Effectiveness of pulse-shape criteria for the selection of dicentric chromosomes by slit-scan flow cytometry and sorting

A method was developed to detect dicentric chromosomes by slit-scan flow cytometry. The two centromeres of dicentric chromosomes are represented by the two dips in the trimodal fluorescence profile. A trimodal profile can, however, also be generated by aggregates of chromosomes. We tested the effectiveness of slit-scan profile criteria that were applied to discriminate between trimodal profiles generated by dicentrics and trimodal profiles generated by artefacts. A Profile-Dip Counter (PDC) module was designed that can assess, in real time, the number of dips in slit-scan profiles. The PDC module was used in combination with a Cytofluorograph System 50 cell sorter for slit-scan sorting of chromosomes prepared from irradiated V79 cells. Chromosomes corresponding to trimodal profiles were sorted individually onto slides for subsequent visual inspection by fluorescence microscopy. The isolated chromosomes were stretched by treatment with trypsin to increase the efficiency for centromere detection. When fixed with glutaraldehyde, chromosomes could be sorted intact on slides. We found that trimodal profiles are generated by dicentric chromosomes as well as by monocentric and aggregated chromosomes. When stringent pulse-shape criteria were applied for the selection of profiles, the yield of dicentric chromosomes was 70% of the sorted chromosomes