Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal residual disease in precursor-B-ALL

During the last two decades, major progress has been made in the technology of flow cytometry and in the availability of a large series of monoclonal antibodies against surface membrane and intracellular antigens. Flow cytome tric immunophenotyping has become a diagnostic tool for the analysis of nor mal and malignant leukocytes and it has proven to be a reliable approach fo r the investigation of minimal residual disease (MRD) in leukemia patients during and after treatment. In order to standardize the flow cytometric det ection of MRD in acute leukemia, a BIOMED-1 Concerted Action was initiated with the participation of six laboratories in five different European count ries. This European co-operative study included the immunophenotypic charac terization and enumeration of different precursor and mature B cell subpopu lations in normal bone marrow (BM). The phenotypic profiles in normal B cel l differentiation may form a frame of reference for the identification of a berrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precu rsor-B-ALL) and may therefore be helpful in MRD detection. Thirty-eight nor mal BM samples were analyzed with five different pre-selected monoclonal an tibody combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/ CD22/CD19, CD19/ CD34/CD45 and TdT/CD10/CD19. Two CD19-immature subpopulations which coexpre ssed B cell-associated antigens were identified: CD34(+)/CD22(+)/CD19(-) an d TdT(+)/CD10(+)/CD19(-), which represented 0.11 +/- 0.09% and 0.04 +/- 0.0 5% of the total BM nucleated cells, respectively. These immunophenotypes ma y correspond to the earliest stages of B cell differentiation. In addition to these minor subpopulations, three major CD19(+) B cell subpopulations we re identified, representing three consecutive maturation stages; CD19(dim)/ CD34(+)/TdT(+)/CD10(bright)/CD22(dim)/ CD45(dim)/CD38(bright)/CD20(-) (subp opulation 1), CD19(+)/CD34(-)/ TdT(-)/CD10(+)/CD22(dim)/CD45(+)/CD38(bright )/CD20(dim) (subpopulation 2) and CD19(+)/CD34(-)/TdT(-)/CD10(-)/CD22(brigh t)/CD45(bright)/CD38(dim)/ CD20(bright) (subpopulation 3). The relative siz es of subpopulations 1 and 2 were found to be age related: at the age of 15 years, the phenotypic precursor-B cell profile in BM changed from the chil dhood 'immature' profile (large subpopulations 1 and 2/small subpopulation 3) to the adult 'mature' profile (small subpopulation 1 and 2/large subpopu lation 3). When the immunophenotypically defined precursor-B cell subpopula tions from normal BM samples are projected in fluorescence dot-plots, templ ates for the normal B cell differentiation pathways can be defined and so-c alled 'empty spaces' where no cell populations are located become evident. This allows discrimination between normal and malignant precursor-B cells a nd can therefore be used for MRD detection.