The in vitro cytokinesis-block micronucleus assay: a detailed description of an improved slide preparation technique for the automated detection of micronuclei in human lymphocytes.

An improved slide preparation technique is described for the automated detection of micronuclei in binucleate cytokinesis-blocked human lymphocytes. This automated system (Discovery Image Analyser, Becton-Dickinson Image Cytometry Systems, Leiden, The Netherlands) searches for the pattern of two touching nuclei in binucleate cells instead of the edges of the cytoplasm. For this purpose several ratios of the fixative mixture, methanol/acetic acid, were checked. After fixation with a ratio of 25:1, touching nuclei were obtained in almost 100% of the binucleate cells. A hydrolysis treatment with 5 N HCl before staining with Romanowsky-Giemsa resulted in binucleate cells with dark-stained nuclei and micronuclei and a vaguely stained cytoplasm. The high visual contrast between cytoplasm and nuclear material obtained by this staining procedure makes an accurate automated detection of micronuclei feasible. Additionally, a 64 h culture time resulted in an optimal yield of binucleate cells. The results of manual micronucleus scoring on slides prepared with the 'manual protocol' and with the 'automation protocol' indicate no significant differences between both sets of data supporting the validity of the automation protocol.