ANALYSIS OF MICRONUCLEATED CELLS BY FLOW-CYTOMETRY .1. ACHIEVING HIGH-RESOLUTION WITH A MALARIA MODEL

Micronucleated cells (MN cells) are present in the blood as rare event s (i.e. about 2 MN cells/1000 total). Scoring MN cells by hand is both time-consuming and tedious, which is the primary reason why only 1000 -2000 total cells (PCEs) are routinely scored for each sample. It is g enerally recognized that scoring larger numbers of cells would improve assay statistics and is desirable, but impractical with hand-scoring. In contrast, automated scoring methods can process large numbers of c ells, thus improving statistical analysis. In order to accurately and quickly evaluate clastogenic activity, we have developed a flow cytome try based method of scoring micronucleated cells. One of the first ste ps in developing an automated assay is to demonstrate the ability of t he method to resolve the cells of interest. In this case, micronucleat ed cells must be resolved from DNA-deficient red blood cells (RBCs). S ince micronuclei are heterogeneous rare events which vary in both size and DNA content, we chose to use a more enriched and homogeneous biol ogical model for optimizing the experimental variables of this assay, leading to high resolution of the rare cells. Experiments are describe d in which the murine malaria parasite, P. berghei, served as a micron ucleus model and facilitated the development of an accurate flow cytom etry based scoring method. This parasite resides in the red blood cell population and endows the cells with a homogeneous (genetically deter mined) DNA component in the micronucleus size range. The conditions de veloped with the malaria parasite are readily applied to the analysis of micronucleus events in blood samples. Bivariate profiles that are o btained with the malaria parasite can be used to define the analysis a rea for high-speed micronucleus scoring.