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 events (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 generally 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 cells, thus improving statistical analysis. In order to accurately and quickly evaluate clastogenic activity, we have developed a flow cytometry based method of scoring micronucleated cells. One of the first steps in developing an automated assay is to demonstrate the ability of the method to resolve the cells of interest. In this case, micronucleated cells must be resolved from DNA-deficient red blood cells (RBCs). Since micronuclei are heterogeneous rare events which vary in both size and DNA content, we chose to use a more enriched and homogeneous biological model for optimizing the experimental variables of this assay, leading to high resolution of the rare cells. Experiments are described in which the murine malaria parasite, P. berghei, served as a micronucleus model and facilitated the development of an accurate flow cytometry based scoring method. This parasite resides in the red blood cell population and endows the cells with a homogeneous (genetically determined) DNA component in the micronucleus size range. The conditions developed with the malaria parasite are readily applied to the analysis of micronucleus events in blood samples.(ABSTRACT TRUNCATED AT 250 WORDS)