A comparison of multiplex and monoplex T-cell receptor gamma PCR.

Abstract

T-cell receptor gamma (TCRgamma) PCR is often used to detect clonal T-cell populations. Because TCRgamma contains a limited number of variable (Vgamma) and joining (Jgamma) regions, a small number of PCR primers can be used to assess T-cell clonality. The seven primers used in the current study were described previously and were split into 2 or 3 multiplex primer sets. In this study, a single 7-primer multiplex (7-plex) PCR reaction was compared with all 12 possible monoplex primer combinations on 18 samples previously analyzed for T-cell receptor rearrangements by TCRbeta Southern blot and/or TCRgamma PCR followed by temporal temperature gradient gel electrophoresis. Using fluorescent Vgamma-region primers, unlabeled Jgamma-region primers, and capillary electrophoresis, we show all TCRgamma rearrangements seen by 7-plex PCR on known positive samples were seen following monoplex PCR. However, additional TCRgamma gene rearrangements were seen in monoplex PCR reactions that were not seen in the 7-plex PCR reactions. Monoplex but not 7-plex PCR of known negative samples occasionally showed TCRgamma gene rearrangements, often with less frequently used Vgamma and Jgamma-region primers, and may have represented false positive results. In summary, the single 7-plex PCR reaction correctly identified specimens with TCRgamma clonal populations and represents an improvement over existing assays that use these same primers split into several smaller multiplex reactions. Monoplex PCR has no advantage over multiplex PCR and has the potential to lead to false positive results.