AUTOMATED BIODOSIMETRY USING DIGITAL IMAGE-ANALYSIS OF FLUORESCENCE IN-SITU HYBRIDIZATION SPECIMENS

Fluorescence in situ hybridization (FISH) of metaphase chromosome spre ads is valuable for monitoring the radiation dose to circulating lymph ocytes. At low dose levels, the number of cells that must be examined to estimate aberration frequencies is quite large. An automated micros cope that can perform this analysis autonomously on suitably prepared specimens promises to make practical the large-scale studies that will be required for biodosimetry in the future. This paper describes such an instrument that is currently under development. We use metaphase s pecimens in which the five largest chromosomes have been hybridized wi th different-colored whole-chromosome painting probes. An automated mu ltiband fluorescence microscope locates the spreads and counts the num ber of chromosome components of each color. Digital image analysis is used to locate and isolate the cells, count chromosome components, and estimate the proportions of abnormal cells. Cells exhibiting more tha n two chromosomal fragments in any color correspond to a clastogenic e vent. These automatically derived counts are corrected for statistical bias and used to estimate the overall rate of chromosome breakage. Ov erlap of fluorophore emission spectra prohibits isolation of the diffe rent chromosomes into separate color channels. Image processing effect ively isolates each fluorophore to a single monochrome image, simplify ing the task of counting chromosome fragments and reducing the error i n the algorithm. Using proportion estimation, we remove the bias intro duced by counting errors, leaving accuracy restricted by sample size c onsiderations alone. (C) 1997 by Radiation Research Society.