Flow cytometric analysis of band 3 Coimbra RBCs.
Cells were analyzed by flow cytometry using murine monoclonal anti–band 3 antibodies that were directly labeled with FITC (BRIC6) or were detected using FITC-labeled goat antimouse (Fab′)2fragments (BRIC71, BRIC90, BRIC200). In each histogram, dotted lines represent results obtained with Coimbra RBCs, and solid lines represent results obtained with the positive control. All 4 antibodies detected a single population of RBCs in the band 3 Coimbra sample. With BRIC6, BRIC90, and BRIC200, fluorescence intensity (FL1) was similar to that of the negative control antibody (BRIC169, directed against an intracellular band 3 epitope) and lower than the fluorescence intensity obtained with positive control cells, demonstrating that the epitopes recognized by these antibodies were absent on band 3 Coimbra RBCs. In contrast, band 3 Coimbra cells incubated with BRIC71 yielded a fluorescence intensity, FL1, that was also lower than that obtained with positive control cells but approximately 10-fold higher than that obtained with BRIC169, demonstrating that the epitope recognized by BRIC71 was expressed at a low level on all the band 3 Coimbra cells.
