Epidemiology, Clinico-Haematological Profile and Management of Aplastic Anaemia: AIIMS Experience.

BACKGROUND: The incidence of aplastic anaemia (AA) is higher in Asia than in the West. The precise incidence of AA in India is not known due to lack of epidemiological study. 20-40% of pancytopenic patients in referral centres are of aplastic anaemia. PATIENTS AND METHODS: This was an analysis of 1501 patients diagnosed with aplastic anaemia over a period of seven and half years (January 2007- June 2014) attending the Aplastic clinic of department of haematology of All India Institute of Medical Sciences, New Delhi. The details regarding medical history, physical examination, complete blood count, bone marrow aspirate and biopsy, treatment received, were retrieved. Inherited bone marrow failure was screened in patients below 35 years. Treatment response was analysed for various treatment modalities. RESULTS: 1501 patients of AA from 20 different states of India were analysed. The bulk of patients were from Uttar Pradesh (28.7%), Bihar (23.6%), Delhi/NCR (20%) and Haryana (7%).The average number of new aplastic anaemia patients enrolled per year 214 (range: 101 -263). The median age at presentation was 25 years (range 2-83),with M;F - 2.3:1. Severity of AA revealed: severe (SAA): 75%, very severe (VSAA): 15%, non-severe (NSAA): 10%. Inherited bone marrow failure syndromes constituted 5% (75 patients) of all aplastic anaemia patients. The most common clinical presentations were pallor (97%), bleeding manifestations (69.6%) and fever (54%). The haematological parameters showed: median level of haemoglobin level: 5.9 gm/dL, WBC: 2700/mm3, ANC: 380/mm3, platelet: 1 0000/mm3. PNH clone was present in 13.5% of patients. 107 patients (7%) were lost to follow up or expired before any treatment was initiated. Only 69 patients (4.5%) received treatment with HLA-matched sibling stem cell transplantation and another 232 (15.5%) patients received ATG plus cyclosporine as immunosuppressive therapy. Seven hundred thirteenpatients (47.5%) received cyclosporine. The overall response to various treatment modalities was: HLA matched sibling haematopoietic stem cell transplant: 75.3%, Anti-thymocyte globulin plus cyclosporine: 58.7%, cyclosporine plus androgen: 45.6%, cyclosporine alone: 32.2%. CONCLUSION: Management of AA is a real challenge in developing countries.This is one of the largest case series from a single centre from India. It is our endeavour to reduce the detrimental outcome by increasing awareness among patients and referring physicians to reduce the delay between diagnosis and treatment.

Antigenic variation of foot-and-mouth disease virus serotype A.

The current measures to control foot-and-mouth disease (FMD) include vaccination, movement control and slaughter of infected or susceptible animals. One of the difficulties in controlling FMD by vaccination arises due to the substantial diversity found among the seven serotypes of FMD virus (FMDV) and the strains within these serotypes. Therefore, vaccination using a single vaccine strain may not fully cross-protect against all strains within that serotype, and therefore selection of appropriate vaccines requires serological comparison of the field virus and potential vaccine viruses using relationship coefficients (r1 values). Limitations of this approach are that antigenic relationships among field viruses are not addressed, as comparisons are only with potential vaccine virus. Furthermore, inherent variation among vaccine sera may impair reproducibility of one-way relationship scores. Here, we used antigenic cartography to quantify and visualize the antigenic relationships among FMD serotype A viruses, aiming to improve the understanding of FMDV antigenic evolution and the scope and reliability of vaccine matching. Our results suggest that predicting antigenic difference using genetic sequence alone or by geographical location is not currently reliable. We found co-circulating lineages in one region that were genetically similar but antigenically distinct. Nevertheless, by comparing antigenic distances measured from the antigenic maps with the full capsid (P1) sequence, we identified a specific amino acid substitution associated with an antigenic mismatch among field viruses and a commonly used prototype vaccine strain, A22/IRQ/24/64.

Foot-and-mouth disease virus epitope dominance in the antibody response of vaccinated animals.

Five neutralizing antigenic sites have been identified on the surface of serotype O foot-and-mouth disease virus (FMDV). A set of mAb neutralization-escape mutant viruses was used for the first time to evaluate the relative use of known binding sites by polyclonal antibodies from three target species: cattle, sheep and pigs. Antibodies to all five neutralizing antigenic sites were detected in all three species, with most antibodies directed against antigenic site 2, followed by antigenic site 1. In 76 % of cattle, 65 % of sheep and 58 % of pigs, most antibodies were directed against site 2. Antibodies specific to antigenic sites 3, 4 and 5 were found to be minor constituents in the sera of each of the target species. This implies that antigenic site 2 is a dominant neutralization immunogenic site in serotype O FMDV and may therefore be a good candidate for designing novel vaccines.

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).

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.

Efficacy and safety of anti-D for treatment of adults with immune thrombocytopenia.

This study was planned to assess the response of anti-D in patients with immune thrombocytopenia (ITP). Twenty adults (8 males: 12 females) with a median age 33.5 years (19-59 years) were included. Nine patients had newly-diagnosed ITP, 6 had persistent ITP and 5 had chronic ITP. The overall response rate was 65%. Patients with newly diagnosed ITP showed response rates of 77% (7/9), persistent ITP had response rates 50% (3/6) and patients with chronic ITP had response rates of 60% (3/5). The median time to response was 3 days (1-11 days). There was no correlation of response with age, sex, severity of bleeding, presenting platelet counts, ABO blood group or prior steroid or IVIG response.

Utilisation of Flow-cytometry in the Diagnosis of Auto Immune Haemolytic Anaemia.

Auto Immune Haemolytic Anaemia (AIHA) is one of the most common types of acquired haemolytic anaemias. Its main cause is auto-antibody mediated rapid destruction of Red Blood Cells (RBCs). Demonstration of a positive Direct Antiglobulin Test also known as Coomb's test, against these autoantibodies is the crucial serological assay in the diagnosis of AIHA. This routinely used test has the disadvantage of low sensitivity and does not detect low levels of red cell auto antibodies leading to false negative results sometimes. Flow cytometry can effectively diagnose such patients with low levels of autoantibodies. This study was carried out in a tertiary care center, where patients with suspected AIHA were studied during 2 years period. Blood samples of suspected patients of AIHA were tested by both Gel Card Test and by Flow-cytometry for detection of RBC bound IgG. A total of 50 patients with suspected diagnosis of AIHA were studied by flow-cytometry as well as by Gel card test for detection of RBC bound IgG. Out of these 50 cases, 41 cases have turned out to be positive and 9 were negative by flow-cytometry. By Gel card test, out of 50 cases, 34 were positive and 16 were negative. Therefore, there were 7 cases which were negative for RBC bound IgG by Gel card test and these were positive by flow-cytometry. Flow-cytometry is a reliable and more sensitive method and can be used as a new routine diagnostic technique for AIHA.

Intracranial haemorrhage in patients with congenital haemostatic defects.

We investigated 52 of 457 patients with congenital factor deficiencies with 57 episodes of intracranial haemorrhage (ICH) between 1998 and 2007. There were 38 severe haemophiliacs, 6 with factor XIII deficiency, 5 with factor X deficiency, 2 factor V-deficient patients, and 1 with type 3 von Willebrand disease (VWD). The median age was 8 years (range 1 month-22 years). Most patients were below 15 years of age (86.5%). All patients with factor X deficiency were between 1 and 5 months of age. ICH was the primary bleeding episode leading to detection of factor deficiency in 19.2% (five patients with severe haemophilia and all patients with factor X deficiency). Trauma caused bleeding in 66%. None of the patients with factor X deficiency had history of prior trauma. Surgery was performed in five patients with subdural haematomas, all of whom survived. Conservative factor replacement with 100% correction for 3 days followed by 50-60% correction for 7 days was possible in 60% patients. Seizures requiring prolonged therapy were noted in eight patients. Death was recorded in 15 patients (29%). Inadequate therapy in the form of delay or insufficient replacement was noted in 7/15 deaths. ICH was seen in 11.3% of all patients with coagulation factor deficiencies. Factor X deficiency presented with ICH at an earlier age. Inadequate replacement therapy including delayed treatment caused nearly 50% of all deaths. Most patients can be managed satisfactorily with adequate replacement therapy alone, with surgery being reserved for those with worsening neurological conditions.

Fournier's gangrene and scrotal ulcerations during all-trans-retinoic acid therapy for acute promyelocytic leukemia.

Scrotal ulcers are a rare manifestation in patients with acute promyelocytic leukemia. Fournier's gangrene (FG) is even rarer. We describe three adolescents and young adults who developed scrotal ulcerations during induction with all-trans-retinoic acid. One patient developed FG. These lesions are predominantly seen in Asian population. A good outcome with supportive management occurred in all the cases.

Evaluation of a monoclonal antibody-based approach for the selection of foot-and-mouth disease (FMD) vaccine strains.

Foot-and-mouth disease (FMD) virus exists as seven serotypes within which are numerous variants necessitating careful selection of vaccine strains. Currently, a serological assay system based on the use of polyclonal vaccine antisera is widely used for this selection. However, inherent variability in the matching antisera used makes the tests poorly reproducible and difficult to interpret. In this study, we have explored the possibility of replacing or supplementing the polyclonal antibody (PAb)-based method with one based on use of monoclonal antibodies (MAb). Panels of MAbs raised against two serotype O vaccine strains were examined for reactivity with 22 field viruses, isolated over a 10-year period between 1991 and 2001. Antigenic site 2 was found to comprise more than one epitope. The sequence variation in capsid protein VP2 harbouring antigenic site 2 was analysed and the amino acid residues at positions 79 and 134 appeared to greatly influence the binding of site 2 MAbs. Prediction of antigenic match based on MAb reactivity did not correlate closely with the results of a PAb-based "gold-standard" method and it was concluded that a wider panel of MAbs are needed that recognise all protective epitopes present on the surface of FMD virus together with a better understanding of those epitopes which are important in conferring protection.

Hairy cell leukemia: clinical, pathological and ultrastructural findings in Asian-Indians.

BACKGROUND: Hairy-cell leukemia (HCL), lymphoproliferative disease of older age, is characterized by projections from surface of abnormal cells. AIM: The aim was to study the clinical presentation and ultrastructural changes in hairy cells (HCs) following cladribine treatment. SETTINGS AND DESIGN: Clinical presentation, peripheral smear, bone marrow aspiration and biopsy of HCL cases diagnosed over a period of three years were reviewed. MATERIALS AND METHODS: Consecutive HCL cases in Hematology clinic of a tertiary care center were enrolled. Tartarate-resistant acid phosphatase (TRAP) test was done to detect HCs and electron microscopy was done to demonstrate initial ultrastructural changes and alterations following cladribine therapy. RESULTS: Fifteen cases of HCL, aged 32-57 years (median 47 years) were studied. The clinical presentation included splenomegaly in 15 (100%), fever in 10 (67%), hepatomegaly and pain abdomen in eight (53%), fatigue in nine (60 %) cases. The commonest laboratory features were monocytopenia in 13 (87%), neutropenia in 12 (80%), anemia in 10 (67 %) and pancytopenia in nine (60%). All patients showed symptomatic improvement on cladribine therapy. Electron microscopy after treatment (three months) showed loss of the finger like projections, characteristic bald lymphocytes, loss of ribosomal lamellar complexes, as well as decrease in mitochondria and vacuoles. CONCLUSIONS: Indian patients with HCL are younger. Cladribine is an effective therapy for these patients and leads to complete response in most of the patients. There is a significant improvement in the ultrastructural features following cladribine therapy.

Spectrum of hemostatic disorders in Indian females presenting with bleeding manifestations.

INTRODUCTION: Hemostatic disorders are often missed in women with bleeding particularly menorrhagia. Preexisting hemostatic disorders are now known as common risk factor for postpartum hemorrhage and prolonged bleeding in puerperium. Females with bleeding complaints constitute an important population referred to hematology clinic. Hence, we aim to evaluate the type and frequency of hemostatic disorders among females presenting with bleeding in a tertiary care hospital and a basic hemostatic laboratory. METHODS: Three-year data were retrospectively analyzed for 200 females with various bleeding complaints. Due to resource constraints, a hemostatic workup was done with prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen assay, clot solubility test, mixing studies, specific factor assays, platelet function test, and von Willebrand factor antigen level. RESULTS: A total of 200 females were investigated to identify the cause of their bleeding. Thirty-five of 200 (17.5%) females were found with an underlying bleeding disorder. Of these 35 females, 65.7% presented with bleeding from more than 1 site. Most common bleeding manifestation was spontaneous bruising in 18 of 35 (51.4%) patients followed by petechiae (48.6%). Inherited bleeding disorders were noted in majority. The most common inherited bleeding disorder identified was von Willebrand disease (VWD) in 34.3% females. Second most common disorder was Glanzmann's thrombasthenia accounting for 22.8%. Rare coagulation factor deficiency, such as factors VII, X, and XIII deficiencies, was noted. Three cases revealed acquired causes of coagulation defects. CONCLUSION: Underlying hemostatic defects should be searched for in women with unexplained bleeding complaints. This will not only help in diagnosis but also in proper management for future hemostatic challenges.

Paraplegia due to extramedullary hematopoiesis in thalassemia treated successfully with radiation therapy.

Spinal cord compression due to extramedullary hematopoiesis (EMH) is a rare complication of thalassemia and generally presents as paraparesis with sensory impairment. Complete paraplegia is extremely rare in EMH due to thalassemia although it is known to occur in polycythemia vera and sickle cell anemia. Treatment options mostly include surgery and/or radiotherapy. Whereas cases presenting with paraparesis have been treated with either surgery or radiotherapy with equal frequency and efficacy, almost all reported cases with paraplegia have been treated with surgery with or without radiation therapy. We hereby report a case of thalassemia intermedia with paraplegia treated successfully with radiotherapy.

Hypercoagulable state in five thalassemia intermedia patients.

Fifty-three patients of thalassemia intermedia and 40 controls were studied for clinical evidence of thrombosis and laboratory evidence of hypercoagulable state. Thrombotic episodes were detected in 5 (9.4%) patients. Two of these 5 patients with thrombosis were splenectomized. Laboratory evaluation showed presence of thrombocytosis in 8 (15%), 5 of these were splenectomized. Platelet hyperaggregation was detected in 12 (22.2%) patients. Although rate of aggregation was slow in 7 (13.2%) patients, degree of aggregation was normal in these 7 patients and platelet hypoaggregation was not detected in any patient. Level of coagulation inhibitors protein C and protein S, and antithrombin III were decreased in 31 (58.4%) patients. There was no correlation between low level of protein C and protein S with hepatic dysfunction and iron overload. Antithrombin III level was decreased only in 8 (15%) patients. There was a statistically significant association between the lower level of this inhibitor and hepatic dysfunction. In conclusion, this study provides evidence for the existence of a chronic hypercoagulable state in patients with beta thalassemia intermedia, and suggests that expression of a procoagulant surface by thalassemia intermedia red blood cells may be the major underlying factor giving rise to platelet and coagulation inhibitor abnormalities in these patients. These alterations are not related to iron overload or hepatic dysfunction.

Autoimmune hemolytic anemia in children.

The clinical and hematological profile and treatment outcome of children with warm autoimmune hemolytic anemia (AIHA) were assessed using retrospective case record analysis. There were 26 (17 idiopathic; 9 secondary) patients with a median age of 11 years. Pallor (100%), fever (39%), and jaundice (59%) were the main presenting complaints. Jaundice was much more common in idiopathic (70%) compared to secondary (44%). Direct antiglobulin test was negative in 3 patients. Oral prednisolone produced remission in 81% patients. Four patients relapsed after a median period of 7 months (2 months to 2 year) after response. All responded to a second course of steroids in median 14 days. One child required cyclosporin A in addition. No correlation was found between response and parameters such as age, sex, jaundice, low pretreatment hemoglobin, reticulocyte count, total leukocyte count, platelet count, subtype of AIHA, and hepatosplenomegaly. Relapse correlated with increased duration between the onset of symptoms and treatment. This study indicates that oral prednisolone is an effective therapy for autoimmune hemolytic anemia. In refractory cases cyclosporine A may be useful.

Is it safe to avoid bone marrow examination in suspected itp?

Two children with suspected ITP are described. One child was treated outside with corticosteroids and was diagnosed acute lymphoblastic leukemia. Another child was fresh and diagnosed as acute myeloid leukemia on bone marrow aspirate examination. Both the children had no physical or peripheral smear finding suggestive of leukemia. We suggest that a bone marrow examination is required in developing countries for evaluation of thrombocytopenia before labeling it an immune thrombocytopenic purpura.

Low Dose Rituximab in Chronic ITP: Still an Option in Resource Limited Settings.

The etiology of ITP remains unknown but its pathogenesis consists of loss of tolerance to platelet antigens. There is a complex dysregulation of the immune system involving both the B cells and the T cells. Splenectomy is the standard second line option in steroid refractory chronic ITP patients. However, costs of surgery and reluctance for surgery in severely thrombocytopenic patients on part of surgeons are major obstacles in resource limited settings. Rituximab has been used in both the standard doses of 375 mg/m2 and low doses of 100 mg/m2 with similar results. We studied the utility of low dose Rituximab (@100 mg/m2 weekly × 4 doses) in resource limited settings. Overall response, complete response (CR) and partial response (PR) rates were 47.6% (10/21), 33.3% (7/21) and 14.3% (3/21) respectively. Median time to response in patients achieving CR was 75 days (range 45-185 days) while in patients achieving PR it was 105 days (range 45-165 days). However, there was no significant difference between males and females achieving CR or PR. We also observed that patients who had earlier responded to any form of treatment were more likely to respond to Rituximab treatment. The cumulative relapse free survival (RFS) at 13 months was 78%. By giving lower dose, six times less than conventional dosing dose, we have been able to demonstrate cost effectiveness in our study population. We were able to administer all the doses in day care without any major adverse events leading to further cost savings on in-patient care.

Molecular Response to Imatinib and Its Correlation with mRNA Expression Levels of Imatinib Influx Transporter (OCT1) in Indian Chronic Myeloid Leukemia Patients

Background and objectives: Imatinib mesylate is approved for the treatment of Chronic Myeloid Leukemia (CML). About 20% of patients with CML do not respond to treatment with Imatinib either initially or because of acquired resistance. In addition to mutated BCR-ABL1 kinase, the organic cation transporter1 (OCT1, encoded by SLC22A1) has been considered to contribute to Imatinib resistance in patients with chronic myeloid leukemia (CML). OCT1 has been reported to be the main influx transporter involved in Imatinib uptake into CML cells. To date, only a few studies have been reported on involvement of influx transporters in development of Imatinib resistance. Therefore this study was aimed to determine the expression level of Imatinib uptake transporter (OCT1) in CML patients and to correlate this level with molecular response. Methods: One hundred fifty eight patients on Imatinib were considered for gene expression analysis study for OCT1 gene. Total RNA was extracted from peripheral blood mononuclear cells. Complementary DNAs (cDNAs) were synthesized and Real Time Polymerase Chain Reaction (RQ-PCR) was performed. Results: High OCT1 expression was present in 81 (51.8%) patients and low OCT1 expression was in 77 (48.7%) patients. Low Sokal risk score group have a significantly high OCT1 expression (p=0.048). The rate of molecular response was higher in those with high OCT1 expression than in those with low OCT1 expression (p=0.05). Both event-free survival and median overall survival were significantly shorter in patients with low OCT1 expressions when compared to the patients with high OCT1 expression (p=0.03 and p=0.05). Conclusions: Our findings demonstrated that the mRNA expression level of OCT1 was significantly correlated with molecular response in CML patients. Based on these findings, present study believes that the pre-therapeutic higher expression of OCT1 may help to predict response to imatinib therapy in CML patients.

Higher rate of central nervous system involvement by flow cytometry than morphology in acute lymphoblastic leukemia.

INTRODUCTION: Central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is diagnosed traditionally by cytopathology (CP) of the cerebrospinal fluid (CSF). Role of flow cytometry (FC) to diagnose CNS involvement has not been extensively investigated. METHODS: We aimed to detect CNS involvement in 42 ALL patients (33 B-ALL, nine T-ALL) at diagnosis by FC and comparing it with CP and to correlate it with known risk factors for CNS disease like Lactate dehydrogenase (LDH). A receiver operating characteristic curve was used to determine the cutoff of LDH to predict CSF involvement. For the analysis of categorical/quantitative variables, Fisher's exact test was used. For the analysis of continuous variables, Mann-Whitney test was used. A P value of <.05 was taken as significant. RESULTS: CP and FC were positive in five (11.9%) and 11 patients (26.14%) respectively with FC detecting a significantly higher level of involvement (P=.001). All CP-positive cases were FC positive. A LDH value of >472 U/L had a sensitivity of 61% and specificity of 62.5% for diagnosis of CSF involvement by FC. CONCLUSIONS: CSF FC detects CNS disease in ALL patients at diagnosis at a rate double than CP alone and is statistically associated with an elevated LDH level. It should be incorporated in the evaluation of CSF to detect CNS involvement.