Micronuclei assay by laser scanning cytometry

Background: The micronuclei (MN) assay is used to assess the chromosomal/mi totic spindle damage induced by ionizing radiation or mutagenic agents in v ivo or in vitro. Because visual scoring of MN is cumbersome semi-automatic procedures that relay either on flow cytometry or image analysis were devel oped: both offer some advantages but also have shortcomings. Methods: in the present study laser scanning cytometer (LSC), the instrumen t that combines,Analytical capabilities of flow and image cytometry, has be en adapted for quantitative analysis of MN. The micronucleation of human br east carcinoma MCF-7 and leukemic HL-60 and U-937 cells was induced by in v itro treatment with mitomycin C. Cellular DNA was stained with propidium io dide (Pl), protein was counterstained with fluorescein isothiocyanate (FITC ). Two approaches were used to detect MN: (a) the threshold contour was set based on the data from the photosensor measuring red fluorescence of PI an d MN were identified on the bivariate PI versus PI/FITC fluorescence distri butions by their characteristic position; (b) the threshold contour was set on the data from the sensor measuring FITC fluorescence which made it poss ible, using the LSC software dedicated for FISH analysis, to assay both the frequency and DNA content of individual MN within each measured cell. Results: The capability of LSC to relocate MN for visual examination was us eful to confirm their identification. Visual identification of MN combined with their multiparameter characterization that took into an account their DNA content and protein/DNA ratio made it possible establish the gating par ameters that excluded objects that were not MN; 93.3+/-3.3 events within th e selected gate were MN. It was also possible to successfully apply FISH so ftware to characterize individual cells with respect to quantity of MN resi ding in them. The percentage of MN assayed by LSC correlated well with that estimated visually by microscopy, both for MCF-7 (r = 0.93) and HL-60 cell s (r = 0.87). Conclusions: LSC can be used to obtain unbiased estimate of MN frequencies. Unlike flow cytometry, it also allows one to characterize individual cells with respect to frequency and DNA content of MN residing in these cells. T hese analytical capabilities of LSC may be helpful not only to score MN but also to study mechanisms by which clastogenic agents induce MN. Cytometry 45:19-26, 2001. (C) 2001 Wiley-Liss, Inc.