Blood group status affects Siglec-2-Fc-QDs binding to Sias on RBCs. (A) Top panel: each Siglec-2-Fc-QD complex appears as a single bright dot on the cell surface. Sialidase treatment (AUS; right panel) abolishes Siglec-2-Fc-QD binding to RBCs; type A RBC is shown as an example. Bottom panel: brightfield image of each cell. Bar represents 1 μM. (B) Box plot of Siglec-2-Fc-QD–binding density on RBCs from 3 type A, 3 type B, and 4 type O volunteers. Binding density was unique to each blood type (P < .0001). (C) Representative results for a single volunteer from each blood group are shown. Enzymatic treatment of type A and B RBCs with A-Zyme (α-N-acetylgalactosaminidase) and B-Zyme (α-d-galactosidase), respectively, changes Siglec-2-Fc-QD density (P < .01) to resemble type O. Statistical significance was determined by an unpaired, 2-tailed t test. n = Number of cells in each group; N/S = not significant. Box plot: Box shows the quartile 25 to quartile 75 range, the line represents the median value, and whiskers show the minimum and maximum values. Images were acquired with Olympus FV1000 confocal microscope. Z-sections were done at 0.15-μM steps. Image analysis and 3-dimensional reconstruction were done with ImagePro Plus software (Media Cybernetics) and ImageJ 1.33K software (NIH).