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FISH
From: Carlos Brito (clbmtoc@telcel.net.ve)
Tue Sep 24 09:20:42 2002
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Perdonen lo extenso, pero me parecio utilINTRODUCTION Conventional prenatal diagnosis enables the study of the chromosomal structure of the fetus (karyotype). It must be done after the culture of amniotic cells obtained by amniocentesis, which leads to a delay in the diagnostic response (7-12 days in a private laboratory). Express prenatal diagnostics is based on a molecular cytogenetic technique (fluorescent in situ hybridization - FISH) which does not require the culture of amniotic cells and which enables the detection of numerical anomalies affecting chromosomes 13, 18, 21, X and Y in the fetus. Express prenatal diagnosis therefore meets a very pressing need for the pregnant woman. It provides an initial, quick diagnostic response, only 24-48 hours after amniocentesis, covering 95% of numerical chromosomal anomalies found at birth, thereby reassuring or providing early warning to the parents regarding the presence of a chromosomal anomaly. Conventional cytogenetic analysis with culture of amniotic cells and preparation of the fetal karyotype is systematically done in parallel in order to provide a complete chromosomal analysis. OPERATING PROTOCOL When the sample arrives in the laboratory, a minimum of 15 mL is systematically set aside for cell culture and conventional cytogenetic techniques. Between 2-5 mL of fluid are set aside for the express FISH technique. This technique is performed on the interphase nuclei of uncultivated amniotic cells, using the AneuvysionTM diagnostic kit. It uses five fluorochromemarked DNA probes (Figure 1), each one recognizing and binding to a specific region of one of the five chromosomes studied to form a hybrid. The hybrid thus formed generates a fluorescent signal observable under a fluorescent microscope, which makes it possible to rapidly identify and count chromosomes 13, 18, 21, X and Y (figures 2 and 3). RESULT OF THE EXPRESS PRENATAL DIAGNOSIS For each of the five chromosomes studied, an average of 100 nuclei are counted after express prenatal diagnosis: • A sample is considered normal when at least 80% of the nuclei observed have a normal number of signals and less than 10% of nuclei show the same abnormal pattern; • A sample is considered abnormal when at least 60% of nuclei show the same abnormal number of signals. A sample in which the same abnormal pattern is found in more than 10% but less than 60% of nuclei analyzed is inconclusive. In these cases the continued search for a possible mosaic (existence of two cell populations with different chromosome structures) is possible by the observation and counting of 200 nuclei. The results of the express prenatal diagnosis are available within 48 hours of receipt of the amniotic sample in the laboratory. A negative result can reassure parents regarding the vast majority of chromosomal anomalies, although the results of the fetal karyotype (7 to 12 days in a private laboratory) are necessary to complete the prenatal diagnosis. RÉFÉRENCES : 1. AMERICAN COLLEGE OF MEDICAL GENETICS. (1993). Prenatal F.I.S.H. Policy Statement . Am. J. Hum. Genet., 53 : 526-527. 2. BRYNDORF T. ET AL. (1997). Prenatal detection of chromosome aneuploidies by FISH : experience with 2000 uncultured amniotic fluid samples in a prospective preclinical trial, Prenat. Diagn., 17 : 333-341. 3. EIBEN B. ET AL. (1998). A prospective comparative study on F.I.S.H. of uncultured amniocytes and standard karyotype analysis, Prenat. Diagn., 18 : 901-906. 4. MERCIER S. ET AL. (1995). Prenatal diagnosis of chromosomal aneuploidies by F.I.S.H. on uncultured amniotic cells. Ann. Genet., 38(3) : 151-157. 5. WARD B.E. ET AL. (1993). Rapid prenatal diagnosis of chromosomal aneuploidies by F.I.S.H. : clinical experience with 4500 specimens. Am. J. Hum. Genet., 52 : 854-865. Sylvie Mercier, Ph.D. Cytogenetics Coordinator, Diagnostic Laboratories
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