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 lymp hocytes. This automated system (Discovery Image Analyser, Becton-Dicki nson Image Cytometry Systems, Leiden, The Netherlands) searches for th e pattern of two touching nuclei in binucleate cells instead of the ed ges of the cytoplasm. For this purpose several ratios of the fixative mixture, methanol/acetic acid, were checked. After fixation with a rat io of 25:1, touching nuclei were obtained in almost 100% of the binucl eate cells. A hydrolysis treatment with 5 N HCl before staining with R omanowsky-Giemsa resulted in binucleate cells with dark-stained nuclei and micronuclei and a vaguely stained cytoplasm. The high visual cont rast between cytoplasm and nuclear material obtained by this staining procedure makes an accurate automated detection of micronulei feasible . Additionally, a 64 h culture time resulted in an optimal yield of bi nucleate cells. The results of manual micronucleus scoring an slides p repared with the 'manual protocol' and with the 'automation protocol' indicate no significant differences between both sets of data supporti ng the validity of the automation protocol.