Quantitative karyotyping of human chromosomes by dual beam flow cytometry

Dual beam flow cytometry of chromosomes stained with Hoechst 33258 and chromomycin A3 has been proposed as a method for quantitative classification of human chromosomes (bivariate flow karyotyping). In this paper we investigate the sources and magnitudes of variability in the mean fluorescence intensities of each chromosome group resolved in bivariate flow karyotypes and study the impact of this variability on chromosome classification. Replicate bivariate flow karyotypes of chromosomes isolated from lymphocytes from 10 individuals demonstrated that person-to-person variability was significantly greater than run-to-run variability. The total variability was sufficiently small that it did not interfere with classification of normal chromosome types except chromosomes 9 through 12 and chromosomes 14 and 15. Furthermore, the variability was generally smaller than 1/600th of the mitotic genome, so that one-band rearrangements should be detectable in bivariate flow karyotypes.