Influence of tissue factor polymorphisms (603A>G and 5466A>G) on plasma tissue factor levels and their impact on deep vein thrombosis risk in young Indian population.

Deep vein thrombosis (DVT) is multifactorial disorder and well known to cause substantial morbidity and mortality. There is sparse data in the Asian population, particularly India regarding association of tissue factor (TF) gene single nucleotide polymorphisms (SNPs) with plasma TF levels in DVT. So, we analyzed the distribution of SNPs (603A>G and 5466A>G) in India, to evaluate their effect on TF levels in DVT patients. Plasma level and SNPs (603A>G and 5466A>G) of TF gene were screened in subjects (100 DVT patients and 100 controls). Patients had significantly higher TF levels than controls (patients: 84.95 ± 17.16 pg/ml, controls: 70.55 ± 15.87 pg/ml, p < 0.001). G allele of 603A>G polymorphism was significantly higher in patients than controls (patients: 40.5% controls: 27.5%, p = 0.004). Subjects with AG and GG genotype had significantly higher TF levels than AA genotype (p = 0.001). After multiple logistic regression analysis, risk of DVT was increased 1.398 fold (95% CI 0.738-2.651) and 4.41 fold (95% CI 1.404-13.884) with AG and GG genotype respectively. Allelic and genotypic frequencies of 5466A>G polymorphism was neither associated with TF levels nor with DVT. We found high TF level in patients with TF 603A>G polymorphism, which is an important predisposing factor in increasing risk of DVT in young Indians. Furthermore, GG genotype of 603A>G polymorphism augments the risk of thrombosis by 4.4 fold, thus highlighting the significance of this polymorphism in the development of DVT. So, we suggest that inclusion of 603A>G polymorphism in prothrombotic work-up may be helpful in making the treatment strategy in DVT patients.

Lymphoma in Rheumatoid Arthritis - Catastrophic Sequela of a Common Disease.

Rheumatoid arthritis (RA) is a common rheumatological condition affecting the joints and has a wide range of extra-articular manifestations. A 69 year old male, known case of rheumatoid arthritis presented to our OPD with right lower limb redness and swelling, and left axillary lymph node swelling. Lymph node biopsy revealed a high grade diffuse large B-cell lymphoma with co-expression of c-myc and bcl-2 (double expressor). RA increases the risk of both Hodgkin's lymphoma (HL) and non-Hodgkin's Lymphoma (NHL). The association with diffuse large B-cell lymphoma (DLBCL) has been found to be particularly strong, however double expressor DLBCL is an extremely uncommon occurrence. High disease activity of rheumatoid arthritis is a major determinant in development of lymphomas.

Value of CD16/CD66b/CD45 in comparison to CD55/CD59/CD45 in diagnosis of paroxysmal nocturnal haemoglobinuria: An Indian experience.

BACKGROUND & OBJECTIVES: Diagnosis of paroxysmal nocturnal haemoglobinuria (PNH), a rare haematopoietic stem cell disorder, is challenging in patients with bone marrow failure (BMF) syndrome like aplastic anaemia (AA). This study was conducted with the aim to test the efficacy of the newly recommended markers viz. anti-CD16 and CD66b antibody over the existing anti-CD55 and CD59 antibody for PNH diagnosis in India. METHODS: This study was conducted on 193 suspected cases of PNH by flow cytometry using lyse wash technique to stain the granulocytes with CD16/CD66b and CD55/CD59. RESULTS: Of the 193 suspected cases, 62 patients showed the presence of PNH clone. Forty six patients were detected by CD55/CD59/CD45, whereas 61 were detected by CD16/CD66b/CD45. CD16/CD66b detected 16 (25.8%) additional patients over CD55/CD59 (P<0.05) and was more sensitive in detecting the PNH clone with higher negative predictive value. Most of the patients (11/16) who were picked up by CD16/CD66b were of AA who had small clone sizes. Further, the PNH clones were more discreetly identified in CD16/CD66b plots than by CD55/CD59. Clone size assessed by CD16/CD66b which reflects the clinical severity of classical PNH (thrombosis/haemolysis), was more representative of the underlying clinical condition than CD55/59. INTERPRETATION & CONCLUSIONS: In our experience of 62 patients of PNH, CD16/CD66b proved to be more efficacious in detecting PNH. The new panel was especially useful in monitoring PNH associated with BMF which had small clone sizes.

Chronic myeloid leukemia with a rare fusion transcript, e19a2 BCR-ABL1: A report of three cases from India.

The µ-bcr breakpoint connects exon 19 of BCR with ABL giving rise to the e19a2 transcript corresponding to the p230 fusion protein (micro-BCR breakpoint) which is rarely seen in chronic myeloid leukemia (CML) patients. Here we report three patients with p230 fusion protein presenting with different clinical presentations and diagnosed as CML-CP. These patients received Imatinib (tyrosine kinase inhibitor-TKI) and are still in remission.

Dyskeratosis Congenita with Acute Myeloid Leukemia, Cryptogenic Liver Fibrosis and Portal Hypertension.

Dyskeratosis Congenita (DC), a 100-year-old known rare hereditary entity, has recently changed its definition as per the pathogenetic model in the last decade. Now it is well known as one of the telomeropathies, pathognomonically characterized by a triad of reticulate pigmentation of the skin, nail dystrophy, and mucosal leukoplakia. It is a progressive systemic disorder which usually presents with involvement of several family members. Malignancies are increasingly reported. Clinical diagnosis is simple once there is a suspicion, but nowadays genetic diagnosis is advocated. Treatment is symptomatic and organ-oriented. We hereby report an adolescent male who presented with the classical mucocutaneous triad of DC with pancytopenia for four months. Bone marrow examination later revealed evolution of acute myeloid leukemia (AML). Liver function tests, imaging, and liver biopsy showed cryptogenic fibrosis with portal hypertension. Chemotherapy was started since hematopoietic stem cell transplantation was not feasible; however, he died very early due to repeated infections before completion of the treatment. AML and liver disease are increasingly reported independently in DC; however, coexistence of both complications in a single patient at first presentation has never been reported earlier. Early age onset of AML is noticeable too.

Ocular graft versus host disease in allogenic haematopoetic stem cell transplantation in a tertiary care centre in India.

BACKGROUND & OBJECTIVES: This study was aimed to report the occurrence of ocular graft versus host disease (oGVHD) in allogeneic haematopoietic stem cell transplantation (allo-HSCT) patients in a tertiary care hospital setting. METHODS: A cross-sectional study of ocular surface of allo-HSCT patients was done. Slit lamp biomicroscopy, symptom score, tear meniscus height, fluorescein tear break-up time, Schirmer's test I, ocular surface staining, dry eye severity, ocular surface disease index score were done. Indications for allo-HSCT, human leukocyte antigen (HLA) matching, GVHD risk factor, systemic manifestation and treatment were also noted. RESULTS: GVHD occurred in 44.4 per cent of 54 allo-HSCT patients (mean age 26.7 ± 12 yr) included in the study. GVHD risk factors identified included female gender, relapse, older age of donor, cytomagelo virus (CMV) reactivation, and multiparous female donors. oGVHD was noted in 31.5 per cent with mean time to occurrence being 17.8 ± 21.9 months after the allo-HSCT and was observed in 89.5 per cent of chronic GVHD cases. Acute GVHD (oral and dermatological) involvement showed a significant association with GVHD in our patients (P< 0.001, 0R 23.0, CI 6.4-82.1). Chronic GVHD was observed to be associated with the occurrence of oGVHD (dry eye) (P<0.001, OR = 24.0, CI 0.02 - 0.29). Of the 34 eyes with oGHVD, dry eye of level 3 severity was seen in 16, level 2 in six, level 1 in 12 eyes. INTERPRETATION & CONCLUSIONS: GVHD occurred in 44.4 per cent of the patients studied in the present study. Acute and chronic GVHD showed a strong association with oGVHD. Dry eye disease due to chronic oGVHD was observed in 17 (31.5%) of 54 allo-HSCT patient with chronic oGVHD occurring in 17 (89.4%) of chronic GVHD cases in allo-HSCT patients. Our study on oGVHD in post allo-HSCT patients in tertiary care centre points towards the fact that ocular morbidity due to dry eye disease as a result of oGVHD is a cause for concern in these patients.

C1236T polymorphism in MDR1 gene correlates with therapeutic response to imatinib mesylate in Indian patients with chronic myeloid leukaemia.

Patients with chronic myeloid leukaemia show an excellent response to treatment with imatinib. However, in some patients, the disease is resistant to imatinib. This resistance may be related to the presence of genetic variations on the drug's pharmacokinetics and metabolism. We therefore studied three polymorphisms (C1236T, G2677T and C3435T) in the human multidrug-resistance gene (MDR1) in 86 patients with chronic myeloid leukaemia treated with imatinib. Imatinib resistance was more frequent in patients with TT genotype at locus 1236 than in those with CT/CC genotypes (p=0.003). For the other two loci (G2677T and C3435T), resistance was seen to be higher for TT genotype when compared to GG/GT and CT/CC but it was not statistically significant (p=0.13 and p=0.099). In conclusion, determination of C1236T MDR1 genotype may help to predict response to imatinib therapy in patients with chronic myeloid leukaemia.

Radiological presentation of transient perivascular inflammation of carotid artery syndrome in a patient with myelodysplasia.

Transient Perivascular Inflammation of Carotid artery syndrome (TIPIC syndrome) is a non-specific inflammatory thickening of the carotid artery. The exact etiology of this syndrome is poorly understood. We will describe the radiological findings of a rare case of TIPIC syndrome in a patient with myelodysplastic syndrome and discuss the potential pathophysiological mechanisms.

Evaluation of Diagnostic Usefulness of CD200 Expression in B-cell Chronic Lymphoproliferative Disorders.

Immunophenotyping by flow cytometry (FCM) is a useful diagnostic tool for the evaluation of mature B-cell neoplasms (MBN). Here, CD200 expression may play a significant role and improve the distinction between various MBNs, but any potential as a prognostic marker is yet to be established. The present prospective study was conducted on all the suspected cases of MBNs. Immunophenotyping was done using a BD FACS Canto FCM using a panel of 4 to 6 color combinations of monoclonal antibodies; CD45, CD34, CD5, CD19, CD20, CD22, CD23, CD79b, FMC7, CD10, CD38, ZAP70, CD200, IgG, IgM, CD25, CD103, CD2, CD3, CD11c as well as κ and λ light chains. CD200 expression was compared in different subgroups. Of the total of 130 cases included in the study, CD200 was positive in 118 cases (90%). CD200 was expressed in 100% of the cases of CLL(86 cases), atypical CLL(06 cases), HCL(14 cases), FL(02 cases), SMZL(04 cases), LPL (01 case), and low-grade NHL (05 cases), with the highest intensity of fluorescence in HCL followed by CLL. All the cases of MCL and PLL were exclusively negative for CD200. In conclusion, the results of the present study support inclusion of this marker in the flow cytometric panels for the differential diagnosis of MBNs.

Flow cytometry in myelodysplastic syndrome: analysis of diagnostic utility using maturation pattern-based and quantitative approaches.

Flow cytometry (FCM) is being increasingly evaluated for the diagnosis of myelodysplastic syndrome (MDS). We employed multiple FCM approaches to assess MDS. Five-color FCM, morphology blind, was done on bone marrow aspirates of 57 suspected MDS and 31 normal controls. Maturation pattern, quantitative FCM for low-grade MDS that awards FCM score, and expression of selected antigens on erythroid cells and CD34(+) blasts were evaluated. FCM results were correlated with clinical and laboratory workup. Patients (n = 57) included proven MDS (n = 14), suspected MDS (n = 13), and non-MDS (n = 30). By pattern-based approach, all proven cases were FCM positive. In suspected MDS, 11 (84.61 %) were positive including morphology-negative cases, and two (15.38 %) were intermediate. In non-MDS cases, 27 of 30 (90 %) were FCM negative, 2 of 30 (6.67 %) intermediate, and 1 of 30 (3.33 %) a hematinic-responsive case, positive. Quantitative parameters that characterized MDS included FCM score of >3, percentage CD34(+) B cells, and expression of CD11b, CD15, and CD56 on myeloblasts. CD71 MFI on CD235a(+) erythroblasts and CD38 MFI on myeloblasts were significantly lower in MDS. The former was present in FCM-intermediate suspected MDS but not FCM-intermediate non-MDS cases. Used in the overall clinical context, both maturation pattern recognition and quantitative approaches, the latter for low-grade MDS, are sensitive methods of diagnosing MDS, including cases negative by morphology and cytogenetics, especially if combined with evaluation of selected antigens, CD71 on CD235a(+) cells and CD38 on CD34(+) cells. The value of FCM in morphology-negative cases needs better definition of specificity through more extensive evaluation of secondary dyspoiesis.

Pulseless right upper limb: an unusual manifestation of invasive pulmonary aspergillosis in acute myeloid leukemia.

Aspergillus is the most common cause of fungal pneumonia in acute leukemia patients receiving chemotherapy or undergoing hematopoietic stem cell transplantation. Despite a high index of suspicion and prompt institution of specific antifungal therapy, it causes significant morbidity and mortality in patients with hematological malignancies. It has to be differentiated from mucormycosis because the treatment differs. Histological confirmation obtained by lung biopsy is ideal, but is difficult to obtain in those patients who often have thrombocytopenia. We report a case of acute megakaryoblastic leukemia with typical manifestations of invasive pulmonary aspergillosis who developed pulseless right arm due to invasion of the right subclavian artery. When total leucocyte counts recovered, patient also developed immune reconstitution inflammatory syndrome and massive pulmonary hemorrhage, which was managed by bronchial artery embolization.

Towards a unified approach for multiple myeloma care in Kenya - proceedings of the Inaugural Multiple Myeloma Congress.

The rising burden of multiple myeloma in Kenya has not been met by a commensurate effort for control. Patients and practitioners struggle with unavailability and unaffordability of diagnostics, drugs and stem cell transplant leading to presentation at advanced stages and under-treatment with increased morbidities and mortality. A concerted effort among stakeholders is urgently needed to develop strategies for myeloma control. The scarcity of providers also carries grave consequences for Kenyan patients. The Academic Model Providing Access To Healthcare (AMPATH) multiple myeloma program organized the Inaugural Virtual Multiple Myeloma Congress to achieve both interactive specialist instruction and stakeholder engagement. Expert presenters and panellists from diverse disciplines were invited to offer in-depth presentations on myeloma care and case studies from panellists´ practice were used to contextualize learning points and form a basis for generating debate on the challenges facing providers and opportunities for care improvement. An audience of health professionals offering care to myeloma patients was invited. The underlying principle of recommendations developed during the congress was collaboration among in-country and international practitioners, researchers and policy experts from private and public sector. This partnership of stakeholders bears the potential of pooling scarce resources and for collective advocacy towards better patient care.

High dose dexamethasone therapy shows better responses in acute immune thrombocytopenia than in chronic immune thrombocytopenia.

Oral high dose dexamethasone (HDD) was given as a single daily dose for four consecutive days, every 14 days for four courses. Twenty-nine patients were enrolled. Overall 20 patients (69%) responded: complete response (CR) was achieved in 16 (55%) patients, partial response (PR) in three (10%) patients and MR in one (3%) patient. In acute immune thrombocytopenic purpura (ITP) response rates after the first, second, third and fourth cycles were as follows: 64% (9/14), 64% (9/14), 79% (11/14), and 85.7% (12/14), respectively. In chronic ITP, overall response rates after the first, second, third and fourth cycles were as follows: 33% (5/15), 40% (6/15), 53% (8/15) and 53% (8/15) respectively. The median time to response was 14 days (4-42 days). Twelve out of 20 patients (5/12 acute ITP and 7/8 chronic ITP) relapsed; median relapse free survival till last follow-up in the remaining eight patients was 130 days (65-365 days).

Efficacy of immunization against hepatitis B virus infection in acute leukemia.

OBJECTIVE: The aim of this study was to assess the antibody response to combined passive-active immunization versus active immunization against hepatitis B in 71 patients with acute leukemia with negative hepatitis B virus serology at presentation. METHODS: The first group (n=28) received a double dose of hepatitis B vaccine at 0, 1, 2 and 6 months and immunoglobin (HBIG) at 0 and 1 month concurrently with vaccine but at a different intramuscular site. The second group (n=43) received double dose of hepatitis B vaccine at 0, 1, 2, and 6 months. HBsAg and anti-HBs titers were determined one month after the 1st, 2nd, 3rd and 4th doses of vaccine. ESULTS: In the vaccine-only group, 2.56%, 8.33%, 14.28% and 34.29% of patients developed anti-HBs titer ≥10 IU/L after the 1st, 2nd, 3rd and 4th doses of vaccine, respectively. In the HBIG group, 91.30%, 91.30%, 69.56% and 73.91% of patients developed anti-HBs titer ≥10 IU/L after the 1st, 2nd, 3rd and 4th doses of vaccine, respectively. Those in the vaccine-HBIG group maintained their anti-HBs titer ≥10 IU/L from the 1st to the 4th doses. In the vaccine-only group, 34.29% of patients gained protective antibody titer after receiving the 4th dose of vaccine. Subgroup analysis of age (pediatric vs adult) and disease (acute lymphoblastic leukemia vs acute myeloid leukemia) groups showed no effect of either on the development of protective antibody titer. The incidence of HBsAg positivity one month after the 4th dose of vaccine was 8.62%. No patient became positive for anti-HCV or HIV antibody before or after chemo therapy. CONCLUSION: Combined HBIG and vaccine may protect acute leukemia patients during the intensive chemotherapy period.

Intracranial hemorrhage in childhood immune thrombocytopenic purpura.

We retrospectively analyzed 750 patients with ITP for development of intracranial hemorrhage (ICH). Seventeen cases with age range of 10 months to 18 years were studied. Ten patients were of acute ITP and seven had chronic ITP. Nine patients developed ICH one month after the onset of ITP and five patients had ICH on presentation. ICH was precipitated by trauma in four patients and possibly the use of NSAIDs in one patient. Median platelets counts at the time of ICH were 12 x 10(9)/L (range 2-50 x 10(9)/L). Most patients were treated with corticosteroids. Four patients (24%) died due to ICH.

Patterns of acquired bleeding disorders in a tertiary care hospital.

Bleeding disorders constitute a large proportion of referrals to hematology departments. Worldwide, acquired causes of bleeding are commoner than inherited ones. To identify the spectrum of these disorders, we evaluated all referrals for bleeding encountered in this tertiary care centre over a one-year period. Of the total 1342 cases, 1040 (77.5%) had underlying exclusively acquired causes, whereas inherited causes constituted 302 cases (22.5%). Amongst acquired causes, disseminated intravascular coagulation was seen in 297 (28.6%), hepatic coagulopathy in 218 (20.9%), neurosurgical causes (intracranial bleeds) in 154 (14.8%), malignancy in 89 (8.6%), and miscellaneous multiple acquired causes including those due to anticoagulant drug overdose in 282 patients (27.1%). Referrals for isolated prolonged prothrombin time or thrombocytopenia were common, but were excluded from this study because not all presented with bleeding. Prompt laboratory work-up and precise identification of acquired causes of bleeding is the key to planning appropriate patient management including transfusion support.

Role of CD138, CD56, and light chain immunohistochemistry in suspected and diagnosed plasma cell myeloma: A prospective study.

INTRODUCTION: Plasma cells (PCs) have conventionally been counted on the bone marrow aspirate, and small focal involvement may be missed even on bone marrow biopsy sections. MATERIAL AND METHODS: We aimed to study the role of CD138, CD56, anti-κ, and anti-λ immunohistochemistry (IHC) to separate PC myeloma from reactive plasmacytosis and to study the utility of these in cases suspected as myelomas and lacking >10% PCs on bone marrow aspirate. The study comprised 35 diagnosed myelomas, 20 reactive plasmacytosis, and 19 M-band positive suspected myelomas. CD138 IHC was performed on all cases along with CD56, anti-κ, and anti-λ IHC. PCs were counted on CD138-immunostained sections by manual count and by image analysis. In addition, CD56 expression was correlated with clinical features in diagnosed myeloma group. RESULTS: In all cases, both manual counts and image analysis, PC counts were significantly higher on the CD138 stained sections than bone marrow aspirates. It was seen that the manual PC counts and image analysis counts were equivalent in diagnosed myeloma cases. CD56 expression was seen in ~62.85% diagnosed myeloma cases while it was negative in cases of reactive plasmacytosis. CD56 expression was significantly higher in patients with lytic lesions (78.26% vs. 21.74%). CD138, anti-κ, and anti-λ IHC also helped classify 11/19 (57.8%) cases correctly. CONCLUSION: The use of CD138 along with the light chain and CD56 IHC adds a high diagnostic value in myeloma patients and suspected myeloma cases. The PCs can be counted manually on the CD138-immunostained sections and correlate well with the counts obtained by image analysis.

T Regulatory Cells in Donor Grafts May Predict the Severity of Acute Graft Versus Host Disease After Matched Sibling Donor Allogenic Peripheral Blood Stem Cell Transplantation.

Acute graft-versus-host disease (aGVHD) and relapse are major issues for patients undergoing allogenic hematopoietic stem cell transplant (allo-HSCT). T-regulatory (Treg) cells in the donor graft are negatively correlated with the incidence of aGVHD without any impact on relapse. In this study to determine the association of Treg cells with aGVHD in allo-HSCT patients. Thirty-two patients with hematological disorders, who underwent allo-HSCT. Twenty-nine patients who achieved engraftment were enrolled in the study. Treg cells were quantified in donor graft by flowcytometry and were assessed for their association with aGVHD and other clinical outcomes. Fifteen of 29 patients developed aGVHD. According to the occurrence and severity of aGVHD, patients were divided into two groups: 20 (68.9%) patients with grade 0-I aGVHD and 9 (31.1%) patients with grade II-IV aGVHD. Treg cells/CD4 ratio was significantly higher in the grade 0-I aGVHD group than in grade II-IV aGVHD group, (p = 0.0002). We could not find the association of CD34 dose (p = 0.55) or CD3 dose (p = 0.57) with the severity of aGVHD. Higher Treg cells/CD4 ratio in donor graft was associated with less severe aGVHD. Though more studies are needed, Treg cells/CD4 ratio may be used as a predictive marker for severity of aGVHD in post allo-HSCT.

Flow cytometric analysis of patients with hereditary spherocytosis - an Indian scenario.

OBJECTIVES: Flow cytometry osmotic fragility test (FC-OFT) was a recently introduced screening test for hereditary spherocytosis (HS). This study was conducted to evaluate the utility of FC-OFT in all newly diagnosed cases of HS, to compare its diagnostic value with conventional OFT and to correlate with clinical disease severity. METHODS: In this study, the percentage of residual red cells (%RRC) was measured using flow cytometer after creating a red cell suspension. Subsequently, this was spiked with deionized water for FC-OFT in all cases of HS (n = 40), healthy subjects (n = 40) and beta-thalassemia traits (BTT) (n = 20). RESULTS: The receiver operator curve analysis defined the optimal cut-offs for FC-OFT-derived indices, such as %RRC value (≤16.29%) and %RRC ratio (>1.72), for HS cases when compared with healthy subjects and BTT (p < 0.05). The FC-OFT (96%) achieved higher test efficiency than the conventional OF test (68.9%). A significant positive and a negative correlation were found between number of spherocytes/hpf and %RRC ratio (p = 0.001) and %RRC values (p = 0.0486). No significant correlation was observed between %RRC value (p = 0.8934), %RRC ratio (p = 0.6348) and HS disease severity score. CONCLUSION: Our results suggest that FC-OFT could be the better screening test for HS cases in developing countries if flow cytometer is available.