Molecular and flow cytometric analysis of the Vβ repertoire for clonality assessment in mature TCRαβ 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 V beta -C beta transcripts and flow cytometry with a V beta antibody panel covering more than 65% of V beta domains was validated using 28 SE-defined clonal T-cell receptor (TCR)alpha beta (+) T-ALL samples and T-cell lines. Next, the diagnostic applicability of the V beta RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal V beta -C beta RT-PCR products were detected in all 47 samples, whereas single VP 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 V beta monoclonal antibody reactivity that was confirmed by molecular data showing the usage of V beta gene segments not covered by the applied V beta 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 V beta -CP products and single V beta 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 antigen-activated cytotoxic T cells. It is concluded that molecular V beta analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper V beta antibody studies can be used as a powerful screening method for the detection of single V beta domain expression, followed by molecular studies in patients with more than 20% single V beta domain expression or large suspect T-cell populations (more than 50%-60%) without V beta reactivity, (C) 2001 by The American Society of Hematology.