How to develop an in-house real-time quantitative cytomegalovirus polymerase chain reaction: Insights from a cancer centre in Eastern India.

Development of a reliable, cost-effective cytomegalovirus quantitative polymerase chain reaction (QPCR) is a priority for developing countries. Manufactured kits are expensive, and availability can be inconsistent. Development of an in-house QPCR kit that is reliable and quality assured requires significant effort and initial investment. However, the rewards of such an enterprise are manifold and include an in-depth understanding of molecular reactions, and expertise in the development of further low-cost molecular kits. The experience of an oncology centre in Eastern India has been shared. Hopefully, this would provide a brief roadmap for such an initiative. Staff with adequate understanding of molecular processes are essential along with vital infrastructure for molecular research and development.

Role of water quality assessments in hospital infection control: Experience from a new oncology center in eastern India.

Water quality assessment and timely intervention are essential for health. Microbiology, total dissolved solids (TDS) and free residual chlorine were measured for water quality maintenance in an oncology center in India. Impact of these interventions over a period of 22 months has been demonstrated with four cardinal events. Pseudomonas in hospital water was controlled by adequate chlorination, whereas high TDS in the central sterile supply department water was corrected by the installation of electro-deionization plant. Contaminated bottled water was replaced using quality controlled hospital supply. Timely detection and correction of water-related issues, including reverse osmosis plant was possible through multi-faceted approach to water quality.

A rapid & sensitive liquid chromatography- tandem mass spectrometry method for the quantitation of busulfan levels in plasma & application for routine therapeutic monitoring in haematopoietic stem cell transplantation.

Background & objectives: Busulfan (Bu) in combination with cyclophosphamide is widely used in myeloablative conditioning regimen prior to haematopoietic stem cell transplantation (HSCT). Its narrow therapeutic range and toxic side effects at high systemic exposure and graft rejection at low exposure emphasize the need for busulfan dose optimization using targeted dose adjustment prior to HSCT. We report here a rapid and sensitive method to quantitate busulfan plasma levels in patients receiving busulfan as part of pre-transplant conditioning. Methods: The method involves simple protein precipitation of the plasma followed by analysis using a high performance liquid chromatography (HPLC) with tandem mass spectrometry - electrospray ionization technique (LC-ESI MS/MS) in positive ionization mode and quantified using multiple reaction monitoring (MRM). Deuterated busulfan (d8-busulf`an) was used as the internal standard. Results: The method was linear for the concentration ranging from 0 to 4000 ng/ml of busulfan with a limit of detection of 2 ng/ml and limit of quantitation of 5 ng/ml. The assay was accurate for serial concentrations of Bu in plasma for five consecutive days and the CV was less than 10 per cent. Conclusion: Using this rapid and sensitive method, busulfan levels were targeted and subsequent doses adjusted at our center in 26 patients receiving high dose busulfan in combination with cyclophosphamide or fludarabine.

Prenatal diagnosis of Glanzmann thrombasthenia.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive disorder of platelet function, which results in major morbidity due to persistent, spontaneous, mucocutaneous bleeding and menorrhagia in women. Platelet transfusions are often needed to control the bleeding. Glanzmann thrombasthenia results from mutations in the genes located on chromosome 17q21-23, encoding the platelet glycoprotein (GP) IIb/IIIa receptor. METHODS: This report describes, for the first time in India, the prenatal diagnosis performed in a family who had a child with GT. As the molecular defect had not been identified at the time of chorionic villus sampling (CVS), prenatal diagnosis was done by linkage assessment. Haplotype analysis was performed using polymorphic markers on chromosome 17q 12-21, which included the dinucleotide repeat polymorphisms (CA)n in BRCA1 gene and locus D17S579 and (CT)n within GP IIIa intron 6, and the known restriction fragment length polymorphism (RFLP) markers Fok I (GP IIb exon 26), Taq I (GP IIIa exon 8) and Sma I (GP IIIa exon 9). The specific mutation in this family was subsequently confirmed. RESULTS: Both parents and the foetus were heterozygous for all the dinucleotide repeat polymorphisms and the affected child was homozygous. Both parents and the affected child were homozygous for Fok I RFLP. The father was heterozygous, and the mother, affected child and foetus were homozygous for Taq I and Sma I. The Fok I RFLP was identical for all the family members and hence did not provide any information for haplotype analysis (foetus not tested). CONCLUSION: The findings from dinucleotide repeat polymorphisms in BRCA1, D17S579, and GP IIIa intron 6 and the Sma I and Taq I RFLPs in GP IIIa strongly suggested that the foetus had inherited the father's mutant and the mother's normal alleles. Hence, the foetus was diagnosed to be a heterozygous carrier of GT by haplotype analysis. A private sequence alteration was later identified in the affected child in GP IIIa IVS1 (-14C --> A). The parents and foetus were heterozygous for this mutation. This confirmed the findings of the haploytpe analysis.