definitely recruit voluntary donors for repeat donation. Recruitment of such donors as repetitive donors will definitely add to main pool of safe donors. Few words of pre donation counseling has a great impact on adverse donation reaction incidences.

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these erythrocytes after transfusion soon behave like senescent cells that undergo untimely eryptosis, leading to the requirement for repeated transfusion at short intervals. Objective Our objective was to investigate the mechanism which makes healthy erythrocytes prone to premature eryptosis after transfusion to Eß thalassemia major patients. Methods Erythrocyte morphology was determined using confocal microscope. Eryptotic changes were assessed from flow cytometric analysis. Results: Analysis of morphological changes in erythrocytes of major phenotypic patients showed loss of discoid shape along with the formation of spheroechinocytes and membrane microvesiculation. Densitometric scan of membrane protein profile revealed alteration in the molar ratio of spectrin / band 3, which is likely to contribute to the spheroechinocytic transformation in erythrocytes. As the changes in membrane proteins could be mimicked by ionophore-mediated Ca2+ influx into healthy erythrocytes, observed structural alterations in transfused erythrocytes may be explained from intracellular Ca2+ accumulation in consequence to increased Ca2+ uptake and decreased activity of Ca2+–Mg2+ ATPase. FACS analysis of erythrocytes from thalassemia patients revealed decrease in cell size and increased surface area:volume ratio, which were also evident in Ca2+ treated healthy red cells. Annexin-V binding studies showed more surface exposed phosphatidyl serine, confirming this to be the pathogenic mechanism for programmed cell death of transfused erythrocytes. Conclusion This study indicates the involvement of elevated intracellular Ca2+ in promoting apoptosis of transfused erythrocytes, thus enhancing the need for repeated transfusion in Eß thalassemia major patients.

OP 39 Intracellular Ca2+ accumulation in transfused erythrocytes contribute to their premature eryptosis raising the need for repeated transfusion in Eßthalassemia major patients Kaustuv Dutta Chowdhury1 · Debabrata Biswas1 · Dijendra Nath Roy1 · Gargi Sen1 · Tuli Biswas1 · Maitreyee Bhattacharyya2 · Malay Ghosh2 1 Physiology Department, Indian Institute of Chemical Biology, 4, Raja S.C. Mulliuck Road, Kolkata - 700 032, India 2 Department of Hematology, N.R.S Medical College, Kolkata e-mail: [email protected] [email protected] Background Transfusion of healthy erythrocytes is the main treatment for Eß thalassemia major patients. However,

OP 40 Comparison of therapeutic efficacy of whole blood derived vs apheresis platelet transfusion in oncology set-up

R. Sawant · A. Marathe · A. Tirlotkar · S. Kannan Department of Transfusion Medicine, ACTREC – Tata Memorial Centre, Navi Mumbai, Mumbai, India

Background There is lack of consensus regarding the clinical efficacy of various types of platelet preparations available for transfusion to patients. Objective To compare the therapeutic efficacy of whole blood derived (RDP) vs Apheresis Platelet (SDP) transfusion in cancer patients.

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Material and methods All platelet units were evaluated for Platelet count, WBC content and volume prior to issue. The CCI was evaluated at 1 hour and 18 or 24 hours. Platelet transfusions were randomized rather than patients. Any adverse reactions, time to next transfusion and outcome of bleeding was recorded. The correlation between platelet dose, day of storage and ABO compatibility with CCI was studied. Results 400 transfusion episodes in 68 patients were studied. SDP’s had higher (p = 0.003) platelet content. 84% of RDP and 27% of SDP transfusions were ABO incompatible. 7% transfusions were therapeutic and bleeding was controlled in all patients irrespective of the platelet preparation used. Transfusion trigger of > 20000 was adhered to in 53% cases. The 1 hour CCI was equal with RDP and SDP transfusion. 24 hour CCI with SDP transfusion was significantly greater (p=0.049). ABO incompatibility affected the 24 hour CCI significantly in case of RDP and SDP’s. Leucocyte content of RDP’s was greater than SDP’s. Allergic reactions and FNHTR’s were associated more (p = 0.002) with RDP transfusions. The interval between platelet transfusions showed no correlation with the platelet type and dose transfused. CCI was not affected by age of the platelet product. Conclusion A significantly greater 24 hour CCI is the only clinically relevant advantage found to be associated with SDP transfusions. A final conclusion regarding the superiority of SDP preparations cannot be drawn based on the above findings. Further studies to evaluate the utility of pooled, leucodepleted RDP transfusions versus RDP and SDP transfusions are needed to derive conclusive evidence in this regard. PP2/1 Carcinoma cell leukaemia (Carcinocythemia) – a rare finding in peripheral smear

Dilip Suryawanshi · Partho Protim Barman · Tilottma Prasad · Ramji Rai Pondicherry Institute of Medical Sciences. Pondicherry - 605 014, India

Background A 49-year-old female patient was brought to emergency in a moribund state and acute onset of breathlessness. O/E- No spontaneous breathing was noted. Doppler study showed large pericardial effusion with diastolic compression of Right ventricle suggestive of cardiac tamponade. Patient expired 2 hrs after admission. Material and methods Complete haemogram (PSLeishman’s stain, PAS), Pericardial fluid aspirate, Immunocytochemistry on smears.

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Result Peripheral smear showed circulating tumor cells arranged in clusters and forming acini. Tumor cells were large with large vesicular nucleus and abundant eosinophilic cytoplasm. Pericardial fluid aspirate showed presence of metastatic tumor cells of the same nature, as in peripheral smear. Conclusion Carcinoma cell leukaemia Carcinocythaemia is a term used to describe tumor cells circulating in blood. Even though tumor cells spread via blood stream, it is extremely rare to find tumor cells on routine peripheral smear examination. On review of literature it was found that most references were case reports. A review of English literature revealed only 8 cases of carcinocythaemia (CCA) (4 patients-oat cell carcinoma, 2 patients-disseminated breast carcinoma, one each a case of adenocarcinoma ileum and abdominal rhabdomyosarcoma). The mean time of survival after discovery of CCA and death was 5 weeks. PP2/2 Haemophagocytic syndrome secondary to tuberculosis: a report of 3 rare cases P. Sharma · S. K. Gupta · R. Kapoor · H. P. Pati · S. Tyagi Haematology Department, All India Institute of Medical Sciences, New Delhi, India e-mail: [email protected] Background Haemophagocytosis as a reactive phenomenon in the bone marrow is usually associated with viral, fungal and Leishmanial infections. Its association with tuberculosis is rare. A literature search reveals 30 prior cases of the haemophagocytic syndrome in patients with tuberculosis. Such co-existence often results in atypical presentations and life-threatening pancytopenia. Case findings Three patients presented acutely with fever, hepatosplenomegaly, lymphadenopathy and variable peripheral blood cytopenias. Bone marrow aspirates revealed florid haemophagocytic activity by histiocytic cells. Tentative diagnoses of infectionassociated haemophagocytic syndrome were considered and serological / biochemical work-up was advised. Bone marrow trephine biopsy sections subsequently showed epithelioid cell granulomata in all 3 cases with acid-fast bacilli in 2 cases. Aspirate smears were negative for AFB in both these latter cases. The third bone marrow was AFB negative on direct microscopy but showed positive PCR for M. tuberculosis. On follow-up 2 of our 3 patients died during hospital stay. Conclusion These cases illustrate the importance of including disseminated tuberculosis in the differential diagnosis of bone marrow haemophagocytosis. The bone marrow biopsy has a vital role in the diagnostic armamentarium for such clinical situations. The role of

Indian J. Hematol. Blood Transfus 23(6–9):86–145

tuberculosis in causing the haemophagocytic syndrome is discussed.

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PP2/4 Bone marrow necrosis in haematological and non haematological disorders

PP2/3 Paget’s disease with myelodysplastic syndrome (RAEB-Type1): a coincidental findings or a rare association?

Partho Protim Barman · Dilip Kumar Suryawanshi · Erli Amel Ivan · Ramji Rai Institute of Medical Sciences, Pondicherry, India e-mail: partho.barman @gmail.com

Background A 81-year-old male came to OPD with h/o polyarthritis for past 10 years, sudden onset of pain and swelling of hand and knee joints. He had bowing of legs, pallor, generalized lymphadenopathy, bilateral pleural effusion, pansystolic murmur and moderate hepatosplenomegaly. Methodology PBS, Bone marrow aspiration and Bone marrow biopsy (HandE, polarizer, Reticulin and Perl’s Stain), X-ray of lower limbs, Serum alkaline phosphatase, LDH, Rheumatoid factor. Results Serum alkaline phosphatase: 419 IU/L (Normal: 50-136), RF: Negative, LDH: 259 IU/L (Normal: 114-240), PBS: Dimorphic anemia with eosinophilia. Bone marrow aspiration: MDS (RAEB-1), Bone marrow biopsy: Paget’s disease of bone Conclusion Paget’s disease with Myelodysplastic Syndrome (RAEB-Type1) Primary MDS and Paget’s both occur in relatively older age group. Their incidence increases with each decade with exponential rise in eighth decade. Both are associated with other malignant and non-malignant conditions. MDS is mainly associated with hematological malignancies, unusual skin lesions and other autoimmune disorders while Paget’s disease is associated with tumour and tumour– like condition of bone. The exact etiopathogenesis of both these disease conditions remain largely unknown. Viral etiology, familial clustering with genetic basis, antiapoptotic oncogene Bcl-2, and chromosomal mutation especially in chromosome 5 have been implicated in both these disease conditions but their coexistence in the same patient has not been reported in literature to the best of our knowledge. We hypothesize that while etiologies may be same in evolution of both these diseases the target cell affected is hematopoetic stromal cell in Paget’s disease and hematopoetic stem cell in MDS with different clinicopathological outcomes, hence rarity of their coexistence in same patient.

Nivedita Ghosh · Tejinder Singh · Neeta Khurana · N. Gupta Dept. of Pathology and Medicine, Maulana Azad Medical College, New Delhi, India Background Bone marrow necrosis (BMN) is defined as “necrosis of myeloid tissue and medullary stroma in large areas of the hematopoetic bone marrow.” On trephine biopsy, BMN is characterised by disruption of the normal bone marrow architecture with a considerable loss of fat spaces. Aim of study To summarize the state of knowledge on BMN, its prevalence, symptoms and signs, underlying disease associations, and the usefulness of diagnostic procedures. Methodology Twenty five cases of BMN diagnosed during life were selected and correlated clinicopathologically. Results BMN seems to be rare as evidenced by the few (25) reports in which BMN was diagnosed during life in our hospital in last 7 years (2001–2007) with a prevalence of 0.005%. Fever (100%) and bone pain (55%) were the most important symptoms. Lymphadenopathy (40%) and hepatosplenomegaly (35%) were the frequent associations on clinical examination. Anaemia (95%) and thrombocytopenia (60%) were the most frequent haematological abnormalities. In 17/25 (768%) cases an underlying malignancy was identified. Haematological malignancies were found in 11/25 (44%) cases and were the most common association. In haematological malignancies, lymphoma (70%) was the leading cause. The association of BMN was observed with non haematological solid tumors in 6/25 (24%) cases. In majority of cases (80%), the primary origin of tumor was not found. Infection as the cause of BMN was seen in 5/25 (20%) patients. Mycobacterium tuberculosis has been the responsible agent in 3/25 (12%) cases and one case was found in a human immunodeficiency virus positive patient. One case of BMN was also seen in association with Leishmaniasis. Conclusion BMN is caused by hypoxemia after failure of microcirculation. Given the high rate of malignancy as an underlying disease association, an extensive search for neoplastic disease (both haematological and nonhematological) is justified whenever BMN is diagnosed. PP2/5 Hematological manifestations of Parvovirus B19 infection Shramana Mandal · Meeta Singh · T. Singh Maulana Azad Medical College, New Delhi, India

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Background Parvovirus B19 (B19V), is a single-strand DNA virus. The most widely known clinical manifestation of B19V infection is erythema infectiosum, a mild viral illness. Parvovirus infection is associated with erythroid hypoplasia and presence of giant proerythroblasts in the bone marrow. Aims and objective To study the various hematological changes in Parvovirus infection. Mateial and methods 35 cases of parvovirus infection were studied. All hematological parameters and bone marrow findings were noted and where possible serological analysis was done. Results Cases demonstrating Giant Proerythroblasts (GPE), in the bone marrow were included since GPE are characteristic of Parvovirus B19 infection. These cases comprised of 11 cases of PRCA, 2 case of Aplastic anaemia, 1 case of Gauchers disease, 2 cases of Congenital dyserythropoietic anaemia and pulmonary Kochs, 1 case of erythroid hyperplasia, 2 cases of acute leukemia patients on chemotherapy, 2 cases of thrombocytopenia, 1 case of CLL, 1 case of Megakaryocytic hyperplasia, 1 case of Tuberculosis and 11 cases of normocellular marrow. The commonest manifestation was PRCA (11) followed by 11 cases of normal marrow. Conclusion Parvovirus B19 infection presents as asymptomatic, mild, nonspecific and cold-like symptoms. Treatment is usually supportive, although some patients may require transfusions or intravenous immune globulin therapy. Most patients recover completely. This study highlights the varied hematological manifestations of Parvovirus B19 infection.

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Materials and methods A retrospective analysis of 160 cases of BMA and BMB performed simultaneously and examined in the Department of Pathology of our Institute was done. The findings were compared. Results The results were divided into four groups. Those that showed positive correlation between BMA and BMB – 98/160 cases (73%). Diagnosis on BMB alone – 20/160 (12.5%). Diagnosis on BMA alone – (1/160) Number of cases where a definite opinion could not be given (20/ 160) 12.5%. The highest positive correlation was in cases given as reactive marrow 37.7 % (37/98) and those given as erythroid hyperplasia 18.8% (22/98). Good positive correlation was also seen in multiple myeloma 88.8% (8/9), leukemias 62.5%(5/9), bone marrow metastasis 50%(3/6). 80% (8/10) cases of tuberculous granulomas, all cases of Hodgkin’s disease (3/3), and 50% of bone marrow metastasis were diagnosed by BMB alone. In 12.5% cases, material was inadequate for opinion. Conclusion As routinely used diagnostic procedures, BMA and BMB can be done simultaneously. Both the procedures are complementary to each other. We found that, BMB was useful for tuberculous granulomas, Hodgkin’s disease, and especially in the diagnosis of unsuspected non haematological malignancy. Employment of proper technique should be kept in mind because in 12.5% of our cases a definite opinion could not be given. PP2/7 Role of bone marrow trephine biopsy in chronic lymphocytic leukemia

PP2/6 Comparative evaluation of simultaneous bone marrow aspiration and bone marrow biopsy – an institutional experience

Ashumi Gupta · P. Arora · D. Jain · N. Khurana · T. Singh Department of Pathology, Maulana Azad Medical College, New Delhi, India

Pampa Ch. Toi · Renu G’boy Varghese · Ramji Rai Pondicherry Institute of Medical Sciences, Kalapet, Pondicherry, India e-mail: [email protected]

Background Chronic lymphocytic leukemia (CLL) is the uncommon type of leukemia in India characterized by progressive accumulation of morphologically mature but immunologically less mature lymphocytes. A bone marrow aspirate (BMA) and biopsy (BMB) are generally not required to diagnose CLL. Prognostic features comprise the clinical, hematologic, biochemical parameters, percentage of lymphocytes in BMA and BM histopathological features including pattern of infiltration and normal hematopoietic reserve. BM histopathology pattern has been shown to be a single prognostic parameter better than any one of the variables employed in current clinical staging systems. Objectives To determine the bone marrow histologic patterns in CLL. Materials and methods Fourty two cases of CLL were studied. Their clinicohematological profile was recorded. The BM histopathology was studied for each of the

Background Bone marrow aspiration (BMA) and bone marrow biopsies (BMB) are done frequently for diagnosis of diseases. These procedures can be done simultaneously, so as to yield maximum material for studying both the cell morphology and cell distribution. Objectives A comparative study of BMA and BMB is done retrospectively where the clinical history is correlated with BMA and BMB results. The advantage of each method is analyzed. Correlation of our findings with that given in the literature is done to give a guideline for both techniques.

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patients. The clinical and hematologic prognostic factors were correlated with BMB infiltration patterns. Results Out of 42 cases studied 16 were <60 years and 26 were >60 years of age. Total leukocyte count varied from 15000 to 2.2 lacs/cumm. 2 cases were coombs positive demonstrating autoimmune hemolytic anemia. 2 cases had pure red cell aplasia associated with parvovirus infection. BMA showed lymphocytosis varying from 55% to 95%. BMB revealed nodular pattern in 8 (19%), interstitial in 10 (24%), mixed pattern in 9 (21%) and diffuse pattern in 15 (36%) cases. Clinicopathological prognostic factors were correlated with BM findings. Follow up of the cases varied from 3 months to 12 years. It was observed that cases with diffuse pattern had a shorter survival than cases with nondiffuse pattern. Conclusion A combined clinicopathological system incorporating the BM pattern, along with the usual clinical variables, may be of clinically predictive value in disease progression of patients. Marker studies and other newer techniques may not be available in all the medical centres. However, bone marrow trephine biopsy is of considerable prognostic value and must be done in all patients diagnosed with CLL. PP2/8 Bone marrow histomorphology and stromal reaction to non hematolymphoid neoplasms

Nidhi Mahajan · Kajal Dhingra · Deepali Jain · Nita Khurana · Tejinder Singh Department of Pathology, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi - 110 002, India

Background Bone marrow examination in cases of malignancies not only helps in staging the tumor, but also provides significant diagnostic and prognostic information. Objectives To study the histological aspects of metastasis in bone marrow. To identify various kinds of bone marrow reactions to metastasis. Materials and methods Bone marrow aspirates, biopsies and case records of 45 patients were retrieved. Various clinicopathological features including age of the patient, primary site, effects of the tumor on bone, marrow stroma and hematopoietic elements were studied. Histopathology of each tumor was recorded. Special stains for mucin, melanin and immunohistochemical studies were performed to categorize the origin of the tumor. A reaction of the marrow within the metastasis, in the interface and in the neighborhood of the metastasis was recorded. Results In adults, most of these carcinomas originated from an unknown primary (12) followed by breast (10), lungs (4), prostate (4), gastrointestinal tract (3) respectively.

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In the pediatric age group, neuroblastoma (4) contributed to the maximum number of marrow metastasis. 15 cases of marrow metastasis had presented with pyrexia of unknown origin. Involvement of the marrow was diffuse in 10 cases and focal in 35. New bone formation was observed in 5 cases. The stromal changes included marrow edema, necrosis, fibrosis, gelatinous transformation and non specific granulomatous reaction. The corresponding bone marrow aspirates demonstrated tumor cells only in 11 cases. Conclusion Histomorphological study of bone marrow biopsy provides an interesting insight into the tumor host interactions. As BMA yields low cellularity due to fibrosis or necrosis, a biopsy is more advantageous. Furthermore, patients with positive bone marrow biopsies are categorized as stage IV, obliviating the need for other staging procedures. PP2/9 Significance of apoptotic leucocytes in peripheral blood smears

Aparna Narasimha · C. S. B. R. Prasad · M. L. Harendra Kumar Sri Devraj Urs Medical College, Kolar, India e-mail: [email protected]

Background Apoptosis is a carefully governed process that brings about cell death in a regulated manner. The morphological changes of apoptosis include contraction of chromatin with nuclear and cytoplasmic budding occurring to form membrane bound apoptotic bodies. After observing apoptotic leucocytes in peripheral blood smears, a clinicopathologic study was undertaken to analyse their clinical significance. Aim of the study To evaluate the morphological features of apoptotic leucocytes in peripheral blood smears, to confirm their presence by Immunofluorescence and to ascertain their clinical significance. Methodology The material for the present study comprised of 100 cases was obtained from the Haematology laboratory at R. L. Jalappa Hospital and Research Centre, attached to Sri Devaraj Urs Medical College. The blood samples were collected in tubes with Tripotassium EDTA as an anticoagulant. Peripheral blood smears were prepared within 1–2 hrs of venipuncture, stained with Leishman stain and examined under oil immersion light microscopy at a final magnification of X 1000. The percentage of apoptotic leucocytes were determined by counting the number of cells showing features associated with apoptosis. The peripheral smears were also stained with Acridine Orange dye and examined under fluoroscence microscope. Clinical details, laboratory

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findings and therapeutic information were reviewed in all cases. Results Neutrophils were the most common apoptotic leukocytes identified followed by lymphocytes and eosinophils. Apoptotic neutrophils were more commonly seen in infections whereas lymphocytes were seen in diabetes and neoplastic diseases. Immunofluoroscence confirmed the presence of apoptotic leucocytes. Conclusion: The presence of apoptotic leukocytes in peripheral blood smears may aid in the differential diagnosis of various disease process. 2) Immunofluroscence can be used as an additional special technique for detecting apoptotic leukocytes.

biopsy sections more often than in aspirate smears. The pattern of infiltration was diffuse in majority of cases and increased marrow fibrosis was also noted. Interpretation and conclusion Chronic lymphoproliferative disorders can present over a wide range of ages. Circulating lymphoma cells were found in nearly one-third cases of non-Hodgkin’s lymphoma. Bone marrow trephine is superior to aspirate in detecting a lymphoma infiltrate in the marrow. PP2/11 Significance of red cell distribution width in pre eclampsia and eclampsia

PP2/10 Blood and bone marrow in chronic lymphoproliferative disorders

Santosh K. Yatnatti · Annam Vamseedhar Sree Siddharta medical college and research centre Tumkur, India e-mail: [email protected]

Amrita Singh · S. Sitalakshmi St Johns Medical College, Bangalore, India e-mail: [email protected]

Background Malignancies of lymphoid neoplasms range from the most indolent to the most aggressive human malignancies. These cancers arise from the cells of the immune system at different stages of differentiation, resulting in a wide range of morphologic, immunologic and clinical findings. Aim of study To study the peripheral blood smear and bone marrow aspiration and biopsy findings in patients with a diagnosis of a chronic lymphoproliferative disorder. Methodology The peripheral smear, bone marrow aspirate and bone marrow trephine biopsy slides of patients diagnosed with chronic lymphoproliferative disorders were studied over a four year period. The study included 200 cases. • Periodic acid Schiff, peroxidase and reticulin stains were done wherever necessary. • The clinical details regarding age, sex, presenting symptoms like fever, and clinical findings like pallor, peripheral lymphadenopathy, hepatomegaly and/or splenomegaly were noted. Results Of the 200 chronic lymphoproliferative disorders studied, non-Hodgkin’s lymphoma comprised the majority of cases (70%), followed by Hodgkin’s lymphoma (19%), chronic lymphocytic leukemia (9%) and hairy cell leukemia (2%). The age group ranged from 3 years to 80 years. A male preponderance noted. Anemia, lymphadenopathy, hepato- and/splenomegaly were common findings at presentation. Abnormal cells were noted in the trephine

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Objective To establish the significance of red cell distribution width (RDW)in a group of normal pregnant women and those with pre-eclampsia and eclampsia. Materials and methods The present prospective crosssectional study was conducted at the central laborratory, Department of Pathology, Sree Siddharta Medical College, Hospital and research Centre,Tumkur. From 1st september 2007 to 31st Janauary 2008. The sample size included 200 pregnant women with thier gestational age in the third trimester. The exclusion criteria included pregnant women with gestational age other than third trimester and pregnant women with known history of hypertenstion,diabetes,or proteinuria.After informed consent,blood samples were collected and analysed on an automated hematology analyser two to six hours after collection, to minimize changes in red cell size. Results Of all total 200 pregnant women included in the study,100 were normal pregnant woman, 50 were preeclampsia and the remaining 50 belonged to the group of eclampsia.The mean RDW in normal pregnant women,preeclampsia and eclampsia were 51.66 6.78, 51.10 6.65 and 68.19 2.13 respectively.The RDW was increased significantly (p < 0.001) in patients with eclampsia while the normal pregnant woman and those with pre eclampsia were in the same range. Conclusion The unexpected rise of RDW in patients with eclampsia suggests increased bone marrow activity.The stimulus is unknown, but as RDW changes are highly significant they may well be a useful indicator of impending parturation.RDW can be used routinely in assessing the red cell status in eclampsia.

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PP2/12 Plasma cell leukemia: case report M. Kiran Kumar · R. P. Swaminathan · V. S. Negi Dept. of General Medicine, JIPMER, India Plasma cell leukemia is an aggressive and rare hematological malignancy involving proliferation of plasma cells. It may appear either de novo (primary form) or as a secondary leukemic transformation of multiple myeloma (secondary form). It is an aggressive disease with extremely poor prognosis when compared to multiple myeloma. We report a case of plasma cell leukemia who had an aggressive disease. Thirty seven year old male patient had come to JIPMER casualty with complaints of generalized fatigue, loss of appetite and dyspnoea on exertion for twenty days. Patient had sustained blunt trauma abdomen twenty days back following which he had four episodes of melena and was treated conservatively in local general hospital. His physical examination revealed tachycardia, severe pallor, ejection systolic murmur in mitral area and hepatosplenomegaly. He didnot have other findings of hematologic malignancy like lymphadenopathy, Waldeyers ring enlargement or sternal tenderness. His initial ultrasound abdomen showed hepatomegaly, splenomegaly with focal lesion suggestive of splenic infarct. In view of history, systolic murmur in mitral area and ultrasound abdomen report of splenic infarct, a provisional diagnosis of infective endocarditis was made and managed with blood transfusions and antibiotics. Later complete hemogram showed a hemoglobin of 3.8 gm/dl, TC of 59,500 cells/μl with DC of N25, E9, L60, MC6 and platelet count of 11,000 cells/μl. Peripheral smear showed – normocytic and normochromic nucleated RBCs, lymphoid leukemoid reaction with myeloid toxic change. He developed progressive renal failure with elevated uric acid level of 10.4 mg/dl. Further, a diagnosis of acute leukemia with spontaneous tumour lysis syndrome was arrived at. His bone marrow aspiration revealed plasma cell leukemia with 44 % plasma blasts. The patient was treated with allopurinol, steroids and dialysis. Patient deteriorated further with rising blood urea/ serum creatinine, uric acid and expired. PP2/13 Neutrophil hypersegmentation in iron deficiency anaemia Aurobindo Prasad Das · Debdatta Basu · Parasappa J. Yaranal Department of Pathology,

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Jawaharlal Institute of Postgraduate Medicine and Research Pondicherry, India Background Hypersegrnentation of neutrophil lobes is an important feature in peripheral blood in rnegalo blasticanemia. It has also been reported as anecdotal case studies in iron deficiency anemia. Methods Fifty cases of iron deficiency anemia and 50 age and sex matched controls were studied for the presence for neutrophil hypersegmentation. Patients with other conditions, which could affect segmentation of neutrophil lobe like infections, pregnancy, drug exosure, renal failure, were excluded. Students unpaired t test was performed for testing the significance between the control and iron deficiency anaemia group. Probability of < 0.05 was considered significant. Results In iron deficiency anemia, the average lobe count was 3.13 while it was 2.48 in controls. Neutrophil hypersegmentation (presence of more than 5% five- lobed neutrophil or a single neutrophil with more than six lobes) were seen in 60% cases of iron deficiency anemia as compared to 4% in the controls.These differences were statistically significant. Conclusion Neutrophil hypersegmentation is common in iron deficiency anemia.The mechanism whereby iron deficiency causes neutrophil hypersegmentation needs to be further studied. PP2/14 Reticulocyte research population data for the detection of target cells on Beckman Coulter Lh 750 David Inbakumar · Kamal · Josphine · U. Sitaram · S.C. Nair Clinical Pathology, Christian Medical College Vellore, India. e-mail: [email protected] Introduction The Coulter LH 750 system uses VCS technology which measures volume, conductivity and light scatter on each cell in a hydrodynamically focused stream. This system also includes an investigation screen that gives statistical information (Mean and SD) of VCS about the main WBC population and also describes about the red cell VCS during reticulocyte analysis. The irregular shape of mature red cells and Reticulocytes produce unpredictable light scatter information when subjected to a laser beam at angle 0 deg to 90 deg. This was used as basis to study the mature red cells in the non – retic area and to correlate with the presence of Poikilocytes in smears and which shows increase in scatter mean and SD of red cells in Non – Retic area.

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Methods The total of 232 samples (K2 EDTA) were processed in the reticulocyte mode on the Coulter LH750 analyser 159 of these samples were having normal blood picture (No poikilocytosis) and 73 samples were having poikilocytes on smear review. We compared the Non-retic Scatter Mean and SD of samples with no poikilocytes to the samples having Ovalocytes/Elliptocytosis and found that the Non-retic Mean Scatter and SD of the sample having ovalocytes/Elliptocytes is higher than the sample with no poikilocytes. Results For the detection of Ovalocytes we found that the best parameters were the Mean reticulocyte Mean Scatter (nretscme) and RDW when the presence of Ovalocytes were not so high (Ovalocytes +) (see table 1). When the presence of ovallocytes was very high (++++) also MCHC and the Reticulocyte Scatter SD (nretscsd) were useful for its detection. (see table 2). For the detection of elliptocytes we found that the best parameters were also Mean reticulocyte Mean Scatter (nretscme) and RDW when the presence of Elliptocytes were not so high (Elliptocytes +) (see table 3). When the presence of Elliptocytes was very high (++++) also MCHC and the Reticulocyte Scatter SD (nretscsd) were useful for its detection. (see table 4). Table 1 Ovalocytes + ROC AUC Sens

spec

Cut-off

nretscme

0.819

86.11

64.62

>56

RDW

0.8425

88.89

63.59

>17.1

Table 2 Ovalocytes ++++ ROC AUC Sens

spec

Cut-off

nretscme

0.8877

100

81.94

>62

RDW

0.9681

100

88.55

>23.1

nretscsd

0.9824

100

96.48

>18

MCHC

0.8265

100

62.56

>33.2

Table 3 Ellitocytes + ROC AUC Sens

spec

Cut-off

nretscme

0.7847

78.26

67.31

>57

RDW

0.8695

91.3

70.19

>18.1

Table 4 Ellitocytes ++++ ROC AUC Sens

spec

Cut-off

nretscme

0.963

100

96.09

>74

RDW

0.9587

100

94.35

>25

nretscsd

0.987

100

98.7

>21

MCHC

0.7413

100

72.17

>33.5

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Conclusion: The mean reticulocyte Scatter was also useful and we have to investigate is also specific of these abnormalities. It is possible today with the instruments of the LH700 series from Beckman Coulter to create rules with the classical parameters MCHC, RDW, etc. but also with the reticulocyte research population data and produce flags that may be will increase the detection of cases with these red cell abnormalities. PP2/15 Clinicopathological profile of paediatric versus adult Hodgkin lymphoma J. Prabhavati · Qutbuddin Chahwala · Debdatta Basu · Bhawana Badhe Department of Pathology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, India e-mail: [email protected] Background and methods Sixty nine cases of paediatric Hodgkin lymphoma and 162 cases of adult Hodgkin lymphoma were diagnosed between January 2000 and December 2007. The age ranged from 2 yrs to 80 yrs. In both, the paediatric and adult groups, number of cases were higher amongst males. The lymph nodes were classified according to the WHO classification. Hodgkin lymphomaNodular sclerosis was the commonest histologic subtype in both paediatric and adult groups. However, amongst females, Hodgkin lymphoma-Mixed cellularity was most common in paediatric age group and Nodular sclerosis in adults. In both the groups Hodgkin lymphoma-Lymphocyte depletion was the least common. Nodular lymphocyte predominanceHodgkin lymphoma was seen in 4.35% and 6.79%, in the paediatric and adult groups respectively. Interfollicular pattern and granulomatous infiltrate were other interesting morphological features seen. Immunophenotyping was done with CD15, CD30, CD20, CD45. EMA and Alk1 were done where indicated. CD15 was positive in 85.11% and 86.74% of paediatric and adult groups respectively. CD30 was positive in 91.49% and 95.92% of paediatric and adult groups respectively. CD15 negativity was seen in 14.89% of paediatric patients. Immunophenotyping helped in classifying Hodgkins lymphoma- Nodular lymphocyte predominance and some cases of Anaplastic large cell NHL. Bone marrow involvement was documented in 13 adult patients but none in the paediatric age group. Conclusion Hodgkin lymphoma-Nodular sclerosis is the commonest in paediatric and adult groups. However, Hodgkins lymphoma-Mixed cellularity is more common amongst females in the paediatric age group as compared to adult females in whom Nodular sclerosis is more common. CD15 negativity is seen in 14.89% of paediatric cases. Bone

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marrow involvement was seen in only in adult patients and none in the paediatric group. PP2/16 Platelet volume indices – an analysis of their utility

E. Babu · Debdatta Basu, JIPMER, No: 5, Old market street, Mudaliarpet, Puducherry - 605 004, India e-mail: [email protected]

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trephine biopsies done, in a period of 8.5 years. A detailed clinical and hematological profile was studied in each case. The diseases associated with marrow necrosis were varied and included metastatic carcinoma, acute leukaemia, post chemotherapy in a case of non-Hodgkin’s lymphoma, tuberculosis and acquired immunodeficiency syndrome. Thrombocytopenia, anemia and leucoerythroblastic blood picture were common hematological abnormalities. Three patients died shortly after the diagnosis was made. Myelonecrosis or bone marrow necrosis is an uncommon finding in bone marrow biopsies. Presence of necrosis in marrow is usually associated with an underlying disease and has a poor prognosis. PP2/18

Platelet volume indices such as platelet large cell ratio (P-LCR), Platelet Distribution Width (PDW) and Mean Platelet Volume (MPV) are estimated by automated blood cell analyzers. This study aimed at understanding the clinical significance of these indices in various conditions with abnormal platelet counts. These indices were studied in 779 patients with normal platelet counts, 74 patients with high platelet counts and 41 cases with low platelet counts. The normal ranges (Mean ± 2SD) of P-LCR, PDW and MPV were 8.8 to 38.4 %, 7.5 to 16.3 fl and 7.4 to 11.8 fl respectively. All three indices were significantly decreased in thrombocytosis but increased in thrombocytopenia when compared to those with normal counts. Among those with high counts, these indices were significantly decreased in reactive thrombocytosis than neoplastic thrombocytosis. In those with low counts, they were increased in destructive thrombocytopenia than those with hypoproliferative thrombocytopenia. While each of these indices was directly correlated to one another, all three indices were inversely correlated to platelet count. Machine counting provides speed, accuracy and feasible utilization of time. This technique may be complementary to a careful examination of peripheral blood smear in the differential diagnosis of abnormal platelet counts. PP2/17 Myelonecrosis – clinicopathological significance of a rare bone marrow lesion

Neha Singh · Parasappa Yaranal · Debdatta Basu · Bhawana Badhe Dept. of Pathology, JIPMER, Pondicherry, India

Myelonecrosis is a rare ante-mortem finding and reported in a wide range of disorders. Ten cases of myelonecrosis were diagnosed, accounting for 0.31% of bone marrow

Establishment of pioneering red cell glycerolization project for armed forces transfusion services in India Col A. Khetarpal · Lt Col Alok Sen Armed Forces Transfusion Centre, Delhi Cantt, New Delhi - 110 010, India e- mail: [email protected] Background India has a population of 1.13 billion and supports nearly one-sixth of the world’s population. Strategic blood reserves are an important component in meeting blood needs and this can be accomplished through the establishment of a frozen blood program. The armed forces transfusion services have taken a leap forward in this direction to augment its inventory of liquid blood. Aim To establish the Pioneering closed system Red cell Glycerolization project for the Armed Forces Transfusion Services in India and to assess the feasibility of launching a frozen blood program. Methods A Red cell glycerolization project was undertaken at the Armed Forces Transfusion Centre (AFTC) New Delhi. 100 units of 450 ml whole blood were collected in Triple blood bags in CPDA – 1 anti coagulant from healthy volunteer donors. Glycerolization of RBCs was carried out within 5 days of blood collection. The packed RBCs were first transferred to 1000 ml PVC bags and then connected to the disposable glycerolization set using the sterile connecting device. Glycerol (57%) W/V) solution was pumped through the disposable glycerolization set using the Haemonetics Automated Cell Processor (ACP 215) to achieve a final glycerol concentration of 40% W/V glycerol. Glycerolized red cell bags were then centrifuged to remove supernatant glycerol and stored at minus 80oC in rigid card board box with a shelf life of upto 10 years according to FDA approved protocol. Results A total of 99 units of glycerolized red cells were successfully prepared. One unit was discarded due to the blood bag leakage during centrifugation.

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Summary/conclusion The first Red Cell Automated glycerolization project was successfully conducted in India. Ninety nine red cell units were glycerolized at AFTC over twenty days using one instrument by a single operator. This is a milestone for the frozen blood program in India

PP2/20 To Gel or not to Gel ... that is the question?

Vanamala Alwar · A. M. Shanthala Devi · S. Sitalakshmi · B. K. P. Prasanna · P. Parimala · R. K. Karuna Department of Clinical Pathology, St John’s Medical College Hospital, Sarjapur Road, Bangalore - 560 034, India e-mail: [email protected]

Background A positive Direct Coombs test (DCT) is the hallmark of diagnosis of Autoimmune hemolytic anemias. The reagent used for the test is the Antihuman globulin (AHG), which may be either ‘Polyspecific’ or ‘Monospecific’. the advent of the gel card systems has made the procedure and interpretation of DCT simpler. Aim of the study To evaluate the various techniques used for the performance and interpretation of DCT. Methods A total of 96 EDTA samples were included in the study. DCT was performed by (i) Polyspecific AHG manual tube method, (ii) Polyspecific AHG Gel card method and (iii) Monospecific AHG (Anti IgG and Anti Complement) manual tube method. In our study we considered positivity by monospecific AHG as the standard for diagnosis. Results Of the total 96 samples evaluated, 44 cases positive by Gel card method, were also positive for either one or both the monospecific AHG reagents. 17 cases positive by Gel card were negative by all manual methods. These false positive cases were attributed to reasons such as increased ESR, macrocytosis and marked leucocytosis. 15 cases that were positive by Gel card and /or monospecific AHG were negative by the manual method using Polyspecific AHG. 9 cases were negative by Gel card but picked up positivity with the Monospecific AHG. The sensitivity of DCT done by the polyspecific AHG Gel card technique was 83.01% and the specificity was 60.46%. Conclusion Use of Gel card technique to perform and interpret DCT is easier than manual tube methods, but positivity by Gel card needs to be correlated with Clinical presentation of the patient and other laboratory findings. Monospecific antisera can be used to confirm cases that are positive by the Gel card systems. Details of presenting author for further communications.

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PP2/21 Serum FasL levels and HIV seroreactivity pattern in blood donors

Anupam Verma · Prashant Pandey · Dheeraj Khetan · Amita Aggarwal · Rahul Katharia · Rajendra Chaudhary Department of Transfusion Medicine, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India e-mail: [email protected]

Background Serum Fas ligand (FasL) levels are reported to be increased in patients with various clinical conditions such as viral infections, malaria, tuberculosis, autoimmune disorders and in apparently healthy persons in certain geographic areas. Aim To correlate serum FasL levels with HIV seroreactivity pattern in blood donors. Methods Serum samples of HIV-1 and 2 ELISA reactive blood donors were stored frozen at –80°C for FasL determination. These samples were further confirmed using Western Blot (WB). Serum FasL was measured by Duoset ELISA kit (sensitivity 31.25 pg/mL). Serum FasL levels from 50 age & gender matched blood donors (nonreactive for HIVELISA) were taken as control. Results Out of 58 ELISA reactive blood donors, 12 were positive by WB. There was a significant increase in serum FasL levels of HIV-ELISA reactive blood donors compared to normal controls [median FasL levels of 345 pg/ml (range 170–620) in HIV- ELISA reactive but WB negative blood donors compared to median FasL levels of 61.2 pg/mL (range non-detectable to 380) in normal controls].However, serum FasL levels (median 107.5 pg/mL, range 57–195) in confirmed HIV positive blood donors (ELISA reactive and WB positive) were comparable to normal controls but significantly (p<0.001) lower than HIV- ELISA reactive but WB negative blood donors. Conclusion We observed no difference in serum FasL levels of normal controls compared to confirmed HIV positive individuals unlike elevated serum FasL levels in HIV patients reported previously. There was a significant correlation between ELISA false reactive samples and increased serum FasL levels indicating a common cause for the occurrence of both. This may be related to latent infections including tuberculosis, malaria or subclinical autoimmune conditions etc which are known to increase FasL levels and cause false reactive ELISA. In conclusion, serum FasL may help in identifying false ELISA reactive blood units.

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Background Blood transfusion is an important mode of transmission of sexually transmitted diseases. Diseases like HIV can be transmitted during the window period without being detected during routine serological testing. Profile of blood donors provides an indirect way of understanding the risk of transmission of these diseases. Objectives To study about the socio-demographic characteristics of the blood donors. To ascertain the knowledge of donors about HIV and its mode of transmission. To find out whether all the donors were well within the Universal Accepted Criteria for blood donation. Study design Descriptive Study Study period 12th August, 07 to 19th August, 07. Study population Candidates who donated blood to the blood bank in district hospital during 1st Jan, 07 to 30th June, 07. Data collection The data was collected from the records of the district hospital blood bank. Result Study included 2073 individuals, males 1786 (86.3%) and females 283 (13.7%). Majority of them 1549 (77.2%) donated voluntarily and 68.7% of the donors were students. Maximum donors were of age group 15 to 29 yrs (73.9%). 96.6% of the donors were locals and 88.2% of them were aware of HIV and 99.6% of them were aware of transmission of HIV through blood. Conclusion All the donors studied were well within the accepted universal criteria of blood donation.

CABG ) has changed the transfusion practices in CABG surgeries.This study was aimed at: To compare blood transfusion practices in patients who underwent CABG with CBP and patients operated by the OP CABG. To study the influence of factors considered as risk factors to require transfusions such as female gender,serum creatinine > 1.8mg/dl,diabetes and left ventricular ejection fraction < 0.3 (LVEF) in both the groups. Methodology 250 patients in each group who underwent CABG for the first time were studied. Patients who underwent CABG with CBP were classified as group A and patients who underwent OP CABG were classified as group B. Anti platelet medication was withdrawn 24 hours before the surgery and none of the patients were given antifibrinolytics Data regarding transfusions,serum creatinine,diabetes,LVEF and gender were taken from the Hospital Information System Results Both the group of patients had comparable age,weight and preoperative Hb (p>0.05).240/250 in group A and 167/250 in group B received transfusions (p<0.001). The average number of transfusions in group A were 7.2 packed red cells, 4.2 FFPs, 2.1 platelets and 1.3 packed red cells, 0.6 FFPs and 0.2 platelets in group B (p<0.001) In patients with s.creatinine >1.8mg/dl,52/58 in group A and 14/16 in group B received transfusions (p<0.001).In patients with LVEF<0.3,96/102 in group A and 92/106 in group B received transfusions (p>0.001). In group A, 46/47 females and in group B 18 out of 26 received transfusions (p>0.001). Conclusion The number of patients and the number of blood components transfused in patients who were operated by the OP CABG were significantly less when compared with the patients who were operated by CABG with CBP. Patients with factors which were considered risk factors for receiving transfusions such as Serum creatinine >1.8mg/dl and diabetes, received significantly lesser transfusions when operated by the off pump method. Off pump CABG if practised widely will reduce the exposure to blood transfusions thereby also preventing the hazards associated with it.

PP2/23

PP2/24

Present transfusion practices in coronary artery bypass graft surgeries

Profile of HIV positives in a VCTC at a district hospital

R. Sudha · S. Shyamala Apollo hospital, Hyderabad, India e-mail: [email protected]

T. Mahima1 · W. Khyati1 · A. Kumar1 · R. Sharath2 1 Department of Community Medicine, Kasturba Medical College, Manipal, India 2 Blood bank Officer, Government District Hospital, Udupi, India

PP2/22 Profile of the blood donors to a blood bank in a district hospital W. Khyati1 · T. Mahima1 · A. Kumar1, R. Sharath2 1 Associate Professor, Kasturba Medical College, Manipal, India 2 Blood bank Officer, Government District Hospital, Udupi, Kasturba Medical College, Manipal, India

Background and aims Transfusion support to patients undergoing Coronary Artery Bypass Graft surgeries with the use of Cardio Pulmonary Pump(CABG with CBP) is complex.The recent technique of Off pump method (OP

Background HIV infection is highly prevalent in our society. By studying the infected individuals who present

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to the hospital, we indirectly study the prevalence and distribution of HIV in Udupi district and the surrounding areas. Objectives To explore the socio-demographic characteristics of HIV positives. To assess the high risk behaviour. To study the needs of the clients with respect to support and counseling required. Study design Retrospective Descriptive Study. Setting VCTC of Udupi district, Karnataka. Participants 293 individuals found positive at VCTC. Time period of study January 15th–April 15th Results Out of 293 positives 203 were males and 90 were females. Majority of them belonged to the age group 3049years. 22% of the studied population was illiterate. 183 were married and 45.5% of these reported partner`s risky sexual behaviour. Most of these individuals were referred for testing by govt. doctors while 74 of them came on their own. Only 91 positives disclosed their status to their spouse and 43 of them got their spouse tested. Conclusion The findings clearly indicate that most of the HIV positives were males in the reproductive age group infected by sexual route. PP2/25 Prevalence of transfusion transmitted diseases among blood donors at Medical College Hospital Ujjain, Madhya Pradesh

G. P. Bhagwat · O. P. Bhargava · V. K. Mahadik · M. Purohit Department of Pathology, R.D. Gardi Medical College, Ujjain (M.P.), India e-mail: [email protected]

Introduction Studies on the trends of transfusiontransmitted diseases in this region are very few. It is mandatory to test the blood for HbsAg, HIV, HCV, VDRL and Malaria in the blood donors. Replacement donors constitute the major bulk amongst the donors. Study was therefore undertaken to find the prevalence of these diseases amongst different donor categories and determine their occurrence. Material and methods A retrospective study was carried out over a period of two years from Jan 2006 to Dec 2007. A total of 9670 blood donors were analyzed. Tests were carried out by ELISA method. VDRL was done by RPR. Result The total prevalence for different markers was 196(2.02%). for HbsAg 154(1.6%), for HIV 12(0.12%) for anti-HCV 8 (0.08% and for VDRL 22(0.23%) respectively. Conclusion: Our results are comparable with the general trends of prevalence. There has been a marginal increase over the 2-year period.

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PP2/26 Retrospective and prospective study of seroprevalence of hiv, hbv, hcv, syphilis and malaria in voluntary and replacement blood donors

R. Gahine · V. Sudershan · V. Kapse · N. Hussain · S. K. Rathia · V. Bombeshwar Department of Pathology, Pt. J.N.M. Medical College, Raipur (C.G.), India

Background Voluntary non-remunerated blood donation is the source of the safest blood suppy to the transfusion service. In the Indian set up where voluntary donations are fewer and poorly structured, safety of blood could still be compromised. Objectives To assess the seropositive prevalence rate of anti HIV, anti HCV, HBsAg, VDRL, and malaria in different types of donor population. To assess the difference in prevalence of seropositivity in replacement donors and voluntary donors.To assess the trend of various coinfections. Material and methods The Study included screening of 59,262 voluntary and replacement blood donors for antibodies of HIV, HCV by commercially available Elisa and rapid test, Hbs Ag by Elisa and card test and VDRL by RPR test. Screening of malaria parasite was done in Leishman stained peripheral blood smear. Results Between 2001 to 2007, a total of 59,262 (Voluntary and replacement) donors were screened. Majority (88.40%) were replacement donors. Males (97.68%) formed a major part of donation in the study. The incidence of HIV was 0.63% in total donors, more in replacement (0.54%) as compared to voluntary (0.09%). The seroprevalence of HBV in total donor was 2.02%, the replacement donors had high incidence (1.92%) as compared to voluntary donors. (0.1%). The seroprevalence of HCV in total donors was 0.21%, more in replacement donors (0.16%) as compare to voluntary donors (0.04%). The incidence of seropostivity for VDRL was 0.04%, more in replacement donor (0.03%) as compared to voluntary donors. (0.003%). The concurrent rates for seroreactivity were highest for HsAg followed by HIV, HCVand VDRL in descending order. Coinfection of HIV with HBV, HCV and VDRL and of HBV with HCV and VDRL were noted. Conclusion The present study emphasizes the continuing need for an effective donor screening programme to protect blood recipients and keep transfusion transmitted diseases to minimum. Recruitment and retention of VD is the key to a safe and sufficient blood supply.

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PP2/27 First report of Hb Jackson in India: a fast moving α chain hemoglobin variant associated with erythrocytosis Roshan Colah · A. Nadkarni · S. Nair · E. D’Suza · A. Bhave · K. Ghosh National Institute of Immunohematology, 13th floor KEM hospital campus, Parel, Mumbai, India e-mail: [email protected] Background The structural hemoglobinopathies are due to globin gene mutations resulting in an abnormal structure or function of the hemoglobin molecule. Majority of them are due to single amino acid substitutions and have an autosomal recessive inheritance. Objective To characterize the abnormal hemoglobin variant associated with erythrocytosis in a family from Maharashtra. Methods: Hematological indices were measured on an automated cell counter (Sysmex-1000). HbA2 and HbF levels were measured on HPLC system (Variant Hemoglobin Testing System, Bio-Rad Laboratories) and conventional cellulose acetate electrophoresis (pH 8.9) was run to look for an altered mobility. DNA sequencing was carried out using the ABI -310 DNA sequencer. Results A family with erythrocytosis was referred to us to look for the possibility of an associated Hb variant leading to polycythemia. The proband and his 4 years daughter had persistently raised hemoglobin levels of 19.1 g/dl and 14.2 g/dl respectively. Cellulose acetate electrophoresis (pH 8.9) showed the presence of a fast moving hemoglobin band (HbX-16%). HPLC analysis showed an abnormal peak in the P3 window (21.5%) with a retention time of 1.78 minutes. DNA analysis revealed a single base substitution AAGAAT at codon 127 in the α globin gene giving rise to a LysAsn amino acid substitution which corresponds to the abnormal hemoglobin Hb Jackson. This rare hemoglobinopathy has not been reported so far in the Indian population. Conclusion Molecular characterization of such haemoglobin variants could help in a better understanding of phenotype-genotype correlations. PP2/28 NF-1 associated pediatric myelodysplastic syndrome in Familial neurofibromatosis Pallavi Bhuyan · Sitaram Mahapatra · Sudhansu Malini Sethy · Smita Mahapatra · Surama Satpathy Dept. of Pathology, S.C.B. Medical College, Cuttack, Orissa, India e-mail: [email protected]

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Introduction Pediatric myelodysplastic syndrome is an extremely rare entity encountered in clinical practice. Its association secondary to familial neurofibromatosis specifically in cases of transfusion dependent refractory anemia in pediatric population is extremely rare. Case Summary A 6-year-old boy with definite history of familial neurofibromatosis reported with transfusion dependent refractory anemia of 6 months duration. Hematological investigation revealed: Hb:5.68 G/dl, Sr. Electrophoresis: AA pattern, G6 PD: Normal activity, Sickling: Negative, OF: WNL, Stool: NAD and PBS revealed macrocytic anemia with neutrophilic pseudo – pelger-huet anamoly and giant platelets. Bone-marrow aspiration showed tri-lineage myelodysplasia. Hence, a final diagnosis of pediatric myelodysplastic syndrome associated with familial neurofibromatosis was made. Conclusion Familial genetic mutations triggering myelodysplastic syndrome in children resulting into transfusion dependent refractory anemia although extremely rare must be kept is mind while dissecting out the differential diagnosis of all cases of difficult and unexplained anemias in clinical practice. PP2/29 Erythrocyte membrane defects in hereditary elliptocytosis

Abhijit Chakrabarty1 · Sutapa Saha1 · Sumanta Basu1 · Dipankar Bhattacharyya1 · T. K. Sabui2 · Maitreyee Bhattacharyya3 · Sanjay Misra3 · Malay ghosh3 1 Structural genomic section, Saha Institute of Nuclear Physics, Kolkata, India 2 Department of Pediatrics, N.R.S Medical College, Kolkata, India 3 Department of Hematology, N.R.S Medical College, Kolkata, India e-mail: [email protected]

Introduction Erythrocyte membrane ultrastructure, the membrane protein profile, cell surface sialylation and levels of PS asymmetry was studied in a case of hereditary elliptocytosis along with three of the patient,s family members. Methods Erythrocytes from peripheral blood of the 8 year old female patient were isolated using percoll density gradient centrifugation and subjected to hypotonic lysis to obtain erythrocyte ghosts. The ghosts were subjected to transmission electron microscopy after staining with 1% phosphotungstic acid.Membrane protein profile was obtained by 2D gel electrophoresis after staining with coomassaie blue. The extent of cell surface sialylation and PS exposure were measured by flowcytometry using FITC-labeled wheat germ agglutinin (WGA ) and Annexin V respectively.

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Results Transmission Electron Micrographs of the sealed erythrocyte ghosts showed impression of discontinuous membrane skeleton. The 2D gel electrophoresis showed membrane protein profile indicating membrane associated globin chains. The Younger cells showed greater cell surface sialylation (MFI 36.7 + 2.5 ) compared to the aged cells (MFI 31.5 + 1.7), The younger erythrocytes also showed higher extent of PS exposed cells (1.7 + 0.2% ), compared to the aged cells (1.4 + 0.04% ), however, the extent of PS exposed cells was low and comparable (1.8 + 0.1% ) with those of normal cells.. The FSc/SSc ratio varied from 1.9 + 0.2 to 2.2 + 0.2 from young to aged cells in normal erythrocytes. However, the ratio decreased significantly in case of the patient and all three family members from 0.62 in aged cells to 0.68 indicating microcytic vesiculation, particularly in the aged erythrocyte fractions Conclusion The erythrocyte membrane is altered in hereditary elliptocytosis in terms of spectrin based membrane skeletal architecture, increased association of globin chains with the membrane skeleton. The extent of sialylation wenr down with aging, however, there were no significant loss of PS asymmetry with aging of the red cells compared to other family members of the patient. Measurement of scattering ratio indicated microcytic vesiculation of red cells in the patient as well as the family members. PP2/30 Quantum dot tagged seeded silver nanoparticles on protein template for the identification of hemoglobinopathy Raib De · Shibsekhar Roy · Jaydeep Bhattacharya · Utpal Choudhuri · Rupak Choudhury · Anjan K. Dasgupta Institute of Haematology and Transfusion Medicine, Medical College and Hospital Kolkata, India e-mail: [email protected] Background Metallic and non metallic nanoparticles are found to interact with proteins and have been used to explore the nanoscale regime. It is found that silver nanoparticles (AgNPs) grown on different protein templates assume different cluster size. The size depends on the protein template as well as the folding status of the protein. Objectives To differentiate the Hemoglobin variants by the Method of Fluorescence activated Cell Sorter (FACS) using characteristic AgNP clustering and farther by using Cadmium sulphide Quantum dots (CdSQDs) for fluorescence enhancement. Materials and methods Haemoglobin obtained from thalassemics and normal volunteers were characterized by HPLC and some by genetic mutation study. The silver

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nanoparticles were synthesized by the seeding method. Cadmium sulphide quantum dots were prepared by standard method. The CdSQDs were added in the protein seeded silver nanoparticle clusters and were analyzed by FACS. Result and conclusion The protein Haemoglobin isolated from different individuals having thalassemia (alpha-thalassemia, Beta thalassemia, E beta thalassemia) and normal controls can be differentiated by the described method. They are separated through FACS by forward scattering and side scattering ratio which characterizes the differential cluster formation in presence of protein template. To develop this method further similar method has been used with Cadmium Sulphide Quantum Dots. The paper describes how this added dimension (fluorescence emission) in the flow cytometric channels help in discriminating different hemoglobin variants. PP2/31 A clinical study of HbE syndrome presenting in a single institute P. K. Gogoi · J. Bhattacharya · A. K. Agarwala Dept of Haematology, Guwahati Medical College and Hospital, India e-mail: [email protected] Background Anaemia due to iron deficiency is a global health problem and the spectrum of anaemia is modified due to prevalence of disorder of hemoglobin structure and synthesis.. The frequency of HbE among indigenous Assamese communities varies from 23% to as high as 78%.Haematology Deptt.GMCH being the premier referral centre for NE India, where HbE is highly prevalent, we got a large no. of HbE disorder presenting with various manifestation. Keeping all these in our mind a humble effort was made to undertake this study. Aims and objective To study the Clinical behavior in HbE syndrome. Material and method One hundred twenty cases of HbE syndrome coming to haematlogy OPD with anaemia during the period of 2005–2007 have been evaluated in this study. RBC indices were measured on, Sysmex XS800i. Hemoglobin typing were done by HPLC method, serum ferritin and TSH value is measured by chemiluminescence method. Result It is observed from the study that relative incidence of HbE disease in patients presenting with different symptoms was 37.50 and that of trait was 41.67. Incidence of HbE-βthalassaemia was found to be 16.67% and compound heterozygous 4.16%. Conclusion Majority of patient maintain a mild lower range of hemoglobin level.Those patient have significant

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lower level of hemoglobin were usually associated with underlying diseases like iron deficiency, hypothyroidism, associated thalassemia or compound heterozygous form.

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PP2/33 Autoimmune hemolytic anemia in beta –thalassemia: report of two cases in children

PP2/32 Different faces of myelophthisis in unsuspected non hematological malignancies

Sajini Elizabeth Jacob · Renu G’ Boy Varghese · Ramji Rai Department of Pathology, Pondicherry Institute of Medical Sciences, Kalapet, Pondicherry - 605 014, India e-mail: sajinijac@ yahoo.com

Background Diagnosing an unsuspected non hematological malignancy by bone marrow examination is not common. There are certain peripheral blood findings which make you suspect the possibility of a malignancy metastasizing to the marrow. Aim of the study To study the state of the peripheral blood in cancers metastasizing to the bone marrow and to try to explain the cause of such peripheral blood changes. Methodology During the period January 2002 to December 2007 a total of 290 bone marrow aspirations and 183 bone marrow biopsies were performed in our hospital. Eight patients were diagnosed to have metastatic malignancy in the bone marrow. Except for one patient all the other seven patients were cases where diagnosis of malignancy was initially done from examination of the marrow. The peripheral blood findings of these seven patients were evaluated. Results The most consistent findings was anemia (5/7 cases). Thrombocytopenia was seen in 57 % (4/7) cases. A leucoerythroblastic blood picture was seen in 42.8% cases (3/7). There was one case with Micro angiopathic hemolytic anemia. One patient showed neutrophilic leucocytosis and another neutrophilic predominance. There was one case showing prominence of microspherocytes. Cytopenias are due to suppressor cytokines released by malignant cells. The reduction in marrow space due to fibrogenic factors secreted by tumor cells cause cytopenias and premature release of erythroblasts and myelocytes leading to a leucoerythroblastic blood picture. It will also cause damage to the cells leading to hemolysis. The leukocytosis can be attributed to release of WBC from storage pools by factors secreted by the tumor. Abnormal metabolites produced by malignant cells can also affect the cell membrane leading to formation of spherocytes Conclusion Leucoerythroblastic blood picture and unexplained cytopenias serve as valuable clues about a possible underlying malignancy.

R. Das1 · I. Panigrahi1 · K. Naranje2 · A. Trehan1 · R. K. Marwaha1 1 Institute of Medical Education and Research, Chandigarh, India 2 Advanced Pediatric Centre, Institute of Medical Education and Research, Chandigarh, India e-mail: [email protected]

Thalassemias are group of disorders characterized by chronic extravascular hemolysis. Acute intravascular hemolysis in a case of thalassemia should prompt to find other associated causes like G6PD deficiency and autoimmune hemolytic anemia (AIHA). There are few case reports of AIHA in beta-thalassemia in literature. In the present report we describe two cases (11 yr and 2.5 yr old respectively) with thalassemia intermedia associated with AIHA and discuss the previous reports in existing literature. One of the patients who was on regular transfusions was put on hydroxyurea after the episode and showed beneficial response with elimination of transfusion requirement. PP2/34 Microangiopathic hemolytic anaemia in a metastatic mucinous adenocarcinoma

A. Dattagupta · S. Jayakumar · A. Mani · Renu G’Boy Verghese Pondicherry Institute of Medical Sciences, Pondicherry, India e-mail: [email protected]

Background and objective A case of metastatic mucinous adenocarcinoma with microangiopathic hemolytic anaemia is being presented. Case A 35-year-old male presented with history of low backache for 3 months and jaundice for one week. On admission was found to be pale and icteric. USG showed hepatosplenomagaly. X-ray Dorsolumbar spine was found to be normal. Blood investigations revealed anaemia with thrombocytopenia and features of hemolytic anaemia in the form of elevated reticulocyte count, increased Lactate Dehydrogenase, hyperbilirubinemia. Patient also had evidence of Disseminated intravascular coagulopathy in the form of prolonged Prothombin time and Fibrin Degradation

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Products and D-dimer positive. Peripheral smear showed leucoerythroblastic picture, thrombocytopenia and plenty of fragmented RBCs. Alkaline Phosphatase was highly elevated. Bone marrow aspirate was dry tap, but biopsy imprint smear showed features of metastatic deposit in the marrow and bone marrow biopsy sections showed clusters of malignant cells arranged in glandular pattern with mucin secretion, suggesting metastatic deposit from a mucnous adenocarcinoma. PP2/35 Pregnancy with thrombotic microangioopathy treated with plasma infusion C. Nandakumar · A. Mani · Renu G. Boy Verghese · R. Vedavalli Pondicherry Institute of Medical Sciences, India e-mail [email protected] Background and objective A case of pregnancy with Thrombotic microangiopathy treated with plasma infusion. Case Twenty seven years pregnant female (7 months of gestation) came with fever and headache for 3 days, altered sensorium for 1 day. On examination she was deeply unconscious, pale with normal vitals. Haemoglobin 5 gm%, normal total leucocyte count, peripheral smear showed normocytic normochromic RBC-plenty of microspherocytes, fragmented cells and good number of polychromatic cells, nucleated RBCs, platelet count 30,000/cu mm, urine Hb positive, LDH 4636 IU/L, Liver profile-normal, Antiphospholipid antibody positive, Anti nuclear antibody negative, urine culture and blood culture showed no growth. A diagnosis of Thrombotic thrombocytopenic purpura was made. She was treated with plasma infusion, packed cells, methyl prednisolone. Patient delivered macerated foetus after 48 hours and became conscious on the same day. Haemoglobin and platelets returned to normal levels in the next few days. PP2/36 Congenital sideroblastic anemia – a case report Sajini Elizabeth Jacob1 · Renu G’ Boy Varghese1 · Soumya Sampath2 · Ramji Rai1 1 Department of Pathology and 2Pediatrics, Pondicherry Institute of Medical Sciences, Kalapet, Pondicherry – 605 014, India e-mail sajinijac@ yahoo.com

Background The sideroblastic anemias are a heterogeneous group of hereditary or acquired conditions characterised by anemia, usually refractory to therapy,

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ineffective erythropoiesis and the presence of ring sideroblasts in the marrow. Congenital sideroblastic anemia is a rare disorder and it has to be differentiated from other commoner causes of microcytic hypochromic anemias in the infancy period. We report a case of a child presenting with Congenital sideroblastic anemia. Case report A one-year-old child presented with severe pallor. She was born as a premature child and there was past history of receiving blood transfusions, the first one at three months of age. The anemia had been chronic and an empiric trial of hematinics had not brought any significant response. Investigations showed hemoglobin (Hb) of 2.4 g/dl with a reticulocyte count of 0.1%. Detailed investigations revealed a serum iron of 216μg/ dl whilst the total iron binding capacity was 286 μg/dl. Peripheral smear showed a dimorphic blood picture with normal red cells and microcytes. Bone marrow examination showed ring sideroblasts.She was started on high dose pyridoxine. But on follow up, there was no significant response. So she at present is on periodic blood transfusions. Conclusion Though congenital sideroblastic anemia is a rare cause for microcytic hypochromic anemias in infancy we should be aware of it and differentiate it from other commoner causes of microcytic hypochromic anemias PP2/37 Case report: association of alpha thalassasemia with sickle haemoglobin S. Bhave · B. Sayaji · V. Melinkeri · A. Kelkar Sahyadri Speciality Hospital, Pune, India

We describe a family of four, hailing from Panchmahal region of Gujrat. Their eldest son had presented with pulmonary hypertension at the age of 17 years and succumbed to CCF. He was found to have sickling test positive. The family of 4 referred to us consisted of father, mother, son and daughter; were referred for Hb HPLC testing. Their laboratory finding revealed various combinations of alpha thalassaemia with sickle cell, which will be presented in details. PP2/38 Myelodyspastic syndrome (MDS) – incidence and prognosis, experience at PGIMS rohtak

P. S. Ghalaut · V. S. Ghalaut · R. Shandilya · Gaurav · Atul · Kamlesh

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PT. B.D. Sharma PGIMS, Rohtak - 124 001, Haryana, India email: [email protected]

Aims To study the incidence and prognosis of cases of Myelodysplastic Syndrome at PGIMS, Rohtak. Material and methods This was a retrospective study of all cases of Myelodysplastic Syndrome admitted or seen at Clinical Haematology Clinic at PGIMS, Rohtak. The case records of patients were analysed over a period of 10 years. Results A total of 50 patients of MDS were seen at Clinical Hematology Clinic of PGIMS, Rohtak over a period of 10 years (1997–2006). This constituted 3.7% of total cases of hematology. Patients were seen in all age groups, however maximum no. of patients were seen between 40–60 years (42%). There were 26 females and 24 males. All the patients presented with marked pallor, generalized weakness. About half the no. of patients had fever (27 patients). 11 patients had bleeding manifestations. According to FAB classification 40% cases had refractory anemia with ringed sideroblasts (RARS) and 32% cases had refractory anemia (RA). There was only one case of CMML. All the patients were followed up for over 6 months to 2 years and patients died in spite of supportive treatment, since there is no definite treatment of the disease. A brief review of literature is also described. Conclusion Myelodysplastic Syndrome was seen in 3.9% of all haematological cases seen at PGIMS during 10 years. The disease occurred at a younger age group as compared to west and had poor prognosis. PP2/39 Case report: CMPD with ONLY minor bcr–abl positivity S. Bhave · S. Apte · A. Karpe · B. Sayaji · V. Melinkeri · A. Kelkar Sahyadri Speciality Hospital, Pune, India

A 60-year-old male presented with dyspnoea on exertion, anemia. O/E Pallor +. Gen Lnpathy, Hsmegaly. Hb 9.2, TC-339500, platelets 49000, blasts – 03% and monocytoid cells 14 % without basophilia and eosinophilia. BM– hypercellular 11% monocytoid cells,No eosinophilia and basophilia. S/O CMPD ? CML Cytogenetics: Ph +ve. RT PCR: Negative for BCR-ABL Major (p210) breakpoint and Positive for BCR-ABL Minor (p190) breakpoint. Patient followed aggressive course requiring repeated admissions and succumbed to his illness within a period of few months. The literature describes only few such cases so far. We wish to present this case in detail for it’s unique presentation.

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PP2/40 Effect of α- gene numbers on the genotype of sickle cell patients Vineeta Sharma · Sanjay Kumar Pandey · Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi - 29, India e-mail: [email protected] Introduction Sickle cell disease results from a specific point mutation (A > T) causing the substitution of glutamic acid by valine at amino acid six in the beta-globin gene. Sickle patients have a very variable clinical phenotype. Some of the possible explanations for the observed variable clinical severity are coinheritance of α-deletions and α- triplications. Objective To determine the frequency of α deletion and triplication in Sickle cell patients and to study their effect on the phenotype of Patients. Material and methods Subjects were 45 Sickle cell patients who attended the Hematology out patient department of the institute. Out of these 10 were sickle cell trait, 25 were sickle/Bthal. And 10 were sickle cell disease (diagnosed by HPLC). Patients were divided into three subgroups according to a scoring system based on seven clinical criteria as mild (score 0–3.5), moderate (score 4–7) and severe (score7.5–10). α deletions and triplication were studied by GAP-PCR and multiplex-PCR respectively. Results: α deletion was found in 8 (17.7%) out of these 8 patients 5 (αα/-α3.7) were from Gp1 and 3 (αα/-α3.7) were from Gp2. α triplication was found in 2(4.4%) both of these were (αα/αααanti-3.7) from Gp3. Conclusion Patients with coexisting α deletion, had higher mean Hemoglobin, required lesser transfusions and had less severe phenotype while patients with α triplication had lower mean hemoglobin, were on frequent transfusions and had severe phenotype. PP2/41 Profile of platelet function defects in adult patients from a tertiary care hospital S. Khodaiji · M. Sethi · S. Ghosh P. D. Hinduja National Hospital and Medical Research Centre, Mumbai, India Background Platelet function testing has played an important role in the diagnosis of hereditary bleeding disorders. It is now being used to monitor patients on ant-platelet drugs. Despite some limitations, platelet aggregometry is considered the gold standard assay for platelet function. It measures real time aggregation of platelets by optical clearing of PRP

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Objective to classify the various platelet function disorders in an adult population of patients referred for platelet aggregometry. The common presenting complaints were epistaxis, bleeding gums, ecchymotic patches, easy bruising and menorrhagia. A few patients on aspirin/ clopidogrel were referred to monitor the efficacy of the drugs Materials and methods A total of 101 patients were studied with ages ranging from 18 to 88 years. The M:F ratio was 2:3. Aggregation was tested with ADP, epinephrine, collagen, ristocetin and arachidonic acid Results Of the 101 patients, 52 were normal, 23 had Glanzmann’s Thrombasthenia (GT), 1 had a Storage Pool Defect, 7 had vWF deficiency/Bernard Soulier Syndrome, 15 were drug induced and 3 were unclassifiable. Reduced aggregation response to only collagen was seen in 8 patients, epinephrine in 5, low concentration of ADP in 5, high concentration of ADP in 1 and combined ADP in 2 patients. These patients probably have reduced levels or absence of receptors for these agonists Conclusion Since platelet aggregometry is subject to many pre-analytical and analytical variables, it is advisable to repeat the assay before conferring a final diagnosis on the patient. It is now known that a number of variants of GT exist in which the surface glycoproteins are present in normal amounts but are functionally defective. Genetic mutations of these glycoproteins have been detected which result in a thrombasthenia phenotype. Heterozygous carriers who are clinically asymptomatic can also be detected by molecular techniques. Further, recognizing polymorphisms in these glycoproteins is important in carrier and prenatal diagnosis Since the last guidelines for platelet function testing were published by the BCHS in the 1980s, many newer methods of testing platelet function have now become available such as flow cytometry and molecular techniques PP2/42 Freelite chain assay in plasma cell dyscrasias: a preliminary analysis

D. K. Mishra1 · V. S. M. Velu Nair2 · Ajay Sharma1 · S. Bhattacharya1 · Satya Das1 · J Kotwal1 · T Verma1 · Rohit Vohra1 1 Department of Pathology and Molecular Medicine and 2 Department of Clinical Haematology and BMT, Army Hospital (Research and Referral), Delhi Cantt. New Delhi, India

Introduction Free light chains (kappa and lambda) are the normal constituents of an immunoglobulin. It is

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metabolized by the kidneys and a small fraction is found in the serum of healthy individuals. Free light chains are increased in plasma cell dyscrasias and are measureable as a diagnostic and prognostic assay in intact immunoglobulin multiple myeloma, non-secretory multiple myeloma, light chain disease and amyloidosis. It may be used to monitor therapeutic efficacy and relapse of disease. Aim The aim of this ongoing study is to evaluate the diagnostic and prognostic potential of freelite chain assay in plasma cell dyscrasias. Materials and methods We studied 42 cases of which 30 cases were of plasma cell dyscrasias and 12 cases were analysed as controls. All multiple myeloma cases (27/30) showed M band on serum protein electrophoresis (SPE) and on immunofixation electrophoresis (IFE) showed monoclonal peaks (IgG 70% and in IgA 30% ) in all of them. Two patients were of light chain myeloma and one primary systemic amyloidosis. Eighteen multiple myeloma, 2 light chain myeloma and one primary systemic amyloidosis were newly diagnosed cases and the rest 09 were in various stages of therapy. The freelite chain assay, i.e Kappa and Lambda light chains were performed using patients’ serum by using freelite (kappa and lambda) kits manufactured by the Binding Site, UK and the Dade Behring BN ProSpec-100 nephelometer. The normal reference range of kappa: lambda ratio as per product insert is 0.26–1.65, (kappa – 3.30–19.40mg/L), (lambda – 5.71–26.30mg/L). Results In the multiple myeloma group the kappa:lambda ratio varied from 0.837 to 137.56. The eighteen newly diagnosed cases of multiple myeloma showed a kappa: lambda ratio of 2.06 to 137.56. Nine cases of multiple myeloma in various stages of therapy showed a kappa: lambda ratio of 0.873 to 12.72. Both cases of light chain myeloma and primary systemic amyloidosis could be diagnosed only by IFE and freelite assay (kappa: lambda ratio 2.64 to 3.14 ). Kappa: lambda ratio in the 12 cases, assayed as controls ranged between 0.548 to 1.83. All these cases had shown polyclonal increase in gammaglobulins on SPE and IFE was negative for any monoclonal patterns. Only one patient showed a ratio of 1.83, which is higher than the normal reference value. Discussion Freelite chain assay is currently the most used diagnostic and prognostic assay in plasma cell disorders. Our study could pick up light chain myeloma and primary systemic amyloidosis only by IFE and freelite assay. The prognostic relevance of serial monitoring of kappa:lambda ratio in multiple myeloma in therapeutic monitoring could be appreciated. However, it may be important to assay more control individuals to arrive at a population specific reference range for the kappa-lambda ratio. Important aspects of this assay will be discussed.

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PP2/43 Flowcytometric immunophenotyping: a rapid diagnostic tool in a case of plasmablastic lymphoma K. Sehgal · P. G. Subramanian · P. R. Tembhare · A. Vazifdar · Y. Badrinath · Ashok Kumar · S. Gujral Hematopathology lab, Tata memorial hospital, Mumbai, India Background Plasmablastic lymphoma was originally described as an AIDS associated B-lineage neoplasm with plasmacytic differentiation, typically presenting in the oral cavity. At present it is considered a variant of diffuse large B-cell lymphoma in the World Health Organization (WHO) classification of lymphoid neoplasms. The cells were designated as plasmablastic due to the blastic appearance of neoplastic cells associated with strong expression of plasma cell associated antigens such as CD38 and CD138 and absence or weak expression of B-cell markers. Case report We present a case of a 12 year old boy who was HIV positive and presented with a mass lesion in the paranasal sinuses with extension into the nasopharynx. FNAC from the mass was suggestive of a Non hodgkins lymphoma. Staging bone marrow examination revealed 42% atypical blast like cells with plasmacytoid morphology along with spill of these lymphoma cells into the peripheral blood. FCM analysis performed on the BM and Peripheral blood samples revealed coexpression for CD 38 and CD 138 along with CD 56 positivity. CD 20, CD 45 and CD 3 were negative. A diagnosis of plasmablastic lymphoma was made and this was confirmed by using relevant IHC markers on the bone marrow biopsy as well as the biopsy from the nasal mass. EBER performed by in situ hybridization was positive. Conclusion Flowcytometric immunophenotyping can be used as rapid diagnostic tool for diagnosing PBL (CD20 –ve, CD138 +ve) and to differentiate it from other variants of DLBL (CD 20 +ve, CD 138 –ve). PP2/44 Granular B-cell acute lymphoblastic leukemia in a sixteen years male patient: a case report P. R. Tembhare · P. G. Subramanian · Ashok Kumar · Y. Badrinath · K. Sehgal · R. Ansari · S. Gujral Background Diagnosis of acute leukemia is mainly done on morphologic and cytochemical evaluation of peripheral

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blood or bone marrow aspirate smears. Cytoplasmic granules in the blasts is one of the well accepted distinguishing feature for myeloid versus lymphoid differentiation. In many centers where immunophenotyping is done, usually a panel of limited markers is advised on the basis of morphology which may lead to erroneous results. Case report We describe a rare case of granular Bcell acute lymphoblastic leukemia in a 16-year-old male patient showing CALLA positive (CD19+, CD10+, CD34+ HLADR+, and cytoCD22+) phenotype with aberrant expression of CD33 and CD117. The blasts were three times of the size of a mature lymphocyte with abundant cytoplasm having numerous azurophilic granules. These granules were completely negative for myeloperoxidase (MPO), acid phosphatase (ACP) and Periodic Acid-Schiff reaction (PAS) but were positive for non-specific esterase (NSE) by cytochemical staining. Conclusion Acute lymphoblastic leukemia (ALL) presenting with numerous cytoplasmic granules in blasts is a rarity and may be misdiagnosed as acute myeloid leukemia especially AML-M3. Hence it is important to use a comprehensive panel of antibodies for Immunophenotypic analysis of leukemia and to be aware of this rare entity. PP2/45 Immunophenotypic pattern analysis by flow cytometry in two cases of myelodysplatic syndrome (RAEB2)

P. R. Tembhare · P. G. Subramanian · Y. Badrinath · Ashok Kumar · K. Sehgal · S. Gujral

Background According to WHO guidelines, the diagnosis of MDS is currently based on morphologic and cytogenetic abnormalities. However, morphologic evaluation is inherently subjective, and cytogenetics, while being objective, identifies abnormalities only in 30% to 40% cases. Hence, an additional objective criteria is needed. Flow cytometric analysis provides a highly reproducible and objective tool for assessing the patterns of expression of multiple antigens in different hematopoietic lineages. Case study We present a flow cytometric study of abnormal surface antigen expression patterns in two casesof MDS. A 53-year-old male patient presented with fever, weight loss and pancytopenia since six months and a second case of 28-year-old female with five year history of Hodgkin’s lymphoma presented with Hb of 7.1g/dL, platelets of 34 × 109/L and TLC of 37.4 × 109/L. Bone marrow examination of both patients revealed increase in blasts percentage and variable dyspoiesis in cells of all three lineages. Immunophenotypic analysis

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revealed the abnormal patterns of surface antigen expression in both patients. In MDS, patterns of antigen expression are distinctly different from that of normal bone marrow aspirates as shown by two examples (will be discussed). Conclusion By comparing patterns of antigen expression on a given cell population with the patterns identified on normal cells of that type, one potentially can identify abnormalities that helps in diagnosis of MDS and if sufficiently great, might substitute for clonality studies. PP2/46 Platelet counts correlation between two hematology analysers P. G. Subramanian · M. R. Tiwari · S. G. Mahadik · S. C. Shinde · S. Gujral Background Platelet counts done by hematology analysers are conventionally counter checked using manual platelet counts and estimated counts from peripheral blood smears. Currently the ICSH reference method for platelet counting is using flowcytometric immunophenotyping to distinguish platelets from cytoplasmic fragments and other particles which interfere with impedance counts. Objective To study the correlation between various platelet counts methodologies used in two different hematology analysers i.e. Cell-Dyn Sapphire and LH500 in the laboratory Materials and methods 73 samples with platelet count less than 100 × 109/L were run in Cell-Dyn Sapphire (Abbot diagnostics) and LH500 (Beckman coulter). Cell-Dyn Sapphire counts platelet using two technologies –1) Impedance method reported as platelet impedance count (PIC) and 2) Optical scatter and fluorescence reported as Platelet Optical Count (POC). The LH500 uses Impedance counting with software corrections for best fit distribution pattern. Statistical analysis was done of the data generated. Results The Pearson correlation between POC and LH 500 count was 0.959; that between PIC and POC of CellDyn is 0.81; and between PIC and LH500 was 0.834. Simple impedance counting is unreliable at very low counts especially < 20 × 109/L. PIC in general overestimate the platelet count as compared to POC. Conclusion The platelet counts between new generation hematology anlysers are reproducible in the range of less than 100 × 109/L. However there is likely to be significant variation between new generation platelet counts and older hematology analysers. This has to be taken into account when making clinical decisions.

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PP2/47 Clinical profile in gelatinous bone marrow transformation

Nisha Marwah · R. Sen · H. Singh · A. Gupta · J. Sen Pt. B.D. Sharma PGIMS, Rohtak, India e-mail: [email protected]

Objective To study the clinical spectrum associated with gelatinous bone marrow transformation (GMT). Methods All subjects whose bone marrow aspiration showed pink purpose material on Leishman stain underwent a detail history, clinical examination and investigation (biochemical/microbiological/radiological). Additionally, in each subject the smear was stained with special stains of Periodic Acid Schiff and Alcian blue. Results Out of total 1498 marrows, 65 showed evidence of GMT. All of these had anaemia. The associated clinical spectra of diseases noticed were: Infection (31 cases), Nutritional deficiency (5 cases), Haematological disorders (Aplastic/toxic depression) (17 cases), Malignancies (3 cases), and Miscellaneous (9 cases). Conclusion Based on the heterogenecity of associated clinical disorders, GMT indicates severe illness and not a particular disease. GMT may be a result of bioregulatory process (which presently needs further prospective studies) that are activated in different pathologic conditions but resulting in similar lesion in the bone marrow and so till then it may be concluded that GMT is a symptom of bone marrow. PP2/48 Profile of childhood erythroblastopenia Sunita Singh · R. Sen · H. Singh · N. Marwah · R. Kalra · Pt. B.D. Sharma PGIMS, Rohtak, India e-mail: [email protected]

40 children aged between 7 months to 12 years with severe anaemia and isolated erythroblastopenia on bone marrow aspiration were evaluated during 2 years period of study, whereas all the patients showed reticulocytopenia, granulocytes and platelets were normal. The conditions were found to be associated with protein energy malnutrition in 35% cases, mixed nutritional deficiency (17.5%), iron deficiency anaemia (12.5%), gastroenteritis (12.5%), respiratory tract infection (12.5%) amongst others. Erythroblastopenia persisted from few days to 10 weeks. The patients revealed recovery characterized by reticulocytosis

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and improvement in haemoglobin in most of the cases following blood transfusion and symptomatic therapy.

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Lok Nayak Hospital and Maulana Azad Medical College, India email: [email protected]

PP1/1 Spectrum of novel mutations in haemophilia A G. Sankari Devi · G. Jayandharan · E. Eunice Sindhuvi · R. V. Shaji · Auro Viswabandhya · Mammen Chandy · Alok Srivastava Department of Haematology, Christian Medical College, Vellore, India e-mail: [email protected] Haemophilia A (HA) is an X linked bleeding disorder caused by heterogeneous mutations in the coagulation factor VIII (F8) gene. The major molecular defects in the F8 gene causing HA are intron 22 and intron 1 inversions responsible for ~50% of HA phenotype. Detection of point mutations and deletions in F8 gene that account for the rest of molecular defects is challenging due to the large size (186kb) of F8 gene. We analyzed 53 unrelated Indian patients with HA for their F8 gene defects from Sep 2006 to May 2008.Out of these 49 were (92.45%) had severe and 4(7.5%) had moderate HA. Intron 22 and Intron 1 inversions were analyzed by PCR based methods. Point mutations were screened in the coding regions, promoter and the polyadenylation region by multiplex PCRs and conformation sensitive gel electrophoresis (MPCR-CSGE) followed by DNA sequencing. Intron 22 inversions were detected in 18(33.9%) patients while 6(11.3%) were positive for intron 1 inversion. MPCR-CSGE could detect mutations in 22/29 patients (75%). These included 4 gross deletions, 4 nonsense mutations, 3 missense mutations, 7 microdeletions, one duplication and 2 splice site mutations. Ten novel mutations were identified. They included microdeletions (n=5), duplication (n=1), nonsense (n=1), splice site (n=2) and missense (n=1) mutations, all of them resulting in severe phenotypes. Mutations were not detected in 7(13.2%) patients presenting with moderate (n=3) or severe (n=4) phenotypes. This data shows that the novel mutations are not uncommon in HA and a comprehensive screening strategy is essential for genetic and prenatal diagnosis of HA in India. In addition, our study implies that elucidation of mechanisms responsible for the mutation negative cases is important for the comprehensive genetic diagnosis of HA. PP1/2 Cerebral venous thrombosis as a consequence of a unique thrombophilic state S. E. Mathews · A. Vyas · N. Gupta · D. Dhanwal

Background Cerebral venous thrombosis (CVT) is an uncommon cerebrovascular disorder that accounts for 0.5% of all strokes, and often affects young adults. CVT poses a diagnostic and therapeutic challenge because of its diverse clinical manifestations and wide spectrum of etiologies. The underlying diseases remain unrecognized in around 25% of cases. We present one such enigmatic case of CVT. Case description A young male patient presented with a three day history of high grade fever, palpitations, headache followed by acute onset of right hemiparesis, focal seizure and disorientation. On examination temperature was 104 degrees Fahrenheit, there was sinus tachycardia and blood pressure was 120/60 mmHg. Meningeal signs were absent and fundus examination was normal. CSF analysis and non contrast CT of the brain did not reveal any abnormality. In view of a past history of hyperthyroidism thyroid function tests were done. TSH was found to be markedly suppressed (0.018 mIU /l) with raised free T4 (57.8 pmol /l) suggestive of a thyrotoxicosis. MRI and MR venography, detected a left parietal venous infarct with superior sagittal sinus and cortical vein thrombosis. Screening for associated thrombophilia and vasculitis did not identify any abnormality, except for a significantly raised factor VIII level of > 160%. Anticoagulant as well as antithyroid drug therapy was instituted, which resulted in complete neurological recovery. Conclusion The pathogenesis of cerebral venous thrombosis is multifactorial. Patients with thyrotoxicosis may be predisposed to the development of CVT through a factor VIII mediated hypercoagulability. Conversely, hyperthyroidism is worth considering as a possible etiological factor in patients with unexplained CVT. PP1/3 Clinico-pathological study on haemophilia: an analysis of 100 cases P. K. Gogoi · J. Bhattacharyya · Damodar Das. Gauhati Medical College and Hospital, Guwahati, Assam, India e-mail: [email protected] [email protected]

Background Patient with joint pain and swelling, bleeding after trauma, hematuria, occasionally intracranial hemorrhage etc. is referred to this institute from different states of north east region and most often consulted with surgical specialties. After evaluation only these patient come for hematology consultation.

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Objectives To study the clinical spectrum of the disease in north east region. Materials and methods Study was conducted in the dept. of hematology Gauhati Medical College and Hospital during the period of 2006–2008. One hundred patients attending with history of recurrent joint swelling, wound bleeding and prolonged bleeding after surgical procedure were included in this study. Diagnosis was made on the basis of history, physical examination and laboratory investigation i.e. B.T, C.T, P.T, APTT, TT and coagulation factor assay. Results Predominant presenting age groups affected were between 2–20 yrs 55%. Recurrent joint swelling was the predominant presenting symptoms (95%), followed by wound bleeding (52%). On evaluation 48% patients had positive family history of bleeding. Results of coagulation screening tests showed that 86 patients had prolonged APTT and 14 had normal. Reduced activity of factor VIII and IX was observed in 74 and 12 Patients respectively. Conclusion Results suggest that both Hemophilia A and B are seen in this part of country and patients is similar to that described in other population. Need better laboratory facilities in this part of country for early diagnosis of patients. PP1/4 Lipid profile and parameters of coagulation in postmenopausal women on hormone replacement therapy (HRT)

Meera Sikka · Usha Rusia · Neerja Goel · Nishi Madan 1 Departments of Pathology and OBG, University College of Medical Sciences and GTB Hospital, Dilshad Garden, Shahdara, Delhi - 110 095, India e-mail: [email protected]

Background With an increase in life expectancy, women spend an increasing proportion of their lives in the postmenopausal state. Besides experiencing distressing symptoms, there is an increased rick of coronary artery disease (CAD). HRT is known to reduce morbidity and mortality due to CAD or stroke. Objective To assess changes in lipid profile and haemostatic function in women on HRT. Materials and methods Thirty postmenopausal women on HRT (conjugated equine estrogen 0.625mg daily and dydrogesterone 10mg from day 1–12 of cycle) and 30 age matched controls included. Complete blood counts, PT, APTT, TT, KCT, plasma fibrinogen, euglobulin clot lysis and lipid profile were done in all patients before and after 3m of therapy.

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Result There was a significant (P<0.02) reduction in total cholesterol, LDL, plasma fibrinogen and shortening of ECL after HRT with a rise in HDL. Conclusion HRT may be used in post menopausal woman to reduce cardiovascular risk factors and improve quality of life. PP1/5 Frequency of (TA)n Polymorphisms in UGT1A1 Gene in Patients with Hyperbilirubinemia

S. V. Rajkumar · E. Eunice Sindhuvi · R. V. Shaji · Mammen Chandy · Alok Srivastava Department of Haematology, Christian Medical College, Vellore, India e-mail: [email protected]

Uridine Glucuronyl Transferase (UGT) is a key enzyme involved in glucuronidation and excretion of bilirubin. The (TA)n polymorphisms in the promoter region of the gene UGT1A1 have been associated with variable levels of gene expression and bilirubin levels. The mutant alleles have been found to be associated with the risk of gallstones in thalassaemia and sickle cell disease, predisposition of individuals to altered drug metabolism and neonatal hyperbilirubinemia. We analyzed 312 unrelated Indian patients with hyperbilirubinemia for UGT1A1 polymorphisms. The promoter region of the gene was analyzed by polymerase chain reaction (PCR) using fluorescent-labeled primers. The PCR products were analyzed on ABI PRISM 310 genetic analyzer and the data was analyzed using Gene scan software. Of the 312 patients analyzed (TA)6/(TA) 6 was present in 18 patients (5.76%), (TA)6/(TA)7 in 49 patients (15.7%), (TA)7/(TA)7 in 243 patient (77.88%) and (TA)7/(TA)8 in 2 patients(0.64%). The mean total bilirubin (mg/dL) levels in (TA)6/(TA) 6, (TA)6/(TA)7. (TA)7/(TA)7 were 3.3 (1.8–5.4), 3.6 (0.4–11.8) and 4.01(0.5–18.4) respectively. The bilirubin levels were higher in the (TA)7/(TA)7 group compared to other groups, but it is not statistically significant (p=0.5). Our data shows that the most common genetic factor for hyperbilirubinemia in Indian population is the presence of the (TA)7 allele present in the homozygous state (78%) or in the compound heterozygous state with (TA)6 (16%). The allele frequency of (TA)7 in individuals with hyperbilirubenemia in our study is in concordance with the previously published data in the population. Our data confirms that UGT1A1 (TA)7 polymorphism is the most common genetic factor for hyperbilirubinemia in Indian population and routine screening of this polymorphism should be the first step in the differential diagnosis of indirect hyperbilirubinemia.

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PP1/6 Renal osteodystrophic changes in bone marrow biopsy M. Murari · R. Pandey Department of Pathology, SGPGIMS, Lucknow, India e-mail: [email protected] Background Patients with end-stage renal disease (ESRD) have a multitude of manifestations of bone disease with more severe changes seen in cases on maintenance hemodialysis (MHD). Renal osteodystrophy may contribute to anemia of ESRD and in advanced cases marrow may become hypocellular and extensively fibrosed. Bone marrow biopsy is not routinely carried out in patients with ESRD. Case summary A 26-year-old male patient with ESRD on MHD presented with pancytopenia (Hb 4.3g/dl, TLC 3100/μl, platelets 77000/μl). Marrow aspiration and biopsy were done as part of pre-transplant work-up. Marrow aspiration yielded small amount of predominantly fatty hypocellular marrow. Marrow trephine biopsy was processed by B5 fixation, decalcified in TCA and stained with HandE, Reticulin and Masson Trichrome. Extensive osteodystrophic changes including irregularly thickened trabeculae, some with prominent resorption bays, peritrabecular and obliterative fibrosis alternating with marrow spaces showing fatty hypocellular marrow were observed. The patient underwent renal transplantation and showed completely normal hematologic and biochemical profile after transplant. Conclusion Use of undecalcified bone biopsy is the recommended technique for study of spectrum of renal bone disease. However, several distinctive osteodystrophic changes can also be documented in decalcified paraffin embedded marrow trephine biopsy.

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Hence, erythrocyte membrane was investigated in a unique case of hemolytic anemia. Aim To study erythrocyte membrane ultrastructure, the membrane protein profile, cell surface sialylation and levels of PS asymmetry in this case. Methodology Erythrocytes from peripheral blood of the 3-year-old male patient were isolated using Percoll density gradient centrifugation and subjected to hypotonic lysis to obtain erythrocyte ghosts. The ghosts were washed thoroughly to get rid of contaminant hemoglobin and subjected to transmission electron microscopy after staining with 1% phosphotungstic acid. Membrane protein profile was obtained by 2D gel electrophoresis (2DGE). The extent of cell surface sialylation and PS exposure were measured by flow Cytometry using FITC-labeled wheat germ agglutinin (WGA) and annexin V respectively. Results Transmission electron microscopy of the sealed erythrocyte ghosts showed significant changes in the ultrastructure with impression of disrupted membrane skeleton and large pores in the red cell membrane. The 2DGE (pH 3–10) showed the presence of large amount of membrane associated globin chains after staining with coomassie blue. Younger cells showed greater cell surface sialylation (MFI 13.16) compared to the older cells (MFI 12.49). The younger erythrocytes surprisingly showed very high extent of PS exposed cells (5.45%) compared to the older cells (2.41%). The ratio of forward scattering to side scattering (FSc/SSc) varied from 0.68 to 0.99 from aged to younger cells, significantly lower than those compared to normal (1.90±0.18 to 2.16±0.15) indicating microcytic vesiculation of erythrocytes. Conclusion The erythrocyte membrane is significantly altered in this type of hemolytic anemia revealing large pores on the membrane and disrupted membrane skeletal architecture. 2DGE and flow cytometry measurements showed increased levels of membrane associated of globin chains and significant loss of membrane asymmetry indicating faster eryptosis of red blood cells.

PP1/7 Porous red cell ultrastructure and loss of membrane asymmetry in a unique case of hemolytic anemia Debasis Banerjee1 · Sutapa Saha2 · Sumanta Basu2 · Dipankar Bhattacharya2 · Abhijit Chakrabarti2 1 Clinical Haematology Service, Park Clinic, 4 Gorky Terrace, Kolkata 700 017, India e-mail: [email protected] 2 Structural Genomics Section, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064, India e-mail: [email protected]

Background Alterations in erythrocyte membrane cytoskeleton are implicated in various red cell disorders.

PP1/8 A rare case of disseminated intravascular coagulopathy occurring four years after endovascular stent grafting of abdominal aortic aneurysm

Vineetha Binoy · Sharat Damodar Narayana Institute of Cardiac Sciences, Bangalore, India

A 78-year-old gentleman was referred to us with extensive bruising and multiple soft tissue haematomas of recent onset. Past medical history included Coronary Artery Bypass grafting and endovascular stent grafting of an infrarenal aortic aneurysm four years previously. Minimal endoluminal leak

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had been noted post stenting of the aneurysm. On admission, his platelet count was 120,000/mm3, APTT was prolonged at 37.8 sec with normal PT and fibrinogen levels. Liver function test, platelet function studies and bone marrow examination were normal. Patient had persistent thrombocytopenia and bleeding manifestations despite cessation of anti platelet medications. Over the course of a few weeks, platelet counts and serum fibrinogen levels dropped progressively and D-dimer level was found to be markedly elevated. Patient showed marked symptomatic improvement with transfusion of FFP and cryo precipitate. He was screened for occult malignancies with bone scan, PSA and CEA levels and tested negative. CT scan of the abdomen revealed significant increase in the endoluminal leak with no interval increase in the dimension of the aneurysm. There was no evidence of graft migration or angulation. He was given regular FFP and cryoprecipitate transfusions to control the bleeding manifestations. Patient underwent successful restenting of the aneurysm with a custom-made fenestrated device about 12 weeks after the diagnosis following which the endoleak and coagulopathy resolved completely and platelet counts normalized. Unfortunately the patient developed renal failure and sepsis after the procedure and died subsequently of these complications. Localized DIC is known to occur with aortic aneurysms. In our patient, as the restenting abolished the endoleak and the resulting DIC, we suggest that an endoleak can cause acute DIC and must be looked for in a patient presenting with coagulopathy post endovascular stenting of an aneurysm. PP1/9 An evaluation of cardiac functional status by MUGA Scan in chronic phase chronic myeloid leukemia patients

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Aims To evaluate the cardiac function status in CML patients on IM therapy Material and methods This is one point observational study done in the year 2007 on CML patients who were on IM therapy for a variable period of time. CML was diagnosed by standard criteria along with cytogenetic. All patients underwent MUGA scan (multiple gated acquisition) and serum BNP levels (in patients with abnormal Muga scan )to assess their cardiac functions. A MUGA Scan is non invasive radionuclide scan, done to asses the cardiac function by assessing ejection fraction and LV contractility in such patients. Results A total of 110 patients (72 males) were included and subjected to MUGA scan after obtaining consent. The median age of the male patients was 38 years (ranges from 16–78) and female patients was 40 years (ranges from 23– 60) The median dose of the IM patient group was 400mg/ day and median time duration of IM therapy was 26 months (range 5–42 months). None of the patient had overt heart failure, however on MUGA scan, 3 patients (3.3%) had evidence of cardiac dysfunction. The size of LV was normal in these patients; however, the ejection fraction was varying between 39–48% with poor LV contractility. Out of three patients, two were males with age of 37 and 47 years. The third patient was a 27-years old female. This patient also underwent transthoracic echocardiography and the findings were confirmed. There were no other contributing factors for heart failure in these patients. Conclusion The prevalence of cardiac dysfunction on MUGA scan in chronic phase CML patients on IM therapy was found to be 2.67%. This finding needs confirmation in a prospective trial in larger number of patients with CML on IM therapy. PP1/10 Idiopathic chronic eosinophilic pneumonia with recurrent massive bilateral pleural effusion

Sushil K. Mahi1 · Pankaj Malhotra1 · Anish Bhattacharya2 · Neelam Varma3 · B. R. Mittal4 · Vikas Suri1 · S. Varma1 1 Dept. of Internal medicine, PGIMER, Chandigarh. 2 Dept. of Nuclear Medicine, PGIMER, Chandigarh 3 Dept. of Hematology, PGIMER, Chandigarh 4 Dept. of Nuclear Medicine, PGIMER, Chandigarh e-mail: [email protected] [email protected]

Background Imatinib mesylate (IM, Gleevac), is a selective tyrosine kinase inhibitor and is now the drug of choice for all phases of chronic myeloid leukemia (CML). The usual side effects of this drug are nausea, gastrointestinal disturbances, superficial edema, rash and myelosuppresion. A recent report suggested development of heart failure in experimental animals who were on imatinib.

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Sudhansu Malini Sethy · Sitaram Mahapatra · Pallavi Bhuyan · Smita Mahapatra · Surama Satpathy. Dept. of Pathology, S.C.B. Medical College, Cuttack, Orissa e-mail: [email protected]

Introduction Idiopathic eosinophilic pneumonia (ICEP) is characterised by sub-acute or chronic respiratory and general symptoms > 2 weeks duration, alveolar and / or blood eosinophilia (alveolar eosinophilia >40% at BAL, blood eosinophilia >1500 / cm) and peripheral pulmonary infiltrates. Pleural effusions are however usually rare, unilateral and small. Case summary A 37-year-old man reported with history of recurrent bilateral pleural effusion of 1.5 year

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duration. Patient did not had any history of drug therapy, asthma or filariasis; but had a specific history of repeated thoracocentesis at peripheral hospital. On investigation chest X-ray showed bilateral massive pleural effusion and CT scan revealed bilateral pulmonary interstitial infiltrates. Hematological investigation revealed DLC-neutrophil – 22%, eosinophil – 62%, lymphocyte – 14%. Bone marrow examination showed highly accelerated eosinophilic granulopoiesis. Pleural fluid analysis revealed eosinophilic pleocytosis (80%) and was sterile on culture. Hence, a final diagnosis of ICEP with recurrent bilateral pleural effusion was made. Conclusion ICEP although an extremely rare entity must be kept in mind while dissecting out the differential diagnosis of cases of recurrent bilateral pleural effusion.

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investigations such as bone marrow examination is a useful tool for diagnosis especially in the absence of other advance techniques. PP1/12 Hemolytic – uremic syndrome with myelodysplasia

Kalpalata Tripathy · Sitaram Mahapatra · Sudhansu Malini Sethy · Pallavi Bhuyan · Smita Mahapatra · Surama Satpathy. Dept. of Pathology, S.C.B. Medical College, Cuttack, Orissa, India e-mail: [email protected]

PP1/11 A case of chronic bronchiectasis coexisting with essential thrombocythemia? A chance finding

Pampa. Ch. Toi · Renu G’boy Varghese · Ramji Rai Pondicherry Institute of Medical Sciences, Kalapet, Pondicherry, India e-mail: [email protected]

Background We report a case of essential thrombocythemia where clinical diagnosis was that of bronchiectasis with no known hematological pathology. Case Description A 60-year-old male referred from another hospital for bronchiectasis developed haemoptysis and melaena. There is history of fever off and on. On examination there is mild splenomegaly. Pulmonary angiogram was suggestive of bronchiectasis. Initial bronchoscopy showed blood clots and mucous in left upper lobe and left lower lobe bronchus. Routine complete blood count revealed a platelet count of 13 lakhs/cu mm which progress gradually to 22 lakhs/ cu mm. Peripheral smear examination showed large platelet clumps and bone marrow examination was advised. The case was diagnosed as essential thrombocythemia as all the diagnostic criteria were met with. The patient was treated with hydroxyurea. Gradually after one month the respiratory symptoms subsided and the platelet count decreased. Conclusion Essential thrombocythemia clinically presenting as bronchiectasis is very rare. It is a myeloproliferative disorder where the clinical diagnosis requires ruling out other causes of thrombocytosis. A timely bone marrow examination in correlation with the clinical history helped to clinch the diagnosis, and the patient was treated. It is therefore important to keep in mind that essential thrombocythemia can present in different ways and basic

Introduction HUS is seen rarely in pediatric practice. Secondary myelodysplasia is still rarely encountered in HUS. Case summary A 5-year-old boy reported with extreme anemia of 15 days duration. Investigation revealed Hb: 4.8 g/dl, Sr. electrophoresis: AA Band, OF: WNL and G-6 PD: Normal activity. PBS examination showed normocytic normochromic anemia with polychromatophilia and a large number of fragmented rbc’s. DLC revealed N – 82%, E – 1%, M-1%, L 16%, TLC – 16,000/cmm, TPC – 1.10 lacs/ cmm and reticulocytes – 22%. Bone marrow examination revealed trilineage myelodysplasia Sr. Bilirubin assay showed unconjugated hyperbilirubinemia. Coomb’s test was negative. Patient subsequently developed features of acute renal failure (urea: 186 mg/dl, creatinine 5.2 mg/dl) and died inspite of treatment. Hence, a final diagnosis of HUS with secondary myelodysplasia was made. Conclusion Secondary myelodysplasia can be encountered in cases of HUS. PP1/13 A comparative study of the diagnostic techniques in malaria

A. Jaisri Southern Railway Headquarters Hospital, Perambur, Chennai - 23, India e-mail: [email protected]

Background The malarial parasite is elusive to detection on the peripheral smear most often and repeated smears are required. Hence several tests were developed. Objectives To compare the accuracy of identification of the malarial parasite by the different methods thick blood

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film (TBF), thin peripheral smear (TPS), quantitative buffy coat (QBC) and immunochromatographic method (ICG). Methods 300 patients with fever and chills were randomly selected and examined for the malarial parasite with the leishman stained TBF, TPS and the QBC technique. 250 were positive for Plasmodium and 50 were negative by all three methods which were used as negative control. 20 cases which were positive and 10 which were negative on all the methods were tested by the immunochromatographic method. Results Out of 250 positive cases, all 250(100%) were positive by QBC, 212(84.8%) were positive on TBF and 192(76.8%) were positive on TPS. All 20(100%) positive cases tested positive by ICG method. Out of the 10 negative cases, 2 were positive by ICG method. The sensitivity was (TBF) 95.5%, (TPS)88.5%, (QBC) 100% and specificity (TBF)89.3%, (TPS)100%, (QBC)83.4%. Conclusion Based on the observations it is suggested that the QBC is a sensitive and effective tool for the rapid identification of the malarial parasite but requires the TPS to confirm its morphology. The immunochromatographic method needs further study. PP1/14 Mesentric and splenic vein thrombosis and the hypercoagulable state: PGIMER, Chandigarh experience

J. Ahluwalia · C. Agarwal · R. Das · M. U. S. Sachdeva · N. Varma Department of Hematology, The Post Graduate Institute of Medical Education and Research, Chandigarh, India e-mail: [email protected].

Background Thrombosis of the mesentric vein is a rare but significant cause of mesenteric ischemia and can be fatal in many cases. 10–15% of mesenteric ischemia is due to mesenteric thrombosis. Splenic vein is also another rare site for thrombosis and a cause of variceal bleeding. Common predisposing conditions for these are trauma, thrombophilias, malignancies, inflammatory conditions including abscesses and pancreatitis, spleenectomy, spleenomegaly etc. About 25–50% cases lack specific diagnosis. We present profile of 40 patients of mesenteric and splenic vein thrombosis in Indian scenario. Methodology This retrospective analysis was carried out in the department of Haematology, PGIMER over a period of twelve years (Jan 1996 to date).Mesentric and splenic vein thrombosis accounted for approx 2 % of patients referred for thrombosis workup to the coagulation lab.

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Etiological factors and the coagulation profile of these patients were evaluated. Various parameters included PT, APTT, Factor V leiden mutation(FVL), anticardiolipin antibodies (ACA) and lupus anticoagulant(LA). Patients were tested for above factors after 6 weeks of acute event or after 4 weeks of cessation of oral anticoagulant therapy. Results Out of 2060 patients referred for workup of prothrombotic states, 40 had mesenteric/splenic vein thrombosis. The median age of the patients was 42.5 years (range 11–75 years )with a 1.5:1 male to female ratio. Out of the various parameters tested Factor V Leiden mutation (heterozygosity ) was found in two patients. Protein C deficiency and Protein S deficiency was seen in five and fifteen cases respectively. Five cases had borderline Protein C and Protein S values. Nine cases had AT III deficiency. Lupus anticoagulant estimation showed borderline value in one patient. Anticardiolipin antibodies were positive in three patients. Conclusion Mesentric vein thrombosis is a rare type of venous thrombosis. Protein S deficiency and AT III deficiency were the most common abnormalities detected in the coagulation profile followed by Protein C deficiency, Anticardiolipin antibody and Factor V Leiden mutation. Significant proportion of mesenteric and splenic vein thrombosis are due to primary hypercoagulable states and underline the need for proper pro-thrombotic workup and followup for thrombosis at other sites. PP1/15 Bone marrow changes in malarial infection: a study of 18 cases

N. Siddaraju · T. Singh · N. Khurana · Z. N. Singh · S. Nigam Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry and Maulana Azad Medical College, New Delhi, India e-mail: [email protected]

Background Malaria is one of the commonest parasitic infections of the tropical countries. The commonest species responsible for malaria in India are P.vivax and P. falciparum Objectives To study the bone marrow changes associated with malarial infection. Material and methods A retrospective analysis of bone marrow aspiration smears (BMA) was done on 18 patients whose peripheral blood films were positive for malarial parasites. All the available clinical and peripheral blood findings were noted and BMA smears were analyzed. Results There were 14 P. falciparum, 3 P. vivax and 1 mixed malarial infections. A prolonged history of fever,

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pallor and hepatosplenomegaly were the prominent clinical manifestations. Four cases had cerebral malaria. Prominent peripheral blood findings noted were anemia with reticulocytosis, relative lymphocytosis and a left shift of neutrophils, with toxic granules. Bone marrow was normocellular to hypercellular. Erythroid hyperplasia was seen in 11 cases. Ten cases revealed normoblastic reaction, while 8 cases showed megaloblastosis. A varying degree of dyserythropoiesis was noted in 6 cases. Myeloid series showed a left shift with toxic granules in 12 cases. Other changes observed were variable increase in histiocytes, eosinophils, and lymphocytes. One case showed erythrophagocytosis. Malarial parasites and varying amount of hemozoin pigment were noted in 8 cases each. No significant change was observed in megakaryocytes. The marrow changes were found to be more prominent in the falciparum malaria. Conclusion A spectrum of marrow changes occur in malarial infection. In the present study, normoblastic to megaloblastic erythropoiesis with variable dyserythropoiesis was the most significant finding seen in the erythroid series; while, a left shift with toxic granules was a prominent finding in the myeloid series of cells in most cases. Megakaryocytes showed no notable change. A variable amount of malarial pigment was encountered in a significant number of cases.

1. HIV infection: - CD4 > 200/:l 2. Immunological AIDS: - CD4 < 200/:l 3. Clinical AIDS: - patients with any AIDS defining illness. Results Anemia was found in 35.64% of patients in Group A and 45.34% in Group B. The prevalence of Anemia was significantly higher in immunological and clinical AIDS i.e. 42.05% and 70.58% respectively in contrast to only 28.57% in asymptomatic HIV infection (P = 0.005). Normocytic, normochromic, anemia was most common (66.5%). Anemia was observed in 34.62% of patients receiving Zidovudine therapy. Eosinophilia was present in 22.9% of cases. Venous thrombosis and Haemophagocytic syndrome was detected in a single patient each. Leucopenia, thrombocytopenia and pancytopenia were seen in 5.88%, 3.74% and 1.6% patients respectively with a slightly higher prevalence in untreated patients. A strong positive correlation (r = 0.739) was observed between TLC and CD4 counts in Group B. The degree of correlation was lower (r = 0.358) in the group receiving HAART. Conclusion Hematological abnormalities are prevalent through all stages of HIV infection, and some may serve as indicators of clinical progression. TLC is a useful surrogate immunological marker in ART naïve patients, but an inadequate substitute to CD4 count for serial monitoring of response once HAART has been instituted.

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Hematological profile of hiv patients with evaluation of total lymphocyte count (tlc) as a surrogate marker of immunological status

Mycobacterial spindle cell pseudotumour of the lymph node – a histopathological mimic

Se Mathews · D. Drivastava · A. K. Agarwal · R. Yadav Dr. Ram Manohar Lohia Hospital, New Delhi, India e-mail: [email protected]

Background Disorders of the hematological system are common in HIV infection. The manifestations are varied and prevalent throughout the course of the disease. Aims To study the hematological profile of HIV patients and its association with clinicoimmunological stage of the disease. Evaluation of total lymphocyte count (TLC) as a surrogate marker of immunological status. Methodlogy 187 HIV patients were classified into two groups: Group A: included 101 patients receiving HAART and Group B: 86 ART naïve patients. The hematological parameters obtained were complete blood counts, TLC, CD4 count and peripheral smears. Comparisons were made between three clinicoimmunological categories:

Femela Muniraj · Debdatta Basu Department of Pathology, JIPMER, Puducherry, India Background Tuberculous lymphadenitis, in immunosuppressed patients may not have a classical morphology of caseating epithelioid cell granulomas. Mycobacterial spindle cell pseudotumor is a rare tumor-like lesion characterized by the proliferation of spindle cells engorged with mycobacterial microorganisms and is seen in HIV positive patients. Case report We report four patients, who presented with cervical and mesenteric lymphadenopathy. Their status of immunosuppression had not been evaluated at the time, when we received the lymph node biopsies. The sections from the lymph nodes showed fascicles of spindle cells along with foamy macrophages, with very ill-defined epithelioid cell aggregates and a few lymphocytes. Stain for acid fast bacilli showed numerous intracellular bacilli. Apart from this, these bacilli were positive for PAS stain, which indicated a strong possibility of atypical mycobacteriosis. On further investigations, all the patients turned out to be HIV positive.

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Conclusion Patients infected with HIV often have unusual manifestations of common infections and neoplasms. Mycobacterial spindle cell pseudotumor may mimic histologically many spindle cell proliferations in the lymph node, both reactive and neoplastic. Awareness of the varied morphological features and a strong index of suspicion are needed for clinching the diagnosis. PP1/18

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PP1/19 Ring chromosome 8 and trisomy 8 in a patient with acute myeloid leukemia

Bibhas Kar1 · B. Nandhini1 · R. Revathi2 Departments of Medical Genetics and 2 Paediatric Haematology Apollo Hospitals, Chennai - 600 006, India e-mail: [email protected]

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Hereditary spherocytosis in North India: a study of clinical profile and longitudinal growth

N. Ahmed · I. Panigrahi · D. Bansal · A. Bhalla · R. K. Marwaha Advanced Pediatric Centre, PGIMER, Chandigarh, India e-mail: [email protected]

Background Hereditary spherocytosis(HS) is a chronic hemolytic disorder characterized by anemia, intermittent jaundice and splenomegaly. Objectives: The main aims were to study clinical characteristics of children with HS and assess their growth profile. Materials and methods Retrospective analysis of cases of HS followed up in pediatric hematology clinic from 1996 to 2007 was done. Data analysis was done on SPSS software. An increase in Hb of >1g/dl or reduction in transfusions by 50% following splenectomy was considered as significant improvement. Results 25 HS cases (M:F–1.27:1), with median age of 6.5 years were studied. History of neonatal jaundice was observed in 28% of which 1 required exchange transfusion. History of transfusion was found in 64%. Family history was positive in 16%, 2 siblings were affected and 12% of parents had undergone splenectomy. At presentation, pallor was present in 68 %, jaundice in 56 %, failure to thrive in 16 % and clinically palpable spleen in 92 %. On anthropometry, 48 % (13/25) had weight for age < 3rd centile and 40% had height for age < 3rd centile. Only 30 % of those with poor growth had low Hb < 8g/dl. Analysis of growth on follow up was also done.Gall stones were seen in only 2 cases. The mean hemoglobin was 9.2 g/dl. Splenectomy was done in 48% at mean age of 8.8 years. Significant improvement following splenectomy was seen in 92%. Conclusion Most cases of HS in children are sporadic. Gallstones are not a major problem in children with HS. Poor growth was seen in significant proportion of HS patients which requires further assessment and appropriate management.

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We describe a child with Acute Myeloid Leukemia (AML M7) with trisomy 8 and ring chromosome 8. Ring chromosome 8 associated with AML is uncommon and is reported to have a poor outcome. The combination of trisomy 8 and ring chromosome 8 has not been previously reported. This15-month-old girl had presented with a history of fever, weight loss of 1 kg, gum bleeds and pallor. Clinical exam revealed no nodes or organomegaly. Investigations revealed pancytopenia and elevated serum LDH. Bone marrow aspirate confirmed the presence of myeloid blasts positive only for CD 41 and CD 61 on flow cytometry. Chromosomal analysis from the bone marrow showed 46, XX [13]/ 47, XX, +8[2]/ 47, XX, +r (8) [5]. The child was treated as per UK MRC AML protocol (ADE 10+3+5). Bone marrow on day 21 post-induction was in morphological remission. Repeat karyotyping revealed 46,XX suggesting that the patient was in cytogenetic remission. Cytogenetic sub grouping in AML patients provides guidelines for the choice of optimal treatment strategy. There was no HLA matched family donor and hence an unrelated donor search was commenced as she was in the group with unfavourable cytogenetics. She developed acute myelofibrosis soon after the second cycle of chemotherapy with swinging fever and rapidly enlarging spleen. The marrow showed 11% blasts with intense fibrosis. She went through a stormy period during conditioning for unrelated stem cell transplantation. She passed away on day 11 post transplantation of veno-occlusive disease of liver and multi-organ failure. This case illustrates the poor outcome in paediatric AML with trisomy and ring chromosome 8. PP1/20 Prothrombotic profile in 11 children with peripheral gangrene J. Ahluwalia · S. Naseem · R. Das · M. U. S. Sachdeva · S. Singh · N. Varma Departments of Hematology and Paediatrics The Post Graduate Institute of Medical Education and Research, Chandigarh, India e-mail: [email protected]

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Background Thrombotic vascular occlusion which ensues from hypercoagulability with a failure of fibrinolytic system to respond to increased activity is the most common cause of gangrene. Gangrene of distal portions of extremities is called peripheral gangrene. Common causes include septicaemia, autoimmune conditions,vasculitides, measles, chickenpox, malignancy, ergotism, protein C, S or antithrombin deficiency. Peripheral gangrene is rare in children. We are reporting 11 children with peripheral gangrene with normal coagulation profile and in which no obvious primary aetiology was noted. Aim of study To study the role of inherited and acquired prothrombotic factors in children with peripheral gangrene. Methodology This retrospective analysis was carried out of in the Department of Haematology, PGIMER with patients referred from the Advanced Paediatric Centre, PGIMER over a period of 4.5 years (January 2004 to date). We evaluated coagulation profile of 11 children with peripheral gangrene. The parameters included PT, APTT, Fibrinogen, Factor V leiden mutation (FVL), Protein C levels(PC), Protein S levels(PS), Antithrombin levels(AT), Anticardiolipin antibodies(ACA) and lupus anticoagulant(LA).All patients were tested for the above factors after 6 weeks of the acute event/or after 4 weeks of cessation oral anticoagulant therapy. PT, APTT, Fibrinogen, PC, PS, AT were tested on automated coagulation analyser STA Compact (Stago Diagnostica), ACA with a commercial ELISA kit (Organtec GmBH), LA by Screen and Confirm Dade Behring kits and FVL by method described by Bertina et al. Results Out of 935 patients referred for workup for prothrombotic states 267(28.6%) were children.Of these 13(1.4% of total and 4.9% of paediatric cases) had peripheral gangrene. Two patients were excluded due to incomplete workup.The median age of remaining 11 patients was 5 years (range 1 month to 17 years) with a 1.8:1 male: female ratio. Two patients had bilateral involvement of limbs and in remaining nine it was unilateral. All the above parameters tested for were found to be in normal limits in all of them. Conclusion Peripheral gangrene in children is a rare condition. Inherited and acquired prothrombotic factors namely PC, PS, AT, ACA, LA and FVL do not contribute significantly to gangrene. Our results would indicate that, in a resource limited setting, testing for these parameters may not be worthwhile. Others factors including workup for fibrinolytic parameters and platelet activation might yield further clues to the causation of this life threatening condition.

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Hematology Department, All India Institute of Medical Sciences, New Delhi - 110 029, India e-mail: [email protected]

Introduction Amegakaryocytic thrombocytopenia (AMT) usually present at birth or within 2 months of age. Cumulative incidence of AML was 55% by age 17 years and progression to Juvenile Myelomonocytic Leukemia (JMML) is very rare. Appx. 10–20% patient of JMML evolve to acute leukemia. Here we report an interesting case of AMT. Case A 3-yr-2-month male child admitted in outside hospital with granting respiration 18 hrs after birth. O/E -petechial rash over the trunk and extremity. Other examinations were normal. On investigation: Hb-12 g/dl, TLC-8200/μL, N48 L45 M5 B2, platelets- 35,000 /μL. BMA showed no megakaryocyte with normal other components. The diagnosis of AMT was made and treated with IVIG, steroid with minimal improvement. Patient was attended in OPD on 4 months of age with bleeding from nose with torticollis of neck with left gaze. O/E- Liver 2 cm, Spleen just palpable. On investigation: Hb-7.9 g/dl, TLC18,400/μL, N01 L41 M42 E3 B2 Myelo08, Meta03, Platelets29,000 /μL. PBS- leucocytosis with myelomonocytic 50%, basophils 3% with NRBC. Relevant maternal investigations were normal. CT Scan of Brain- small occipital encephalocele with vermain hemorrhage with hydrocephalus. Echocardiography - dextrocardia. USG abdomen- situs inversus with mild hepatosplenomegaly. BMA was suggestive of JMML and patient lost to follow up and took desi medicine. Patient again presented in OPD at 3 years of age with on-off spontaneous bleeding from gum, nose and skin. O/E – moderate anemia with 4 cm spleen, 6 cm liver. On investigation: Hb-7.9 g/dl, TLC – 17,600/ul, N09, L66, M20, E03, Blast01, Meta01, NRBC – 03/100 WBC. Platelet count-6,000/ul. BMA and biopsy suggestive of AML-M6a. Conclusion Child was presented with AMT and within 4 months he developed JMML and finally within 3 years of age he was progress to AML-M6a. To our knowledge and literature it was very uncommon type of progression in a short period of time. PP1/22 Profile and outcome of Hodgkin’s disease managed in the institute of child health, Chennai

PP1/21 Interesting case of amegakaryocytic thrombocytopenia

Tuphan Kanti Dolai · R. Bhargava · M. Mahapatra · P. Mishra · T. Seth · H. P. Pati · S. Tyagi · R. Saxena

Hemchand Krishna Prasad · V. Thilagavathy · Venkatadesikulu Institute of Child Health, Egmore, Chennai, India e-mail: [email protected]

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Background Hodgkin’s disease is a lymphoreticular reticular malignancy where there is replacement of nodal architecture by inflammatory exudates containing Reed Sternberg Cell or their variants. At the Hematology OPD of this institute, where both malignant and benign hematological problems are dealt with, Hodgkin’s disease forms an important component of the malignant disorders presented. Aim To study the complete clinical profile and outcome of Hodgkin’s disease treated at our centre Methodology Retrospective descriptive study was done by reviewing the case records of the children who were treated and being followed up for Hodgkin’s disease. Parameters like Clinical features, investigations, treatment and follow-up details were recorded. Results 112 children were registered between Jan 2001 and Dec 2007. Out of them 11, 59 and 42 children fell in the < 5, 5–10 and 10–12 year age groups respectively. The M: F Ratio was 2.7. 23 of these children were started on ATT prior to referral. Common Clinical presentations were fever, neck swelling, breathlessness, Hepatosplenomegaly and cervical lymphadenopathy. Chest X-Ray revealed mediastinal widening in 18 and Ultrasound Para aortic nodes in 12 of these 112 children. 3 children were HBSAg positive and 3 showed cardiac lesions like Mitral Regurgitation and Mitral Valve Prolapse on Echo screening. Children with Nodular Sclerosis, Mixed Cellularity, Lymphocyte predominant and lymphocyte depleted were 4.46%, 62.5%, 29.46% and 3.57 % respectively. Children with Stage I, II, III and IV were 8%, 34.8%. 36.6% and 20.5% respectively. Of the 112 Immunohistochemistry was possible in 12 and 102 had B symptoms. 70 children were managed with ABVD, remission attained in 3 cycles in 6, 6 cycles in 63, 11 relapsed and 1 was refractory to management. 37 children were managed with COPP, remission attained in 3 cycles in 5, 6 cycles in 24, 8 cycles in 6, 5 relapsed and 2 were refractory to management. On follow-up we have 8, 6, 13, 12, 11 children from 2002, 2003, 2004, 2005 and 2007 respectively. 16 children were managed for relapse and 3 children were refractory to treatment. Complications noted on follow-up were Jaundice, Tuberculosis, Nephrotic syndrome, CSOM, Chicken pox and meningitis. Conclusion Hodgkin’s disease has a male preponderance, late childhood presentation, advanced stage presentation and mixed cellularity the most common sub-type. As 15.7% managed with ABVD regimen and 13.5 % managed with COPP regimen had relapse, our centre has similar experience with both the regimens. These children need to be followed up for progression of the disease and development of complications relating to treatment. PP1/23 Iron deficiency and behavioral disorders in children Usha Rusia · Garima Mahajan · Meear Sikka · M. S. Bhatia Dept. of Pathology, University, College of Medical Sciences and GTB Hospital,

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Dilshad Garden, Shahdara, Delhi - 110 095, India e-mail: [email protected]

Background The brain has a higher concentration of iron than any other metal. It is an important nutrient and an essential element involved in myelin formation and neurotransmitter synthesis. Deficiency of iron is reported to cause several behavioural disturbances in children. Objective To study the iron status in children with behavioural disorders and document any improvement following iron therapy. Materials and methods A prospective study on 44 children in the age group of 3–12 years with behavioural disorders were enrolled for the study alongwith 30 age matched healthy controls. Complete Hemogram and iron profile and scoring on child behavior check list (CBCL) was done on all cases and those found to be iron deficient were treated and reevaluated for Haematological and behavioural improvement. Result Iron deficiency was found in 73% of children. There was a significant improvement with iron supplementation (P<0.001)both in the Hematological parameters and in behavioural disorders specially in patients with pica (P<0.001). Conclusion Iron Deficiency results in behavioural disorders which show clinical improvement after therapy. PP1/24 Acute myeloid leukemia developing over a case of monosomy 7 syndrome - case report S. Tripathy · Samir Behera · K. L. Dei · A. Chaudhary · G. Mishra Department of Pathology, M.K.C.G Medical College, Berhampur, Orissa, India Background Juvenile myelomoncytic leukemia encompasses chronic myeloid leukemia and monosomy-7 syndrome. They show many common features including aggressive clinical course and excessive myelomoncytic proliferation in the bone marrow. But monosomy-7 differs from juvenile chronic myeloid leukemia by normal or slightly increased fetal hemoglobin. Case study A 10-month-old child presented with anemia and receiving blood transfusion since 5 months. On examination he has mild hepato-splenomegally, bleeding gums and perpuric spots. Peripheral smear shows leukocytosis and features of chronic myeloid leukemia with decreased platelets. Fetal hemoglobin was normal. Bone marrow shows features of acute myeloid leukemia. Conclusion A Diagnosis of acute myeloid leukemia developing in monosomy-7 was made on the basis of

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1- Refractory anemia, 2-Chronic myeloid leukemia blood picture on peripheral smear, 3- Normal fetal hemoglobin, 4Acute myeloid leukemia in bone marrow. Patient survived for one week after diagnosis. PP1/25 Juvenile myelomonocytic leukemia – a case report P. Jain · A. Gupta · R. Saxena · D. Nath · R. Gautam Department of Pathology, MLB Medical College, Jhansi, India Juvenile Myelomonocytic Leukemia represents about 1.5 percent of childhood leukemias. The rarity of disease has prompted us to report this interesting case. 3-year-old male was brought with the complaints of poor weight gain, weakness, fever and skin rashes for last three months. On examination, he was found to have severe pallor, spleen palpable 5 cm below costal margin and liver palpable 3 cm below costal margin. Laboratory evaluation showed Hb 3.6 g/dl, PCV 34.5, MCV 101.8 fl, MCH 32 pg, MCHC 30.5 g/dl and peripheral blood smear revealed normocytic hypochromic erythrocytes along with occasional late normoblasts. Total Leucocyte count was raised (48000/μL) and Differential count showed Polymorphs 36%, Lymphocyte 6%, Monocyte 11%, Eosinophil 13%, Basophil 3%, Immature myeloid cells 31%. Platelet count was reduced (75,000/μL), morphology was normal. No blood parasite was seen. Bone marrow examination revealed hypercellular marrow with M:E ratio of 15:1. There was predominance of maturing Myeloid cells along wih Monocytoid cells. Myeloblasts were < 2%. Eosinophils and Basophils along with their precursors were increased. Megakaryocytes were reduced in number. Other tests: Fetal haemoglobin was increased to 6.5% Leucocyte Alkaline Phosphatase score was reduced. Features were consistent with the diagnosis of Juvenile Myelomonocytic Leukemia. After knowing the prognosis of the disease patient’s attendant opted for conservative management. Patient was given 2U of blood transfusion and received paracetamol with cephelexin as supportive treatment. PP1/26 Spontaneous hematomyelia in a child with hemophilia A: a case report

R. Aulakh · I. Panigrahi · K. Naranje · S. Sharda · R. K. Marwaha

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Background Haemorrhagic complications of the nervous system in hemophiliacs are uncommon. Spinal column and the spinal cord are rarely the sites of bleed. Case report A known case of hemophilia A, sevenyear old, presented with sudden onset of back pain radiating to both lower limbs for 4 days with no preceding trauma. He had flaccid paraparesis with hypoactive deep tendon reflexes and impairment of all sensory modalities below C5 dermatome. MRI revealed intramedullary expansion and altered signal intensity involving the spinal cord (iso to hyperintense on T1 W and heterogeneously hyperintense on T2 W) from D5 to lower border of D12 vertebra suggestive of hematomyelia. He received cryoprecipitate during the first 7 days to maintain a level of around 100%. There was gradual improvement on conservative treatment. Conclusion Treatment in hemophilia should be based on prompt and prolonged replacement therapy to ensure haemostatic levels of the deficient coagulation factors as the occurrence and development of neurological dysfunction are related to the length of time between onset of symptoms and factor replacement. PP1/27 Bilirubin crystals in peripheral blood neutrophils in neonatal hyperbilirubinemia with a fatal outcome

P. Kalaivani · Jeevana Nilkund · Debdatta Basu Jawaharlal Institute of Postgraduate Medical Education and Research, India e-mail: [email protected]

Background Sepsis is an important cause of unconjugated hyperbilirubinemia in neonates. There are very few reports of bilirubin crystals, seen in the peripheral blood neutrophils in cases of neonatal hyperbilirubinemia. Case report Two infants presented with severe jaundice and features of septicemia. Peripheral blood smear examination made from EDTA blood in both cases showed many golden colored crystals of bilirubin in the cytoplasm of neutrophils. The outcome in both the infants was fatal. Conclusion Bilirubin crystals are seen in neutrophils in severe neonatal unconjugated hyperbilirubinemia and are associated with a poor prognosis. A careful examination of the blood smear is necessary to detect this uncommon finding.

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Induction chemotherapy given in two patients of CML with blast crisis

Essential thrombocythemia and interstitial deletion of 3p

N. Gupta · P. Madhumita · S. Matthew · N. Sinha · A. V. Kulkarni Department of Medicine Maulana Azad Medical College and Lok Nayak Hospital, New Delhi, India e-mail: [email protected]

Bibhas Kar1 · K. G. Dhanalakshmi1 · R. Varadarajan2 1 Department of Medical Genetics 2 Department Haematology, Apollo Hospitals, Chennai - 600 006, India e-mail: [email protected]

Background Chronic myeloid leukemia (CML) in blastic phase is the transition of CML to an acute leukemia, characterized by ≥30% blasts in the bone marrow or peripheral blood, or the development of extramedullary disease outside of the spleen. Although the definition of CML in blastic phase has not changed from the pre- to postimatinib era, an increasing proportion of new patients with CML with blast crisis will have been managed exclusively with imatinib. Objectives Achievement of remission in patients of CML with blast crisis using induction Chemotherapy regimen of Daunorubicin and Cytarabine Materials and methods The first patient aged 40 years was diagnosed as CML in Oct 2007 and started on Hydroxyurea. She did not follow up for 5 months but continued to take the medication and presented to the hospital again with generalized lymphadenopathy and massive hepatospenomegaly. Repeat bone marrow aspirate and biopsy showed 75 % blasts. Patient had never received imatinib. FNAC and biopsy from the lymph nodes confirmed infiltration with leukemic cells with myeloid differentiation. The second patient also aged 40 was diagnosed as CML in Nov 2007 and also started receiving imatinib at the same time, within 3 months however the patient developed pancytopenia and persistent hepatosplenomegaly and imatinib was discontinued. Repeat bone marrow examination showed 65 % blasts. Both patients were given induction chemotherapy 100 mg/m2 BSA of cytarabine for 7 days and 50 mg /m2 of daunorubicin for 3 days. The patients became aplastic within 10 days of chemotherapy. The patients were then assessed for hematological and clinical remission. Results Historically, therapy for patients with CML in blastic phase has been disappointing because response rates have been far lower than those achieved with standard induction regimens for de novo acute leukemia AML or ALL.Both patients achieved remission by day 8 and day 10 respectively of chemotherapy. Follow up results awaited. Thus, the establishment of treatment algorithms for patients with CML blast crisis is essential.

Essential thrombocythemia is a clonal myeloproliferative disorder that involves primarily the megakaryocytic lineage. Clonal karyotypic aberrations are relatively uncommon in essential thrombocythemia, being reported in less than 80 cases. Abnormal karyotypes are found in only 5–10% of cases. The first case of a novel interstitial deletion of 3p in essential thrombocythemia was reported in 2004. We present a case of essential thromobocythemia with interstitial deletion of 3p and to the best of our knowledge; this is the second case with 3p abnormality. A 49-year-old man with a history of pain in left hypochondrium for one year, had under gone cholecystectomy for chronic calculus cholecystitis. He had a history of excessive bleeding during laproscopy. On examination he has suffused conjuctiva, moderate firm splenomegaly and fever. He was clinically diagnosed as myeloproliferative disorder, ?? CML, ?? Essential thrombocythemia and the necessary investigations were done. Complete blood count (CBC) done has revealed gross thrombocytosis and mild neutrophilic leucocytosis. Bone marrow biopsy histopathological examination has shown hypercellular marrow with predominant megakaryocytic hyperplasia. Normal megakaryocyte morphology and mild megaloblastic change is suggestive of myeloproliferative disorder. Peripheral blood leucocyte alkaline phosphatase (LAP) score done was increased. Chromosomal analysis from bone marrow specimen using GTG banding technique showed 46,XY,del(3)(p14p21) and was negative for Ph chromosome. RT PCR for BCR/ABL fusion was found to be negative. The available clinical and laboratory data taken in conjunction with the interstitial deletion of 3p suggests possible ‘Essential Thrombocythemia’. He was treated with oral Hydroxyurea 500mg once on alternate days after food for 1 month along with Tab Ecospirin 75mg OD and was advised to increase the fluid intake. His splenomegaly regressed significantly and his platelet count decreased to normal range. We did a search for the genes present in this deleted region using pubmed genome search tool. This region contains a lot of genes including PTPRG gene. PTPRG has been considered a potential tumor suppressor gene based on its function, antagonizing activity of protein tyrosine kinases that often function as oncoprotein. Deletion

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or inactivation of PTPRG is involved with renal cell carcinoma, lung carcinoma, colon carcinoma, cutaneous T-Cell lymphoma and melanoma. Lower expression level of PTPRG has been reported for a number of cancerous tissues. Interestingly a group has reported that in studying the features of the PTPRG gene with the aim of providing a method for the diagnosis of myeloproliferative diseases, the applicants unexpectedly discovered that its expression in the peripheral and medullary blood in patients affected by CML and essential thromobocythemia is very low or absent compared to healthy patients. We suggest that diagnostic techniques should be improved in such a way that a patient presented with essential thrombocythemia should be checked for the mutation of PTPRG gene. Further study is required to find out the relationship between the PTPRG gene and essential thromobocythemia MITF gene is present in 3p14.2–p14.1, which is a microphthalmia associated transcription factor. This is also deleted from the proband. It is very important to note that the patient was suffering from conjuctiva. Even though it is not having any association with the cause of essential thrombocythemia, it is an important event. PP1/30 A retrospective non-randomized comparative study of response rate between the VAD (Vincristine, Adriamy cine,dexamethasone) and Thalidomide/dexamethasone regimen in patients with newly diagnosed Multiple Myeloma(MM)

S. L. Cyriac · S. Banik · P. K. Gogoi · A. K. Adhikari · D. J. Borah · J. Bhattyacharya Gauhati Medical College, Guwahati, Assam, India e-mail: [email protected]

Background Multiple Myeloma represents malignant proliferation of plasma cells derived from a single clone. Treatment of MM has evolved over the years. Introduction of Thalidomide and its analogues, newer molecules like Bortezomib and the Farsenyl transferase inhibitors have changed the treatment scenario worldwide. Objectives To compare retrospectively, the response rate of VAD vs. Thal/Dexa regimen in patients with newly diagnosed multiple myeloma. Materials and methods 57 patients, newly diagnosed as MM over 4 yrs were assigned to either VAD (6 cycles, n=24) or Thal/Dexa (4 cycles, n=33) regimen along with supportive care and response rate was analyzed at 4m and 6m with a follow-up for 1 yr. Results Majority were presented between 5th–7th decade with a M:F ratio 3:1.Most common symptoms were bone pain, fever and fatigue with mean duration of 5m.The mean

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Hb-9.14, ESR-117, S.creat-1.51,M-Band value 2.94, Bone Marrow Plasma cell- 38%.Final response rate was 58% for VAD and 60% for Thal/Dexa regimen at the end of 6m. Conclusion Thal/Dexa regimen has similar response rate as compared to the standard VAD regimen in newly diagnosed cases of MM. PP1/31 Chronic myeloid leukaemia in blast crisis – unusual association and presentations P. Madhumita · S. Matthews · N. Sinha · A. V. Kulkarni · A. Vyas · N. Gupta Department of Medicine, Maulana Azad Medical College and Lok Nayak Hospital, New Delhi, India e-mail: [email protected]

Background Chronic myeloid leukemia (CML) in blastic phase is the transition of CML to acute leukemia, characterized by ≥30% blasts in the bone marrow or peripheral blood, or the development of extramedullary disease outside of the spleen. Consistent with the early stem cell nature of CML, blastic transformation may be myeloid, lymphoid, or undifferentiated/mixed. Myeloid blast crisis is about two times more common than lymphoid. Association with other co-morbid diseases may have bearing on nature, behavior and understanding of CML. Objectives To study the unusual association/ presentation of CML in blast crisis in two patients, the first presenting with large generalized lymph node masses infiltrated with myeloid leukemia cells and the second with newly diagnosed rheumatoid arthritis with emphasis on influence of RA on CML and vice versa and correlation with treatment of either conditions and disease progression. Materials and methods The first patient aged 40 years was diagnosed as CML in Oct 2007 on treatment with Hydroxyurea and presented again 5 months later with generalized lymphadenopathy involving bilateral cervical, axillary, and inguinal regions and massive hepatosplenomegaly. Blood counts showed 35% blasts on peripheral blood smear. Patient had never received imatinib. The FNAC and biopsy from the lymph nodes confirmed infiltration with leukemic cells with myeloid differentiation. The second patient also aged 40 was diagnosed as CML in Nov 2007 and started on treatment with imatinib. Within 3 months, she developed pancytopaenia and persistent hepatosplenomegaly. Imatinib was discontinued in April 2008. A repeat bone marrow examination showed 65 % blasts. There was history of symmetrical pain involving the small joints of hand and feet with morning stiffness for previous 2 years, clinically suggestive of rheumatoid arthritis. Rheumatic factor was positive, and radiological changes and joint deformities were consistent

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with rheumatoid arthritis. Patient was started on oral methotrexate and hydroxychloroquine, before she was diagnosed having a blastic crisis. Both the patients were given induction chemotherapy using cytarabine 100 mg/m2 BSA for 7 days and daunorubicin 50 mg /m2 for 3 days. Results Extra medullary presentation of blast crisis is rare. The first patient presented with myeloid blast crisis involving multiple lymph node regions. The second had RA and was given methotrexate and HCQS in addition to imatinib. However no alteration in disease progression was noted despite treatment of either condition. PP1/32 Characteristics of chronic myeloid leukemia in elderly in Indian setting

Parimala Puttaiah · Karuna Rameshkumar · S. Sitalakshmi · A. M. Shanthala · A. Vanamala · B. K. P. Prasanna · Cecil R. Ross · G. S. K. Siddhi St. John’s Medical College Hospital, Bangalore, India

Background The age range of occurrence of chronic myeloid leukemia (CML) in India is 30–50 years, hence the course in elderly has paucity of documentation. Objectives To study the sub population of CML in older patients aged > 60years. Materials and methods Newly diagnosed patients from January 2005 to December 2007 which included 30 patients (9 patients > 60years, median 61 years; range 60 to 70 years and 21 patients < 60 years median 38 years range 20 to 58 years) were studied. The diagnosis of CML was based on characteristic peripheral blood smear and bone marrow examination findings. CML score was calculated at the time of presentation for risk stratification which included age, spleen size, blast count, platelet count, basophil and eosinophil count. All patients were put on Imatinib from 2005 February and are being followed up. Results Male:female ratio was 4:5.All elderly patients had a high score of more than 1480 placing them on a high risk category. Splenomegaly ranged from 6cms to 20cms with a median of 12 cms. However the range was much wider in the other group ranging from 4cms to massive splenomegaly. The blast count did not show much variation between the two groups. Two of the patients in the elderly had high thrombocytosis > 6lakhs / cubic mm which was not observed in the other group. One of them had thrombocytopenia placing the patient on MDS/ MPD category. Only one patient in the younger category had an accelerated phase. All elderly patients are doing well till to date. Conclusion The characteristics and the course of CML may not be different in the elderly and they achieve same response rate and survival in spite of high risk profile. Large

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scale studies are required for ratification of the comment. The present study is being continued. PP1/33 Clinical presentation and outcome of primary mediastinal lymphomas – a single institute experience

D. Biswajit · Rejiv Rajendranath · T. G. Sagar Department of medical Oncology, Cancer Institute W.I.A., Chennai, India e-mail: [email protected]

Background Primary mediastinal Lymphomas are rare tumors and there is a paucity of literature in this subset. Aim The aim of the study was to analyze the clinical profile, histopathology and survival outcome in this group. Methodology 93 patients of primary mediastinal tumours were retrospectively analysed from 1993–2006. Of which 28 patients with Primary mediastinal lymphomas were identified. Actuarial method was used to estimate the overall survival. Results Primary mediastinal tumors formed 20% of all the primary mediastinal tumors. The mean age at presentation was 31 years with male to female ratio of 3.6: 1. SVCO was seen in 12 patients. 4 patients required thoracotomy for a diagnosis. The various histologic subtypes found were Primary mediastinal B cell lymphoma 5(17.8%), Anaplastic large cell lymphoma 1(3.5%), Non Hodgkin lymphoma (NOS) 7(25%), Hodgkin disease +6 (21.4%) and Lymphoblastic lymphoma 9(32%). All patients received chemotherapy and involved field Radiotherapy. The chemotherapy regimens used were CHOP for Non Hodgkin lymphoma, ABVD/ Hybrid for Hodgkin disease and Intensive ALL like protocol for lymphoblastic lymphoma. Complete remission was seen in 70% of the patients.6 patients had a relapse. The5 year overall survival was 51%. Conclusion The outcome of primary mediastinal lymphoma is poorer as compared to other aggressive Non Hodgkin lymphomas. Alternative aggressive chemotherapy regimens may be incorporated to improve the outcome. PP1/34 Acute leukemia masquerading as a painful gluteal mass Renu G’Boy Varghese · Sajini Elizabeth Jacob · P. Partho · Barman · Ramji Rai Pondicherry Institute of Medical Sciences, Pondicherry, India

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Background A 53-year-old male presented with complaints of swelling in the left gluteal region for 10days. The swelling was associated with pain and was insidious in onset, increasing in size gradually, to involve the entire gluteal region. General examination of the patient was within normal limits. The left gluteal region was diffusely swollen with tenderness. The induration extended to involve the pararectal tissues. His total count was initially only 17300/cmm, which increased during his stay in the hospital to 61,000. Blasts were seen in the peripheral smear. Bone marrow aspiration and a biopsy of the gluteal mass were done simultaneously. Both showed features of Acute Myeloid Leukemia. Conclusion It is imperative that routine haematological investigations should be done in for every case. A case like this will be missed if these investigations are not done. PP1/35 Tolerance and toxicity profile of high dose methotrexate regime in children with acute lymphoblastic leukemia: cancer institute (wia) experience Arun R.Warrier · D. Biswajit · T. G. Sagar Dept of Medical Oncology, Cancer Institute, Chennai, India e-mail: [email protected] Background High dose methotrexate(HDMtx) is an essential component of various. ALL protocols including BFM 86 Marked variability exists in the tolerabity of HDMtx Data from Indian population is virtually non existent with HDMtx. Understanding the factors influencing HDMtx will help in maintaining dose intensity, better treatment outcomes with minimal toxicities. Aims To analyse the toxicity profile of high dose methotrexate. To ascertain the variables influencing toxicity profile of high dose methotrexate. To compare the data between 3g/m2 and 5 gm/ m2 of high dose methotrexate Patients and methods Acute Lymphoblastic leukemia patients between age 3–25 undergoing BFM 86 protocol treatment at Cancer Institute, Adyar between July 2006 and June 2008 All patients had received high dose methotrexate (HDMtx) as part of consolidation therapy. Methotrexate level estimation was mandatory. Approximately 320 methotrexate infusions were delivered in 80 ALL patients Clinical parameters like mucositis, infections, etc occurring after HDMtx were analysed Hematological parameters, RFT, LFT derangements, Methotrexate values were studiedInfluence of variables like age, sex, concurrent medications were analysed Tolerability of 3 and 5 gm/ m2 are compared Delays in treatment schedule due to HDM and factors involved are studied Statistical analysis done

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Conclusion Hematological toxicities and LFT derangements and mucositis were noted.6 MP dose reduction and delays in between courses led to reduction of dose intensity. Rare neurological toxicities was also seen in a subgroup. With adequate hydration and Methotrexate level monitoring, renal functions remained normal in the majority. PP1/36 Overview of immunological profile of chronic lymhoprolifrative disorders of patients referred to tertiary health care centre from western India

N. Sujata1 · Manisha Madkaiker1 · R. Maya1 · Farah Jijina2 · K. Ghosh1 1 National Institute of Immunohematology(NIIH), 13th floor, New MSB, KEM, Parel, Mumbai, India 2 Dr J. C. Patil Dept of Haematology, Seth GSMC and KEM Hospital, Mumbai - 12, India e-mail: [email protected]

Background Chronic lymhoprolifrative disorders (CLPD) are a heterogeneous group of neoplastic disorders characterized by different clinical behaviour and therapeutic responses. In patients with overlapping clinical features and in whom histopathological data cannot be obtained at the time of diagnosis, immunophenotyping plays a important role in defining specific disease entity. We evaluated the reactivity of immunological markers and their accuracy in distinguishing various CLPDs Objectives To establish a role of immunophenotyping in diagnosing CLPD. Correlation of various marker profiles with the clinical and lab findings. Materials and method 62 cases of clinically diagnosed CLPD, referred to NIIH for immunophenotyping were evaluated retrospectively from Jan-06 to May-08. Immunophenotypic analysis was performed by using a panel of MoAb and three color immunofluorescence staining methods by flow cytometry. Results Immunophenotyping results were evaluated and 60 cases were diagnosed as mature B-cell leukemia and 2 as T-PLL. B-CLL was diagnosed in 52 cases(84%). CD5 and CD23 positivity was the most reliable marker profile which helped in distinguishing B-CLL from other B-cell lymphoproliferative conditions, although CD5- B-CLL was noted in 5 cases (9%). Follicular lymphoma was diagnosed in 5 patients and CD10+ and CD23 (weak/-) was seen in all cases. CD103 positivity was seen in all cases of Hairy cell leukemia, whose diagnosis was further substantiated by positivity of CD11c and FMC-7. Conclusion It is concluded that immunophenotyping plays an important role in diagnosing CLPDs. In addition, various marker expressions can be correlated with clinical

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presentation and thus can help in predicting disease outcome. PP1/37 The Clinical profile of chronic myeloid leukemia A. K. Singh · A. Dey · P. K. Gogoi · B. P. Chakravarty · D. J. Borah · J. Bhattacharrya Gauhati Medical College and Hospital, Guwahati, Assam, India e-mail: [email protected] Background CML is a clonal myeloproliferative disease affecting principally adults, of all age groups. Philadelphia chromosome(Ph) is closely associated with pathogenesis of the disease. Majority of patients present in chronic phase. Diagnosis can be done on a peripheral blood smear examination. Molecular analysis should be done in all cases to look for Ph chromosome positivity. Data from NorthEast India on CML is lacking. Objectives To study the clinical presentation and laboratory features of CML including cytogenetic and molecular analysis. Materials and methods 50 patients, meeting the inclusion criteria were evaluated with history, physical examination and laboratory tests including bone marrow study, cytogenetic(Ph) and molecular(bcr-abl) analyses. Duration of study was 1 year. Results Mean age was 38 yrs. M:F ratio was 2:1. Symptoms-fatigability (34%), weight loss (52%), abdominal fullness (36%) Signs- splenomegaly (98%), pallor(94%), hepatomegaly (78%). TLC>1lac/mm3(54%). Myelocyte (74%), metamyelocyte (70%). Low LAP (100%). 100% were Ph positive. 88% simple and 12% complex translocation. 92% in chronic phase. Conclusion CML is a common hematological malignancy in our region. Clinical presentation is wide. Timely diagnosis is possible with available resources.

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comparison with western study group probably due to high tumor burden, poor supportive care and different biology of the disease. In order to improve the results, major cancer centers use more intensive protocols like MCP841 and BFM 86. Aim To study predictors of outcome in BFM86 protocol. Methods 139 newly diagnosed ALL patients treated with BFM 86 protocol between August 2005 and April 2008 were included in the study. Retrospective analysis of case records were done to study the predictors of outcome. Induction failure, induction death and relapse were taken as poor outcome. Statistical analysis was conducted using chi square test. Results Of 139 patients included in the study, 129 patients achieved remission induction, 5 cases (3.6%) had induction failure and 5 cases (3.6%) died during induction. Relapse was noted in 25 cases (19%). Most common site of relapse was Bone marrow (60%), followed by CNS (32%). Testicular relapse was noted in 1 patient. Most of the relapses (66%) occurred during maintenance, especially in the first 6 months. Among the various factors analysed as predictors for relapse, absence of rapid early Response on day 8 and 15 in peripheral blood and male sex were found to be predictors of poor outcome. Conclusion Lack of rapid early response on day 8, 15 in peripheral blood and female sex were predictors of poor outcome. Identifying these cases, monitoring for minimal residual disease status and maintaining dose intensity may be required to prevent relapses. PP1/39 Acute promyeloctic leukemia – highly curable and a challenge to medical oncologist – tertiary cancer center experience over 10 years C. N. Patil · Rejiv Rajendranath · T. G. Sagar Dept of Medical Oncology Cancer Institute (Wia) Chennai, India e-mail: [email protected]

PP1/38 Predictors of outcome in children with acute lymphoblastic leukemia treated in a regional cancer centre in south India Arun Seshachalam · Biswajit · T. G. Sagar Dept of Medical Oncology, Cancer Institute, Chennai, India e-mail:[email protected] Background In India, treatment results in children with Acute lymphoblastic leukemia have been suboptimal in

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Introduction Depending on geographic variations, APL accounts for 5 to 20 percent of cases of AML. It is the most curable subtype of acute myeloid leukemia. Aim APML though curable is a rare subset. The aim of the study was to analyze epidemiology clinical presentation and outcome in our institute. Methodology A retrospective analyses of the case records of 286 patients diagnosed as AML at Cancer instititute Chennai was done. Of which 17 patients of APML were identified. Actuarial method was used to estimate the over all survival. Results APML subset constituted 6 % of AML. Mean age of presentation was 30 years and had a female

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preponderance M:F (1:1.3). High risk constituted 35% of the patients Induction therapy constituted. ATRA and anthracycline based chemotherapy in majority of the patients. ATRA syndrome (64), infections (35%), bleeding (14 %) were the major complications encountered during induction therapy. Complete remission after induction was achieved in 86 % of the patients. 60 % of patients after protocol continues to be in molecular remission. Conclusion APML a rare subset of acute myeloid leukemia presents in relatively younger patients with unique complications and comparatively high cure rates with standard molecular remission monitoring during follow up. PP1/40 Analysis of transfusion associated adverse events

S. Basu · R. Kaur · G. Kaur · P. Kaur Govt. Medical College and Hospital, Chandigarh, India

Background Unfavorable events occurring during or after transfusion of blood/blood components is not uncommon and constitute adverse transfusion events. In order to ensure safe transfusion practices it is necessary to identify and minimize them. Identification of these events is necessary not only for patient care but also to rectify existing blood bank protocols and practices. Objectives To study the incidence of adverse transfusion events and analyze their spectrum in a tertiary care hospital. Methods Adverse transfusion events occurring in our hospital were analyzed over 4 years (June 2004 to June 2008). Data was retrieved from transfusion reaction work up files and from patient records. Results During the study period a total of 19,853 whole blood units, 17,038 packed red cell units and 33,726 other component units (platelet concentrates, platelet rich plasma and FFP) were transfused to 22,412 patients. A total of 283 adverse transfusion events were reported, all were of the immediate type. The incidence of adverse transfusion events ranged from 0.81% to 0.45% and the commonest was allergic followed by FNHTR. Among the acute hemolytic reactions, majority were due to improper storage and faulty administration. There were 2 instances of immune hemolytic transfusion reactions and 3 cases of transfusion associated cardiac overload. Acute reactions due to contaminated products were identified in 4 patients.. However no delayed reactions were reported. Conclusion The decreasing trend in the incidence of transfusion reactions over the years was probably due to

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increase in the usage of blood components. Blood Banks need to modify existing management protocols so as to avoid all immune hemolytic transfusion reactions and identify delayed hemolytic transfusion reactions which are presently missed. Majority of acute hemolytic transfusion reactions occurred due to improper handling and storage. These can be prevented by creating an awareness of good beside transfusion practices. PP1/41 The use of imatinib in pediatric chronic myeloid leukemia – an Indian experience Sharmila Ghosh1 · Reetu Agarwal2 · S. H. Advani1 PD Hinduja Hospital and Research Centre and 2 Jaslok Hospital Mumbai, India

1

Objectives Chronic myeloid leukemia accounts for less than 5% of all childhood leukemias. We present our experience with imatinib in a small group of pediatric CML patients Materials and methods A total of ten patients with age ranging from 2–20 years were treated with imatinib between January 2005 and October 2007. There were 8 males and 2 females with a median age of 13.6 years. Eight patients had received imatinib upfront and two patients had received hydrea for one month prior to starting therapy. Eight patients were in the chronic phase, one in accelerated and one in the blastic phase. The dose of imatinib used was 200 mg in the age group 2–8 years, 600 mg in the patients with advanced disease and 400mg in all other patients. Results At the end of a median follow up of 21.6 months (2–33 months) we found that all the patients had complete hematologic response. The follow up bone marrow showed morphologic remission (5 patients), transformation to blastic phase (2 patients) and dilute in two patients. Of the seven evaluable marrows at the end of 6 and 12 months, two had complete cytogenetic response, two had major cytogenetic response, two had minor response and one had no response. The common side effects were changes in skin pigmentation (4 patients), retention of fluid (2 patients) and weight gain (2 patients). The accelerated phase patient developed pancytopenia after three months of imatinib requiring withdrawal of drug which was introduced at a lower dose at a later date. Two patients (AP-1, CP-1) progressed to lymphoid blast crisis and died. The other patients are doing well. Conclusion Imatinib is a good therapeutic option in children and has an acceptable toxicity profile. While the hematologic and morphologic bone marrow responses were fairly uniform, the cytogenetic responses were variable.

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PP1/42

PP1/43

HbH disease mimicking clinically KalaAzar

Exflagellated microgametes of Plasmodium Vivax in human peripheral blood: a case report and review of literature

Anita Tahlan · Ujjwal · Sandeep Chauhan · Anshu Palta Department of Pathology and Medicine Government Medical College and Hospital, Sector-32, Chandigarh, India e-mail: [email protected] A 22-year-old male presented with history of generalized weakness since one month, yellowish discoloration of urine for the last two weeks and high-grade fever since eight days. The patient was a laborer, a resident of West Bengal. On general physical examination, he was pale, had mild haemolytic facies. Ultrasonography abdomen revealed an enlarged spleen spanning 18.9cm. A possibility of Kala azar was kept clinically.On admission, his hemoglobin was 5.6 gm%, TLC 8700/ ml, Platelet count 70,000/ml, 26 nucleated RBCs/100WBCs and a corrected TLC of 6904 /ml, MCV 76.8 fl, MCH 21.0pg, MCHC 27.3%. His ESR was 47mm at 1st hr..The peripheral blood film showed presence of microcytes, macrocytes, elliptocytes, teardrop cells, fragmented cells and polychromatophils. Basophilic stippling and Cabot rings were also seen. Neutrophils showed nuclear hypersegmentation. The reticulocyte preparation showed 14% reticulocytes and an occasional golf-ball like inclusion in the RBCs. Bone marrow aspirate was hypercellular and showed erythroid hyperplasia, moderate megaloblastosis and dyserythropoiesis. No L.D bodies were seen. Cellulose agar gel electrophoresis at PH 8.6 showed a fast moving band in the region of Hb H along with a band in HbA region. HbH disease is seen most common in Asian population (SE Asia, Mediterranean and parts of middle east). The instability of HbH is a major cause of anaemia, as precipitates of oxidized HbH form in older red cells, which are then removed by splenic macrophages leading to hemolysis. The most sensitive method for detection of HbH consists of incubation of peripheral blood cells for 1–2 hours at 37°C in presence of supravital dye, which induces precipitation of HbH as inclusion bodies. Determination of a globin genotype may be useful for prognosis of HbH disease, being that non deletional forms are more severe than deletional forms. Patients with HbH disease should undergo splenectomy if excessive anaemia or transfusion requirement develops. Oxidative drugs should be avoided. Iron overload leading to death can occur in more severely affected patients. Thereby necessitating the need for correct and rapid diagnosis. HbH disease shows considerable variability in clinical and hematologic severity. This possibility should be kept in mind in view of the migrating world population even in non-endemic areas.

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P. R. Tembhare · S. A. Shirke · N. D. Deshpande · K. Sehgal · S. C. Shinde · P. G. Subramanian · S. Gujral Dept. of Pathology, Tata Memorial Centre, Mumbai, India Background Malaria causes by Plasmodium vivax constitutes about 60–65% of total malaria cases in India. Peripheral blood smear examination is the most specific as well as the commonest test done for its diagnosis. Exflagellation of microgametocytes in the life cycle of malarial parasites is only seen in the definitive host – mosquito and it is very unusual to see during the developmental phases in the humans. Hence its presence in human peripheral blood may create diagnostic confusion with organisms such as spirochetes and trypanosomes due to similar microscopic appearance as well as clinical presentation. Case Report We report a extremely rare case of a 40-yearold male patient presented with Plasmodium vivax infection with multiple exflagellated microgametes in the peripheral blood smear with review of literature. Conclusion It is important to recognize these exflagellated microgametes in peripheral blood smear to avoid such misdiagnosis. PP1/44 Autologous haematopoietic stem cell transplantation: army hospital (research and referral) experience

Col V. Nair · Col A. Sharma · Surg Cdr S. Das · Col A. Khetarpal · Col A. Sen · Lt Col T. Verma · R. Dabas Dept of Hematology and Bone Marrow Transplantation, Army Hospital (Research and Referral), New Delhi, India

We present retrospective data of 35(09 females) patients who underwent Autologous Haematopoietic Stem Cell Transplant (Auto-HSCT) from Jan 2002 to Jun 2008. All the transplants were carried out in High Efficiency Particulate Air (HEPA) filtered units. The mean age was 37 years (range: 10-60). The indications were acute myeloid leukemia (AML)-15 and Multiple myeloma(MM)-20. Peripheral blood was used as a source of stem cells for all patients.In two patients with AML, in addition Bone Marrow was also harvested because of inadequate harvest of peripheral blood stem cells. In the AML group, conditioning was done with standard busulfancyclophosphamide (Bu-Cy) based protocols for 7 patients

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Idarubicin-Busulfan (Ida-Bu) for 8 patients. Melphalan was used for conditioning in MM patients.The median cell dose was 6.5 × 108 MNC/ Kg (3.1–11.7). The median day of neutrophil engraftment (ANC > 500/ mm3) was 10 days (818) and for platelets (plt>20,000/μL) it was 13 days (8-32). Further analysis of HSCT revealed that only 2/35 (5.7.1%) patients developed mild veno-occlusive disease (VOD). The overall mortality was 6/35 (17.41%). This included only one transplant related mortality (TRM) i.e. 1/35 (2.86%) and 5 due to relapse of underlying disease (AML-3; MM-2).TRM was due to sepsis in an AML patient. Two patients died due to unrelated causes (one due to heat stroke and one due to head injury following RTA). Two patients have been lost to follow up. Overall survival was 25/33 (75.76%).with median follow up of 472 days (6–2378) and disease free survival (DFS) is 20/35 (57.14%). PP1/45

143 Table 1 Chronic GVHD S.No. Site of Chronic GVHD

PBSCT

BMSCT

1

Skin(19)

18

01

2

Oral(12)

11

01

3

Eye(9)

09

-

4

Liver(5)

05

-

5

Lung(3)

03

-

Table 2

Overall Mortality Sepsis

VOD

Acute GVHD

Chronic GVHD

Total

TRM-100 days 06

03

03

-

12

TRM>100 days

01

-

01

02

04

Relapse

-

-

-

-

10

Total

07

03

04

02

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Allogeneic haematopoietic stem cell transplantation: army hospital (research and referral) experience Velu Nair · Ajay Sharma · Satyaranjan Das · Ridhima Dabas · D. K. Mishra · J. Kotwal · S. R. Mehta · O. P. Mathew Army Hospital (Research and Referral), New Delhi, India Haematopoietic stem cell transplantation (HSCT) is being offered as definitive therapy for various haematological disorders. A number of centers are coming up in India, although the facility is available to a very small proportion of eligible population in the country. We present retrospective data of 74 (29 females) patients who have undergone allogeneic HSCT from Jan 2002 to June 2008. All the transplants were carried out in High Efficiency Particulate Air (HEPA) filtered units. The mean age was 20 years (range: 2–54). The indications were chronic myeloid leukemia (CML)-19; acute myeloid leukemia (AML)-15; Thalassaemia major-17; myelodysplastic syndrome(MDS)01; acute lymphoblastic leukemia (ALL)-09; juvenile myelomonocytic leukemia (JMML)-02; pure red cell aplasia (PRCA)-02 and severe aplastic anemia (SAA)09. All recipients received stem cells from HLA matched siblings. Bone marrow (BMSCT) was used as a source of stem cells for 27 patients and peripheral blood (PBSCT) for 46 patients. Cord blood and bone marrow was used for one patient. The conditioning was done with standard busulfancyclophosphamide (Bu-Cy) based protocols for leukemia; fludarabine, cyclophosphamide and anti thymocyte globulin (ATG) for SAA and Bu-Cy-ATG for thalassemia. The median cell dose was 6.0 × 108 MNC/ Kg (2.2–9.7). The median day of neutrophil engraftment (ANC > 500/ mm3) was 10 days (7–15) for PBSCT and 12.5 days (9–22) for BMSCT. Similarly, for platelets it was 12 days (9–27)

for PBSCT and 17.5 days (13-37) for BMSCT. Detailed analysis of allogeneic HSCTs Further analysis of HSCT revealed that 12/74 (16.22%) patients developed veno-occlusive disease (VOD). Of these, 05/74 (6.76%) had severe VOD, 03 of whom died. 05/74 (6.76%) patients developed hemorrhagic cystitis who responded to conservative management. Incidence of grade III/IV acute graft versus host disease (GVHD) was 9/74 (12.10%) out of which 04/74 (5.41%) died. Amongst these 09 patients, 07/ 74 (9.46%) were PBSCT and 2/74 (2.70%) were BMSCT. Of these 09 cases, 02 were Thalassemia major patients both of whom underwent BMSCT while the remaining 07 were haematological malignancies (CML-04; AML-01; ALL-02) who underwent PBSCT. Of the 74 allo-SCT cases, 20 were noted to have chronic GVHD (27.02%). Interestingly, there was a strikingly higher incidence of chronic GVHD i.e. 19/ 46(41.3%) in the PBSCT group compared to 1/28 (3.57%) in the BMSCT group which was statistically significant (p=0.001) (Table 1). The haematological disorders comprising these 20 patients with chronic GVHD were CML-08; AML-04; ALL-03; SAA-04 and MDS-01. Out of the 08 CML patients, only one underwent BMSCT while the rest underwent PBSCT. The overall mortality was 26/74 (35.14%). This included 10 deaths due to relapse of underlying disease and transplant related mortality (TRM)-16/74 (21.62%). In the TRM group, 11 (14.87%) were PBSCT cases while 05 (6.76%) were BMSCT cases. The study revealed a significantly higher incidence of chronic GVHD in the PBSCT group with an overall survival of 48/74 (64.87%) on median follow up of 388 days (30–2267 days). HSCT is an established form of therapy for an expanding range of haematological malignancies and genetic disorders.

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PP1/46 Molecular prognostic factors in acute promyelocytic leukemia

S. G. Mahadik · P. G. Subramanian · S. D. Banavali · S. C. Shinde · S. Gujral Department of Pathology, Tata Memorial Hospital, Parel, Mumbai, India

Background Acute promyelocytic leukemia is distinct subtype of AML, associated with a unique abnormality resulting in fusion transcript like PML-RARA and PLZFRARA. Recognition of these subtypes of leukemia is important because of its potential for life threatening DIC, dramatic response to ATRA. PML-RARA is a good prognostic factor whereas PLZF-RARA is a poor prognostic factor. The Wilm’s tumor gene (WT1) has been shown to regulate the transcription of several growth factors and receptor genes. WT1 encodes for a zinc finger transcription factor. It is located on short arm of chromosome 11 and contain 10 exons. WT1 inhibits apoptosis by interacting directly with p53. FLT3 is the single most commonly mutated gene in AML. FLT3 ligand is a type I transmembrane protein that is expressed on surface of cells. The most common mutations are internal tandem duplication (ITDs). These mutations occur due to DNA replication errors. Detection of other molecular factors such as WT-1 and FLT-3 ITDs by PCR driven assays provides important prognostic information to treating clinicians. Materials and methods Newly diagnosed 21 APML patients were taken for study. RNA and DNA was extracted, cDNA was synthesized and PCR was carried out using specific primers for PML-RARA, PLZF-RARA, WT-1 and FLT-3. Results Among 21 APML patients, 20 (95.2%) showed PML-RARA fusion transcript. A single case which is negative for PML-RARA showed PLZF-RARA fusion transcript. Of these patients, 4 (19%) were positive for WT1and 3 (14.3%) showed FLT3-ITD mutations. Conclusion: This pilot study shows that WT1 and FLT3ITD occur in a significant percent of APMLs.

Indian J. Hematol. Blood Transfus 24(3):86–145

Bhattacharya1 · Col J. Kotwal1 · Ridhima Dabas1 · Atul Kotwal1 · Lt Col Vinod Raghavan1 1 Department of Clinical Haematology and BMT and 2 Department of Pathology and Molecular Medicine, Army Hospital (Research and Referral), Delhi Cantt. India

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the balanced reciprocal translocation (9:22). The resulting fusion gene, the BCRABL, is responsible for oncogenesis. Imatinib mesylate is a novel molecule, which inhibits the protein product of this fusion gene and hence has been used in the treatment of CML. The present study evaluates 100 consecutive patients with CML-CP (chronic myeloid leukemia in chronic phase) treated with imatinib mesylate (Indian generic molecule) during the period from Oct 2006 - Mar 2008. Patients in CML-CP received imatinib mesylate in the dose of 400 mg PO daily. Monitoring of response and relapse carried out by FISH and qualitative PCR at entry and thereafter by quantitative PCR (RQ-PCR) at 6 and 12 months of therapy. Of the 100 patients with chronic phase, 85 patients could be followed up for 12 months and remaining 15 were lost to follow-up. All 100 patients (100 %) achieved complete hematological response. 22 patients (28 %) achieved complete molecular response, 15 patients (18%) achieved major molecular response and 48 patients (54%) were non-responders. Two patients showed molecular response followed by hematological relapse. Non detectable BCRABL: ABL ratio was taken as complete molecular response and ratio < 0.1 % is considered as major molecular response. The molecular response pattern conforms to all the published literature on the subject. Hypo-pigmentation (40%), wt gain(15%), leucopenia (11%), muscle cramps (10%), facial puffiness (10%), skin rashes (9%), fullness of stomach (6%), anemia (5%), raised trans-aminases (5%), pedal edema (3%), mucosal bleeding (2%), raised uric acid levels (2%) and decreased libido (1%) were toxicities encountered during our study. We conclude that the Indian generic of imatinib mesylate is effective and safe first line therapy for CML-CP. PP1/48

PP1/47 Monitoring response to therapy with imatinib mesylate in chronic myeloid leukemia using indian generic molecule of imatinib

Col Velu Nair2 · Col Ajay Sharma1 · Maj Bhikshapathy1 · Col D. K. Mishra1 · Surg Cdr. Satyaranjan Das1 · Brig A. D. Mathur1 · Col S.

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Prevalence, incidence and clinical significance of P-loop mutations (Y253H and E255N) in imatinib mesylate resistance CML patients. (First Indian study)

A. B. Rashid Mir · Sudha Sazawal · Bharti Sharma · Rekha Chobey · Bijender Bohra · Ravi Kumar · Anita Choudry · Shyam Rathi · U. A. Sheikh · Nitin · Pravas Mishra · Renu Saxena All India Institute of Medical sciences, New Delhi, India

Indian J. Hematol. Blood Transfus 23(6–9):86–145

Objectives To study the incidence of p-loop mutations by ASO-PCR in newly diagnosed CML patients. To study the prevalence of P-loop mutations in relapsed CML patients. Methods Two hundred consecutive patients who presented with a diagnosis of CML had blood examined by RT-PCR for BCR-ABL transcripts. They were given imatinib therapy. Early screening for the P-loop mutations (Y253H and E255N) were performed by allele specificOligonucleotide-PCR (ASO-PCR). Results All patients were monitored for hematologic and molecular responses, time to progression, survival and toxicity. Results: Patients were divided into two groups on the basis of their original presentation. Group A included 190 CP-CML patients who started imatinib at 400mg/day. After a median follow-up of 36 months (range 3 to 5 years 60 (31.6%) 30/190 patients had developed molecular relapse and hematologic progression. P-loop mutations were detected about (66%) {20/30 (Y253H-18/30 and E255N 19/30} of these patients. 15/ 30 responded to high dose Imatinib for some time but 17 progressed to advanced stage and 7 died. Group B included 10 patients in advanced phase (AP or BC) at presentation. All were treated with imatinib at 600 to 800 mg/day. After 6 months of treatment 8 (80%) had evidence of P-loop mutation and 2 died. In total 20% to 24% (30+8) 200 of relapsed CML patients were positive for Y253H/E255N mutation; those originally in chronic phase progressed rapidly to advanced phase (14 months ). No Y253H/ E255N mutations were detected in patients in early-CP. There was a significant difference in the survival rate of patients with or without p-loop mutations. Conclusion ASO-PCR is a more sensitive than mutation detection by sequencing. Early detection of P-loop

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mutations proved to be helpful in clinical management of therapeutic decisions. The speed, simplicity, and sensitivity of ASO-PCR assays will be useful for routine monitoring of mutations. The detection of P-loop mutations was associated with imatinib resistance and poor prognosis. ASO-PCR very economical assay for detection of P-loop mutations. PP1/49 Chediak Higashi Syndrome – a case report of a rare disease Rajeev Sen · Ruchi Goyal · Sant Prakash · Sonia Chhabra · Anita Sharma · Kundan Mittal Pt. B.D. Sharma PGIMS, Rohtak, India e-mail: [email protected] A 3-years-old girl presented with fever, cervical and inguinal lymphadenopathy, patches of hypopigmentation in the region of head and neck and hepatosplenomegaly. She had been having recurrent episodes of febrile illness with chest infection since birth. Peripheral blood revealed severe anaemia with thrombocytopenia. Bone marrow showed dysplastic features in trilineage hematopoeitic precursors. Peripheral blood and bone marrow revealed Sudan black positive granules characteristic of Chediak Higashi Syndrome. Negative staining for metachromasia on toluidine blue ruled out metachromatic leukodystrophy.

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