Bromodeoxyuridine labelling as an alternative method to identify 6-thioguanine-resistant mutant lymphocytes in humans

6-Thioguanine-resistant (TG(R)) mutant lymphocytes in human blood are usual ly enumerated by the cloning assay which allows the molecular characterisat ion of the HPRT mutations to be detected. A "short-term" alternative approa ch is provided by the anti-bromodeoxyuridine (anti-BrdU) technique in which TG(R) lymphocytes are identified immunocytochemically by their ability to synthesise DNA in the presence of 6-thioguanine (TG). We have evaluated the influence of various experimental factors that could affect the frequency of TG(R) lymphocytes. A standard protocol is proposed, based on 24-h cold s torage of isolated lymphocytes at 4 degrees C and 40-h culture with and wit hout TG, the last 16 h with BrdU. The harvested cells are treated with hypo tonic (0.075 M) KCl, fixed with methanol:acetic acid (3:1) and put on micro scopic slides. For the TG cultures, all cells are prepared on the slides, w hile slides from the control cultures are made by a 1/50 dilution. DNA is d enatured by formamide, and the BrdU label is identified by anti-BrdU antibo dy detected by immunoperoxidase staining using a peroxidase-conjugated seco ndary antibody with diaminobenzidine as substrate. In 10 donors, the freque ncy of TG(R) lymphocytes (variant frequency, V-f) detected by this protocol ranged from 69.65 X 10(-6) to 83.45 X 10(-6), and split measurements showe d a relatively small intra-assay variation in V-f values of each donor. Brd U in DNA was also detected by immunofluorescence using a fluorescein-conjug ated anti-BrdU monoclonal antibody. This method, facilitating easy identifi cation of positive cells and rapid microscopic scoring, may serve as a basi s for an automated analysis of TG(R) lymphocytes. V-f values detected by th e anti-BrdU assay are higher than mutant frequencies obtained by the clonin g assay, which has been assigned to the presence of non-mutant phenocopies considered to represent spontaneously cycling lymphocytes. Although the ant i-BrdU assay is rapid and easy and has been shown to respond to genotoxic e xposures, its hue value could be evaluated only when it can be ascertained that phenocopies do not significantly contribute to the V-f values obtained . (C) 1999 Elsevier Science B.V. All rights reserved.