A comparative analysis of protocols for detection of T cell clonality in formalin-fixed, paraffin-embedded tissue—implications for practical use

Analysis of T cell receptor (TCR) gene rearrangements is an important tool for the diagnosis of T cell non-Hodgkin lymphomas (NHL). A number of PCR-based T cell clonality protocols with increasing complexity in primer design have been published in the last decades. The multiplex TCRγ and TCRβ assays developed by the BIOMED-2 consortium have shown superior sensitivity for the detection of clonality in T-NHL. However, they have mainly been tested on fresh frozen tissues and may be difficult to interpret due to their complex design with multiple product size ranges. In this study, two relatively simple, first-generation TCRγ PCR protocols published by McCarthy et al. (Diagn Mol Pathol 1:173–179, 1992) and Trainor et al. (Blood 78:192–196, 1991) were compared with the BIOMED-2 TCRγ and TCRβ assays, using fluorescence-labeled primers and GeneScan analysis. FFPE samples of 52 peripheral T-NHL and 55 controls, including 20 B-NHL, were included. A 50-case subset including all samples with false negative or false positive results with the two TCRγ protocols was analysed additionally with BIOMED-2 TCRγ and TCRβ assays. With the combined BIOMED-2 assays, clonality was detected in four out of six previously false negative T-NHL and increased sensitivity for this selected subgroup to 92%, as compared to 64% (McCarthy) and 72% (Trainor). The overall specificity of 80% for BIOMED-2 assays was comparable to Trainor (84%) and McCarthy (88%) protocols, but incomplete TCRβ DJ rearrangements were identified in four out of ten B-NHL cases. In conclusion, BIOMED-2 TCRγ and TCRβ assays show superior sensitivity for the detection of T cell clonality. However, the complexity of BIOMED-2 protocols requires stringent quality control and experience in interpreting GeneScan patterns.