analysis of micronucleated cells by flow cytometry. 3. Advanced technology for detecting clastogenic activity

Under optimum conditions, flow cytometry (FCM) can provide a powerful technology for analyzing rare micronucleated cells in the peripheral blood. Our efforts in this endeavor have been directed toward a careful and meticulous optimization of experimental conditions, in order to achieve high resolution and high accuracy before introducing biological variation. We have achieved high resolution (Tometsko et al., 1993a) wherein the micronucleus signal is moved 100-fold upfield and away from the DNA deficient red blood cell (RBC) peak. In addition, we have demonstrated the high accuracy of our flow cytometry method in scoring rare micronucleated cells (Tometsko et al., 1993b). In the course of our studies, we rigorously pursued conditions which minimized experimental noise, demanding that FCM-scoring accuracy should approach theoretical limits. Thus, we laid the foundation for detecting clastogen activity with great sensitivity. The experiments described herein extend the previous studies by using high-speed flow cytometry to detect a clastogen-induced increase in MN cells in the total erythrocyte population. Methyl methanesulfonate (MMS) served as a model clastogen in these studies. This manuscript describes our development of a suitable blood-sampling regimen, the advantages of obtaining initial blood samples before dosing, the sex-linked difference in background micronucleus levels in BALB/c mice, and the analysis of a clastogen-induced biological response in male and female mice. As described, our flow cytometry method is able to provide a quantitative analysis of the net change in micronucleated cells ( ΔMN) for each mouse.