HUMAN HEMATOPOIETIC-CELL LINES - A MODEL SYSTEM FOR STUDY OF MINIMAL RESIDUAL DISEASE DETECTION TECHNIQUE IN ACUTE-LEUKEMIA

Double immunofluorescence studies using both surface and cytoplasmic a ntigens were performed on cells of some human hematopoietic lines. We tested several permeabilization protocols in order to optimize, improv e and simplify flow cytometric assay to detect the combinations of two markers present in one cell which could be regarded as leukemia-relat ed markers. It was found, that buffered formaldehyde-acetone (BFA) fix ation renders the cell membrane permeable without destroying surface a ntigens so that intracellular and cell surface markers could be measur ed simultaneously by flow cytometry. Cell lines used for the experimen ts reported here included MOLT4 T cell line, mature B cell lines DAUDI and U-266, and early B cell line REH-6. Results from our studies demo nstrated, that in the absence of CD3 antigen on the surface membrane o f viable MOLT4 blast cells, double labeling of fixed, permeabilized ce lls revealed 97% mCD7+, cCD3+ double positive cells. Two color stainin g with anti-CD19 and anti-CD22 monoclonal antibodies (MoAbs) in DAUDI cells showed, that larger part of cCD22+ cells expressed mCD19 antigen . CD22 antigen was absent on DAUDI cell membrane. Of great interest wa s the finding, that the marker detected by anti-CD19 MoAb which was ab sent on the membrane of U-266 cells was detected in their cytoplasm. D ouble staining of these cells revealed, that the number of mCD22+, cCD 19+ double positive cells was 80%. Cytoplasmic CD22 antigen along with surface membrane CD19 was used to define early B cell line REH-6 as w ell. Our results demonstrate majority of double positive cells among t ested population (mCD19+, cCD22+). To our knowledge the presence of cy toplasmic IgM detectable by flow cytometry in REH-6 cells, which could be so regarded as a precise and adequate counterpart to pre-B acute l eukemia cell phenotype in children, is an original finding. Immunologi cal typing plays an important part in the multiple marker analysis of hematopoietic malignancies. Through these surface and cytoplasmic mark er combinations minor neoplastic cell populations could be detected. H uman hematopoietic cell lines could serve as a reliable model system f or monitoring minimal residual disease in acute leukemia patients.