A RAPID AND RELIABLE PCR METHOD FOR DETECTING CLONAL T-CELL POPULATIONS

Aims-To establish a reverse transcription polymerase chain reaction (R T-PCR) for the detection of clonal T cell populations, and to evaluate the sensitivity and reliability of the technique. Methods-After rever se transcription of the target RNA with a consensus T cell receptor (T CR) beta constant (C) region primer, consensus C, variable (V), divers ity (D) and joining (J) region primers were used to amplify across var ious portions of the TCR beta V-D-J-C junction. Results-In normal T ce lls the polyclonal rearrangements produce a ladder of PCR bands repres enting the different sized junction fragments. The presence of a T cel l clone leads to over-representation of one junction fragment, hence a disproportionately brighter band in the PCR ladder. In a series of 16 patients the RT-PCR detected nine of nine shown to have a clonal TCR beta rearrangement by Southern blotting and for six of seven patients, it confirmed the presence of a clone indicated by histology or immuno phenotyping with FAGS analysis, but which was undetectable (five patie nts) or not investigated (two patients) by Southern blotting. Investig ations mixing RNA from normal lymphocytes and the Jurkat TCR-V beta 8 T cell line suggested that the method was more sensitive than Southern blotting. Conclusions-All PCR methods are faster and easier than Sout hern blotting, but RT-PCR also improves detection of clonal T cell pop ulations, is reliable and produces results that are easy to interpret.