Flow cytometric determination of the frequency of circulating endothelial cells (CECs) in patients with MM compared with healthy control subjects. (A) Peripheral blood mononuclear cells were triple-labeled with anti-CD11b-Cy, anti-CD105-FITC, and either anti-CD34-PE or anti-CD146-PE; 100 000 cells were acquired and analyzed by flow cytometry. Forward-scatter and side-scatter plots were used to identify the mononuclear cell population. Measurements for FITC were carried out using the fluorescence-1 (FL1) detector (530/30 nm), PE was detected using the FL2 detector (585/42 nm), and CyChrome was detected using the FL3 detector (640 nm). Percent positive cells was compared with the percent of cells positive in staining with PE- or FITC-labeled IgG isotype controls; polyfluorescent beads (DAKO FluoroSpheres; DAKO, Glostrup, Denmark) were used to standardize the cytometer. The percentages of the CD11b^- cell population expressing the indicated double antigens from a representative patient and a control subject are shown in each quadrant. In statistical analyses, CD11b^- cells expressing CD105 and CD146 were considered CECs. (B) The percentages of CECs in PBMCs from 26 patients with MM (closed circles) and 12 healthy control subjects (open circles) are shown. Horizontal bars indicate the mean values for each group. Results are the mean of at least 2 FACS determinations performed at least 24 hours apart for each person. Group comparisons were made by Student t test, 2-tailed.