Diagnosis of aneuploidy with fluorescence in situ hybridization (FISH); value in pregnancies with increased risk for chromosome aberrations

Abstract

In the case of abnormal ultrasound findings, abnormal serum-screening 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 management. With fluorescence in situ hybridization (FISH) on uncultured amniotic fluid cells the detection of the most common aneuploidies, which account 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 karyotyping after cell culturing could replace other methods like diagnostics form umbilical cord blood or placental biopsies. For the FISH assays commercially available directly with fluorochromes labelled DNA-probes (Vysis, Stuttgart) were used. FISH assays were performed on amniotic fluid samples from pregnancies at risk for fetal chromosome aberrations parallel to standard cytogenetic analysis. The method was performed on 230 samples of amniotic fluid. We tried to optimize the method concerning preparation of the cell material, the denaturation- and hybridization-steps and well as stringency of post-hybridization washes. All trisomies 13, 18, 21 and the sex chromosome aneuploidies (n = 34) which were diagnosed by conventional cytogenetics were identified correctly by FISH analysis with the exception of one case of trisomy 21 mosaicism, in which hybridization failed. As structural chromosome aberrations and mosaicisms cannot be detected with this method, six additional chromosome aberrations were identified exclusively by cytogenetic analysis. The mean frequency 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 scored. Maternal contamination was found in 17.4% of the samples. In clinical cases with a high risk for an abnormal fetal karyotype and the need of quick clinical consequences, methods which make possible 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 sample with cells of maternal origin. In comparison with methods which permit rapid karyotyping from umbilical cord 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 cell culturing and standard cytogenetic analysis.