PubMed2005
Malignant and reactive lymphoproliferations in most cases can be distinguished by histology and immunohistology alone; however, in T-cell lymphoproliferations and lymphoproliferations of unknown origin, histology often is inconclusive, and there is no reliable protein marker of malignancy. At the genomic level T-cell neoplasms clonally rearrange T-cell receptor (TCR) genes that can serve as clonal markers. In comparison with Southern blot analysis, polymerase chain reaction (PCR) techniques increasingly are being used to detect these rearrangements because PCRs are rapid, easy to perform, and can be used to amplify poor-quality deoxyribonucleic acid (DNA) from paraffin-embedded formalin-fixed biopsies. Nevertheless there are a number of problems associated with the detection of gene rearrangements using PCR. The foremost of these is improper primer annealing that will lead to false-negative or false-positive PCR results that may arise from poor primer design, especially for TCRB genes with an extensive variable (V), diversity (D), and joined (J) gene repertoire. There also can be difficulty in discriminating between clonal and polyclonal PCR products unless specific methods such as heteroduplex analysis or gene scanning are used. In this chapter, we describe methods, derived from a recent European collaborative BIOMED-2 program, for the detection of TCRG, B, and D rearrangements. TCRB VJ and DJ gene rearrangements are detected using 23 VB primers, 13 JB primers, and 2 DB primers in 3 multiplex tubes. TCRG VJ gene rearrangements are detected with four VG and two JG primers in two multiplex tubes, and TCRD VJ, VD, DJ, and DD rearrangements are detected with six VD primers, four JD primers, and two DD primers in one multiplex tube. Gaussian distributions of polyclonal PCR products are seen at specific size ranges for both TCRB and TCRG. Interpretation of TCRD rearrangements is more complex because of the wide range in PCR product size and often low numbers of TCRGD cells in target tissue. For all loci, some indication of gene usage can be ascertained by labeling the primers with different fluorochromes and gene scan analysis of PCR products. Furthermore the complementarity of the TCR loci affords an unprecedented high clonal detection rate. In addition, we also describe a set of control gene primers designed to amplify amplicons of 100, 200, 300, 400, and 600 bp for the assessment of the integrity and amplifiability of DNA.