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 b lood. Our efforts in this endeavor have been directed toward a careful and meticulous optimization of experimental conditions, in order to a chieve 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 fro m the DNA deficient red blood cell (RBC) peak. In addition, we have de monstrated the high accuracy of our flow cytometry method in scoring r are micronucleated cells (Tometsko et al., 1993b). In the course of ou r studies, we rigorously pursued conditions which minimized experiment al noise, demanding that FCM-scoring accuracy should approach theoreti cal limits. Thus, we laid the foundation for detecting clastogen activ ity with great sensitivity. The experiments described herein extend th e previous studies by using high-speed flow cytometry to detect a clas togen-induced increase in MN cells in the total erythrocyte population . Methyl methanesulfonate (MMS) served as a model clastogen in these s tudies. This manuscript describes our development of a suitable blood- sampling regimen, the advantages of obtaining initial blood samples be fore dosing, the sex-linked difference in background micronucleus leve ls in BALB/c mice, and the analysis of a clastogen-induced biological response in male and female mice. As described, our flow cytometry met hod is able to provide a quantitative analysis of the net change in mi cronucleated cells (DELTAMN) for each mouse.