53rd National Conference of Indian Society of Hematology ...

ABSTRACTS

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

� Indian Society of Haematology & Transfusion Medicine 2012

Acute Leukemias

Abstract P 001

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

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

1Drug Development/Diagnostic & Biotechnology Division, Indian

Institute of Chemical Biology (CSIR), 4 Raja S.C. Mullick Road,

Kolkata-700032, West Bengal, India

Objective: The aim of the present study was intended on cancer

because they are the most responsible disease for causing signifi-

cant morbidity, mortality, and incurring healthcare costs worldwide.

Recent data have expanded the concept that many cancers arise

from sites of infection, chronic irritation and inflammation. These

observations imply that anti-inflammatory agents should have a

potential in both the prevention and treatment of cancer. These

drugs can also be used as adjuvant to the currently available

chemotherapy and radiotherapy. Lornoxicam is used for the

treatment of various types of pain, especially resulting from

inflammatory diseases of the joints, osteoarthritis, surgery, sciatica,

and other inflammations but on cancer no work done so far. We

have studied the anti-proliferative, cytotoxic and apoptotic activity

of Lornoxicam on human leukemic cell line and human hepato-

cellular liver carcinoma cell line. Method: We studied cell viability

by Trypan blue exclusion, cytotoxicity study by MTT assay,

morphological study by fluorescence microscopy and DNA frag-

mentation was studied by gel electrophoresis on U937 cell line and

HepG-2 cell line. Result: Lornoxicam significantly inhibited the

cell viability and cytotoxicity (MTT) in a time and concentration

dependent manner. The fluorescence showed characteristic features

of membrane blabbing, chromatin condensation the sign of early

and late apoptotic changes in the cells after treatment with

Lornoxicam. Gel electrophoresis study showed fragmented DNA in

the form of ladder. Conclusion: The present study reveals that the

Lornoxicam possesses potent anti-leukemic activity. Studies are in

progress to identify the mechanism of anti-leukemic activity of

Lornoxicam.

Abstract P 002

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

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



Kolkata-700032, India; 2Department of Chemistry, Indian Institute

of Chemical Biology, Council of Scientific and Industrial Research,

4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India;3Employee State Insurance Hospital Sealdah, 301/3, A.P.C. Road,

Kolkata-700009

Objective: In the present scenario, the demand for herbal products

is growing exponentially throughout the world. This enthusiasm

seems to be a result of people all over the world looking to various

alternative systems of medicine, especially herbal drugs which are

claimed to be safe, equally effective in comparison to allopathic

drugs and which provide some answer to some of the chronic dis-

eases like cancer. Ruellia tuberose leaf has a wide range of

biological activities which includes anti-diabetic, anti-inflammatory,

antipyretic, analgesic etc. Therefore, we have studied the anti-leu-

kemic activity on AML patient’s cell and compared with normal

healthy human leukocytes with methanol and methanol–water

extracts of Ruellia tuberosa leaves. Methods: Collection, identifi-

cation and extraction of Ruellia tuberosa leaves and designated as

RTLE. AML PBMNCs was isolated by Histopaque (Sigma), cell

viability by Trypan blue exclusion and cytotoxicity study by MTT

assay in both the cells. Morphological study was determined by

Fluorescence microscopy and gel electrophoresis of fragmented

DNA was observed. Results: The cell viability and MTT assay

showed the methanol and methanol–water extracts of RTLE sig-

nificantly inhibit the PBMNCs of AML patients in a time and

concentration dependant manner but no toxicity towards normal

lymphocytes after 24 h treatment. The Fluorescence microscopic

images show early and late apoptogenic changes in the leukemic

cells than control, treated with IC50 doses. DNA bands confirmed

apoptosis with both the treatment of RTLEs. Conclusion: The

present study reveals that both the extracts of Ruellia tuberosa leaf

have potent antileukemic activity.

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

DOI 10.1007/s12288-012-0199-y

Abstract P 003

Apoptotogenic Activity of Sulfonoquinovosyldiacylglyceride(SQDG): A Constituent of Azadirachta indica (Leaves) AgainstPBMNCs of AML and CML Patients

Shila Elizabeth Besra1, Moumita Ray1, Subhadeep Roy1,Sayantan Dey1, Aparna Gomes1, Sukdeb Banerjee2, Nirup BikasMondol2, Prithvish Banerjee3, Santanu Bose3



Kolkata-700032, West Bengal, India; 2Chemistry Division, Indian


Kolkata-700032, West Bengal, India; 3Employee State Insurance

Hospital Sealdah, 301/3, A.P.C. Road, Kolkata-700009

Objective: Cancer, a dreadful disease characterized by uncontrolled

growth and spread of abnormal cells without apoptosis, is a leading

cause of death worldwide. Elimination of cancer cells through

apoptosis is the key target of cancer therapy. A sulfonoglycolipid

identified as a sulfonoquinovosyldiacylglyceride (SQDG) isolated

from leaves of Azadirachta indica showed significant anti- leukemic

activity in U937 and K562 human leukemic cell lines. Therefore, we

studied the apoptogenic activity of SQDG against PBMNC of ALL

and AML patients. Methods: In vitro cell proliferation assay were

done by MTT assay, morphological determination of the cells

undergoing apoptosis by Fluorescent dye staining, DNA fragmenta-

tion by gel electrophoresis, quantization of apoptosis by flow

cytometric analysis and PBMNCs isolated by lymphocyte separating

fluid Histopaque (Sigma). Results: After treatment, SQDG signifi-

cantly inhibited the metabolically active cell growth and cytotoxicity

of both the AML and CML patient’s cells. The effect of SQDG on

normal human peripheral blood mononuclear was studied by cell

viability and cytotoxicity and was found to be lower than that on

AML and CML cells, indicating its specificity towards cancer cells.

The morphological study showed the characteristic features of

apoptotic changes in the treated cells than control cells after 24 h. The

induction of apoptosis was confirmed by using Annexin-FITC/PI

staining by flow cytometric analysis and fragmented DNA was found

in the form of ladder after treatment with SQDG. Conclusion: Study

reveals that, sulfonoquinovosyldiacylglyceride (SQDG) may be used

as novel chemotherapeutic agent in future for better treatment of

cancer without systemic toxicity.

Abstract P 004

Treatment Related Complications in Patients with AcuteLymphoblastic Leukemia

Rajesh Kashyap, Mukul Agarwal, Pradeep Kumar, GarimaAgarwal

Department of Hematology, SGPGIMS, Lucknow, UP

Aim: To study the different non-haematological complications

occurring during the late phase (2 weeks of induction therapy to

remission) treatment of ALL patients. Materials and Methods:Patients diagnosed with ALL and being treated at department of

haematology, SGPGIMS were subject of the study. The diagnosis of

ALL was made based on complete hemogram, bone marrow exami-

nation with cytochemistry and immunophenotyping by flow

cytometry. The clinical records of the patients were analysed for

occurrence of non-haematological complications occurring 2 weeks

after the start of therapy. The patients were treated as per the BFM

protocol (BFM 90 of BFM 95 protocol). Result: One hundred and one

patients had 147 events of non-hematological complications. Gas-

trointestinal toxicity was the most frequent complication and occurred

predominantly as hepatitis. Hyperglycemia was seen in 25 cases

(24.7 %) and was most frequent in patients above the age of 20 years.

Neurological complication was predominantly seen in patients below

the age of 20 years (76.2 %) and seizures was the most common

presentation. Osteonecrosis involving the hip joints was seen two

adult young males. Conclusion: Non-hematological complications

are quite frequent during the late phase of treatment of ALL patients.

The Gastrointestinal tract, nervous system and endocrine system most

frequently affected. Many of these events are missed because of low

levels of clinical suspicion. This study highlights the incidence of

these complications as they are associated with high morbidity and

mortality.

Abstract P 005: Poster Presentation

Acute Erythroid Leukaemia: A Clinico-Hematological Reviewof 5 Case Series

Nidhi Rai, M Deepak Nayak1, Chethan Manohar, Sushma VBelurkar

Department of Pathology, Kasturba Medical College, Manipal;1Department of Pathology, Melaka Manipal Medical College,

Manipal

Introduction: Acute erythroid-leukaemia is a rare form of acute

myeloid leukaemia (AML), characterized by abnormal proliferation

of erythroid precursors (proerythroblasts and basophilic erythro-

blasts). It comprises \5 % of AML cases. Objective: To review the

clinico-hematological features of erythroleukaemia cases with review

of literature. Materials and Methods: We report 5 such cases seen in

our institution over a period of 1 year. 4 out of these 5 cases presented

with pallor, easy fatigability and hepatosplenomegaly, while one case

reported with non-classical symptoms. Peripheral smear examination

and complete haemogram revealed pancytopenia with circulating

blasts ([20 %) in all five cases. Bone marrow study yielded [50 %

proerythroblasts and basophilic erythroblasts and dyspoiesis in other

lineages. Results: Final diagnosis of Erythroleukaemia (erythroid/

myeloid) was made in four cases and one case with non-classical

presentation was typed morphologically as Pure Erythroleukaemia.

Conclusion: Erythroleukaemia is an uncommon hematopoietic neo-

plasm. Among these, pure erythroleukaemia is seldom reported in

literature and is known to have poor response to standard chemo-

therapy. In the present case series, clinical presentation did not differ

from other types of acute myeloid leukaemia cases. These five cases

presented in the most unobtrusive manner. Thus awareness of its

hematological and morphological features is necessary to avoid a

diagnostic dilemma since treatment protocols and prognosis are

varied.

Abstract P 006

Clinical, Hematological, Cytogenetic & Molecular Profileof Acute Myeloblastic Leukemia

J Latha Fathima, S Sitalakshmi, Parimala Puttaiah, PoornimaD Rao, A Vanamala, AM Shanthala Devi, Karuna Ramesh Kumar

Department of Clinical Pathology, St. John’s Medical College

Hospital, Bangalore;

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

123

Introduction: Acute myeloid leukemia is a disease resulting from the

clonal expansion of myeloid blasts in the peripheral blood, bone

marrow or in other tissue. It is a clinically, morphologically, geneti-

cally and prognostically heterogeneous disease. In India the incidence

of AML is 2.8 to 3.5 cases per 100,000 population per year. The

course and prognosis of the disease in the western countries and India

differs. Objectives: 1. To study the clinical and hematological fea-

tures of AML. 2. To study the cytogenetic and molecular

abnormalities of AML. 3. To compare the course and prognosis of the

disease with western countries. Methods: This retrospective study

was done from the year 2010 to July 2012 for a period of 30 months

in the department of Clinical Pathology, St.John’s Medical College

Hospital, Bangalore. The clinical details were retrieved from the

patient records in the medical records department. The laboratory

parameters were obtained from the department of Clinical Pathology.

Result: The total number of cases were 131. The age ranged from 2 to

84 years. The peak incidence was in fourth decade. The most com-

mon presentation was fever. Morphologically AML-M2 was the

common subtype followed by acute promyelocytic leukemia.

Immunophenotypically, CD 13 was the most commonly expressed

(62 %) myeloid marker. CD 7 was aberrantly expressed in 28 %.

PML-RARA translocation t(15:17) was the common cytogenetic

abnormality. One case of therapy related AML was also encountered.

Conclusion: The common subtype is AML M2 followed by APML.

The commonest cytogenetic abnormality noted is PML-RARA

translocation.

Keywords Acute myeloid leukemia, Acute promyelocytic leukemia,

Cytogenetics

Abstract P 007

Extensive Extramedullary Involvement in a Child with AcuteMyeloid Leukemia

Anand Prakash1, Athira Ramakrishnan2, Balasubramaniam2,Suravi Mohanty3, Anuradha Ananthamurthy3

1St Johns Medical College Hospital, Department of Pediatrics,

Bangalore, India; 2St Johns Medical College Hospital, Department

of Otorhinolaryngology, Bangalore, India; 3St Johns Medical College

Hospital, Department of Pathology, Bangalore, India

Purpose: Extramedullary involvement can occasionally be the pre-

senting feature of Acute Myeloid Leukemia in children. We report a child

who presented with features of extensive paranasal, orbital, lymph node

and dural involvement of chloromas with facial nerve paralysis. Method:Case Report: A 12 year old girl presented with fever, loss of hearing,

proptosis and headache of 2 months duration. Clinical examination

revealed mild left orbital proptosis with painless cervical lymphade-

nopathy. She had no hepatosplenomegaly. Cranial imaging showed dural

enhancement in the left frontoparietal region with extensive maxillary,

ethmoid and sphenoid sinus obliteration. There were minimal deposits in

both retro-orbital areas. Peripheral blood film and bone marrow aspirate

revealed myeloblasts of 10 %. A biopsy of the paranasal mass showed

features of a granulocytic sarcoma. Immunohistochemistry was strongly

positive for Myeloperoxidase and CD117 and was negative for CD20 and

CD3. The Ki 67 index was about 50 %. CSF was negative for malignant

cells. Conventional Cytogenetics was attempted but unsuccessful.

Results: The patient has completed chemotherapy (induction with cyt-

arabine and daunorubicin and consolidation with three cycles of high

dose cytarabine). She had a complete resolution of her proptosis, facial

palsy and the paranasal sinus mass. She is currently well and in remission.

Conclusion: Extensive extramedullary manifestations of AML can

present with paranasal sinus extension and cranial nerve palsy.


CD133 and MLL in the Same Cup

G Smeeta1, Anita Chopra1, C Jagan1, Sameer Bakhshi2,Sunu Lazar Cyriac2, Rajive Kumar1

1Laboratory Oncology Unit, 2Department of Medical Oncology,

Dr. B.R.A Institute Rotary Cancer Hospital, All India Institute

of Medical Sciences, Ansari Nagar, New Delhi

Introduction: CD133 positivity has been described in, but is rarely

ever emphasized as a facet of mixed lineage leukemia (MLL)+ pro-B

acute lymphoblastic leukemia (ALL). Nuclear cupping is a feature

that hardly ever finds mention outside acute myeloid leukemia

(AML). We present two cases where these coexisted. Case Sum-mary: The first patient, a 5-year old girl, diagnosed B-lineage ALL

4 years back and treated on UK-MRC-ALL-2003 protocol till 2010,

and symptom-free for 2 years, presented to our institution in June

2012 with fever and lymphadenopathy. Peripheral blood smear

showed 80 % blasts; with over 80 % having prominent cup-like

nuclear invagination. The second patient was a 10-month old male,

presented with fever and splenomegaly. Peripheral blood smear

showed 90 % blasts, 20 % exhibiting cup-like morphology. In both

patients, blasts were positive for CD34, CD45, CD19, HLA DR,

CD133, CD38, CD15, cCD79a, CD117dim and negative for CD10,

cCD22, CD20, CD13, CD33, CD56, CD2, CD4, CD64, CD14, cCD3

and cMPO. NG2 was positive in 10 and 100 % blasts in first and

second case, respectively. Both the patients were diagnosed as pro-B

ALL. MLL gene rearrangement was identified in both by fluorescent

in situ hybridization using a dual color, break apart rearrangement

probe. Conclusion: We believe ours is the first case showing florid

nuclear cupping in association with CD133 positivity and MLLrearrangement in a setting of pro-B phenotype. We conclude that

nuclear cupping, even when present in nearly every blast cell, may not

necessarily mean AML and may be a pointer toward CD133 positive

MLL translocated pro-B ALL. CD133 should be evaluated as part of a

B-lineage ALL flow cytometric panel with a view to define its role as

a reliable indicator of MLL rearrangement.

Keywords Cupping, CD133, MLLL

Abstract P 009: Oral Presentation

Acute Promyelocytic Leukemia—from Morphology to MolecularDiagnosis

Tathagata Chatterjee1, Srishti Gupta2, Ajay Sharma2

1Armed Forces Medical College, Pune-40; 2Army Hospital (R&R),

Delhi Cantt-10

Introduction: Acute promyelocytic leukemia (APL) is a distinct

subtype of acute myeloid leukemia (AML) with typical clinico-

hematological features. Cytogenetically, it is predominantly charac-

terized by balanced reciprocal translocation between chromosome 15

and 17 which results in fusion between promyelocytic leukemia

(PML) gene and Retinoic acid receptor a (RARa) gene. There are 3

possible isoforms caused by these translocations. The breakpoint in

chromosome 17 is consistently found in intron 2, but varies in

chromosome 15. The 3 breakpoints on the PML gene can occur at

intron 3 (L-long form), intron 6 (S-short form), and exon 6 (V form).

The study on molecular characterization was undertaken due to lack

of sufficient data in Indian patients. Aims and Objectives: (1) To

study the clinic-hematological and morphological profile in APL

patients. (2) To study molecular characterization of BCR subtypes in

Indian APL patients. Materials and Methods: A prospective study of

Indian J Hematol Blood Transfus (Oct-Dec 2012) 28(4):191–256 193

123

fifteen APL patients presenting to Army Hospital (R&R) were taken

for study between November 2010 to March 2012. The clinical fea-

tures, hematological parameters and morphology were analyzed.

Peripheral blood and bone marrow aspirate were stained with

Leishman-Giemsa, myeloperoxidase (MPO) and chloroacetate ester-

ase (CAE) and non specific esterase (NSE) using Merck’s Diagnostic

reagents. Flowcytometric evaluation was done on bone marrow

aspirate/peripheral blood using Beckman Coulter FC 500, 4 color

flow cytometer using standard lyse wash technique. For molecular

studies, peripheral blood sample was collected and Real Time PCR

was performed using Fusion Quant� kits for bcr-1, bcr-2 and bcr-3

and Rotor GeneTM 3000 software. Results: Patients presented with

fever, loss of appetite, bleeding manifestations (petechial, conjunctiva

hemorrhage, gum bleed, vaginal bleed and bleeding per rectum) and

pallor. Median age was 42 years, TLC—4,500/mm3, Hb—7.5 g/dl

and platelet—35,000/mm3. Three patients were micro granular vari-

ants and rest were hyper granular variants. All cases stained for MPO

and CAE and 33.33 % for NSE. Flowcytometric analysis revealed

classic high SSC with dim CD45 scatter with hyper granular variants

and low SSC with dim CD45 with micro granular variants. Molecular

analysis revealed BCR1 in 6/15 (40 %), BCR2 in 3/15 (20 %) and

BCR 3 in 6/15 patients (40 %). Conclusions: In our study no cor-

relation was found between age, sex, TLC, hemoglobin and platelet

counts with different BCR isoforms. There was no significant increase

in particular isoform in Indian population unlike published data.

Morphological, cytochemical, flow cytometry and molecular diag-

nosis were achieved within 48 h of admission.

Abstract P 010

Trisomy Chromosome 6 as a Sole Cytogenetic Abnormalityin Acute Myeloid Leukemia

Monika Gupta, Nita Radhakrishnan, Manoranjan Mahapatra,Renu Saxena

Department of Haematology, All India Institute of Medical Sciences,

New Delhi, India

Abstract: Identification of cytogenetic abnormalities plays an impor-

tant role in the diagnosis and prognosis of leukemias. Isolated trisomy 6

is a rare abnormality, the prognostic significance of which is not well

established. We report one case of acute myeloid leukemia (AML M5

variant) with trisomy 6 as the sole cytogenetic abnormality. Previously,

trisomy 6 has been reported in aplastic anaemia, myelodysplastic syn-

drome and AML and they are usually associated with hypocellular

marrow. However our patient had a very short history and a hypercel-

lular marrow infiltrated with blasts. We report this case due to the rarity

of the condition. More studies are required to ascertain the role of

trisomy 6 in the development of leukemia as well as in prognosis.

Abstract P 011

Clinical and Immunophenotypic Characterisation of Early T CellPrecursor Acute Lymphoblastic Leukemia: a First Studyfrom India

G Smeeta1, Anita Chopra1, C Jagan1, Lalit Kumar2,Atul Sharma2, Sameer Bakhshi2, Rachna Seth3, Ajay Gogia2,RM Pandey4, Rajive Kumar1

1Laboratory Oncology Unit, 2Department of Medical Oncology,

Dr. B.R.A. Institute Rotary Cancer Hospital, 3Department of

Paediatrics, 4Bioststistics, All India Institute of Medical Sciences,

Ansari Nagar, New Delhi

Introduction: Early T cell precursor acute lymphoblastic leukemia

(ETP-ALL), a newly identified subtype of T-ALL, is characterized by a

strikingly distinct immature immunophenotype: CD5-, CD1a, CD8-

and expression of C1 myeloid or stem cell markers (CD117, CD34,

HLA-DR, CD13, CD33, CD11b, and/or CD65) on C25 % of lympho-

blasts. It has been reported to be associated with poor prognosis. A very

few studies, none from India, have evaluated the clinical and prognostic

significance of ETP-ALL cases. Objective: To determine the incidence

and clinical significance of ETP-ALL. Methods: Fifty five cases of

T-ALL were retrieved from the records of Laboratory Oncology,

AIIMS. The clinical and immunophenotypic characteristics of all cases

were noted. The immunophenotypic markers used were: CD3, CD45,

CD5, CD8, CD1a, CD13, CD33, CD117, HLA-DR, CD34, CD65 and

CD11b. The cases were subclassified based on expression of CD5:

CD5+ and CD5-. These were further subclassified into CD8+/CD1a+

and CD8-/CD1a- subgroups. In all these groups, presence of myeloid

and/or stem cell markers were also noted. The remission rate, relapse

rate and overall survival of these groups were compared. Results: CD5

was positive in 48 and negative in 7 cases. In the CD5+ group, 31 cases

were CD8+/CD1a+ (myeloid/stem cell markers: positive 19 and nega-

tive 12) and 17 were CD8-/CD1a- (myeloid/stem cell markers:

positive 16 and negative 1). All CD5- cases (n = 7) were CD8-/

CD1a- and myeloid/stem cell markers positive. All CD5- cases

(12.7 %) fulfilled the criteria for ETP-ALL. ETP-ALL cases had a poor

remission rate as compared to non- ETP-ALL cases (40 vs. 86.7 %;

p = 0.042). Death rate was also higher in ETP-ALL group (28.6 vs.

6.25 %; p = 0.02). Conclusion: ETP-ALL is a distinct, high risk

subtype of T-ALL, that must be identified by correctly designed im-

munophenotyping panels, so that therapy appropriate to the disease and

distinct from the non-ETP- type T-ALL can be instituted. Recognition

of the disease provides a fresh and meaningful perspective on the

nebulous concept of lineage infidelity of acute leukemias.

Abstract P 012

Biphenotypic Acute Leukemia: A Study of Clinical,Hematological & Immunophenotypic Profile

Man Updesh Singh Sachdeva1, Manupriya1, Neelam Varma1,Subhash Varma2, RK Marwaha3

1Department of Hematology, 2Department of Internal Medicine,3Department of Paediatrics, Postgraduate Institute of Medical

Education & Research, Chandigarh

Background: Acute biphenotypic leukemia (BAL) is a rare neoplasm

comprising of blasts showing more than one lineage on multi-colour

flow cytometry. This prospective study was designed to identify cases

of biphenotypic acute leukemia and study their clinical and hemato-

logical profiles. Methodology: EDTA anticoagulated bone marrow

aspirate/peripheral blood samples of patients diagnosed as acute

leukemia on the basis of morphology were utilized for immunophe-

notyping. A comprehensive panel of fluorochrome labeled monoclonal

antibodies was used to identify the lineage of leukemic cells on flow

cytometry. The patients diagnosed to have BAL, on basis of WHO 2008

classification, were selected for analyses of their clinical, hematological

and immunophenotypic profile. Results: BAL represented 2.99 % (15/

501) of all cases of acute leukemia over 2 years. 47 % (7/15) were

children, all males, mean age of 5 years. 53 % (8/15) were adults,

M:F = 6:2, mean age of 21.4 years. 53 % (8/15) were diagnosed as

B/Myeloid and 47 % (7/15) were T/Myeloid. No correlation was

observed between age and immunophenotype of BAL. Fever and


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lymphadenopathy were more frequently observed in BAL than ALL

and AML, respectively. Thrombocytopenia was less frequent as com-

pared to ALL or AML. On morphology, (73 %) 11/15 were diagnosed

as AML and 27 % (4/15) were diagnosed as ALL. Conclusions: BAL is

a rare type of acute leukemia (2.99 % of all our AL cases). Most of the

clinical, hematological and morphological parameters fail to clearly

predict its occurrence and a comprehensive panel of antibodies should

be used to identify this neoplasm known to have a poor outcome.

Abstract P 013

Isolated CNS Relapse of in a Case of AML-M2 MimickingPosterior Fossa Intracranial Haemorrhage—a Case Reportand Brief Review of Literature

K Kishore, UK Nath, SS Roy, P Chakrabarty, U Chaudhuri

Institute of Hematology and Transfusion medicine, Kolkata

Objective: This report is intended to stress the importance of looking

for subtle clinical signs of CNS RELAPSE during follow-up visits even

when the blood counts are normal in AML patients. Methods: This 17/f

a case of AML-M2, 46XX, NPM1-FLT3-ITD/Asp835 Negative patient

achieved CR after one 7 + 3 and was further treated with 3 cycles of

HIDAC. She was in CR post consolidation. During her follow up visit,

in spite of normal counts, she complained of mild headache and nausea.

Her CT scan of the brain revealed increased attenuation in the midline

posterior fossa with compressed IV ventricle and a provisional diag-

nosis of intracranial haemorrhage was given. In view of normal

coagulation parameters we reviewed the plates again and went in for

MRI and CSF analysis. Results: MRI showed an heterogeneous area in

the posterior fossa in superior vermis region suggestive of a mitotic

mass lesion. CSF also shows blasts confirming an isolated CNS relapse

as bone marrow is also in remission now. Conclusion: The patients who

were treated with only local therapy (intrathecal chemotherapy with or

without radiation therapy) had an overall survival rate of 31.5 %

compared to 21.4 % in patients treated with systemic therapy. The

AML subtype, CNS-1 status at diagnosis, age at relapse, cytogenetic

characteristics and the initial CT picture makes the present case unique.

Abstract P 014

Erythroleukemia (Erythroid/Myeloid): A Case Report

T Santosh, RK Bhola, A Choudhary, AK Bal, MK Patro, J Naik,B behera, S Paradhan

Department of Pathology, MKCG Medical College, Berhampur,

Odisha

Background: Acute erythroid leukemia is a rare form of acute

myeloid leukemia (AML) with predominant erythroid lineage pro-

liferation. It is a heterogeneous entity amongst AML that can occur at

any age, including childhood, and comprises less than 5 % of AML.

It’s defined as ‘‘a proliferation of more than 50 % erythroblast and

[20 % myeloblasts within nonerythroid cells.’’ AML-M6 is a het-

erogeneous disease with poor response to standard chemotherapy that

carries a poor prognosis. The new WHO classification subdivides

acute erythroid leukemia into erythroleukemia (erythroid/myeloid)

and pure erythroid leukemia. Allogeneic bone marrow transplant

should be considered upfront for appropriate candidates once remis-

sion is achieved in AML-M6, as the risk of relapse and mortality is

very high with this disease. Case Report: A 36 year Hindu female

presented with fatigue, generalized body aches, pain since last

6 weeks. The patient’s peripheral blood showed a leukoerythroblastic

blood picture with Hb of 3.4 g/dL, TLC 6,400/mm3, and Platelet

count of 89,000/mm3. Bone marrow aspirate and biopsy revealed a

markedly hyper cellular bone marrow replaced by erythroid precur-

sors, representing approximately 53 % of the marrow cells and

Myeloblasts representing 49 % of non erythroid cells. The erythroid

precursors displayed dysplastic morphology, including megaloblastic

features, multinucleation, nuclear lobation and budding, and they

were periodic acid-Schiff (PAS)–positive. So a diagnosis of acute

erythroid leukemia (erythroid/myeloid) was made.

Keywords Erythroid leukemia, Erythroid/myeloid

Abstract P 015

Study of Clinico-Hematological and Immunophenotypic Profilein Patients with Acute Lymphoblastic Leukemia in SCB MedicalCollege & Hospital, Cuttack, Odisha

Bhattacharyya Debmalya1, Das Sidhartha1, RK Jena2

1Department of Medicine, SCBMCH, 2Department of Clinical

Hematology, SCBMCH

Aims and Objectives: To study clinical, hematological and immuno-

phenotypic profile of patients with Acute Lymphoblastic Leukemia (ALL)

and their association with mortality and remission. Methods: 60 naıve

consecutive cases of adult ALL (C15 years) were taken for study. Sec-

ondary and relapse ALL cases were excluded. Detailed history taking,

clinical examination, hematological parameters, bone marrow study and

Immunophenotyping using bone marrow sample or peripheral blood by

Flow cytometer (BD FACS CALIBUR) were done in all cases. Patients

who survived were treated with MCP-841 protocol and hematological

remission was evaluated after completion of induction phase (1 month).

Results: 68.3 % cases were suffering from B-ALL and 31.7 % from

T-ALL. 58.3 % of cases were young adults (between 15 and 24 years of

age). Bleeding manifestation was significantly associated with B-ALL

(p\0.01) and lymphadenopathy was associated with T-ALL (p\0.01).

FAB-L1 and FAB-L2 morphology in bone marrow was found in 38.3 and

61.7 % of cases respectively. In patients with B-ALL, cCD79a was

positive in 100 % of cases, followed by CD19 (80.49 %), CD10

(85.37 %), CD34 (43.9 %). In patients with T-ALL, cCD3 was positive in

100 % cases, followed by CD7 (94.73 %), CD5 (63.16 %) and CD34

(21.05 %) 0.21.67 % of cases had myeloid co-expression, of which CD13

was the most common (53.84 %), followed by CD33 (38.46 %) and

CD117 (7.69 %) 0.24.4 % of B-ALL and 15.8 % cases of T-ALL had

myeloid co-expressions. 25 % of cases died before completion of induc-

tion phase and 46.7 % cases achieved complete remission (CR) after

4 weeks of induction phase. 21.7 % cases had partial remission and only

6.7 % cases had no remission. 60.97 % of B-ALL cases could achieve CR

whereas only 15.78 % of T-ALL cases could achieve CR (p\0.002).

Conclusion: B-ALL is more prevalent than T-ALL. For Immunophe-

notyping a panel of CD markers is required. Prognosis of B-ALL is better

than T-ALL.

Abstract P 016

Pattern of Occurrence of Childhood Leukemias in a TertiaryCentre in Lucknow: A Eleven Year Study

R Kushwaha, A Kumar, US Singh, K Archana

King George Medical University. Lucknow

Introduction: Pattern of childhood leukemia is known to vary through

out the world. We present clinico-pathological profile of children


123

presenting with Leukemia in our centre during a span of 10 years from

January 2001 to August 2012. This reflects the leukemia pattern in

Eastern U.P from where no such data is published till date. Materialsand Methods: A retrospective and prospective study of children pre-

senting with Leukemia was done from January 2001 to August 2012.

Clinical details like demographic data, age, sex and presenting symp-

toms were noted. Peripheral smear was made and bone marrow

aspiration was done. Smears were stained with Giemsa stain and were

studied for morphology of cells. FAB classification was used to classify

Leukemias based on morphological basis. Results: A total of 888 cases

of Leukemia were studied. Distribution of cases males and females

showed that it was more common in males (72.86 %). Youngest patient

of the series was 2 month child presenting with Chronic Myeloprolif-

erative Disorder, which is very rare in this age group. Acute Leukemia

cases were 89.86 %. Chronic Leukemia was very rare (10.13 %).

Among Acute Leukemia 64.53 % cases were of Acule Lymphoblastic

Leukemia, 32.20 % cases were of Acute Myeloid Leukemia and 3.25 %

case were of Acute Undifferentiated Leukemia. Cases of Acute

Leukemia were equally distributed in two age groups, i.e. 0–5 years and

6–10 years. ALL-L2 was the commonest type of ALL (59.7 %).

ALL-L3 was seen only in 3.37 % of cases. In acute myeloid leukemia

AML-M2 was more common. Conclusion: There is geographic vari-

ation in the incidence of leukemia. In our series we have observed that

ALL-L2 is the most common type of childhood leukemia. Cases were

equally distributed from 0 to 10 years.

Abstract P 017

A Rare Clinical Antithesis of Hope and Despair: A Case Report

P Pujari Ganesh, LS Raut, VV Bohara, GV Badarkhe, SS Ray

Institute of Hematology & Transfusion Medicine, Kolkata

Abstract: Myeloid sarcoma is an extramedullary tumour of immature

cells of granulocytic series, generally occurring in approximately 2 %

of patients with acute myeloid leukaemia. Myeloid sarcoma occurs

mostly in adults aged 45–55 years, and it has a predilection for the

bone, soft tissue, and skin. We here report a 28 year old female,

presenting with fever, menorrhagia and gum hypertrophy. She was

diagnosed as Acute Myeloid Leukaemia (M4) and was treated with

7 + 3 induction therapy. On day 14 post-induction, multiple nodular

skin lesions developed gradually over both lower extremities. The

biopsy from the lesions revealed Leukemia cutis. Post-induction day

14 marrow revealed no evidence of disease and medullary remission

was documented on day 28. However, the skin lesions increased.

With high dose Arabinoside and Mitoxantrone (HAM) the lesions

completely disappeared after 10 days. However, patient succumbed to

septic shock on day 18 of HAM. This case highlights the extremely

unusual concurrent medullary remission and extramedullary relapse

in a case of acute myeloid leukemia treated on induction therapy.

Keywords Myeloid sarcoma, Induction, Failure, Leukemia cutis

Abstract P 018

Presence of FLAER Negative Population in Acute Leukemia

Kotteeswari Kathirvel, Bargavi Balakrishnan, HarikrishnanBabu, Ansu Abu Alex, Rayaz Ahmed, Aby Abraham, AuroViswabandya, Biju George, Vikram Mathews, Alok Srivastava

Department of Haematology, Christian Medical College, Vellore

Background: Fluorescent-labeled bacterial aerolysin (FLAER) is the

single most reliable marker to monitor small PNH clones especially in

conditions such as myelodysplastic syndromes when traditional GPI-

linked surface marker expression can be significantly altered. Studies

with FLAER have described a sensitivity ranging between 0.5 and

1 % in identifying GPI-negative WBCs in samples from aplastic

anemia patients (Cytometry B Clin Cyto, 2007). Also it has been

shown that 5-15 % of PNH patients develop leukocyte dyscrasias

which invariably are acute myelogenous leukemia (Leuk Lymphoma.

1999). Objective: To detect a FLAER negative population in acute

leukemia samples by a single tube multiparameter flowcytometric

assay. Methods: Peripheral blood and bone marrow samples of

patients with newly diagnosed acute leukemia received for immu-

nophenotyping are taken for this study. In a single tube assay, we

have combined FLAER with CD33, CD14 and CD45 for the detection

of PNH clone on blast and neutrophils of patient samples and neu-

trophils of healthy control samples simultaneously. 20,000 events

were acquired and analyzed for all the cases using FACS Calibur.

Blasts and neutrophils were gated by CD45 versus side scatter (R1)

and CD33 versus side scatter (R2) as shown in Fig. 1a. Results: We

have analyzed 31 newly diagnosed acute leukemia among which 13

cases of AML and ALL each (41.9 %) and 5 cases of APL (16.12 %).

The median age of AML, APL and ALL cohorts are 29 years (range:

13–73), 27 years (range: 12–37) and 3 years (range: 0–28) respec-

tively. The healthy control reference range for FLAER expression on

neutrophils was 99.98 % (range: 99.79–100). The FLAER negative

clone was detected on both blasts and neutrophils of patient samples.

In AML the median range of FLAER-ve cells on blasts is 74.4 (range:

0.92–90.18) which is significantly higher than in ALL of 13.56

(range: 4.13–91.22) (p = 0.006) (Fig. 1b) whereas on neutrophils are

11.31 (range: 1–59.06) and 0.62 (range: 0–8.65) respectively

(p = 0.0003) (Fig. 1c). Conclusion: In our study FLAER negative

population were more common in acute leukemia and significantly

higher in AML than ALL. This preliminary observation warrants a

more detailed study on the biology, relevance and prognostic impact

of this population on treatment outcomes.

Abstract P 019

Study of Incidence of Recurrent Genetic Translocations in AdultAcute Myeloid Leukemia

Shano Naseem1, Neelam Varma1, Prateek Bhatia2, JogeshwarBinota1, Subhash Varma3, Pankaj Malhotra3

1Departments of Hematology, 2Paediatrics, 3Internal Medicine;

PGIMER, Chandigarh

Background: World Health Organisation (WHO) classification of

acute myeloid leukemia (AML) incorporates morphologic, immuno-

phenotypic, genetic and clinical features in its classification scheme

and is believed to be of more clinical relevance then the FAB clas-

sification as it defines entities that are biologically homogeneous and

prognostically relevant. The latest 2008 WHO classification

includes a separate category of AML with balanced translocation/

inversion, which comprises of 2 provisional and 7 categories of

characteristic genetic abnormalities, including: (i) t(8;21);RUNX1-

RUNX1T1, (ii) t(15;17);PML-RARA, (iii) inv(16);CBFB-MYH11,

(iv) t(9;11);MLLT3;-MLL, (v) t(6;9);DEK-NUP214, (vi) inv3;RPN1-

EVI1 and (vii) t(1;22);RBM15-MKL1. Of these, t(8;21);RUNX1-

RUNX1T1, t(15;17);PML-RARA and inv(16) CBFB-MYH11 have

been reported in higher frequency than other translocations. Western

literature quotes the incidence of fusion transcripts to be around

40–45 % in AML. However the data from Indian sub-continent is

scarce. We, therefore planned this study to detect the incidence of

common translocation/chimeric fusion transcripts in adult patients

with AML for t(8;21);RUNX1-RUNX1T1, t(15;17);PML-RARA, and


123

(inv16); CBFB-MYH11 using a single multiplex RT-PCR assay.

Materials and Methods: The present study was carried in the

department of Hematology, PGIMER, Chandigarh from May 2010 to

May 2012. Cases diagnosed as AML by bone marrow morphology,

cytochemistry and flow cytometry immunophenotyping were enrolled

in the study. For detection of fusion transcript, a single multiplex

RT-PCR assay was carried out using primers specific to the transcript

being tested. Results: During the study period 125 cases (male:

female ratio = 1.2:1) of AML were tested for above mentioned

fusion transcripts. Of these, 86 (68.8 %) cases were negative and 39

(31.2 %) cases were positive for any of the fusion transcript.

t(15;17);PML-RARA was most common seen in 19/125 (15.2 %)

cases, followed by t(8;21);RUNX1-RUNX1T1 in 17/125 (13.6 %)

cases and inv(16);CBFB-MYH11 in 3/125 (2.4 %) cases. Conclu-sion: This study evaluated the incidence of common recurrent genetic

translocations/chimeric fusion transcript in 125 cases of adult AML.

The incidence of various fusion transcripts was 31.2 %, with

t(15;17);PML-RARA and t(8;21);RUNX1-RUNX1T1 being more

frequent, seen at a frequency of 15.2 and 13.6 % cases respectively.

inv(16);CBFB-MYH11 was less common. Identification of these

transcripts provides therapeutically and prognostically relevant clin-

ical information.

Abstract P 020

A Study of Hematological and Genetic Profile of PrecursorB Lymphoblastic Leukemia/Lymphoma Harbouring BCR-ABLRecurrent Genetic Abnormality

Anita Tahlan1, N Varma1, S Naseem1, J Binota1, D Bansal2,P Malhotra3, RK Marwaha2, S Varma3

Departments of Hematology1, 2Pediatric Hemato-Oncology and3Internal Medicine, PGIMER, Chandigarh

Introduction: Approximately 3 % of children and 25–30 % of adults

with B lymphoblastic leukemia/lymphoma (B-ALL) are reported to

harbour t(9;22)(q34;q11) and/or BCR-ABL positivity. This subset of

patients is considered a poor risk subgroup. Aim: To determine the

hematological and genetic profile of precursor B-ALL with

BCR-ABL positivity. Materials and Methods: The study was con-

ducted from June 2010 to June 2012, at PGIMER, Chandigarh.

Peripheral blood and bone marrow examination and immunopheno-

typing were done. Molecular analysis by reverse transcriptase PCR

(RT-PCR) for BCR-ABL hybrid transcripts was performed on bone

marrow or peripheral blood samples. Results: A total of 302 patients

were diagnosed to have ALL, of which 257 were B-ALL. Seventeen

patients (6.6 %) positive for BCR-ABL transcripts were included in

the study. Median age was 26 years (range 1 year to 49 years). 13 out

of 17 patients were males (76.4 %) and four females (23.5 %). Seven

patients (41 %) were below 18 years of age. The total leucocyte count

ranged from 38.6 to 404.9 9 109/L (median 49.5 9 109/L). Bone

marrow morphology showed a heterogeneous population of immature

cells in 13 cases with prominent nucleoli, irregular to indented

nuclear membrane and cytoplasmic blebbing/vacuolations. The

immunophenotypic profile was positive for the lymphoid markers in

all cases. In addition five cases showed positivity for myeloid markers

CD13 and CD33, One case showed positivity for CD13, CD33 and

CD 117. RT-PCR for BCR-ABL transcript showed e1a2 in 12 cases

(70.5 %), b2a2 in 3 cases and b3a2 in 2 cases (29.4 %). Conclusions:The overall prognosis improves with the addition of tyrosine kinase

inhibitors, in the treatment protocols of BCR-ABL positive B-ALL. In

a resource constraint setting, an attempt should be made to evaluate

BCR-ABL if the morphology is heterogenous, cytoplasmic blebbing/

vacuolations are noted, along with aberrant positivity for myeloid

markers CD13 and CD33.

Keywords B-ALL. BCR-ABL transcripts

Abstract P 021

The Clinical Outcome of Children with Philadelphia chromosome(Ph) Positive Acute Lymphoblastic Leukemia (ALL) in a TertiaryCare Centre

KG Srinivas, L Appaji, Aruna Kumari, KC Lakshmaiah

Department of Medical and Pediatric Oncology, Kidwai Memorial

Institute of Oncology, Bangalore, Karnataka

Objective: To evaluate the clinical outcome of children with

Ph positive ALL who received chemotherapy without tyrosine kinase

inhibitors (TKIs) from 2004 to 2009. In the current era of TKIs this

study serves as historical reference to evaluate the therapeutic impact

of TKIs on the outcome of Ph positive ALL. Methods: 28 children,

age less than 15 years with Ph positive ALL, registered at Kidwai

Institute of Oncology, from 2004 to 2009, who received standard

chemotherapy alone, according to MCP 841 protocol were analysed

retrospectively. Outcomes were compared with 28 Ph negative ALL

children who have taken same treatment during the same period.

Matching was done for appropriate parameters. Philadelphia chro-

mosome positivity was confirmed by translocation t(9:22) by

cytogenetics. Results: In our study. 19 (67.8 %) were males and 9

(32.2 %) were females (M:F 2.1:1). 21 (75 %) were less than 9 years

and 7 (25 %) were between 9 and 15 years. Complete remission was

attained in 82.1 % of children after induction treatment (Day 28). 10

(35.7 %) had early relapse, among them 7 (25 %) had early bone

marrow relapse, 2 (7.1 %) had early CNS relapse, 1 (3.5 %) had early

testicular relapse. 3 (10.7 %) had late bone marrow relapse. There

were three deaths during induction due to sepsis. The Event free

survival (EFS) and Overall survival (OS) at 3 years was 42.8 and

52 % respectively. Median OS was 38 months. In Ph negative ALL,

EFS and OS at 3 years was 73 and 80 % respectively. The median

duration of follow up is 37 months (range 2 months to 90 months).

Fig. 1 Analysis of FLAER-ve subset in newly diagnosed acute

leukemias. a Representative gating strategy to quantify GPI deficient

populations in AML. b FLAER-ve (%) on CD45dim population in

newly diagnosed acute leukemias. c FLAER-ve (%) on neutrophils in

newly diagnosed acute leukemias


123

Conclusion: Children with Ph positive ALL have inferior outcome

with respect to EFS and OS when compared to Ph negative ALL

when given standard chemotherapy alone. Due to limited resources,

none of our children could afford transplantation. With the

encouraging results of combining Imatinib (TKI) with standard che-

motherapy in recent studies, these high risk children should be offered

combined modality of treatment.

Abstract P 022

Novel NPM Mutation in the 30-Untranslated Region Identifiedin a Patient with Acute Myeloid Leukemia

Vinodhini Kumaraswamy, Ajay Abraham, Ashok KumarJeyavelu, M Sathya, Vivi M. Srivastava, Biju George, AlokSrivastava, Vikram Mathews, Poonkuzhali Balasubramanian


Abstract: Mutations in nucleophosmin (NPM) gene are known to

occur most frequently in normal karyotype acute myeloid leukemia

(NK-AML). The frequency of exon 12 NPM mutations in adult AML

patients’ ranges between 25 and 35 %, accounting for more than 50 % in

adult NK-AML. NPM is a nucleo-cytoplasmic shuttling protein initially

known to be involved in rearrangements in leukemia and lymphoma. The

NPM exon 12 mutations are mostly 4 bp insertions, which disrupts the

nucleolar localization signal (NoLS) at the C terminus of the NPM pro-

tein causing cytoplasmic accumulation of truncated NPM protein. The

most common among NPM mutation is type A, with the insertion of

tetranucleotide TCTG in the exon 12 seen in 75 to 80 % of cases. Type B

and D mutations (CATG and CCTG insertions respectively) are observed

in about 10 and 5 % of NPM mutated AML, other NPM mutations are

very rare. We report here a novel deletion detected in exon 12 of NPM

gene identified from a 41 year old male diagnosed with AML. Peripheral

blood smear of this patient revealed total WBC count of 1,200/mm3 with

7 % blasts. There was anemia (Hb 8.5 mg%) and thrombocytopenia

(Platelet counts 42,000/mm3). Bone marrow examination revealed FAB

AML-M2 based on morphology and immunophenotyping. He had tri-

somy 8 on cytogenetic analysis which placed him in the intermediate risk

category of AML. FLT3 ITD and NPM mutation detection was done

using genomic DNA sample at diagnosis by PCR followed by GeneScan

analysis. This patient was FLT3 ITD negative but there was a 5 bp

deletion seen in the NPM exon 12 based on GeneScan electropherogram

(Fig. 1). For further characterization of this mutation, the same sample

was PCR amplified and subjected to automated sequencing using ABI

genetic analyzer. DNA Sequencing revealed the presence of a novel 5

base pair (ATTTC) deletion in the exon 12 (Fig. 2). Unlike other exon 12

mutations, which are mostly insertions, this deletion is in the 30

un-translated region (30UTR) region, 45 bp downstream to the stop codon

(TAA) and hence will not result in the formation of a truncated protein.

This mutation was further confirmed by cDNA sequencing as well and

is in the position 930 from translational start site of the cDNA. NPM

RNA expression for this patient was checked by RQPCR and was found

not different from that of representative NPM type A, type B, type D and

NPM wild type subjects. Flowcytometric evaluation of the diagnostic

marrow showed 95 % positivity for CD34, which is against the previous

reports suggesting NPM mutation is associated with low CD34

expression. Bioinformatic analysis (http://www.microrna.org/) of NPM

30UTR region, where the mutation occurred, revealed potential loss of

binding site for miR208 due this 5 bp deletion. This patient underwent

conventional chemotherapy with daunorubicin and cytosine and

achieved CR1, received consolidation and is in complete remission

now. He did not develop any toxicity. This study reports a novel 30UTR

NPM mutation, which unlike the common NPM exon 12 mutations,

does not disrupt open reading frame but possibly affects the stability of

mRNA. Further characterization of this mutation is needed before we

comment on its clinical significance. The study also highlights the

importance of NPM gene sequencing in addition to GeneScan analysis

in order to identify the specific NPM mutation.

Abstract P 023

Impact of Cytogenetics on Outcomes of Pediatric Acute MyeloidLeukemia: Our Experience from a Tertiary Care Cancer Centrein South India

KS Rachan Shetty1, B Guruprasad1, L Appaji2, KC Lakshmaiah3

1Department of Medical Oncology, 2Department of Paediatric

Oncology, 3Department of Medical Oncology, Kidwai Memorial

Institute of Oncology, Bangalore 560029, India

Aim: To study the prognostic factors of paediatric Acute Myeloid

Leukemia (AML) with chemotherapy. Materials and Methods: 37

patients who had pathologically proven diagnosis of AML and had

received treatment at our institute from January 2007 to December

2010 were included for analysis. Patients were diagnosed based on

morphology, cytochemistry, cytogenetics and immunophenotypic

studies, molecular risk stratification was not done. AML patients

received 7 + 3 induction with Cytarabine and Daunomycin, followed

by 4 cycles of high dose Cytarabine consolidation, except for APML

(acute promyelocytic leukaemia) patients who received All Trans

retinoic acid (ATRA) with Daunomycin induction followed by 2

cycles of Daunomycin consolidation and maintenance with ATRA,

Methotrexate and 6-mercaptopurine. All patients were analyzed for

oncological outcome and these outcomes were correlated with initial

total leukocyte count and cytogenetics. Results: Majority of patients

had favourable cytogenetic abnormality. At the median follow-up at

24 months, (range 4–46 months), induction death rate was 27 and

73 % achieved complete remission. The relapse rate was 30 % and

event free survival rate was 43 %. Two year survival was 66.7 % for

favourable cytogenetics, 50 % for patients with intermediate risk, 0 %

for those with unfavourable cytogenetics. High tumor burden with

intermediate and unfavourable cytogenetics had significant correla-

tion with outcome. Conclusion: Tumor burden measured by initial

total count and WHO cytogenetic risk marker are the most important

prognostic factors even in paediatric AML. Even with advent of

molecular risk stratification cytogenetics remains the most important

risk factor.

Keywords Acute myeloid leukaemia (AML), APML (acute pro-

myelocytic leukemia), world health organisation(WHO) cytogenetic

risk matter


123

http://www.microrna.org/

Abstract P 024

Coding Variants of DNA Repair Genes and Its Associationwith De Novo Acute Promyelocytic Leukemia

GC Gaur1,2, SK Hasan1,2, T Ottone1,2, V Mantovani3,D Centonze4, F Lo-Coco1,2

1Department of Biopathology, University of Rome ‘‘Tor Vergata’’,

Rome, Italy; 2Laboratorio di Neuro-Oncoematologia, Fondazione

Santa Lucia, Rome, Italy; 3Centre for applied Biomedical Research,

St. Orsola-Malpighi University Hospital, Bologna, Italy; 4Clinica

Neurologica, Department of Neuroscience, University of Rome

‘‘Tor Vergata’’, Rome, Italy

Background: DNA is essential to life, but it is subject to damage

from interaction with various endogenous and exogenous agents.

Double-strand DNA breaks are arguably the most serious form of

DNA damage and are predominantly repaired through either the

homologous recombination (HR) or non-homologous end-joining

(NHEJ) pathways. Single nucleotide polymorphisms (SNPs) in dou-

ble-strand break repair genes may alter DNA repair capacity and, in

turn, confer predisposition to leukemia. We analyzed polymorphic

variants of DNA repair and detoxification genes in patients with acute

promyelocytic leukemia (APL). Methods: Using MassARRAY high-

throughput DNA analysis with matrix-assisted laser desorption/ioni-

zation time-of-flight mass spectrometry, we initially genotyped 26

APL patients and 561 healthy blood donors for 210 SNPs of 22 genes

mostly involved in DNA repair and drug detoxification. Based on

results of initial screening, we further analysed BRCA2, XRCC5,

NBN (formerly NBS1) and LIG4 gene in another 40 de novo APL

patients using DNA sequencing. Results: Based on our complete

cohort analysis, we identified 2 genes which were significantly

associated with risk of development of APL. We observed a differ-

ence in risk allele frequency between APL and Healthy controls for

NBN (rs1063045, NG_008860.1:g.6881G[A): 32.5 % and 45.5 %,

p = 0.003 and LIG4 (rs1805386, NG_007396.1:g.10970T[C):

12.1 % and 18.36 %, p = 0.07. The association of homozygous

variants of NBN yielded higher risk of APL (OR: 3.042, p = 0.003),

whereas T allele of LIG4 was found to be a susceptible allele in APL

(OR: 1.630, p = 0.07). Conclusions: Susceptibility to develop APL

may be linked to genetic variations in DNA repair genes that result in

inefficient repair of chemical or radiation induced genetic damage.

SNP rs1063045 of NBN gene could be an important factor which

plays an important role in NHEJ pathway repair in case of APL.

Abstract P 025

Sub Categorisation of T Cell Acute Lymphoblastic LeukemiaUsing Who Criteria—A Retrospective Analysis

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

Department of Hematology, Post Graduate Institute of Medical

Education and Research, Chandigarh

Introduction: According to the 2008-WHO CLASSIFICATION,

T-Acute Lymphoblastic Leukemia (T-ALL) has been subclassified

into PRO T-, PRE T-, CORTICAL T- and MEDULLARY T-ALL

based on immunophenotyping. Some studies indicate prognostic

significance of these subcategories. Aim: This study attempts to

subcategorize T cell acute lymphoblastic leukemia cases to assess

utility of WHO criteria for the subcategorization. Methodology: All

cases diagnosed as acute leukemia over a period of 3 years in the

department of hematology were screened for T cell ALL cases. An

attempt was made to subcategorize all T-ALL cases according to

WHO criteria and also to assess expression of aberrant markers.

Results: Out of 1740 acute leukemia cases diagnosed in 3 years,

29.1 % (507 cases) were of B-ALL and 3.1 % (54 cases) were of

T-ALL. Among the 54 T-ALL cases, a complete T-cell panel was

done in 33 cases. On applying stringent WHO criteria, 54 %

(18 cases) were classifiable and 45 % (15 cases) were not classifiable.

Among the classifiable cases, Pro T-ALL were 11.11 % (2/18), Pre

and Medullary T-ALL were 5.5 % (1/18) each, and Cortical T-ALL

were 77.77 % (14/18). In addition, aberrant expression of other

lineage markers including CD79a (5.5 %), CD13 (7.4 %), CD33

(7.4 %), CD117 (11.11 %), CD 19 (5.5 %), CD10 (18.5 %) were seen

in variable combinations. In addition, analysis of T Cell Receptor

subtypes done in 36 cases, showed 13.8 % of cases expressing TCR

alpha/beta and 50 % cases expressing TCR gamma/delta subtypes.

Conclusion: Among the cases examined, only 54 % of cases could be

subclassified following the present WHO criteria, indicating its lim-

ited utility in routine diagnostic use.

Abstract P 026

Clinicopathologic Profile of Leukemias in Infants—an IndianScenario

S Munot1, PG Subramanian1, S Gujral1, Y Badrinath1,A Kumar1, S Shinde1, S Mahadik1, P Amare-Kadam2, B Arora3,S Banavali3

1Hematopathology Laboratory, 2Department of Cytogenetics,3Department of Medical Oncology, Tata Memorial Centre, Mumbai

Introduction: Acute and chronic leukemias are rare in infants world-

wide, with very limited Indian data on their clinical and hematological

profile. We studied 50 consecutive cases of infant leukemias and the

clinical, morphology, cytochemistry, immunophenotyping and cytoge-

netic data was correlated. Objective: To study the incidence and

clinicopathological profile of leukemias in infants. Methods: A retro-

spective study was undertaken in our tertiary care cancer institute. The

data of all newly diagnosed cases of leukemias in infants from the year

2005 to 2012 was obtained from electronic medical records. Cases were

diagnosed either on peripheral blood, bone marrow aspirate or biopsy.

Results: Of the total 8,223 cases of newly diagnosed acute leukemias in a

7 year period, 49 were diagnosed in infants (0.5 %) It included 26 cases of

acute lymphoblastic leukemia, 10 cases of acute myeloid leukemia, 3

cases of juvenile myelomonocytic leukemia and1 case ofchronic myeloid

leukemia. 9 cases could not be subtyped for want of additional samples.

Only 2/49 cases occurred in neonates. There was a male preponderance

(34/49). A detailed immunophenotypic and cytogenetic analysis will be

presented in the study. Conclusion: This study provides important

insights in the spectrum and biological behavior of infant leukemias.

Abstract P 027

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

Sreeja Karathedath, Ajay Abraham, Savitha Varatharajan,Biju George, Alok Srivastava, Vikram Mathews, PoonkuzhaliBalasubramanian


Background: Conventional chemotherapy with Cytarabine (Ara-C)

and Daunorubicin (Dnr) can cure about 25 % of patients with


123

Acute Myeloid Leukemia (AML) but majority fail to achieve long

term remissions. Drug resistance and relapse are considered major

causes of treatment failure. The toxic side effects of chemothera-

peutic drugs make therapy intolerable and inefficacious especially

in older patients. This emphasizes the need of developing new

therapeutic strategies to improvise the treatment both in terms of

cost and efficacy. Chemotherapeutic agents like Arsenic Trioxide

(ATO) brings cell kill by inducing Reactive Oxygen Species

(ROS). ATO has proved very effective in inducing remissions in

de novo acute promyelocytic leukemia (AML M3), but has shown

to have reduced activity in other AML subtypes. The redox sen-

sitive transcription factor, Nrf2 (Nuclear Factor Erythroid 2 Related

Factor) regulates oxidative stress in normal cells is an important

candidate which determine ROS mediated apoptosis. NRF2 trans-

criptionally regulates the levels of antioxidant response genes

((NADPH Quinone Oxidoreductase (NQO1), Heme Oxygenase

(HO-1), Glutamate Cysteine ligase (GCL) and drug efflux trans-

porters like Multi drug resistance related protein-2 (MRP2)) and

averts ROS mediated cytotoxic effects. Pharmacological modulation

of Nrf2 by Nrf2 inhibitors could be well exploited to provide better

treatment opportunities. We hypothesize that ATO insensitivity in

non-M3 AML cells is due to increased Nrf2 levels which could be

modulated. Materials and Methods: In vitro cytotoxicity to ATO

was done in NB4 (AML-M3), U937 (non-AML-M3) and

KASUMI1 (AML cell line with t(8;21)) cell line and primary AML

cells (n = 32) using MTT Assay. This assay was extended to cell

lines pretreated with pharmacological concentration (10 lM) of

Nrf2 Inhibitor, Luteolin. Nuclear protein extraction was done using

NE-PER kit (Pierce). Nuclear Nrf2 protein level was detected using

Western blot and normalized to b-lamin. RNA extraction from

AML cell lines and primary AML samples at diagnosis (n = 32)

was done using Trizol method and the expression of the down-

stream target genes (NQO1, HO1, GCLC, GCLM, MRP2) of Nrf2

was done using qRT-PCR. Results and Discussion: Based on the

MTT results, U937 was resistant compared to NB4 and Kasumi1

cell lines to ATO (IC 50 values; Table 1). Nuclear expression of

Nrf2 protein by western blot showed U937 cell line to have higher

nuclear Nrf2 compared to NB4 cell line (Fig. 1). To further iden-

tify the functional role of Nrf2, qRT-PCR analysis was done for the

downstream Nrf2 target genes NQO1, HO1, GCLC and GCLM.

ATO resistant U937 cell line showed 1.3, 3, 3.3 and 2.4 fold

increase in the mRNA levels of Nrf2 downstream target genes

NQO1, HO1, GCLC and GCLM respectively compared to ATO

sensitive cell line NB4 (Fig. 2). Pharmacological inhibition of Nrf2

by Luteolin pretreatment efficiently reduced the IC50 of ATO

resistant U937 cell line (Fig. 3). Nrf2 downstream target genes-

NQO1, HO1, GCLC and GCLM mRNA levels were analyzed in

primary AML cells by qRT PCR. Patients with ATO IC50 more

than the median (n = 16, IC 50 [ 2.37) had higher mRNA

expression for the above mentioned Nrf2 target genes when com-

pared to ATO sensitive patients (n = 16, IC 50 \ 2.37) though not

reaching statistical significance, probably due to low sample num-

ber. Conclusion: This pilot study extends the possibility of using

pharmacological inhibitors of Nrf2 to modulate ATO resistance in

AML.

Abstract P 028

Synchronous Presentation of Squamous Cell Carcinomawith Acute Promyelocytic Leukemia: Report of a Rare Case

Ayushi Sahay, Meena Desai, Vijay Hirani2, Pankhi Dutta1

SevenHills Hospital, Mumbai, 2Modern Haematology and

Chemotherapy Centre, Kolhapur, 1Kokilaben Dhirubhai Ambani

Hospital, Mumbai

Objective: Though rare, squamous cell carcinoma (SCC) has been

seen to occur in acute promyelocytic leukemia (APML) patients as a

consequence of treatment with arsenic trioxide. However, there is no

case reported in literature of simultaneous occurrence of these two

malignancies. We report a highly rare case. Method—Case Report:A 47 year old male presented with ulceration of the left inner cheek.

There was no other significant past history or treatment history. There

was history of regular tobacco chewing. Examination revealed a

buccal ulcer which was non tender along with a palpable and hard,

left submandibular lymph node. A biopsy done from the cheek ulcer

revealed a moderately differentiated SCC with nests and sheets of

moderately large malignant squamous cells irregularly infiltrating the

submucosa and muscle layer. A routine hematological evaluation at

this time revealed pancytopenia with Hb: 7.9 mg%; WBC: 660 cells/

mm3; platelet count: 53,000/mm3. A bone marrow (BM) examination

was done and showed highly cellular aspirates with near total

replacement of the marrow by abnormal, hypergranular promyelo-

cytes along with classical faggot cells. The corresponding BM

trephine biopsy showed near total replacement by sheets of immature

myeloid cells. A diagnosis of APML was made. PML-RARA testing

by gel PCR was positive for the bcr1 isoform. The patient refused any

specific treatment and expired after 8 days at home. Conclusion: To

the best of our knowledge, this is the first case in literature of syn-

chronous presentation of SCC with APML and highlights the

importance of bone marrow examination in carcinoma patients with

pancytopenia.

Abstract P 029

Mixed Phenotype Acute Leukemia—a Growing Concern

Shelly Poddar, SM Sethy, P Mohanty, RK Jena

Department of clinical Hematology, Department of Pathology,

SCB Medical College

Aim of the study: To study the incidence of Mixed Phenotype Acute

Leukemia (MPAL) out of 253 cases of Acute Leukemia studied.

Objective: To correlate the haematoclinical presentation with

cytomorphology, cytochemistry, immunophenotyping and treatment


123

outcome. Materials and Method: We evaluated 253 cases presenting as

Acute Leukemia. Each of these were completely evaluated by a

complete blood count, bone marrow aspiration, MPO &PAS positiv-

ity, multiparametric flow cytometry. Then they were evaluated for the

treatment response. Observation: Out of 253 cases of Acute Leu-

kemia 5 cases came out to be of Mixed Phenotype Acute Leukemia

(MPAL). Of these 5 cases morphologically 3 were AML and 2 were

ALL. They received a treatment plan of 3 + 7 CT followed by

MCP841. In all of these cases outcome was poor, all patients died

either before or after induction therapy. Discussion: MPAL accounts

for about 2 % of acute leukemias and can occur in both children and

adults but more common in adults. It can present morphologically as

either AML or ALL. So to distinguish these group of patients we

need to go for a immunophenotyping. In our study we have seen that

there is 100 % mortality rate. We can conclude that this leukemia has

a poor prognosis.

Slno.

Age/sex

PSC BM Immuno-phenotyping

Treatment Outcome

1. 1.8 mn/M

[80 %blast AML-M6

MPAL MCP841 Expired duringinduction

2. 27 years/M

Acleukemia

ALL MPAL 3 + 7CT IncompleteCR expiredbeforenext CT

3. 70 years/F

[50 %blast AML-M5

(B + T)AntiMPO-ve

Oral CT Expiredduringinduction

4. 51 years/M

Acleukemia

AML-M1

MPAL Planned for3 + 7 CT

Expired beforeinduction

5. 32 years/F

Acleukemia

ALL MPAL Received3 + 7CTfollowedbyMCP841

Cr after 2ndCT butexpireddue toinfectionafter4 months

Abstract P 030

Immunophenotypic Analysis of Transient Abnormal Myelopoiesisin 5 Children with Down Syndrome

V Baloda1, S Gujral1, PG Subramanian1, Y Badrinath1,A Kumar1, P Amare Kadam2, S Banavali3, B Arora3

1Department of Hematopathology, 2Cytogenetics Department,3Department of Medical Oncology, Tata Memorial Hospital, Mumbai

Objective: To study the immunophenotypic profile of transient

abnormal myelopoiesis (TAM). Materials and Methods: Retro-

spective analysis of cases reported as TAM during 2007-2012.

Results: 5 children (3 females, 2 males) with Down Syndrome with

age between 5 and 45 days presented with a high WBC counts and

high percentage of blasts in peripheral blood and/or bone marrow.

Blasts were MPO negative. Immunophenotypic analysis using multi

color flow cytometry was performed. Table 1 delineates the expres-

sion pattern of various markers on the blast population. The blasts did

not express any of these B or T cell markers—CD19, CD10, CD20,

CD3 and CD2 (2/2). Blasts expressed CD4 in one case. Conclusion:Transient abnormal myelopoiesis presented in Down syndrome chil-

dren within 45 days of birth in our series. CD34, CD13, CD33,

CD117, CD41, CD61, CD7 and HLA-DR are useful markers for

characterization of blasts of TAM.

Abstract P 031

Volume, Conductivity, Scatter: Simply Ornaments or DiagnosticTools of Acute Leukaemias: A Retrospective Analysis of 108Cases of Acute Leukaemias in a Tertiary Oncology Centre

Monali Gupta, DK Mishra, Mammen Chandy

Department of Lab Hematology, Tata Medical Centre, Kolkata

Introduction: The Automated Hematology analyser Coulter LH-780

uses a combination of three measurements Volume, Conductivity, Scatter

(VCS) to identify WBCs in their near native state but it could take

advantage of these parameters to evaluate their morphologic changes.

Objectives: The aim of this study was to investigate whether VCS can

become a new high-throughput screening method not only for the detection

but also for the exact categorization of Acute Leukaemias. Materials andMethods: VCS parameters of 108 diagnosed cases of acute leukaemias

analysed by Beckman Coulter LH 780 were studied retrospectively. The

CBCs, scatterplots, flags were correlated with VCS to calculate their

sensitivity and specificity alone and in combination. VCS trends and

characteristic cluster patterns were identified in 67 cases of ALLs, 7 AP-

MLs, 34 Non APMLs, 6 HLA-DR negative AMLs, 3 cases each of AML

with myelodysplasia related changes and Acute leukaemia of ambiguous

lineage. Results: High MLV, SD volume and high MLS and SD scatter

was the characteristic VCS trend observed in 61 cases of ALLs while 5

cases deviated from the trend. AMLs showed high MNV and SD but low

mean and SD scatter. APMLs exhibited least mean and SD scatter values in

spite the hypergranularity of abnormal promyelocytes. Interestingly AML

M5 cases exhibited high Monocyte mean scatter and normal SD scatter

despite the fact that the monoblasts are agranular! A tight merging cluster

pattern was seen in all 3 cases of HLA-DR negative AMLs. The current

study also found ‘‘cluster patterns’’ in 66 cases of ALLs and their associ-

ation with presence of aberrant myeloid antigen marker on

immunophenotyping was statistically significant (p value \0.001).

Conclusions: VCS alone proved to be a good diagnostic tool in ALLs as

the sensitivity was 91.8 % and specificity was 80 % while in AMLs the

sensitivity was 94.1 %. There were no AML cases wherein peripheral

blood did not have blasts, so specificity could not be calculated.

Keywords Beckman coulter LH 780, VCS, Mean neutrophil volu-

me(MNV), Mean Lymphocyte volume (MLV), Mean lymphocyte

scatter(MLS)

Abstract P 032

Minimal Residual Disease in Paediatric B Lineage AcuteLymphoblastic Leukemia: Comparison of Mid—InductionPeripheral Blood and Bone Marrow Samples Using Six ColourFlow cytometry

BK Karthik Bommannan1, MUS Sachdeva1, Parveen Bose1,Neelam Varma1, Deepak Bansal2, RK Marwaha2

1Department of Hematology, 2Department of Paediatrics, Post

Graduate Institute of Medical Education and Research, Chandigarh

Introduction: There is strong correlation between MRD levels and

risk of relapse in childhood leukemias. Bone marrow aspirate sample

on day 15 of induction is commonly used in paediatric cases to assess

MRD levels for further management decisions. Peripheral blood

sample might be used for the same if proven to yield similar MRD

levels. Aim: To compare MRD levels in paired peripheral blood and bone

marrow samples of paediatric B-ALL patients on day 15 of induction

using six colour flow cytometry. Methodology: Five newly diagnosed,

CD19 and CD10 dual positive, paediatric B lineage ALL patients were


123

analyzed on day 15 of chemotherapy for peripheral blood and bone

marrow MRD levels. Lyse-stain-wash technique was used to process

single 6 colour tube (CD19PECy7/CD10APC/CD20PerCP/CD34PE/

CD45APCH7/Syto13). One million events were acquired on BDFACS

Canto II and analysed by BDFACS Diva software. Results: MRD levels

of[0.01 % (range 0.02–0.81 %) were seen in all 5 bone marrow sam-

ples. All but one peripheral blood samples showed presence of

MRD [ 0.01 % (range 0.04–0.36 %). Although, the levels of MRD vary

slightly between bone marrow and peripheral blood samples, presence of

peripheral blood MRD in all but one case, may favour using peripheral

blood on day 15 for MRD detection instead of invasive bone marrow

procedure. Conclusion: There is good concordance (4 out of 5 cases) of

MRD level detection between mid-induction samples of peripheral blood

and bone marrow aspirate for paediatric B-ALL cases. This result needs

to be supported by more number of cases.

Abstract P 033

Monosomy 7 in Acute Myeloid Leukemia in India

S Yuvarani1, Usha Sitaram3, Rayaz Ahmed2, Aby Abraham2,Auro Viswabandya2, Biju George2, Vikram Mathews2,Alok Srivastava2, Vivi M. Srivastava1

1Cytogenetic Unit, 2Department of Haematology, 3Department

of Transfusion Medicine and Immunohaematology

Background: Monosomy 7 is seen in 5–10 % of all acute myeloid

leukemia (AML) and is associated with a poor prognosis. We describe the

cytogenetic features of monosomy 7 in AML. Patients and Methods:G-banded karyotypes of all patients with AML and monosomy 7 seen in

the Department of Haematology, Christian Medical College, Vellore

between January 2003 and December 2009 were studied. Cytogenetic

findings were correlated with clinical and laboratory features. Results:Monosomy 7 was seen in 71 patients (5.4 % of 1314 patients with AML),

52 of whom were males (73 %). There were 57 adults (median age:

39 years, range: 1–72). The median hemoglobin was 7.95 g/dl (range:

2.5–15.8); median WBC count, 6.4 9 109/L (range: 0.6 -271); median

platelet count, 25.5 9 109/L (range: 3–401). The most common AML

subtypes were AML-M2 (27 %) and AML, not otherwise specified

(29 %). Dysplasia was seen in 46 % of cases while 10 % of these cases

were of secondary AML. Monosomy 7 was the solitary abnormality in 19

patients (27 %). Complex karyotypes (two or more additional abnor-

malities) were seen in 38 (53 %). These included monosomy 5 (15 %),

deletion 5q (8 %), trisomy 8 and monosomy 17 (7 %), and trisomy 21 and

inversion 3 (6 %). Follow-up data was available for 20 of these patients.

Only two of them survived more than 8 months. Conclusion: This is the

first report of a large series of AML patients with monosomy 7 from India.

While the incidence (5.4 vs. 3–10 %) and poor outcome are similar to

other reports in the literature, these patients are younger (median age: 39

vs. 45–55 years) and show a higher male preponderance (2.3:1 vs. 1.5).

Myelo proliferative Neoplasms

Abstract P 034

JAK2 (V617F) Negative Myeloproliferative Neoplasms—Prevalence and Points to Ponder

Puttaiah Parimala, Karuna Rameshkumar and Cecil Ross1

1Department of Clinical Pathology and Hematology, St John’s

Medical College Hospital, Bangalore

Introduction: Myeloproliferative neoplasms (MPN) are characterised

by clonal proliferation of one or more of myeloid lineages and it is

known mutations in JAK 2 signaling contribute to pathogenesis.

There is a significant group of patients who are JAK2 (V617F) neg-

ative. Aims and Objectives: To study prevalence, clinical and

laboratory features of Philadelphia chromosome negative and JAK2

(V617F) negative myeloproliferative neoplasms. Materials andMethods: From January 2011 to July 2012, patients who fulfilled

2001 WHO criteria of MPN were included. Clinical features and

laboratory profile including complete hemogram, bone marrow aspi-

ration and biopsy were analysed. FISH for Philadelphia chromosome

and JAK2 mutation by allele specific PCR were done for further

characterization. Results: Among 44 patients diagnosed as MPN, 27

patients were diagnosed as JAK2 (V617F) negative. Polycythemia

Vera was most frequent (59 %) followed by idiopathic myelofibrosis

(26 %) and essential thrombocythemia (15 %). Male predominance

was noted (92 %). JAK 2 positive patients were older and had higher

frequency of splenomegaly. In comparison, JAK2 negative patients

had a higher platelet count in ET (11.6 ± 2.4 9 109/L) and isolated

erythrocytosis (19.4 ± 1.38 g/dl) in 94 % of patients with PV. Two

of them had presented with thrombosis. Comment and Conclusions:Mutations at exon 12 associate with erythropoietin signaling have

been reported in JAK 2 (V617F) negative PV in literature. The

present study reinforces the findings of our previous study. In addi-

tion, it highlights need to look at other mutations among JAK2

(V617F) negative myeloproliferative neoplasms, which may be

involved in activation of other signaling pathways.

Abstract P 035

Myelofibrosis—an Unusual Manifestation of HIV Infectionin a Child

Rohini Gupta2, Gunjan Mahajan1, Jagdish Chandra2, SunitaSharma1, Anju Seth2, Bhavna Dhingra2, Praveen Kumar2

1Department of Pathology, 2Department of Paediatrics, Lady

Hardinge Medical College and associated Hospitals, Delhi

Introduction: Myelofibrosis is a myeloproliferative disorder in which

abnormal type of bone marrow stem cells cause fibrosis. Secondary

Myelofibrosis can be associated with haematological malignancies,

infections, endocrine and connective tissue disorders. However,

Myelofibrosis in HIV infected children is very rare. Case Details: A

female child admitted postnatal for umbilical cord bleeding was given

blood transfusion in 2004. Patient remained asymptomatic till 4 years

of age when she presented with anasarca, acute gastroenteritis and

sepsis. On examination, patient had severe anemia, hepatospleno-

megaly and bilateral crepitations on auscultation. On investigations,

patient had normocytic anemia (Hb-6.6 g/dl, MCV-84.6 fl), throm-

bocytopenia (platelet-61 9 103/ll) and normal total leucocyte count

(TLC-6.7 9 103/ll). X-ray chest showed bilateral infiltrates in lungs.

HIV serology was positive. A probable diagnosis of tuberculosis with

HIV was made. CD4 counts were 602/mm3 (CD4 % = 22 %).Patient

was put in HIV clinical stage 3 and started on ATT. On further follow

up, the anemia and thrombocytopenia worsened, leucopenia devel-

oped and CD4 counts reduced to 255/mm3 (15 %). ART was thus

started on 11/01/2011, but pancytopenia of patient did not improve.

Serum vitamin B12 and folate levels were normal. Bone marrow

aspiration was diluted. Bone marrow biopsy was hypocellular for age

and showed focal islands of hematopoetic cells. Megakaryocytes were

reduced in number. On reticulin stain bone marrow showed grade 3

fibrosis. Von Gieson stain showed focal areas of fibrosis. Thus a

diagnosis of Myelofibrosis secondary to HIV infection was made.

Patient was started on thalidomide and steroids for 3 months and then

continued only on steroids. The peripheral blood count improved (Hb-

12.7 g/dl, TLC-5.8 9 103/ll, platelet-141 9 103/ll). She remained

asymptomatic till date and did not require blood transfusion.


123

Conclusion: Myelofibrosis as presenting feature in HIV infection is

rare. Although a few case reports of Myelofibrosis secondary to HIV

infection in adult patients are present in Indian literature, none has

been documented in children to the best of our knowledge.

Stem Cell Transplant


Stemming Role of Stem Cell Transplantation in MultipleMyeloma: Who, When, and What Type?

Deepak Bansal

Maharishi Markandeshhwar Institute of Medical Science & Research,

Mullana

Abstract: High dose chemotherapy with autologous stem cell trans-

plantation has shown improved progression-free survival (PFS) over

the conventional chemotherapy regimens. In the era of novel agents

for myeloma in conjunction with the evolution of hematopoietic stem

cell transplantation, many new questions arise. How to incorporate

these novels into the transplant paradigm? Should transplant be

delayed until relapse? In earlier eras, autologous transplantation was

generally reserved for patients younger than 60 or 65 years. In this

modern era, should there be an age limit for myeloma transplant?

Now that medicine is individualized for every patient, chronologic

age alone should not drive decisions regarding transplantation. What

type of transplant (i.e., autologous compared with reduced intensity

allogeneic transplant) is best? Several large international trials have

demonstrated conflicting results in regard to an overall survival (OS)

benefit with the allogeneic approach. The role of allogeneic transplant

remains under study especially in the high-risk population, which has

high relapse rates with traditional autologous approaches. Future

directions to reduce relapse include post-transplantation consolidation

and maintenance therapy with either approved agents or new agents

and immunotherapy, either vaccine based or natural killer (NK) and

T-cell based.

Abstract P 037

Effect of GSTA1*B Polymorphism on Pharmacokineticsof Intravenous Busulfanin Patients Undergoing HematopoieticStem Cell Transplantation

S Gopinath, M Ezhil Pavai, Biju George, Vikram Mathews,Alok Srivastava, B Poonkuzhali


Background: Busulfan in combination with cyclophosphamide is

widely used conditioning regimen in Hematopoietic cell transplan-

tation (HSCT) for various malignant and non-malignant disorders.

Wide inter-patient variability in busulfan systemic exposure is

observed with oral busulfan due to unpredictable intestinal absorption

and differences in hepatic metabolism. To overcome the problems

associated with oral administration, intravenous (i.v) busulfan was

introduced in early 2000. Busulfan is mainly metabolized in the liver

by glutathione S-transferases (GSTs). We have shown that GSTA1*B

polymorphism explained the inter-patient variability in busulfan

exposure and clearance in homozygous beta thalassemia patients

receiving oral busulfan (ASH 2009, ASH 2010). There is no report on

pharmacokinetics of i.v busulfan from Indian patients undergoing

HSCT. The aim of the present study is to document the PK of i.v

busulfan and to compare the first dose AUC and clearance in with the

GSTA1*B polymorphism. Methods: Sixty patients receiving i.vbusulfan once daily (n = 44) or four times daily (n = 16) as part of

their conditioning regimen before transplantation between June 2010

and July 2012 at the department of hematology, Christian Medical

College, Vellore were included in this study. Genomic DNA was

isolated from the peripheral blood MNCs before transplantation and

GSTA1*B polymorphism was analysed using PCR–RFLP method.

Peripheral blood samples were collected at different time points (0, 3,

4, 5 and 7 h for Q24h and 0, 1, 2, 3 and 4 h for Q6h) after busulfan

infusion. Busulfan plasma levels were measured using previously

reported LC–MS/MS method. The area under the concentration ver-

sus time curve (AUC) and clearance of busulfan after the first dose

was calculated as reported previously (Desire et al., IJMR, in press).

Based on the AUC of the first day, subsequent doses were adjusted to

achieve the target AUC. The AUC and clearance of busulfan were

compared with the GSTA1*B genotype using Mann–Whitney U test.

Results: Patient demographics and busulfan AUC and GSTA1*B

genotypes are listed in Table. There are contradictory reports on the

influence of GSTA1*B polymorphisms on i.v busulfan AUC and

clearance in adult patients as well as in children. We report here that

GST polymorphism does not significantly influence the first dose

AUC or the clearance of i.v busulfan, though in a small number of

patients. This is the first report from Indian population comparing

GST polymorphism and pharmacokinetics of i.v busulfan. We con-

clude that, along with GST polymorphisms, there may be other

genetic variants explaining the inter-individual variability in busulfan

systemic exposure and clearance, which needs to be evaluated.

Table 1 Immunophenotypic profile of blasts

Case 1 Case 2 Case 3 Case 4 Case 5

Age at

presentation

5 days 45 days 11 days Neonate 40 days

Sex M M F F M

Blast % 80 (BM) 57 (BM)

77 (PB) 59

(PB)

20 (PB)

MPO - - - - -

CD 34 + + + + +

CD 13 - - + - -

CD 33 + + + + +

CD 117 + + + - +

Anti MPO ND - + ND -

CD 41 + - + WK + Inconclusive

CD 61 + - + WK + Inconclusive

CD 14 - ND - ND ND

CD 235a - - ND ND ND

CD 7 - + + - +

CD 56 + - + - -

HLA DR - + - - -

M male, F female, ND not done, WK+ weak positive, BM bone

marrow, PB peripheral blood; +, positive; -, negative


123

Diagnosis N = 60 Bu

dosing

GST

A1*B

AUC (lmol) Clearance

(L/h/kg)

AML 19 Q24H (n = 44)

Median age—32 years

(1–58 years)

Variants—19

Median = 4,026(1,713–10,456)

Median—0.186(0.108–0.510)CML 9

CMML 1

MDS 8

PreB-ALL

1 Wildtype—21

Median = 3,554(1,990–10,450)

Median—0.240(0.097–0.494)

Severe AA 1 p value 0.3313 0.2361

PNH 2

Ph +ALL

1

JMML 1

SCID 1 Q6H(n = 16)

Median age—4 years

(2–7 years)

Variants—07

Median = 875(285–1,456)

Median—0.245(0.163–0.444)

THAL 16 Wildtype—09

Median = 670

(353–1,059)

Median—0.383(0.189–1.869)

p value 0.4079 0.2105

Abstract P 038

Targeted Dose Adjustment of i.v Busulfan in Patients UndergoingHematopoietic Cell Transplantation—CMC Vellore Experience

Ezhilpavai Mohanan, Poonkuzhali Balasubramanian, SalamunDesire, S Gopinath, Mammen Chandy, Vikram Mathews, AlokSrivastava, Biju George


Background: Busulfan is a bi-functional alkylating agent used in

combination with cyclophosphamide or fludarabine as a conditioning

regimen prior to hematopoietic stem cell transplantation (HSCT).

Intravenous (i.v) busulfan is preferred over oral busulfan due to reduced

inter-individual variation and better predictability in pharmacokinetics.

Busulfan has a narrow therapeutic window, with toxic side effects such

as sinusoidal obstruction syndrome (SOS) occurring at high systemic

exposure and graft rejection or relapse at low exposure, thereby sig-

nificantly influencing clinical outcome. We share our experience in

targeted dose adjustment of i.v busulfan in patients undergoing HSCT

for various hematological diseases in the past 2 years. Patients andMethods: Sixty patients diagnosed with various haematological dis-

orders receiving high dose i.v busulfan as combination chemotherapy

between June 2010 and July 2012, were included in this study. Forty

three patients received single daily (Q24H) whereas 17 had 6 hourly

dose of (Q6H) busulfan. Peripheral blood samples were collected at

different time points and plasma were immediately separated. Busulfan

levels were analyzed using a rapid and sensitive LC-ESI MS/MS

method with deuterated internal standard (d8-Bu) and quantified using

Multiple Reaction Monitoring (MRM). The results are interpreted as

ng/ml. Inter and intra-day variability of the standards and controls was

less than 10 %.An AUC of 5,000–5,500 lmol (cumulative AUC target:

20,000–22,000 lmol) in Q24H or 900–1,350 lmol in Q6H were tar-

geted. Results: Diagnosis of the patients, busulfan dosing interval and

plasma AUC on day 1 and 3 is listed in Table. Figure shows the first dose

and dose adjusted AUCs after Q24H and Q6H respectively. Five

patients (8 %) achieved targeted levels after first dose and required no

further dose adjustment, while 39 (65 %) patients achieved targeted

levels after first dose adjustment. However, in 16 (27 %) patients the

dose had to be further adjusted on day 3. Eight (13 %) of these patients

did not achieve the targeted AUC even after increasing the dose the

second time and in 8 patients (13 %) the dose had to be reduced from the

initial rise. However, the dose was not increased beyond 20 % at any

given time. When we analyzed the regimen related toxicity in these

patients, 13 (22 %) developed mucositis Grade III-IV; 7 developed

SOS. Three patients rejected the grafts and all of the three had primary

graft failure. Conclusion: The target level busulfan for Indian patients

undergoing HSCT with various hematological conditions has not been

studied. There is also no documented pharmacokinetics (PK) of i.vbusulfan in our population. From our experience with targeted dose

adjustment of i.v busulfan, we observed that majority of these patients

required dose escalation, which is very different from previous reports.

The reason for this needs further PK and pharmacogenetic studies in our

population.

Abstract P 039

Hematopoietic Stem Cell Transplantation in Acute Leukemias:Long Term Results, Impact of Conditioning Regimensand the Role of Prognostic Factors from a Tertiary CancerCenter in India

Jayant Gawande1, Ravi Thippeswamy1, Bhausaheb Bagal1,Sadhana Kannan2, Manju Sengar1, Hari Menon1, Reena Nair1,Navin Khattry1

1BMT Unit, Department of Medical Oncology, 2Department

of Biostatistics, ACTREC, Tata Memorial Centre, Mumbai

Objectives: Hematopoietic Stem Cell Transplantation (HSCT) is the

most curative option in acute leukemias. We retrospectively ana-

lyzed our data for survival outcomes, impact of conditioning

regimens on outcomes and role of possible prognostic factors.

Methods: One hundred and fourteen patients (101 allogeneic, 13

autologous) who underwent transplant for acute myeloid leukemia

(AML-81) and acute lymphoblastic leukemia (ALL-33) between

March 1983 and August 2011 were evaluated. Poor risk cytogenetics

were seen in 84 and 38 % patients of ALL and AML respectively.

Seventy nine patients (69 %) received full intensity (FI; cyclo-

phosphamide-total body irradiation Cy-TBI and busulphan-

cyclophosphamide Bu-Cy) and 21 patients (18 %) received fludar-

abine based reduced intensity (RI) conditioning. Prognostic factors

evaluated for overall survival (OS) and relapse free survival (RFS)

were WBC count at presentation, baseline cytogenetics, disease

status at transplant, dose of cytarabine consolidation (18 vs. \18 g/

m2) in AML, conditioning regimen (Cy-TBI vs. Bu-Cy and FI vs.

RI), time from diagnosis to transplant, duration of relapse free

interval (RFI) pretransplant and chronic Graft versus Host disease

(cGVHD). Univariate and multivariate comparisons of survival

times for potential prognostic factors were made using log-rank test

and cox regression analysis respectively. Results: Median age at

Diagnosis N=60 Bu dosing AUC (µMoles) Dose adjustment

AML 19

Q24H (n= 44)Median age : 32 yrs

(1-58 yrs)

Day 1Median = 3776(2407-10456)

Increase : 30Median – 15%(4% to 47%)

CML 9 Decrease : 09Median – 25%

(5%-38%)CMML 1

MDS 8Pre B-ALL 1

Day 3Median = 4986

(3220-7661)

Severe AA 1PNH 2 No Change : 05

Ph+ ALL 1

JMML 1SCID 1

Q6H (n= 16) Median age : 4 yrs

(2-7 yrs)

Day 1Median = 671

(285-1456)

Increase : 13Median – 19%(2% to 39%)

THAL 16 Day 3Median = 995

(557-1712)

Decrease : 03Median – 8%

(4%-13%)

Q24H

Day 1

AUC

Day 3

AUC0

5000

10000

15000P = 0.0397

AU

C (

uM

ole

s/m

in)

Q6H

Day 1

AUC

Day 3

AUC0

500

1000

1500

2000P = 0.0138

AU

C (

uM

ole

s/m

in)


123

transplant was 26 years. Median time from diagnosis to transplant

was 7 months. At the time of transplant, 59 % were in first remis-

sion (CR-1), 26 % in second remission (CR-2), 10 % in refractory

state and in 5 % status at transplant was not known. Median follow

up time was 46 months. The incidence of acute and chronic GVHD

was 46 and 36 % respectively. The transplant related mortality

(TRM) was 30 %. The TRM was significantly higher (P = 0.001) in

the pre 2007 period (42 %) compared to the post 2007 period

(10 %). The cumulative probabilities of OS and RFS at 8 years were

37 and 35 % for the whole group, 35 and 32 % for AML, 29 and

20 % for ALL respectively. On multivariate analysis, Cy-TBI con-

ditioning (P = 0.01; P = 0.012)) and chronic GVHD (P = 0.009;

P = 0.004) each were prognostic factors for both better OS and

RFS. On univariate analysis, CR at transplant (P = 0.029) and

RFI [ 9 months pre transplant (P = 0.017) were also associated

with better OS and RFS respectively while RI conditioning showed

a trend towards better OS (P = 0.087). Conclusion: Our long term

results suggest that at least one-third of patients with acute leukemia

are cured with HSCT. Patients with cGVHD and those receiving

Cy-TBI had better survival. Reduced intensity conditioning is an

option in patients with acute leukemias with at least comparable

results.TRM has reduced significantly over recent years probably

due to better supportive care.

Abstract P 040

Bacterial Contamination of Peripheral Blood Stem Cell Harvest:Incidence, Risk Factor and Clinical Outcomes

Bhausaheb Bagal1, Sumathi SH2, Rajib De1, Vijay Patil1, JayantGawande1, Shashank Ojha2, Vivek Bhat3, Preeti Chavan4, RohiniKelkar3, Navin Khattry1

1BMT Unit, Department of Medical Oncology, 2Department

of Transfusion Medicine, 3Department of Microbiology, 4Department

of Laboratory Medicine, Tata Memorial Centre, Mumbai, India

Introduction: Microbial contamination is a potentially serious

complication of peripheral blood stem cell (PBSC) harvest. Data on

incidence, organism type, outcomes after infusion and risk factors

is scarce. Methods: We studied medical records of 195 patients

(autologous) and donors (allogeneic). Bacterial cultures of PBSC

harvest were obtained immediately after PBSC collections, prior to

freezing and after thawing of cryo-preserved PBSC. Risk factors

analyzed for positive culture were type of transplant, method of

mobilization, use of CVC versus peripheral venous access for

apheresis, number of days of collections and total blood volume

processed. Results: 155 subjects underwent apheresis yielding 334

PBSC bags (112 from healthy donors and 222 patients). Apheresis

of adequate PBSC required median of 2 days (range 1–5). Median

volume of blood processed per collection was 10.7 l. The incidence

of microbial contamination was 9.8 %. Microorganism cultured

were skin commensals (n = 19), gram negative pathogens (E. coli-4, Kleibsiella-8 and Proteus mirabilis-2). Seven post thaw samples

grew an organism, all of which were skin commensals. Only one

patient receiving culture positive PBSC harvest developed fever

with rigors during infusion. No microorganism present in the PBSC

was recovered from blood cultures collected in post-transplantation

period. Total blood volume processed greater than 10.7 liters was

associated with higher likelihood of positive culture in PBSC

harvest (p = 0.01). Conclusion: Our experience suggests infusion

of contaminated PBSC graft does not play a significant role as a

source for infections in the HSCT setting and higher blood vol-

umes processed during apheresis increases the risk of bacterial

contamination.

Abstract P 041

Breast Cancer Stem Cells (BCSCs): An Implication to Therapyin Chemoresistant Breast Cancer

Preetha Bhadra, S Gangopadhyay, S Sengupta, A Mukhopadhyay

Netaji Subhas Chandra Bose Cancer Research Institute, 16A Park

Lane, Kolkata-700016, West Bengal, India

Objective: Invasive and mesenchymal property of BCSCs with

CD44+/CD24low/ALDH1+ phenotype has made them promising tar-

get for targeted treatment. Chemotherapy treatment uses medicine to

weaken and destroy cancer cells in body, including cells at original

cancer site and any cancer cells that may have spread to another part

of body. Chemotherapeutic drugs for breast cancer are not well

defined yet. Combination of drugs is also not fully known. Our

objective is to define chemotherapeutic drugs and its action in breast

cancer. Methods: Total of 20 early chemo-naıve patients with biopsy

proven triple-negative metastatic breast cancer in age group of

18–36 years were selected randomly and tested for CD44/CD24 cell

analysis. Isolated BCSCs were cultured for in vitro drug sensitivity

towards Platinum, Anthracyclin, Docetaxel, Rapamycin, Sunitinib,

Sorafenib, Gefitinib. Correlation was drawn between cell differenti-

ations, % of stem cells and drug response. Accordingly chemotherapy

was designed for particular patient. % of BCSCs in pre and post-

chemotherapeutic condition was further compared. Results: We have

detected BCSCs in 90 % of cases. Among positive sample shows

in vitro drug sensitivity 4 (20 %) to Rapamycin, 1 (5 %) to Sunitinib,

1 (5 %) to Sorafenib, 1 (5 %) to Gefitinib, 3 (15 %) to Platinum, 1

(5 %) to Anthracyclin, 1 (5 %) to Docetaxel and rest shows no sen-

sitivity to any drug. Conclusion: Thus primary aim to target BCSCs

at onset of tumors in breast cancer patients to control metastasis and

relapse of disease was somewhat obtained. We further plan to cor-

relate ratio of selected markers present in pre and post-

chemotherapeutic condition with time to recurrence, mortality, mor-

bidity and progression free survival. If no BCSCs prevail after

chemotherapy, patients would be kept under observation and if traces

are found, we would proceed to targeted therapy.

Abstract P 042

Infections in Hematopoietic Stem cell Transplant Recipients—an12 Year Single Centre Experience of 207 Cases

N Grover, Nair Velu, S Bhattacharya, A Sharma, AK Sahni,S Das, L Singh, GS Chopra, S Sharma, R Kapoor, V Behera

AFMC, Pune

Background: Infections are the main cause of mortality and mor-

bidity in haematopoietic stem cell transplant (HSCT) patients

despite prophylaxis and newer anti microbial drugs. Aim: To study

the various infections in HSCT recipients at a large tertiary care

hospital of India between Feb 1998 to March 2010. Study Design:Retrospective case series. Methods: Total 207 patients underwent

HSCT with proper sterile precautions. Infections were diagnosed

according to the results of clinical examination or specific diagnostic

procedures and were defined based on CDC criteria. Bacterial

infections were documented on positive cultures, fungal infections

documented as per CDC criteria and viral infections diagnosed by

RT-PCR results. Proper antibiotic, antiviral and antifungal therapy

given whenever infection was suspected/documented. Results:164/207 patients (79.2 %) had one episode of fever and 35/207


123

(16.9 %) had second episode of fever. Second episode of fever was

more frequent in patients who underwent allogenic (23.3 %) than

autologous (4.2 %) and who nursed under clean side room (18/28)

versus protected environment (17/179) with p \ 0.001. Total 267

documented infections occurred in 207 transplants in which 134

(48.9 %) were bacterial, 98 (35.7 %) viral, 38 (14 %) fungal and 1

had plasmodium falciparum. Pathogens identified were more in

allograft than autograft transplants. Gram negative infections were

identified more in which the most common was E. coli. CMV was

the most common viral and candida the common fungal infection

documented. Death occurred in 72 patients in which 50 were post

allogenic. Conclusion: Overall incidence of all infections and feb-

rile episodes was high and were comparable to both western and

Indian studies. Infections were commoner in allograft than in

autograft patients. Major causes of death included fungal pneumonia

and bacterial pneumonia.

Abstract P 043

Long Term Results of Autologous Transplants for Hodgkin’sLymphoma—a Retrospective Multi-Center Analysis from India

Reetu Jain1, Navin Khattry2, Ravi Thippeswamy3, AdwaitaGore4, Bharat Bhosale5, Nandish Jeevangi6, Sadhana Kannan7,Anjana Sainani8, Ganapathy Bhat9, Reena Nair10, TapanSaikia11, On Behalf of Indian Study Oncology Group Trust

1,8,9Department of Medical Oncology, Jaslok Hospital and Research

Centre, Mumbai; 2,3,5,6,7,10BMT Unit, Department of Medical

Oncology, ACTREC, Tata Memorial Center, Mumbai;4,11Department of Medical Oncology, Prince Aly Khan Hospital,

Mumbai; 1Dr.G.Deshmukh Marg, Mumbai 26, India

Introduction: Autologous transplantation is the standard of care for

patients of relapsed and refractory Hodgkin’s lymphoma (HL). We report

the results of transplants from three centers in Mumbai and role of possible

prognostic factors. Methods: One Hundred and three patients underwent

transplant for HL from August 1994-May 2011. All patients received 2–3

cycles of DHAP, ESHAP, MINE, Mini-BEAM, GDP or ICE with or

without rituximab as salvage chemotherapy. Cheson’s response criteria

was applied for assessing response pre and post transplant at day 100.

Forty-eight percent of patients received BEAM (carmustine, etoposide,

ara-c and melphalan), while 52 % received LACE (lomustine, ara-c,

cyclophosphamide and etoposide) regimen. Ninety-two percent received

peripheral blood stem cells. Prognostic factors evaluated for progression

free survival (PFS) and overall survival (OS) were stage at diagnosis,

presence of B symptoms at diagnosis, time from diagnosis to transplant,

number of lines of chemotherapy pretransplant, remission status at trans-

plant, conditioning regimen, pretransplant salvage chemotherapy and

serum albumin at transplant. Univariate and multivariate comparisons of

survival times for potential prognostic factors were made using log-rank

test and cox regression analysis respectively. Results: The median age at

transplant was 23 years. B symptoms were present in 47 % at diagnosis.

The median time from diagnosis to transplant was 1.9 years. Fifty-seven

percent of patients had stage III and IV disease at diagnosis. At transplant,

41 % were in complete remission (CR) Sixty percent of patients who

received platinum- based salvage achieved CR compared to 30 % of those

who received ifosfamide (P = 0.012). Median number of lines of che-

motherapy pretransplant was 2 and median serum albumin at time of

transplant was 4 g/dl. Transplant related mortality was 11 %.The cumu-

lative probability of OS and PFS at 3 years were 65 and 49 % respectively.

Serum albumin[4.0 g/dl (P = 0.03) and CR at transplant (P = 0.025)

were associated with better OS while CR at transplant was associated with

improved PFS in multivariate analysis. Conclusion: Our data suggest that

serum albumin and CR at transplant are important factors affecting

survival. Platinum based pre-transplant salvage regimens achieved better

CR rates than predominantly ifosfamide based regimens.

Abstract P 044

Role of Reduced Intensity Conditioning Allogeneic Stem CellTransplant for Young Adults with Acute Myeloid Leukemiain First Complete Remission

Chepsy C Philip1, Abhijeet Ganapule1, Rayaz Ahmed1,Aby Abraham1, Biju George1, Kavitha M Lakshmi1, AlokSrivastava1, Vikram Mathews1

1Department of Clinical Hematology; CMC Vellore

Introduction: Allogeneic hematopoietic stem cell transplantation

(HSCT) is an effective treatment for patients with Acute Myeloid Leu-

kemia (AML). In AML, graft versus leukemia contributes significantly to

durable remission. Reduced intensity conditioning (RIC) is frequently

indicated in the elderly or those with co-morbidities. There is limited data

on clinical outcomes in young adults with AML with a RIC allogeneic

HSCT. Methods: From 2005 we have been using reduced intensity

conditioning with Fludarabine and melphalan in patients with AML in

CR1. The reason to change from myeloablative conditioning was our

concern about the high treatment related mortality with our earlier my-

eloablative regime. We performed an analysis of 46 consecutive patients

who received RIC. Kaplan Meir method was used to generate event free

survival (EFS) and overall survival (OS). Results: The median age of the

recipients was 34 (11–63). All patients engrafted. Median time to

ANC C 500 and platelets C20,000 was 14 and 17 days respectively.

Acute GVHD was seen in 24 patients [Grade 2–4: 20 (43.4 %); Grade

3–4: 09 (19.5 %)] and Chronic GVHD in 28 recipients [Limited: 21

(45.6 %); Extensive: 07 (15.2 %)] 0.4 recipients (8.7 %) relapsed. There

were 12 deaths. Treatment related mortality was as follows

(B100 days = 6.5 %, n = 03; B1 year = 17.3 %, n = 08). The esti-

mated EFS was 68.6 % and OS was 71 % at 2 years with a mean follow

up of 51 and 53 months respectively. Conclusion: The use of reduced

intensity conditioning is a reasonable option to consider in young adults

with AML in CR1.

Aplastic Anemia

Abstract P 045: Oral presentation

Pancytopenia: A Clinico Hematological Study

Vijai Tilak1, Madhukar Rai2, Vikram Singh3

Department of Pathology, Institute of Medical Sciences, B.H.U.,

Varanasi; 2Department of Internal Medicine, Institute of Medical

Sciences, B.H.U., Varanasi; 3Department of Internal Medicine,

Vir Chandra Singh Garhwali Govt. Institute of Medical Sciences

& Research, Srinagar, Pauri Garhwal, Uttarakhand

Objectives: (1) To study the different causes of pancytopenia. (2) To

evaluate the incidence of various disorders causing pancytopenia in chil-

dren and adults. (3) To explore a cost effective and rapid test to differentiate

between Megaloblastic anemia from megaloblastosis of myelodysplastic

syndrome (MDS). (4) To compare our data with other studies on pancy-

topenia. Material and Methods: The present study was conducted in the

Division of Hematology, Department of Pathology, Institute of Medical

Sciences over a period of 45 months on 428 patients of pancytopenia.

Results: The age of the patients ranged from 3 to 73 years. There was male


123

preponderance. The overall most common cause of pancytopenia was

Aplastic anemia followed by Megaloblastic anemia. Aplastic anemia and

acute Leukemia often bleed whereas Megaloblastic anemia rarely bleed.

Patients with aplastic anemia and acute Leukemia were often children.

Conclusions: Childhood pancytopenia is a distinct entity with a grave

prognosis vis-a-vis adult and elderly pancytopenia. Serum LDH is a cost

effective and rapid test to differentiate megaloblastosis of vitamin B12 and/

or folate deficiency from MDS. Bone marrow examination can be deferred

in pancytopenic patients with hypersegmented neutrophils; it can be per-

formed later if there is no response to hematinics.


Chronic Benign Neutropenia—a Case Report

Rachana Garg

KMC Manipal

Introduction: Chronic benign neutropenia is defined as absolute

neutrophil count (ANC)\1,000 cells/mm3 in infants and\1,500 cells/

mm3 in children and adults, persistently for more than 3 months. CaseHistory: 1 year old male child presented with fever for 5 days and

upper respiratory tract infection. Clinical examination was normal. His

peripheral blood smear showed ANC of 120 cells/mm3 with eosino-

philia and monocytosis. Bone marrow aspiration showed myeloid

hyperplasia with increase in precursor cells. IgG was raised. Child was

evaluated and no acquired immunodeficiency was seen. He was given

3 doses of G-CSF following which neutrophil counts improved,

however counts done 4 days after G-CSF again showed ANC -252

cells/mm3. Conclusion: Chronic benign neutropenia is a rare disorder,

but commonest cause of neutropenia in children in age group

(3–30 months). Careful evaluation is required to avoid misdiagnosing

it as leukemia or other hematological malignancies. In these cases

close follow up of patient is needed to assess the course of the disease.

Abstract P 047

A Clinico-Pathological Study of Cases of Pancytopenia—aHospital Based Study

Abhijit Phukan, Jina Bhattacharyya, DK Saikia, PK Gogoi

GMCH

Objective: This study was conducted to evaluate. (1) The disease

patter in different age and sexes. (2) Their clinical presentation. (3)

Various causes of pancytopenia and their relative frequencies in this

region. (4) Haematological parameters and bone marrow finding in

various causes. Methods: The study was conducted in the Depart-

ment of Pathology, GMCH, Guwahati, extending over a period of one

year from 1st July 2011 to 30th June 2012. The criterion of selection

of the patients was: (1) Hb \ 9 g. (2) Total leucocyte count \4,000/

mm3. (3) Platelet count\1 lakh/mm3. All the patients were subjected

to PBS study, bone marrow aspiration and biopsy. We also did special

stains, flow cytometry and cytogenetics as and when necessary.

Results: From the study of 88 cases, various important results can be

summarised as (1) Maximum cases Pancytopenia are observed in the

age group of 31–40 years, with male predominance (61 %). (2)

Common presenting features are generalized weakness (100 %),

bleeding (23 %) etc. (3) Among the various causes, Megaloblastic

anaemia is found to be the most prevalent (54 %). (4) Haematological

parameters vary according the causes. (5) Hypercellularity of marrow

is a feature in most of the cases (84 %). Conclusion: This study will

help in finding the disease scenario in this part of the world.

Abstract P 048

Antilymphocyte (ALG)/Antithymocyte Globulin (ATG)for Severe Aplastic Anemia—Single Centre Experience

Venkatesh S Ekbote, Biju George, Vikram Mathews,Aby Abraham, Riyaz Ahmed, M Kavitha, Alok Srivastava

Department of Hematology, Christian Medical College, Vellore,

Tamil Nadu

Objective: To analyze response to immunosuppressive therapy with

ATG/ALG in children and adults with severe and very severe aplastic

anemia treated between 1985 and 2012. Methodology: We conducted a

single institution retrospective analysis of severe and very severe aplastic

anaemia patients treated with ATG-based IST from 1989 to 2012.

Response, based on EBMTR guidelines, was determined at 3 and

6 months following ATG. Results: Two hundred ninety six patients with

Severe and Very Severe Aplastic Anaemia with median age of 38 years

(range 3–83 years) were studied, including 198 (66.9 %) males and 98

(33.2 %) females. The overall objective response (CR + PR) to ALG/

ATG CSA in this cohort was 50 %. Patients in younger age group

(1–15 years) had poorer response rate—34 % compared to adults who

had comparable response across all age groups; (16–29 years)—55 %,

(30–50 years)—56 % and more than 50 years—59 %. Also, 32

(10.8 %) patients died of infection within first month of ATG/ALG

treatment. On follow up, 14 patients (4.7 %) lost response while 13

(4.3 %) underwent clonal evolution to paroxysmal nocturnal haemo-

globinuria, myelodysplastic syndrome or acute myeloid leukemia. Seven

(2.4 %) patients underwent allogeneic stem cell transplantation. The

overall survival in this cohort of patients was 57 %. It was 51, 52, 59 and

71 % in age groups: 1–15, 16–29, 30–50 and more than 50 years,

respectively. Conclusion: This study suggests that ATG is a viable

option in adult Indian patients even more than 50 year old. The poorer

response seen in Indian children needs to be studied further.

Abstract P 049

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

Priti, Anil Sood, KS Rana, N Varma

Department of Hematology, Post Graduate Institute of Medical

education and Research, Chandigarh

Introduction: Fanconi Anemia (FA) is the most common chromo-

somal breakage (CB) syndrome and is the prototype of inherited bone

marrow failure syndrome (BMFS). FA cases present during childhood

or at times later in adulthood. Other chromosomal instability syn-

dromes not characterized by BMFS include Nijmegen breakage

syndrome (NBS), Ataxia telangiectasia (AT), Bloom’s syndrome and

Xeroderma pigmentosa. Clinical heterogeneity of these disorders

often leads to an incorrect or missed diagnosis. Although clinical

suspicion index may be high for these disorders, definitive diagnoses

(many times) based upon chromosomal breakage studies (CBS) are

not well documented in India. This study was carried out to document

the spectrum of these syndromes observed in patients with and

without BMFS, at PGIMER, Chandigarh. Materials and Methods:The study included consecutive patients referred for work up of

chromosomal breakage syndromes, between Jan 2011 and August

2012. In our department, appropriate phytohemagglutinin (PHA)

stimulated peripheral blood lymphocyte cultures (PBLC) are set up

for patients and controls, with and without clastogens [1. diepoxy-

butane (DEB) and 2. mitomycin C (MMC) for Fanconi Anemia; 1.


123

DEB induced CBS, 2. X-irradiation and 3. treatment with bleomycin

at G2 phase of cell cycle for Ataxia telangiectasia; and 1. PHA

stimulated PBLC: (a) spontaneous breakage (b) G-banding for

translocations, 2. DEB induced CBS, 3. MMC induced CBS 4.

X-irradiation and 5. treatment with bleomycin at G2 phase of cell

cycle for NBS]. Result: Out of 104 patients studied, 11 were diag-

nosed as Fanconi Anemia, 2 as Ataxia telangiectasia, whereas NBS

was diagnosed in just 1 patient. Median age of FA patients was

8 years (range 5–32 years). Both the AT patients were males. Patient

with Nijmegen breakage syndrome was an 18 months old boy. As

highlighted earlier, work up for chromosomal breakage syndromes is

extremely time consuming, though clinically very rewarding. Con-clusion: Approximately 130 cases of Ataxia telangiectasia have been

reported worldwide so far. Nijmegen breakage syndrome patient

included in this study is the second case reported in India. The exact

incidence of AT and NBS in India is not known. FA cases are being

diagnosed at few centers in India (including our department), however

the facilities to undertake specialized chromosomal breakage studies

for patients without BMFS need to be widely established. This is

required for appropriate genetic counseling and patient management.

Unless specific confirmatory tests using a battery of clastogens, are

performed, such cases will continue to remain under-diagnosed.

Chronic Leukemia

Abstract P 050

A Case of Early Ocular Manifestation of Maculopathyin a 37 year Old Male Patient of CLL

Sufia Khan, Syed Riaz Mehdi, Sharique Ahmad, Parul Gupta

Era’s Lucknow Medical College, Lucknow, UP

Abstract: We are presenting a case of 37 year old male patient

referred from medicine OPD for routine Complete Blood Count

(CBC). His Total Leukocyte Count (TLC) came out to be alarmingly

high .His general blood picture (GBP) and bone marrow examination

revealed it to be a case of Chronic Lymphocytic Leukemia (CLL).

Later on immunophenotyping was also performed. His cervical lymph

node biopsy was reported as a case of chronic lymphocytic leukemia/

small lymphocytic lymphoma. Subsequently, on his ophthalmic

examination maculopathy was detected. CLL in young male has not

yet been reported from India.

Abstract P 051

Clinico-Haematological Analysis of Haematological Malignancy,A Hospital Based Study

Rahul Varshney, Muktanjali Deka, Jina Bhattacharya, PK Gogoi

GMCH

Objective: Early diagnosis of haematological malignancy is important in

clinical practice to prevent mortality. This study was conducted to evaluate

their relative frequencies in different ages and sexes in this region.

Methods: The present study was conducted in the Department of

Pathology and Haematology, Gauhati Medical College & Hospital, Gu-

wahati from 1st July 2011 to 30th June 2012. Patients were examined and

blood tests including haemoglobin estimation, total and differential leu-

kocyte count, platelets count, reticulocyte count and PBS study, Bone

marrow aspiration and biopsy, special stains, flow cytometry and cyto-

genetics was done in each cases. Results: Out of 450 patient attending

Haematology OPD 236 were having malignancy. About 29.66 % patient

had chronic myeloid leukaemia while 1.69 % patient had chronic

lymphocytic leukemic. Among acute leukaemia, acute myeloid leukaemia

outnumbered acute lymphoblastic leukaemia, (21.19 %against 7.2 %).

Multiple myeloma was seen in 20.33 %. Non Hodgkin’s lymphoma was

seen in 10.59 % while Hodgkin’s disease 6.6 %, MDS 1.69 %, MPN and

LCH 1 %. Male to female ratio in haematological malignancies was

2.87:1. Maximum cases were observed in 51–60 years. Low grade fever,

progressive pallor, weakness and body aches were the commonest

symptoms (70 % cases) while pallor was the frequently observed sign.

Conclusion: The prevalence of haematological malignancy is

52.44 %.This study may help in finding out their relative distribution in

this region.

Abstract P 052

Monitoring of Response to Therapy in Chronic MyeloidLeukaemia Patients by Quantitative Real Time PCRon the International Scale: Our Experience in CML Patientsin a Tertiary Care Centre in India

J Kotwal, BK Chakrabarty, R Kapoor, V Nair, V Dutta

Armed Forces Medical College, Pune

Background: With advent of Tyrosine kinase inhibitors (TKIs),

treatment and prognosis of Chronic myeloid leukaemia (CML) has been

revolutionized. The patients go into complete haematological, cytolo-

genetic (CCyR) and major molecular remission (MMR) with therapy.

This is highly desirable monitoring reliability of RQ-PCR assay should

be optimized in different laboratories. The IRIS group has advised

cytogenetic remission but Adelaide group has favoured molecular

monitoring to be more effective as cytogenetic remission corresponds to

3 log reduction at molecular level. In our laboratory in 2005 Bcr-Abl

transcription was reported as copies/ml. Afterwards Bcr-Abl/Abl ratio

expressed as percentage was started using the M BCR fusion Quant kit

by Ipsogen in Rotor gene 3000. Base line value of Bcr-Abl/Abl ratio was

calculated by taking median of 30 newly diagnosed cases of CML. With

further improvements now Bcr-Abl Mbcr kit, compliant with updated

international recommendations and contains an IS-MMR calibrator,

aligned with international scale is used for converting NCN results to

the International scale and report MMR. Methods: RQ-PCR on Light

cycler 480 is used for monitoring of patients. WHO validated and

accredited reference reagent and IS MMR calibrator is used in each run

to calculate MMR on the IS scale. Results: Our experience related to 50

CML patients on therapy will be presented. Conclusion: Goals of

therapy are to reach major molecular remission. It seems desirable that

use of RQ-PCR for monitoring response to treatment for leukemia

should follow the same pattern to satisfy criteria for clinical use as

required by the appropriate international regulatory agencies.

Keywords Chronic myeloid leukemia, Complete molecular response,

Major molecular response, RQ-PCR, IS scale

Abstract P 053

Incidence of Different BCR-ABL Transcript Variants AmongstCML Patients: Their Correlation with Presenting Features,Relative Risk and Response to Treatment with Imatinib Mesylate

Pratik Deb, Prantar Chakrabarti, Utpal Chaudhuri,Shila Chakrabarti

Institute of Hematology and Transfusion Medicine, Medical College,

Kolkata


123

Objective: To establish how different transcript variants of BCR-

ABL play their role in the disease process of Chronic Myeloid Leu-

kemia in our patient population. Method: RNA extraction, RTPCR,

Spectrophotometry and agarose gel electrophoresis, Statistical anal-

ysis. Results: Amongst 80 patients (56 male, 24 female) 56.25 %

patients showed the presence of b3a2 and 41.25 % patients showed

the presence of b2a2 transcript variant. The rest 2.5 % (numerically, 2

cases) showed the expression of rare e19b2 variety. Patients with

b2a2 transcript variant presented with higher Sokal, Hasford and

EUTOS score than the patients with b3a2 transcripts. Difference

between different parameters like Platelet count, TLC, Hemoglobin,

Organomegaly was minor. After receiving almost 18 months of I-

matinib therapy, patients with b2a2 reached hematological and

cytological remission more than their counterpart. Conclusion: CML

patients in our population with b2a2 variants tend to present with

higher risk scores but respond better to Imatinib therapy.

Abstract P 054

Standardisation of RTPCR for Confirmatory Diagnosisof Chronic Myeloid Leukemia: First Time in a Tertiary HealthCare and Teaching Hospital in West Bengal

Rajarshi Aich, Pratik Deb, Utpal Choudhuri, Shila Chakrabarty,Prantar Chakrabarti

Institute of Haematology and Transfusion Medicine, Kolkata

Objectives: Our challenge was to standardise a RTPCR protocol, for

the first time in a tertiary health care system of West Bengal, for the

confirmatory diagnosis of CML patients, and, at the same time, keeping

the procedure as costeffective as possible. Methods: Total 80 patients

were selected amongst which, 60 were diagnosed cases and 20 were

suspected cases of CML. Total RNA extraction was done from the

blood of all the patients and cDNA was made from it by reverse

transcriptase PCR using random primers. PCR amplification of the

BCR-ABL gene of cDNA was done using primers of standardized PCR

protocol of BIOMED1. The PCR products were subjected to 2 %

agarose gel electrophoresis and observed under UV light. Result: The

PCR reaction was able to successfully amplify the target products of

BCR-ABL. All 20 suspected cases of CML were diagnosed to be car-

rying BCR-ABL through our test and later 18 of those diagnosed cases

were confirmed by bone marrow cytogenetics (BMC). The 2 failed

cases were confirmed to be CML through FISH, thus establishing RT-

PCR’s higher sensitivity than BMC. All the established cases gave

positive results. Conclusion: Our present study has led us to the

development of a standardised RTPCR protocol for confirmatory

diagnosis of CML. It had also affirmed that the primer pair combination

used in Europe is properly working in our population.

Abstract P 055

Induction of Apoptosis in Chronic Myelogenous HumanLeukemic Cells K562 and U937 by Trichosanthes dioica LeafExtract

Nilanjana Deb1, Moumita Ray1, Biswajit Chakraborty2, ChinmayChowdhury2, Sugato Banerjee 3, Aparna Gomes1, Shila ElizabethBesra1



Kolkata-700032, West Bengal, India; 2Department of Chemistry,

Indian Institute of Chemical Biology, (C.S.I.R), 4 Raja S. C. Mullick

Road, Kolkata -700 032, West Bengal, India; 3Gupta College

of Technological Sciences, Ashram More, Asansol-713301

Objective: Plants have been an immense source of therapeutics. Cancer,

a dreadful disease is a leading cause of death worldwide. Synthetic anti-

cancer agents used in chemotherapy are generally toxic or immunosup-

pressive. But compounds from natural sources tend to have more

immunostimulants properties. We have studied the apoptosis on CML

cell lines along with the immunomodulatory effects of the leaf extracts of

Trichosanthes dioica. Method: We studied cell viability, cytotoxicity

and apoptosis on two CML cell lines. Cell morphology was determined

by Fluorescence and Confocal microscopy. DNA fragmentation was

studied by gel electrophoresis. Effect of TDLE on NO production was

studied in RAW264.7 cell line. Result: TDLE significantly inhibited the

cell viability and cytotoxicity (MTT) in a time and concentration

dependent manner. The fluorescence and Confocal study showed char-

acteristic features of membrane blabbing, chromatin condensation the

sign of early and late apoptotic changes in the cells after treatment with

TDLE. Gel electrophoresis study showed fragmented DNA in the form of

ladder. It was found that TDLE was able to induce NO production in

resting RAW264.7 cell line. But when TDLE was used in combination

with recombinant interferon-c, there was a marked cooperative induction

of NO production in RAW264.7 cells. Conclusion: The present study

reveals that the TDLE possesses potent anti-leukemic with immuno-

modulatory activity. Trichosanthes dioica constitute a major part of our

every day diet. The compounds from TDL can be used as immunother-

apeutic agents for cancer treatment. Studies are in progress to identify the

mechanism of anti-leukemic activity of TDLE.

Abstract P 056

Concordance Between ZAP-70 and IgVH Mutation Statusin Chronic Lymphocytic Leukemia

Ritu Gupta1, Lata Rani1, Nitin Mathur1, Atul Sharma2,Lalit Kumar2, Vinod Raina2

1Department of Laboratory Oncology, Dr. B.R.A. IRCH, AIIMS,

New Delhi, India-110029; 2Department of Medical Oncology,

Dr. B.R.A. IRCH, AIIMS, New Delhi, India-110029

Objective: Following gene expression profiling, ZAP-70 has emerged

as the most promising surrogate marker for the IgVH mutation status, a

robust prognostic marker in CLL. Western studies using flow cytom-

etry for ZAP-70 with 20 % cutoff value, showed an overall correlation

between ZAP-70 expression and IgVH mutational status ranging from

77 to 90 % concordance. However, there are no such data from Indian

population. We aimed to investigate the optimal cut off of ZAP-70

with IgVH mutational status and its concordance in CLL patients from

India. Methods: Peripheral blood samples from 100 B-CLL patients

were assessed for ZAP-70 and IgVH mutation status. ZAP-70

expression was analyzed on CD5+CD19+ CLL cells using multipara-

metric flow cytometry. For IgVH mutation status, RNA was extracted

from peripheral blood MNC, transcribed to cDNA which was ampli-

fied by PCR according to BIO-MED-2 protocol, using VH1-VH6

family FR1 primers and 30JH consensus primer. PCR products were

purified and sequenced with an automated DNA sequencer. Ig-BLAST

was used for comparing rearranged IgVH segments; cases with C98 %

identity to germline were classified as unmutated (UM) while cases

with\98 % identity were considered mutated (M). Results: Among a

total of 100 patients, the number of mutated and unmutated cases were

62 and 38 respectively. Using ROC analysis, the optimal cut-off value

for ZAP-70 was found to be C22 % (83 % sensitivity, 81 % speci-

ficity). Using this cut off value, 59 patients were ZAP-70 negative

while 41 patients were positive for ZAP-70. Out of 59 ZAP-70 neg-

ative, 52 (88.1 %) were mutated and 7 (11.8 %) were unmutated. Out

of 41 ZAP-70 positive patients, 31 (75.6 %) were unmutated and 10


123

(24.3 %) were mutated. Therefore, a total of 83 % cases were con-

cordant for ZAP-70 cut off and IgVH status (ZAP-70+UM, ZAP-

70-M) whereas 17 % cases were discordant (ZAP-70-UM, ZAP-70+

M). Conclusion: A cut off value of C22 % for ZAP-70 correctly

predicted the IgVH mutation status in 83 % cases in Indian population.

Abstract P 057

VEGF in CML: significance and Correlation with Phaseof Disease

Purvi Mathur1, Usha Rusia1, Meera Sikka1, RK Grover1

1Department of Pathology, University College of Medical Sciences

& GTB Hospital, Delhi-110095, India; 2Delhi State Cancer Institute

Background: Current data suggests that angiogenesis plays a significant

role in the progressive growth and metastasis of many tumors, including

hematological malignancies. Vascular endothelial growth factor (VEGF)

is a key cytokine involved in the angiogenic process in tumors leading to

acceleration of tumor growth rate, invasion and metastasis. The current

study focused on evaluating the serum levels and bone marrow expres-

sion of this cytokine in Chronic myeloid leukemia. Aims andObjectives: The present study aimed to document the serum level of

vascular endothelial growth factor (VEGF) in different stages of Chronic

myeloid leukemia (CML), to correlate the levels with clinicopathological

factors and to study the immunohistochemical expression of VEGF in

bone marrow in these patients. Materials and Methods: Serum levels of

VEGF were evaluated in 40 Philadelphia chromosome positive patients

of Chronic myeloid leukemia (CML) and 40 age and sex matched healthy

controls by using commercially available ELISA kits. Bone marrow

biopsies were obtained from 50 patients of CML. Biopsies from 10

patients suspected of lymphoma which were morphologically normal

were used as controls. Immunohistochemical staining of VEGF was

performed by the labeled streptavidin avidin biotin method to establish

it’s cellular distribution in bone marrow. Results: Serum VEGF levels

were found to be significantly (p \ 0.0001) elevated in patients as

compared to controls and in patients in blast and accelerated phase of

CML as compared to those in the chronic phase (p = 0.02).Correlation of

serum VEGF levels with established prognostic factors showed a positive

correlation between serum VEGF levels and total leucocyte counts

(p = 0.04), blast percentage (p = 0.006), Hasford score (p = 0.02) and

a positive trend with splenomegaly (p = 0.06). The cellular distribution

of VEGF in the bone marrow was found to be similar in both patients and

controls, being positive in megakaryocytes and negative in erythroid cells

and neutrophils. The myeloid precursors showed a variable positivity for

VEGF. Conclusion: Serum VEGF levels may play a role as an important

independent prognostic factor in patients of CML. The marked elevation

of VEGF levels in patients of blast crisis as compared to the chronic phase

indicates that raised VEGF levels could signify advanced disease stage.

The findings demonstrated in the study suggest that antiangiogenic

therapy could potentially be successful in treatment of these patients.


The Effect of Imatinib Mesylate Therapy on Bone MarrowMorphology in Chronic Myeloid Leukemia Patients—anImmunohistochemical Study

Kamal Preet1, Usha Rusia1, Meera Sikka1, RK Grover2


& GTB Hospital, Delhi, India; 2Delhi State Cancer Institute

Background: Chronic myeloid leukemia (CML) is the commonest

leukemia occurring in Indian adults. Imatinib Mesylate is now being

used in these patients as first line of treatment. There is scarcity of

data on the effect of this drug on bone marrow morphology from

Indian subcontinent. Moreover there is no study which has objectively

tried to measure the effect of Imatinib Mesylate on stem cells and

proliferation in Indian patients of CML. Aims: To evaluate the

morphological changes in the peripheral blood, bone marrow aspi-

rates and biopsies in patients of CML before and after treatment

(6 months) with Imatinib Mesylate and to study the effect of Imatinib

on proliferating marrow stem cells using CD34 and PCNA. Materialand Methods: Morphological features were studied on peripheral

blood, bone marrow aspirates and biopsies in 30 patients of CML at

presentation and at the end of six months of Imatinib therapy.

Results: The M:F ratio was 2.2:1 and the median age was 30 years.

Common complaints at presentation were splenomegaly (97.7 %),

anaemia (82 %), weakness (51 %), pain abdomen (53 %) and fever

(40 %). At the end of 6 months of therapy, 96 % of the patients

achieved complete hematological remission. The bone marrow aspi-

rates and biopsies showed reduction in overall cellularity, reduction in

the M:E ratio, reappearance of erythroid colonies and relative

decrease in myeloid precursors in all patients except one. Megakar-

yocytes showed normalization of morphology and numbers.

Myelofibrosis post therapy was seen in 48 %patients. Only one

patient had significant myelofibrosis (grade 2-3). A statistically sig-

nificant reduction in the number of CD34+ progenitors from 20 to 50

cells/hpf pre therapy to 3-35 cells/hpf post therapy was observed. Post

therapy PCNA counts were 42-126 cells/mm2 which were markedly

lower than the initial counts. Conclusion: Imatinib mesylate is an

effective drug as first line treatment of CML patients. Apart from

causing haematological remission, it also effectively acts on the

CD34+ stem cells and proliferating fraction in CML. Sequential bone

marrow biopsies can be useful in monitoring disease remission and

progression in CML patients, hence bone marrow biopsies need to be

an integral part in the follow up and management of CML patients.

Abstract P 059

Different Clinical and Haematological Features in CML PatientsDuring First Presentation—a Study of 166 Patients Seenat Referral Centre

Chetan Jain, Palash Kumar Mandal, Nirmal KumarBhattacharya, Swapan Kumar Sinha

Medical College, Kolkata

Introduction: Chronic myelogenous leukaemia (CML) is a common

myeloproliferative disorder (MPD) seen in our country and world

over. The condition has varied clinical presentations and haemato-

logical features but demonstration of Philadelphia chromosome is the

surest test for a confirmation at present.CML may be seen in one of

three stages-chronic phase (CP) or accelerated phase (AP) or blast

phase (BP), each having different features. Objectives: To analyze

the different clinical and hematological features at presentation of

patients diagnosed as chronic myelogenous leukaemia (CML). This

study was conducted in Medical College, Kolkata from July 2009 to

June 2012. A total of 166 patients of chronic myeloid leukaemia were

included in the study. The diagnosis was made on clinical history,

physical examination, complete haemogram, bone marrow examina-

tion and cytogenetic study. Results: Out of 166 CML patients, males

predominated, with a M:F ratio of 3:2. The youngest patient was

8 year old and the oldest patient was of 77 years old with a mean age

of 43 years. The peak age incidence was 53 years. Cytogenetic study

showed Ph chromosome to be positive in 160 cases while 6 patients


123

showed BCR-ABL1 positivity by RT-PCR. Symptoms of patients

included fatigue (79 %), low grade fever (18 %), body ache (38 %),

weight loss (40 %), arthralgia (17 %), bleeding manifestations

(10 %), dragging sensation in left loin (15 %) etc. Common

signs were splenomegaly (98 %), sternal tenderness (71 %),

lymphadenopathy (42 %), hepatomegaly (40 %) and purpura

(12 %).Haematological features were marked leucocytosis with

basophilia (2–17 %), thrombocytosis/thrombocytopenia (40,000–

8,00,000/lL), anemia (Hb6.1–10.4 g/dl). Marrow was hypercellular

in all, blasts varied from 1 to 7 %. Conclusion: Though CML may

have variety of clinical findings a correct approach to diagnosis with

proper management may prolong the life of these patients.

Keywords Chronicmyelogenousleukaemin, Clinical features,

Haematological features

Abstract P 060

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

Lalit Raut, Vinay Bohara, Girish Badarkhe, Ganesh Pujari,Uttam Kumar, SS Ray, Prantar Chakrbarti, Utpal Chaudhuri

Institute of Haematology & Transfusion Medicine, Kolkata 700073

Aim: The aim of the study was to evaluate the characteristics at

presentation and the treatment outcome in CML in children and

adolescents age group. Methods: Retrospective analysis was carried

out at a single centre in India. Thirteen patients (B17 years) attending

CML outdoor from April 2008 to August 2012 were included in the

analysis. Results: CML-CP was the most common phase at presen-

tation. Maximum patients belonged to the 14–17 years age group.

Disease was common in male sex. Splenic discomfort and asthenia

were the most common symptoms and splenomegaly was the most

common sign. The median WBC count at presentation was 65 9 109/

L. In majority (69.23 %) of patients the WBC count was between

20 9 109/L to 99 9 109/L. The median hemoglobin at presentation

was 9.5 g/dL. The median platelet count at presentation was

462 9 109/L. CHR was documented in 11 out of 12 (91.66 %)

evaluable responses. At 18 months of treatment MMR was achieved

in 3 out of 5 evaluable patients. In one of the remaining two patients

MMR was documented at 24 months while in the other patient less

than MMR was documented at 12 months. Conclusion: The pre-

senting features of CML in the children and adolescent age group are

similar to that shown in other studies. The treatment with Imatinib

was effective and well tolerated.

Abstract P 061

Does the Concomitant Use of Antitubercular Drugs Alterthe Natural History of CML?

V Badarkhe Girish, Lalit Raut, Vinaykumar Bohra, GaneshPujari, Prantar Chakrabarti, SS Ray, Uttamkumar Nath, UtpalChaudhuri

Institute of Haematology & Transfusion Medicine Kolkata-73

Objective: We report the outcome of CML in five patients receiving

Imatinib and anti tubercular therapy. Methods: Four CML-CP patients

(C17 years) attending CML clinic developed tuberculosis (2 pulmon-

ary and 2 extra pulmonary) while on Imatinib and another one was

diagnosed with Pulmonary tuberculosis and CML upfront and started

on ATD and Imatinib concomitantly. All the four patients who devel-

oped tuberculosis later had achieved CCyR (Complete Cytogenetic

Response) prior to developing tuberculosis. Four drug ATT was started

under RNTCP. Result: Four out of five (80 %) patients progressed to

Accelerated Phase/Blast Crisis while on antitubercular therapy, while

IRIS data has showed that only 7 % progressed to Accelerated Phase/

Blast Crisis. The exact cause is not known, but drug interaction might

have played an important role in reducing the therapeutic efficacy of

Imatinib as the interaction of Imatinib with Rifampicin is well known.

Conclusion: The concomitant use of Imatinib and antitubercular

therapy may lead to disease progression in CML. A systematic registry

should be maintained to document the outcome of CML patients on

antitubercular therapy.

Abstract P 062

Clinical and Biological Features of Chronic LymphocyticLeukemia: A Single Tertiary Centre Experience

Punit Jain, Bargavi Balakrishnan, Ansu Abu Alex, Biju George,Aby Abraham, Rayaz Ahmed, Alok Srivastava, Vikram Mathews

Department of hematology, Christian Medical College, Vellore

Objective: To evaluate the clinical and biological features of chronic

lymphocytic leukemia (CLL) at presentation, indication for treatment

and comparison with reported data. Methods: We undertook a

descriptive retrospective study to evaluate cases diagnosed to have

CLL over the last 5 years at a tertiary care centre in India and review

its literature. Results: 125 cases were diagnosed to have CLL in this

period. The median age was 60 years (range; 34–85). 45 (36 %)

patients were \55 years (young CLL), while 3.2 % were \40 years.

We found a male to female ratio of 3:1. 52 % of the cases were

incidentally detected, while 29 % presented with swellings, followed

by anemia requiring transfusions in 5 % and fever in 1.6 % of cases.

Hepatomegaly was seen in 47.2 % and splenomegaly was seen in

40.8 %. 46.4 % patients had anaemia with autoimmune hemolytic

anaemia being seen in 3.2 % patients. Thrombocytopenia (\1 9 109/

L) was seen in 21.6 %. The median white cell count and the absolute

lymphocyte count were 40 9 109/L and 33 9 109/L, respectively.

Majority of patients presented in Rai stage II (25.6 %) followed by

stage IV (24 %), stage I (19.25 %), stage III (18.4 %) and lastly stage

0 (11.2 %). 68 (54 %) patients received treatment, out of which fol-

low up was available in 53 patients (range 3–45 months). Out of

these, 16 % had stable disease, 13.6 % had partial remission, 7.2 %

had progressive disease, while 5.6 % reached complete remission. In

our series, we also found CD 38 positivity in 42.4 % of the patients.

Of the patients treated, progressive disease was noted in 2.7 %

patients in CD38 negative patients as compared to 13.2 % patients in

CD 38 positive patients. Conclusions: Our findings are similar to data

reported internationally, except that we had a larger number of

younger patients. Most of our patients presented in an advanced stage.

Chlorambucil continues to be a widely used therapeutic option.

Abstract P 063

Profile of Chronic Lymphocytic Leukemia (CLL)—Single CentreStudy from Eastern India

Sandeep Saha, Basab Bagchi, Tuphan Kanti Dolai, PrakasKR Mandal, Ashutosh Panigrahi, Meet Kumar, MaitreyeeBhattacharyya, Shyamali Dutta, Rajiv De, Malay Ghosh

Hematology Department, NRS Medical College and Hospital,

Kolkata

Objective: To determined the demographic and clinical profile of CLL

patients from Eastern India. Methodology: Prospective demographic,


123

clinical, investigations including immunophenotype (IPT) data were

collected from patients attending hematology services of NRS Medical

College, Kolkata. Period: October 2009 to March 2012. Results:Among the 38 CLL, 29 were male and 9 female. Median age of pre-

sentation were 57 (range 37–78) years. Eight (21.05 %) patients were

asymptomatic at presentation and 73.68 % (28/38) with fever,

78.94 % (30/38) with pallor and 57.89 % (22/38) with infection (RTI).

On examination 28 (73.68 %) had generalized nontender lymphade-

nopathy, lymph node size average 2–4 cm and maximum 6 cm. Ten

(26.32 %) had no peripheral lymphadenopathy (clinically) which were

revealed by CECT or USG. Twenty six (68.42 %) patients presents

with both splenomegaly and hepatomegaly. One had extra nodal

involvement over skin (nodular lesion) below the right eye. Fifteen

(39.47 %) presents with TLC [50,000, 30 (78.94 %) with anemia

(normocytic normochromic anemia) and 23 (60.52 %) thrombocyto-

penia. Six (15.78 %) had positive Coombs test. BM involvement with

interstitial pattern was found in 17 (44.73 %), nodular in 8 (21.05 %),

mixed in 6 (15.78 %) and diffuse pattern in 7 (18.42 %). Myelofibrosis

was found in two cases. Immunophenotyping revealed CD5+ in all

cases, CD23+ and CD20+ in 36 (94.73 %), FMC7 negative in 21

(55.26 %) and sIgM positive in 23 (60.52 %) cases. Thirteen

(34.21 %) were CD 38 positive. Twenty two (57.89 %) patients had

advanced disease (Binet C) at diagnosis. Conclusions: (1) Majority of

CLL presents at fifth decades. (2) Poor prognostic feature (CD 38

positive) were found in 34.21 % subjects. (3) One third patients had

positive FMC7 and negative sIgM. (4) 57.89 % presents with

advanced stage of disease.

Abstract P 064

Spectrum of BCR-ABL Kinase Domain Mutations in Patientswith Chronic Myeloid Leukemia Receiving Imatinib MesylateTherapy

Senthamizh Selvi, Preetha Markose, C Ezhilarasi, EuniceS Edison, Sachin Jain, Biju George, Alok Srivastava, VikramMathews, B Poonkuzhali


Abstract: Imatinib mesylate is the first line therapy for chronic

myeloid leukemia (CML) and induces durable complete haemato-

logical, cytogenetic responses. Despite the impressive responses, a

proportion of patients show resistance to imatinib. This has been

attributed to several mechanisms of which point mutations in the

BCR-ABL kinase domain is the most common mechanism. To date,

approximately 90 mutants in the BCR-ABL kinase domain conferring

different levels of resistance to imatinib have been identified. The aim

of the present study was to analyze the spectrum of BCR-ABL kinase

domain mutations in patients with clinical resistance to imatinib. A

total of 162 CML patients who failed to achieve major molecular

response (in the international scale—IS \ 0.1 %) were included in

the study. RNA was extracted from peripheral blood leucocytes,

cDNA was synthesized. BCR-ABL transcripts were quantified using

real time quantitative PCR (RQ-PCR) using ABL as control gene.

Mutations in BCR-ABL kinase domain was evaluated by nested PCR

followed by direct sequencing. Mutations were identified in 54 of 162

(33.33 %) cases. Nineteen were in chronic phase (CP), 26 in accel-

erated phase (AP) and 9 in blast crisis (BC). Twenty two different

mutations were identified and the distribution of these mutations is

shown in Figure. T315I mutation, which is associated with resistance

to first and second generation tyrosine kinase inhibitors, was the most

common mutation seen [11/54; 20.37 %] followed by the p-loop

mutation M244V [12.96 %]. Among the other reported mutations,

p-loop mutations (Q252H, G250E and Y253H), activation loop

mutation (L384M) and mutations in STI 571 contact site (F317L)

were also observed. In addition, one novel mutation (codon 246–273

indel) in p-loop was identified in two cases. The functional conse-

quence remains to be characterized. The median BCR-ABL IS ratio in

54 patients who had a mutation was 21.43 [0.57–82.42] compared to

16.32 [0.156–74.99] in patients who did not have a mutation which

was not statistically significant (p = 0.2). The frequency of ABL

kinase domain mutations seen in this study suggests that the emer-

gence of a mutation may not be the major mechanism of imatinib

resistance in this cohort. Other mechanisms of resistance including

pharmacogenetic variations, plasma sequestration of the drug or over

expression of BCR-ABL are being evaluated in an ongoing study.

Abstract P 065

A Study to Analyze the Hematological Parameters in NewlyDiagnosed CML Patients with b2a2 Type of BCR-ABLTranscripts

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

1Department of Pediatrics, 2Department of Internal Medicine, Post

Graduate Institute of Medical Education and Research, Chandigarh

Introduction: Chronic myelogenous leukaemia (CML) is the com-

monest leukemia in Asia. However very few studies have been

published from India, documenting the frequency of BCR-ABL fusion

transcripts, possibly due to diagnostic techniques not being widely

available in our country. The sine qua non of CML, t(9;22)(q34;q11)

and/or BCR-ABL positivity must be detected in all cases of CML.

Major bcr (M-bcr) is almost always involved in CML patients, resulting

in b2a2 and b3a2 mRNA transcripts, p210 chimeric protein, and clas-

sical CML phenotype. Variable frequencies of BCR-ABL fusion

transcripts have been reported from different parts of the world. b3a2

and b2a2 type of BCR-ABL transcripts are detected in approximately

95 % of CML cases, b3a2 transcript being more common than b2a2

transcript. The aim of the present study was to analyze the hemato-

logical parameters at presentation in CML patients with b2a2 type of

transcripts. Materials and Method: A retrospective and prospective

analysis of CML cases was undertaken over a period of 3 years (Sep

2009–August 2012). Multiplex RT-PCR was performed to determine

the BCR-ABL transcripts in all the newly diagnosed CML cases. The

hematological parameters studied in CML cases with b2a2 transcript

were total leucocyte count (TLC), platelet count, absolute basophil

count and LAP score. Results: A total of 410 new CML cases were

0

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Mutations in ABL Kinase Domain

DISTRIBUTION OF ABL KINASE MUTATION


123

diagnosed over the study period. The incidence of b2a2 transcript was

26.3 % (108/410). The age ranged from 3.5 to 72 years with median age

of 36.9 years and M:F ratio was 1.6:1. The TLC ranged from 5.8 9 109

to 924.4 9 109/L with median of 160.2 9 109/L and 26 % of the cases

had a very high TLC of more than 200 9 109/L at presentation. Platelet

count ranged from 7 9 109 to 1644 9 109/L with a median

370.3 9 109/L. 5.6 % cases had a platelet count \100 9 109/L and

28.3 % [450 9 109/L. Absolute Basophil count ranged from

0.069 9 109 to 57.05 9 109/L. Severe basophilia ([1000 9 109/L)

was noted in 74 % cases. Discussion and Conclusion: The incidence of

b2a2 transcript in India has been reported to be 28–30 %. The present

study shows a slightly lower incidence of b2a2 transcript in CML cases

in our region. The median ranges for all the hematological parameters

are comparable to that described in western literature and slightly higher

than few of the studies reported from Indian subcontinent.

Keywords BCR-ABL transcripts, CML, Hematological parameters

Abstract P 066

Isolated Central Nervous System Blast Crisis in a Patientof Chronic Myeloid Leukemia on Imatinib Mesylate Therapy

Sanjeev, Ruchi Gupta, Soniya Nityanand

Department of hematology, Sanjay Gandhi Postgraduate Institute

of Medical Sciences, Lucknow

Objective: Imatinib mesylate is the treatment of choice in all phases of

chronic myeloid leukemia (CML). However, the drug or its active

metabolites have a poor penetration in the central nervous system (CNS).

Thus CNS acts as a sanctuary site for malignant cells in these patients. We

report an unusual case of CML on Imatinib therapy who presented with

an isolated CNS blast crisis and peripheral neuropathy. Case-Report: A

25 year old male patient, a known case of CML, presented to the

Department of Hematology, SGPGI, in Aug 2011 with complaints of

headache, double vision, vomiting and tingling sensation of both lower

limbs for past 3 months. He was on Imatinib therapy since February

2011. Salient examination findings were neck rigidity, bilateral flexor

plantar reflexes and absent deep tendon reflexes. The patient was in

hematological remission as confirmed by peripheral blood and bone

marrow examination. Nerve conduction study of lower limbs was sug-

gestive of axonal neuropathy. The CSF showed a turbid appearance with

protein—153 mg/dl, sugar—8 mg/dl, chloride—690 meq/l and TLC-

12,800/cmm. Giemsa staining of CSF smears showed 98 % blasts and

flow cytometry revealed a mixed phenotype blast crisis (B-Myeloid). The

BCR-ABL transcript was 51.4 %. Patient was started on Dasatinib.

Triple drug intra-thecal chemotherapy was given till three samples of

CSF were negative for blasts. The patient 4 months later developed a

systemic blast crisis and expired. Conclusion: Isolated CNS blast crisis

has been rarely reported in CML. Thus in a CML patient, who presents

with CNS symptoms like headache, vomiting, diplopia, etc., even if in

hematological remission, a suspicion of isolated CNS blast crisis should

arise and the patient should be investigated accordingly.

Abstract P 067

A Comparative Study of Sokal Versus Hasford Scoring Systemsin Chronic Myeloid Leukemia Patients in a Tertiary CareHospital in Eastern India

Lopamudra Chakravarty1, Prantar Chakrabarti1, Uttam KumarNath1, Siddhartha Sankar Ray1, Utpal Chaudhuri1

1Institute of Haematology & Transfusion Medicine (IHTM), 3rd floor,

MCH Building, Medical College, 88 College Street, Kolkata 700073, India

Introduction: Chronic myeloid leukaemia (CML) is one of the

commonest leukemias in India. Patients of CML are subjected to

risk stratification at the time of diagnosis according to the available

scoring systems, namely the Sokal and Hasford scores. Objective:Comparison of the Sokal and Hasford scoring systems for predicting

progression free survival and overall survival in patients with CML.

Methods: The data of 52 consecutive patients (n = 52) with diag-

nosis of chronic myeloid leukemia were analyzed retrospectively.

The Sokal score and Hasford score at diagnosis were compared in

terms of prediction of progression-free survival (PFS) and overall

survival (OS) at one year after diagnosis. Results: The median

patient age was 38 years (range 11–72). Thirty percent, 38 and 32 %

patients belonged to the Sokal low, intermediate and high-risk

groups respectively. Forty two percent, 35 and 23 % had low,

intermediate and high-risk Hasford scores, respectively. The PFS

was 90 % and OS was 94 % at one year after diagnosis. Both the

Sokal and Hasford scores predicted better PFS for low-risk patients.

However, there was no significant difference between the two scores

in predicting PFS or OS. Conclusion: In our study there was no

significant difference between the Sokal and Hasford scores in

predicting survival in CML. Late presentation of patients may be

one of the reasons for the higher prevalence of intermediate and

high-risk groups in the study.

Abstract P 068

Characterization of Genes Associated with Disease Progressionin Chronic Myeloid Leukemia by DNA Microarray: AIIMSExperience

Sudha Sazawal, Sunita Chhikara, Rekha Chaubey, RekhaKataria, Manoranjan Mahapatra, Pravas Mishra, Renu Saxena

Department of Hematology, All India Institute of Medical Sciences,

New Delhi-110029

Background: Imatinib resistance is associated with point mutation

in tyrosine kinase domain in 20–40 % of CML patients. The

studies on genetic events that cause the progression of chronic

phase to blast crisis CML in the remaining patients are very lim-

ited. In view of this, we explored gene expression profiling by

DNA microarray in CML patients. Objectives: To identify pre-

dictive genes that might prove informative for stage progression in

CML. Methods: Patients of CML in chronic, accelerated and blast

phase were the subjects of this study. RNA was isolated from

peripheral blood and QC checked using Agilent Bioanalyzer (Ag-

ilent Technologies, Palo Alto, CA). The samples were labelled,

hybridized, scanned and the data was extracted according to the

manufacturer protocol (Agilent Technologies). The data was ana-

lyzed using Agilent Genespring 12.0 software. Results: A total of

14 CML patients (M::8, F::6), median 40 years (range

20–60 years), chronic phase (n = 6), accelerated phase (n = 3),

blast crisis (n = 5) were analysed for changes in gene expression.

A set of genes involved in apoptosis (DAPK1, STAT protein, TNF

and PDCD1), cell adhesion (TNXB, VWF, CD6) and MAPK

pathway were significantly (p B 0.05) under expressed in all the

accelerated phase CML patients. Patients with blast crisis (n = 5)

showed significantly elevated expression of all the genes except

DAPK1. SOCS-2 showed elevated expression both in accelerated

(2/3) and blast phase (3/5) CML. The above genes showed no

expression in the chronic phase patients. The reported mutations

were not observed in any of our patients. Conclusion: Thus, under

expression or elevated expression of the above genes is associated

with the disease progression in CML.


123

Abstract P 069

Correlation of Multidrug Resistance Gene Polymorphism(T1236C) with Imatinib Response in Chronic Myeloid LeukemiaPatients: AIIMS Experience

Sunita Chhikara, Sudha Sazawal, Rekha Chaubey, RekhaKataria, Manoranjan Mahapatra, Pravas Mishra, Renu Saxena


New Delhi-110029

Background: Imatinib mesylate (IM) is a selective tyrosine kinase

inhibitor used for treatment of patients with chronic myeloid leukemia

(CML), however; some patients develop resistance to it. Several gene

polymorphisms have been associated with the resistance and one such

polymorphism is MDR1. Its role has been reported differently among

different populations and different ethnic groups. So far there are no

reported studies from India. Aim of the study: To study the associa-

tion of MDR1 (T1236C) polymorphism in Indian CML patients and

to correlate with response to imatinib. Material and Method: CML,

chronic phase, patients treated with imatinib (400 mg/day) for

24 months were analyzed for T1236C polymorphism. DNA was

extracted from peripheral blood and subjected to PCR–RFLP using

restriction enzyme HaeIII. Results: A total of 100 CML chronic

phase, patients at presentation were included (M:F ratio 2:1) mean

age 40 (range 20–60 years). Of these, 66 patients showed response to

imatinib and 34 patients showed resistance. Among 66 responders the

frequency of three different alleles of T1236C locus was as follows;

CC-43 (86 %), CT-18 (69.3 %), TT-5 (21 %) and in non responders

(n = 34): CC-7 (14 %), CT-8 (30.7 %), and TT-19 (79 %). Resis-

tance to imatinib was higher in patients who were homozygous for the

TT allele in contrast to patients with CT/CC genotype (79 vs. 19.7 %,

P B 0.001). Thus, prevalence of TT genotype seems to be higher in

imatinib resistant patients compared to those who responded to the

drug. Conclusion: It was observed that homozygous TT allele was

associated with higher risk of imatinib resistance.

Abstract P 070

Effect of Long-Term Treatment with Imatinib on Bone Health:Single Institution Experience from Jaipur

Hemant Malhotra1, Bharti Malhotra2, Om Singh Rathore2, AjayYadav1, Shipra Bhargava2, Pratibha Sharma2, Ashwin Mathur1,Sandeep Jasuja1

1Division of Medical Oncology, Department of Medicine, 2Advanced

Research Laboratory, SMS Medical College & Hospital, Jaipur, India

Background: Standard treatment for newly diagnosed patients of

Chronic Myeloid Leukemia (CML) today is the bcr/abl tyrosine

kinase inhibitor, Imatinib. The drug has to be continued for several

years, possibly life-long. Long-term toxicity of the drug is not

established yet. In the present study, we report the effects of the

Imatinib on bone health as assessed by serum bone markers, serum

vitamin D3 levels and bone mineral density by the DEXA scan.

Design and Methods: We evaluated 104 CML patients who had

received Imatinib for more than 1 year at the Birla Cancer Center,

SMS Medical College Hospital, Jaipur. The serum calcium, magne-

sium and 25-hydroxyvitamin D3 levels were analyzed. The Bone

Mineralization Density (BMD) of AP spine in these patients was

measured using DEXA scan. Results: Eighty six percent (86 %,

n = 89) of the total patients (n = 104) received 400 mg IM daily

while 15 % (n = 11) of the patients received 600 mg IM. Eighty

percent (80 %) (n = 82) of the patients received IM for more than

3 year, 12 % (n = 13) received for more than 2 years while 8 %

(n = 9) received IM for more than one year. Eleven out of the 104

patients (11/104) (10 %) and 7/104 (6 %) patients were found to have

low serum calcium and low serum magnesium levels respectively.

The levels of 25-hydroxyvitamin D3 were found to be deficient in

24 % (n = 26) and insufficient in 39 % (n = 40) of the total patients.

Twenty three percent (23 %, n = 30) of the patients were found to be

osteopenic while 8 % (n = 11) were found to have osteoporosis

according to their T score values of AP spine as assessed by DEXA

scan. Conclusion: Our findings suggest that long-term Imatinib may

have an effect on bone health. Periodic monitoring for serum markers

of bone health (vitamin D3, calcium and magnesium) and for osteo-

porosis (by DEXA scan) should be considered in all patients on

long-term Imatinib, especially younger patients. Appropriate supple-

mentation with Vitamin D and/or calcium should be considered for

the deficient patient.

Abstract P 071

Detection of bcr/abl Kinase Domain Mutations by PCR–RFLPand Sequencing in Imatinib Resistant CML Patients

Hemant Malhotra1, Bharti Malhotra2, Shipra Bhargava2,Pratibha Sharma2, Om Singh Rathode2, Ashwin Mathur1,Sandeep Jasuja1

1Division of Medical Oncology, Department of Medicine, 2Advanced

Research Laboratory, SMS Medical College & Hospital, Jaipur, India

Background and Objective: BCR-ABL oncogene mutations are

responsible for the failure of Imatinib (bcr/abl tyrosine kinase

inhibitor (TKI)) treatment in patients with chronic myeloid leukemia

(CML). Moreover, the second generation TKI to be given after

Imatinib resistance depends on the type of mutation present. Patients

with the T315I mutation are resistant to second generation TKIs drugs

like Dasatinib and Nilotinib. Therefore it is important to routinely test

mutation in CML patients who have relapsed or have a sub-optimal

response to Imatinib. Sequencing has been widely used to screen for

bcr/abl kinase domain (KD) mutations in CML patient as a gold

standard but it is expensive and time consuming. PCR RFLP can be

used for early detection of the critical mutations like the T315I and

the technique can provide rapid and inexpensive detection of

important specific mutations. Our study was planned to compare PCR

RFLP and sequencing in CML patients who have relapsed after or are

resistant to Imatinib treatment. Methods: Forty CML patients who

have relapsed after or are resistant to standard Imatinib therapy after

one year were included in the study. PCR–RFLP of Thr315Ile

mutation was done using Dde1 restriction enzyme and ABL KD

mutations were screened by semi-nested reverse transcription

PCR followed by sequencing on ABI 3500 Dx Genetic Analyzer.

Results: Seven different point mutations were detected in the 7/40

(17.50 %) TKI resistant patients. These mutations were located at the

four hot spots of ABL kinase domain: one at the P-loop (Q260H), two

at the imatinib binding site (T315I, T317I), two at the catalytic

domain (M351, E355G) and two at the active A loop (V379E,

D381N). Dde1 RFLP showed wild-type pattern characterized by 2

fragments of 171 and 36 bp in thirty nine patients and one patient had

T315I mutation which showed 207 bp uncut band. Interpretation and

Conclusion: Screening of mutation by PCR RFLP helps in identi-

fying T315I mutation in non responders at an early stage; this is one

of the most important mutation as these patients do not respond to

dasatinib and nilotinib. However sequencing needs to be done in


123

patients not having this mutation to help in therapeutic decisions

regarding the choice of the second generation TKI.

MDS

Abstract P 072

Methylation Status of Fragile Histidine Triad (FHIT) Geneand its Impact on Prognosis of Patients with MyelodysplasticSyndrome-AIIMS Experience

Rekha Chaubey, Sudha Sazawal, Sunita Chikkara,Manoranjan Mahapatra, Renu Saxena


New Delhi-29

Background: Myelodysplastic syndromes (MDSs) are clonal hema-

tologic disorders that frequently represent an intermediate disease stage

before progression to acute myeloid leukemia (AML). Although several

cytogenetic abnormalities have been associated with this heterogenous

disorder little is known about the genetic changes responsible for the

disease. Deregulated epigenetic mechanisms are likely involved in the

pathogenesis of MDS. Thus, we evaluated the FHIT methylation in

MDS patients and correlated with the prognosis. Aim of the Study: To

investigate the frequency of FHIT gene methylation and its correlation

with the prognosis in MDS patients. Methodology: DNA was extracted

from bone marrow/peripheral blood leucocytes of MDS patients.

Bisulphite modified DNA was amplified by methylation specific PCR.

All the baseline hematological and clinical parameters were correlated

with the FHIT methylation status. Results: A total of 100 MDS patients

were analyzed (M:F ratio 2:1) mean age 46 years (range

9–84 years).Forty three patients (43 %) showed methylation with sig-

nificantly high frequency in RAEB-2 subtypes (P \ 0.02). On

comparing the methylation status with other prognostic factors, FHIT

gene methylation was inversely associated with low WHO risk

(P \ 0.006). The overall survival (4 years) was significantly shorter in

patients with methylated FHIT gene when compared to the patients with

unmethylated FHIT gene (P \ 0.005). Conclusion: These results

suggested that aberrant methylation of the FHIT gene might be involved

in the disease progression and be an adverse prognostic factor in MDS.

Abstract P 073

A Rare Case of Transformation of MDS to AML

Abhisek Saini, Sima Chauhan, Sukumar Chakravarty,Raghumani Mohanty

Department of Pathology, Institute of Medical Science & SUM

Hospital, Bhubaneswar

Abstract: The MDS, represent a spectrum of stem cell malignancies

that manifest dysplastic and ineffective blood cell production in one

or more of the major myeloid cell lines compounded by a variable risk

of transformation to Acute Myeloid Leukemia (AML). MDS occurs

principally in older adults with a median age of 70 years, with a non-

age corrected annual incidence of 3–5/100,000 persons but rising to

[20/100,000 among those over the age of 70 years. MDS usually

arises denovo with the risk increasing proportionate to age. Although

progression to AML is the natural course in MDS, only 10–15 % of

MDS develop AML, and rest progress to BM failure. The percentage

of patient who progress to AML varies substantially in various sub-

types. A higher percentage of MDS with increased myeloblasts

transforms to AML, the biologic course in RA, RARS is prolonged

and indolent with a very low incidence of evolution to AML. A

50 year old female patient presented with Bicytopenia (anemia,

thrombocytopenia) and diagnosed to be a case of RCMD. After a span

of 4 months she transformed into AML which is quite rare.

Keywords MDS, AML, Myelodysplasia

Coagulation Abnormalities

Abstract P 074

Type 2 B Von Willebrand Disease—A Case Report

Sushma Belurkar, Chethan Manohar

KMC, Manipal

Introduction: Von Willebrand Disease (VWD) is the most common

human bleeding disorder. Most of the patients have simple quantitative

deficiency of von Willebrand factor (VWF) but around 20 % can have

qualitative defects in the VWF and are referred to as VWD type II. Type

II is further classified into IIA, B, M and N types. Type IIBVWD is a rare

type of VWD that is characterized by enhanced affinity of VWF to

aspecific platelet receptor, glycoprotein Ib-IX complex. The charac-

teristic functional abnormality in this variant is demonstrated by

ristocetin induced platelet aggregation (RIPA). In type II B VWD very

low doses of ristocetin, which have no effect on normal platelet rich

plasma result in full aggregation. Case report: A 7 year old child

presented with maculopapular rash on the body with bleeding tendency.

Patient also had history of prolonged bleeding following tooth fall.

There was no history of bleeding from any other sites. There was no

positive family or sibling history. Laboratory investigations revealed a

prolonged bleeding time ([15 min) and mildly prolonged APTT

(40.4 s). Platelets were mildly reduced in number. RIPA showed

[50 % aggregation with low dose ristocetin (0.5 mg/ml) and VWF

assay was also mildly reduced (38 %). Hence a diagnosis of type II-

bVWD was considered. Hyperaggregation with low dose ristocetin was

not corrected by adding normal platelets but was corrected by adding

normal plasma, hence platelet type of VWD was ruled out. Conclusion:Type IIBVWD is a rare subtype of VWD which can be easily distin-

guished from other subtypes of VWD due to its characteristic feature of

hyperaggregation with low dose ristocetin however it needs to be dif-

ferentiated from platelet type pfVWD by doing proper mixing studies.

Abstract P 075

Clinical and Haemostatic Profile in Suspected FemaleHaemophilia—A Carriers

S Kate1, A Saxena2, S Aggarwal1, N Gupta1

1Department of Medicine, MAMC, New Delhi; 2Department

of Biochemistry, MAMC, New Delhi

Background: Haemophilia A is an X-linked recessive bleeding disorder

resulting from deficiency of Factor VIII. Hence screening asymptomatic

women carriers is important for an effective informed decision at their

pregnancies. Objective: To study the hereditary transmission in families

with hemophilia A and to conduct screening coagulation tests amongst the

suspected carriers. Methods: Sixty one suspected carriers in 25 families of

haemophilia A were subjected to pedigree analysis and coagulation studies

including prothrombin time, activated partial thromboplastin time and


123

Factor VIIIc levels. Ten families (10 patients and 31 suspected carriers) were

subjected toRFLPlinkageanalysisusingbcl1 (Intron18)and HindIII (Intron

19) markers. Results: On the basis of pedigree analysis, Fourteen suspected

carriers could be classified as Obligate (22.9 %) and 47 as Possible (77.0 %).

Six out of 61 suspected carriers (10 %) had an abnormal APTT correcting

with normal plasma whereas 53 (86.8 %) had an abnormal mean Factor

VIIIc activity with an average of 25.47 ± 16.3 %. None of the 8 suspected

carriers with normal FVIIIc was obligate carrier or had a history of bleeding.

Eight out of 25 families (32 %) had a family history of haemophilia. The

suspected carriers with a positive family history (inherited case) had a sig-

nificantly lower Factor VIIIc levels as compared to those without, i.e.

sporadic case (p = 0.04). Molecular genetic analysis in 10 families using

bcl1 and hind III RFLP markers gave noninformative results. Conclusions:Suspected women carriers of hemophilia are more likely to have lower

Factor VIIIc levels where there is a positive family history.

Abstract P 076

Genetic Diagnosis of Protein C Deficiency in Two UnrelatedFamilies from India

G Sankari Devi1, S Rajkumar 1, E Sumitha 1, P Shenbagapriya2,SC Nair#, V Mathews 1, GR Jayandharan1, A Srivastava1

Departments of 1Hematology, 2Immunohematology and Transfusion

Medicine, Christian Medical College, Vellore, Tamil Nadu, India

Introduction: Protein C (PC) is a vitamin K dependent coagulation

factor which regulates the activity of factor (F) V and FVIII. The

incidence of clinically significant protein C deficiency has been

estimated to be 1 in 20,000. Only *322 mutations in the protein C

gene (PROC) have been reported as being disease-causative in the

literature. Identification of additional mutations is important for

understanding the biology of protein C deficiency as well as offering

genetic testing to families affected by this disorder. Material andMethods: Two unrelated families diagnosed with hereditary PC

deficiency at Christian Medical College, Vellore were investigated.

PC activity and antigen levels were measured. Mutation analysis was

performed by amplification of the PROC gene using 9 pairs of

primers designed by Primer3 software. A direct DNA sequencing

analysis of the amplicons was carried out in an ABI 3130 genetic

analyzer. In silico analyses, including PolyPhen-2, SIFT, multiple

sequence alignment, splicing prediction were performed to predict the

consequences of each variant identified. Results and Discussion: In

family 1 the proband, a 21 year old female had lost two children due

to purpura fulminans and a spontaneous abortion. The Protein C

levels was 19.2 % (proband/mother) and 24.2 % (husband) reduced in

both the parents, consistent with a heterozygous phenotype and sug-

gesting a autosomal recessive pattern of inheritance. Mutation

analysis identified a p.Cys160Arg missense substitution in the epi-

dermal growth factor domain of Protein C n a heterozygous state in

both the parents. Cys160 is conserved across various species (Homosapiens, Bos Taurus, Canis lupus familiaris, Gallus gallus, Musmusculus, Pan troglodytes, Rattus norvegicus) and a mutation at this

site is predicted to affect the disulphide linkage between Cys160–

Cys147 and thus affect the tertiary structure of Protein C. This family

subsequently underwent antenatal diagnosis. The foetus was found to

be only a carrier of this mutation and the family continued with this

pregnancy. In the second family, a 6 day old, full-term neonate born

from a consanguineous marriage presented with progressive ischemia

and gangrene of multiple body parts. Excluding the common infective

causes, purpura fulminans secondary to a thrombotic cause or sepsis

was suspected. Protein C level in this patient was found to be 1 %. On

genetic analysis, a novel c.1048 A ? T transversion at codon 350 of

the PROC gene was identified in the proband in a homozygous state.

This nonsense mutation predicted premature termination of transla-

tion (Lys350Stop) in the catalytic domain of PROC to result in a

severe phenotype. Further studies confirmed that both the parents

were heterozygous carriers of Lys350Stop mutation. Subsequently,

the mother presented to us for a prenatal diagnosis during her next

pregnancy. The foetus was homozygous for the Lys350Stop mutation

in the PROC gene and thus affected by this disorder. Conclusions:This is the first report describing mutations in PROC gene from

southern India and the mutation data was helpful in offering genetic

diagnosis to families affected by this disorder.

Abstract P 077

Diagnostic Value of a Quantitative Immunoturbidimetricd-Dimer Assay in the Evaluation of Oncology Patients who havea Moderate Pretest Probability of Thrombosis

J Kotwal, MK Patra, A Kotwal, BN Kapoor, V Dutta, V Nair

Armed Forces Medical College, Pune

Objective: Malignancies may present with life-threatening compli-

cations like Deep vein thrombosis (DVT) and consequent pulmonary

embolism (PE). Unfortunately, diagnosis based on history and

examination alone is often unreliable. The d-dimer test has a high

sensitivity and low specificity in diagnosis of thrombosis. Earlier

evaluations studies were based on ELISA for d-dimer which is not

easily applicable for emergency evaluation of patients. The goal of

this study was to test the efficacy of automated quantitative immu-

noturbidimetric d-dimer assay on Coagulometer. Subjects andMethods: Patients at the oncology centre at a tertiary care centre with

moderate probability of DVT for 3 years were evaluated. 210 such

cases were studied. All (210) were in intermediate risk for DVT as per

Well’s score (score 2–4) and 105/210 (50 %) were in intermediate

score for PE as per the revised Geneva score. D-dimer was evaluated

by the automated immunoturbidimetric d-dimer assay (Lia test D-Di

from Stago diagnostica) on the STA compact. Results: 210 cancer

patients were evaluated. Taking the value of d-dimer \0.5 ml (FEU)

as negative, 50 patients were negative and 160 were positive. How-

ever, out of the 160 d-dimer positive cases only 16 were detected to

have DVT/PE. ROC curve was drawn and the max sensitivity and

specificity was at a cut off of 0.80 ml (Sens 75.4 %. Spec 73.8 % and

AUC 0.815). We observed that all the 16 cases of DVT/PE had

d-dimer [ 1.0 |ag/ml. Thus as per our study a d-dimer \1.0 fj.g/ml

would rule out DVT/PE in intermediate risk cancer patients. Con-clusion: The results seem to support the use of a quantitative d-dimer

assay on a coagulometer as a first-line test in evaluation for DVT/

pulmonary embolism in cancer patients when the clinical probability

of the presence of DVT/PE is intermediate.

Keywords d-dimer, Immunoturbidimetric, Coagulometry

Abstract P 078

Assessment of Protein C, Protein S and Prothrombin Levelin Gastrointestinal Carcinoma Patients with and WithoutMetastasis

Ashutosh Kumar1, Mohd. Yusuf1, MLB Bhatt2, Surya Kant3,Abhijit Chandra4

1Department of Pathology, 2Department of Radiation Oncology,3Department of Pulmonary Medicine, 4Department of Surgical

Gastroenterology, King Georg’s Medical University, Lucknow,

Uttar Pradesh, India


123

Background: A hypercoagulable or prothrombotic state of malig-

nancy with metastasis occurs due to the ability of tumor cells to

activate the coagulation system. Material and Method: A total of 80

Patients with gastrointestinal carcinoma such as malignant carcinoma

rectum, malignant carcinoma oesophagus and malignant carcinoma

colon with and without metastasis were studied in order to evaluate

the presence and extent of hemostatic abnormalities in case of gas-

trointestinal carcinoma. Results: The mean average prothrombin time

in patients of gastrointestinal carcinoma was less than normal value.

Similarly, the average activated partial prothrombin time was also

less than the normal value. The mean level of Protein C ranged from

55 to 90 %. The mean level of Protein S ranged from 48 to 95 %.Out

of the total 80 patients, 7 were Protein C deficient and 5 were Protein

S deficient. However, 3 were Protein C and S both deficient. The

Protein C level was significantly lower (p \ 0.0001) in Protein C

deficient patients with metastasis compared to without metastasis.

Similarly, the Protein S level was significantly lower (p \ 0.0001) in

protein S deficient patients with metastasis as compared to without

metastasis. The Protein C and S levels were also lower in those who

were deficient with metastasis. Conclusion: Our study conclude that

APC resistance in Gastrointestinal Carcinoma with metastasis may

contribute thrombotic episodes in these patients. Cancer patients

including GI malignancy are at increased risk for the development of

thrombotic events that contribute significantly to the morbidity and

mortality of malignancy in these patients.

Abstract P 079

Changes in the Coagulation Profile During InductionChemotherapy in Childhood Acute Lymphoblastic Leukemia

Shivali Sehgal, Sunita Sharma, Jagdish Chandra1, Anita Nangia

Department of Pathology and Paediatrics1, Lady Hardinge Medical

College and Kalawati Saran Children’s Hospital

Introduction: Acute lymphoblastic leukemia (ALL) is the most

common childhood malignancy. The risk of thrombosis in children

with ALL varied from 1.1 to 36.7 % in literature. Two important

factors triggering hypercoagulability are tumor cells themselves and

chemotherapeutic drugs, primarily l-asparaginase. Aims and Objec-tives: To study the effect of induction chemotherapy on coagulation

parameters in paediatric ALL patients. Materials and Methods:Thirty seven newly diagnosed patients of ALL up to 18 years of age

were evaluated along with 30 age and sex matched controls. At the

time of diagnosis (day 0), various coagulation parameters (PT, APTT,

fibrinogen, d-dimer, Protein C, Protein S, Antithrombin, tPA, PAI-1)

were tested. These were sequentially analysed on day 14 (after the

completion of l-asparaginase doses) and on day 28 of therapy (after

the completion of induction). Results: No major change in PT and

APTT was observed during chemotherapy, however, fibrinogen levels

declined significantly (p = 0.04) following l-asparaginase treatment.

D-dimer levels were significantly raised at diagnosis and throughout

induction therapy. PC, PS and AT were reduced in the initial part of

induction followed by a rise in the second half of therapy, reaching

their respective baseline levels (p \ 0.05). The tPA levels were sig-

nificantly reduced in the patients at diagnosis and throughout therapy

(p \ 0.001). PAI-1 levels were comparable to controls at presentation

and showed a rising trend during therapy. Conclusion: The coagu-

lopathy in ALL is multifactorial. The thrombin activation during

induction therapy coupled with deficiency of anticoagulants and

decreased fibrinolysis shifts the balance towards hypercoagulability.

This may enhance the risk of thrombosis in these patients.

Abstract P 080

Hypobaric Hypoxia Alters Coagulation and FibrinolysisPathways

Neha Gupta1, Tathagata Chatterjee2, Srishti Gupta2, MohammadZ Ashraf1

1Genomics Group, Defence Institute of Physiology & Allied

Sciences, Lucknow Road, Timarpur, Delhi, 110054 India;2Department of Pathology, Army Hospital (R&R), Delhi Cantt,

New Delhi-110010 India

Introduction: Studies have indicated that high altitude facilitates the

occurrence of thromboembolic disorders (TED). However, the exact

mechanism still remains to be understood. The present study was

designed to understand the role of HH in TED using rat model of deep

vein thrombosis. Methods: Deep Vein thrombosis was induced by

flow restriction model via ligation of inferior vena cava just below the

renal veins. Hypoxic animals were exposed to simulated altitude of

15,000 ft (429 mmHg). Histopathological studies were conducted in

thrombus, lungs and heart tissue. Expression levels of fibrinolytic

phase protein including PAI-1, tPA were detected by western blotting.

Results: 24 h of hypobaric hypoxic exposure resulted in animal death

and there were indications of Disseminated Intravascular coagulation

(DIC) in HH exposed animals which led to excessive bleeding

resulting in animal death. Histopathological studies revealed the

presence of fibrin thrombi in lungs tissue of thrombotic animals on

exposure to HH. At a HH exposure of lower time point (6 h)

thrombus formation could be captured in IVC of rats. Moreover, the

presence of fibrin thrombi in coronary heart vessels suggests that

thrombus formed in the IVC would have detached and migrated to the

lungs via pulmonary circulation. Levels of fibrinolytic phase proteins;

PAI-1, tpA were found to be altered under hypoxic conditions.

Conclusion: The in vivo experiments conducted so far have sug-

gested that hypobaric hypoxia leads to the development of

hypercoagulation followed by hypofibrinolytic state which leads to

thrombo embolism and ultimately resulting in animal mortality.


Screening for Lupus Anticoagulant in Women with SpontaneousFetal Loss and its Association with Anticardiolipin Antibodies

Akanksha Rawat1, Meera Sikka1, Usha Rusia1, Kiran Guleria2

1Department of Pathology, 2Department of OBG, University College

of Medical Sciences and GTB Hospital, Delhi

Introduction: Recurrent pregnancy loss affects 1 % of pregnant

women. Several causes of RPL have been observed. However, the

potential cause remains unexplained in several cases which must be

accurately identified for appropriate treatment. Aims: To study the

prevalence of lupus anticoagulants (LA) and anticardiolipin (aCL) anti-

bodies in women with spontaneous fetal loss using a combination of tests,

and to confirm presence of LA by using a platelet neutralization proce-

dure. Methods: Hundred women with spontaneous/recurrent fetal loss

and fifty healthy controls were included in the study. The following tests

were done: complete blood counts (LH 500), PT, APTT, APTT using LA

sensitive reagent, TT, KCT, dRVVT (screening and confirmatory) for LA

detection and ELISA for IgG and IgMaCL antibodies. Results: PT and

TT were not prolonged in any patient or control. APTT of patients was

prolonged in 8 (8 %) patients. Use of LA sensitive reagent identified 13

additional patients suspected of having LA. dRVVT screening time was

found to be prolonged in 16 (16 %) patients and 15/16 patients showed


123

confirmed positivity. Thus, the prevalence of LA in women with recur-

rent fetal loss was 15 %.A combination of LA sensitive APTT and LA

screening test was found to be most sensitive and specific in identifying

LA. IgG and IgM anticardiolipin antibodies were estimated in 78 patients

and all controls. One (1.2 %) patient was positive for aCLIgG antibodies

and 3 (3.8 %) patients were positive for aCLIgMantibodies. Thus anti-

cardiolipin antibodies were detected in 4 (5.1 %) patients. Conclusions:Screening for antiphospholipid antibodies must be done in women with

spontaneous/recurrent fetal loss even in absence of other clinical mani-

festations. The use of sensitive tests like LA sensitive APTT and

confirmatory and specific test like dRVVT can diagnose a significant

number of these underdiagnosed patients.

Abstract P 082

Coagulation Aberration in HIV Infected Patients

Swapan Kumar Sinha, Soumit Mondal

Department of Pathology, Medical College, Kolkata

Objective: To study disorders of haemostasis in untreated cases of

HIV/AIDS. Total Platelet Count, prothrombin time (PT), activated

partial thromboplastin time (APTT) was performed on 53 diagnosed

patients of HIV. Methods: Citrated blood (1:9) samples were col-

lected for P.Time, APTT, FDPs and EDTA blood for Complete Blood

Count ART Centre of Medical College and School of Tropical

Medicine. PTimre and APPT was performed as per validated methods

with Internal Quality Control. Platelet count was done by making

blood smears of individual samples and counting under light micro-

scope. Results: Among them 19/53 pts. (35.84 %) had decreased plt.

count. Only 7/53 pts. (13.2 %) had elevated PTimer, 6/53 (11.32 %)

had elevated APTT and 9/53 (16.98 %) had elevated PT, APTT and

Low platelet. Conclusion: Thrombocytopenia may be due to

increased destruction commonly immune mediated, increased con-

sumption due to microangiopathy, TTP or decreased production.

Thromboplastin like substances, Factor X activation by angiopathy or

due to CMV infection may lead to activation of coagulation system.

Acquired Factor V Leiden may lead to hypercoagulable states

including APLAS. A detail study in HIV patients for coagulation

disorders will be more informative.

Abstract P 083

F8 Gene Mutation Analysis in Inversion Negative HaemophiliaA Patients—Identification of 29 Novel Mutations

Preethi Satish Nair, Shrimati Shetty, Kanjaksha Ghosh

Department of Haemostasis, National Institute

of Immunohaematology (I.C.M.R), K.E.M Hospital campus,

Parel, Mumbai-400012, India

Objective: Identification of causative mutations in HA is very

important for genetic diagnoses, assessing clinical manifestations and

establishing a mutation database, enabling an accurate genetic diag-

nosis in all affected families. Method: We analyzed 108 inversion

negative cases (37 severe, 36 moderate and 35 mild), by multiplex

PCRs and CSGE technique followed by DNA sequencing. The

pathogenicity of these mutations was assessed using various predic-

tion softwares and genotypic–phenotypic correlation was studied.

Results: We identified mutations in 82 patients with 56 unique

mutations (29 novel and 27 recurrent). 35 missense, 3 nonsense, 1

donor splice site, 10 deletions and 6 insertional mutations were

detected in the present study. A homozygous mutation in a classical

female hemophilic and heterozygous mutation in another female

bleeder was detected. Double mutations were detected in 2 cases (1

familial and 1 unrelated). 8 CRM (+) and 6 CRM reduced cases were

identified, p.R531C was observed to be associated with both CRM (+)

and reduced cases, whereas p.F309L was observed in 2 unrelated

CRM (+) cases. Conclusion: High heterogeneity of F8 gene muta-

tions, with unique mutations in most of the patients analyzed,

suggests the need for maintaining a mutation database in India.

Presence of double mutations gives an indication for a total gene scan,

even when one mutation is identified. Presence of a female bleeder

suggests that all carriers should be screened for FVIII levels, as;

occasionally the heterozygote carriers may have very low levels of

FVIII, most probably due to extreme lyonization.

Abstract P 084

Procoagulant Microparticles and Their Associationwith Pregnancy Loss

Rucha Patil, Kanjaksha Ghosh, Shrimati Shetty

National Institute of Immunohaematology (ICMR), Mumbai

Objective: To study and analyze the role played by the circulating

microparticles in women suffering from pregnancy loss (PL) with

both recurrent miscarriage (RM) and unexplained fetal loss (UFL) and

characterize their cellular origin. Methods: Twenty six women with

RM and 16 normal healthy control women were analyzed in the

present study. Methodology for analysis of microparticles has been

standardized on BD FACS Aria flow cytometer, using the ‘Megamix

beads’ by participating in the ‘‘Vascular Biology SSC workshop:

standardization of FCM-based PMP enumeration’’. Results: The total

annexin MP and activated endothelial (CD 62e) in patients with

recurrent miscarriages of all categories (early, late and early and late)

were higher than in women in the control group (P \ 0.05). Differ-

ences in platelet (CD41a), non activated endothelial (CD 146),

leukocyte (CD45) and erythrocyte (CD 235a) MP levels were not

statistically significant. Conclusion: Our findings suggest that mi-

croparticles may have a role in the pathogenesis of recurrent

miscarriages. One of the proposed causes of recurrent miscarriages is

uteroplacental thrombosis, and MPs may be associated with this. In

addition, presence of elevated total annexin and endothelial micro-

particles at a distance from the adverse pregnancy event suggest a

continued chronic endothelial damage or activation which might be

one of the causes of recurrent pregnancy loss. Although none of the

patients in this study had any thrombotic episode, it is possible that

this might become apparent in pregnancy.

Abstract P 085

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

Sachin David1, Vikram Mathews1, Shashikant Apte3, GiridharaR. Giridhara R. Jayandharan1, Sukesh C. Nair2, Dolly Daniel2,Suresh Kumar1, Ansu Abu Alex1, Saravanan Ganesan1, KannanSubramanian3, Rayaz Ahmed1, Aby Abraham1, AuroViswabandya1, Biju George1, Alok Srivastava1

Departments of 1Haematology, 2Immunohaematology and

Transfusion Medicine, Christian Medical College, Vellore,

Tamil Nadu; 3Department of Haematology, Sahyadri Speciality

Hospitals, Pune


123

Introduction: Inhibitors or neutralizing antibodies against therapeu-

tic FVIII is a severe complication affecting the treatment of

hemophilia. A number of environmental and genetic factors have

been reported to be associated with the risk of inhibitor development.

We undertook a community based study to evaluate the prevalence of

inhibitors among patients with severe hemophilia A and to study the

predisposing environmental and genetic factors. Material andMethods: Samples from 312 patients were collected which fulfilled the

inclusion criteria (severe hemophilia A and at least 5 life time exposures

to factor replacement). A detailed questionnaire was administered to all

subjects to obtain demographic, clinical and treatment details. Genomic

DNA was used for PCR–RFLP’s (IL4Ra Ile50Val; Arg551Gln, IL4-

590C/T, IL5 746T[C, CTLA4 49G/A), allele specific PCR’s for

identifying cytokine polymorphisms (TNF a-308G/A, TGF b 1 codon

10T/C; codon 25C/G, IL6 -174C/G, IL10 -592A/C; -819C/T;

1082A/G, IFNc +874T/A) was done using a CYTGEN cytokine

genotyping trays from One Lambda (Canoga Park, CA, USA), VNTR’s

in IL5Ra 30UTR by gene scan. HLA Class II typing was done using

standard kit (AllSet+ SSP PCR kits. Dynal, Merseyside, UK). The

immunophenotype of the Treg population was analyzed by flow

cytometry using co-expression of the CD4+, CD25+ markers. Results:Sixty patients (19.2 %) had evidence of inhibitors. Of these 22 patients

(36.6 %) had high titre inhibitors (C5 BU). Table 1 compares the

demographic and laboratory variables among cases that had inhibitors

versus those that did not. We could not find a correlation with envi-

ronmental factors. While all patients in this study had FVIII levels

\1 %, patients with inhibitors had a significantly lower FVIII level than

those that did not (Table 1). Cases with inhibitors were significantly

more likely to have siblings with inhibitors (Table 1). Of the evaluated

immune-regulatory gene polymorphisms a significant protective asso-

ciation was noted with the heterozygous IL4-590 C/T allele and

development of inhibitors (RR = 0.22; 95 % CI 0.108–0.442:

P = 0.000). This protective effect was retained in the high titre subset

when low titre inhibitor positive cases were excluded from the analysis.

There was also a trend to decreased risk of inhibitor formation among

those that were HLA-DRB1-07 positive and this achieved statistical

significance among the inhibitor cases that were high titre positive

(RR = 0.24; 95 % CI 0.055–1.05: P = 0.047). It is recognized that

there is significant heterogeneity in allele distribution of DRB1-07 and

DRB1-13 in the Indian population (www.ebi.ac.uk/imgt/hla). There

was no significant association with T-reg levels and inhibitors. Con-clusions: In this series there was no apparent association with any of the

environmental parameters. There was a significant association with

IL4-590 C/T polymorphism, HLA-DRB1-13 and a trend with HLA-

DRB1-07. This along with the increased incidence of inhibitors in

siblings with hemophilia suggests that inherited parameters may have a

more significant pre-disposing effect than environmental factors in

inhibitor formation in this population.

Abstract P 086

Hematological Parameters with Perinatal Outcomein Preeclamptic Toxaemia—a Study in a Tertiary Care Hospitalof Eastern India

Koyela Mondal1, Swapan Kumar Sinha1, Palash Kumar Mandal1,Bandana Biswas2, Swati Bhattacharyaa3, SanghamitraChakraborty3

1Department of Pathology, Medical College, Kolkatta, Rabindra nath

Mandal, Dehanadapur, Hooghly. 2Department of Gynaecology

& Obstetrics, Medical College, Kolkatta, Rabindra nath Mandal,

Dehanadapur, Hooghly. 3Department of Biochemistry, Medical

College, Kolkatta, Rabindra nath Mandal, Dehanadapur, Hooghly.

Introduction: Preeclampsia and eclampsia are important causes

of morbidity and mortality in pregnant women in developing

countries. Hemolytic anaemia and related disorders like TTP,

HELLP and HUS are commonly seen during pregnancy associ-

ated with preeclampsia and eclampsia and may cause further

complications. Objectives: To study haematological parameters,

coagulation profile and liver enzymes with perinatal outcomes in

preeclasmpsia and eclampsia. Methods: Out of 10,782 number of

antenatal mothers attending the labour room during the period of

August 2011 to July 2012, 636 were found to have severe pre-

eclampsia and eclampsia. Physical examination of patients with

routine urine and blood examination was done in all the patients.

Complete hemogram with coagulation profile (PT, APTT, FDP)

and liver function tests of all 636 mothers were noted. Results:Patients were in age range of 21–38 years. The criteria for

severe preeclampsia included sustained blood pressure of at least

160/110 mmHg or higher with persistent proteinuria on urine

dipstick or elevated creatinine ([1.2 mg/dL). Out of 636, 305

(48 %) mothers were anaemic, fifty cases (8 %) of thrombocy-

topenia alone. Out of 305 anaemic mothers, 57 (19 %) presented

with HELLP syndrome. PT, APTT, FDP was raised in 19, 45

and 9 cases respectively while combined PT and APTT in 50

cases. Still birth was found in 31 cases (5 %) and 190 cases

(30 %) were of small for gestational age. Conclusion: Early

detection of toxaemia and also hemolytic disorders in antenatal

patients may in preventing morbidity and mortality of mothers

and newborns.

Abstract P 087

Genetic Determinants of Warfarin Dosage in Indian Patients

Tejasvita Gaikwad, Shrimati Shetty, Vrinda Kulkarni1,Cecil Ross2, Shanaz Khodaiji3, Kanjaksha Ghosh

National Institute of Immunohematology (ICMR), KEM Hospital,

Mumbai; 1Nair Hospital, Mumbai; 2St.John’s Hospital, Bangalore;3P.D Hinduja Hospital, Mahim

Introduction: Warfarin, a widely prescribed anticoagulant for

prevention of thrombosis, is associated with narrow therapeutic

window where even small variations in dosing may result in

hemorrhagic or thrombotic complications. Several polymorphisms

in VKORC1, CYP2C9, CYP4F2, EPHX1, GGCX, factor IX, factor

II and factor VII have been shown to be associated with warfarin

dose requirement. Material and Methods: 120 warfarin treated

patients were screened for CYP2C9*2, CYP2C9*3, VKORC1-

1639G/A, EPHX1 T113C (rs1051740) by PCR–RFLP and allele

specific PCR method. Results: See Table. (1) CYP2C9 and

VKORC1 promoter region polymorphisms: Overall 44.4 % studied

patient population was found to be carriers for any of the three

warfarin sensitive alleles, requiring low warfarin dose as compared

to wild type, i.e. 3.1 mg versus 6.25 mg (±1.3 mg) OD. Of this,

approximately 62 % patients manifested with bleeding problems

while on warfarin. (2) EPHX1 gene polymorphisms: Patients with

CC genotype require approximately 1 mg/day less warfarin than

patients with TT genotype. Conclusions: Approximately 45 % of

the patients carry any one of other 3 strongly linked warfarin

sensitive polymorphisms, suggesting the need for identifying the

warfarin genotype before starting the dose. The strongest deter-

minant of warfarin dosage was shown to be VKORC1 AA,

followed by cytochrome 3 and cytochrome 2 polymorphisms.

EPHX1 polymorphism showed a minor effect on warfarin dosage.

Keywords Warfarin genotype, VKORC1, CYP2C9, EPHX1,

Warfarin adverse effect


123

http://www.ebi.ac.uk/imgt/hla

Association of different variants with warfarin dose requirement and

over anticoagulation

No. Mean warfa-

rin

dose

Patients

with

INR \ 3

Wild type allele carriers

VKORC1

GG + CYP2C9*1/

*1 + EPHX1 TT

31 6.2 7 (22.5 %)

One variant carriers

VKORC1 variant carriers

VKORC1

GA + CYP2C9*1/

*1 + EPHX1 TT

9 4.0 6 (66.6 %)

VKORC1

AA + CYP2C9*1/

*1 + EPHX1 TT

1 2.0 1 (100 %)

Cytochrome variant

carriers

VKORC1

GG + CYP2C9*1/

*2 + EPHX1 TT

3 4.41 1 (33.3 %)

VKORC1

GG + CYP2C9*1/

*3 + EPHX1 TT

5 3.36 3 (60 %)

VKORC1

GG + CYP2C9*3/

*3 + EPHX1 TT

1 2 1 (100 %)

VKORC1

GG + CYP2C9*2/

*3 + EPHX1 TT

1 3.5 1 (100 %)

EPHX1 variant carriers

VKORC1

GG + CYP2C9*1/

*1 + EPHX1 TC

30 5.19 8 (26.6 %)

VKORC1

GG + CYP2C9*1/

*1 + EPHX1 CC

10 5.02 3 (30 %)

Combined variant carriers

VKORC1

GG + CYP2C9*1/

*3 + EPHX1 TC

9 2.94 4 (44.4 %)

VKORC1

GG + CYP2C9*1/

*3 + EPHX1 CC

3 3.16 2 (66.6 %)

VKORC1

GA + CYP2C9*1/

*2 + EPHX1 TT

1 3.5 1 (100 %)

VKORC1

GA + CYP2C9*1/

*3 + EPHX1 TT

1 2.5 1 (100 %)

VKORC1

GA + CYP2C9*1/

*1 + EPHX1 CC

1 2.5 1 (100 %)

VKORC1

GA + CYP2C9*1/

*1 + EPHX1 TC

14 2.98 8 (57.14 %)

Abstract P 088

FXI Deficiency—a Rare Cause for APTT Prolongation

Archana Suman, SK Bose, Joseph, Alaknanda, C Batra,J Ahluwalia, S Varma1, N Varma

Department of Hematology and Internal Medicine1, Postgraduate

Institute of Medical Education and Research, Chandigarh

Introduction: FXI deficiency is a rare bleeding disorder character-

ized by a hemorrhagic syndrome of variable degree of severity. FXI is

involved in intrinsic coagulation pathway. Here we present a case

report of patient with FXI deficiency. Case Report: A 16 year old

Muslim girl, presented with an episode of hemochezia 2 years back

and epistaxis since 2 years. She had no family history of bleeding or

previous transfusion. She was born of a consanguineous marriage.

Laboratory tests revealed normal PT and prolonged APTT which was

corrected by normal pooled plasma. Mixing studies showed correc-

tion of APTT with Factor VIII and IX deficient plasma and no

correction with FXI deficient plasma. The platelet count was normal.

Factor XI assay revealed marked deficiency (1 %) in patient. Family

screening revealed low FXI activity in the mother and father, 40 and

39 % respectively. Her sister was asymptomatic and had normal FXI

levels. Conclusion: FXI deficiency is a rare cause of bleeding and

must be looked for in patients with isolated APTT prolongation. The

diagnosis is usually delayed in females with prolonged APTT, since a

commoner cause—Von Willebrand disease needs to be excluded.

This case reiterates the fact that as has been described earlier, the

factor levels do not coincide with symptom severity. Less than 150

cases of FXI deficiency have been reported so far in India.

Abstract P 089

The Laboratory Profile of the Thrombotic AntiphospholipidSyndrome

J Ahluwalia, Joseph, SK Bose

Department of Hematology, PGIMER Chandigarh

Objective: The antiphospholipid antibody syndrome (APS) is the

commonest acquired cause of thrombosis). We attempted to

determine the prevalence of APS in patients with thrombosis and to

study the pattern of positivity in the laboratory tests. Methods andPatients: Patients with thrombosis undergo 3 tests viz Lupus

anticoagulant testing (clot based screening and confirmation) and

anticardiolipin and anti beta 2 Glycoprotein 1 antibody assay (both

by EIA) as a part of workup of the APS. Laboratory data of

patients reporting for the investigation for thrombophilia for a

thrombotic event was reviewed and examined for the persistent

positivity of these tests at an interval of at least 3 months. Results:In the period of 3 years, 991 cases were tested for the APS. 889

cases were available for analysis. In 186 (20 %) of these any one

of the APS antibodies were positive at least once. In 30 (3.3 %) the

antibodies persisted for at least 12 weeks and they qualified for the

APS. Only 2 cases had persistent positivity for all 3 antibodies. In

22 cases only 1 antibody was positive. Anti beta 2 GP 1, Anti-

cardiolipin and LA antibodies were positive in 28, 9 and 4 cases

respectively. Fifteen of the 30 cases had CNS vascular obstruction,

whereas 6 had limb DVT. Conclusions: Positivity for all 3 anti-

bodies is rare. Anti beta2 GP 1 antibodies were the commonest

ones encountered. Testing for LA is less frequently positive pos-

sibly because of inability to reconfirm the initial positive test due

to start of anticoagulation.


123

Abstract P 090

Molecular Basis of 19 Severe Factor XIII Deficient Cases

Sharda Shanbhag, Shrimati Shetty, Kanjaksha Ghosh

National Institute of Immunohaematology (ICMR), Mumbai

Introduction: Congenital Factor XIII (FXIII) deficiency is a rare

autosomal recessive disorder affecting 1 in 1–5 million individuals,

with a higher prevalence in countries where consanguineous mar-

riages are common. It is a serious bleeding diathesis, generally

manifested as umbilical stump bleeding, post-injury prolonged

bleeding, intra cranial bleeding, and spontaneous abortions in women.

FXIII deficiency is usually attributed to mutations in F13A gene, on

chromosome 6. Material and Methods: We analyzed 19 FXIII

deficient patients, diagnosed on the basis of their clinical history,

normal screening coagulation and clot solubility assay. Genomic

DNA was extracted by the phenol chloroform method, and mutations

were detected by direct DNA sequencing of F13A gene. 14 of 19

patients had history of primary consanguinity. Results: 17 mutations

were detected in 19 FXIII deficient patients, of which 8 were missense

(5 novel, 3 recurrent), 6 were nonsense (3 novel, 3 recurrent), and 2

patients showed a novel single base pair deletion; one patient showed

a splice site mutation in exon 14. A large deletion in exon 3 is sus-

pected in 2 unrelated patients because of repetitive failure of PCR

amplification of this exon. Seven polymorphisms were detected in

these patients, of which one is novel. Conclusions: We have identi-

fied F13A gene mutations in all the 19 FXIII deficient patients, and

observed a high heterogeneity in the mutation profile. The data

obtained would assist in establishing a National Mutation Database,

and enable an accurate carrier and antenatal diagnosis in affected

families by direct mutation analysis.

Abstract P 091

Variations of PT Reagent to FX Deficiency

Shenbagapriya, Ramya, Jennifer, Suresh, Vandana Kamath,Usha Sitaram, Sukesh C Nair

Department of Transfusion Medicine & Immunohaematology,

Christian Medical College, Vellore, India

Abstract: FX deficiency is a rare bleeding disorder which results in

variable bleeding tendency, but patients with severe FX deficiency

tend to have severe bleeding manifestations among the rare bleeding

disorders. Prothrombin time reagents may vary in sensitivity to FX

deficiency and congenital variants have been identified in which PT or

APTT being normal. We have a case of 31 year old female with

complaints of menorrhagia for the past 3 months, easy bruisability

and prolonged bleeding. Laboratory Findings: Plasma clotting test

and Global haemostatic test were done and the results are as follows:

Plasma Clotting test: PT PT (Innovin:Recombinant Thromboplastin):

Pt: [ 2 min Normal range: 10.0–12.5 s INR: [10.0;PT (Thromborel

S:Human Placental Thromboplastin): Pt: 28 s;aPTT: Pt:52.4 s Nor-

mal range: 25.0–34.8 s;� PT with � control plasma: 11.4 s;� PT

with � adsorbed plasma :23.1 s;� PT with � aged serum: 11.0 s;TT:

Pt:13.8 s Normal range: 12–16 s;FVIII: 367.6 % FIX:315.4 % FXI:

195.8 % FV: 167.0 %; FX: \1 % (Innovin:Recombinant Thrombo-

plastin); FX: 5.7 % (Thromborel S:Human Placental Thrombo-

plastin); Lupus Anticoagulant (DRVVT): Negative Pt: Screen: 64.5 s

Screen Mix: 40.2 s Control Screen: 37.2 s. Global Haemostatic test

also showed normal clotting parameters. Marked prolongation of PT

([2 min–132.5 s) to recombinant Thromboplastin which also resulted

in its assay system revealing severe deficiency of FX. This could be

either a laboratory phenomenon or FX variant.

Abstract P 092

The Epidemiology of FVIII Inhibitors in Indian HaemophiliaA Patients

Patricia Pinto, Preethi Nair, Priyanka Kasatkar, TejasvitaGaikwad, Shahnaz Ali, Anshul Jadli, Rucha Patil, BipinKulkarni, Kanjaksha Ghosh, Shrimati Shetty

Department of Haemostasis & Thrombosis, National Institute

of Immunohaematology, (Indian Council of Medical Research),

Mumbai, India

Objective: A serious complication of replacement therapy in patients

with bleeding disorders, is the development of specific antibodies or

‘inhibitors’ to the deficient coagulation factors, particularly in haemo-

philia A patients. This leads to an increase in the management cost,

morbidity and mortality, especially post-operatively in case of FVIII

inhibitors. The mechanism of FVIII inhibitor development is quite

complex and it is difficult to predict their development, but a prompt and

accurate diagnosis is critical as early therapy can save lives. The aim of

this study was to screen patients with bleeding disorders for inhibitors,

and analyse the incidence of inhibitors in different regions in India.

Methods: Samples were collected in sodium citrate vacutainers from

patients in different cities in India for coagulation and inhibitor screening

assays. The Bethesda assay was performed in inhibitor positive samples

for confirmation and quantification of the FVIII Inhibitor titre. Patient

Table 1 Comparison of cases with and without inhibitors

Demographic parameters Inhibitor posi-

tive: N = 60

N (%)/median

(range)

Inhibitor neg-

ative: n = 252

N (%)/median

(range)

P value

Age (years) 18,.5 (3–59) 18 (2–75) 0.647

Age at diagnosis of

hemophilia (years)

1.5 (0–55) 1.5 (0–32) 0.718

FVIII:C (%) 0.25 (0.04–0.99) 0.35

(0.10–0.60)

0.007

No: of exposures in first

year of life

0 (0–4) 0 (0–25) 0.696

Life time number of

exposures

30 (2–336) 23.5 (5–1,000) 0.164

FVIII (life time) total

factor replacement

used (IU)

13,200

(1,000–241,000) 9,200

(500–425,600) 0.133

Number of joints affected 2 (0–7) 3 (0–7) 0.505

Parental consanguinity 18 (30) 56 (22.2) 0.237

Familial hemophilia 38 (63) 152 (60.3) 0.769

Sporadic hemophilia 22 (36.7) 100 (39.7) 0.769

Siblings with inhibitors 4 (6.6) 5 (1.96) 0.016

Discordance between

siblings for inhibitors

7 (11.6) 12 (4.76) 0.044

Chronic Illness 6 (10) 19 (8) 0.596

Past history of surgical

procedure

7 (11.7) 52 (20.6) 0.142


123

details were recorded in a clinical proforma. A total of 800 patients with

bleeding disorders from different cities of India were screened for

‘Inhibitors’. Results: Out of the 800 samples screened, 710 were Hae-

mophilia A patients, out of which 51 were positive for ‘FVIII Inhibitors’.

Conclusion: The highest incidence of FVIII Inhibitors, i.e. 20.48 % was

seen among the Chennai samples, followed by Hyderabad, Mumbai and

Guwahati, which showed an incidence of 12.5, 11.02, and 8.51 % FVIII

Inhibitors respectively, with respect to the samples analysed. The other

regions showed an Inhibitor incidence\7 %. The overall FVIII Inhibitor

incidence in the haemophilia A samples studied was 7.18 %.

Keywords FVIII Inhibitors, epidemiology, haemophilia A

Abstract P 093

Analysis of FVIII Polymorphism Haplotypes as Risk Factorsfor FVIII Inhibitor Development in Indian Severe HaemophiliaA Patients

Patricia Pinto, Kanjaksha Ghosh, Shrimati Shetty

Department of Haemostasis & Thrombosis, National Institute

of Immunohaematology, (Indian Council of Medical Research),

Mumbai, India

Objective: ‘Factor VIII (FVIII) inhibitor’ development, a serious

complication of FVIII replacement therapy in haemophilia A patients,

leads to an increase in management cost, morbidity and mortality,

especially post-operatively. Recently, four non-synonymous single-

nucleotide polymorphisms (SNPs) in the F8 gene, G1679A [R484H],

A2554G [R776G], C3951G [D1241E], and A6940G [M2238V],

whose haplotypes encode six wild-type FVIII proteins (H1–H6), have

been implicated as risk factors for FVIII inhibitor development due to

mismatched FVIII transfusions. The aim of the study was to deter-

mine if mismatched FVIII transfusions could significantly contribute

to FVIII inhibitor development in Indian haemophiliacs. Methods:We analyzed the above four SNPs, in 60 Indian severe hemophilia A

patients, i.e. 30 inhibitor positive and 30 inhibitor negative control

patients, by direct DNA sequencing of the relevant regions of the F8gene. Results: The prevalence rates of the H1 and H2 haplotypes,

were found to be 1.00 and 0.00 among the inhibitor positive patients,

and 0.94 and 0.06 among the inhibitor negative patients, respectively.

These two haplotypes (H1 and H2) match the recombinant FVIII

products Kogenate (Bayer) and Recombinate (Baxter), used clini-

cally. Conclusion: The F8 polymorphism haplotypes are probably

not a risk factor for FVIII inhibitor development in Indian haemo-

philiacs, but could explain the lower inhibitor incidence. Further

studies, in a larger patient cohort, with regard to association with F8mutations and other genetic risk factors of inhibitor development,

could provide useful insights into the FVIII immune response.

Keywords FVIII Inhibitors, F8polymorphisms, Haemophilia A

Abstract P 094

Effect of Antitubercular Therapy on Haemostasis IncludingCoagulation Factor VIII in Patients with Tuberculosis

Aditya Kutiyal, Naresh Gupta, Sandeep Garg

Department of Medicine, Maulana Azad Medical College,

New Delhi-110002, India

Objective: To study the effect of antitubercular therapy on haemo-

static parameters including coagulation Factor VIII in patients with

different types of tuberculosis. Methodology: Adult patients over

12 years of either sex with newly diagnosed tuberculosis of different

sites categorized as pulmonary, CNS, disseminated and others were

included. Detailed clinical assessment included the haemostatic

parameters namely Prothrombin Time, Activated Partial Prothrombin

Time, Factor VIII, fibrinogen and D-dimers were done in all, before

and after 2 months of intensive standard antitubercular therapy.

Results: Mean age of the 128 subjects was 31.55 ± 15.03 years,

ranging 12–75 years. Pulmonary, CNS, disseminated and other types

of tuberculosis comprised 30.5, 28.9, 17.2, and 23.4 % patients

respectively. Abnormal PT and APPT at baseline was seen in 64

(50 %) and 23 (18 %) cases respectively whereas the mean coagu-

lation Factor VIII level was 125.08 % ± 72.83. Factor VIII was

deranged in 45 (35.15 %) cases, being high in 28.12 % and low in

7.03 %. Baseline fibrinogen levels were altered in 73 (57 %), with 62

being elevated beyond 400 mg%. D-dimers were high in 57.8 %

patients. Platelets were disturbed in 66 (51.5 %), with thrombocyto-

paenia in 47 and thrombocytosis exceeding 400 9 109/l in 19 cases.

Following 2 months ATT, the PT became normal in statistically

significant 32 (50 %) of 64 abnormal at baseline. Factor VIII levels

did not change significantly after ATT (125.08 ± 72.83 vs.

126.21 ± 37.65) whereas fibrinogen decreased significantly from

391.43 ± 167.34 to 272.33 ± 92.66 mg% (p \ 0.01). Cases with

elevated D-dimers came down to 12.5 %. Interestingly, all the platelet

disturbances reverted to normal within 2 months of ATT. Subgroup

analysis of different categories of tuberculosis mirrored these results

except the PT in disseminated group. Conclusions: Elevated fibrin-

ogen, D-dimers, and Factor VIII levels were observed in patients with

different types of tuberculosis, favouring a state of hypercoagulability

which improved with anti-tubercular therapy.

Keywords Tuberculosis, Haemostasis, Coagulation factor VIII,

Hypercoagulability

Hemoglobino Pathies

Abstract P 095

Evaluation of D-10 Hemoglobin Testing System for Diagnosisof Beta Thalassemia Carrier State

Debajyoti Singha Roy

Ramakrishna Mission Sevapratisthan and Vivekananda Institute of

Medical Science, 99 Sarat Bose Road, Kolkata-26

Objective: Prevention is the only way to reduce the burden of thal-

assemia through beta thalassemia carrier state detection. The most

reliable method is High Performance Liquid Chromatography

(HPLC). The conventional HPLC instrument (VARIANT, BIO-RAD)

is very expensive ([3 million).The present study examines the fea-

sibility of using a relatively less expensive HPLC instrument D-10

HEMOGLOBIN TESTING SYSTEM, BIO-RAD which was initially

launched for HbA1c estimation by HPLC. After a thorough search we

could not find any comparative study between the VARIANT and

D-10. Methods: Study population- Patients who are diagnosed as beta

thalassemia carrier by D-10 in RKMSP and VIMS. Exclusion criteria:

1. Children \ 6 months; 2. Patients with prior blood transfusion; 3.

Co existing hematinic deficiency and borderline HbA2 value. Data

collection: same blood samples of the cases diagnosed as beta thal-

assemia carrier by D-10 are tested in SYSMEX KX21 cell counter for

red cell indices and also in VARIANT, BIO-RAD. Some retained

samples are also run in D-10 on day 7. Analysis: statistical analysis

done to establish correlation between results obtained from D-10 and

VARIANT. Results: Sample size 96. (1) Between D-10 and


123

VARIANT. (2) HbF value- 99 % correlation. (3) HbA2 value—83 %

correlation. (4) Not a single case misdiagnosed. (5) 16 samples run on day

1 and 7 in D-10. (6) HbF—95 % correlation. (7) HbA2—97 % correla-

tion. Conclusions: After obtaining a good correlation value it can be

concluded that D-10 HEMOGLOBIN TESTING SYSTEM is a reliable,

low cost instrument for beta thalassemia carrier state detection.

Abstract P 096

Detection of Pattern of Hemoglobin in Patients AdvisedHPLC—A Hospital Based Study

Aishwarya Raj, Jina Bhattacharya, UC Dutta, PK Gogoi

Department of Hematology, Gauhati Medical College and Hospital,

Guwahati, Assam

Objective: Study was conducted to detect the pattern of hemoglobin

and relative distribution of different variants in patients attending the

Hematology department, GMCH. Methods: Present study was con-

ducted in Department of Haematology, Gauhati medical college and

hospital from 1st December 2010 to 31st July 2012. Geographical

distribution of cases predominantly included parts of Assam. Patients

presenting with clinical features and blood examinations indicating

haemoglobinopathies were subjected to HPLC. Further tests were

carried out in selected cases (sickling test, acid elution test, osmotic

fragility test etc.). Result: Out of total 783 patients subjected to HPLC,

394 cases (50.38 %) were having normal Hb A. Among the 49.62%

cases with abnormal hemoglobin, the most prevalent pattern was het-

erozygous Hb E (21.18 %) followed by homozygous Hb E (15.07 %).

Compound heterozygous Hb E-b was the next commonly observed

variant (6.13 %). 2.29 % had sickle cell disease; 0.26 % had sickle cell

trait, 0.38 % had Hb S-b trait, while two cases (0.24 %) with double

heterozygous Hb S-Hb E were observed. A single case (0.12 %) of

hereditary persistence of fetal hemoglobin was noted. 0.89 % cases

suffered from beta thalassemia major, while 3.06 % cases were het-

erozygous for beta thalassemia. Conclusion: Study shows that the

prevalence of abnormal hemoglobin pattern is 49.62 %, of which the

most common is noted in Hb E (36.25 %) in this part of India.

Abstract P 097

Trials of Hydroxyurea in Sickle Cell HemoglobinopathiesPatients of Eastern India

Prasanta Purohit, Dilip Kumar Patel1, Siris Patel, SnehadhiniDehury, Subasa C Bishwal, Satyabrata Meher, BidyapatiPradhan, Kishalaya Das

Sickle Cell Clinic & Molecular Biology Laboratory, VSS Medical

College, Burla, Sambalpur, Odisha; 1Department of Medicine, OSCP,

NRHM, V.S.S. Medical College, Burla, Odish

Introduction: Increased level of HbF (22.3 ± 6.9) was found to have

protective effect against painful crisis, osteo-necrosis, ACS and

splenic dysfunction in Sickle cell disease patients of Odisha (Mashon

RS, 2009). Low dose Hydroxyurea (10 mg/kg body-wt/day) effec-

tively increased the level of HbF in this patients (Patel DK, 2012).

Unfortunately, the use of HU is extremely limited in India, because of

its high cost and the apprehension of its toxicities. Objective: To

assess the clinical and haematological response of low dose

Hydroxyurea in patients of sickle cell hemoglobinopathies in eastern

India. Materials and Method: The study was undertaken at Sickle

Cell Clinic & Molecular Biology Laboratory, V.S.S. Medical College,

Burla, Odisha, India, from 2006 to 2012. 1887 patients were enrolled

including 1738 HbSS (35.7 % paediatrics and 64.3 % adults), 125

HbS-b thalassemia (33.6 % paediatrics and 66.4 % adults), 18 HbSD

and 6 HbSE. The indication were[3 VOC or[2 blood transfusion in

last 12 months of presentation. Detailed baseline studies, i.e. CBC,

HPLC, Bio-chemical, liver function test were done and cases were

followed up at three month intervals. Here we analysed the data of

364 cases under regular follow-up for more than 2 years of HU

therapy. Result: After 2 years HU therapy %HbF increased signifi-

cantly from 18.6 ± 6.9 to 22.5 ± 7.3 but the proportionate rise of

HbF varied from case to case. MCV, MCH and MCHC level also

increased significantly in all cases. The frequency of painful crises

reduced significantly after HU therapy. In paediatric cases response

rate was 71.5 % where as in adults it was 91.2 %. Following HU

therapy, about 95.0 % patients became transfusion independent.

Transient bone marrow suppression (Absolute Neutrophil Count

\2,500/lL, platelet count \80,000/lL) was occurs in 6.96 % cases.

Discussion: With a minimal dose (10 mg/kg body-wt/day) of HU,

most of the patients showed an impressive improvement in clinical

and haematological parameters. In resource poor country like India,

low dose HU therapy provides a suitable therapeutic option for a vast

number of untreated sickle cell hemoglobinopathies patients.

Abstract P 098

Clinical Hematological and Molecular Characterization of SickleCell HbDPunjab (HbSD) in Eastern India: The Largest seriesin world

Subasa C Bishwal, Dilip Kumar Patel1, Siris Patel, SnehadhiniDehury, Prasanta Purohit, Saytabrata Meher, Bidyapati Padhan,Kishalaya Das

Sickle Cell Clinic, VSS Medical College & Hospital, Burla,

Sambalpur, Odisha-768 017; 1Department of Medicine,

V.S.S. Medical College, Burla, Odisha

Introduction: Although HbDPunjab[b-121(GH4)Glu ? Gln] is the

commonest haemoglobinopathy in northern India, HbSD, the com-

pound heterozygote state with sickle gene is rare. Hereby we report

detailed profile of 36 cases Hb SD, the largest number hitherto reported

in world literature. Objectives: To describe the clinical, hematological

and molecular profile of 36 HbSD patients from Eastern India. Mate-rials and Methods: HbSD disease was diagnosed by HPLC. All cases

were subjected to detailed clinical, radiological, biochemical exami-

nation and CBC. HbS mutation was confirmed by ARMS-PCR,

HbDPunjab mutation was confirmed by EcoR1-RE digestion following

PCR, RFLP and Xmnl polymorphism were studied by analyzing six RE

sites as described by Orkin et.al (1982). a-Thalassaemia was diagnosed

by GAP-PCR. Results: The mean age of patients was

21.63 ± 11.7 years and majority belonged to a particular caste namely

the Agharia, which is a predominant caste of Western Odisha. Highest

number (30.55 %) of patients was from Sundergarh district. The

commonest clinical presentation was blood transfusion (in 69.44 % of

patients) followed by painful crisis (66.67 % of patients). 32.0 % of

patients had splenomegaly and hepatomegaly. 18.18 % had AVN of the

femur head, 12.0 % had cholelithiasis, whereas 8.0 % had splenic

atrophy. The mean Hb, MCV and MCH were 8.4 ± 2.59 g/dL,

88.54 ± 11.0 fl and 29.4 ± 4.34 pg respectively in the HbSD patients.

The mean HbF concentration was 19.05 ± 9.95 % and HbD was

41.46 ± 3.89 %. 81.18 % were heterozygote for Xmnl polymorphism

whereas 18.82 % were homozygotes and 5 cases (15.0 %) had dele-

tional a-thalassaemia. Conclusion: We report the detail profile of 36

cases of HbSD patients who were thoroughly investigated and their

detailed molecular characterization was accomplished.


123

Abstract P 99

Epidemiology of Beta Thalassemia Trait in Western Odisha

Satyabrata Meher, Dilip Kumar Patel1, Siris Patel, PrasantaPurohit, Snehadhini Dehury, Subasa C Bishwal, BidyapatiPadhan, Kishalaya Das


Sambalpur, Odisha-768 017; 1Department of Medicine & Project

coordinator, OSCP, NRHM, V.S.S. Medical College, Burla, Odisha

Introduction: Heterozygote state of Beta thalassaemia (Beta thalas-

saemia trait or BTT) is benign and asymptomatic. Detection of BTT

has been very important in the effort of prevention of the disease by

prenatal diagnosis. Microcytic hypochromic red cells and an elevated

HbA2 fraction is hallmark to detect BTT. Objective: To study the

epidemiology of BTT in Western Odisha from our Centre. Materialand Method: Total of 5690 blood samples were collected from cross

sectional study from different parts of Western Odisha were studied at

the Sickle Cell Clinic and Molecular Biology Laboratory, V.S.S.

Medical College, Burla, Odisha, India. Samples were subjected to

CBC and CE-HPLC to quantify haemoglobin fractions. Cases with

MCV \ 80 fl and MCH \ 27 pg were suspected for BTT (Colah

et al. 2007). Cases with HbA2 [ 3.5 % (Madan et al. 2010) and

HbF \ 3.0 % were considered as BTT. Results: From a total of 5690

cases, 213 (3.7 %) individuals were found with BTT. The mean age

of males and females were 32.1 ± 14.1 and 31.7 ± 14.6 years

respectively. 3 (1.16 %) cases of BTT were found among a total of

259 ANC cases screened. 97.65 % of BTT were Hindu by religion

and only 2.35 % were from other religions. Among Hindus, 44.6 %

fall under OBC and 24.9 % under SC category, whereas 30 cases each

were from ST and general category. While BTT was found prepon-

derant among individuals from various districts of Odisha; Cases of

BTT were also detected in individuals from West Bengal, Chhattis-

garh and Andhra Pradesh. Conclusion: The prevalence of beta

thalassemia gene in Western Odisha was 3.7 %. The finding that the

religious groups like Muslims and Christians have lower frequency of

BTT differs from the observation (Sur and Mukhopadhyay 2006)

from West Bengal. The trend observed in this study though is unique

in its representation of epidemiology of BTT from Eastern India; a

larger population-based investigation is required to map the spread of

b-thalassaemia gene in Eastern India.

Abstract P 100

Clinical and Molecular Characterization of 194 cases of Sickle-beta Thalassaemia in Western Odisha and Their Responseto Hydroxyurea Therapy

Snehadhini Dehury, Dilip Kumar Patel, Siris Patel, PrasantaPurohit, Subasa C Bishwal, Satyabrata Meher, Bidyapati Padhan,Kishalaya Das


Sambalpur, Odisha-768 017; 1 Department of Medicine, OSCP,

NRHM, V.S.S. Medical College, Burla, Odisha-768017

Introduction: Hemoglobin Sickle-b-thalassaemia is a variant form of

sickle cell disease, resulting from the inheritance of HbS and b-thalassemia

genes. Objective: To study the clinical, hematological and molecular

profile of sickle-b-thalassaemia double heterozygotes in western Odisha.

Materials and Methods: 194 cases of HbS-b-thalassemia were studied at

Sickle Cell Clinic, VSS Medical College Hospital, Burla, Odisha, India.

Cases were diagnosed by CE-HPLC (BioRad Variant II) and family study

and confirmation was done by ARMS-PCR. a-Thalassaemia was studied by

GAP-PCR. Cases with severe clinical features were treated with Hydroxy-

urea at a dose of 10 mg/kg body wt/day orally. Results: Three b-

thalassaemia mutations [IVS1-5(G-C), FS41/42(-CTTT), codon 15

(G[A)] accounted for 97 % of all HbS-b-thalassaemia cases. IVS1-5(G-

C) was the commonest mutation (184/194, 94.85 %). The mean %HbA2,

%HbF and %HbS were 5.02 ± 0.86, 19.04 ± 8.89 and 69.84 ± 9.91

respectively, whereas the mean RBC, MCV and MCH were found to be

3.90 ± 0.98 9 103/ll, 71.68 ± 9.48 fl and 23.72 ± 8.94 pg respectively.

Commonest clinical presentation was splenomegaly (39.69 %) followed by

hepatomegaly and AVN (23 and 7.2 % respectively). 57.21 % had severe

anaemia (Hb below 9 g/dL). Deletional a-thalassemia was found in 25 % of

cases. 64.43 % patients with VOC more than 3/year and BT more than

2/year were treated with hydroxyurea.Conclusion: IVS1-5(G-C) mutation

was the commonest and found in 94.85 % of cases. FS41/42(-CTTT) and

codon 15 (G[A) mutations were reported for the first time from this region.

a-Thalassaemia had no significant effect on the clinical and hematological

features except that HbF was significantly high in a-thalassaemia group.

Low dose Hydroxyurea had an ameliorating affect on the clinical and

hematological profile of HbS-b-thalassaemia patients in western Odisha.

Abstract P 101

The ‘Odisha Sickle Cell Project’—a New Horizon of Hopefor Sickle Cell Aggrieved Patients in Odisha

Siris Patel1, Dilip Kumar Patel, Prasanta Purohit, SnehadhiniDehury, Subasa C Bishwal, Satyabrata Meher, BidyapatiPradhan, Kishalaya Das

Sickle Cell Clinic & Molecular Biology Laboratory, VSS Medical

College, Burla, Sambalpur, Odisha; 1V.S.S. Medical College, Burla,

Odisha

Introduction: Sickle cell disease (SCD) is a serious health problem in

Odisha with significant morbidity and mortality. It is a huge burden for

the patients and his families and a serious challenge to the medical

fraternity. However the major constraint for management of this

problem is non-availability of health facility in remote areas and lack of

awareness amongst health care professionals about the various treat-

ment of this disease. In view of this, National Rural Health Mission

(NRHM), Govt. of Odisha sponsored Odisha Sickle Cell Project.

Objective: Screening of population for sickle cell haemoglobinopa-

thies and develop an infrastructure for investigation, registration,

treatment, follow-up and counselling of all patients with SCD of

Odisha. Materials and Method: The Odisha Sickle cell Project

(OSCP) was started in April-2010, under this project Sickle cell unit

were constructed at six DHHs of Western Odisha. Counselling and

diagnosis of sickle cell patients at peripheral hospitals is done by a field

worker and trained Laboratory technician. A state-of-art Molecular

Biology & Haematology Laboratory has been developed at the referral

centre, V.S.S. Medical College & Hospital, B urla. Cases referred from

periphery are examined, counselled, registered here. This laboratory

has facility for Hb electrophoresis, CBC, Biochemical test, CE-HPLC,

PCR & Flow Cytometry on a routine basis. Severe cases of SCD are

started Hydroxyurea therapy at a low dose (10 mg/kg body wt/day).

2073 individuals have been screened for sickle cell haemoglobinopa-

thies in 14 health camps organised in various districts of western

Odisha. Simultaneously 10 CMEs have been coordinated for creating

awareness among the doctors. Results: Till date the number of patients

examined, counselled, diagnosed to have sickle cell haemoglobinopa-

thies at VSS Medical College & Hospital, Burla are 28321, 13720 and


123

5840 respectively. At six DHHs the number of new cases of sickle cell

haemoglobinopathies detected are 1381, 737, 568, 1005, 578 and 1020

at Bolangir, Bargarh, Sambalpur, Jharsuguda, Deogarh and Sunder-

garh, respectively. Of the 5840 cases registered 5335 HbSS, 194 HbS-bthalassemia, 36 HbSD, 56 HbAD, 13 HbSE, 18 HbAE, 2 HbSC, 16 b-

thalassemia major, 160 b thalassemia trait, 5 Gc(Acdb)0 thalassemia

and 5 HPFH. 1997 no. of cases has been started Hydroxyurea (10 mg/kg

body wt/day). It has been observed that low dose Hydroxyurea is

effectively reducing the frequency of VOC and blood transfusion.

Conclusion: Under OSCP we have diagnosed thousands of new cases

of Sickle cell haemoglobinopathies, counselled them and have created

awareness among the doctors of Western Odisha for better management

and treatment of this Sickle Cell Disease.

Abstract P 102

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

Ritam Chakrabarty, Suvro Sankha Datta, Biplabendu Talukder,Somnath Mukherjee, Prasun Bhattacharya, KrishnenduMukherjee

Department of Immunohematology & Blood Transfusion, Medical

College Hospital, Kolkata-73

Objective: Delayed hemolytic transfusion reaction (DHTR) occurs

3–21 days after blood transfusion. It usually presents as fever, anaemia

with mild jaundice and sudden drop in haemoglobin level few days after

transfusion. Here we present a case of DHTR in a 26 years old E-Beta

thalassemic female, with blood group of O positive, caused by the

alloantibody to c antigen. Methods: The patient was transfusion

dependent since 2006. Her haemoglobin dropped to 4.6 g/dl, from

6.4 g/dl, within 2 weeks after two units of O positive packed red cell

transfusion. A serological work-up was performed to detect the cause of

this sudden drop in haemoglobin level. The direct antiglobulin test

(DAT), autocontrol and antibody screening by three and eleven cell

panel were performed in polyspecific coombs card (anti IgG + C3d).

Results: DAT and autocontrol showed no agglutination, but antibody

screen with commercial cells (BIORAD, ID, Diacell) showed aggluti-

nation (4 +) in Nos. 2 and 3 of the three cell panel and in Nos. 3–11 of the

11 cell panel (Tables 1, 2). Conclusion: The alloantibody to ‘c’ antigen

was detected and c antigen negative O positive packed red cell was

given for safe transfusion. Alloantibody is the major cause of DHTR in

transfusion dependent patients. So an extended Rh (C,E,c,e) and Kell

phenotyping should be performed in these patients before the first

episode of blood transfusion and antibody screening should be con-

sidered routinely if there is any evidence of DHTR.

Abstract P 103

Hemoglobin J: A Case Report

Anurag Sharma1, Amit Kumar Adhya1, Santosh Kumar Panda2,Manas Nayak3

1Department of Pathology, KIMS, Bhubaneswar, Odisha; 2KIMS,

Bhubaneswar, Odisha; 3Department of Paediatrics, KIMS, Odisha

Introduction: Haemoglobin J is a very uncommon hemoglobinopa-

thy. The clinical presentation and follow up of such a case is

presented here. Case: A term baby boy, birth wt 2.2 kg delivered to

G5L1A3 mother by LSCS. Baby looked pale and had abdominal

distension due to hepatosplenomegaly. His hemogram revealed Hb-

9.6 g/dl, TWBC-12000/CC, TPC-60,000/CC. Peripheral smear was

suggestive of hemolysis. Reticulocyte count was 10 % and DCT was

negative. Baby developed jaundice on day one of life and Liver

function test showed conjugated hyperbilirubinemia with elevated

transaminase and mild raised ALP. On D2, baby had respiratory

distress secondary to PDA, managed conservatively. TORCH anti-

body screening was negative. During NICU stay baby never looked

sick looking. USG Abdomen showed severe hepatosplenomegaly and

no other obstructive pathology in biliary tract and Neurosonogram

was normal. Hb electrophoresis revealed fast moving Hb variant (Hb

J variant). For severe anaemia packed cell transfusion was given. On

10 week follow up, Baby is gaining weight and cholestasis is

decreased. Discussion and Conclusions: There are more than 50

hemoglobin J variants described in the literature. They all have an

electrophoretic mobility ‘‘faster’’ than ‘‘A’’ on cellulose acetate, in

common. All are classified under ‘‘variants of the alpha- or beta-

chains’’ or ‘‘hemoglobins with more than one amino acid substitution

in the alpha chain’’. The clinical presentation and prognosis of the

patients is variable and is determined by the presence or absence of

other beta chain variants. Further follow up is required in our case to

fully delineate the course of the disease.

Table 2

Cell Rh-hr Kell Duffy Kidd

D C E c e K k Kpa Kpb Fya Fyb JKa JKb

1 CCDee R1R1 + + 0 0 + 0 + 0 + + 0 + +

2 CCDee R1R1 + + 0 0 + + + 0 + 0 + + 0

3 ccDEE R2R2 + 0 + + 0 0 + 0 + + 0 0 +

4 Ccddee rIr 0 + 0 + + 0 + 0 + + 0 + 0

5 ccddEe rIIr 0 0 + + + 0 + 0 + + 0 0 +

6 ccddee rr 0 0 0 + + + + 0 + 0 + + 0

7 ccddee rr 0 0 0 + + 0 + + + 0 + 0 +

8 ccD.ee R0r + 0 0 + + 0 + 0 + 0 0 + 0

9 ccddee rr 0 0 0 + + 0 + 0 + 0 + + +

10 ccddee rr 0 0 0 + + 0 + 0 + 0 + + 0

11 ccddee rr 0 0 0 + + 0 + 0 + + 0 0 +

Cell Lewis P MNS LUTH. Result

Lea Leb P1 M N S s Lua Lub IS 37�C AHG

1 CCDee R1R1 0 0 + 0 + + + 0 + – – –

2 CCDee R1R1 0 + + + 0 + 0 + + – – –

3 ccDEE R2R2 + 0 + + 0 0 + 0 + – 4+ 4+

4 Ccddee rIr + 0 + + + 0 + 0 + – 4+ 4+

5 ccddEe rIIr + 0 + + + 0 + 0 + – 4+ 4+

6 ccddee rr 0 + 0 0 + 0 + 0 + – 4+ 4+

7 ccddee rr + 0 + + + + + 0 + – 4+ 4+

8 ccD.ee R0r + 0 + + + + 0 0 + – 4+ 4+

9 ccddee rr 0 0 0 0 + + 0 0 + – 4+ 4+

10 ccddee rr 0 + 0 + + + + + + – 4+ 4+

11 ccddee rr 0 + + + 0 + 0 0 + – 4+ 4+

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

Table 1

Cell Rh-hr Kell Duffy Kidd

D C E c e K k Kpa Kpb Fya Fyb JKa JKb

1 CCDee R1R1 + + 0 0 + 0 + 0 + + + + +

2 ccDEE R2R2 + 0 + + 0 + + 0 + 0 + 0 +

3 ccddee rr 0 0 0 + + + + 0 + + 0 + 0

Cell Lewis P MNS LUTH Result

Lea Leb P1 M N S s Lua Lub IS 37�C AHG

1 CCDee R1R1 0 + + + 0 + 0 0 + – – –

2 ccDEE R2R2 + 0 + + + + + + + – 4+ 4+

3 ccddee rr 0 + + 0 + 0 + 0 + – 4+ 4+

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


123

Abstract P 104

A Novel 9 bp Denovo Deletion [HBB: c.132_141del9bp] inb-Globin Gene Causing Heinz Body

K Neelakandan, Neeraj Arora, Sathish Kumar1, Usha Sitaram2,Alok Srivastava, Eunice Sindhuvi Edison, RV Shaji

Department of Haematology, Christian Medical College, Vellore;1Department of Child Health, Christian Medical College, Vellore;2Department of Transfusion Medicine & Immunohaematology,

Christian Medical College, Vellore

Abstract: The ‘‘Heinz-body anaemias’’ are a group of hemolytic

syndromes of diverse etiology with common morphologic charac-

teristics. One of the rare causes for Heinz-body anaemias is dominant

b-thalassaemia (b-thal). Dominant b-thal presents with moderate to

severe haemolytic anaemia with jaundice and splenomegaly and is

associated with a thalassemia intermedia phenotype in the heterozy-

gous state. We describe a case of dominant b-thal with severe

hemolytic anaemia caused by a novel denovo deletion in b-globin

gene. The patient was a 3 year old girl who presented with transfusion

dependent anaemia and hepatosplenomegaly (liver-4 cm; spleen-

8 cm). She had marked reticulocytosis (19 %) and elevated LDH

levels. Both direct and indirect coombs test were negative. The blood

picture and other common tests (G6PDH, autohaemolysis, sickling

preparation, Hams and sucrose lysis test, osmotic fragility) done for

haemolytic anaemia were unremarkable, but Heinz bodies test was

positive. Interestingly, peripheral blood smear stained with brilliant

cresyl blue showed numerous RBCs containing hemoglobin H (HbH)-

like inclusions. However, the eight common alpha (a)-globin dele-

tions prevalent in our population screened by a multiplex PCR were

absent. We performed sequencing of globin genes to identify possible

mutations that produce unstable haemoglobin causing haemolytic

anaemia. We identified a heterozygous state of a novel 9 bp deletion

(TCC TTT GGG) in the exon 2 of the beta globin gene (HBB:

c.132_141del9bp). This region of the b globin is situated in or around

the heme pocket and the mutations that destabilize heme binding has

been found to be associated with extremely unstable hemoglobins.

Both parents had normal HbF, HbA2, MCV and MCH values and had

normal b globin gene sequences. Most of the mutations that cause

dominant b-thalassaemia reside in the exon 3 of the b globin gene,

although rare cases with mutations in other exons 1 and 2 have also

been reported. Our case serves to underscore the importance of

considering dominant b thalassaemia as a cause of moderate to severe

hemolytic anaemia in children even without a significant family

history. DNA based diagnostics can readily detect and confirm these

uncommon b globin gene mutations, and help implement appropriate

treatment planning and genetic counselling.

Abstract P 105

Profile of Liver Fucntion Test in Sickle Cell Patients

Mitali Madhumita Rath, MK Panigrahi, Sudha Sethy, RajibNayak, Pranati Mohanty, Kalyani Prava Gouda, Sujata Pujari,SR Mohapatra, RK Jena

Department of Pathology & Clinical Hematology, S.C.B. Medical

College & Hospital, Cuttack

Introduction: Abnormal liver function tests are common in patients

with sickle cell anaemia, even in the absence of liver disease.It may be

hemolytic, cholestatic, hepatocellular and mixed pattern. There is a

paucity of data on liver function in sickle cell disease (SCD) from this

region. Aims and Objective: The present study aims at determinig the

prevalance and the profile of abnormal liver fuction test in sickle cell

patients. Material and Mathods: One hundred consucutive patients of

sickle cell disease (homozygous)attainding the out patients department

of hematology, S.C.B. Medical College from January 2012 to July 2012

were included in this study. Detailed clinical history was taken and

subjected for complete blood count, hemoglobin profile, liver function

tests, viral markers, ultrasonography of abdomen and pelvis. Results:The 100 patients included in the study ranged in age from 8 to 62 years

(mean 30.8 ± 9.11 years) and included 64 males and 36 females. Mean

episode of vaso-oclusive crisis was 2.65 ± 1.82 times and mean units of

blood transfusion was 3.74 ± 1.75. Mean hemoglobin concentration

was 8.2 ± 2.3 g/dl. Abnormality in liverfuction test was found in 86 %

of the study participants. The mean serum total bilirubin, AST, ALT,

ALP were 8.4 ± 6.5 mg/dl, 104 ± 43 U/L, 68 ± 27 U/L, 258 ± 76

U/L respectively. 49 % had hemolytic, 12 % had obstructive 25 % had

mixed pattern of LFTs. On ultrasonography 24 % had hepatomegaly,

46 % had spleenomegaly, 11 % cholelithiasis and 2 % had both cho-

lelithiasis and choledocholithiasis. Conclusion: Majority of sickle cell

homozygous patients had abnormal liver fuction. One fourth of the

patients had mixed pattern of LFTs that can not be explained by either

hemolytic or obstructive component.

Abstract P 106

Profile of Hb E/beta Thalassaemia Patients After Splenectomy—Experience of a Thalassemia Care Centre in Eastern India

Prakas Kumar Mandal, Basab Bagchi, Sandeep Saha, TuphanKanti Dolai, Sanjay Misra, Malay Kumar Ghosh, MaitreyeeBhattacharrya

Department of Hematology, N.R.S. Medical College and Hospital,

Kolkata-700014

Objective: The study was done to evaluate various complications

following splenectomy in HbE/beta thalassaemia (EBT). Methods:A total of 72 splenectomised EBT were studied retrospectively.

Detail history including age of splenectomy, transfusion require-

ment (before and after splenectomy) was taken. Physical

examination was done for facial deformities, pubertal growth.

Baseline Hb %, serum ferritin level, evidence of pulmonary

hypertension (PHTN), thrombo-embolic manifestations (VTE),

extramedullary hematopoiesis were noted. Results: Out of 1380

registered EBT patients, 72 (5.22 %) underwent splenectomy. Ten

(13.9 %) were diagnosed \1 year, 24 (33.3 %) 1–5 years, 23

(31.87 %) 5–15 years and 15 (20.83 %) [ 15 years. Thirty five

(48.57 %) started transfusion \ 5 years and one at 39 years. 19

(26.4 %) underwent splenectomy \ 10 years, 38 (52.7 %) between

10–20 years and 15 (20.8 %) [ 20 years of age. Mechanical dis-

comfort was the leading cause for splenectomy in 37 (51.39 %)

followed by increased transfusion requirement in 15 (20.83 %).

Mean baseline Hb level of 5.43 g/dl with 48 (66.6 %) pre-sple-

nectomy and majority had Hb % of 6.8 g/dl Post-splenectomy.

Mean transfusion requirement was remarkably reduced from

18.1 unit/year to 7.8 unit/year after splenectomy. Four (5.5 %)

received single transfusion till date. Mean serum ferritin level was

increased from 907.58 ng/ml (before) to 1091.6 ng/ml (after) ins

pite of reduced transfusion requirement after splenectomy. 48


123

(66.6 %) and 31 (43.05 %) patients had facial deformities and

delayed pubertal growth respectively. Four (5.5 %) had evidence

of extramedullary hematopoiesis, 15 (20.83 %) had VTE and 7

(9.7 %) developed PHTN. Though 48 (66.67 %) was started with

iron chelation therapy, majority 38 (52.7 %) stopped and 10

(13.89 %) are continuing till date. Conclusions: Though splenec-

tomy reduces requirement of transfusion; it does not prevent

skeletal abnormalities and delayed puberty. VTE are increased.

Patients should be encouraged for effective Iron chelation therapy.

Abstract P 107

Optimizing the Dose of Hydroxyurea in Thalassemia Intermedia

Vinaykumar V Bohara, Lalit Raut, Girish Badarkhe, GaneshPujari, P Chakrabarti, S Ray, U Nath, Utpal Chaudhuri

Institute of Hematology and Transfusion Medicine (IHTM), Kolkata

Introduction: The optimum dose of hydroxyurea in the manage-

ment of thalassemia patients is an area of gray zone. We report the

results of study carried out on patients of E-b thalassemia. Aims and

objectives: To determine the optimum dose of hydroxyurea in E-bthalassemia patients and to assess its efficacy and safety. Methods:This was a prospective, single center study in which E-b thalassemia

patients with no or minimum transfusion needs were enrolled and

randomised to receive 10 (group A) and 20 mg/kg/day (group B)

hydroxyurea. Complete response (CR) was defined as transfusion

independence or rise of haemoglobin (Hb) by [1.5 g%. Partial

response (PR) was rise in Hb by 0.5 to 1.5 g% or reduction in

transfusion needs. Anything less was defined as no response (NR).

Results: Median age in group A (n = 32) and B (n = 31) was 12.5

and 12 years, respectively (p [ 0.05). The median follow up was 9

and 6 months in groups A and B respectively (range 3–9 months).

In group A response was seen in 65.7 % (20 out of 32). Of this CR

and PR was seen in 32.3 % (n = 10) and 33.4 % (n = 11). The rise

in Hb in group A was statistically significant at 3 and 6 months. In

group B there was NR in 83.9 % (26 out of 31) of patients. There

was no CR and eight (25.8 %) patients discontinued hydroxyurea at

6 months due to adverse effects requiring intervention. High base-

line HbF was associated with higher response in group A but not in

group B. In Complete responders IVS 1-5(G-C) was the commonest

mutation. Conclusion: Hydoxyurea is effective and well tolerated at

10 mg/kg/d dose in E-b thalassemia (Thalassemia Intermedia) in

Eastern India.

Abstract P 108

Association of Genetic Modulators of b Thalassemia Linkedto Raised Fetal Hb and its Effect on Clinical Severity

Pooja Dabke, Roshan Colah, K.Ghosh, Anita Nadkarni

National institute of Immunohematology, 13th Floor NMS Building,

KEM hospital Campus, Parel, Mumbai-12

Objective: Study of genetic modifiers linked to raised HbF and its

effect on clinical severity of b thalassemia. Methods: Study group

consisted of 104 patients of hemoglobinopathies and 50 healthy

individuals. HbA2 and HbF levels were measured using HPLC System.

XmnI polymorphism, Ac-d intergenic haplotypes, (AT)x(T)y motif,

Pre G c haplotype, b globin LCR motifs were studied by PCR–RFLP,

Gene scan analysis and DNA sequencing. Results: Clinically 40

patients were classified as thalassemia major and 39 as thalassemia

intermedia. 13 different b thalassemia mutations were encountered.

IVS1nt5 (G ? C) was the prevalent mutation (56.96 %). 25 % of the

thalassemia intermedia chromosomes showed presence of (AT)9(T)5

motif. The Pre G c TAG haplotype associated with higher HbF levels

was present in homozygosity in 48.57 % of the thalassemia intermedia

cases as against 12.82 % of the thalassemia major cases. 80.55 % of

the milder patients showed presence of the Ac-d intergenic haplotype

T. All the 11 sickle cell anemia patients showed homozygosity for Pre

G c TAG and Ac-d intergenic T haplotypes showing their linkage with

Arab-Indian haplotype. In b LCR region the HS2 motif were well

spread among the patients. The HS3 motif did not show any change

with respect to the reference sequence. Conclusion: As 33.33 % of our

milder cases showed presence of 4 modifiers linked to higher HbF

(XmnI, (AT)9(T)5, TAG Pre G c, Ac-d intergenic T haplotype), it

appears that in the Indian scenario these secondary modifiers of bthalassemia seem to contribute to ameliorate the disease severity.

Abstract P 109

A Hospital Based Study of Thalassemia from Kolkata

Deboshree M. Bhattacharyya, Jayasri Basak,Soma Mukhopadhyay, Ashis Mukhopadhyay



Objective: The present study represents a population of Eastern

Indian state of West Bengal. This is a hospital based study where in the

last 3 years 242 individuals have been screened for their carrier status.

The main objective was to study the genotype-phenotype correlation

of b thalassemia cases, and finally to identify different b thalassemia

mutations prevalent in West Bengal. Method: After obtaining written

consent from each individual or parents of individuals under the age of

18 years, 3 ml of peripheral blood was drawn from each subject and

stored in EDTA vials. At first complete blood count was done followed

by High Performance Liquid Chromatography. DNA is then extracted

from the remaining blood following Millers method (1988). ARMS

PCR and GAP PCR were done to detect beta and alpha mutations

respectively. Results: Total of 242 subjects were studied which

included 22.31 % b thalassemia carriers, 2.1 % of b thalassemia major

1.24 % sickle beta thalassemia, 6.2 % Eb thalassemia, 8.3 % HbE

carriers and HbE 2.1 % homozygous. 10 Eb patients responded to

hydroxyurea treatment and required less blood transfusion whereas 5

Eb patients did not respond to the treatment and required regular blood

transfusion. The severity of the Eb mutations depends on the genotype

of the beta mutation. Apart from these we have identified families

having mutations for HPFH, Fanning Lubbock and a alpha thalasse-

mia. Conclusion: Genotype phenotype correlation plays a significant

role in determining the severity of thalassemia. Among the studied

samples the prevailing beta mutations are IVS 1-5, codon 15 and

codon 30. Compound heterozygosity of HbE with the above men-

tioned beta mutation produces mild to severe anemia. The above study

gives an idea of the thalassemia status of Kolkata.


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Abstract P 110

Role of NESTROFT in Mass Screening of Thalassemia

Nabamita Pal, Deboshree M. Battacharyya, AshisMukhopadhyay, Abhijit Chakraborty, Swati Dasgupta,Priyabrata Das, Soma Mukhopadhyay, Jayasri Basak

Department of Molecular Biology, Netaji Subhas Chandra Bose

Cancer Research Institute, 16 A, Park Lane, Kolkata-700016,

West Bengal, India

Objective: Thalassemia represents a major health problem worldwide.

Present carrier status of Thalassemia in West Bengal according to Sur

D et al. is 20.47 %, which is comparatively higher than other states of

this country. Our main aim is to arrange Mass Awareness and

Screening camps among both urban and rural West Bengal and screen

people to know their thalassemia carrier status. Method: In the present

study from January 2009 to January 2011 screening was carried out

among various populations of West Bengal including tribal. With their

individual consent 3 ml of peripheral blood was collected in EDTA

vials. NESTROFT was done on spot using 0.36 % Saline Buffer

solution (Sodium chloride, Sodium dihydrogen phosphate, Disodium

hydrogen Phosphate). Complete Blood Count was performed within

24 h of collection. HPLC (High Performance Liquid Chromatography)

was done to identify the beta samples. Result: In this period total 6482

individuals have been screened. Among them 25 % were NESTROFT

positive and 50 % are negative. Rest of them were doubtful cases.

NESTROFT result was compatible with the respective HPLC reports.

Most of the NESTROFT positive were either carriers of beta thalas-

semia, sickle cell anemia, carrier of alpha thalassemia and victims of

Eb thalassemia. The doubtful cases of the test contributed to mostly E

carriers and individuals suffering from iron deficiency anemia. The

efficiency of the NESTROFT test is 71.29 % among the general

population of West Bengal. The specificity and sensitivity of the test

are 61.1 and 72 % respectively. Conclusion: Hence NESTROFT is

both cost-effective and cost-efficient test in Thalassemia Mass

Screening. It plays a significant role in thalassemia carrier detection

but HPLC is mandatory in the area where HbE is prevalent.

Abstract P 111

Role of Wheat Grass Juice as an Iron Chelator in IntermediateThalassemia Patients

Abhijit Chakraborty1, Priyabrata Das1, Manoj Kar2, SomaMukhopadhyay1, Suvra Mondol3, Ashis Mukhopadhyay1

1Netaji Subhas Chandra Bose Cancer Research Institute, 16A Park

Lane, Kolkata-700016, West Bengal, India; 2N.R.S Medical College

& Hospital, Kolkata-700014, West Bengal, India; 3National Research

Institute for Ayurvedic Drug Development, Kolkata-700091,

West Bengal, India

Objective: The aim of the study is to observe the effect of wheat grass

juice in reducing ferritin level and increasing the hemoglobin level in

intermediate Thalassemia patients. We will also study the biochemical

properties of wheat grass juice. Methods: We performed a study on

236 patients of intermediate thalassemia during the period from March

2009 to March 2012. Of these, 48 transfusion dependent intermediate

thalassemia patients whose mean age is 6.42 ± 1.71 were selected.

30 ml of fresh juice from 5-7-day-old wheat grass leaves including

stems was given daily to all 48 patients regularly for 6 months. Wheat

grass juice was analyzed by cation exchange column chromatography.

Deoxyribose degradation assay was performed to study the iron che-

lating activity of wheat grass. Result: Wheat grass juice is found to be

rich in active ingredients which play an important role in dietary

absorption of iron from intestine. It is also found to contain unique

active ingredients with iron chelating property. The mean serum fer-

ritin level of the patients before treatment was 2,250 (range

650–3,100), which reduced to 950 (range 68–1680) (p \ 0.0001) after

treatment. The mean levels of hemoglobin before starting wheat grass

juice were 6.2 g%. After 6 months of wheat grass therapy the mean

value for hemoglobin was 7.8 g% (p \ 0.005). The performance status

was improved from 60 to 80 % (Karnofsky) after wheat grass treat-

ment. The mean interval between transfusions was also found to

increase. Conclusions: Hence, wheat grass juice is an effective iron

chelator and it is an effective alternative of blood transfusion. Its use in

intermediate thalassemia patients should be encouraged.

Abstract P 112

Prevalence of Thalassemia in West Bengal with Special Referenceto Different Tribes

Jayasri Basak, Deboshree M Bhattacharyya, SomaMukhopadhyay, Swati Dasgupta, Abhijit Chakraborty,Priyabrata Das, Nabamita Pal, Sukanta Koner,Ashis Mukhopadhyay

Department of Molecular Biology, Netaji Subhash Chandra Bose

Cancer Research Institute, Kolkata

Introduction: Thalassemia is a hereditary anemia resulting from

defect in hemoglobin production and it is the most common genetic

disorder worldwide. Prevalence of Thalassemia (both alpha and beta)

is high in West Bengal, especially among the scheduled tribes. To

reduce the Thalassemia burden from West Bengal we have taken a

project for Thalassemia carrier screening through awareness and

screening program among the rural and urban populations. Our target

population were tribes, e.g., Toto, Rabha, Munda, Oraon, Kerketa,

Sardar etc. residing in remote rural areas. Materials and Methods:From January 2009 to December 2011, we have organized 73 camps

in different districts of West Bengal. After taking written consent,

2–3 ml peripheral blood samples were collected from each interested

people attending the mass awareness camp. NESTROF, CBC, HPLC

were done for all samples. To detect alpha and beta mutations GAP-

PCR and ARMS-PCR were performed following the standard pro-

tocol. Results: During the above mentioned period, we have screened

on total 8387 individuals (4,869 male; 3,518 female) for Thalassemia

carrier detection through camps, organized in 14 districts of West

Bengal. Out of 8,387 screened individuals, tribes were 2,885 and

general caste including scheduled castes was 5502. Among tribes

HbE carrier and homozygous percentage are 14.6 and 4.01 respec-

tively while percentage of sickle cell anemia and beta Thalassemia

carriers are 1.25 and 4.19 respectively. Total beta carrier percentage

among general caste and scheduled caste is 6.94. Our result also

revealed that alpha Thalassemia carrier among Oraon and Sardar

tribes is very high. Conclusion: Incidence of Thalassemia is high in

different rural populations especially among tribes. So, large scale

awareness and preventive program should be taken.


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Abstract P 113

Spectrum of Beta Globin Gene Mutations in North IndianPatients with Thalassemia Major: Application in PrenatalDiagnosis

Reena Das, Jasbir Kaur, Sanjeev Chabra, Jasmina Ahluwalia,Amita Trehan1, Subhash Saha2, Rashmi Bagga2, Deepak Bansal1,Inusha Panigrahi1, RK Marwaha1, G Garewal

Departments of Hematology, Pediatrics1 and Obstetrics

& Gynaecology2, PGIMER, Chandigarh 160 012, India

Objectives: Thalassemia major (TM) is an autosomal recessive dis-

order where the parents are asymptomatic beta thalassemia carriers

(bTT). The prevalence of bTT is 3.5 % in north Indians. We analyzed

the spectrum of mutations in 1500 TM alleles and its application to offer

prenatal diagnosis to couples who were both bTT. Method: ARMS-

PCR and beta globin gene sequencing from genomic DNA from the

peripheral blood was carried out. Prenatal diagnosis was offered to 570

pregnancies in the last 14 years using molecular analysis. Results:Common five Indian mutations were found in 86.8 % alleles; com-

monest being IVS 1,5 (G-C) [29.4 %], followed by Fr 8/9 (+G) [21 %],

619 bp deletion [17 %], IVS 1,1 (G-T) [8.5 %] and Fr 41/42 (-TTCT)

[10.8 %]. Codon 16 (-C) was seen in 4 % and uncommon b++ muta-

tions -88 (C-T) and Cap + 1 (A-C) was 2.6 and 2 % respectively. Rare

mutations constituted 4.5 % alleles and many were identified by beta

globin gene sequencing. Only 0.1 % alleles remained uncharacterized.

Results of the prenatal diagnosis of 570 pregnancies showed that in

25.7 % cases the fetuses were normal, 49.8 % cases were bTT, 23.2 %

were TM and in 1 % cases we were unable to distinguish between bTT

and TM. Seven cases had twin pregnancies and all except one case

showed similar normal/bTT/TM status. Conclusion: This application

has helped to prevent the birth of 132 children with thalassemia major in

the region and alleviated the anxiety of 430 pregnancies to have children

who were either normal or carriers.


Clinico-Hematological Analysis of Hb Variants and Spectrumof Hemoglobinopathies on HPLC: a 3 Year Study from a TertiaryCare Centre of North India

Poojan Agarwal, Snigdha Goyal, Aneesha Mohanpuria,Vijay Kumar, Sadhna Marwah, A.S.Nigam, Gurdeep Buxi

Department of Pathology, PGIMER, Dr. Ram Manohar Lohia

Hospital, New Delhi

Objective: To analyse and characterize various Hb variants and he-

moglobinopathies using high performance liquid chromatography

(HPLC). Methods: The present retrospective study was carried out at

PGIMER, Dr. RML Hospital, by analysing the data obtained from

September 2009 to August 2012. The clinical and haematological

records of patients suspected of hemoglobinopathies were retrieved

and studied with the HPLC pattern. Family studies carried out in

indeterminate cases were also reviewed. Results: Abnormal haemo-

globin fractions on HPLC were seen in 121 of the 1846 cases studied.

Of these, beta thalassemia trait was the predominant abnormality with

a total of 69 cases (57.02 %). HbS was the next most common Hb

variant, observed in 18 cases (14.8 %). Of these, four cases were

homozygous for sickle cell disease. Other hemoglobins observed were

Hb D Punjab (7 cases; 5.78 %), alpha thal (7 cases; 5.78 %) and Hb E

(5 cases; 4.13 %). Hb J Meerut, HPFH and combined Hb E/beta thal

inheritance had 3 cases each (i.e. 2.3 % each). 5 cases also showed

peak in the C window. 2 cases (1.6 %) were each of Hb Q India, Hb

Lepore trait and beta thal major. Conclusion: Our study highlights that

high performance liquid chromatography (HPLC) is a simple yet rapid

and reliable tool for the detection of haemoglobin variants.

Abstract P 115

Determination of Allelic Frequency of SNPs at Quantitative TraitLoci (QTL)s of Fetal Hemoglobin

Charu Batra1, Jasbir Kaur1, Amita Trehan2, Jasmina Ahluwalia1,Reena Das1

1Department of Hematology, 2Department of Pediatrics, Postgraduate

Institute of Medical Education and Research, Chandigarh

Introduction: Fetal Hemoglobin (HbF) is considered a ‘quantitative

trait’ (QT) wherein multiple genes together with a small environmental

component determine the value measured in any given individual. High

HbF levels are associated with milder disease progression and fewer

complications in patients with Sickle cell Disease (SCD) and b thal-

assemia. Recently conducted Genome-Wide Association Studies

(GWAS) in European and African American populations and patients

with hemoglobinopathies, have identified single-nucleotide polymor-

phisms (SNPs) from chromosomal loci that contribute to varying

expression levels of HbF and other clinical traits. They include the

Xmn1-Gc polymorphism and SNPs at BCL11A and HBSB1L-cMYBinter-region loci. To the best of our knowledge, the allelic frequency of

SNPs at BCL11A and HBS1L-MYB intergenic region in normal North

Indian population and their association with thalassemia in India has not

been studied. Objective: Determination of prevalence of polymor-

phism at SNP rs11886868 in BCL11A exon 2, SNP rs4895441 in the

HBSB1L-cMYB inter-region and SNP rs74822144 in b globin gene

cluster (The Xmn1-Gc polymorphism) in normal North Indian popula-

tion. Methods: Single-nucleotide polymorphism (SNP) analysis was

performed by using polymerase chain reaction (PCR)/restriction

enzymes on genomic DNA extracted from peripheral blood leukocytes

of fifty healthy normal children[3 years of age. Enzymatic digestion

was performed by XmnI, MboII, and RsaI for, rs74822144, rs11886868

and rs4895441 respectively. Results: Minor allele frequencies for

rs11886868 and rs4895441, rs74822144 were 0.35, 0.13 and 0.26 in

normal North Indian children. We plan to determine the allelic fre-

quency at these loci in children with thalassemia intermedia and to

correlate the SNPs with the clinical phenotypes

Abstract P 116

Non-Transfusion Dependent Thalassemia; Management Issues

Maitreyee Bhattacharyya, Asutosh Panigrahi

N.R.S Medical College, 138, A.J.C Bose Road, Kolkata 700014

Objectives: Non transfusion dependent thalassaemia (NTDT) usually

do not require regular RBC transfusions for survival but associated with a

variety of serious clinical complications that require proactive and

comprehensive management. Methods: We investigated clinical and

biochemical profile of non-transfusion dependent thalassemia patients

attending thalassemia clinic of N.R.S medical College, Kolkata. Results:Out of 179 patients studied, 158 were HbE b thalassemia and rest Sb or

Hb E disease or b thal intermedia. Mean age of diagnosis was 15 year

(range 1–48 years). Mean Hb % was 7.4 g/dl (range 4–10 g/dl). Mean

age of 1st BT was 17 years 20 (11 %) patients didn’t have splenomegaly,

47 (26 %) without skeletal deformity (ineffective erythropoiesis


123

indicator). Growth failure observed in 87 patients, 95 were with jaundice,

5 had delayed puberty. Predominant complications observed was Gall

stone in 1 patient, thyroid dysfunction in 5 patients VTE in 1. We didn’t

observe diabetes, pulmonary hypertension, in any patients. Serum ferritin

was above 1000 in 16 [with normal development in 4 (2 %) and normal

puberty in 6 (3 %)] and 122 patients had s.ferritin between 300 and 1,000.

Management provided were transfusion [required 98 (55 %), frequent in

7 (4 %) and not required in 81 (45 %)], splenectomy in 3 (1.6 %), hy-

droxylurea 2 (1 %), thromboprophylaxis in 1 (0.5 %) iron chelation in 17

(9 %) and monitoring. Conclusion: Results from our study shows that

effective management of NTDT patients in our genetic background can

give a far better quality of life.

Abstract P 117

Profile of Hemoglobinopathies in the State of Odisha

Anasuya Lenka, Sudha Sethy, Rajeeb Nayak, RK Jena, PranatiMohanty

Department of pathology and department of clinical hematology,

S.C.B. Medical College and Hospital, Cuttack

Objectives: To determine the clinical and hematological profile of

different hemoglobinopathies. Materials and Methods: Patients with

clinical features of chronic hemolytic anemia without history of blood

transfusion in last three months attending the department of clinical

hematology of SCB Medical College & Hospital, Cuttack from

November 2007 to February 2012 are included in the study. About 2 ml

of blood was collected from each patient which was investigated in fully

automated capillary zone electrophoresis. Screening tests like sickling

test and complete blood count by five part cell counter (sysmax 2000I)

were done in all cases. Results: Out of total 6,195 cases hemoglobin-

opathy was found in 3,263 cases. Beta-thalassemia constitutes 28.1 %

cases with 21.02 % of trait, 4.51 % thalassemia intermedia and 2.57 %

of thalassemia major cases. Sickle cell disorder was found in 27.03 %

cases with sickle cell trait in 23.17 % and sickle cell disease in 3.86 %

cases. Sicke-beta thal was 21.88 %. Hb E was found in 2.69 % cases and

E-beta thal in 9.65 % cases. Conclusion: Hemoglobinopathy is com-

mon in almost all parts of Odisha. They cause high degree of morbidity

and mortality along with socio- economic burden. In this study diag-

nosis could not be confirmed in 8.8 % of cases which needs further

investigations like parental evaluation and globin chain mutation

analysis.

Keywords Hemoglobinopathies, Sickle cell, Thalassemia, Hb-E,

Odisha state

Lymphomas

Abstract P 118

Plasmablastic Lymphoma of the Retroperitoneumin an HIV-Negative Patient

Nishad Dhakate1, Soniya Nityanand2

1S.G.P.G.I, Lucknow; 2Department of Hematology, SGPGI, Lucknow

Abstract: Plasmablastic lymphoma (PBL) is an aggressive non-

Hodgkin lymphoma classically occurring in individuals infected with

HIV. Plasmablastic lymphoma has a predilection for the oral cavity

and jaw. However, recent case reports have shown plasmablastic

lymphoma in the stomach, lung, nasal cavity, cervical lymph nodes

and jejunum in HIV negative individuals. We report what is, to the

best of our knowledge, the second case of plasmablastic lymphoma

occurring in the retroperitoneum of a HIV-negative man. Case

Report: A 57 years old male, known case of hypertension presented

with chief complaints of swelling in the right thigh since 3.5 months.

MRI pelvis revealed a soft tissue lesion in adductor compartment of

right thigh. FDG PET scan showed a soft tissue swelling in retro-

peritoneum extending into the pelvis along bilateral psoas muscle, on

the right extending into right inguinoscrotal region, inguinofemoral

region and in adductor compartment. Pathological examination

showed tumour cells with eccentrically placed nucleus and moderate

amount of eosinophilic cytoplasm consistent with plasmablastic

lymphoma. Immunohistochemistry was diffusely and strongly posi-

tive for vimentin and CD 138 and focally and weakly positive for

LCA, CD 38 and CD 79a consistent with plasmablastic lymphoma.

His LDH was 5864, creatinine 2.8 mg%, Na 132 meq, K 6.5 meq,

uric acid: [ 20.0 mg, phosphorus: 5.9 meq and calcium 8.8 mg%

suggestive of tumour lysis syndrome which required dialysis. He was

HIV and EBV negative. Serum protein electrophoresis was normal.

Serum free kappa light chains of 42.8 mg/L (3.3–19.40) and serum

free lambda light chains of 760 mg/L (5.71–26.30). Bone marrow

Aspiration and Biopsy showed no e/o myeloma or lymphoma. Our

case was started on standard CHOP (cyclophosphamide, doxorubicin,

vincristine, and prednisone) chemotherapy. In view of minimal

response he was shifted to salvage chemotherapy (ESHAP). But

patient expired due to neutropenic sepsis.

Abstract P 119

Primary Bone Marrow Lymphoma: A Diagnostic Dilemma

M Deepak Nayak1, Lakshmi Rao, Sushma V. Belurkar,Chethan Manohar

1Department of Pathology, Melaka Manipal Medical College,

Manipal University, Manipal; Department of Pathology, Kasturba

Medical College, Manipal University, Manipal

Introduction: Bone marrow involvement by non Hodgkin Lymphoma

(nHL) has conventionally been considered as a systemic dissemination

of a nodal malignancy. Primary Bone Marrow Lymphoma (PBML) is a

rare exception to this rule. This entity accounts for less than 50 cases in

published literature; thereby highlighting its diagnostic novelty and

clinical significance. Apart from the exclusion of a leukemia originating

in the bone marrow, the current criteria include ruling out a nodal or an

extranodal involvement (including the bone). We present one such

unique case in a 41 year old male patient presenting with isolated

thrombocytopenia. Case Report: The peripheral smear showed

abnormal large lymphoid cells; also reflecting in the bone marrow

aspirate. This led to consideration of alternative diagnoses such as acute

lymphoblastic leukemia and lymphoma. The clinico-radiological work

up showed an absence of organomegaly and lymphadenopathy; thus

necessitating a review of the bone marrow study. The trephine biopsy

revealed interstitial and paratrabecular infiltrates of abnormal lymphoid

cells with brisk mitotic figures. The immunohistochemistry panel for

lymphoma showed a strong positivity for CD10, CD 20 and BCL 2 in

the lymphoid cells. The other markers such as CD34, CD99 and BCL6

were negative. Considering all the above information, a diagnosis of

PBML was rendered. A 5 month follow up has thus far showed a

favourable response to chemotherapy. Conclusion: Primary Bone

Marrow Lymphoma is an uncommon lymphoid malignancy, often

overshadowed by its more common neoplastic counterparts. An

awareness of this entity is essential to establish an accurate diagnosis

and adequate therapy.


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Abstract P 120

Gastric Outlet Obstruction in a Child—a Rare Manifestationof Burkitts Lymphoma

Anand Prakash1, Kanishka Das2, Usha Kini3

1St Johns Medical College Hospital, Department of Pediatrics,

Bangalore, India; 2St Johns Medical College Hospital, Department

of Pediatric Surgery, Bangalore, India; 3St Johns Medical College

Hospital, Department of Pathology, Bangalore, India

Purpose: Burkitt’s lymphoma may arise in many atypical locations

which on rare occasions can include stomach. A case of one such

primary non-endemic gastric Burkitt’s lymphoma is described here in

a non-HIV child who presented with features of gastric outlet

obstruction and had a dramatic resolution of symptoms with che-

motherapy. Method: Case report: A 6 year old undernourished girl

with history of abdominal pain and persistent vomiting of 2 months

duration presented with a large, firm, irregular epigastric mass. The

CT-scan revealed a gastric mass with diffuse uniform thickening of its

wall involving the body and antrum with near complete obliteration of

the pyloric orifice. A pelvic lymphnodal mass lesion measuring

5.0 9 4.5 cm along with multiple perigastric and mesenteric lymph

nodes, were also seen. A tru-cut biopsy of the gastric mass showed a

high grade diffuse mature B cell lymphoma with positivity for CD10,

CD 20, CD 79a and Ki67 index of 100 % and negative expression for

Tdt, diagnostic of Burkitt’s lymphoma and Stage III as bone marrow

aspiration biopsy and CSF were negative for lymphoma cells.

Results: The patient was started on NHL-902 protocol. Within a

week of commencing therapy of cytoreduction with vincristine,

cyclophosphamide and prednisolone her symptoms of gastric outlet

obstruction resolved. She continued therapy as per protocol with

COPAD and COPADM and is currently well. Conclusion: The case

presented here highlights an uncommon presentation of a common

pediatric lymphoma and demonstrates the prompt resolution of gastric

outlet obstruction with chemotherapy.


A Case Series of Three Patients of Hematolymphoid Malignancywith Primary Presentation of Renomegaly

Bidish Kumar Patel, Pritinanda Mishra, Debdatta Basu, RakheeKar

Department of Pathology, JIPMER, Puducherry-6

Introduction: Extramedullary renal involvement in hematological

malignancies is a rare occurrence and may lead to diagnostic dilem-

mas. Report: We report a series of three patients, two children, a girl

aged seven and a boy of 11 years and an adult male aged 37 years, all

of whom on initial presentation had renomegaly and were investigated

for a suspected renal disease. Two of them underwent a renal biopsy.

Subsequently, based on further clinical evaluation, peripheral blood

and bone marrow examination and lymph node biopsy in one; all the

three patients were diagnosed with high-grade hematolymphoid

malignancies. Both the male patients had precursor B cell Acute

Lymphoblastic Leukemia with Renal infiltration while the girl was

diagnosed as a primary Renal T cell Acute Lymphoblastic Lymphoma

with infiltration of the bone marrow. Conclusion: This series of three

cases highlight the fact that hematolymphoid malignancies can at

times masquerade as renal disease. Leukemias and lymphomas can

have myriad clinical manifestations which can mislead the clinical

judgment necessitating a high index of clinical suspicion and a good

clinicopathological correlation to arrive at a final diagnosis.


Hodgkin Lymphoma: Immunohistochemical Featuresand Association with Epstein-Barr Virus

Debdatta Basu, S Muthu

Department of Pathology, JIPMER, Pondicherry

Background and Objectives: Hodgkin Lymphoma (HL) shows

variation in epidemiological, immunohistochemical features and

association with Epstein-Barr virus (EBV). Immunohistochemical

features as well as the EBV status of HL patients have an impact on

the prognosis. The objectives of this study were to classify HL based

on histomorphology and immunophenotype, to determine the asso-

ciation of EBV with HL and to compare the histological subtype,

proliferation index, and clinical features between EBV positive and

EBV negative cases. Materials and Methods: There were 82 patients

of HL diagnosed over a period of 5 years. The histomorphological

features were analyzed. Immunohistochemistry was done with a panel

of markers including CD3, CD20, CD15, CD30, LCA, EBV LMP-1

and PCNA. The clinical, histomorphological and immunohisto-

chemical parameters were correlated and statistical analysis were

performed using Fischers exact test and Chi-square test. Results:There were 80 cases of classical Hodgkin Lymphoma (CHL) and two

cases of Nodular Lymphocyte Predominant Hodgkin Lymphoma

(NLPHL). Nodular sclerosis was the most common subtype of CHL

(48 cases—60 %). CD30, CD15, CD20 were positive in 100, 83.8 and

10 % cases respectively. Group A immunophenotype (CD30+,

CD15+, CD20-) was the most common (75 %) followed by Group B

(CD30+, CD15-, CD20-). Both cases of NLPHL were CD20+,

CD45+, CD15-, CD30- and negative for EBV. EBV LMP-1 was

positive in 46 CHL cases (57.5 %). EBV association was more

common in children, males, mixed cellularity and lymphocyte

depleted subtypes, and in patients with bone marrow infiltration, high

clinical stage and B-symptoms. PCNA expression was high in almost

all cases irrespective of their EBV status. Conclusion: The immu-

nohistochemical features of HL in our study population was similar to

that of the Western countries. EBV positivity in HL was more often

associated with adverse prognostic factors. However the overall

impact of these differences on the clinical outcome of these patients

need to be studied in large scale clinical studies.


Diagnosis of Hematolymphoid Malignancies in SerousFluids—a Spectrum of Three Cases

S Prasath, NG Rajesh, Debdatta Basu

Department of Pathology, JIPMER, Pondicherry

Background: Involvement of serous fluids by hematolymphoid

malignancies is a common manifestation across the spectrum of

indolent as well as high grade lesions. At times, involvement of

serous cavities is the primary manifestation of lymphoma/leukemia

thus presenting a challenge in accurate diagnosis. Case Series: We

present a series of three cases presenting with pericardial, pleural

and peritoneal effusions. Our first patient, a 25 year old male pre-

sented with massive pericardial effusion to the casualty. His

pericardial fluid cytology, revealed numerous atypical lymphoid


123

cells which were positive for PAS stain. His peripheral smear, bone

marrow aspirate and biopsy with immunohistochemical work up of

CD3, Tdt and CD 10 positive lymphoid cells confirmed the diag-

nosis of acute lymphoblastic leukemia. Our second patient presented

with history of ischemic heart disease and bilateral pleural effusion

and was suspected to have congestive cardiac failure. His pleural

fluid cytology revealed sheets of mature lymphoid cells and scat-

tered mesothelial cells. His peripheral smear and bone marrow

aspirate showed lymphocytosis (80 %) with CD20+, CD5+ and

CD23+ nodular to interstitial deposits of lymphoid cells in biopsy.

Our third patient a 5-year-old child presented with ascites and

ultrasound revealed retroperitoneal nodes. Ascitic fluid cytology

showed CD 10+, Tdt-, CD20+ lymphoid cells with basophilic

cytoplasm, prominent vacuolation and high mitotic activity. We

diagnosed Burkitt lymphoma and child is on treatment. Conclusion:Knowledge of various manifestations of hematolymphoid malig-

nancies in serous fluids is crucial in accurate diagnosis and timely

management.


Primary Extranodal non Hodgkin’s Lymphoma of the Headand Neck—a Study from a Tertiary Care Centre in South India

Pritinanda Mishra, Sreeya Das, Debdatta Basu, Rakhee Kar,Bhawana Badhe, Sajini E Jacob

Department of Pathology, Jawaharlal Institute of Postgraduate

Medical Education and Research, Pondicherry

Introduction: Primary extranodal non Hodgkin’s lymphoma (NHL)

of the head and neck accounts for 10–20 % of all cases of NHL. This

study was undertaken to ascertain the anatomic distribution, histo-

logical subtypes and sites of extranodal NHL presenting in the head

and neck region. Materials and Methods: This was a descriptive

study which included record review of the departmental archives over

a period of 3 years (2010—2012). Clinicopathologic features of 25

cases were studied in detail. Results: Of the 25 cases involving head

and neck 16 were males and nine were females. Mean age was

50 years. The most common sites of involvement were Waldeyer’s

ring, followed by nose, brain, orbit, palate and thyroid. Histologically

majority of the cases (80 %) were B cell origin of which diffuse large

B cell lymphoma was the commonest subtype (15 cases), followed by

plasmablastic lymphoma (2 cases) and a single case each of small

lymphocytic lymphoma, lymphoblastic lymphoma and low grade

marginal zone lymphoma of the mucosa associated lymphoid tissue

(MALT) type. T cell lymphomas constituted 20 % of the cases.

Conclusion: This retrospective study in a tertiary care centre in South

India illustrates the pattern of clinical and histological distribution of

various common and uncommon subtypes of extranodal NHL in the

head and neck region.

Abstract P 125

Gemcitabine Induced Skin Rash in a Boy with Hodgkin Disease

Vikas Dua1, Jai Bhagwan Sharma2

1Department of Pediatric Hematology Oncology, Action Cancer

Hospital, Delhi, India; 2Department of Medical Oncology, Action

Cancer Hospital, Delhi, India

Objective: Gemcitabine is used in various carcinomas like lung

cancer, pancreatic cancer, bladder cancer and breast cancer in adults.

It is considered to be a well-tolerated drug with little known side

effects. The reported toxic effects of gemcitabine include myelo-

suppression, altered liver function tests, flu-like syndrome,

bronchospasm, rash, itching, and fever. Gemcitabine has not been

frequently used in pediatric malignancies and to our knowledge

cutaneous reaction has never been reported in children. Method: A

8-year-old boy was admitted in our hospital because of fever and

multiple swellings on both sides of his neck in March 2012. On

examination multiple bilateral cervical lymph nodes were palpable.

Abdominal examination showed hepatosplenomegaly. Biopsy of

cervical lymph node suggested Hodgkin’s disease. Diagnosed as stage

III B Hodgkin’s disease, he was treated with ABVD based chemo-

therapy. Re evaluation following four cycles of chemotherapy

revealed progressive disease, so patient was put on ifosfamide,

gemcitabine, vinorelabine and prednisolone (IGV) based chemother-

apy. Results: On Day 3 of treatment child developed a

maculopapular, itchy skin rash. The rashes involved the neck, chest,

back, upper arms and abdominal wall. The rash subsided in severity

within 4-5 days with the use of oral antihistamine. However, it

reappeared again on Day 5 on repeat challenge with gemcitabine

during second cycle of chemotherapy. The skin lesions were again

easily managed with oral antihistamines. Conclusion: Doctors deal-

ing with chemotherapy drugs should be aware of this kind of side

effect in order to avoid a misdiagnosis and identify the cause of rash.

Abstract P 126

A Retrospective Analysis of Patterns of Distributionof Lymphomas in Elderly Patients in a Tertiary Care Hospitalin South India Over a Period of 5 years

Parimal Sarda1, Marie Therese Manipadam1, Sheila Nair1,Auro Viswabandya2

1Department of Pathology, Christian Medical College, Vellore;2Department of Hematology, Christian Medical College, Vellore

Introduction: This study aims to analyse the pattern of distribution of

lymphoid neoplasms in elderly patients (age more than 60 years) in a

single tertiary care centre in South India using WHO 2008 classifi-

cation. Material and Methods: This is a retrospective analysis of 359

cases diagnosed as lymphoma in elderly patients during the period of

Jan 2007 to Dec 2011. Results: A total number of 359 elderly patients

with diagnosis of lymphoma were included in this study. There were

245 males and 114 female patients. The mean age was 66.72 years.

Of these, non Hodgkin lymphomas (NHL) accounted for 89.97 %

(n = 323), Hodgkin lymphoma (HL) 9.75 % (n = 35) and post

transplant lymphoproliferative disorder 0.28 % (n = 1) respectively.

In NHLs, B cell neoplasms accounted for 90.09 % (n = 291) and T

cell neoplasms 9.91 % (n = 32). In B cell neoplasms, mature B cell

NHL accounted for 99.65 %. In mature B-NHL, indolent lymphomas

accounted for 43.10 % (n = 125) and aggressive lymphomas

56.90 % (n = 165). The subtypes of non Hodgkin lymphomas were

as follow: diffuse large B cell lymphoma (n = 161, 50 %), follicular

lymphoma (n = 33, 10 %), chronic lymphocytic leukemia/small

lymphocytic lymphoma (n = 17, 5.26 %). In T cell neoplasms,

mature T cell NHL accounted for 96.7 % and precursors T-LBL 3 %.

Among the mature T cell NHLs, the pattern of distribution was;

peripheral T cell lymphoma, not otherwise specified in 14 (4.34 %)

patients, angioimmunoblastic T cell lymphoma in 9 (2.78 %) and

anaplastic large cell lymphoma in 4 (1.23 %). Mixed cellularity was

the major subtype of classical Hodgkin lymphoma found in 13 (38 %)

where as Nodular lymphocyte predominant HL constituted 3 %

(n = 1). As compared to the distribution pattern of lymphomas

including all age groups in our institution the striking feature was that


123

B cell NHLs are more common in elderly patients. T-LBL and ALCL

are more common in the former group, but this included childhood

lymphoma also, hence when these are excluded overall frequency

becomes similar. Frequency of EBV + DLBCL of elderly will be also

presented. Conclusions: This is the first study from India showing the

pattern of distribution of lymphoid neoplasms in the elderly patients.

Abstract P 127

Lymphoma Involving Spleen and its Histopathological Patternin a Tertiary Care Hospital in South India: A Five Year Study

Subramaniam Kandasamy, Marie Therese Manipadam,Sheila Nair

Department of Pathology, Christian Medical College, Vellore

Introduction: Lymphoma involving spleen is heterogeneous array of

disease whose accurate diagnosis is essential in further management.

The aim of this study is to analyze the lymphoma involving spleen and

its histopathological pattern in a South India tertiary care centre using

WHO 2008 classification. Material and Methods: A retrospective

study of all splenic lymphomas diagnosed in Christian Medical Col-

lege Hospital, Vellore during a 5 year period (Jan 2007 to Dec 2011).

Results: This study includes 69 cases diagnosed as splenic lymphoma

from January 2007 to December 2011. This includes 50 males and 19

females. The mean age was 47 years (range from 7 to 74 years). Non-

Hodgkin lymphoma (NHL) was more common 88.4 % (n-69), than

Hodgkin lymphoma (HL) 11.6 % (n = 69). In NHL, B cell neoplasms

accounted for 73.8 % (n = 61) and T cell neoplasms 26.2 % (n = 61).

The subtypes of NHL were as follow; splenic marginal zone lym-

phoma (n = 13; 28.8 %), diffuse large B cell lymphoma (n = 12;

26.6 %), hairy cell leukemia (n = 6; 13.3 %), follicular lymphoma

(n = 4; 8.8 %), T cell/histiocyte-rich large B cell lymphoma (n = 2;

4.4 %) and one each of mantle cell lymphoma (2.2 %), lymphoplas-

macytic lymphoma (2.2 %) and chronic lymphocytic leukemia/small

lymphocyte lymphoma (2.2 %). 3 cases of low grade B cell lym-

phoma, unclassifiable. Among T cell NHL NK/T cell lymphoma

accounts for 37.5 % (n = 6/16) followed by hepatosplenic T cell

lymphoma (n = 5/16; 31.25), Peripheral T cell lymphoma (PTCL)

(n = 3/16; 18.25 %) and one case of T cell large granular lymphoma.

Splenic marginal zone and diffuse large B cell lymphomas were the

commonest types. As compared to western literature, follicular lym-

phomas were infrequent and peripheral T cell lymphomas including

Hepatosplenic T cell lymphomas and NK/T cell lymphomas were

more common. Conclusions: This study shows the frequency and

pattern of lymphomas involving spleen in a tertiary care centre.

Abstract P 128

Mature T/NK-Cell Lymphoproliferative Disorders: A TertiaryCentre Flow Cytometry Experience

Neeraj Arora, Bhargavi Balakrishnan, Ansu Abu Alex, RayazAhmed, Aby Abraham, Biju George, Alok Srivastava, VikramMathews

Department of Haematology, Christian Medical College and Hospital,

Vellore 632004

Abstract: The classification of the T cell malignancies is complex

and based on clinical features and laboratory investigations, which

include morphology, histology, immunophenotype, HTLV-I serology

and molecular genetics. Mature T/NK-cell lymphoproliferative dis-

orders are rare disorders with morphologic heterogeneity, and lack of

specific immunophenotypic markers for clonality. We retrospectively

analyzed immunophenotypic data from consecutive cases of mature

T/NK cell lymphoproliferative disorders diagnosed in our flow

cytometry laboratory from 2009 to August 2012. These cases were

diagnosed based on immunophenotyping of PB and BM aspirates and

morphology as per WHO 2008 guideline. The gating strategy used

included FSC/SSC, CD45/side scatter and CD3 gating. The initial

panels evaluated for acute leukemia or mature lymphoma depending

on the morphology. In all patients a T cell specific secondary panel

was used for further characterization using CD2, CD3, CD4, CD5,

CD7, CD8, CD10, CD16, CD19, CD25, CD34, CD56, CD57, TCR-

ab, TCR-cd, CD1a, HLA-DR and TdT (BD Biosciences, USA) in

most cases. Correlation was carried out with immunohistochemistry

on tissue (wherever available) such as BM, lymph node and skin

biopsies, serological tests for HTLV1 and other laboratory investi-

gations. Of the total 285 mature lymphoid neoplasms, T/NK-cell

lymphoproliferative disorders constituted 9.5 % (n = 27) and B cell

lymphoproliferative disorders including 12 cases of Burkitts lym-

phoma constituted 90.5 % (n = 258). Plasma cell disorders were not

included in the study. The commonest mature T/NK-cell lympho-

proliferative disorder in this study was hepatosplenic lymphoma

(HSL) (n = 10, 37.1 %) followed by T-large granular leukaemia (T-

LGL) (n = 4, 14.8 %), Adult T cell leukaemia/lymphoma (ATLL)

(n = 3, 11.1 %), Mycosis/Sezary syndrome (MF/SS) (n = 3, 11.1 %)

and T cell prolymphocytic leukaemia (T-PLL) (n = 1, 3.7 %).

Table 1 Immunophenotypic patterns in Mature T/NK cell lympho-

proliferative disorders

HSL (%) T-LGL (%) MF/SS (%) ATLL (%)

CD2 10 (100) 3/3 (100) 3 (100) 3 (100)

CD3+ 10 (100) 4 (100) 2 (66) 3 (100)

CD5+ 1 (10) 2/3 (67) 3 (100) 3 (100)

CD4+ CD8- 0 0 2 (66) 3 (100)

CD4- CD8+ 4 (40) 4 (100) 0 0

CD4- CD8- 6 (60) 0 0 0

CD4+ CD8+ 0 0 0 0

CD7+ 10 (100) 3/3 (100) 1 (33) 0

CD16+ 6 (85) 2 (50) 0 0

CD25+ 0 0 1 (33) 3 (100)

CD56+ 3 (30) 2/4 (50) 0/1 0

CD57 0/8 1/3 (33) ND 0

TCRab+ 1 (10) 3/3 (100) 1/1 (100) 2 (66)

TCRcd+ 9 (90) 0 0/1 0

Total cases 10 4 3 3

NKCLPD(%)

T-PLL(%)

AggressiveNK- cellleukaemia (%)

T-CLPDu (%)

CD2 1 (100) 1 (100) 2 3 (100)

CD3+ 0 1 (100) 0 3 (100)

CD5+ 0 1 (100) 0 3 (100)

CD4+ CD8- 0 1 (100) 0 2 (67)

CD4- CD8+ 1 (100) 0 1 (50) 1 (33)

CD4- CD8- 1 (100) 0 1 (50) 0

CD4+ CD8+ 0 0 0 0

CD7+ 1 (100) 1 (100) 1 (50) 2 (67)

CD16+ 1 (100) 0 2 (100) 0

CD25+ 0 0 0 1 (33)

CD56+ 0 1 (100) 2 (100) 0

CD57 0 0 1 (50) 0

TCRab+ 0 0 1 (50) 3 (100)

TCRcd+ 0 0 0 0

Total cases 1 1 2 3


123

Mature NK-cell disorders (n = 3, 11.1 %) included 2 cases of

aggressive NK-cell leukaemia and a case of chronic NK cell lym-

phoproliferative disorder (NK-CLPD). Because of the lack of clinical

details a definite diagnosis could not be made in 3 cases and these

cases were labeled as T cell lymphoproliferative disorders unclassi-

fiable (T-CLPD u).The different expression patterns of T cell markers

in various subtypes are summarized in Table 1. To conclude with an

appropriately selected panel, phenotypic analysis using FCM is very

useful for the diagnosis of T cell and NK-cell lymphoproliferative

disorders.

Abstract P 129

Result of Paediatric Non Hodgkin’s Lymphoma with IntensifiedShort Duration Chemotherapy

Prattusha Sengupta, P Gupta, S Biswas, A Mukhopadhyay



Objective:Aim of our study was to observe result of intensified

short duration chemotherapy in pediatric Non-Hodgkin’s Lympho-

mas (NHL). Methods: We included consecutive 240 pediatric NHL

patients in pediatric haematooncology department of Netaji Sub-

hash Chandra Bose Cancer Research Institute during period from

2000 to 2011. Inclusion criteria: patients \18 years of age with a

diagnosis of NHL. Exclusion criteria: Patients with [25 % blasts in

the bone marrow. Each patient received three cycles A and three

cycles B of MCP 842 protocol of INCTR. Response was assessed

at the completion of 2 cycles of chemotherapy (1 each of A and B)

and 6 cycles of chemotherapy. Result: 70 (29.17 %) patients-

Lymphoblastic Lymphoma (LL), 98 (40.83 %)-Burkitt Lymphoma.

60 (25 %)-diffuse Large B Cell Lymphoma (DLCL), 12 (5 %)-

Anaplastic Large Cell. The abdomen was the most common site in

80 (33.3 %) of involvement followed by mediastinum in 38

(15.83 %). 202 (84.17 %) patients achieved complete response after

2 cycles of therapy. 20 (8.33 %) achieved partial response, 10

(4.17 %) had no response, 10 (4.17 %) were not evaluable. With

median follow up of 4 years a total of 64 (26.67 %) patients (28

LL, 24 Burkitt Lymphoma, 8 DLCL and 4 ALCL) had died.

Causes of death were progressive disease in 48, infection in 12,

hepatitis in 2 and unknown 2. 176 (73.33 %) patients are alive and

disease free. Patients tolerated chemotherapy well. Grade-IV febrile

Neutropenia was seen in 52 patients. Conclusion: Result of short

duration chemotherapy MCP842 is promising. We intend to con-

tinue the same protocol for next few years.

Abstract P 130

Multiple CNS (Only) Relapse of a Case of Extra Nodal NonHodgkin’s Lymphoma with Repeated Complete Remissions

Deba Dulal Biswal, Dinesh Pendharkar, Akshay D. Shah,Suresh H. Advani

Asian Institute of Oncology, Mumbai

Abstract: Relapse of Non Hodgkin’s Lymphoma (NHL) in CNS

though occurs, is a rare phenomena. Relapse of extra nodal NHL in

CNS multiple times with complete response to conventional therapy

each time is very rare. We report a case of multiple CNS (Only)

relapse of a case of extra nodal Non Hodgkin’s Lymphoma with

repeated complete remissions. A 41 years male patient, who was a

known case of NHL, DLBCL, CD20 Positive, of Maxilla and Eth-

moid with mild extension to intracranial, but not involving the brain

parenchyma presented to TMH, Mumbai in 2003. This was an extra

nodal NHL in the face around nose, nearer to cranium. Following 6

cycles of CHOP chemotherapy and local Radiotherapy, he showed

complete response. About 3 years later again presented to the same

hospital with first time brain relapse only (ataxia, brain lesions on

MRI), received treatment as per CNS protocol. Post treatment scan

showed complete response and disappearance of lesion. Two years

later follow up MRI showed normal brain. Later he presented to our

hospital with multiple (two times) only CNS relapses at a gap of

4 years and 10 months respectively with complete response to

treatment.

Abstract P 131

Comparison of Efficacy and Safety of Rituximab (Mabthera�)and its Biosimilar (Reditux�) in Diffuse Large B Cell Lymphoma(DLBCL) Patients Treated with Chemo-Immunotherapy:A Retrospective Analysis

Partha Sarathi Roy, Shiji John, Sadhana Kannan, Sumeet Gujral,Jayant Gawande, Bhausaheb Bagal, Navin Khattry, ManjuSengar, Hari Menon, Reena Nair

Hematolymphoid Disease Management Group, Department

of Medical Oncology, Tata Memorial Centre, Mumbai, India

Objective: Rituximab with CHOP regimen (R-CHOP) has been the

standard of care for DLBCL. Reditux�, a biosimilar molecule of

Rituximab, was in use in India since 2007. The objective of this

retrospective audit is to compare the efficacy, safety and toxicity of

Mabthera� with Reditux�. Methods: Two hundred twenty-three

patients aged C18 years who received 4-8 cycles of R-CHOP

regimen between January 2004 to June 2010 were included.

Complete response (CR), Partial response (PR)] in both groups

were evaluated as per Cheson’s criteria and compared by Chi-

Square test. Overall survival (OS) and progression-free survival

(PFS) were estimated by Kaplan–Meier method and compared by

two-sided log rank test. All grade 3–4 hematological and non-

hematological toxicities were recorded. Results: One hundred one

patients received Mabthera�, 72 received Reditux�. In 17 patients,

the brand used was unknown and 33 patients received both.

Baseline characteristics at presentation were similar in both groups.

Median number of treatment cycle received was 6 in each group.

Observed CR rate was 75 % in Mabthera� group and 81 % in

Reditux� group. OS at 5 years was 84 % (Mabthera� 83 % and in

Reditux� 90 %; P = 0.214). PFS at 5 years was 67 % (Mabthera�

71 % and Reditux� 78 %; P = 0.579). We also evaluated patients

who had received C4 cycles of Mabthera� or Reditux� in their

respective groups, and found no differences in efficacy and out-

comes. Three patients died during treatment in each group. Grade

3–4 febrile neutropenia was observed in 23 % (Mabthera�) and

20 % (Reditux�); grade 3–4 oral mucositis and diarrhea was seen

in 27 % (Mabthera�) and 20 % (Reditux�). No difference in in-

fusional reactions was observed. Conclusion: In this retrospective

analysis we found Reditux� is as efficacious and safe as

Mabthera�.


123

Abstract P 132

Utility of Flow Cytometry in Bone Marrow Staging of Lymphoma

K Ghodke, T Shet1, E Sridhar1, S Ghogale, Y Badrinath, S More,PG Subramanian, S Gujral

Department of Hematopathology, 1Department of Histotopathology,

Tata Memorial Centre, Mumbai

Introduction: Immunophenotypic (IPT) analysis is an established

tool in the diagnosis and classification of many hematolymphoid

disorders; however, the role of flow cytometry (FC) in detecting

bone marrow (BM) involvement for the staging of lymphoma is

yet to be defined. Objectives: To evaluate the role of FC im-

munophenotyping of BM in the staging of lymphoma and its

correlation with morphology on bone marrow aspirates (BMA)

and trephine biopsy (BM Bx) with immunohistochemistry.

Materials and Methods: A retrospective review was done of 193

consecutive staging BM over a period of 2 month (June–July

2012). Of these, FC analysis was asked for in 60 cases (as per

clinicians request). Morphologic and FC IPT analyses were per-

formed and results were compared with BM Bx findings. FC IPT

analysis was done by six color antibody panel on FACSCANTO

II. Results: As seen in following table, FC helped in detection of

BM involvement in 5 % (3/60) cases in which morphological

analyses on BM failed to detect the involvement. Conclusion: FC

IPT analysis on staging marrow contributed upstaging in 5 % of

cases.

Comparison of FC IPT analysis and morphological analysis in 60

cases

FC IPT

analysis

Morphological analysis Total

BMA (i)

BMBx

(i)

BMA (i)

BMBx

(ui)

BMA

(ui)

BMBx

(i)

BMA (ui)

BMBx

(ui)

i 14 0 7 3 24

ui 0 0 4 32 36

Total 14 0 11 35 60

i involved, ui uninvolved)


Flowcytometric Immunophenotypingof T Cell Leukemia/Lymphoma

Jyoti Garg, Poojan Agarwal, Vijay Kumar, Sadhna Marwah,AS Nigam, G Buxi

Department of Pathology, PGIMER & Dr. Ram Manohar Lohia

Hospital, New Delhi

Objective: To evaluate the role of flowcytometric immunophenotyping

in T cell leukemia/lymphoma. Methods: Flowcytometric analysis was

carried out on peripheral blood samples of 46 patients suspicious of

acute leukemia. Cases expressing T cell lineage markers were further

characterized with the help of a panel of antibodies comprising of TdT,

CD3, CD4, CD8, CD7, CD5, CD2, CD1a, CD56 and CD45 using BD

FACS Calibur. Results: Of the 10 cases expressing T cell lineage

markers, 8 cases were classified as T-lymphoblastic leukemia/lym-

phoma. All of these cases showed bright positivity for TdT, cCD3 and

CD7. Variable expression of CD1a, CD2, CD4, CD8 and CD5 was seen.

2 cases were classified as peripheral (mature) T cell leukemia/lym-

phoma. These cases showed bright positivity for CD45 and T lineage

markers with aberrant loss of some markers. Conclusion: Flowcy-

tometric immunophenotyping is a useful tool in identifying and

classifying T cell leukemia/lymphoma.

Plasma Cell Dyscrasias

Abstract P 134

sFLC Assay: Establishing National Reference Range—A Single Tertiary Care Centre Experience

Sarika Singh, Rajive Kumar, Vinod Raina, Lalit Kumar

All India Institute of Medical Sciences, New Delhi

Introduction: Serum free light chain (sFLC) assay measures levels of

free j&k immunoglobulin light chains. It is recommended to be used as

a screening test for all plasma cell dyscrasias (PCD).Therefore, inclu-

ded in both the guidelines for diagnosis as well as uniform response

criteria during therapy by International myeloma working group. To

conduct any study on sFLC we take a cue from reference range data

provided by west, due to non availability of Indian data. Aims andObjectives: This study was aimed to establish normal reference range

of sFLC in healthy donors and its comparison with other non myeloma

cohorts to validate the hypothesis that prevalent subclinical infections

and inflammation can result in higher j/k and to establish if any ethnic

variations exist in these cohorts compared to west. Materials andMethods: We studied 135 cases of healthy blood donors, 25 cases of

tuberculosis, autoimmune disorders, chronic renal failure (CRF) and

MM each during 2009–2010. sFLC assay was performed by nephelo-

metric bioassay. Results: j levels ranged from 6.8–70.7, 8.9–77.7, and

5.9–5420 mg/L in healthy donors, non myeloma cohort and MM cases

respectively. k levels were 3.3–66.7, 4.3–125, and 0.002–669.1 mg/L

levels in healthy donors, non myeloma and myeloma cohort respec-

tively. The j/k was 0.6–3.1 mg/L in healthy donors while was

0.4–1.1 mg/L in autoimmune disorder, 0.6–6.9 mg/L in CRF,

0.61–1.66 in TB patients. Conclusion: In contrast to west, all the

parameters were higher in Indian population, more so was the j/k. Since

this deviation is not observed in other non myeloma cohorts, this has a

bearing on the ethnicity rather than subclinical infections/inflammation

which was hypothesised to be the cause.


Immunohistochemical Profile and Bone Marrow Angiogenesisin Multiple Myeloma with Reference to its Clinical Significance

Sarah Grace Priyadarshini, Debdatta Basu, Rakhee Kar,TK Dutta1

Departments of Pathology and Medicine1, Jawaharlal Institute

of Postgraduate Medical Education and Research, Puducherry

Aims of the Study: To study the histomorphological features of bone

marrow in multiple myeloma and to evaluate the immunohisto-

chemical profile and angiogenesis using a panel of markers including

CD38, kappa and lambda light chain, CD56, Cyclin D1, Ki67 and CD

34 and to correlate immuno-hematological profile with various clin-

ical parameters. Materials and Methods: The study includes 48

cases of myeloma diagnosed over the period of 5 years. The


123

histomorphological features like plasma cell morphology, percentage

of plasma cells and pattern of infiltration were studied. Angiogenesis

was assessed by calculating the microvessel density (MVD) using

immunohistochemistry for CD34. Proliferation was assessed using

both Ki67 and CD38 highlighted cells. Immunohistochemistry for

CD56 and Cyclin D1 was also done. Results: A significant associa-

tion was seen between the plasma cell morphology and the pattern of

infiltration (p = 0.0067). The percentage of plasma cells showed a

significant association with the clinical staging. The MVD was sig-

nificantly associated with plasma cell morphology (p = 0.04) as well

as pattern of infiltration (p \ 0.001). The proliferation index was also

significantly associated with the plasma cell morphology (p = 0.003).

Both the Ki67 and MVD showed an increasing trend with clinical

staging and MVD was significantly associated with serum albumin

(p = 0.02). CD56 negativity was associated with circulating plasma

cells and also with higher clinical stage. Cyclin D1 positivity was not

seen in poorly differentiated morphology and the mean Ki67 was

lower in the cyclin D1 positive group. It was also found that the cases

with kappa light chain restriction had significantly less of poorly

differentiated morphology, diffuse pattern of infiltration, lower MVD

and Ki67. Conclusion: The study of bone marrow morphology and

angiogenesis with the aid of immunohistochemistry is useful for

prognosticating patients with multiple myeloma.

Abstract P 136

A Case of Hemophagocytic Syndrome with ReactivePlasmacytosis in a 16 Years Old Immunocompromised Patient

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

Era’s Lucknow Medical College and Hospital, Lucknow

Abstract: Hereditary and sporadic cases of hemophagocytic syndrome

(HPS) are primarily reported in children. We present a case of hemo-

phagocytic syndrome with reactive plasmacytosis in a 16 years young

male. The patient presented to us with persistent fever, hepatospleno-

megaly, lymphadenopathy, anaemia, leucopoenia and marked

thrombocytopenia with elevated triglycerides level. For all the above

complaints he was advised bone marrow examination which revealed

hypocellular smears with marked erythrophagocytosis by histiocytes

and his NK cell activity was reduced which was determined by Flow

cytometry. He was diagnosed as a case of hemophagocytic syndrome

but before any treatment could have been started, he expired. This

experience showed that HPS may be a life-threatening condition. Its

rarity combined with non-specific clinical features, such as fever and

lymphadenopathy, makes it a diagnosis that can be easily missed.

Keywords Hemophagocytosis, Plasma-cytosis, Immunocompromised

Abstract P 137

Prevalence of Autoimmune Hemolytic Anemia in MultipleMyeloma: A prospective study

Rajesh Kashyap1, Abhay Singh2, Pradeep Kumar1

1Department of Hematology, Sanjay Gandhi Postgraduate Institute

of medical Sciences, Lucknow, Uttar Pradesh, India; 2Department

of Transfusion Medicine, Sanjay Gandhi Postgraduate Institute

of medical Sciences, Lucknow, Uttar Pradesh, India

Objective: Autoimmune hemolytic anemia (AIHA) is often associ-

ated with B cell lymphoproliferative disorders. Patients with

malignant lymphoma often demonstrate a positive antiglobulin test,

but they rarely develop overt clinical symptoms of AIHA. AIHA

has rarely been documented in patients with multiple myeloma. We

conducted a prospective study to detect the presence of AIHA in

patients with multiple myeloma and its impact on clinical presen-

tation and outcome of disease. Study Design and Method: A total

of 66 patients diagnosed to have MM over a period of 18 months

were the subjects of the study. Patients with (a) severe anemia

(hemoglobin \ 6 g/dl), (b) requiring frequent blood transfusions and

(c) having clinical and laboratory features of hemolysis, were

screened for AIHA, by performing direct and indirect antiglobulin

(Coomb’s) test. Results: Of the total of 66 patients of multiple

myeloma, seventeen patients were screened for AIHA. Seven

(10.6 %) were found to be complicated with AIHA and carried

auto-antibodies in their sera. Five of these patients had denovo

multiple myeloma and two had multiple myeloma relapse. The IgG

subclass of the antibody binding to red cell membrane was com-

pared with that of M-protein and these findings were showing full

coincidence in all the seven patients. Six patients (85.7 %) had

stage IIIA and one patient (14.3 %) having stage IIIB disease. All

of these patients were positive for subtypes of IgG with IgG1

(n = 2), IgG2 (n = 4), IgG3 (n = 1) and IgG4 (n = 1). One patient

had simultaneous positivity for IgA and IgG2, with presence of cold

antibodies in the serum. Conclusion: We found that autoimmune

hemolysis is a frequent finding in patients with multiple myeloma

and failing to rule out this phenomenon may overburden the man-

agement protocol in form of multiple blood transfusion and related

complications.

Abstract P 138

A Case of Waldenstrom Macroglobulinemia Treatedwith Plasmapheresis

Suvro Sankha Datta, Biplabendu Talukder, Ritam Chakrabarty,Somnath Mukherjee, Prasun Bhattacharya, KrishnenduMukherjee

Department of Immunohematology & Blood transfusion, Medical

College Hospital, Kolkata-73

Objective: Therapeutic plasma exchange is a conjunctive modality

of treatment to decrease paraproteinemias associated with hyper-

viscosity. Here we narrate our experience in treating a diagnosed

case of Waldenstrom macroglobulinemia in 70 years old man with

moderate anaemia and severe features of hyperviscosity syndrome

by serial TPE and chemotherapy. Methods: Three therapeutic

plasma exchanges (TPE) were performed by intermittent cell sepa-

rator (Hemonetics, MCS+, USA). His first two exchanges were done

on alternate day followed by chemotherapy and third TPE was done

six weeks after the second TPE. Results: Initially his serum IgM

was 11.3 g/dl on serum protein electrophoresis. At the end of second

TPE the patient was relieved of his weakness and syncope. Before

third TPE his serum IgM again became 9.901 g/dl. After twenty

four hours of third TPE, his serum IgM level came down to 3.13

g/dl and the patient became asymptomatic. Conclusion: The rela-

tionship between blood viscosity and abnormal immunoglobulin

concentration is exponential, such that removal of relatively small

amount of immunoglobulin by TPE will result in a large reduction

in serum viscosity. So TPE must be used in conjunction with che-

motherapy for symptomatic relief in case of Waldenstrom

macroglobulinemia.


123

Abstract P 139

Crystalline Inclusions in Myeloma Cells: A Case Report

Shweta Sushmita, Amit Kumar Adhya

Department of Pathology, KIMS, Bhubaneswar, Odisha, 751024

Introduction: The presence of crystalline inclusions in plasma cell

myeloma is a rare phenomenon and cases have been reported with

rod, needle, and rectangular shaped crystals. Here, we present a case

of plasma cell myeloma with needle and rhomboid shaped intracel-

lular crystalline inclusions and extracellular crystal depositions in the

bone marrow. Case: A 56 year old male patient presented with low

back pain. On investigation he was found to have multiple lytic bone

lesions. Serum M band was positive. Bone marrow study revealed

90 % myeloma cells with numerous intracellular and extracellular

needle and rhomboid shaped crystalline inclusions. The inclusions

were negative for MPO, SBB but positive for NSE. Discussion:Crystalline inclusions are believed to be due to accumulation of

cytoplasmic immunoglobulins secondary to a block in the protein

synthetic pathway Since rhomboid crystal depositions can be seen in

other clinical conditions such as pseudogout, this case invites con-

sideration of plasma cell myeloma in the differential diagnosis of

patients with rhomboid crystalline deposition in the bone marrow and

in sites/organs other than the bone marrow. This case and the review

of similar cases presented in the literature suggest that extracellular

rhomboid crystals in the bone marrow and extramedullary sites such

as articular spaces or renal parenchyma should raise suspicion of a

plasma cell neoplasm, yet other differential diagnoses such as gran-

ulocytic sarcoma or reactive processes should also be considered.

Abstract P 140

Multiple Myeloma Presenting as Proptosis: A DiagnosticDilemma

Pattanayak Lucy1, Padhi Sanjukta1, SN Senapati1, SamantaraySagarika2, Rout Niranjan2

1Department of Radiation Oncology, 2Oncopathology, AHRCC,

Cuttack

Background: Plasma cell neoplasms are characterized by monoclo-

nal proliferation of plasma cells or terminally differentiated B cells.

They may present as solitary plasmacytoma, which can affect bone or

soft tissues, or they can manifest with systemic involvement as

multiple myeloma. Extramedullary presentation of multiple myeloma

is uncommon and the orbit is involved in less than 0.25 % cases till

date. We describe an unusual case of multiple myeloma that presented

to us with proptosis. Case: A 45-year-old male presented with gradual

proptosis of the right eye for a period of 15 days. Local examination

revealed a forward and outward protrusion of the eyeball, restricted

movements and loss of vision in the right eye. On palpation there was

a firm mass 2.5 9 2.5 cm in size, nontender in the superior orbit of

the eye. Hb was 7 g% and ESR was 110 mm in the 1st hour. Scrape

cytology from the mass showed sheets of binucleate and multinu-

cleate plasma cells suggestive of plasmacytoma. Bone marrow

aspiration and cytology showed oval cells with pale basophilic

cytoplasm, perinuclear halo, nuclei with cartwheel chromatin sug-

gesting plasma cells which were [30 %. Serum Bens Jones protein

was positive and electrophoresis showed a sharp M band in the

gamma region confirming the diagnosis of Ig G multiple myeloma.

Skeletal survey was normal. Conclusion: Orbital myeloma with

negative skeletal survey is infrequently encountered. Cytological

picture from the lesion as well as bone marrow aspiration cytology is

confirmatory in the absence of bony lesions. Associated features like

anemia help in staging and prognostication.

Keywords Plasmacytoma, Multiple Myeloma, Orbit, Proptosis,

Abstract P 141

Primary Amyloidosis Associated with IgM k ProteinemiaPresented as Nephrotic Syndrome

Upasana Das, SM Sethy, RK Jena, P Mohanty

Department of Clinical haematology, Department of Pathology,

S.C.B. Medical College, Cuttack, Odisha

Introduction: Primary amyloidosis presents with systemic or localised

form without any evidence of preceding or coexisting disease, para-

proteinemia or plasma cell dyscrasias. The amyloid of primary

amyloidosis is made by plasma cells in bone marrow. The L chains are

secreted into the serum and they undergo partial lysosomal proteolysis

and get deposited as insoluble amyloid filament. Three fourth of

immunoglobulin light chains are of the k group. The organs most

commonly involved are the kidneys, heart, blood vessel walls. Renal

amyloidosis may manifests in many forms. Out of all presentations

nephritic syndrome is one of them. Usually the mean age is 43.9 years.

This is a case of presented in young male. Case Report: A 20 year male

boy presented with bilateral pitting pedal edema, puffiness of face and

loss of weight for last 6 months. Serum albumin was 1.5 g/dl, s. Protein

2.9 g/dl, urine albumin +++. The case was diagnosed to be nephrotic

syndrome. The capillary zone electrophoresis showed the presence of M

band, serum immunofixation revealed monoclonal IgG k type. Bone

marrow aspiration 5 % marrow plasma cells. The core needle biopsy of

kidney showed presence of glomerular and vascular deposition of

amyloid. Renal manifestation in amyloidosis carry a grave prognosis.

Keywords Primary Amycoidoses, Nephrotic Syndrome, 1Gms 2chain

Abstract P 142

Secondary Malignancy (AML) in Case of Post Chemotherapy(MPV) Multiple Myeloma

S Burma, P Bhuyan, S Sethi, RK Jena

Department of Pathology & Department of Clinical Hematology,

S C B Medical College, Cuttack, Odisha

Introduction: The multiple myeloma patients may develop AML or

MDS as a secondary malignancy either due to a complication of

Serial

no.

Pre-exchange

hematocrit

Total

blood

volume

processed

Replacement

fluid

1 37.10 % 2,139 ml *5 % albumin:850 ml

+ FFP:900 ml

+ ACD:220 ml

2 36.80 % 2,040 ml *5 % albumin:800 ml

+ FFP:750 ml

+ ACD:187 ml

3 35.30 2,087 ml *5 % albumin:900 ml

+ FFP:600 ml

+ ACD:260 ml

*Albumin is reconstituted with normal saline, FFP: Fresh Frozen

Plasma, ACD: Acid citrate dextrose


123

chemotherapy or may be in the absence of any chemotherapy. We

reported only two such cases out of 145 multiple myeloma receiving

MPV therapy from July 2010 to July 2012. Case Report: We have

reported two cases of multiple myeloma who developed AML 2 years

after the diagnosis. They had been treated with oral melphalan,

prednisone and vincristine (MPV) for MM. The patients were

achieved complete remission. But after 2 years later, one case pre-

sented with fever, generalized weakness. BM examination reveals

[50 % myeloblasts with[10 % maturation and diagnosed as AML-

M2. But the patient was died after 2 days of initiation of CT. Another

case, 1� years later, presented with fever, sternal tenderness and

mucocutaneous bleeding and diagnosed as AML-M4. The patient

expired due to thrombocytopenia before initiation of any chemo-

therapy. Conclusion: Incidence of secondary myeloid leukemia in the

setting of multiple myeloma is very uncommon. In our series it is

\2 %. Such leukemias are typically acute myeloid leukemia (AML).

The myelomonocytic subtype is particularly common.

Abstract P 143

Stomach Involvement in a Relapse Case of Multiple Myeloma

Priyadarshini Dehuri, Sudha Sethy, Rabindra Kumar Jena,Pranati Mohanty

Department of Clinical Haematology and Department of Pathology,

S.C.B Medical College, Cuttack

Introduction: Multiple myeloma is a neoplastic proliferation of

monoclonal plasma cells usually seen in the bone marrow. Relapse

cases with extramedullary presentation in the form of stomach lesion is

only rarely reported. Case Report: A 60 year female post chemother-

apy for multiple myeloma presented with painful swelling in abdomen,

fever and anaemia. The USG revealed a gastric mass involving fundus

and body. With a clinical suspicion of adenocarcinoma, biopsy was

advised. The biopsy reports were suggestive of either high grade lym-

phoma or undifferentiated carcinoma. Immunohistochemistry was

suggested for definite diagnosis. The IHC stains proved all lymphoma

markers to be negative. There was pan cytokeratin positivity. Most

importantly CD 56 positivity was noted. In the light chain analysis,

kappa to lambda ratio was 3:1. Thus all the investigations favoured a

diagnosis of a monoclonal plasma cell expansion. Conclusion: Relapse

cases of multiple myeloma with extramedullary presentation being very

rare, need attention for their further management.

Keywords Multiple Myeloma, Extramedullary, Stomach, CD56

Abstract P 144

Multiple Myeloma with CNS Involvement: A Rare Case Report

Surabhi, KL Tripathy, P Bhuyan, A Mishra, BP Das

Background: Involvement of CNS is extremely rare in multiple

myeloma, observed in approximately 1 % of the patients. CaseReport: A 35 year old female presented in Neurosurgery OPD with

rapidly progressive scalp swelling over left side of head since

4 months and weakness of right upper and lower limbs for last

1 month. On examination, the swelling measured 10 9 10 9 6 cm

and was tense with variegated consistency. X-ray skull showed a bony

erosion in left frontoparietal region with soft tissue swelling. CT scan

of brain showed a solid SOL in frontal lobe, 8 9 7 cm, with erosion

of left frontal bone and involvement of scalp. With differential diag-

nosis of meningioma with malignant transformation, lymphoma the

patient underwent debulking surgery. Result: The histopathological

study showed features of Plasmacytoma. On bone-marrow aspiration,

the case was diagnosed to be multiple myeloma supported by M-Band

in serum electrophoresis.

Platelet Disorders

Abstract P 145

Profile of Vascular Thrombotic Events in EssentialThrombocythemia

A Krishna Prasad, M Shetty, AMVR Narendra, VR Srinivasan

Nizam’s Institute of Medical Sciences, Hyderabad, AP

Introduction: Essential thrombocythemia (ET) is a chronic myelo-

proliferative disorder characterized by the clonal proliferation of

primarily megakaryocytes, resulting in thrombocythemia. Complica-

tions, often thrombotic, are more common in patients with high

platelet counts with an incidence of 84 %. Profile of vascular

thrombotic events in ET both at presentation and on follow up was

reported below. Aim: Study of vascular thrombotic events in ET

patients. Materials and Methods: Vascular thrombotic events

information, from the case records of ET patients attending General

Medicine department, was retrospectively analyzed. Period of col-

lection was 14 years. All cases satisfied the 2008 WHO criteria for

diagnosis of ET. Results: Total cases were 15. Males were 8 and

females 7. Mean age was 49 years (range of 24–75 years). Mean

platelet count at presentation 13.7 l/ll (range of 5–40 l/ll). All cases

presented with vascular events. Total thrombotic events were 25

(arterial 17 and venous 9). Mean platelet count in arterial thrombosis

was 11.1 l/ll. Arterial thrombosis noted in cerebral vessels (6), cor-

onaries (4), digital arteries (2), renal artery (1), anterior spinal artery

(1), aorto-iliac vessels (1), ulnar artery (1) and tibial artery (1). Mean

platelet count in venous thrombosis was 8.6 l/ll. Venous thrombosis

noted in portal veins (6), deep venous system of lower limbs (4),

spleenic vein (2) and superior mesenteric vein (2). Surgeries were done

in 7 cases [CABG (1), amputation of limbs (2), splenectomy (4)]. JAK2

positivity was noted in 3 out of 5 cases. Conclusions: Arterial events

were more than venous events. Recurrent vascular thrombosis was

noted in 11 cases due to poor control of the disease (twice in 9 and thrice

in 2 cases). Thrombotic complications affect the quality of life.

Abstract P 146

Study of the Cytokine Profile of Type 1 and Type 2 T-Cellsin Children with Acute Immune Thrombocytopenia (ITP)

Vanita Bhaskar1, Anita Nangia1, Sunita Sharma1, JagdishChandra2, Anju Seth2

Department of 1Pathology and 2Paediatrics, Lady Hardinge Medical

College and Associated Kalawati Saran Children’s Hospital,

New Delhi

Objectives: To study pretreatment and post treatment cytokine profile

of Type 1 (Th1 = IFNc, IL-2) and Type 2 (T2 = IL-4, IL-10) T-cells

in children with acute ITP after immunomodulatory treatment.

Method: 30 newly diagnosed ITP patients below 18 years of age,

were subjected to Complete Haemogram with platelet count, Periph-

eral smear and bone marrow aspirate (BMA). Serum cytokine level for

IFNc, IL-2 (Th1 subset) and IL-4, IL-10 (Th2 subset) were performed


123

in all using ELISA. Results: The mean values of Th1 cytokines (IFNy,

IL-2) were higher and Th2 cytokines (IL-4, IL-10) were lower in all

the patients pre treatment. Decrease in mean levels of Th1 and an

increase in mean values of Th2 cytokines post treatment were seen

with p value statistically significant. Conclusion: The present study

found an increase in Th1 cytokines (IFNc and IL-2) and decrease in

Th2 cytokines (IL-4 and IL-10), in all paediatric patients of acute ITP

at time of diagnosis emphasising the T cell dysregulation as the early

event in pathophysiology of acute ITP. The decrease in Th1 cytokines

and increase in Th2 cytokine post treatment with normalisation of Th1/

Th2 ratio is an excellent indicator of response to treatment.

Abstract P 147

T Regulatory Cells in Acute Immune ThrombocytopenicPurpura—Study from a Tertiary Centre

Pratibha Dhiman, Arnab Chattopadhyay, Utpal Chaudhuri

Institute of Haematology & Transfusion medicine (IHTM), Medical

College Kolkata

Introduction: Immune thrombocytopenic purpura (ITP) is charac-

terised by enhanced platelet destruction due to platelet autoantibodies.

There is an increasing evidence that regulatory T lymphocytes play an

important role in pathogenesis. Objective: To determine the fre-

quency of CD4+ C425+ CD127low T regulatory cells by flow

cytometry and to investigate the differences in their expression during

periods of disease activity and remission in acute ITP. Materials andMethods: A total of 30 patients with acute ITP (12 males and 18

females), aged 5–40 years were enrolled in the study. Diagnosis of

ITP was based on presence of thrombocytopenia only with no evi-

dence of red cell and white blood cell abnormalities, normal or

increased number of megakaryocytes in bone marrow smears and

exclusion of other causes of thrombocytopenia. Age and sex matched

30 control are also enrolled. All patients and controls are subjected to

clinical evaluation, complete blood count, direct coombs’ test, anti-

nuclear antibody detection, serology testing and morphological

examination of bone marrow samples (in case of patients only). T

regulatory cells were identified by flow cytometry after separation of

peripheral blood mononuclear cells using cell preparation tubes. T

regulatory cells are defined as CD4+ CD25+ CD127low lymphocytes.

Results: The frequency of T regulatory cells ranged from 1.4 to

3.4 % in acute ITP patients at the time of diagnosis. Patients who

achieved remission, the T regulatory cells were increased ranging

from 6.5 to 9 %. Among controls, the levels range from 7.2 to

10.5 %. Conclusion: The study imply one of the possible funda-

mental role of T regulatory cells in pathogenesis of ITP patients.

Abstract P 148

A-3 Haplotype of EPCR Gene: Is it a Risk Factorfor Recurrent Miscarriage in Indian Population

Amit Sharma, Ravi Ranjan, Suhail Akhter, Kamal Kishor,Hareram Panday, Renu Saxena


New Delhi, India

Background: Recurrent miscarriage affects 1–5 % pregnancies and in

50 % of these women, the cause of the preceding miscarriages is

unknown. It is multi-factorial in origin and the role of inherited throm-

bophilia and gene polymorphisms of coagulation and anticoagulation

factors such as-thrombomodulin and endothelial protein C receptor

(EPCR) are involved in the pathogenesis of recurrent miscarriage. The

EPCR facilitates PC activation by the thrombin-thrombomodulin com-

plex. In plasma soluble form of EPCR (sEPCR) inhibits both

anticoagulant activity of activated protein C (APC) by blocking its

binding to phospholipids and protein C (PC) activation by competing for

PC with membrane-associated EPCR. sEPCR levels suggested under

genetic control. There are 13 known polymorphisms in EPCR gene which

are in complete linkage disequilibrium; these defined three haplotypes:

A-1, A-2, A-3. Out of them, A-3 was most studied and found strongly

associated with high sEPCR levels in deep vein thrombotic patients in

different population. However role of A3 haplotype of EPCR is a risk

factor for recurrent miscarriage remains controversial. Objective: The

aim of this study is to identify the role of EPCR haplotype (A3) in the

pathogenesis of recurrent miscarriage. Methods: 100 consecutive

patients with recurrent miscarriage and equal number of age and sex

matched healthy controls were the study subjects. All study subjects were

genotyped for A3 haplotype tagging SNP (A6936G) using PCR–RFLP.

Results: Homozygosity of A-3 genotype did not show any significant

association (P = 0.21, c2 = 1.55) while the allelic frequency of the

tagging SNP (A6936G) showed a trend of association (P = 0.07,

c2 = 3.10) with the recurrent miscarriage. Conclusion: A trend of

association in the allelic frequency of the tagging SNP (A6936G) reveals

that in a large sample size this A-3 Haplotype may be found associated

with the recurrent miscarriage in Indian population.

Abstract P 149

Immune Thrombocytopenic Purpura in a Girl with AcuteLymphoblastic Leukemia—an Unusual Association

Vikas Dua1, Jai Bhagwan Sharma2

1Department of Pediatric Hematology Oncology, Action Cancer

Hospital, Delhi, India; 2Department of Medical Oncology, Action

Cancer Hospital, Delhi, India

Objective: Lymphomas have been associated with Immune thrombocy-

topenic purpura (ITP) but it is rare to see ITP in patient with acute

lymphoblastic leukemia (ALL). Nine cases of ITP in children with ALL

have been reported so far, three of them were acute ITP, and rest were

chronic ITP. Only one such case has been reported from India. Method: A

15-year-old girl was admitted in our hospital because of fever, increasing

pallor and bleeding spots over the body in July 2010. Peripheral blood

smear showed 54 % blasts. The bone marrow was consistent with pre-B

ALL. Patient was started on chemotherapy using BFM-95 regimen. Bone

marrow showed remission on the 33rd day of treatment. After phases of

Protocol-I, Protocol-M, and Protocol-II, patient was started on mainte-

nance therapy including 6-mercaptopurine and methotrexate. After

20 months into therapy, the patient was noted to have a platelet count of

38,000/mm3. Results: Therapy was stopped and a suspicion of relapse was

kept and bone marrow was performed. An adequate number of megakar-

yocytes with findings of ALL in remission were detected. The patient was

started on prednisone at 2 mg/kg orally daily. After 3 weeks of prednis-

olone, she had improvement of her platelet count to 78,000/mm3. Her last

platelet count in July 2012 was 88,000/mm3.Secondary causes of ITP were

ruled out. Conclusion: The presence of thrombocytopenia in patient of

ALL does not always means relapse of ALL or as a result of chemotherapy

but the possibility of ITP should also be considered.


123

Abstract P 150

Comparison of Platelet Counts by Sysmex XE 2100 and LH 750with the International Flow Reference Methodin Thrombocytopenic Patients

Tina Dadu, Sehgal Kunal, Shaikh Anjum, Khodaiji Shanaz

Department of Hematology, P.D.Hinduja Hospital & Medical

Research Centre, Mumbai

Background: There are several methods for counting platelets, of which

The International Flow Reference method is considered to be the gold

standard. We compared the platelet count given by this method to the

count given by automated analyzers using other methods. Aims: To

compare the platelet counts obtained by XE2100 (Impedance, optical

florescence and reported based on switching algorithm) and LH 750

(Impedance) with the international flow reference method in thrombo-

cytopenic blood samples. To calculate the sensitivity, specificity, PPV

and NPV of various technologies at the clinically relevant transfusion

thresholds of 10,000 and 20,000/ll. Materials and Methods: A total of

118 blood samples with platelet count of\50,000/ll were selected for the

study. Platelet counts of all samples were analyzed by all methods using

the Sysmex analyzer, LH750 and IRM in parallel within 6 h of collection.

Results and Conclusions: Sysmex-R had the least Bias and 95 %LA

range and thus correlated best with IRM values. LH-750 had a higher Bias

compared to Sysmex-O and Sysmex-R but a strikingly similar 95 %LA

ensures similar results in all three methods. In fact, in the oncology subset,

it had the narrowest 95 % LA which made it the best performer in this

subgroup. Of the three Sysmex results, Sysmex-I had the highest Bias,

widest 95 % LA, and highest potential risk of over transfusion. Hence

Sysmex-R and LH750 were found to be reliable tools for estimation of

platelet count in thrombocytopenic patients.

Abstract P 151

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

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

1Department of Intensive Care, P. D. Hinduja National Hospital and

MRC; 2Department of Research, P. D. Hinduja National Hospital and

MRC; 3Department of Laboratory Medicine, P. D. Hinduja National

Hospital and MRC

Objective: Heparin Induced Thrombocytopenia (HIT) is an immune

mediated complication of heparin therapy. Our objective was (1) to

compare various laboratory assays for HIT against clinical probability

(4-T score) and 14C-Serotonin Release Assay (SRA) which was the

composite gold standard and (2) to determine the incidence of HIT in the

ICU. Methods: The study Group (n = 217) consisted of consecutive

ICU patients with heparin exposure followed by thrombocytopenia.

The clinical probability (4-T score) was applied to the study group.

Enzyme Linked Immunosorbent Assay (ELISA), Particle Gel Immu-

noassay (PGIA), SRA and Platelet Aggregation Assay (PAA) were

performed. Results: The 4-T score showed that 1/217 patients had high

probability, 48 had intermediate probability and 168 had low proba-

bility for HIT. One patient was positive by SRA, 3 by PGIA and 33 by

ELISA. The incidence based on a combination of clinical features and

laboratory findings was 1.8 %. Conclusions: A greater number of false

positives were observed by ELISA than by PGIA when compared to a

composite gold standard of SRA and clinical probability. The incidence

of SRA positive HIT was 0.46 % (1/217).

Abstract P 152

Coexistence of Macro Thrombocytopenia and GlansmannThrombasthenia—a Case Report

Shenbagapriya, Ramya, Jennifer, Suresh, Vandana Kamath,Usha Sitaram Sukesh C. Nair

Department of Transfusion Medicine & Immunohaematology,

Christian Medical College, Vellore, India

Abstract: Congenital platelet disorders associated with thrombocytope-

nia often pose special diagnostic challenges to clinicians and diagnostic

laboratories. Glansmann thrombasthenia (GT) is a rare autosomal reces-

sive disorder characterized by mucocutaneous bleeding, normal platelet

counts, morphology, and prolonged bleeding time and absent clot retrac-

tion. Here we present a case of 10 year old female who presented to us with

the history of mucocutaneous bleeds requiring transfusions. Laboratory

investigations revealed mild thrombocytopenia with giant platelets (MPV:

15fL), prolonged bleeding time, good clot retraction, platelets failed to

aggregate with ADP, Collagen, AA and Epinephrine and aggregation seen

with Ristocetin. Flow cytometric analysis of GpII bIIIa also showed

findings consistent with GT. An unusual finding of macrothrombocy-

topenia with Glansmann thrombasthenia was identified.

Abstract P 153

Spectrum of Platelet Function Disorders Presenting with BleedingManifestations to a Tertiary Care Centre

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

Institute of Haematology and Transfusion Medicine (IHTM), Medical

College, Kolkata

Introduction: Platelet aggregometry serves as an important tool for

evaluation of the patient with bleeding manifestations especially when

coagulation profile is normal. IHTM has been performing platelet

aggregometry as a clinical service for the last 10 years. Here, we are

presenting the data of 82 bleeders referred in the last 1 year for platelet

aggregometry using common platelet agonists. Aim: (1) Determination

of type of platelet dysfunction among patients with bleeding symptoms.

(2) To understand the correlation between bleeding time and platelet

aggregation. Method: The detailed clinical notes of the patients referred

for platelet aggregometry were analyzed. Tests were performed using

ADP, Collagen, Arachidonic acid and Ristocetin (standard and low dose)

by Chrono log Model-700. Results: Patients were referred because of

more skin-related bleeding symptoms rather than wet purpura. Using

platelet aggregometry, only 33 % demonstrated characteristic platelet

dysfunction. 7 patients had Glanzmann thrombaesthenia. Bleeding time

was prolonged ([7 min) in only 15 % patients who had platelet dys-

function on aggregometry. Impairment of aggregation with collagen and

arachidonic acid translates into prolonged bleeding time (p \ 0.05).

8.5 % patients showed hyperaggregation though they had bleeding

manifestations. Conclusion: Platelet aggregometry is a useful tool for

evaluation of a bleeding disorder but may remain inconclusive in 67 %

cases if not supplemented by other tests. Bleeding time is not a sensitive

test for detecting platelet dysfunction.


123

Others


Systemic Mastocytosis—A Rare Entity

Ketan Mallya2, Bikash Singhania1, K Annamma2, B Sushma1,R Lakshmi1

1Kasturba Medical College, Manipal University, Manipal; 2Melaka

Manipal Medical College, Manipal

Introduction: Mastocytosis encompasses a rare and heterogeneous

group of clinical disorders characterised by abnormal growth and

accumulation of mast cells in various organs, commonly skin and bone

marrow. Here we report a case of systemic mastocytosis with bone

marrow involvement as the first manifestation. Specific histocytologi-

cal techniques were needed to establish the diagnosis. Case Report: A

52 year old lady presented with severe lower and upper back pain of

8 months duration. On local examination severe tenderness was elicited

over dorsal and lumbar spine. X-ray and MRI for the spine showed lytic

lesions at the L1 to L5 vertebrae. Bone scan was suggestive of osteo-

blastic changes. Gastroscopy demonstrated pangastritis. Metabolic and

endocrinological etiologies were excluded because laboratory evalua-

tion was unremarkable. Iliac crest bone marrow biopsy demonstrated

infiltration by mast cells, arranged in sheets and whorls. Extensive areas

of marrow fibrosis was seen along with bone destruction. Special stains:

AFB—Negative. Toluidine blue—positive in mast cells. Immunohis-

tochemistry: CD117—positive in mast cells. Conclusion: The

diagnosis of mastocytosis requires a great degree of clinical suspicion as

it is a rare disease that may go unrecognized or misdiagnosed because of

the non specificity of many of its symptoms, particularly in cases with

visceral involvement and absence of characteristic skin lesions of

urticaria pigmentosa. Bone involvement in mastocytosis is related to

infiltration of bone marrow by mast cells. Bone marrow biopsy and

histocytological techniques are the key for diagnosis.

Abstract P 155

A Template Based Reporting System for Peripheral SmearInterpretation

Shabnam Roohi1, Venkatesha Joshi2, Devendra Prasad2,Padam Kafle3

1Department of Haematology and Clinical Pathology,

Apollo Hospitals Bangalore; 1Apollo Hospitals Bangalore;3Akhil systems Pvt.Ltd

Objective: The peripheral smear examination and interpretation is an

important part of a pathologist’s day to day activity. A template based

reporting system was developed to help reduce the turnaround time

for preparation of an accurate, comprehensive and concise peripheral

smear report. Methods: The knowledge base of previously reported

cases, references from text books and the internet were used to

develop these templates. These templates were then uploaded onto to

the hospital information system using Microsoft visual studio 2005

and visual basic 6.0. A guide for appropriate template to be uploaded

based on the haematology analyzer output was developed. The

technicians were trained to upload the appropriate template based on

the analyzer output. The pathologist reviewed these for accuracy and

completeness after slide review. Results: Of 504 uploaded peripheral

smear templates screened for accuracy 242 were normal studies, 262

were abnormal, out of the abnormal studies 25 required to be modified

and there was no change in the normal study templates. Therefore the

error rate was 4.96 %. Implementation of this system reduced the

turnaround time for report preparation by 58 % and increased the

capacity of the lab by 30 %. Conclusion: A template based reporting

system for reporting peripheral smear interpretations is an effective

tool in reducing the turnaround time and the error rate of the lab for

reporting peripheral smears.

Abstract P 156

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

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

Era’s Lucknow Medical College and Hospital, Lucknow

Abstract: Hereditary and sporadic cases of hemophagocytic syn-

drome (HPS) are primarily reported in children. We present a case of

hemophagocytic syndrome with reactive plasmacytosis in a 16 years

young male. The patient presented to us with persistent fever, hepa-

tosplenomegaly, lymphadenopathy, anaemia, leucopoenia and marked

thrombocytopenia with elevated triglycerides level. For all the above

complaints he was advised bone marrow examination which revealed

hypocellular smears with marked erythrophagocytosis by histiocytes

and his NK cell activity was reduced which was determined by Flow

cytometry. He was diagnosed as a case of hemophagocytic syndrome

but before any treatment could have been started, he expired. This

experience showed that HPS may be a life-threatening condition. Its

rarity combined with non-specific clinical features, such as fever and

lymphadenopathy, makes it a diagnosis that can be easily missed.

Keywords Hemophagocytosis, Plasmacytosis, Immunocompromised


VCS Parameters in Viral Infections

Mohammed Musheb, Chethan Manohar

Kasturba Medical College, Manipal, Udupi-576104

Objectives: To study the utility of WBC research population data

(RPD) provided as VCS parameters on the Coulter LH750 Hema-

tology analyzer for the screening of viral blood samples against

normal controls. Materials and Methods: The WBC differential

system of Coulter LH750 consists of a dedicated flow channel based

on the ‘Coulter principle’ & identifies the WBC types according to

three physical measurements: (1) Impedance method applied to direct

current-Cell volume (V). (2) Radiofrequency method—nuclear size

and density-conductivity (C). (3) Light scatter of a laser beam–

cytoplasmic granularity (S). Study groups: 1. 106 healthy blood

donors (controls). 2. 118 presumed viral infections—cases (with

bacterial cultures and serology negative, autoimmune serology neg-

ative). Samples were run on the Coulter LH750 on DC mode and their

VCS parameters, i.e. mean (M) and standard deviation (SD) of vol-

ume (V), conductivity (C) and scatter (S) for the different leukocyte

populations (N,L,M,E) were studied. Results: Studying the various

VCS parameters between 2 groups, it was found that mean and

standard deviation parameters for volume, conductivity and scatter for

neutrophils, lymphocytes and monocytes were significantly different

between the controls and cases. LSdV, NSdS in group 2 showed

significant difference and ROC curve analysis showed best AUCs for

mean monocyte volume-MMC (sensitivity 80% -specificity 57%

increasing to 74% for a sensitivity of 72% at a value of 166.3). The

scatter parameters proved to be non predictive. Conclusion: In


123

conclusion, the leukocyte VCS parameters is moderately predictive in

the detection of viral infections and further studies with serologically

proved viral infections would be useful.

Abstract P 158

Anti M Antibodies Complicating Transfusion Therapy: A Reportof Two Cases

Meenakshi Rao, Sangeeta Pahuja, P Lalita Jyotsna, Manjula Jain

Department of Pathology & Blood Bank, Lady Hardinge Medical

College, Delhi

Introduction: Anti M is a naturally occurring antibody usually active at

temperatures below 37�C and thus is of no clinical significance. However,

rarely the antibody agglutinates red cells at 37�C or at the antiglobulin

phase of testing and can lead to haemolytic transfusion reactions and

haemolytic disease of newborn. Case 1: A 3 year/F with no history of

blood transfusion (BT) presented with severe infection and anemia. For-

ward grouping showed AB positive; reverse grouping was inconclusive.

Blood grouping at different temperatures showed panagglutination at 22�Cwhich enhanced at 4�C and resolved at 37�C. DAT was negative. Antibody

identification using 11 cell panel suggested presence of anti-M antibodies.

DTT treatment of serum suggested presence of IgG + IgM type of anti-M

antibodies. Case 2: A 7 year/M with no history of BT presented with

severe anemia with impending CHF. Blood group (BG) was A positive.

Antibody screen was positive. Reverse BG at different temperatures

showed agglutination with all 3 cells at 22�C which enhanced at 4�C. Only

B cells showed reaction at 37�C. DAT was negative. Antibody identifi-

cation with 11 cell panel suggested presence of anti-M antibodies. DTT

treatment of serum suggested presence of IgM antibodies. Both children

were successfully transfused with M-negative compatible blood units

according to their antigen profile. Conclusion: Although considered

clinically insignificant, anti-M antibodies have been implicated in HDN

and delayed haemolytic transfusion reactions. They can assume signifi-

cance in cases of clinically induced hypothermia. Also, these are the cause

of incompatible cross-match results causing delay in blood transfusion.

Hence, it is extremely important to carefully interpret the results of blood

grouping and antibody screening.

Abstract P 159

Acute Megaloblastic Anemia Presenting as a Severe FulminatingPyrexial Illness. A Case Series

Vineet Behera1, Velu Nair2, Rajan Kapoor2, C Agrawal1

1Department of Medicine, AFMC Pune; 2Medicine & Hematology,

AFMC, Pune

Background: Megaloblastic anemia is a common illness known to have

diverse and protean manifestations. But megaloblastic anemia presenting

as a severe fulminating pyrexial illness is rarely reported. Objective: To

study the clinicopathological profile of megaloblastic anemia presenting as

an acute pyrexial illness. Materials and Methods: This is a retrospective

study in which the clinical, laboratory and therapeutic profile of patients of

megaloblastic anemia presenting as a acute febrile illness was studied.

Results: A total of 14 cases were included. The average age of patients was

31.8 years with 09 (64.28 %) vegetarians. All patients presented with an

acute febrile illness (mean duration 9.1 days) with 71 % having high grade

fever with chills or rigors. All had features of symptomatic anemia.

Jaundice was seen in 12 patients (85.71 %), splenomegaly in 10 (71.42 %),

hepatomegaly in 8 (57.14 %) and features of peripheral neuropathy in 4

(28.47 %) patients. Investigations revealed mean hemoglobin of 4.1 g/dl

(range 2.6–7.4), leucopenia and thrombocytopenia in all, mean ANC

1100/mm3 (ANC\500 in 4 patients) with mean MCV being 113 (max-

imum 132) with macrocytes and hypersegmented neutrophils seen in all.

The mean serum bilirubin was 3.88 mg/dl (unconjugated) with normal

enzymes, mean LDH 1733.42. Serum B12 levels were low (\140 pg/ml)

(mean—112) in all patients with folic acid levels low in 10 patients (71 %).

Bone marrow examination showed cellular reactive bone marrow with

megaloblastoid changes in all. All patients responded dramatically to

therapy with parenteral vitamin B12, folic acid and other supportive

therapy with gradual recovery and normalization of investigations. Con-clusion: Megaloblastic anemia should be considered as a differential

diagnosis of acute pyrexial illness in all patients having a similar presen-

tation to enable early diagnosis and treatment of this easily treatable

disorder.

Abstract P 160

High Incidence of Zidovudine Induced Anaemia in HIV InfectedPatients in Southern Odisha

Kaibalya Ranjan Dash, Lalit Kumar Meher, PK Hui, SPMohanty, SN Nayak

ART Center, Department of General Medicine, MKCG Medical

College & Hospital, Berhampur, Odisha

Aims and Objectives: This study was conducted with an aim to

determine prevalence of ZDV induced anaemia in HIV infected patients

initiated on ZDV containing ART regimen and also to find out corre-

lation with any factor for causing ZDV induced anaemia. Materialsand Methods: This is a retrospective study carried out in ART center,

MKCG MCH, Berhampur between 2009 Jan to 2011 Dec. HIV infected

patients registered at ART center were treated according to NACO

guidelines. Patients (n = 1221) with Hb [ 8 g/dl were prescribed ZDV

based ART regimen. Patients having anaemia (\8 g/dl) were excluded

from the study. Correlation of baseline characteristics (age, sex, weight,

Hb lebel, CD4 count, WHO clinical stage) with risk of developing

anaemia was also calculated. Results: 178 (14.6 %) patients on ZDV

regimen developed anaemia and 6.8 % (n = 83) of these developed

severe Anaemia (\6.5 g%). Patients with low CD4 count were more

prone to develop Anaemia. Age, sex, weight, WHO clinical stage had no

relation to development of Anaemia. Interpretation and Conclusion:Incidence of ZDV induced Anaemia is very High and patients having

low CD4 count were more susceptible to develop anaemia.


Validation of Automated ESR Machines with ModifiedWestergren Method

Asha Patil1, M Deepak Nayak2, Sushma V. Belurkar3,Seemitr Verma3

Manipal College of Allied Health Sciences, Manipal; 2Melaka

Manipal Medical College, Manipal; 3Kasturba Medical College,

Manipal

Aims and objectives: The study was conducted with the primary

objectives of comparing the following methods of determining ESR.

The new Ves MATIC cube 80TM instrument with modified Wester-

gren method. Ves MATIC cube 80TM with the Ves matic 20TM

instrument, which is currently used in our laboratory. Ves matic 20TM

with modified Westergren method. Materials and Methods: The Ves

MATIC cube 80TM and Ves matic 20TM ESR analyzer were compared

with modified Westergren method. A paired, simultaneously collected

venous blood sample was obtained from 200 random patients who had

a request for both CBC and ESR; that arrived at the sample collection

center, Kasturba Hospital, Manipal. All the samples were evaluated


123

using the three methods in our clinical laboratory as per the meth-

odology for each of the individual methods. Statistical Analysis:

Bland and Altman statistical analysis was applied for evaluating the

automated ESR analyser against the modified Westergren method.

Results: The analysis revealed a low degree of agreement between

manual and automated method especially for higher ESR values

([60 mm/h); whereas a reasonable concordance was observed

between values of 20–60 mm/h. When ESR values were\20 mm/h a

good concordance was seen with all the three methods. We went a

step further comparing Ves MATIC cube 80TM with Ves matic 20TM

and found that the discrepancy existed for higher values of ESR.

Conclusion: The fully automated Ves MATIC cube 80TM and Ves

matic 20TM for ESR measurement tend to underestimate the manual

ESR reading. Hence it is recommended that a correction factor should

be applied for a range of ESR values while using these machines.

Further studies & validation are needed.

Abstract P 162

Primary Diagnosis of an Unsuspected non-HaematologicMalignancy by Bone Marrow Biopsy Examination

Krishnendu Halder1, Palash Kumar Mandal1, Swapan KumarSinha1, Debdas Bose1, Debasis Banerjee2

1Department of Pathology Medical College; 2Department

of Pathology, Vivenkanda Institute of Medical Sciences, Kolkata

Objective: To diagnose the primary site of metastatic non-haematologic

malignancies by bone marrow trephine biopsy. Methods: A prospective

observational study was done at a tertiary care hospital of eastern India

from august 2009 to july 2012. Patients advised bone marrow biopsy

examination for causes like pancytopenia, anemia, leucoerythroblastic

blood picture on peripheral blood smears were reviewed during this period.

Patients with metastatic non-haematologic malignancies were selected out

of which only unsuspected malignancy with metastasis to bone marrow

formed the study group. Results: Sixteen cases of metastatic malignancy

with unknown primary site were noted. The age range of the patients was

10–65 years. The most common histologic subtype was adenocarcinoma

and the commonest site identified later was colon. Four cases remained

undiagnosed till the end. Conclusion: Bone marrow biopsy was fruitful

for initial diagnosis of a fair number of patients with occult malignancy. A

detailed history with ancillary investigations like endoscopy, advanced

imaging and PET scan could diagnose the primary site in most of the cases.

However prognosis remained poor due to disseminated malignancy.

Abstract P 163

Gelatinous Transformation of Bone Marrow: A ProspectiveTertiary Center Study, Indicating Varying Trendsin Epidemiology and Pathogenesis

S Singh1, R Sen1, M Gupta1, G Singh1, S Chabbra1, R Verma1,Abhinav2

1Department of Pathology, Pt.Bhagwat Dayal Sharma P.G.I.M.S,

Rohtak; 2Department of Hospital Administration, DCRUST, Murthal,

Sonipat

Introduction: Gelatinous transformation of bone marrow charac-

terized by fat cell atrophy, focal loss of hematopoietic cells and

deposition extracellular gelatinous material histochemically proven

to be mucopolysaccharides rich in hyaluronic acid. Objective: To

study the spectrum of causes of gelatinous transformation in bone

marrow. Materials and Methods: Bone marrow aspirates were

examined for a period of 1 year (1.09.2010 to 31.08.2012).

Gelatinous transformation was confirmed by Alcian blue stain (pH

2.5).This was correlated with the clinico-hematological profile of

the patient. Bone marrow cellularity and ratio of hematopoietic

cells: non hematopoietic cells (plasma cells, mast cells and stromal

cells), increased vascularity (capillary cores) was assessed. Results:A total of 732 samples of bone marrow aspirate were received

1 year period of which 35 showed gelatinous bone marrow

transformation (GBMT). Incidence was nearly 4.8 % Mal-

e:Female = 2:1 (23:12). Fourteen patients (40 %) were in pediatric

age group (\15 years), 13 (37 %) were adolescents and young

adults (15 to 40 years) 4 each (23 %) were in 5th and 6th decades

respectively. The bone marrow was hypocellular in 23 (66 %),

normocellular in 9 (25 %) and hypercellular in 3 (9 %) cases.

Conclusion: Capillary fronds in the bone marrow aspirates, irre-

spective of an increased number of lymphocytes, plasma cells,

mast cells and stromal cells was associated with specific infections

like TB, Leptospirosis and non-specific infectious conditions. 5

study cases (2 children) progressed to aplastic anaemia. The

presence of gelatinous transformation in bone marrow along with

relative increase in lymphoid, plasma, mast and stromal cells and

the paucity of capillary fronds could be a sign of impending

aplastic anaemia, moresoever in children.

Abstract P 164

Follicular Dendritic Cell Tumour in a Patient of Chronic MyeloidLeukemia: A Rare Association

Ragini Singh1, Rajeev Sen1, Nisha Marwah1, PS Gahlaut2,Soumik Chaudhuri3, Nisha Sharma1, Jatin Ahuja4, Kanika1

1Department of Pathology, PGIMS, Rohtak; 2Department of Medicine

& Haematology, PGIMS, Rohtak; 3Department of Haematology,

PGIMS, Rohtak; 4Department of Medicine, PGIMS, Rohtak

Abstract: Follicular dendritic cells, also known as dendritic retic-

ulum cells, are important components of the immune system and are

essential for functions of antigen presentation. Malignancies arising

from these cells are rare and the first such primary neoplasm arising

from follicular dendritic cells was reported only in 1986. The

commonest presence of these follicular dendritic cell sarcomas are

in the lymph nodes, especially of the cervical, axillary and medi-

astinal region but extra-nodal sites such as head and neck and

gastro-intestinal tract may be present in up to a third of patients. The

etiology remains an enigma with a possible link to the Epstein-Barr

virus. Immunohistochemical studies assume great importance in

differentiating this from its close congeners. We describe a case of

Follicular Dendritic Cell Sarcoma, arising in a patient of Chronic

Myeloid Leukemia (CML) on treatment with Imatinib mesylate for

the past 6 years. The Histopathological features of diffuse efface-

ment of the lymph nodes with spindle cells which were positive for

CD 23, CD 10, LCA and vimentin, showed the closest resemblance

to follicular dendritic cell sarcoma. This case deserves reporting due

to the rarity of the disease and hitherto unreported association with

CML. Furthermore, the pathological diagnosis is challenging and

requires a close-knit effort between the pathologist and the

haematologist.


123

Abstract P 165

Myelonecrosis—a Clinicopathological Study from a TertiaryCare Centre in South India

J Sree Rekha, Debdatta Basu, Rakhee Kar


Medical Education and Research (JIPMER), Pondicherry

Introduction: Myelonecrosis is a relatively uncommon clinicopatho-

logic entity. The etiology of myelonecrosis is diverse, and malignancy,

especially hematopoietic in origin, is the most common underlying dis-

ease. Objective: To describe the causes, diagnostic features and clinical

significance of myelonecrosis. Methods: 21/5679 bone marrow biopsies

showing myelonecrosis were identified from a retrospective review of

trephine biopsies done between January 2000 to July 2012. Clinical

findings, peripheral smear and bone marrow findings of these were

reviewed. Results: Among 21 patients, 16 (76.2 %) were males and five

(23.8 %) were female. Fever was the most common presenting complaint

seen in 12 cases (57.1 %). Pallor was the most common clinical sign seen

in 8 cases (38.1 %). Five patients were retropositive. Peripheral smear

examination showed pancytopenia in six cases (28.5 %), leucoery-

throblastic blood picture in six (28.5 %) and leukaemia in four (19.0 %).

Sickle cells were seen in peripheral smear in one case. Myelonecrosis was

identified on bone marrow aspiration smears in five cases. Among 21

cases 12 cases (57.1 %) were due to a neoplastic cause, four due to

tuberculous infection (three in retopositive patients), one due to sickle

cell disease. In three cases the cause could not be identified. Among the

neoplastic causes, acute leukemia was seen in 6 cases (4—ALL, 2—

AML), non-hodgkin lymphoma in two, metastatic adenocarcinoma in

two. Hodgkins disease and malignant melanoma accounted for one case

each. Conclusion: Though conditions associated with myelonecrosis are

varied, malignancy is the commonest cause. Haematological findings are

often present and may give clue to the underlying disorder.

Keywords Myelonecrosis, Bone marrow malignancy


Neutrophilic Cytoplasmic Inclusions and Their Utilityin Diagnosis of Diseases

Y Sharmila, Sajini Elizabeth Jacob, Debdatta Basu

Department of Pathology, JIPMER

Introduction: Qualitative changes in white blood cells have diag-

nostic significance in certain situations. These include changes like

cytoplasmic vacuolation, abnormal granulation, abnormalities in

nuclear segmentation etc. A detailed study of a peripheral smear,

stained by a standardized Romanowsky method can provide diag-

nostic clues which when correlated with the clinical findings can

clinch the diagnosis. Report: We report a series of eleven cases

where peripheral smear examination of neutrophils helped us to

diagnose varied clinical entities. There were five cases where the

leucocytes showed abnormal giant cytoplasmic granules. Correlating

with the clinical findings, a diagnosis of Chediak Higashi syndrome

was made. Examination of the peripheral smear in four cases of

neonatal sepsis showed neutrophils with golden yellow crystals in the

cytoplasm which are indicative of bilirubin crystals. One patient with

history of fever showed brownish pigmentation in neutrophils.

Detailed search of the smear picked up an occasional gametocyte of

Plasmodium falciparun. The last case was of a 2 year old male child

who presented with hepatomegaly and icthyosis. Peripheral smear

shows vacuolation in the neutrophils which on Sudan black staining

was optically clear. Oil red O staining shows reddish granules con-

firming Jordan anomaly. This finally was a case of Dorfman Chanarin

syndrome. Conclusion: Detailed study of neutrophils in an optimally

stained peripheral smear is a simple, though valuable diagnostic

procedure.


Tumors of non Haematopoietic Malignancies of Unknown Originin the Bone Marrow: A 5 Year Study from a Tertiary CareCentre in South India

Sreeya Das, Pritinanda Mishra, Rakhee Kar, Debdatta Basu,Sajini E Jacob


Medical Education and Research, Pondicherry

Introduction: Tumors of unknown origin represent a common presen-

tation of malignancy and account for 3 to 10 % of cancers. They are

infrequently reported and account for up to 12 % of all cancers detected

by bone marrow biopsy. The study was undertaken to assess the

involvement of bone marrow with tumors of unknown origin and its

significance in establishing primary diagnosis. Materials and Methods:This was a descriptive study which included record review of the

departmental archives for the last 5 years (January 2007 to December

2011).A bone marrow tumor of unknown origin was defined as a meta-

static cancer in the bone marrow in which a primary site was not apparent

at the time of clinical evaluation. A total 2,426 of bone marrow exami-

nation was done in this period of time, of which 15 cases were non

haematological malignancy of unknown malignancy. Results: Bone

marrow metastases of solid tumors were identified in 15 cases (0.6 %). Of

the 15 cases in 13 cases a definitive primary site could be identified by

correlating clinicoradiological findings with morphology and immuno-

histochemistry. In the pediatric population there was one case each of

neuroblastoma, rhabdomyosarcoma and metastatic small round cell

tumor. In the adult population adenocarcinoma of gastrointestinal tract

(41.7 %) was the commonest. Conclusion: Bone marrow metastasis can

masquerade a primary haematopoietic disorder; however its detection

has both therapeutic and prognostic significance. Immunohistochemistry

is an useful adjunct to morphology in reaching a definitive diagnosis.

Abstract P 168

Haematological Profile in HIV Patients Before and After Haart

PS Ghalaut, P Mehta, R Singh, S Dash, V Singh

Department of Clinical Haematology, Pathology & Biochemistry,

Pt. B.D. Shrama PGIMS, Rohtak

Introduction: A number of haematological abnormalities are repor-

ted in HIV patients such as anemia, neutropenia, thrombocytopenia.

Some of these are reversible with Highly active antiretroviral therapy

(HAART).The present study was undertaken to see the effect of

HAART in patients of HIV admitted at PGIMS, ROHTAK. Aims andObjectives: The present study was to evaluate the haematological

profile in HIV patients before and after HAART. Methods: A Total

newly diagnosed (by ELISA) 100 HIV positive patients, in age group

16-64 were taken for the study. 50 patients were put on HAART and

other 50 patients were not on HAART. Various haematological

parameters like Hb, TLC, DLC, APC, BT, CT, PTI, APTT, serum

ferritin, B12 level and serum folate level, CD4 levels and bone

marrow was done base line and after 3, 6 months post HAART level.

Statistical analysis was done by Chi square test. Observation: The


123

most frequent haematological abnormality was found to be normo-

cytic normochromic anaemia. (80 %) with mostly normo cellular

bone marrow. The observed value of mean Hb on HAART in study

group showed rise from 10.16 ± 2.30 g/dl to 10.96 ± 1.47 g/dl after

6 months which was statistically significant (p \ 0.01).in non HA-

ART group it show a fall in mean Hb from 9.84 ± 3.03, and to

9.42 ± 2.28 after 6 month follow up. The WBC count increased in

study group from 5110.64 ± 4401 to 6220 = -140 which was sig-

nificant (p \ 0.05), as compared to non significant rise from

5433 ± 4408 to 5888 ± 1547 in control group. The mean platelet

count in study group shows raised from 2.25 ± 0.737 to 2.52 ± 0.433

which was significant than the controlled group with non significant

rise from 2.19 ± 84 to 2.35 ± 6111. There were no significant

findings in other parameters. Conclusion: The study demonstrates

that normocytic normochromic anaemia was most common haema-

tological abnormality in HIV patient’s. There was significant

improvement in haemoglobin level, white blood cell level, platelet

level, absolute neutrophil count, and bone marrow picture in the

patients who were on HAART and rise was statistically significant.

Abstract P 169: Paper Presentation

Clinico-Haematological Analysis of Haematological Malignancy,a Hospital Based Study

Rahul Varshney, Muktanjali Deka, Jina Bhattacharya, PK Gogoi

Department of Haematology, GMCH

Objective: Early diagnosis of haematological malignancy is important

in clinical practice to prevent mortality. This study was conducted to

evaluate their relative frequencies in different ages and sexes in this

region. Methods: The present study was conducted in the Department

of Pathology and Haematology, Gauhati Medical College & Hospital,

Guwahati from 1st July 2011 to 30th June 2012. Patients were

examined and blood tests including haemoglobin estimation, total and

differential leukocyte count, platelets count, reticulocyte count and

PBS study, Bone marrow aspiration and biopsy, special stains, flow

cytometry and cytogenetics was done in each cases. Results: Out of

450 patient attending Haematology OPD 236 were having malignancy.

About 29.66 % patient had chronic myeloid leukaemia while 1.69 %

patient had chronic lymphocytic leukemic. Among acute leukaemia,

acute myeloid leukaemia outnumbered acute lymphoblastic leukae-

mia, (21.19 %against 7.2 %). Multiple myeloma was seen in

20.33 %. Non Hodgkin’s lymphoma was seen in 10.59 % while

Hodgkin’s disease 6.6 %, MDS 1.69 %, MPN and LCH 1 %. Male to

female ratio in haematological malignancies was 2.87:1. Maximum

cases were observed in 51-60 years. Low grade fever, progressive

pallor, weakness and body aches were the commonest symptoms

(70 % cases) while pallor was the frequently observed sign. Conclu-sion: The prevalence of haematological malignancy is 52.44 %.This

study may help in finding out their relative distribution in this region.

Abstract P 170

To Study Spectrum of Morphological Findings on Bone MarrowExamination in Cases of Visceral Leishmaniasis

Anshu Palta1, Sanjeev Garg1, Anita Tahlan1, Atul Sachdev2

1Department of Pathology, 2Department of Medicine, Govt. Medical

College and Hospital, Sector 32, Chandigarh

Objective: To evaluate various morphological findings on bone marrow

aspirate and trephine biopsies in cases of visceral leishmaniasis. Method:This is a retrospective analysis of 15 cases of visceral leishmaniasis

diagnosed in the Haematology section of Pathology Department,

GMCH-32B, Chandigarh over a seven year period (2004–2011). The

detailed clinical data, MGG stained slides of bone marrow aspirate and

H&E stained slides of trephine biopsies done in all the cases were

retrieved from archival material. Results: 15 cases diagnosed with vis-

ceral leishmaniasis were in the age group of 5–50 years with M:Fratio of

1.1:1. Sign and symptoms included fever (100 %), splenomegaly

(93.3 %), hepatomegaly (86.7 %) and lymphadenopathy (6.7 %). Pan-

cytopenia was noted in 73.3 % of cases while the remaining cases had

bicytopenia. Bone marrow hypercellularity was noticed in 53.3 % cases.

Main bone marrow aspirate findings were increase in plasma cells

(66.7 %), increase in histiocytes (66.7 %), increase in eosinophils with

precursors (40 %), hemophagocytosis (33.3 %), increased bone marrow

iron[ 2 + (40 %) and PelgerHuet cells (13.3 %) of cases. Additional

features noted in trephine biopsy were focal benign lymphoid aggregate

(6.7 %), gelatinous transformation (6.7 %), marrow fibrosis (13.3 %) of

cases.. The average parasite density in smear was 3+ and range of posi-

tivity was 1+ to 5+. Conclusions: A note of such aforementioned

observations together or individually should warn a pathologist to do

more vigorous search for L.D. bodies.


Endothelial Dysfunction in Young Patients of Coronary ArteryDisease in a Tertiary Care Setup

Shipra Garg1, Satendra Sharma1, Meera Sikka1, RajnishAvasthi2, Neelam Wadhwa1, Gopesh Mehrotra3


& Guru TegBahadur Hospital, Delhi; 2Department of Medicine,

University College of Medical Sciences & Guru TegBahadur

Hospital, Delhi; 3Department of Radiology & Imaging, University

College of Medical Sciences & Guru TegBahadur Hospital, Delhi

Aims and Objectives: A case-control study was planned to determine

the association between occurrence of CAD in young patients with

endothelial nitric oxide synthase polymorphism (eNOS) Glu298Asp

alone and in combination with TNF-a activity and Flow mediated dila-

tation (FMD) of brachial artery. Methods: The study enrolled 30

diagnosed young male patients of CAD below 40 years as cases and 30

healthy age matched male individuals without history of CAD as the

control group. Polymerase chain reaction- restriction fragment length

polymorphism (PCR–RFLP) analysis for detection of the eNOS-

Glu298 ? Asp (G894T) variant was carried out along with TNF-aactivity by ELISA technique in cases and controls. Endothelial dys-

function was also assessed by non-invasive brachial artery FMD through

ultrasonography. Results: The distribution of GG, GT genotypes in

young CAD was observed to be 73, 27 % and 80, 20 % in cases and

control subjects respectively with no mutant TT found in both study

groups. There was lack of significant association between T allele

inheritance (GT + TT vs GG) in young CAD patients (p = 0.76) with

similar ‘‘T’’ allele frequency in cases and controls. In premature CAD

patients increased TNF-a activity was found to be statistically significant

(p = 0.008) with strong association in young patients with eNOS

Glu298Asp polymorphism GT + TT (p = 0.004). Two-third of cases

with reduction in FMD brachial artery showed significant association

with eNOS Glu298Asp GT + TT genotype than GG (p = 0.02). Con-clusion: Although no significant association of eNOS Glu298Asp

polymorphism was found in young CAD, the observation of GT genotype

in almost one fourth cases of CAD may indicate a need to demonstrate

this polymorphism as a useful genetic marker with it’s possible role to

augment the risk of premature atherosclerosis and subsequent events in

younger patients with few conventional risk factors. The association of

elevated TNF-a activity and decreased FMD brachial artery in young


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CAD with eNOS Glu298Asp polymorphism may indicate the probable

role of endothelial dysfunction and inflammation in pathophysiology of

atherosclerosis in young patients.

Abstract P 172

Role of Radiation in the management of Mycosis Fungoides

DK Parida

Department of Radiation Oncology, North Eastern Indira Gandhi

Regional Institute of Health & Medical Sciences (NEIGRIHMS),

Shillong

Abstract: Mycosis Fungoides is a low grade, chronic lymphoproliferative

disorder of the skin caused by abnormal proliferation of CD4+ T cells. The

clinical manifestation ranges from Premycotic, Macule, Plaques, Tumos

and Erythrodermic phase/stage. The leukemic variant is known as Sezary

Syndrome. In order to bring all the lymphoproliferative disorders of skin

the term ‘‘Cutaneous T-Cell Lymphoma’’ was introduced by Eldelson. The

overall incidence of mycosis fungoides is about 4 per 100,000 population.

Because of unavailability of diagnostic facilities many times there is

underreporting of the disease. Majority of the patients present between 40

and 60 years of age. From among the spectrum of treatment modalities,

radiotherapy treatment produces the best therapeutic response in terms of

cure and palliation. The important prognostic factors were (1) type of the

lesions, (2) extent of cutaneous involvement, (3) Involvement of lymph

node at the time of presentation and (4) Presence of lymphoma cells in

peripheral circulation. We treated 14 patients of mycosis fungoides patients

between the age of 27–82 years between 1985 and 1998. Total Skin

Electron Irradiation Treatment was given to the patients by Linear

Accelerator. The dose range was between 8 and 36 Gy. It was also

observed that the end response was directly proportional to the total dose of

radiation treatment delivered to the patient. Subsequently the treatment

techniques were innovated and modified. The further radiation treatment

was delivered by high dose rate mode. This innovation brought down the

treatment time from 2 h to 15 min, hence became very much patient as

well as machine compliant. By this method we could deliver total dose of

36 Gy to all the patients. Seven patients were treated between 1998 and

2000. The major radiation related toxicities were skin blisters, swelling of

joints, desquamation of skin etc. These adverse effects were also dose

dependant. Because of these problems the treatment has to be interrupted

and the total duration of treatment was stretching beyond 10-12 weeks of

time. The ultimate response of radiation treatment depends upon the (1)

total radiation dose and (2) total treatment duration. If the treatment

duration is unnecessarily prolonged, the effect of radiation on the disease

becomes poor resulting in either residual or quick recurrence of the disease.

We modified the treatment schedule from daily to alternate day radiation

treatment schedule beyond third week, which produced the desired result.

Four patients were treated by this method between 2000 and 2004. Skin

reactions were visibly less for which no treatment interruptions were

warranted. The total dose of 36 Gy could be delivered within a stipulated

time period which produced best of disease control. Out of 25 patients, 20

patients remained disease free at the end of 5 years, 5 patients had

recurrent disease. Two patients had progressive disease and 3 patients died.


Eosinophilia: An Approach to Management

Maya Gopalakrishnan, Karyampudi Arun, Kiran Kumar Matta,KK Nisar, TK Dutta

Jawaharlal Institute of Postgraduate Medical Education and Research

(JIPMER), Puducherry

Background: Peripheral blood eosinophilia is a common and

interesting presentation of various hematological and non-hemato-

logical disorders. Despite extensive evaluation, up to 50 % of

patients with eosinophilia remain undiagnosed. Objective: To

evaluate patients presenting with peripheral blood eosinophilia using

a diagnostic algorithm. Methods: We report a prospective case

series of patients presenting with peripheral blood eosinophilia. A

structured diagnostic algorithm was used, focusing on empiric

treatment of filarial and helminthic infections even after negative

test results owing to their high prevalence in south India. Results:Five patients presenting with different clinical features and absolute

eosinophil count ranging from 2,530 to 32,000 per mm3 were

diagnosed using the algorithm. A 47 year old businessman pre-

senting with low backache was diagnosed to have indolent systemic

mastocytosis. Another 54 year old shopkeeper with bleeding gums

was diagnosed as having myeloproliferative disorder. An asymp-

tomatic agricultural labourer aged 50 years who was referred for

eosinophilia improved with empiric antihelminthic therapy. A

48 year old labourer with wheeze for 2 months was found to have

tropical pulmonary eosinophilia. Whereas another 55 year old driver

presenting with dry cough and reticulonodular shadowing on X-ray

was ultimately diagnosed to have allergic bronchopulmonary

aspergillosis even after positive filarial serology. Conclusion: A

finding of eosinophilia is related to a wide range of disorders.

Therefore, a structured diagnostic algorithm suited to local preva-

lence of diseases may be useful in effective diagnosis and treatment

of patients with eosinophilia.

Abstract P 174

Anemia in Zidovudine Treated Patients in HIV/AIDS

Parna Bhaumik1, Swapan Kumar Sinha1, Pramit Ghosh1,Indranil Dhar2, Bibhuti Saha2, Mimi Gangopadhyay3, JyotirmoyPal3

1Medical College, Kolkata; 2School of Tropical Medicine, Kolkata;3North Bengal Medical College, SusrutaNagar, Darjeeling

Introduction: Anemia is common in HIV/AIDS patients primarily

due to HIV infection as well as due to secondary infections (CMV,

Parvo virus B19). Hemolytic anemia, anemia due to GI bleeding,

Vitamin B12 deficiency due to malabsorption, deranged Iron

metabolism anemia of chronic disease etc. are different types of

anaemia encountered in HIV/AIDS patients. HAART particularly

Zidovine (AZT) induced secondary anemia is commonly associated

with marrow toxicity. Inhibition of thymidine kinase and DNA

chain translocation may lead to anemia. However other lineages of

cells like neutrophils and platelets are affected. Aims and Objec-tives: The aim of the study was to find significant relation between

incidence of anemia and AZT therapy in Aids patients. Materialsand Method: 930 patients of HIV/AIDS were registered in Anti-

retroviral Centre, School of Tropical Medicine, Kolkata from July

2011 to June 2012 and 780 patients were registered in North

Bengal Medical college ART Centre. Out of these 1083 patients

were initiated with AZT and 325 patients developed anemia

(Hb \ 8.00 g/dl). EDTA blood samples were collected and CBC

was performed. Conclusion: The present study with limited num-

ber of patients show anaemia is commonly associated with AZT

HAART. Further study on pathophysiology of anemia including

types of anaemia e.g. Immunohaemolytic, Microangiopathic,

Nutritional, due to Marrow Suppression/dyserythropoietic anemia

may be necessary.


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Abstract P 175

Bacterial Profile in Haematological Malignancies

Anjali Akhuj1, Lalit S. Raut2, Swagnik Roy1, Kalpana Karak1,Subrata Bhattacharya1, Prantar Chakrabarti2, UtpalChaudhuri2, Barun Saha Dalal1

1Department of Microbiology, KPC Medical College & Hospital,

Jadavpur, Kolkata; 2Institute of Haematology and Transfusion

Medicine, Medical College & Hospital, Kolkata

Introduction: The choice of empiric antimicrobial therapy in febrile

patients with haematological malignancies is based on susceptibility

pattern of locally prevalent pathogens. Aim: To review the bacterial

etiology and antimicrobial susceptibility pattern of blood culture isolates

in these patients. Method: In this one year retrospective study, blood

samples were processed by Rapid culture automated Bac T/Alert 3D

systems (Biomerieux). Results: Out of 458 blood samples, 18.34 % were

positive; gram negative organisms constituted 57.14 % and gram posi-

tive organisms 42.87 %. Gram-positive isolates were coagulase negativeStaphylococcus (CoNS) 77.77 %, Staphylococcus aureus 13.88 % and

Enterococci 8.33 %. There were 20 % Methicillin Resistant Staphylo-coccus aureus (MRSA). Methicillin sensitive CoNS were 64.28 %. All

gram positive isolates were sensitive to glycopeptides and linezolid. Most

common gram negative isolates were Pseudomonas aeruginosa 39.58 %

followed by Escherichia coli 22.99 %, Klebsiella pneumoniae 16.66 %,

Nonfermenting gram negative bacilli 8.33 % and Acinetobacter sp.

4.16 %. Ralstonia paucula, Providencea sp. and Cedeceae neteri were

isolated in 2, 1 and 1 cultures, respectively. In Enterobacteriaceae,

71.42 % were ESBL producers. Amongst the gram negative isolates

Fluoroquinolone resistance was noticed in 66.67 %. All gram negative

isolates were sensitive to Polymyxin and Colistin. Multidrug resistant

Pseudomonas aeruginosa sensitive only to Polymyxin and Colistin were

15.78 %. Conclusion: Due to emergence of multidrug resistant patho-

gens, monitoring antimicrobial resistance pattern locally is crucial in

making decisions of empirical therapy.

Abstract P 176

A Comparative Study of Cord Blood Hemoglobin, Iron, Ferritinand TIBC Level in Babies of Primigravida and Multigravidawith Relation to APGAR Score in a Tertiary Care Hospital

Madhumita Mondal1, Ranapratap Ojha1, Swapan Kumar Sinha1,Utpal Chowdhury2

1Department of Pathology, Medical College Kolkata; 1I.H.T.M.,

Medical College Kolkata

Objectives: (1) To compare the values of hemoglobin and iron profile

of newborns with APGAR score. (2)To establish relation between iron

profile and level of hemoglobin of newborn. Methods: We conducted a

prospective observational study over a period of 1 year. The study

population consisted of 48 pregnant women and their single live-birth

baby. Among the mothers 19 were primigravida and 29 were multi-

gravida. All the babies were delivered normally. Cord blood was

collected from all the cases in both EDTA and clotted vials. EDTA

blood was used for hemoglobin estimation and serum was used for

estimation of iron, ferritin and TIBC. APGAR score of all the newborns

at 5 min was measured. All the results were noted and analyzed by

slandered statistical methods. Results: APGAR score results showed 16

babies were in moderate to severe depression. Preterm babies were

proportionately more depressed than term ones. Hb concentration

varied from 13 to 18.7 g/dl. The iron profile of all the newborns were

within normal limit though case to case variation was present.

Conclusion: No statistical significant ‘p’ value was obtained from the

study. So there was no significant correlation between the cord blood

hemoglobin and iron profile with the APGAR score of the newborns.

Abstract P 177

Plasmapheresis in a Case of Severe Acute Hemolysis

Meenu Bajpai, Suman Lata1, Raju Singh

Department of Transfusion Medicine, 1Department of Nephrology,

Institute of Liver and Biliary Sciences, New Delhi

Introduction: Acute hemolysis is a difficult situation to treat and

usually leads to acute renal failure due to acute tubular necrosis. The

patient’s vascular system is loaded with free haemoglobin and toxic

effects of the same are seen. Here we present a case of acute hemolysis

of unknown origin treated successfully with plasmapheresis. Case: A

22 year old male presented at the emergency unit with fever, jaundice

for 2 days associated with progressive shortness of breath and altered

mutation for one day. The patient was labelled as Acute severe hemo-

lysis with jaundice and put on symptomatic therapy while awaiting

investigations. The patient was unresponsive to symptomatic therapy

and plasmapheresis was suggested as a therapeutic modality in con-

sultation with nephrology and transfusion medicine. Two sessions of

plasmapheresis were done on alternate days starting from day 2 of

admission in which one volume of plasma was exchanged using fresh

frozen plasma as replacement. Post plasmapheresis there was

improvement in the patient’s parameters. Investigation for all possible

causes of hemolysis was done. The patient was negative for all viral

hepatitis markers, leptospira, HIV, mycoplasma. Blood and urine cul-

ture were negative as were Indirect and direct Coombs test. The patient

gave no history of intake of any poisonous substance or animal bite. The

cause for the acute hemolysis remained an enigma. The patient suffered

from acute tubular necrosis of the kidneys from which he recovered and

was discharged. Conclusion: Therapeutic plasmapheresis was a life

saving intervention in the above case and should be considered when

routine therapies fail to bring about the desired outcome.

Abstract P 178

ABO Blood Groups Differentially Allow Plasmodium falciparumGrowth

Vrushali Pathak, Roshan Colah, Kanjaksha Ghosh

Objective: Plasmodium falciparum malaria has an impact on the

distribution of ABO blood groups in humans. The ratio of group O to

A is higher in geographic regions where malaria is currently or was

previously endemic. It is known that, during infection with P. falciparum,

group O offers a survival advantage, however the mechanism by

which the survival advantage is translated is not known. This study

was undertaken to correlate the P. falciparum parasitemia with the

ABO blood groups in an in vitro culture system. Methods: We used

an in vitro culture system that involved co-culturing of P. falciparum(strain 3D7) infected erythrocytes with uninfected different blood

group erythrocytes. A fixed number of parasites were allowed to grow

in fixed number of erythrocytes without addition of fresh cells for up

to 5 days. Mean percent parasitemias (Percent parasite-infected

erythrocytes) were calculated after every 24 h. Results: Percent

parasitemia (mean ± SD) in A, B and O group cultures on fourth day

were 15.4 ± 0.7, 13.3 ± 1.3 and 19.9 ± 3.2 respectively. Parasite-

mia was found to be significantly higher in O blood group when

compared to A and B blood groups (P \ 0.01). Conclusion: This

work demonstrated that blood groups differentially allow


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P. falciparum growth. The resistance of O group hosts in vivo is not

due to low parasitemia some other mechanisms need to be explored.

Abstract P 179

Diagnosis of Malaria on the XT 2000i

Pankhi Dutta, Chitrangi Navadkar, Sharyu Awate1, SandhyaBastian2

Kokilaben Dhirubhai Ambani Hospital, Mumbai; 1SevenHills

Hospital Mumbai; 2Sysmex India Pvt Ltd

Objective: Detection of malaria on different fully automated 5-part

cell counters have been reported with varying sensitivities and

specificities. This study was conducted to evaluate the efficiency of

the XT 2000i (Sysmex) in diagnosing malaria. Method: The com-

plete blood counts including the various scattergrams of 446 febrile

patients suspected to have malaria were analyzed. Thick and thin

blood smears were examined by two observers and the cases were

divided into malaria positives and malaria negatives. In both groups,

abnormal scatter patterns were looked for in the WBC, WBC/Baso

and reticulocyte scattergrams respectively. Results: Malaria positive

cases were 276 while 170 were negative. In the malaria positive

group, additional cluster on the WBC scatter, complete voting out of

the differential, flag for ‘abnormal WBC scatter’, extended ‘ghost’

cluster in the WBC/Baso scatter and abnormal events in the reticu-

locyte scatter were seen in 93, 39.9, 27.5, 78.3 and 93.9 % of the

cases respectively while the same were seen in only 4.12, 0.6, 0.6, 0.6

and 7.6 % respectively, in the malaria negative group. In predicting

malaria positivity, the sensitivity and specificity of presence of an

additional cluster on WBC diff scatter were 92 and 96 % respectively

while it was 78 and 99 % respectively for an extended ghost cluster

on the WBC/Baso scatter. Presence of abnormal events in the retic

scatter had a sensitivity and specificity of 93.6 and 94 % respectively.

Conclusion: It is possible to detect malaria on the XT 2000i in an

automated manner with reasonable sensitivity and specificity.

Familiarization with the various abnormal scatter patterns will be

helpful in increasing the overall sensitivity of malaria diagnosis.

Abstract P 180

Haematological and Inflammatory Parameters in Patientswith Different Types of Tuberculosis and Their Responseto Antitubercular Therapy

Aditya Kutiyal, Sandeep Garg, and Naresh Gupta

Department of Medicine, Maulana Azad Medical College, New Delhi

Aim: To study the haematological parameters and markers of

inflammation in patients with tuberculosis involving different sites/

organs and to correlate these abnormalities following antitubercular

treatment. Methodology: Adult patients [12 years age of either sex,

newly diagnosed tuberculosis ATT naıve were studied. Four sub-

groups were analyzed stratified on the site of tuberculosis namely

pulmonary, CNS, disseminated and others. Haematological parame-

ters like Hb, WBC, platelet count, ESR, LDH, CRP and albumin were

assayed at baseline and 2 months after ATT. Results: Mean age

amongst the 128 patients was 31.55 years. Pulmonary, CNS, dis-

seminated and other types of tuberculosis comprised 30.5, 28.9, 17.2,

and 23.4 % patients respectively. At baseline, mean Hb, WBC,

platelet, ESR, LDH and albumin was 10.87 g% ± 2.17, 9.24 9 109/

l ± 4.27, 256 9 109/l ± 1.38, 61.43 mm ± 21.10, 753.92 iu ±

183.39 and 3.45 g% ± 0.65 respectively. Anaemia was present in

75.78 %, leukocytosis in 49.21 % and leukopenia in 3.9 %. Throm-

bocytosis and thrombocytopenia was encountered in 12.5 and

38.28 % respectively. ESR levels were raised in 98.43 %. Hypoal-

buminemia was seen in 75 % patients. CRP was elevated in 13

patients. Follow-up comparison after 2 months ATT revealed mean

Hb, WBC, platelet, ESR, LDH and albumin to be 11.72 g% ± 1.79,

7.93 v ± 1.4, 207 9 109/l ± 0.33, 8.34 mm ± 4.29, 205.70 iu ±

42.11 and 3.83 g% ± 0.38 respectively. The p values for change in

all the parameters were highly significant (\0.01). This was true

across the four subgroups of different tuberculosis. CRP was no

longer elevated in any patient after ATT. Conclusion: Tuberculosis

presents with varied haematological and inflammatory abnormalities

in majority which revert with 2 months of ATT. A normal ESR is

highly unlikely and anemia is very likely in tuberculosis at presen-

tation. Leucocytosis may occur in up to half of the cases.

Keywords Tuberculosis, Haematology, Inflammation, Acid phase

reactarts

Abstract P 181

Immature Granulocyte Percentage (IG%) in the Diagnosisof Sepsis

Pankhi Dutta1, Shashikala Shivaprakasha, Sharyu Awate,Monisha Sethi2

1Kokilaben Dhirubhai Ambani Hospital, Mumbai; SevenHills

Hospital, Mumbai; 2Sysmex India Pvt Ltd

Objective: Immature Granulocytes is a new, fully automated, FDA

approved parameter available on some cell counters from Sysmex,

Japan. We evaluated the role of IG% as an aid in the diagnosis of sepsis

as compared to the traditional parameters like absolute neutrophil count

(ANC) and total leukocyte count (TLC). Method- The complete blood

count (CBC) of 166 consecutive patients in whom sepsis was suspected

and for whom blood cultures were ordered, were analyzed on the XT

2000i for the IG%, TLC and ANC. Blood culture (BC) positivity was

considered diagnostic of sepsis. The counts in the BC positive group

versus the culture negative group were compared. The Mann–Whitney

test was applied to find the ‘p value’. The sensitivity and specificity of

these parameters in predicting sepsis were also calculated. Results: Of

166 patients (107-males, 59 females, aged 9–98 years), 62 were BC

positive and 104 were negative. In the BC positive group, the mean IG%

was 2.55 (range: 0–6.2 %) versus a mean of 1.46 (range: 0–3.35) in the

BC negative group (p = 0.0025). The BC positive group had higher

TLC and ANC values as compared to the negative group. The sensi-

tivity and specificity of IG% in predicting BC positivity was 61 and

59 % respectively while it was 87 and 29 % respectively for ANC and

84 and 38 % respectively for TLC. Conclusions: The IG % had a lesser

sensitivity but higher specificity versus ANC & TLC in predicting BC

positivity. It is automated, available at the time and cost of a routine

CBC, and can be a useful parameter in the diagnostic workup of sepsis.

Abstract P 182

Haematological Profile, Serum Iron and Ferritin Level in Anemiaof Inflammation: A Prospective study

Nibedita Sahoo, AK Dash, P Sahu, B Mishra, G Rath, SP Pradhan

Department of Pathology, MKCG Medical College, Berhampur

Introduction: Anemia of Chronic Disease (ACD) is the most common

anemia found in hospitalised patients and it is the SECOND most


123

prevalent after Iron Deficiency Anemia (IDA). ACD seen in patients

with chronic infections, malignancy, autoimmune disorders. Condition

also termed as ‘‘Anemia of Inflammation’’. Aim and Objective:Combined clinical history, haematological evaluation, serum iron and

ferritin estimation to reach diagnosis of ACD. Material and Methods:Patients of chronic infection, Autoimmune disorder or malignancy

admitted to different Department s of M.K.C.G.M.C & Hospital are

included in the study during period of 2010–2012. Routine haemato-

logical profile, ESR, serum iron, serum ferritin and bone marrow

aspiration with special emphasis to iron study was performed in 70

patients. Results: Prevalence of ACD was 71 %, IDA 13 % and non

ACD 16 %.Among the patients with ACD mild to moderate anemia

present in 74 % cases. On peripheral smear examination normochro-

mic normocytic anemia in 46 %, normocytic hypochromic 12 %,

dimorphic 16 %, microcytic hypochromic 26 % cases observed. Sig-

nificant correlation of degree of anemia with ESR was obtained. All

cases showed either increase or normal bone marrow iron store.

Conclusion: An increase serum Ferritin, increase Erythrocyte Sedi-

mentation Rate (ESR) and marrow iron staining results indicating

plentiful iron store distinguish people with ACD from IDA.

Keywords Anemia of inflammation (A1), Anemia of chronic disease

(ACD), Irondeficiency anemia (IDA)


Richter’s Transformation : A Case Report

Ipsita Dhal, Sudha Sethy, Rajeeb Nayak, Rabindra Kumar Jena

Department of pathology and Department of clinical haematology,

SCB Medical College, Cuttack

Introduction: Richter’s transformation or Richter’s syndrome is a

complication of B cell chronic lymphocytic leukemia (CLL) in which

the leukemia changes into a fast growing diffuse large Bcell lymphoma

(DLBCL). Richter’s syndrome affects nearly about 5 % of all CLL

patients at some point during their lives. Case History: A 56 year old

male presented with generalised lymphadenopathy and splenomegaly.

His haematological parameters were haemoglobin 7.5 g/dl, total leu-

cocyte count 50,000/cmm with 80 % atypical lymphocytes. He was

diagnosed as a case of CLL/Small lymphocytic leukemia and was put on

CVP regimen (4 cycles). He attended remission. After one and half

years he came with complaints of fever, myalgia and generalised

lymphadenopathy. His peripheral blood smear showed features of CLL.

Biopsy from cervical lymph nodes showed features of non Hodgkin’s

lymphoma (NHL), which was subsequently confirmed by immuno-

histochemical stains showing positivity for B lineage markers (CD 20,

CD 23). Thus the final impression was DLBCL. Conclusion: we report

this case because of its rarity.

Abstract P 184

Treatment Related Complications in Patients with AcuteLymphoblastic Leukemia

Rajesh Kashyap, Mukul Agarwal, Pradeep Kumar, GarimaAgarwal

Department of Hematology, Sanjay Gandhi Postgraduate Institute of

medical Sciences, Lucknow, Uttar Pradesh, India

Objective: To study the different non-haematological complications

occurring during the late phase (2 weeks of induction therapy to remis-

sion) treatment of ALL patients. Material and Methods: Patients

diagnosed with ALL and being treated at department of haematology,

SGPGIMS were subject of the study. The diagnosis of ALL was made

based on complete hemogram, bone marrow examination with cyto-

chemistry and immunophenotyping by flow cytometry. The clinical

records of the patients were analysed for occurrence of non-haemato-

logical complications occurring 2 weeks after the start of therapy. The

patients were treated as per the BFM protocol (BFM 90 of BFM 95

protocol). Result: One hundred and one patients had 147 events of non-

hematological complications. Gastrointestinal toxicity was the most

frequent complication and occurred predominantly as hepatitis. Hyper-

glycemia was seen in 25 cases (24.7 %) and was most frequent in patients

above the age of 20 years. Neurological complication was predominantly

seen in patients below the age of 20 years (76.2 %) and seizures was the

most common presentation. Osteonecrosis involving the hip joints was

seen two adult young males. Conclusion: Non-hematological compli-

cations are quite frequent during the late phase of treatment of ALL

patients. The Gastrointestinal tract, nervous system and endocrine system

most frequently affected. Many of these events are missed because of low

levels of clinical suspicion. This study highlights the incidence of these

complications as they are associated with high morbidity and mortality.

Abstract P 185

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

Shivangi J. Harankhedkar, Sudha Sethy, Pranati Mahanty,BP Das, RK Jena, SR Mahapatra

SCB Medical College and Hospital, Cuttack

Background: PNH is a consequence of non malignant clonal

expansion of one or several hematopoietic stem cells due to a somatic

mutation in PIG-A gene. Resultant deficiency of glycosyl phospha-

tidyl inositol-anchored proteins (GPI-APs), CD55 and CD59,

accounts for the primary manifestation of intravascular hemolysis.

PNH may also arise in association with aplastic anemias and MDS.

Aims and Objectives: (1) Diagnosis of PNH by using multiparameter

flow cytometry with antibodies specific for GPI-APs, CD55 and

CD59, and assess severity of hemolysis. (2) Detect PNH clones in

patient of aplastic anemia and MDS, and correlate with therapy and

prognosis. Materials and Methods: 253 patients of pancytopenia and

refractory anemia (Coombs negative) were screened for PNH clones.

Using 2 ml of peripheral blood (with EDTA), CD55 (DAF) and CD59

(MIRL) deficient clones were detected on granulocytes by multipa-

rameter flow cytometry. Other supportive routine investigations were

done. Results: Out of 253 patients, classical PNH: 18. PNH clones in

50 aplastic anemia patients: 17. PNH clones in 5 MDS patients: 2. All

cases presented with anemia. Pancytopenia (80 %), hyperbilirubine-

mia (20 %), reticulocytosis and evidence of hemolysis (75 %).

Conclusion: (1) Flow cytometry is considered to be gold standard for

diagnosis of PNH. (2) Association of PNH with aplastic anemia and

MDS is identified as better prognostic indicator.

Abstract P 186

‘Bleeding’ and ‘transfusion Support’ in Acute Myeloid LeukemiaPatients: The Challenges

Rahul Chaurasia, Priti Elhence, Sonia Nityanand, AnupamVerma

Department of Hematology, Others Department of Transfusion

Medicine, SaNjay Gandhi PGIMS, Lucknow


123

Background: Bleeding due to severe thrombocytopenia in acute

myeloid leukemia (AML), may occur due to the disease process itself

or because of the chemotherapy. Prophylactic platelet transfusions are

given to these patients to prevent the occurrence of bleeding.

Occurrence of bleeding in these patients is therefore an indicator of

presence of factors whereby patient’s response to transfusion is sub-

optimal. Bleeding may be classified according to WHO grading

system and management of bleeding is done according to bleeding

grade. There is however insufficient data to validate this approach.

Objectives: To study the WHO bleeding grades and therapeutic

transfusion support in AML patients undergoing chemotherapy and

prophylactic platelet transfusions. Materials and Methods: A pro-

spective study of 19 patients suffering from AML was carried out at

our center. Patient details, clinical details, bleeding grades as per

WHO, the platelet transfusion data including the indication, trigger

dose and the response to the platelet transfusion were recorded and

analyzed. Results: 256 bleeding days was observed of the total 1302

patient days. WHO Bleeding grades observed were Grade

1 = 115 days, Grade 2 = 86 days, and Grade 3 = 55 days, no grade

4 bleeding was observed. GI bleeding (28.5 %) was the most common

site of the bleed followed by oropharyngeal bleed (19.1 %), epistaxis

(17.1 %), mucocutaneous bleeding (16.4 %), genitourinary (14.4 %)

and others (4.3 %). Mean pre transfusion Hb was 7.82 ± 1.32

(4.3–12.3) and platelet count was 17.54 ± 12.24 (6–46) for which

total 135 LPRBC units (mean 0.53 per episode) and 835 RDP units

(mean 3.26 per episode) was transfused. Clinically bleeding was

stopped after platelet transfusion. No bleeding related mortality was

noted. Conclusion: Despite the prophylactic platelet transfusion

19.6 % of observation days, majority of III bleeding episodes were in

patients who started as grade I or Grade II bleedings therefore this

management approach needs further improvement.

Abstract P 187

Sickle Cell Anaemia—Community Control Programme AmongstTribal Groups from Satpuda Hilly Ranges in Maharashtra, India

SL Kate, Gunvant H. Yeola, Prashant N. Dalvi,Girish T. Kulkarni, Yogesh S. Prabhune

Maharashtra Arogya Mandal, Hadapsar, Pune-411028

Abstract: Amongst all the tribal area of Maharashtra Sickle Cell

Disorder is very common amongst Bhill and Pawara tribal population

groups residing in the Satpuda hilly ranges (trait prevalence 1:5).

Considering counselling is the only alternative, to control the disease,

we have established a community control centre in high risk area

located between 3rd and 4th hilly ranges of Satpuda hills (at Roshmal

Budruk, Tal. Dhadgaon, Dist. Nandurbar Maharashtra state. We

provided accurate diagnosis, possible treatment, follow-up counsel-

ling. We have now 1501 sickle cell patients under our medical

supervision. Counselling is provided to patients, parents and public

from this area. Details will be described in the presentation.

Abstract P 188

Systemic Mastocytosis: A Rare Entity

M Ketan, Bikash Singhania, K Annamma, B Sushma, R Lakshmi

1Kasturba Medical College, Manipal University, Manipal; 2Melaka

Manipal Medical College, Manipal

Introduction: Mastocytosis encompasses a rare and heterogeneous

group of clinical disorders characterised by abnormal growth and

accumulation of mast cells in various organs, commonly skin and bone

marrow. Here we report a case of systemic mastocytosis with bone

marrow involvement as the first manifestation. Specific histocytological

techniques were needed to establish the diagnosis. Case Report: A

52 years old lady presented with severe lower and upper back pain of

8 months duration. On local examination severe tenderness was elicited

over dorsal and lumbar spine. X-Ray and MRI for the spine showed lytic

lesions at the L1 to L5 vertebrae. Bone scan was suggestive of osteo-

blastic changes. Gastroscopy demonstrated pangastritis. Metabolic and

endocrinological etiologies were excluded because laboratory evaluation

was unremarkable. Iliac crest bone marrow biopsy demonstrated infil-

tration by mast cells, arranged in sheets and whorls. Extensive areas of

marrow fibrosis was seen along with bone destruction. Special stains:

AFB—negative; Toluidine blue—positive in mast cells. Immunohisto-

chemistry: CD117—positive in mast cells. Conclusion: The diagnosis of

mastocytosis requires a great degree of clinical suspicion as it is a rare

disease that may go unrecognized or misdiagnosed because of the non

specificity of many of its symptoms, particularly in cases with visceral

involvement and absence of characteristic skin lesions of urticaria pig-

mentosa. Bone involvement in mastocytosis is related to infiltration of

bone marrow by mast cells. Bone marrow biopsy and histocytological

techniques are the key for diagnosis.

Abstract P 189

Flowcytometric Characterization of B-ALL: A 3 Year Studyfrom a Tertiary Care Centre

Nabhajit Mallik, Vijay Kumar, Anjali Sharma, Gurdeep Buxi

Department of Pathology, PGIMER & Dr. Ram Manohar Lohia

Hospital, New Delhi

Objective: To evaluate the expression of Common Acute Lympho-

blastic Leukemia Antigen (CD10), CD34, HLA-DR and myeloid

antigens in all cases of B-ALL over a period of 3 years in a tertiary

care centre in North India. Methods: The study was carried out at the

Department of Pathology, PGIMER & Dr. RML hospital, by ana-

lysing the flow cytometry data over a period of 3 years, from

September 2009 to August 2012. All cases of B-ALL were studied to

look for the expression of CD10, CD34, HLA-DR and myeloid

antigens (CD13, CD33). Results: A total of 36 cases of B-ALL were

retrieved, of which 27 (75 %) patients were male, and 9 (25 %) were

female. The youngest patient was 3 months old, while the oldest was

48 years. 29 cases (80.5 %) showed CALLA positivity, while 7

(19.5 %) were CALLA negative. CD34 positivity was seen in 32

cases (88.9 %) while 4 cases (11.1 %) were negative for CD34. Only

1 case (2.7 %) was negative for HLA-DR, and the remaining 35 cases

(97.3 %) were positive. A total of 13 cases (36.1 %) showed aberrant

expression of myeloid markers in the form of CD13 positivity.

Conclusion: The present study illustrates the role of flow cytometry

in immunophenotypic characterization of B-ALL, especially with

respect to markers that carry prognostic significance for the patient.


Hairy Cell Leukemia—A Rare Case Report

Sugatha Sahu, S Behera, SP Pradhan

Department of pathology, MKCG Medical college, Berhampur,

Odisha, India

Introduction: Hairy cell leukaemia (HCL) is a rare, clonal, chronic

lymphoproliferative disorder commonly seen in males in the middle years

of life. It represents 2 % of all leukemia’s. Pancytopaenia with moderate


123

to massive splenomegaly is the most common clinical presentation. The

diagnosis is based upon the recognition of the characteristic ‘‘hairy’’

nature of the leukemic lymphoid cells in the peripheral blood smears and

the typical appearance and pattern of infiltration in the bone marrow

biopsies in association with increased reticulin and eventual fibrosis of the

marrow. Case Report: We report a case of 54 year male who presented

with pallor, fever, abdominal distension. On physical examination

splenomegaly was detected. Peripheral blood examination showed pan-

cytopenia and lymphocytes with abundant cytoplasm which spread into

hair-like processes. The bone marrow aspirate was a dry tap. The trephine

biopsy has the characteristic features of a honey comb appearance. The

cytochemistry was positive with tartrate resistant acid phosphatase

(TRAP).Hence diagnosis of hairy cell leukemia was made. Conclusion:HCL is an easy diagnosis to make on the morphology of well made blood

and bone marrow smears. Although there are no specific markers for

HCL, cytochemistry and flow cytometry readily confirm the diagnosis.


Niemann-Pick Disease Presenting with Hepatosplenomegalyand Thrombocytopenia

Mitali Swain, SK Behera, SP Pradhan

Department of Pathology, MKCG Medical College, Berhampur,

Odisha, India

Introduction: Niemann-Pick disease (NPD) is an autosomal recessive

genetic disorder resulting in abnormal lipid metabolism. NPD is a clini-

cally and biochemically heterogeneous disorder, with four variants. Type

A and B have a generalized sphingomyelinase deficiency, whereas types C

and D have normal sphingomyelinase levels. Case Report: We report two

cases of Niemann-Pick disease in children, one aged 9 months and the

other of 2 years, complaining of abdominal distension and recurrent fever.

The older child also complained of epistaxis and melena. Both the children

had coarse facial features and growth retardation. The abdominal ultra-

sound confirmed the clinical findings of hepatosplenomegaly. Bone

marrow and liver biopsy examination revealed presence of foamy histio-

cytes, suggestive of Gauchers or Niemann-Pick. Confirmatory study by

lysosomal enzyme from leucocytes was normal for b-Glucosidase and

sphingomyelinase specific for Gauchers and Niemann Pick type A or B

respectively. So, a diagnosis of Niemann-Pick disease (types C or D) was

made. Conclusion: Niemann-Pick disease in children is very rare, with a

reported incidence of 1:1, 20,000 to 1:1, 50,000 live births. Prenatal

diagnosis with amniocentesis or chorionic villus sampling is available.

Keywords Niemann-Pick disease, Sphingomyelinase,

Hepatosplenomegaly

Abstract P 192

A Prospective Study to Test the Usefulnessof an Immunophenotype-Based Scoring System to DistinguishBetween T-ALL with Myeloid Markers [T-ALL(M)] and AMLWith T-Lymphoid Markers [AML(T)]

Vishal Mehrotra, Manisha Ramani, Janmejay Yadav, PallaviGujarathi, Santosh Parab, Archana Vazifdar and Amar DasGupta

Sections of Hematology and Flow Cytometry, SRL Limited, Mumbai

Introduction: From a retrospective analysis of 1874 cases of acute

leukemia we proposed a scoring system in 2010 to distinguish between

cases of AML(T) and T-ALL(M) on the basis of scores assigned for

expression of surface antigens that indicated myeloid phenotype of

blast cells because of their strong association with typical cases of

AML, e.g. CD5 negativity: score 2; CD10 negativity: 1; CD13 posi-

tivity: 1; CD117 positivity: 0.5 and HLA DR positivity: 1. Cases of

T-ALL(M) had a score\2.5 and those of AML(T) [ 3.5. In this pro-

spective study we tested the sensitivity and specificity of this scoring

system. Materials and Methods: Immunophenotyping was performed

in 1574 additional cases of acute leukemia from May 2010 to July 2012

using a primary panel consisting of antibodies against CD 3, 5, 7, 10,

13, 19, 20, 22, 33, 34, 45, 117 and HLA-DR. Additional markers such

as CD2, 4, 8, 14, 15, 41, 61, 64, 99 and glycophorin were tested as

indicated. The presence of cytoplasmic (c) myeloperoxidase, cCD79a

and cCD3 was examined in all cases with aberrant expression of CD

markers by blast cells. Results: 126/130 cases of AML(T) had a score

of C3.5 (sensitivity and specificity = 97 %) while 71/73 cases of

T-ALL(M) had a score B2.5 (sensitivity 97; specificity 98.5 %). There

was no case of true mixed (T and myeloid) leukemia. Conclusions: The

scoring system proposed by us allows accurate distinction between

AML(T) and T-ALL(M) in most cases at a much lower cost than that

involved in the use of expensive reagent panels, including cytoplasmic

markers, and advanced flow cytometry software.

Abstract P 193

CD200 Expression in B-Chronic Lymphocytic Leukemia(B-CLL)—it is not an all or None Phenomenon

Manisha Ramani, Vishal Mehrotra, Janmejay Yadav, PallaviGujarathi, Santosh Parab, Amar Das Gupta

Hematology & Flow Cytometry Sections, SRL Limited, Mumbai

Introduction: Immunophenotypic distinction between B-CLL and non-

CLL B-chronic lymphoproliferative disorders (B-CLPD) can be chal-

lenging and is sometimes impossible due to overlapping features among

these entities. However, a firm diagnosis is necessary for proper man-

agement of different types of CLPD. Recently CD200, a B-lymphoid

antigen has been shown to be useful in this regard by virtue of its close

(100 %) association with B-CLL and its absence in the lymphoma cells in

non-CLL B-CLPD, except hairy cell leukemia and B-lymphoblastic leu-

kemia/lymphoma. Hence CD200 has been proposed in recent studies as a

diagnostic marker for B-CLL. We wanted to verify this claim by checking

the specificity and sensitivity of this marker for the diagnosis of B-CLL.

Materials and Methods: Twenty eight cases of B-CLL and 28 cases of

other types of B-CLPD (splenic marginal zone lymphoma 7; mantle cell

lymphoma 10; ‘hairy cell’ leukemia 3; others 8) were immunophenotyped

between April and August 2012 using a large panel of antibodies against

CD3, 5, 10, 11c, 19, 20, 22, 23, 25, 38, 103, 200, FMC7, HLA-DR,

immunoglobulin light chains and surface immunoglobulins. Results:Lymphoma cells in all 28 cases of B-CLL were CD200 positive (sensitivity

100 %), while 9/28 cases of non-CLL B-CLPD (‘hairy cell leukemia: 3/3;

mantle cell lymphoma: 2/10; others: 4/8) also were CD200 positive indi-

cating a relatively low specificity (68 %). Conclusions: Our study

suggests that although CD200 is a highly sensitive marker for B-CLL, its

low specificity would limit its utility in the differential diagnosis between

this entity and the other types of B-CLPD.

Abstract P 194

Study of Treatment Outcome of Different Art Regimen Basingon CD4 Count

Vandana Raut, P Kote, RK Bhola, SK Behera, S Pradhan

Vandana Raut, MKCG Medical College, Brahmapur


123

Introduction: HIV is the leading infection in southern Orissa.

37.8 % of total HIV Population of Orissa is contributed by Ganjam

district alone, and the most probable reason being; unemployed

people from coastal districts like Ganjam migrate to industrial areas

where HIV cases are more. We have a functional ART centre since

2006 and mainly four different regimens are followed consisting of

two nucleoside reverse transcriptase inhibitors (NRTIs) and one

either a non-nucleoside reverse transcriptase inhibitor (NNRTI), or a

protease inhibitor (PI). Aims and Objectives: (1) To know the

efficacy of each of these regimen to restore the immune function (as

indicated by the CD4 cell count). (2) Compare the treatment out-

come of patients in relation to their base line cd4 count at onset of

ART. (3) Response to ART in HIV patient’s co infected with TB.

Materials and Method: The study included 464 HIV cases which

were followed over a period of 1 year. Efficacy of different ART

regimens is studied in terms of CD4 count. Result: Patients age

ranges from 15 to 60 years with mean age of 37.4 years and M:F is

1.4:1. Among 464 cases 220 patients are in regimen I(d4T, 3TC,

NVP), 60 patients are in regimen II(d4T, 3TC, EFV), 100 patients

are in regimen III(AZT, 3TC, NVP) and 84 patients are in regimen

IV(AZT, 3TC, EFV). % increase in CD4 count is maximum with

regimen I. Conclusion: From this we conclude that increase in CD4

count is best achieved with regimen I and presence of TB impairs

the response to ART.

Abstract P 195

Essential Thrombocythemia Transforming into Acute Leukemia:A Case Report

Shabnam Roohi1, Jyothsna Krishnappa2, PP Bapsy3, CN Patil3,Manish Chugh1

1Department of Haematology and Clinical Pathology; 2Department of

Internal Medicine; 3Department of Medical Oncology, Apollo

Hospitals, Bangalore

Introduction: The transformation of essential thrombocythemia into

myelofibrosis is a rare and late complication. The recent advances in

the management of this disease and its complications have raised a

hope for better control. In this case report we present a case of an

elderly patient having essential thrombocythemia with rapid trans-

formation to myelofibrosis and leukemia. Case Description: A

79 year old female case of essential thrombocythemia detected in

2007, on symptomatic treatment, presented to Apollo hospitals in

April 2012 with fever, low back ache, easy fatigability and loss of

appetite. She was thin, pale and had massive splenomegaly on

examination. Peripheral smear examination revealed a leukoery-

throblastic blood picture with severe thrombocytopenia. Bone

marrow aspirate was aparticulate with scattered immature myeloid

cells. The bone marrow biopsy revealed grade four fibrosis on a

scale of 0–4. A diagnosis of Essential Thrombocythemia trans-

forming to Myelofibrosis was made. Considering her high risk

status, regular follow-up with blood transfusion was initiated. In

august 2012 she presented with worsening general weakness and

breathlessness. Peripheral smear showed 24% blasts, suggestive of

leukemic transformation probably myeloid. Flow cytometry report is

awaited. Discussion: Progression of essential thrombocythemia to

Myelofibrosis is 2.8 and 2.3% for acute leukemia with a median of

11 years. In this case the progression to acute leukemia was within

5 years of the initial diagnosis. Conclusion: Transformation of

essential thrombocythemia to Myelofibrosis and acute leukemia can

be rapid. Therefore the use of targeted therapy even for high risk

patients needs to be explored.

Abstract P 196

Evaluation of ADVIA 2120 Haematology Analyzer

Shabnam Roohi, BC Sangamesh, LB Manigantan

Department of Haematology and Clinical Pathology, Apollo

Hospitals, Bangalore, Karnataka, India

Objective: To evaluate performance of Siemens ADVIA 2120 fully

automated haematology analyzer, compare it with the Beckman

Coulter ACT i 5 Diff and manual reference methods. Methods: The

precision, accuracy and Bias of the equipment were evaluated using

standard methods. The Linearity and Carryover of the equipment was

validated. The routine haematological parameters generated by the

ADVIA 2120 were compared to those obtained from ACTi 5 Diff.

The automated differential counts, platelet counts, n RBC and retic-

ulocyte percent were also compared to manual methods. Results: The

precision, accuracy and Bias of the equipment of the equipment were

within acceptable limits. Linearity and carryover were within the

limits established by the manufacturer. There was statistically sig-

nificant difference between the two equipments for platelet count and

eosinophil count, the difference for the other routine parameters was

not statistically significant. The leukocyte differential counts indi-

cated an excellent correlation with the manual reference method for

Neutrophils, Lymphocytes and Eosinophils, a good correlation for

Monocytes and a poor correlation for Basophils. The platelet count

and reticulocyte count indicated an excellent correlation with the

manual reference method, the enumeration of nucleated red blood

cells by the automated method showed moderate correlation with the

manual reference method. Conclusion: The results generated by the

ADVIA 2120 analyzer are comparable to the existing equipment and

the reference methods. The potential of new parameters generated by

the analyzer in extending the clinical role of haematological studies

needs to be explored.

Abstract P 197

Cytogenetic Abnormalities in Acute Leukemia Cases—SingleCentre Study from Eastern India

Basab Bagchi1,Tuphan Kanti Dolai1, Prakash KR Mondal1,Ashutosh Panigrahi1, Sandeep Saha1, Meet Kumar1, MaitreyeeBhattacharyya1, Shyamali Dutta1, Rajiv De1, Malay Ghosh1,Bani B Ganguli2

1Hematology Department, NRS Medical College and Hospital,

Kolkata; 2Genetics Centre, Navi Mumbai, Maharashtra

Objective: Cytogenetics has emerged as an effective tool in diag-

nosis, classification, risk stratification, prognostication and as a guide

for providing appropriate therapy in acute leukemia cases. However

there is not enough data from India. This study was conducted to

determine the incidence and common cytogenetic abnormalities in

AML and ALL. Methodology: Prospective collection of cytogenetics

data from patients attending hematology services of NRS Medical

College, Kolkata. Demographic data, diagnosis data collected, cyto-

genetics analysis done in all patients. Period: July 2009 to July 2012.

Results: Of the 235 patients 152 were males and 83 were females.

118 had ALL and 117 had AML. Among ALL(118) cases 86 patients

were \18 years of age and 32 patients were C18 years of age. Nor-

mal Karyotype was observed in 31/118 (26.3 %) patients.

Hyperdiploidy was the commonest cytogenetic abnormality observed

in 38/118 (32.2 %) of ALL patients, followed by t(9;22) detected in

15/118 (12.7 %) of patients. The next common abnormality was

t(4;11), detected in 9/118 (7.6 %) of ALL patients. Among the ph


123

positive patients 12/15 (80 %) were C18 years. Among AML 38/117

(32.5 %) had normal karyotype. t(15;17) was the commonest cyto-

genetic abnormality [13/117 (11.1 %)] followed by trisomy 8

(10.25 %) and t(8;21)9.4 %. Conclusions: 1. Cytogenetic abnor-

malities were present in 73.7 % of ALL and 67.5 % of AML. 2.

Hyperdiploidy was the most common cytogenetic abnormality in

ALL followed by t(9;22). 3. Among the Philadelphia positive patients

80 % were C18 years. 4. t(15;17) was the most common cytogenetic

abnormality in AML followed by trisomy 8 and t(8;21).

Abstract P 198

Coagulation Aberration in HIV Infected Patients

Swapan Kumar Sinha, Soumit Mondal

Department of Pathology, Medical College, Kolkata

Objective: To study disorders of haemostasis in untreated cases of

HIV/AIDS. Total Platelet Count, prothrombin time (PT), activated

partial thromboplastin time (APTT) was performed on 53 diagnosed

patients of HIV. Methods: Citrated blood (1:9) samples were col-

lected for P.Time, APTT, FDPs and EDTA blood for Complete Blood

Count ART Centre of Medical College and School of Tropical

Medicine. PTimre & APPT was performed as per validated methods

with Internal Quality Control. Platelet count was done by making

blood smears of individual samples and counting under light micro-

scope. Results: Among them 19/53 pts. (35.84 %) had decreased plt.

count. Only 7/53 pts. (13.2 %) had elevated PTimer, 6/53 (11.32 %)

had elevated APTT and 9/53 (16.98 %) had elevated PT, APTT and

Low platelet. Conclusion: Thrombocytopenia may be due to

increased destruction commonly immune mediated, increased con-

sumption due to microangiopathy, TTP or decreased production.

Thromboplastin like substances, Factor X activation by angiopathy or

due to CMV infection may lead to activation of coagulation system.

Acquired Factor V Leiden may lead to hypercoagulable states

including APLAS. A detail study in HIV patients for coagulation

disorders will be more informative.

Abstract P 199

Genotypic and Phenotypic Characterisation Bernard-SoulierSyndrome Patients from India

Objective: Molecular characterization of BSS patients from India.

Introduction: Bernard Soulier Syndrome (BSS) is a rare autosomal

recessive bleeding disorder caused by an absence of or defects in the

platelet membrane glycoprotein GP/IX/V complex. Methods: The diag-

nosis of 10 patients was based on low platelet count, presence of giant

platelets in the peripheral smear, platelet function and platelet receptor

study, flow cytometry study to assess the surface expression of GP1b/IX/V

complex showed reduced expression, genomic DNA was screened for

mutations in GP1BA, GP1BB and GP9 through direct sequencing.

Results: We studied 10 patients diagnosed with BSS with classical

bleeding symptoms. Bleeding diathesis measured by the World Health

Organisation bleeding scale (grade 0, no bleeding; grade 1, petechiae;

grade 2, mild blood loss; grade 3, gross blood loss; grade 4, debilitating

blood loss) ranged from 1 to 4. All subjects suffered from frequent

ecchymoses, epistaxis and gum bleeding, the three menstruated females

had severe menorrhagia that required treatment. All the patients had low

platelet count ranging from 6 9 109/L to 55 9 109/L determined auto-

mated cell counter (XT-2000i, Transasia Bio-Medical Ltd,

India).Thrombocytopenia is always more severe when platelets were

measured by a cell counter, because it does not recognises and enumerate

large platelets, as to mean platelet volume (MPV)/platelet size, in none of

the patients the instrument could report the MPV due to its platelet

abnormality. Ristocetin (normal and low dose) induced platelet aggrega-

tion was absent in all patients except one, which was unavailable for study

(BSS1.01), whereas it was normal in controls. Sequencing analysis of the

GPIba, GP1bb and GP9 gene revealed homozygous changes in the pro-

bands. Of the 6 mutations; 4 were found out to be novel mutations

including one nonsense changes in GP1bb (p.Cys32X) and two missense

changes in GP9 (p.Thr95Asp) and (p.Cys135Tyr) and one frameshift

change in GP1ba (p.Met338AsnfsX13). The known mutations found out to

be one missense change in GP9 (p.Cys24Arg) in one patient with severe

bleeding diathesis and one frameshift change in GP1ba (p.Val485Val-

fsX12). Conclusion: The molecular data presented here is a new data on

BSS patients from India apart from the existing data, adding significantly to

the mutation database of this condition and also useful for its genetic

diagnosis in India.

Abstract P 200

Comparative Evaluation of Bone Marrow Trephine Biopsieswith Bone Marrow Aspirates: A Tertiary Care InstitutionalExperience

Usha R Singh, Surbhi Goyal, Usha Rusia

University College of Medical Sciences & Guru Teg bahadur

Hospital, Delhi

Introduction: Bone marrow examination is an important diagnostic tool

to evaluate various neoplastic and non neoplastic hematological diseases.

Aims: To assess correlation between bone marrow biopsy and aspirate.

Correlation of lymphoma positivity with trephine biopsy length. Meth-ods: Diagnostic correlation was done between bone marrow aspirates &

bone marrow trephine biopsies in 518 cases received in the department of

pathology from Jan 2011 to Feb. 2012. Proportion of biopsy showing

marrow infiltration by lymphoma cells was studied in relation to total

trephine length. Biopsies were fixed in formalin & length measured. After

EDTA decalcification they were processed routinely and H&E stained.

Special stain & immunohistochemistry was done if needed. Results: We

found a positive correlation of 88.6 % for acute leukemias and plasma

cell dyscrasias, 60% for nonhematopoietic metastases, 53.7 % for NHL,

38.5 % for aplastic anemia, 5% for Hodgkin’s lymphoma, and 0 % for

granulomatous infections and myelofibrosis. The maximum proportion

of positivity in lymphoma was attained at a length of 1.3 cm. Conclu-sion: Granulomatous infections and myelofibrosis could only be

diagnosed on biopsy. Though both are complimentary, biopsy is more

sensitive and informative in detection of nonhematopoietic metastases,

assessing marrow involvement in lymphomas and plasma cell deposits in

multiple myeloma. In acute and chronic leukemias, immune thrombo-

cytopenic purpuras and nutritional anemias biopsy is a useful adjunct to

aspirate in diagnosis and follow up. A biopsy length of C1.3 cm was

adequate for assessing lymphoma infiltration.


Anticardiolipin Antibodies in Women with Spontaneous FetalLoss

Meera Sikka1, Akansha Rawat1, Usha Rusia1, Kiran Gulleria2

1Department of Pathology; 2Department of OBG, University College

of Medical Sciences & GTB Hospital, Delhi, India


123

Introduction: Spontaneous fetal loss is a frequent complication of

pregnancy. Recurrent pregnancy loss (RPL) i.e. occurrence of

three consecutive pregnancy losses during the first trimester

affects 1 % of pregnant women. Several causes of RPL have been

reported . Antiphospholipid syndrome, an autoimmune disorder

characterized by RPL, arterial and/or venous thrombosis and

elevated titers of antiphospholipid antibodies is a frequent cause

of RPL. Anticardiolipin antibodies (ACA) are one of the signifi-

cant antibodies detected in this syndrome. Aim: To ascertain the

prevalence of anticardiolipin antibodies in Indian women with

spontaneous/recurrent fetal loss. Methods: Seventy eight women

with spontaneous/ recurrent fetal loss in the first trimester were

included in the study. A detailed history and physical examination

was done. Anticardiolipin antibodies, IgG and IgM were deter-

mined by ELISA using commercially available kits. Results: IgG

antibodies were detected in 1 (1.2 %) woman and IgM in 3

(3.8 %). These women had no other clinical manifestation. Other

pregnancy related complications were significantly more in ACA

positive women as compared to ACA negative women. Conclu-sions: Presence of ACA defines a population at risk for fetal loss

and other pregnancy related complications. Women with RPL

must be screened for these antibodies even in the absence of other

clinical symptoms as on treatment these women have a better

chance of a subsequent successful pregnancy.

Abstract P 202

Non Immunoglobulin CMYC Translocation in High Grade B CellNHL: The Gray Zone Between Diffuse Large B Cell Lymphomaand Burkitt’s Lymphoma

M. Parihar1, A. Gupta1, A. Yadav1, S. R. Arun1, S. Bhave2,V.Radhakrishnan2, A. Chakrapani2, D. K. Mishra1, M. Chandy2

1Departments of Cytogenetics and Lab Hematology; 2Clinical

Hematology, Tata Medical Center, Kolkata, India

Background: High grade B cell non Hodgkins lymphoma (NHL) with

features between diffuse large B cell lymphoma (DLBCL) and Bur-

kitt’s lymphoma (BL) have been in the grey zone of diagnostic

uncertainty. Although immunoglobulin gene CMYC translocations

define BL, the CMYC gene may be involved in DLBCL as a secondary

hit to the BCL6 or the BCL2 gene translocations. We present a case of

non immunoglobulin gene (Ig) CMYC translocation,t(3;8)(q27;q24)

juxtaposing the CMYC gene to the BCL6 gene in a patient with features

of Burkitts lymphoma . Case report: Our patient a 29 year old male

presented with weakness and low backache following surgery for an

ileocaecal mass operated outside and diagnosed as DLBCL. Clinical

examination revealed no lymphadenopathy or hepatosplenomegaly. A

complete blood count showed low platelet counts with other parameters

being normal. The bone marrow aspirate showed 90 % L3 lympho-

blasts which on flowcytometry expressed CD43, CD10, CD19, CD38,

cCD79a and CD22 with surface immunoglobulin expression. The

trephine biopsy showed infiltration by sheets of abnormal cells with

grade 2 fibrosis. Immunohistochemistry showed positivity for

CD20,BCL6 and negativity for MUM-1 and BCL2 with 100 % Ki67

staining. Karyotyping showed 46,XY,t(3;8)(q27;q24)+2mar[20].

Metaphase FISH using breakapart CMYC probe confirmed the CMYC

gene rearrangement with the partner chromosome being 3q27.

Discussion: The non-IG-MYC translocations have been said to be

almost exclusively secondary events and rarely occur in typical BL.

From clinical point of view these cases with MYC rearrangements but

without an mBL signature are associated with a poor clinical outcome

and classified as intermediate BL/DLBCL by the WHO.

Abstract P 203

A Novel t(2;12) in Precursor T Cell ALL Involving the ETV6Gene

M. Parihar1, A. Gupta1, A. Yadav A1, S. R. Arun1,A. Bhattacharyya2, D. K. Mishra1, M. Chandy3

1Departments of Cytogenetics and Lab Hematology; 2 Paediatric

Oncology; 3Clinical Hematology, Tata Medical Center, Kolkata,

India

Background: Rearrangements of the short arm of chromosome 12 (12p),

that harbors the ETV6 and the CCND genes have been reported in acute

lymphoblastic leukemia (ALL) .Although the role of ETV6 in precursor

B cell ALL has been extensively studied only rare cases of ETV6 gene

involvement in paediatric T cell ALL have been described where the

CCND gene is more commonly involved. We report a case of early

precursor T cell ALL in a 13 year old male with t(2;12)(p13;q31)

involving the ETV6 gene. There have been no reports to date on t(2;12) in

ALL. Case Report: The patient presented with history of fatigue and

irregular fever for the past 6 months. On examination severe pallor,

generalized lymphadenopathy and hepatosplenomegaly were present.

Investigations revealed low hemoglobin, normal white blood cell count

and low platelet counts. Peripheral blood showed 14 % blasts. The bone

marrow showed 90 % L2 lymphoblasts with bright expression of cCD3

and CD7, and moderate expression of CD2, CD5, HLA DR and Tdt. The

karyotype showed 46,XY,t(2;12)(q31;p13) [20]. The involvement of

ETV6 gene was confirmed by metaphase FISH. The patient received the

UKALL 2003 Regimen B protocol. He was in complete remission (CR)

with negative MRD by flowcytometry on day 29 and remains to be in CR

post consolidation therapy. Discussion: The t(2;12)(p13;q31) results in

the relocation of the ETV6 present on 12p13 to 2q31 locus that harbors

the class 1 homeobox gene (HOX) cluster D. The homeobox genes are

expressed during early stages of T cell development and the exact

mechanism of activation of these HOX genes remains to be explored. Our

report highlights the importance of these genes in leukemegenesis of T

cell ALL by probable developmental arrest at specific T cell stages.

Abstract P 204

Cytogenetic Profile of Hematolymphoid Neoplasms in a NewlyOpened Tertiary Care Center: The First Year Experiencefrom East India

M. Parihar1, A. Gupta1, A. Yadav1, S. R. Arun1, S. Bhave2,V. Radhakrishnan2, A. Chakrapani2, A. Bhattacharyya3,D. K. Mishra1, M. Chandy2

1Departments of Cytogenetics and Lab Hematology; 2Clinical

Hematology; 3Paediatric Oncology, Tata Medical Center, Kolkata,

India

Introduction: Conventional cytogenetic analysis of hematolymphoid

neoplasms provides valuable diagnostic and prognostic information.

We describe the cytogenetic profile of hematolymphoid neoplasms

from East India. Material and methods: All patients that were

referred for karyotyping at Tata Medical Center, from August 2011 to

July 2012 were studied. The bone marrow findings and diagnosis was

recorded. G banded karyotypes were reported as per the ISCN.

Results: A total 221 bone marrow samples were studied. The turn-

over time ranged from 2 to 10 days with a median TAT of 5 days. The

disease distribution was as follows 108 patients with Acute Lym-

phoblastic leukemia (ALL),53 with acute myeloid leukemia

(AML),22 with myelodysplastic syndrome (MDS),16 with Non

hodgkins lymphomas(NHL), 10 with myeloproliferative neoplasms

(MPD), 9 with chronic myeloid lelukemia (CML), and 3 with


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multiple myeloma(MM). Metaphases were available in 212 cases

with a success rate of 96.5 %. Among the ALL patients 65 % had

abnormal karyotypes, hyperdiploidy being the commonest abnor-

mality in children (21/72, 30 %) and t(9;22) in adults(9/36, 25 %).

The other cytogenetic abnormalities seen were MLL gene rear-

rangements (3 %), hypodiploidy (2 %),t(1;19)(3 %), del6q(6 %) and

t(12;21)(2 %). The following cytogenetic abnormalities were seen in

patients with AML (58 % abnormal Karyotypes): t(15;17) in 7

patients, (8;21) in 5,trisomy 8 in 3,3q abnormalities in 3,monosomy 7

in 4,del 9q in 2, MLL gene rearrangements in 1 and complex

karyotype in three. Abnormal karyotypes were seen in 32, 62 and

30 % of patients with MDS,NHL and MM. Conclusion: The inci-

dence of hyperdiploidy in paediatric ALL patients is similar to what

has been reported in western literature and higher than some reports

from India. The incidence of t(9;22) in adult ALL,t(8;21) and t(15;17)

in AML is similar to what has been reported from India and west.

Abstract P 205

Jumping Translocations in AML: A Role in LeukemogenesisAlong with Tumor Progression

M. Parihar1, A. Gupta1, A. Yadav1, S. R. Arun1, A.Bhattacharyya2, D. K. Mishra1, M. Chandy3

1Departments of Cytogenetics and Lab Hematology; 2Paediatric


India

Background: Jumping translocations (JT) are rare cytogenetic phe-

nomenon resulting from translocation of the same segment of the

donor chromosome on to various recipient chromosomes creating

multiple related clones. Previous reports of JT in myeloid malig-

nancies have implicated its role in disease progression in patients with

myelodysplastic syndrome or myeloproliferative disorders that evolve

into acute myeloid leukemia (AML). We present a rare case of JT in a

1 year old female with de novo AML M5. Case report: The patient

presented with fever and purulent discharge from the left ear. On

examination pallor, facial edema, proptosis of the right eye, mass in

the maxillary region and hepatosplenomegaly were seen. Investiga-

tions revealed low hemoglobin, normal white blood cell count and

low platelet counts. There were no blasts in the peripheral blood. The

bone marrow was completely replaced by blasts having morphology

akin to monoblasts with expression of CD13, CD33, CD34, HLA-DR,

CD117, CD64, CD4 and MPO negativity on immunophenotyping.

Karytotyping showed a JT with the long arm of chromosome 1 (1q12)

as the donor fragment and short arms of chromosome 12 (12p13) and

13 (13p12) as the recipient chromosomes in 24 and 5 metaphases

respectively. The patient died within few days of admission. Dis-cussion: The most commonly involved chromosomal segment in JT

in myeloid malignancies is 1q and is associated with a poor prognosis.

However its presence here in a de novo pediatric AML indicates its

role in leukemogenesis unlike previous reports that have implicated

its role only in tumor progression.

Abstract P 206

Loss of Y Chromosome in Acute Lymphoblastic Leukemia:Constitutional or Neoplastic

S. R. Arun1, M. Parihar1, A. Gupta1, A. Yadav1,A. Bhattacharyya2, D. K. Mishra1, M. Chandy3

1Departments of Cytogenetics and Lab Hematology; 2Paediatric


India

Introduction: Loss of chromosome Y chromosome is a well estab-

lished cytogenetic abnormality in myeloid neoplasms and has been

reported with varying frequency ranging from 6.3 to 16.4 % in var-

ious hematological disorders. There have been no reports of a

neoplasia associated loss of Y in acute lymphoblastic leukemia (ALL)

and occasional reports in adult ALL have suggested it to be an age

related phenomenon rather than a neoplastic one. We report a case of

Philadelphia positive mixed phenotype ALL with loss of Y as a

secondary associated abnormality. Case Report: The patient a 4 year

old male child presented with a history of low back ache, bone pain,

and inability to walk. Systemic examination showed hepatospleno-

megaly. His blood counts revealed low hemoglobin, raised total

leucocyte count with 57 % blasts and low platelets. Bone marrow

examination showed 72 % L1 lymphoblasts. Immunophenotyping

showed mixed lineage phenotype with bright expression of CD10,

CD19, CD34, cCD22, and cCD3, TdT, HLA-DR and aberrant CD13.

Cytogenetic analysis (karyotype and FISH) showed two clones,one

showing t(9;22) with additional philadelphia chromosome (ph) and

loss of Y chromosome in 16 metaphases and the other showing t(9;22)

with t(1;19) in 4 metaphases. Phytohaemagglutinin stimulated

peripheral blood cultures showed a normal karyotype ruling out

constitutional loss of Y chromosome. Conclusion: Loss of Y chro-

mosome can be seen in ALL as a part of genetic evolution. The other

interesting findings were presence of additional Philadelphia chro-

mosome and t(1;19) as secondary abnormalities in genetic evolution

in a case of t(9;22) ALL.

Dr. J.C. Patel Award Session

Abstract P 207

RNA Expression of Genes Involved in Cytarabine Metabolismand Transport is an Independent Predictor of Ex-vivo CytarabineResponse in Acute Myeloid Leukemia

Ajay Abraham, Savitha Varatharajan, J Ashok Kumar, K Sreeja,Vivi M Srivastava, RV Shaji, Rayaz Ahmed, Aby Abraham, BijuGeorge, Mammen Chandy, Alok Srivastava, Vikram Mathewsand Poonkuzhali Balasubramanian


Abstract: Wide inter-individual variation in terms of treatment

outcome exists among AML patients receiving chemotherapy with

cytarabine (Ara-C) and daunorubicin. The pre-requisite for the

cytotoxic action of Ara-C is its enzymatic conversion to active tri-

phosphorylated form Ara-CTP. The ex vivo cytotoxicity to Ara-C

in AML patients exhibited a wide inter individual variation, and

were stratified into 3 groups based on IC-50 values

(\5.4 lM, [5.4 lM and [80 lM of Ara-C respectively). DCK

and hENT1 RNA levels were significantly higher in Ara-C

ex vivo sensitive samples compared to those with intermediate

sensitivity and resistant patients. Ara-C resistance index (RI) was

proposed based on candidate gene expression data where,

RI = DCT (dCK 9 ENT1)/DCT CDA. RI values were signifi-

cantly higher in resistant and intermediately sensitive compared to

sensitive samples (median 5.428 and 5.278 vs. 3.475; p \ 0.0001).

The median RI for patients who presented at relapse was signif-

icantly higher compared to that of ex vivo sensitive and resistant

samples at diagnosis. Molecular markers including FLT3-ITD,

NPM mutation, AML-ETO and inv16, as well as MN1, BAALC,

ERG1 and CD34 expression, did not show any significant asso-

ciation with Ara-C RI or IC50. This study suggests RI as an

independent predictor of ex vivo Ara-C sensitivity irrespective of

cytogenetic and molecular risk factors.


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Abstract P 208

Comparative Evaluation of the Coagulation Parameters Beforeand After Chemotherapy and/Radiotherapy in Solid Tumours

Supriya Dhar1, Swapan Kumar Sinha2, Pranab Kumar Biswas2,Shibashish Bhattacharya3

1Department of Transfusion Medicine, Tata Medical center, Kolkata;2Department of Pathology, Medical College, Kolkata; 3Department of

Oncology, Medical College, Kolkata

Objective: To evaluate the coagulation abnormalities in patients with

solid tumors and changes in their coagulation profile following che-

motherapy and or radiotherapy. Materials and Methods: A

prospective observational study was carried out in the Department of

Pathology, Medical College, and Kolkata. Forty one patients between

15 and 70 years with solid neoplasm were evaluated prior to therapy

and following treatment with radiotherapy and or chemotherapy

during the period between November 2010–November 2011. Results:In our study 14 % of cervical cancer patients showed prolonged PT

and APTT before therapy which normalized after therapy. Around

62 % of breast cancer patients showed prolonged PT and APTT

before therapy. After therapy, 37.5 % showed prolonged PT and 12 %

show shortening of APTT. Around 44 % lung cancer patients showed

pre therapy increased PT and APTT. After therapy both PT and APTT

were shortened in 33.3 and 44 % cases respectively. No coagulation

abnormality was seen in gastric cancer patients. Around 44 % lung

cancer patients had increased FDP levels before therapy which nor-

malised after therapy. In cervical cancer patients 14.2 % showed

increased FDP and all cases normalised after therapy. Plasma FDP

level was normal in all gastric cancer patients. All ovarian cancer

patients had increased pre therapy FDP and all cases normalized with

therapy. 62.5 % breast cancer patient had increased FDP which came

down to 12.5 % after therapy. Conclusions: Thus prolongation of PT

and APTT was seen in many untreated solid tumor patients, after

therapy few normalized, some showed shortening while others remain

prolonged. Pre therapy there was a 34 % increase in FDP which came

down to 2.4 % after therapy.

Abstract P 209

Indian MDS: Do Molecular Abnormalities Alter its Biology:AIIMS Experience

Rekha Chaubey, Sudha Sazawal, Manoranjan Mahapatra,Renu Saxena


New Delhi-110029

Background: MDS in the Western countries are mainly found in the

elderly population but is being increasingly seen in young adults in

India. Also, the Indian patients are more severe at presentation and the

response to treatment in these patients is poorer than West. There may

be some molecular factors that may be responsible for the poor

prognosis of these patients. The present study is an attempt to look for

its effect on biology of Indian patients. Aim: To study the effect of

molecular factors on the biology of Indian MDS patients. Materialand Methods: Cytogentics, molecular mutations of RAS and FLT3

gene, hTERT expression, telomerase activity and methylation of

TSGs were analyzed in 100 MDS patients and correlated with disease

severity, progression and survival. Results: There were 67 % male

and 33 % female patients with Mean age at presentation 46 years and

median 48 years. The frequency of patients with age \60 years was

high as compared to the patients with age C60 years (75 vs. 25 %).

26/51 (51 %) patients had normal cytogenetics and 25/51 (49 %)

patients had chromosomal abnormalities. The frequency of N-RAS

mutation was 3 % and K-RAS mutation was 9 %. FLT3-LM and

FLT3-TKD-mutations were not observed at all. The increased telo-

merase activity (TA) was found in 17/100 (17 %) cases. hTERT

expression was present in 17/100 (17 %) cases. Forty patients (40 %)

had p15 INK4b gene methylation. Fifty three patients (53 %) had

SOCS-1 gene methylated. Forty three patients (43 %) had FHIT gene

methylated. Fifty eight patients (58 %) showed calcitonin gene

methylation. In multivariate analysis, of all the molecular factors

studied, only p15INK4b gene methylation was found as an important

predictor for progression of disease in MDS patients. Conclusion:The Indian patients showed some difference from the patients from

West in terms of molecular factors like rare chromosomal aberrations,

absence of FLT3 gene mutations, increased telomerase activity cor-

related with increased hTERT expression, and in methylation

pattern.


Immunohistochemical Profile and Bone Marrow Angiogenesisin Multiple Myeloma with Reference to its Clinical Significance

Sarah Grace Priyadarshini, Debdatta Basu, Rakhee Kar,TK Dutta1

Departments of Pathology and Medicine1, Jawaharlal Institute of

Postgraduate Medical Education and Research, Puducherry

Aims of the Study: To study the histomorphological features of bone

marrow in multiple myeloma and to evaluate the immunohisto-

chemical profile and angiogenesis using a panel of markers including

CD38, kappa and lambda light chain, CD56, Cyclin D1, Ki67 and CD

34 and to correlate immuno-hematological profile with various clin-

ical parameters. Materials and Methods: The study includes 48

cases of myeloma diagnosed over the period of 5 years. The histo-

morphological features like plasma cell morphology, percentage of

plasma cells and pattern of infiltration were studied. Angiogenesis

was assessed by calculating the microvessel density (MVD) using

immunohistochemistry for CD34. Proliferation was assessed using

both Ki67 and CD38 highlighted cells. Immunohistochemistry for

CD56 and Cyclin D1 was also done. Results: A significant associa-

tion was seen between the plasma cell morphology and the pattern of

infiltration (p = 0.0067). The percentage of plasma cells showed a

significant association with the clinical staging. The MVD was sig-

nificantly associated with plasma cell morphology (p = 0.04) as well

as pattern of infiltration (p \ 0.001). The proliferation index was also

significantly associated with the plasma cell morphology (p = 0.003).

Both the Ki67 and MVD showed an increasing trend with clinical

staging and MVD was significantly associated with serum albumin

(p = 0.02). CD56 negativity was associated with circulating plasma

cells and also with higher clinical stage. Cyclin D1 positivity was not

seen in poorly differentiated morphology and the mean Ki67 was

lower in the cyclin D1 positive group. It was also found that the cases

with kappa light chain restriction had significantly less of poorly

differentiated morphology, diffuse pattern of infiltration, lower MVD

and Ki67. Conclusion: The study of bone marrow morphology and

angiogenesis with the aid of immunohistochemistry is useful for

prognosticating patients with multiple myeloma.


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