Diagnosis of aneuploidies by fluorescence in situ hybridization (FISH); its value in pregnancies at risk for chromosomal aberrations

Background: In the case of abnormal ultrasound findings, abnormal serum-scr eening and age-risk in advanced pregnancy a rapid diagnosis or exclusion of a chromosomal aneuploidy of the fetus is of great value for the clinical m anagement. With fluorescence in situ hybridization (FISH) on uncultured amn iotic fluid cells the detection of the most common aneuploidies, which acco unt for about 2/3 of all chromosomal aberrations [1], is possible within 24 hours. The aim was to evaluate if the FISH-technique in combination with karyotypi ng after cell culturing could replace other methods like diagnostics from u mbilical cord blood or placental biopsies. Materials and methods: For the FISH assays commercially available directly with fluorochromes labelled DNA-probes (Vysis, Stuttgart) were used. FISH a ssays were performed on amniotic fluid samples from pregnancies at risk for fetal chromosome aberrations parallel to standard cytogenetic analysis. Th e method was performed on 230 samples of amniotic fluid. We tried to optimi ze the method concerning preparation of the cell material, the denaturation - and hybridization-steps as well as stringency of post-hybridization washe s. Results: All trisomies 13, 18, 21 and the sex chromosome aneuploidies (n=34 ) which were diagnosed by conventional cytogenetics were identified correct ly by FISH analysis with the exception of one case of trisomy 21 mosaicism, in which hybridization failed. As structural chromosome aberrations and mo saicisms cannot be detected with this method, six additional chromosome abe rrations were identified exclusively by cytogenetic analysis. The mean freq uency of nuclei with abnormal signal pattern in the aneuploid cases was 89% . A minimum of 50 nuclei for each DNA-probe could be counted in 86% of the samples. The results of 12 cases were classified as uninformative, because only less than 15 hybridized nuclei or no hybridization signals could be sc ored. Maternal contamination was found in 17,4% of the samples. Conclusions: In clinical cases with a high risk for an abnormal fetal karyo type and the need of quick clinical consequences, methods which make possib le a karyotyping within shortest time should be preferred to amniocentesis and FISH-analysis, because Chromosomal mosaicism and structural aberrations, which represent up to 20% of all chromosomal abnormalities in this group, cannot be detected, uninformative cases can occur in up to 15% of all investigated samples and There is a risk for false-negative results through contamination of the sam ple with cells of maternal origin. In comparison with methods which permit rapid karyotyping from umbilical co rd blood or placental biopsies, a delay in the diagnostic procedure has to be accepted, when the result of the FISH-analysis has to be confirmed by ce ll culturing and standard cytogenetic analysis.