Maya Gupta

Advances in autoimmune lymphoproliferative syndromes.

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis. It is characterized by non‐malignant lymphoproliferation autoimmunity mostly directed toward blood cells and increased risk of lymphoma. Majority of patients with ALPS harbor heterozygous germline mutations in the gene for the TNF receptor‐family member Fas (CD 95, Apo‐1) which are inherited in an autosomal dominant fashion. Somatic Fas mutations are the second most common genetic etiology of ALPS. Additionally mutations in the genes encoding Fas‐ligand (FASLG), caspase 10 (CASP10) and caspase 8 (CASP8), NRAS and KRAS have been identified in a small number of patients with ALPS and related disorders. Approximately one‐third of patients with ALPS have yet unidentified defect. ALPS was initially thought to be a very rare disease, but recent studies have shown that it may be more common than previously thought. Testing for ALPS should therefore be considered in patients with unexplained lymphadenopathy, cytopenias, and hepatosplenomegaly. There have been significant advances in the understanding of the pathophysiology of ALPS in last few years which has resulted in the development of new diagnostic criteria and a number of targeted therapies. This review describes the clinical and laboratory manifestations found in patients with ALPS, as well as the molecular basis for the disease and new advances in treatment.

Flow cytometric osmotic fragility—An effective screening approach for red cell membranopathies

Among the red cell membrane disorders, hereditary spherocytosis (HS) is one of the most common causes of inherited hemolytic anemia. The aim of this study was to compare the flow‐cytometric approach for screening of red cell membrane disorders based on osmotic fragility with the eosin‐5‐maleimide (E5′M) dye test. A group of β‐thalassemia heterozygotes were also studied.

Ex vivo expansion of umbilical cord blood stem cells using different combinations of cytokines and stromal cells.

Umbilical cord blood is a promising source of hematopoietic stem cells (HSC) for allogeneic transplantation. However, graft rejection and delayed engraftment remain major limitations, both of which are related to a limited number of stem cells in the cord blood graft. Ex vivo expansion of HSC has been suggested as one of the ways of overcoming the challenges caused by a limited hematopoietic cell number from cord blood stem cell transplantation. In this study, we quantified and characterized an ex vivo expansion capacity of cord blood-derived HSC in a liquid culture system under different conditions. These conditions included: the combinations and concentrations of hematopoietic growth factors [stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, IL-6 and erythropoietin (EPO)], placental conditioning medium (PCM), and stromal cell support. During culture, the mean nucleated cell count, the mean CD34+ cell count, fold expansion, viability, clonogenic assays and immunophenotypic characterization were performed on day 0, day 7, day 12 and day 14 on the expanded cellular product. The maximum expansion was achieved using GF2 (SCF + IL-3 + GM-CSF) with stromal cell support. The mean CD34+ cell expansion on days 7 and 12 was 16.25- and 21.4-fold (5.2–32), respectively, and the mean nucleated cell expansion was 15.1- and 21-fold (18.1–23.2). The mean nucleated cell viability on day 12 was 87.9% (85.6–92.5). After 12 days, granulocyte-macrophage colony-forming units CFU-GEMM showed a 20.4-fold increase. A 21.4-fold increase in the CD34+ cells and a 20-fold increase in the CFU-GEMM should provide enough cells from a single cord blood unit to reduce the period of cytopenia after single unit cord blood transplantation. Even if there was some doubt about the long-term repopulating capacity of the expanded cells part of the collected umbilical cord cells (25%) could be expanded till day 12 after transplanting the major part (75%) of the collection.

Comparison of in-vitro and in-vivo response to fetal hemoglobin production and γ-mRNA expression by hydroxyurea in Hemoglobinopathies

BACKGROUND: Hydroxyurea, which induces Fetal hemoglobin (HbF) synthesis, is the only drug widely used in different hemoglobinopathies; however, the response is very variable. We compared the efficacy of hydroxyurea in-vitro in erythroid cultures and in-vivo in the same patients with different hemoglobinopathies to induce HbF production and enhance γ-messenger RNA expression. MATERIALS AND METHODS: A total of 24-patients with different Hemoglobinopathies were given hydroxyurea and their response was studied in-vivo and in-vitro on mononuclear cells collected from them simultaneously. RESULTS: A total of 57.7% of patients (responders) showed no further crisis or transfusion requirements after hydroxyurea therapy with a mean increase in fetal cells (F-cells) of 63.8 ± 59.1% and γ-mRNA expression of 205.5 ± 120.8%. In-vitro results also showed a mean increase in F-cells of 27.2 ± 24.7% and γ-mRNA expression of 119.6% ± 65.4% among the treated cells. Nearly 19.0% of the partial-responders reduced their transfusion requirements by 50% with a mean increase in F-cells of 61.2 ± 25.0% and 28.4 ± 25.3% and γ-mRNA-expression of 21.0% ± 1.4% and 80.0% ± 14.1% in-vivo and in-vitro respectively. The non-responders (15.3%) showed no change in their clinical status and there was no significant increase in F-cells levels and γ-mRNA expression in-vivo or in-vitro. CONCLUSION: Thus, this method may help to predict the in-vivo response to hydroxyurea therapy; however, a much larger study is required.

Paroxysmal nocturnal haemoglobinuria: diagnostic tests, advantages, & limitations

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal disorder of haematopoietic stem cells. The molecular defect in PNH is mutation in the phosphotidylinositol glycan complementation class A (PIGA gene) causing defect in glycosylphosphatidylinositol anchored proteins (Cell, 73, 1993, 703). The deficiency of these GPI‐anchored proteins on the membranes of haematopoietic cells lead to the various clinical manifestations of PNH. Clinically PNH is classified into classic PNH, PNH in the setting of another specified bone marrow disorder and sub clinical PNH. Size of the PNH clone differs in these different subtypes. The management of PNH has been revolutionized by the advent of monoclonal antibody, eculizumab. Thus, today it is important to have sensitive tests to diagnose and monitor the clone size in patients of PNH. Before 1990, diagnosis of PNH was made using complement based tests. However in the last decade, flowcytometry has become the gold standard diagnostic test as it has increased sensitivity to detect small clones, ability to measure clone size and is not affected by blood transfusions. This review is aimed to focus mainly on the different methods available for the detection of PNH clone and the recent advances and recommendations for the flowcytometric diagnosis of PNH.

Clinical profile of leukocyte adhesion deficiency type I

Leukocyte adhesion deficiency type I (LAD-I) is a rare, inherited immunodeficiency with defect in the recruitment of leukocyte to the site of inflammation. Patients with severe LAD-I have absent or markedly reduced expression of CD18 and CD11. Here we report clinical profile of 7 cases of LAD-I diagnosed at our center over a period of 3 years. Recurrent skin and mucous membrane infections were the major presenting manifestations. All children had a history of delayed cord separation.

X-linked hyper IgM syndrome: Clinical, immunological and molecular features in patients from India☆☆☆

BACKGROUND X-linked hyper-IgM (XHIM) is a primary immunodeficiency disorder characterized by recurrent infections, low serum IgG and IgA and normal or elevated IgM. It results from mutations in the CD40 ligand (CD40L) gene. Confirmation of diagnosis with identification of underlying molecular defect is important for the initiation of appropriate therapeutic interventions, including immunoglobulin replacement, antibiotics and bone marrow transplantation. METHODS To investigate the molecular basis of XHIM, we evaluated 7 patients with suspected XHIM and abnormal CD40L expression on activated CD4(+) T lymphocytes. The entire coding region and intronic splice sites of the CD40L gene were sequenced from the genomic DNA of the patients. RESULTS 7 mutations; 3 nonsense (c.172delA, c.A229T, c.C478T), 1 missense (c.A506G) and 3 splice sites [c.346+2(T→C), c.289-1(G→C), c.346+1(G→T)] were identified, out of which 5 were novel. CONCLUSION A wide heterogeneity in the nature of mutations has been observed in Indian XHIM patients in the present study. Identification of mutations in this rare disorder will help in genetic diagnosis in affected families which could be further useful in prenatal diagnosis.

A Modified NK Cell Degranulation Assay Applicable for Routine Evaluation of NK Cell Function

Natural killer (NK) cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a) is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC) or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA) and calcium ionophore (Ca2+-ionophore) instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (p < 0.0001). It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL) with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells.

Molecular characterization of leukocyte adhesion deficiency-I in Indian patients: Identification of 9 novel mutations

PURPOSE Leukocyte adhesion deficiency type-I (LAD-I) is caused by mutations in the ITGB2 gene, encoding the β2-subunit of β2-integrin (CD18) which leads to markedly reduced expression of CD18 on leukocytes resulting into recurrent life threatening infections. Here we aim to identify the molecular defects underlying LAD-I in Indian patients and correlate with the clinical presentation. METHODS Blood was collected from 30 patients and their parents for absolute neutrophil count, expression of CD18 and CD11 by flow cytometry and DNA extraction. PCR and DNA sequencing of the ITGB2 gene was done for mutation characterization. RESULTS Phenotypically, 22 patients were LAD-I(0), 1 was LAD-I(-) and 7 were LAD-I(+) showing no expression and reduced expression of CD18 respectively. Nine novel mutations in 15 patients and 11 known mutations in 16 patients were detected. Prenatal diagnosis was performed for 5 families. CONCLUSION In this study 30 patients were phenotypically and genotypically evaluated for a less known disease LAD-I. Unavailability of curative options to majority of the patients and high cost of supportive care emphasize the need to increase awareness about a suspicious case so that timely management can be given to the patient and prenatal diagnosis can be offered to their families.

Predominant Neurologic Manifestations Seen in a Patient With a Biallelic Perforin1 Mutation (prf1; p.r225w)

Neurological manifestations in familial hemophagocytic lymphohistiocytosis (FHL) are common, seen in up to 73% of patients in their course of disease. However, in majority of the cases central nervous system manifestations are associated with other clinical and laboratory parameters of hemophagocytic lymphohistiocytosis. We report here a case with FHL2 in whom hemophagocytic lymphohistiocytosis was a presenting manifestation which responded to specific therapy, however, there was isolated central nervous system relapse while patient was in remission and off therapy. FHL2 was confirmed on the basis of reduced perforin expression and homozygous mutation in PRF1at codon 637 in exon 3 (c.673C>T p.Arg225Trp).