Validation of the human T-lymphocyte cloning assay - ring test report fromthe EU concerted action on HPRT mutation (EUCAHM)

The T-cell cloning assay, which enables the enumeration and molecular analy sis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been ext ensively used for studying human somatic gene mutation in vivo. However, la rge inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental m ethodology, and development of an optimal but easy uniform cloning protocol . As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have car ried out two Ring tests for the T-cell cloning assay. For each test, duplic ate and coded samples from three buffy coats were distributed to five labor atories for determination of MF using sis different protocols. The results indicated a good agreement between split samples within each laboratory. Ho wever, both the cloning efficiencies (CEs) and MFs measured for the same bl ood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remar kable difference in the level of MF, A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring te st. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation. but decreased the CE in laboratories traditionally usi ng priming. Adjusted for donor, use of different protocols contributed sign ificantly to the overall variation in InCE (P = 0.0004) and InMF(P = 0.03), but there was no significant laboratory effect on the InCE (P = 0.38) or I nMF (P = 0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free mediumn X-Vivo 10 and PMA was tested in one la boratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations. (C) 1999 Elsevier Science B.V. All rig hts reserved.