AUTOMATED SCORING AND ANALYSIS OF MICRONUCLEATED HUMAN LYMPHOCYTES.

摘要

Physical and chemical mutagens and carcinogens in our environment produce chromosome abberations in the circulating peripheral blood lymphocytes. The abberations, in turn, give rise to micronuclei when the lymphocytes proliferate in culture. In order to improve the micronucleus assay as a method for screening human populations for chromosome damage, I have (1) developed a high-resolution optical low-light-level micrometry expert system (HOLMES) to digitize and process microscope images of micronuclei in human peripheral blood lymphocytes, (2) defined a protocol of image processing techniques to objectively and uniquely identify and score micronuclei, and (3) analysed digital images of lymphocytes in order to study methods for (a) verifying the identification of suspect micronuclei, (b) classifying proliferating and non-proliferating lymphocytes, and (c) understanding the mechanisms of micronuclei formation and micronuclei fate during cell division.;For the purpose of scoring micronuclei, HOLMES promises to (a) improve counting statistics since a greater number of cells can be scored without operator/microscopist fatigue, (b) provide for a more objective and consistent criterion for the identification of micronuclei than the human observer, and (c) yield quantitative information on nuclear and micronuclear characteristics useful in better understanding the micronucleus life cycle.;My results on computer aided identification of micronuclei on microscope slides are gratifying. They demonstrate that automation of the micronucleus assay is feasible. Manual verification of HOLMES' results show correct extraction of micronuclei from the scene for 70% of the digitized images and correct identification of the micronuclei for 90% of the extracted objects.;Moreover, quantitative analysis on digitized images of lymphocytes using HOLMES has revealed several exciting results: (a) micronuclear DNA content may be estimated from simple area measurements, (b) micronuclei seem to originate not only from chromosome fragments or from whole chromosomes, but also from dicentrics, (c) distributions of DNA content may serve as a means to identify the type of chromosome damage that had been incurred (i.e. ionizing radiation or chemicals), and (d) texture analysis coupled with feature analysis may allow us to estimate the number of proliferating lymphocytes and in turn compute the extent of chromosome damage more accurately.