Immunophenotyping of B lymphocytes by multiparametric flow cytometry in bone marrow aspirates of healthy adults.

Establishing reference ranges by multiparametric immunophenotyping of mature B cells in bone marrow of healthy adults is of interest because the detection of bone marrow infiltration and persistence of light chain restriction, as well as discrimination between reactive and malignant lymphocytes are important applications of B-cell immunophenotyping. To determine the pattern of antigens as expressed by malignant mature B lymphocytes in the present study, bone marrow aspirates of healthy adults were investigated for the presence and percentage frequency of those antigens as defined for immunophenotyping of B cells by the REAL Classification. For this purpose, analysis of CD19-positive B lymphocytes by 'live gate' analysis was performed. The established two-color as well as three-color stainings will serve as a basis for future investigations designed to test multiparametric analysis of B lymphocytes in bone marrow aspirates. All investigated antibodies stained with varying percentage frequency on B-cell subtypes, and no statistical significant difference was found between bone marrow aspirates of women and those of men. On the basis of this analysis, all the reported lineage antigen combinations are present both in malignant B lymphocytes and in normal B cells in considerable percentage frequency. These findings are of importance for follow-up investigations of patients with non-Hodgkin's lymphomas by multiparametric immunophenotyping.