Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia (GT) is an uncommon cause of bleeding in children. We diagnosed two siblings as having GT on the basis of flow cytometric studies. Both had cutaneous bleedings and epistaxis since early childhood. Hematological investigations revealed prolonged bleeding time and a normal platelet count. Both the patients had absence of aggregation of platelets with the agonist adenosine diphosphate. Absence of the GPIIb/IIIa receptor was confirmed by flow cytometry. A short review of the disorder is presented.

Anti-myeloperoxidase antibodies in patients with microscopic polyangiitis.

BACKGROUND & OBJECTIVES: Anti-neutrophil cytoplasmic antibodies in active necrotizing and crescentic glomerulonephritis are associated with systemic vasculitides like Wegener's granulomatosis, Microscopic polyangitiis and Churg Strauss Syndrome. This study shows the incidence of ANCA with specificities to Myeloperoxidase and Proteinase3 in MPA cases and gives the correlation of ANCA with Birmingham Vasculitis Activity Score. MATERIAL & METHODS: Eighteen cases of MPA were diagnosed as per Chapel Hill Consensus Criteria. ANCA was detected by indirect immunofluorescence microscopy using fluorescence and Confocal Laser Scanning Microscopes. Anti-MPO and anti-PR3 were identified by commercial ELISAs and anti-MPO subclass and IgG isotypes were also detected. RESULTS: MPA patients showed a male preponderance with BVAS ranging from 17-30. Systemic involvement was seen in 88.9%, lower respiratory tract involvement in 77.8% and upper respiratory tract in only 33.3% cases. All these patients had perinuclear pattern on IIF, where titers ranged from 80-640 and ELISA showed anti-MPO; values ranging from 20-80 units/ml. IIF and ELISA showed a good correlation (r=0.77). Two patients having FPGN had dual specificities and had both anti-MPO and anti-PR3 which could be picked up only by ELISA. A good correlation (r=0.78) was observed between BVAS and ANCA levels as well. IgG ANCA was detected in 88.7% and 11.1% had IgG+IgM and IgG1+IgG4 ANCA was detected in 50% patients. CONCLUSION: p-ANCA with anti-MPO is highly specific for MPA; both IIF and ELISA should be carried out for true positivity and to identify rare cases of dual specificities. Confocal laser scanning microscopy is useful in identifying ANCA patterns especially when ANA is also positive. ANCA testing with BVAS assessment will surely help in early diagnosis and estimating the severity of this life threatening disease.

Prenatal diagnosis of beta-thalassemia among Indians using denaturing gradient gel electrophoresis.

We have offered first trimester prenatal diagnosis to 55 couples at risk for beta-thalassemia, originating from various parts of India, using polymerase chain reaction and denaturing gradient gel electrophoresis. Apart from the six common mutations, codon 30 (CAG-->CAA), Cap site +1 (A-->C), as well as three uncharacterized mutations were seen among the parents. In the majority of cases, the diagnosis was possible by scanning only one fragment (B) where most of the Indian mutations are situated. In 18 out of 55 cases, framework analysis could also have been used to offer prenatal diagnosis without characterizing the beta-thalassemia mutations. In the two cases where the mutations were uncharacterized, prenatal diagnosis was done only on the basis of the anomalous denaturing gradient gel electrophoresis patterns seen in the parents and in previously affected children. This is the first attempt of prenatal analysis using denaturing gradient gel electrophoresis in the extremely diverse Indian population where the profile of mutations has not yet been fully elucidated.

Potential of denaturing gradient gel electrophoresis for scanning of beta-thalassemia mutations in India.

Over the last few years, substantial progress has been made in developing strategies for the detection and characterization of various mutations causing beta-thalassemia. The Indian population comprises of numerous endogamous caste groups and beta-thalassemia is seen in almost all of them. Knowledge of the spectrum of beta-thalassemia mutations in the population is a prerequisite for successful implementation of a prevention programme. Among the different approaches available today, Denaturing Gradient Gel Electrophoresis (DGGE) offers a valid technical approach which is applicable for screening of known mutants and polymorphisms as well as in locating regions of DNA bearing unknown mutations. We analysed 356 unrelated beta-thalassemia heterozygotes by DGGE and detected 30 anomalous DGGE patterns. Fifteen mutations were characterized after sequencing 25 anomalous patterns. Of these, codon 10(GCC --> GCA) is a recently reported novel beta-thalassemia mutation while -28(A --> G) and codon 121(G --> T) are being reported for the first time in the Indian population. HbS and HbE also showed two anomalous DGGE patterns each. Framework (FW) linkage studies showed that four mutations were associated with different beta-globin gene frameworks. Linkage of IVSI-5(G --> C) and cap site +1(A --> C) to FW2 and 619-bp deletion to FW1 is being observed for the first time. Multiple DGGE patterns corresponding to the same mutation is one of the major drawbacks of this technique. In spite of this, if sufficient preliminary work has been carried out to compile a comprehensive catalogue of DGGE patterns; this is a powerful approach to characterize the mutation or to localize a small region of DNA in the case of rarer mutations.