Molecular and flow cytometric analysis of the Vb repertoire for clonality assessment in mature TCRab T-cell proliferations

Clonality assessment through Southern blot (SB) analysis of TCRB genes or polymerase chain reaction (PCR) analysis of TCRG genes is important for diagnosing suspect mature T-cell proliferations. Clonality assessment through reverse transcription (RT)–PCR analysis of Vb-Cb transcripts and flow cytometry with a Vb antibody panel covering more than 65% of Vb domains was validated using 28 SB-defined clonal T-cell receptor (TCR)ab1 T-ALL samples and T-cell lines. Next, the diagnostic applicability of the Vb RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal Vb-Cb RT-PCR products were detected in all 47 samples, whereas single Vb domain usage was found in 31 (66%) of 47 patients. The suspect leukemic cell populations in the other 16 patients showed a complete lack of Vb monoclonal antibody reactivity that was confirmed by molecular data showing the usage of Vb gene segments not covered by the applied Vb monoclonal antibodies. Nevertheless, this could be considered indirect evidence for the “clonal” character of these cells. Remarkably, RT-PCR revealed an oligoclonal pattern in addition to dominant Vb-Cb products and single Vb domain expression in many T-LGL proliferations, providing further evidence for the hypothesis raised earlier that T-LGL derive from polyclonal and oligoclonal proliferations of antigenactivated cytotoxic T cells. It is concluded that molecular Vb analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper Vb antibody studies can be used as a powerful screening method for the detection of single Vb domain expression, followed by molecular studies in patients with more than 20% single Vb domain expression or large suspect T-cell populations (more than 50%-60%) without Vb reactivity. (Blood. 2001;98: 165-173)