Comparison of Multiplex Versus Non-Multiplex Primer Sets for the Detection of T-Cell Receptor- Gene Rearrangement
KP Patel, Q Pan, RW Maitta, J Du, H Ratech. Montefiore Medical Center, Bronx, NY
Background: Multiplex PCR assays are often associated with decreased sensitivity due to a competition between the primers. This can be compensated by including a non-multiplex (simplex, single pairs) primer set in the test panel. We evaluated clinical utility of combining a non-multiplex PCR assay with a recently described multiplex assay (BIOMED-2 protocol) for T-cell receptor (TCR)- gene rearrangement (GR).
Design: A total of 144 consecutive samples received for TCR GR were subjected to PCR analysis using two sets of primers: (1) Multiplex BIOMED-2 primers for TCR- and TCR- (van Dongen et al, 2003) and (2) Simplex primer sets for TCR- in a home-brew assay (Jaffe et al, 1995). PCR analysis for immunoglobulin heavy chain (IgH) GR was also performed on 62/144 cases. The results of TCR- GR by simplex and multiplex protocols were compared against those of TCR- GR. Clinical and histological correlations were included in the interpretation of GR studies.
Results: The samples included skin biospies (101/144), bone marrow (19/144), blood (9/144) and other tissue (15/144). The results of TCR- GR were considered as the gold standard for the evidence of TCR clonality. TCR- showed clonal GR in 51/144 cases. The multiplex and simplex primers detected clonality in 33/51 and 37/51 TCR- positive cases respectively, with 64.7% (multiplex) and 72.5% (simplex) sensitivity. Taken together, simplex and multiplex primers identified 41/51 TCR- positive cases (sensitivity=80%). Overall, multiplex and simplex primers were in agreement in 121/144 (84%) cases. Out of 23/144 discrepant cases, clonality was detected in 8 cases with multiplex and in 15 cases with simplex primers. Out of 15 cases positive with simplex assay, 8/15 cases showed clonal TCR- GR and 1/15 cases showed clonal IgH GR. The multiplex and simplex assay identified TCR- clonality in 16 and 19 cases respectively in TCR- negative (93/144) cases. Analysis of discordant cases showed a wide range of presentations emphasizing the need to interpret the GR studies in the context of clinical and histological presentation.
Conclusions: Combining the multiplex BIOMED-2 primers with a non-multiplex primer set for TCR- increased the sensitivity of the T cell clonality assay. In addition, an independent confirmation of a positive or a negative multiplex assay by a set of non-multiplex primers adds to the clinical utility of TCR GR assay. Our study also highlights the real-life experience with cases showing ambiguous clinicohistologic findings.
Tuesday, March 10, 2009 11:00 AM
Platform Session: Section G2, Tuesday Morning