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.

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.

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.

Serological Detection of Antibodies to Peste des Petits Ruminants Virus in Large Ruminants.

Peste des petits ruminants (PPR) is an economically important disease of small ruminants with a rapidly expanding geographical distribution. Peste des petits ruminants virus may manifest in a variety of ways with disease ranging from acute to subclinical. We investigated the exposure of large ruminants to PPRV in areas where the virus is endemic in the small ruminant population by assessing the serological status of groups of animals. This study focused on the Punjab province of Pakistan as an area where the virus is endemic and where mixed farming practices occur enabling close interactions between small and large ruminant populations. An overall PPR seropositivity was detected in 10.0% of cattle and 14.16% of buffaloes. Following an assessment of serological profiles in large ruminants within different age groups, a maximum seroprevalence was observed in cattle (17.5%) and buffaloes (22.5%) over 2 years of age indicating the potential utility of sampling large ruminant populations for PPR serosurveillance. The large ruminants sampled between one and two years of age had similar levels of seropositivity within populations with 11.2% and 16.2% of animals being seropositive, respectively. Current PPR vaccination strategies do not enable the differentiation between infected and vaccinated small ruminants, and as such, the serological surveillance of sheep and goats is of little value. When considering eradication programmes for PPRV, this factor is of great significance. However, where large and small ruminants are farmed together, serological surveillance of large ruminants may provide a snapshot of virus infection within populations where mild disease is present or where small ruminants are regularly vaccinated.

Re-emergence of Peste des Petits Ruminants virus in 2015 in Morocco: Molecular characterization and experimental infection in Alpine goats.

Peste des Petits Ruminants (PPR) is a transboundary viral disease of small ruminants that causes huge economic losses in Africa, The Middle East and Asia. In Morocco, the first PPR outbreak was notified in 2008. Since then no cases were reported for seven years, probably due to three successive vaccination campaigns during 2008-2011 and close surveillance at the border areas. In June 2015, the disease re-emerged in Morocco, raising questions about the origin of the virus. The PPR virus was confirmed by qRT-PCR and virus was isolated from clinical samples on VeroNectin-4 cells. The disease was experimentally reproduced in Alpine goats using both sheep and goat derived outbreak isolates. Molecular characterization of the 2015 Moroccan PPR isolate confirmed the identity of the virus as lineage IV, closely related to the 2012 Algerian (KP793696) and 2012 Tunisian (KM068121) isolates and significantly distinct from the previous PPRV Morocco 2008 strain (HQ131927). Therefore this study confirms a new incursion of PPR virus in Morocco during 2015 and highlights the urgency of implementation of a common control strategy to combat PPR in Maghreb region in North Africa.

Value of Quantitative assessment of Myeloid Nuclear Differentiation Antigen expression and other flow cytometric parameters in the diagnosis of Myelodysplastic syndrome.

INTRODUCTION: The diagnosis of myelodysplastic syndrome (MDS) based on morphology is particularly difficult in low-grade MDS. Thus, the role of myeloid nuclear differentiation antigen (MNDA) and other flow cytometric (FCM) parameters in MDS was evaluated. METHODS: Bone marrow aspirates (BMA) collected from 52 patients with unexplained persistent cytopenias were divided into three groups: (i) proven MDS (n = 12) based on morphology and/or cytogenetics; (ii) suspected MDS (n = 6), noncontributory morphology, and cytogenetics; and (iii) non-MDS (n = 34). Sixteen control BMA were studied. Cases were analyzed for MNDA expression (on granulocytes, blasts, monocytes, and lymphocytes) and for seven quantitative parameters: CD34(+) myeloblasts % in nucleated cells, CD34(+) B-cell progenitor% in CD34(+) cells, lymphocyte/myeloblast CD45 MFI ratio, granulocyte/lymphocyte SSC peak channel ratio and the proportion of CD34(+) myeloblasts expressing CD15, CD11b, and CD56. A score of 1 was given to each parameter beyond the cutoff, and score ≥3 was considered FCM positive. RESULTS: MNDA expression on granulocytes and blasts was significantly lower in proven MDS and suspected MDS vs. non-MDS. Quantitative FCM parameters successfully distinguished MDS and suspected MDS from non-MDS. CONCLUSION: MNDA expression is an independent marker for the evaluation of dyspoiesis and may be added to the standard panel for quantitative assessment by FCM.

Emergence of Lineage IV Peste des Petits Ruminants Virus in Ethiopia: Complete Genome Sequence of an Ethiopian Isolate 2010.

Isolates of peste des petits ruminants virus (PPRV) can be segregated genetically into four lineages. For decades, lineages I-III have been reported across Africa whilst lineage IV has predominantly circulated across Asia. However, the lineage distribution is currently changing in Africa. Importantly, full genome sequence data for African field isolates have been lacking. Here, we announce the first complete genome sequence of a field isolate of peste des petits ruminants virus (PPRV) from East Africa. This isolate was derived from the intestine of a goat suffering from severe clinical disease during the 2010 outbreak in Ethiopia. The full genome sequence of this isolate, PPRV Ethiopia/2010, clusters genetically with other lineage IV isolates of PPRV, sharing high levels of sequence identity across the genome. Further, we have carried out a phylogenetic analysis of all of the available African partial N gene and F gene PPRV sequences to investigate the epidemiology of PPRV with a focus on the emergence of different lineages of PPRV in Africa.

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.

Peste des petits ruminants.

Peste des petits ruminants virus causes a highly infectious disease of small ruminants that is endemic across Africa, the Middle East and large regions of Asia. The virus is considered to be a major obstacle to the development of sustainable agriculture across the developing world and has recently been targeted by the World Organisation for Animal Health (OIE) and the Food and Agriculture Organisation (FAO) for eradication with the aim of global elimination of the disease by 2030. Fundamentally, the vaccines required to successfully achieve this goal are currently available, but the availability of novel vaccine preparations to also fulfill the requisite for differentiation between infected and vaccinated animals (DIVA) may reduce the time taken and the financial costs of serological surveillance in the later stages of any eradication campaign. Here, we overview what is currently known about the virus, with reference to its origin, updated global circulation, molecular evolution, diagnostic tools and vaccines currently available to combat the disease. Further, we comment on recent developments in our knowledge of various recombinant vaccines and on the potential for the development of novel multivalent vaccines for small ruminants.

CD34 immunohistochemistry in bone marrow biopsies for early response assessment in acute myeloid leukemia.

INTRODUCTION: Acute myeloid leukemia is a heterogenous disease with respect to prognosis. Early response assessment has an established role as predictor of remission rate, and overall and disease-free survival. Assessment of blast percentage on bone marrow aspirate smears at this stage has its own limitations. MATERIALS AND METHOD: In this study, a total of 100 AML cases that were positive for CD34 at the time of diagnosis were included in the study. Blast percentage obtained in bone marrow aspirate smears by morphology was compared with that obtained in bone marrow biopsy using CD34 immunohistochemistry. RESULTS: Bone marrow aspirate and biopsy were discordant in 19% of the cases. In 15% of the cases, bone marrow aspirate blast count was ≤ 5% and bone marrow biopsy blast percentage was >5%. CONCLUSION: Early response assessment plays an important role in management of acute myeloid leukemia. In patients with CD34-positive blasts, the CD34 IHC can improve the detection of residual blasts on Day 14 bone marrow biopsy in comparison with morphological assessment of blast percentage in bone marrow aspirate.

Prognostic relevance of aberrant SOCS-1 gene promoter methylation in myelodysplastic syndromes patients.

INTRODUCTION: The inactivation of suppressor of cytokine signaling SOCS-1, a negative regulator of cytokine pathways, by hypermethylation was shown in hematological malignancies including Myelsplastic Syndromes. So far, its prognostic relevance in myelodysplastic syndromes (MDS) patients has not been understood. METHODS: Methylation status of SOCS-1 gene was analyzed in series of 100 patients using methylation-specific PCR (MS-PCR) and correlated with disease severity, progression, and survival by comparing prognostic factors such as hematological, clinical, and cytogenetics. RESULTS: Of the total of 100 MDS patients analyzed, methylation of SOCS1 gene was found in 53% patients. Also, the frequency of patients with poor and intermediate cytogenetics was observed significantly high in methylated group (P < 0.001). Moreover, the patients with methylated SOCS-1 gene had significantly more frequent disease progression as compared to the patients with unmethylated SOCS-1 gene (P < 0.006). Both progression-free survival and median overall survival were significantly shorter in patients with methylated SOCS-1 gene when compared to the patients with unmethylated SOCS-1 gene (P = 0.006 & P = 0.001, respectively). CONCLUSION: This study for the first time showed that the mathylation of SOCS-1 gene plays an important role in the disease progression and is associated with poor survival especially among the high-risk patients. This may be due to high association between SOCS1 methylation and higher risk subtypes of MDS (such as RAEB) in this study.

Clinicohematologic Profile of Patients With Factor VIII Inhibitors: A Case Series.

Factor VIII (FVIII) inhibitors present major clinical challenge as a complication of hemophilia A in patients on treatment with FVIII concentrates and as acquired autoantibodies in patients without hemophilia A. We aimed to study the prevalence of FVIII inhibitors in Indian settings, risk factors involved in early development of inhibitors in patients with hemophilia, differences in their clinical behavior, and approach to treatment, in comparison to patients with acquired hemophilia. The overall prevalence of FVIII inhibitors in patients with severe hemophilia A was found to be 22.3%. Two cases of acquired hemophilia were reported. Due to heterogeneity of our study population, cases have been discussed individually. We observed that the early development of FVIII inhibitors in patients with hemophilia A is dependent upon an interplay of several risk factors that need to be studied in a multivariable analysis to bring out significant correlation with response to treatment. Also, they differ from patients without hemophilia A entirely in terms of presentation and management.

Clinico-hematological profile and outcome of acute promyelocytic leukemia patients at a tertiary care center in North India.

OBJECTIVES: Acute promyelocytic leukemia (APL) is the only acute leukemia amenable to targeted therapy. However, there is limited Indian data on APL. We aimed to analyze data of APL patients treated with all trans retinoic acid (ATRA) and anthracycline based chemotherapy. MATERIALS AND METHODS: A total of 34 cases of APL were treated at our center over 4 years. Induction chemotherapy consisted of a combination of ATRA and daunorubicin. RESULTS: Most of our patients were intermediate risk (50%) followed by high risk (41.17%). Induction mortality was 14.7%. We observed a high incidence of febrile neutropenia (91%) and 50% of our patients developed ATRA syndrome. Four patients (11.76%) relapsed during follow-up (median - 15 months, range: 13-33 months). There was no correlation between risk status and death or relapse or ATRA syndrome. Median event free survival (EFS) duration was not reached however mean duration was 38.45 ± 3.84 months. Median overall survival (OS) duration was also not reached at 53 months of follow-up. The 4 year OS and EFS were 75.45% and 64.5% respectively. On multivariate analysis, only disseminated intravascular coagulation (DIC) significantly correlated with a poor OS and EFS. DISCUSSION: Our data reflects that APL remains a highly curable malignancy with good response to ATRA plus anthracycline based chemotherapy even with a greater number of high and intermediate risk patients. Only DIC during induction chemotherapy bore an impact on survival in our patients.

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.

Skin manifestations associated with mantle cell lymphoma: a case report.

Mantle cell lymphoma (MCL) is a distinct non-Hodgkin's lymphoma type that commonly affects extra nodal sites. The most often affected sites are bone marrow, gastrointestinal tract and Waldeyer's ring, being the skin rarely involved. We report a case of 56 year-old man with MCL, exhibiting multiple large maculopapular skin rashes and skin ulcers. Histopathological examination had not shown direct infiltration by any atypical cells. He had significant improvement of skin lesions with combination chemotherapy and debridement. Awareness of skin manifestations of MCL is crucial for dermatologists and haematologists to establish the early diagnosis and timely administration of appropriate treatment.

Audit of peripheral stem cell transplantation for aplastic anemia in multitransfused infected patients.

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation is a curative modality for aplastic anemia; the preferred stem cell source is bone marrow. However, allogeneic peripheral blood stem cell transplantation (PBSCT) used in high-risk patients is associated with higher risk of chronic graft-versus-host disease (GVHD). Our center receives multitransfused, alloimmunized, infected, late referrals for transplant. METHODS: Forty-one patients of median age 22 years (range 8-37) received allogeneic-PBSCT from human leukocyte antigen (HLA)-matched sibling donors. The median time since diagnosis was 12 months (range 4-65) and median pretransplant transfusions were 37 (range 6-160). Six patients were platelet refractory and one alloimmunized for pan-red blood cell (RBC) antigens. Several patients had pretransplant icterus or renal dysfunction and 26 (63.4%) had unresponsive bacterial/fungal infections. Our conditioning regimen included fludarabine 30 mg/m(2) for 6 days (days -10 to -5), cyclophosphamide 60 mg/kg/d for 2 days (days -6 to -5), and antithymocyte globulin (ATGAM) 30 mg/kg/d for 4 days (day -4 to -1), which was reduced to 2 days in 2 patients. We used standard GVHD prophylaxis with cyclosporine and methotrexate on days 1, 3, 6, 11. RESULTS: The median follow-up period was 29 months (range 6-78) and median engraftment time 10 days (range 8-17). Thirty-one patients (75.6%) were treated for infections, with 20 of these on antifungals for preexisting infections. There were two graft rejections and 10 (24.4%) deaths, with three intracranial hemorrhages, two rejections with infection, three cases of refractory GVHD (acute/overlap syndrome) with cytomegalovirus reactivation, and two invasive fungal infections. Overall incidence of acute GVHD was 39% with 2 grade IV cases. Ten (25%) cases developed chronic GVHD, with extensive GVHD in four. CONCLUSION: With more experience using shortened course of ATGAM, HLA-matched donor transfusions, and availability of newer antifungals, we have been able to decrease PBSCT-related mortality. Further improvement will be possible with early referrals.