Manisha Madkaikar

Venous thromboembolism in young patients from western India: a study.

The goal of this article is to study the association of known markers of thrombophilia with venous thrombosis in young patients (< 45 years) from the Western part of India. A prospective study of 432 patients (252 males and 180 females, age 1-45 years) was conducted between 1994 and 2000 (6 years). The diagnosis was confirmed in all the patients by ultrasound with Doppler or by a computed tomograph (CT) scan of the brain with or without contrast depending on the case. Detailed clinical examination, and family history was taken to establish recurrent thrombosis and familial occurrence of thrombosis. The markers studied were protein C, protein S, antithrombin (AT) III, factor V Leiden mutation, prothrombin gene G20210A polymorphism, and the thermolabile MTHFR variant C677T polymorphism, using appropriate techniques. Lupus inhibitor was tested in the first 72 patients using Dilute Russel Viper Venom Time (DRVVT) test, and anticardiolipin antibodies were tested by enzyme-linked immunosorbent assay. Protein C, protein S, and AT III deficiency was detected in 9.5%, 6.5%, and 2.6%, respectively, among the patients. Anticardiolipin antibody was present in 9.9% of the patients, whereas lupus anticoagulant was present in 8.3% of patients; factor V Leiden mutation was detected in 3% of patients; thermolabile variant of MTHFR C677T polymorphism was present in 14.9% of patients with 1.2% homozygotes. Prothrombin G20210A polymorphism was not detected in any sample in this population. One hundred and four patients of 432 (24.9%) had recurrent attacks of thrombosis without any proximate precipitating cause, whereas 7.5 % of the patients had another close member of the family with a history of deep venous thrombosis. Eighty-six members from 28 families (out of 32 families giving family history of thrombosis) were investigated and found to have protein C and protein S deficiency in seven each; factor V Leiden was present in 6, and MTHFR C677T polymorphism was present in 5 cases. Hence, 25 of 86 members (28%) from the family of patients with familial history deep venous thrombosis had positive markers for thrombophilia. Thus, we could show that in young patients presenting with thrombosis, at least 34% of them had a demonstrable cause for thrombophilia. Prothrombin gene polymorphism G20210A seems to be nonexistent in our population and AT III deficiency also appears to be low compared to other markers of thrombophilia. There is a high prevalence of variant MTHFR C677T in our series, but the incidence of MTHFR C677T in our general population is also high. Hence, the significance of this finding in our cases of deep venous thrombosis remains to be seen, but we did not see any homozygotes when we tested 70 randomly selected asymptomatic persons, whereas in the present series, 1.8% of the patients had homozygosity for the MTHFR C677T polymorphism.

Hematologically important mutations: Leukocyte Adhesion Deficiency (first update)

Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, mutations are found in ITGB2, the gene that encodes the β subunit of the β(2) integrins. This syndrome is characterized directly after birth by delayed separation of the umbilical cord. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Le(a) and Le(b) blood group antigens. Finally, in LAD-III (also called LAD-I/variant) the conformational activation of the hematopoietically expressed β integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells that is involved in the regulation of β integrin conformation.

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.

First‐time development of FVIII inhibitor in haemophilia patients during the postoperative period

Summary. Development of inhibitor to FVIII in haemophilia patients is well‐known and is not uncommon. However, their development for the first time during the postoperative period has hardly been reported. In a developing country such as India, where resources are limited, development of such an eventuality may prove disastrous. However, as many of our patients are sparingly treated, therefore, even if they test negative for the inhibitor preoperatively, they may get the requisite FVIII antigenic stimulation during the preoperative and immediate postoperative period, leading to the development of inhibitors during this critical time of wound healing. We describe here six patients who developed such an inhibitor, from a group of 35 patients with haemophilia A who underwent various surgical procedures (19%). We stress that such an eventuality may not remain rare in developing countries as more patients of severe haemophilia undergo surgery and are therefore challenged for the first time in their life with large amounts of FVIII concentrate during their preoperative period.

Intracranial haemorrhage in severe haemophilia: prevalence and outcome in a developing country.

Summary.  Intracranial haemorrhage (ICH) is a common cause of morbidity and mortality in haemophilic patients all over the world. From 1995 to 2004, we have investigated 37 patients with 43 episodes of ICH at our Comprehensive Haemophilia Care Center from a total of 600 registered patients. Diagnosis of ICH in the patients was confirmed by clinical, haematological and computed tomographic imaging data. Three patients died despite replacement therapy while one child who had a ventriculo‐atrial shunt for acute hydrocephalus also died before further intervention. One of the four patients who died also had severe aplastic anaemia for 6 years in addition to severe haemophilia. Detailed history obtained from 143 families with haemophilia attending the Genetic Diagnosis Clinic at our Center showed a positive history of cerebral bleed in 39 episodes in 37 patients. Sixteen families gave a history of death in the family of haemophilic patients due to ICH, while in the remaining 21 families, the patients had survived the episode after treatment elsewhere. However, the ICH was not confirmed by image data in these cases. The treatment protocols were also not available in these cases. Conservative factor replacement therapy 100% correction for 3 days followed by 50–60% correction for 7 days) coupled with the epsilon amino caproic acid, the antifibrinolytic agent at least for 30 days led to a mortality (10.8%) similar to that of the western countries and almost no morbidity. Surgery was not required in any of these patients except in one elderly patient with HIV infection on antiretroviral therapy.

Autoimmune lymphoproliferative syndrome caused by a homozygous null FAS ligand (FASLG) mutation

BACKGROUND Autoimmune lymphoproliferative syndrome (ALPS) is characterized by chronic nonmalignant lymphoproliferation, accumulation of double-negative T cells, hypergammaglobulinemia G and A, and autoimmune cytopenia. OBJECTIVES Although mostly associated with FAS mutations, different genetic defects leading to impaired apoptosis have been described in patients with ALPS, including the FAS ligand gene (FASLG) in rare cases. Here we report on the first case of complete FAS ligand deficiency caused by a homozygous null mutant. METHODS Double-negative T-cell counts and plasma IL-10 and FAS ligand concentrations were determined as ALPS markers. The FASLG gene was sequenced, and its expression was analyzed by means of Western blotting. FAS ligand function was assessed based on reactivation-induced cell death. RESULTS We describe a patient born to consanguineous parents who presented with a severe form of ALPS caused by FASLG deficiency. Although the clinical presentation was compatible with a homozygous FAS mutation, FAS-induced apoptosis was normal, and plasma FAS ligand levels were not detectable. This patient carries a homozygous, germline, single-base-pair deletion in FASLG exon 1, leading to a premature stop codon (F87fs x95) and a complete defect in FASLG expression. The healthy parents were each heterozygous for the mutation, confirming its recessive trait. CONCLUSION FAS ligand deficiency should be screened in patients presenting with ALPS features but lacking the usual markers, including plasma soluble FAS ligand and an in vitro apoptotic defect. An activation-induced cell death test could help in discrimination.

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.

Hemolytic anemia and distal renal tubular acidosis in two Indian patients homozygous for SLC4A1/AE1 mutation A858D

Familial distal renal tubular acidosis (dRTA) can be caused by mutations in the Cl2/HCO32 exchanger of the renal Type A intercalated cell, kidney AE1/SLC4A1. dRTA-associated AE1 mutations have been reported in families from North America, Europe, Thailand, Malaysia, Papua-New Guinea, Taiwan, and the Philippines, but not India. The dRTA mutation AE1 A858D has been detected only in the context of compound heterozygosity. We report here two unrelated Indian patients with combined hemolytic anemia and dRTA who share homozygous A858D mutations of the AE1/SLC4A1 gene. The mutation creates a novel restriction site that is validated for diagnostic screening.

Fractures of long bones in severe haemophilia

Dear Editor, Patients with severe haemophilia in developing countries still get crippling arthropathies and joint deformities. This has been highlighted in several previous studies [1]. The Comprehensive Hemophilia Care Center, Mumbai, has 700 patients of hemophilia registered. Out of these, 500 patients have severe haemophilia. None of these patients are on the prophylactic factor replacement therapy. For many of these patients, joint bleeds are treated with only symptomatic measures like immobilization, local ice application and analgesics owing to either non-availability or non-affordability of the factor concentrates. Out of these 500 cases reviewed over the last 5 years, 20 patients had long-bone fractures (22 episodes). These patients were treated in consultation with orthopaedic surgeons, haematologists and physiotherapists, depending on the site of fracture, nature of fracture, and inhibitor status of the patient. Twenty out of the 500 cases of severe haemophilia sustained fracture during the last 5 years. two patients had two episodes of fracture each. The average age at the time of sustaining fracture was 28 years (14–46). Fifteen patients were <35 years of age. All these patients had severe haemophilia, 19 with factor VIII <1% and one with factor IX <1%. Nine patients had inhibitors positive ranging from 4.2 to 465 BU mL. The details of the site of fracture, nature of injury, inhibitor status and time taken or fracture union are given in Table 1. Seventeen patients had fractured femur (two had fractured neck, five supracondylar and 10 shaft femur); three had fracture of upper end of tibia; one had fracture humerus; and one had fracture of radius and ulna. Six patients with fractured femur required surgery for internal fixation, whereas others were managed conservatively with closed reduction and plaster cast. Liver function tests, including total and conjugated bilirubin, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase and serum albumin, were normal in all the 20 patients, indicating the absence of active liver disease, either acute or chronic. Bone density assessment by DEXA (Dual Energy Xray Absorptiometry) scan was done in six of these patients. They showed bone mineral density (BMD) (Z score) of )2.5 to )4.9 in lumbar spine and )1.4 to )3.8 in the femoral neck. Osteoporosis of lumbar spine was observed in all the six patients. Both the patients who sustained fracture of neck femur had osteoporosis of the hip area. Osteopaenia of the hip was observed in the remaining four patients. Table 2 shows the results of DEXA scan in these six cases. The severity of osteoporosis can be gauged from Figs 1 and 2. The review of literature on fractures in haemophilia showed that there is very little information on the magnitude of this problem, and most of the literature is on the management of these cases [2]. Spontaneous fracture with trivial trauma is normally not seen in young healthy individuals. All the patients, except four, in this group sustained fracture after a fall from standing position following tripping, much like the patients of old age with fracture of femoral neck. In a recent study in patients with severe haemophilia in children, the mean BMD z score was )0.92, which was significantly reduced, compared with that for control subjects [3]. It has also been shown that weight-bearing exercises are crucial in acquiring adequate bone mass in childhood [4]. Correspondence: Dr Kanjaksha Ghosh MD, MRCP, FRC Path, FACP, FNASc, Deputy Director, Institute of Immunohaematology, 13th Floor, NMS Bldg, KEM Hospital Campus, Parel, Mumbai – 12, India. Tel.: 24138518/19; fax: 24138521; e-mail: kanjakshaghosh@hotmail.com

Systemic Capillary Leak Syndrome Preceding Plasma Cell Leukaemia

We report a patient with plasma cell leukaemia with systemic capillary leak syndrome, a rare disorder often associated with monoclonal gammopathy. In this patient, the manifestation of capillary leak syndrome antedated the diagnosis of plasma cell leukaemia by 5–6 months. During that time, he was repeatedly admitted to the hospital with weight gain, congestive cardiac failure, cough and anasarca in the presence of normal renal function, liver function and normal echocardiography. On presentation, a serum protein electrophoresis showed monoclonal IgG; the blood smear showed 60% plasma cells with a total count of 4.4 × 109/l. A bone marrow aspirate showed replacement of the normal marrow by sheets of immature plasma cells. His systemic capillary leak syndrome initially responded to decongestive therapy with terbutaline and aminophylline but later on he became refractory to them and responded to vincristine, doxorubicin and dexamethasone (VAD) combination therapy only transiently. Danocrine and oxypentiphylline, added during VAD chemotherapy, did not produce a durable response in capillary leak syndrome, which finally responded to autologous peripheral blood stem cell transplantation (PBSCT). After PBSCT, he remained free of capillary leak for 10 months without terbutaline, oxypentiphylline corticosteroids, aminophylline or danocrine. His disease relapsed without recurrence of the capillary leak. He died 15 months after PBSCT and 20 months after the diagnosis of plasma cell leukaemia.