The selection of various T-cell receptor (TCR) gene families and complex rearrangements during intra-thymic differentiation provide the basis for the expression of antigen specificity by mature T cells. TCR beta variable (TCRBV) transcripts can be identified by RT-PCR, but multiple reactions are required to detect all genes of the TCRBV subfamilies. We describe here a multiplex PCR method that amplifies 46 functional genes comparing 23 TCRBV families in 5 reactions where each reaction contains 4 to 7 specific primers together with a single fluorescence-tagged TCR beta constant region primer. Between 8 and 10 distinct subtypes within each of the 23 TCRBV families can be identified by analysis of the CDR3 length. Multiplex PCR products isolated from agarose gels can be subjected to direct sequencing for confirmation and definitive clonotyping if necessary. The data illustrated here show that the multiplex PCR technique is useful for screening TCRBV usage and can be easily adapted for analysis of clonal composition in T-cell populations.

Detection of clonal T cell receptor ␥ (TCRG) gene rearrangements by PCR is widely used in both the diagnostic assessment of lymphoproliferative disorders and the follow-up of acute lymphoblastic leukaemia (ALL), when residual positivity in excess of 10 −3 at morphological complete remission is increasingly recognised to be an independent marker of poor prognosis. This is largely based on specific detection of V-J rearrangements from childhood cases. We describe rapid, multifluorescent V␥ and J␥ PCR typing of multiplex amplified diagnostic samples, as applied to 46 T-ALL. These strategies allow selected analysis of appropriate cases, immediate identification of V␥ and J␥ segments in over 95% of alleles, improved resolution and precision sizing and a sensitivity of detection at the 10 −2 -10 −3 level. We demonstrate preferential V-J combinations but no difference in V-J usage between children and adults, nor between SIL-TALI-negative and -positive cases. A combination of fluorescent multiplex and V␥-J␥-specific monoplex follow-up, as described here, will allow detection of both significant clonal evolution and of the diagnostic clone at a level of prognostic significance, by techniques which can readily be applied to large-scale prospective studies for which real-time analysis is required.

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