Differentiation of Fanconi anemia from idiopathic aplastic anemia by induced chromosomal breakage study using mitomycin-C (MMC).

This study was conducted to differentiate between Fanconi anemia (FA) and "idiopathic" aplastic anemia on the basis of induced chromosomal breakage study with mitomycin C (MMC). MMC-stress test was conducted on peripheral blood lymphocytes from 29 patients with aplastic anemia. Ten patients with very high percentage of chromosomal breakage and four patients exhibiting somatic mosaicism were diagnosed as FA on the basis of chromosomal breakage study. Six of these patients exhibited congenital anomalies at presentation while another eight lacked such anomalies or had minor physical problems.The present study illustrates that MMC stress test provides an unequivocal means of differentiation between Fanconi anemia and 'idiopathic' aplastic anemia. Further, the study, first of its kind from India, stresses on the need for conducting this test in all aplastic anemia cases, even those without congenital anomalies, for accurate and timely diagnosis of Fanconi anemia to implement appropriate therapy.

Evaluation of minimal residual disease in patients with chronic myeloid leukaemia on IFN-alpha 2b therapy using conventional cytogenetics and fluorescence in situ hybridization.

BACKGROUND: Chronic myeloid leukaemia (CML) is a haematopoietic malignancy characterized by the presence of the Philadelphia (Ph) chromosome that results from balanced reciprocal translocation between chromosomes 9 and 22 leading to the formation of the bcr/abl fusion gene. Studies have shown that interferon-alpha (IFN-alpha) therapy induces both cytogenetic (reduction in Ph+ cells) and molecular response (reduction in the bcr/abl positive cells) in a large proportion of patients, thereby improving their prognosis and survival. There are no reports available from India on the clinical management of CML patients using IFN-alpha therapy and molecular methods for the evaluation of residual disease. We evaluated the efficacy of IFN-alpha 2b therapy bysequential cytogenetic and molecularanalysis. METHODS: Karyotypingwas done from G-banded metaphases obtained from 24-hour culture of bone marrow aspirates of 45 patients. Cytogenetic analysis was repeated at intervals of 4-6 months during the course of IFN-alpha therapy. Dual-colour fluorescence in situ hybridization (FISH) analysis using specific probes for bcr and abl genes was done to assess the molecular response. RESULTS: Eight patients achieved complete cytogenetic response with no Ph+ cells. Using FISH analysis, 4 of these patients were negative for the fusion gene implying a complete response, while the remaining 4 patients showed bcr/abl fusion signals that represent residual disease. CONCLUSION: Our study emphasizes the need for sequential cytogenetic and molecular analysis in the management of patients with CML and for the evaluation of minimal residual disease in patients on IFN-alpha therapy.

Detection of human aneuploidies in prenatal and postnatal diagnosis using molecular cytogenetics.

Chromosomal aneuploidies especially trisomies 13, 18, 21, monosomy X and 47, XXY account for up to 95% of live born cytogenetic abnormalities. The diagnosis of aneuploidies usually done by conventional cytogenetic analysis (CCA) is associated with technical difficulties and requires about 1-3 weeks for providing a result, especially in prenatal diagnosis. In the present study, Fluorescence In Situ Hybridization (FISH) was used on interphase cells for rapid prenatal and postnatal detection of aneuploidies. The frequent indications of high pregnancies included for prenatal diagnosis were previous child with chromosomal abnormalities, abnormal ultrasound scan and advanced maternal age (> 35 years). Interphase FISH was done using probes specific for chromosomes 13, 18, 21, X and Y on uncultured chorionic villi and amniotic fluid samples. All samples were analyzed subsequently using conventional cytogenetics. The analysis of aneuploidies for chromosomes 13, 15, 16, 18, 21, 22, X and Y using FISH was extended to abortuses from spontaneous abortion cases. In cases where cytogenetics was not informative, a diagnosis could be made using interphase FISH. For postnatal diagnosis, interphase FISH was done to confirm low-level mosaicism in patients with primary amenorrhea, suspected cases of Klinefelter syndrome, and mental retardation using probes specific for various autosomes, X and Y chromosomes. FISH was also done using probe specific for the sex-determining region (SRY) on the Y chromosome in cases with ambiguous genitalia. The SRY region could be identified in cases that lacked the Y chromosome on conventional cytogenetic analysis thereby emphasizing on the high resolution of FISH technique in detecting sub-microscopic rearrangements. To conclude, interphase FISH decreases the time interval between sampling and diagnosis. This is of tremendous value in prenatal diagnosis of urgent high-risk pregnancies, management of ambiguous genitalia and low-level mosaicism where result can be obtained within 24 hours.