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 fl ow cytometry. The two centromeres of dicentric chromosomes are represe nted 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 ap plied to discriminate between trimodal profiles generated by dicentric s 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 wi th a Cytofluorograph System 50 cell sorter for slit-scan sorting of ch romosomes prepared from irradiated V79 cells. Chromosomes correspondin g to trimodal profiles were sorted individually onto slides for subseq uent visual inspection by fluorescence microscopy. The isolated chromo somes were stretched by treatment with trypsin to increase the efficie ncy for centromere detection. When fixed with glutaraldehyde, chromoso mes could be sorted intact on slides. We found that trimodal profiles are generated by dicentric chromosomes as well as by monocentric and a ggregated chromosomes. When stringent pulse-shape criteria were applie d for the selection of profiles, the yield of dicentric chromosomes wa s 70% of the sorted chromosomes.