Differences in Siglec2-Fc binding do not reflect RBC aging. Human RBCs of A, B, and O blood groups were double labeled with Siglec-2-Fc, followed by secondary antibody (goat anti–mouse FITC), and with thiazole orange, which interacts with RNA that is found only in the young reticulocytes. Cells were analyzed by flow cytometry, and the fluorescence intensity of Siglec-2-Fc labeling is shown. (A) Siglec-2-Fc/FITC binding on type A RBCs (gray) was higher than that measured on type B (black) and type O (dark gray). (B) Thiazole orange–positive cell fraction comprising 1% to 1.5% of total RBCs (reticulocytes, black) was slightly shifted to the right, but the width of the histogram is similar to that of the thiazole orange-negative cells (mature RBCs, gray). Fifty thousand events were collected on a FACSCalibur flow cytometer with CellQuest software. Postcollection analysis was done with FlowJo software (TreeStar). The experiment was repeated 3 times with blood from 9 volunteers.