Flow cytometric determination of HPRT-variants in human peripheral blood lymphocytes.
Hypoxanthine guanine phosphoribosyl transferase (HPRT) deficient human peripheral blood lymphocytes are usually enumerated either by the cloning assay or by the autoradiographic short-term assay. The short-term approach presented here is based on flow cytometric (FCM) scoring of 6-thioguanine (6-TG) resistant lymphocytes. HPRT-variants are enumerated on the basis of both DNA synthesis (by use of immunofluorescent detection of incorporated 5-bromo-2-deoxyuridine, BrdU) and total DNA content (by propidium iodide (PI) incorporation) of proliferating cells, i.e. the cells must both be labelled with BrdU and reside in late-S or G2 phase in order to be scored as a HPRT-variant. This approach is combined with a stringent discrimination of false-positive events, minimising occurrence of phenocopies or other non-specifically labelled cells that might falsely be scored as true HPRT-variants. The HPRT-variant frequency (V(f)) found by the presented method varied between 0.8 x 10(-5) and 5.8 x 10(-5) for healthy male and female donors aged between 20 and 74 years. There was no significant gender difference in V(f). A strong linear correlation was found between HPRT-variant frequency and age, showing an increase of 0.56 x 10(-6) per year of age (r(2)=0.62, P<0.001). The frequencies of false-positive events found showed a mean of 0.22 x 10(-5) in comparison with a pooled mean V(f) of 2.87 x 10(-5). There was no significant age effect on the frequency of false events (r(2)=0.15, P<0.095). The method presented here may provide a rapid and sensitive alternative to the autoradiographic technique for the short-term enumeration of HPRT-variants.
Kotova
Natalia
N
Department of Radiobiology and Environmental Medicine, International Sakharov Environmental University, Dolgobrodskaya 23, Minsk 220009, Belarus.
Grawé
Jan
J
D000328:Adult; D000368:Aged; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D014644:Genetic Variation; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008214:Lymphocytes / Q000187:drug effects / Q000502:physiology*; D008297:Male; D008875:Middle Aged; D013866:Thioguanine / Q000494:pharmacology
D000964:Antimetabolites, Antineoplastic; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine; D001973:Bromodeoxyuridine