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

Abstract

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 cytometric immunophenotyping has become a diagnostic tool for the analysis of normal and malignant leukocytes and it has proven to be a reliable approach for the investigation of minimal residual disease (MRD) in leukemia patients during and after treatment. In order to standardize the flow cytometric detection of MRD in acute leukemia, a BIOMED-1 Concerted Action was initiated with the participation of six laboratories in five different European countries. This European co-operative study included the immunophenotypic characterization and enumeration of different precursor and mature B cell subpopulations in normal bone marrow (BM). The phenotypic profiles in normal B cell differentiation may form a frame of reference for the identification of aberrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precursor-B-ALL) and may therefore be helpful in MRD detection. Thirty-eight normal BM samples were anal- yzed with five different pre-selected monoclonal antibody combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/ CD22/CD19, CD19/CD34/CD45 and TdT/CD10/CD19. Two CD19⁻ immature subpopulations which coexpressed B cell-associated antigens were identified: CD34⁺/CD22⁺/CD19⁻ and TdT⁺/CD10⁺/CD19⁻, which represented 0.11 ± 0.09% and 0.04 ± 0.05% of the total BM nucleated cells, respectively. These immunophenotypes may correspond to the earliest stages of B cell differentiation. In addition to these minor subpopulations, three major CD19⁺ B cell subpop- ulations were identified, representing three consecutive maturation stages; CD19^dim/CD34⁺/TdT⁺/CD10^bright/CD22^dim/ CD45^dim/CD38^bright/CD20⁻ (subpopulation 1), CD19⁺/CD34⁻/ TdT⁻/CD10⁺/CD22^dim/CD45⁺/CD38^bright/CD20^dim (subpopulation 2) and CD19⁺/CD34⁻/TdT⁻/CD10⁻/CD22^bright/CD45^bright/CD38^dim/ CD20^bright (subpopulation 3). The relative sizes 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 childhood 'immature' profile (large subpopulations 1 and 2/small subpopulation 3) to the adult 'mature' profile (small subpopulation 1 and 2/large subpopulation 3). When the immunophenotypically defined precursor-B cell subpopulations from normal BM samples are projected in fluorescence dot-plots, templates for the normal B cell differentiation pathways can be defined and so-called 'empty spaces' where no cell populations are located become evident. This allows discrimination between normal and malignant precursor-B cells and can therefore be used for MRD detection.