Novel flow-cytometric analysis based on BCD5+ subpopulations for the evaluation of minimal residual disease in chronic lymphocytic leukaemia.

We describe a new flow-cytometric analysis using quadruple labelling with anti-CD19, CD20, CD5, CD79b monoclonal antibodies and sequential gating. We determined a novel criteria defined by BCD5+CD79b-/low/total BCD5+ cells ratio (BCD5+R), and compared it with the previous definition of phenotypic remission, based on CD19+CD5+ coexpression, and with complementarity-determining region 3 polymerase chain reaction (CDR3 PCR) and clonotypic PCR (cPCR). A series of 54 peripheral blood samples from 21 chronic lymphocytic leukaemia (CLL) patients in complete haematological remission and a series of 16 from normal volunteers were analysed. In normal controls, the BCD5+R was always < 0.2. The sensitivity of the BCD5+R was 1 x 10-4vs 5 x 10-2 for CDR3 PCR and 1 x 10-5 for cPCR. Among the 54 CLL samples, 35 had a BCD5+R < 0.2 and showed polyclonal CDR3 PCR, whereas the cPCR was positive in 12 out of 20 tested. In the remaining 19 samples, BCD5+R was > 0.2, CDR3 PCR was monoclonal in 16 out of 19 and cPCR positive in 14 out 14 tested, including one out of three samples with polyclonal CDR3 amplification. Even though cPCR remains the most sensitive method to evaluate MRD, this new, sensitive and specific flow cytometric parameter, the BCD5+R, is more suitable than CDR3 PCR for routine clinical MRD assessment in CLL.