Slit Scan Flow Cytometry Of Isolated Chromosomes Following Fluorescence Hybridization: An Approach Of Online Screening For Specific Chromosomes And Chromosome Translocations

Abstract

The recently developed methods of non radioactive in situ hybridization of chromosomes offer new aspects for chromosome analysis. Fluorescent labelling of hybridized chromosomes or chromosomal subregions allows to facilitate considerably the detection of specific chromosomal abnormalities. Formany biomedical applications (e.g. biological dosimetry in the low dose range), a fast scoring for aberrations (e.g. dicentrics or translocations) in required. Here, we present an approach depending on fluorescence in situ hybridization of isolated suspension chromosomes that indicates the feasibility of a rapid screening for specific chromosomes or translocations by slit scan flow cytometry. Chromosomes of a Chinese hamster x human hybrid cell line were hybridized in suspension with biotinylated human genomic DNA . This DNA was decorated with FITC by a double antibody system against biotin. For flow cytometry the chromosomes were stabilized with ethanol and counterstained with DAPI or propidium iodide (PI). An experimental data set of several hundred double profiles was obtained by two parameter slit scan flow cytometry and evaluated automatically. The evaluation algorithm developed allowed a classification of chromosomes according to the number of centromeres and their chromosomal positions in less than 1 msec per individual profile. Approximately 20% of the measured DAPI profiles showed a bimodal distribution with a significant centromeric dip indicating a “ normal” chromosomal morphology and a correct alignment in the flow system. In many cases, profiles of a “normal” bimodal fluorescence distribution of the DNA stain (DAPI, PI) were correlated with a “ normal” FITC profile. Due to their centromeric indices these profiles agreed well to the expected human chromosomes of the cell line. In some cases of “ normal” DAPI (PI) profiles, “aberrant” FITC profiles were observed. These were interpreted as interspecies translocation chromosomes. For all results, there was a good agreement between flow cytometry and microscopic observations (digital image analysis)