Flow cytometric analysis of small DNA content differences in heterogeneous cell populations: human amniotic fluid...

Accurate flow cytometric determination of nuclear DNA fluorescence has been attempted with human amniocentesis materials employing a detergent nuclear isolation protocol and 4,6-diamidino-2-phenylindole (DAPI) staining. Assay of native fluids yielded inadequate fluorescence histograms, whereas analysis of cultured amniocytes yielded unimodal histograms in 80 of 83 samples. Three fluids displayed unexpected bimodality, which may have resulted from the interaction between detergent and chromatin in rare cell types. Using a standardized protocol for preparation, staining, and assay, including brief periods of cocultivation of a human triploid reference standard, the percent experimental error in the flow cytometric DNA content estimate was as low as 0.2% in repeated assays of a single sample, and 0.4% in assays of a given genotype at different occasions. In two series of blindly assayed specimens, the percent standard error of the DNA content estimate ranged from 0.6 to 1.0%.