Siglec-2-Fc and SNA form clusters on RBC membrane. (A) Electron micrographs of Siglec-2-Fc (top panel) and SNA (bottom panel) on plasma membranes of RBCs. Siglec-2-Fc and SNA were both detected using QD655-conjugated secondary antibody (gray ovals, indicated by black arrow). The membranes were also labeled with A-, B-, and H-specific antibodies, detected with gold 5-nm–conjugated secondary (black dots, indicated by white arrow). Siglec-2-Fc and SNA bound the membranes in defined patches. The area of these patches was calculated using ImageJ 1.33 (NIH) software, by marking a polygon around each patch. The software calculated the area and center of mass for each polygon. The distance between each A and B antigen associated with the cluster center was easily derived. Bar indicates 50 nm. (B-C) Box plot of the area of Siglec-2 and SNA clusters, respectively. Enzymatic treatment of type A and B RBCs with A- and B-Zyme, respectively, reduced Siglec-2 and SNA cluster size significantly (P < .005) to resemble type O. (D) The percentage of A antigen (■) and B antigen (□) localized in the patch center. Error bars indicate SD. (E-F) Number of clusters per μM2 was calculated for Siglec-2-Fc and SNA, respectively. The number of Siglec-2-Fc clusters on blood type B membranes was reduced by 58% compared with type A (P = .01) and 43% compared with type O (P = .002). Average ± SD is shown. Statistical significance was determined by an unpaired, 2-tailed t test. N/S = not significant. Images were acquired with Philips CM10 transmission microscope.
