Cell-surface expression of CD44-3v, -6v, and -9v containing isoforms on normal myeloid cells. (A) Expression of CD44-6v on CD34⁺ cells. CD34⁺ cells were isolated from normal BM by specific immunoadsorption, as described in Materials and Methods. Histogram 1 defines the morphologic gate. Cells were double-stained with the anti-6v MoAb VFF18 plus GAM-PE and FITC-conjugated HPCA-2 MoAb directed to CD34 (histogram 2). Quadrant limits defining positivity and negativity have been set up using cells labeled with PE-conjugated and FITC-conjugated control antibodies (histograms 3 and 4), as described in Materials and Methods. Adjustment of the crossover fluorescence was obtained by compensation of the two single-stained samples to limit superposition of the fluorochrome emission spectra. (B) CD34⁺ cells and monocytic cells. CD34⁺ cells, very immature hematopoietic cells; MP, monocytic precursors (CD34^neg, CD14⁺ BM cells); Mature monocytes, circulating CD14⁺ cells. Mature monocytes from PB were identified according to their forward angle and side scatter.37 CD34⁺ cells and MP were double-stained with (1) an FITC-conjugated MoAb directed to CD34, which defines very immature hematopoietic cells, and CD14, a monocyte-specific antigen, respectively and (2) MoAbs directed at CD44-3v (BBA11), -6v (VFF-18), and -9v (11-24), plus PE-conjugated goat anti-mouse IgG. Negative controls (gray lines) were cells labeled with FITC and PE-conjugated isotype-matched control antibodies. Flow cytometric analysis was performed as described in Materials and Methods. Four independent experiments gave similar results. HPC were negative for CD44-3v and CD44-9v proteins.