Flow cytometric determination of HPRT-variants in human peripheral blood lymphocytes

Abstract

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 G 2 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 0.8×10 −5 and 5.8×10 −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 0.56×10 −6 per year of age ( r 2=0.62, P<0.001). The frequencies of false-positive events found showed a mean of 0.22×10 −5 in comparison with a pooled mean V f of 2.87×10 −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.