SOME EARLY DIFFERENTIATION MARKERS DETECTED IN CYTOPLASM OF PRE-B CELLS BY FLOW-CYTOMETRY

Immunofluorescent staining of cytoplasmic IgM (heavy chains) and CD24 as well as their simultaneous staining with surface B cell markers was used to study immunophenotype changes in B cell differentiation. Huma n hematopoietic B cell lines P3HR1 and RAJI were used. We found that I gM and CD24 cell markers while absent on cell membrane could be detect ed in their cytoplasm (c). The presence of cIgM in cell lines RAJI, P3 HR1 indicates their early pre-B differentiation stage. The presence of cCD24 simultaneously with mCD22 and cIgM is the evidence that hematop oietic cell lines dr leukemias may not accurately reflect normal diffe rentiation pathway. Combinations of cIgM, cCD24 with surface B cell ma rkers CD10, CD19 on these cell lines can be considered as leukemia ass ociated phenotypes. Some of them were shown in bone marrow and periphe ral blood of pre-B ALL and B-CLL patients and can be used for the dete ction of minimal residual disease. Different fixation/permeabilization methods were tested in order to choose the optimal one for simple det ection of cytoplasmic markers or their simultaneous detection with sur face markers by flow cytometry. They included ''one-component-methods' ' (methanol - M, saponin - S), methods combining these components with paraformaldehyde (P+M, P+S) or buffered formaldehyde acetone (BFA). T he choice depended on individual marker detected. General parameters l ike the proportion of debris, cell aggregation, cell loss and the chan ges of scatter parameters FSC and SSC were taken into consideration. T he priorities of combined methods P+S, P+M1 and BFA over one-component methods are demonstrated.