Determination of 6-thioguanine resistant lymphocytes in human blood by immunohistochemical antibromodeoxyuridine staining

An immunohistochemical method for the determination of 6-thioguanine resistant (TGr) lymphocytes in human blood samples has been developed. The new technique is a modification of the autoradiographic assay and uses labelling of DNA synthesizing cells by bromodeoxyuridine (BrdU) instead of tritiated thymidine. The label is detected immunohistochemically by using a monoclonal antibody against BrdU in single-stranded DNA. Thawed cryopreserved isolated mononuclear cells are cultured with (TG cultures) and without (control cultures) TG for 40 h, the last 16 h with BrdU. The cells are harvested, fixed and put on microscopic slides; for the TG cultures, all material is used, while slides from the control culture are prepared at a dilution of 1:300. The immunohistochemical staining of the slides involves denaturation of DNA in formamide, incubation with anti-BrdU-DNA antibody, followed by a secondary antibody conjugated with peroxidase and staining with diaminobenzidine. Nuclei that have incorporated BrdU appear brown while all other material remains unstained. The number of all labelled nuclei is scored similarly in control and TG slides and the frequency of TGr lymphocytes is obtained by dividing the number of labelled nuclei in TG slides with that (multiplied by the dilution factor) in control slides. The anti-BrdU method will much simplify and speed up the microscopical evaluation of the TG variants and is very suitable for automation.