GPA, the major surface sialoglycoprotein on erythrocytes, engages Siglec-9 via sialic acids. (A) Treated (NaIO4/MTSC), mock treated (MTSC only), and untreated (DPBS) erythrocytes were evaluated for the binding of Siglec-9-Fc and analyzed by flow cytometry. Isotype control (goat anti-human IgG); n = 4. (B) Five human recombinant Siglec-Fcs were measured for their ability to bind immobilized GPA by enzyme-linked immunosorbent assay; n = 3. Statistics were analyzed by ordinary 1-way ANOVA. ****P < .0001 versus control values considered statistically significant. (C) Smears prepared immediately after drawing whole blood (left) or those prepared from buffy coat (right), fixed and costained for the erythrocyte GPA (green), Siglec-9 on neutrophils (red), and nucleus (Hoechst). Clustering (Merge) of GPA with neutrophils Siglec-9 upon contact (1 representative image of each shown). Scale bar, 10 μm. (D) Smears were stained similarly as in panel C and analyzed by confocal microscopy. Whole-blood smears (left) confirm polarized clustering of Siglec-9 on neutrophils toward GPA-covered red blood cells (4 representative patterns are shown). One staining pattern in which GPA colocalizes with Siglec-9 (lower left); individual GPA and Siglec-9 channels are in black and white. Scale bars, 5 µm. (E) Isotype controls were stained with streptavidin-PE, mouse IgG1-FITC, and Hoechst stain. Scale bar, 10 μm. (F) Siglec-9 clustering and nonclustering in neutrophils from epifluorescent blood smears were counted and normalized to the total number of Siglec-9 positive neutrophils. Representative images from panel C or pooled data percentage (normalized to Siglec-9 positive cells) are shown (+/− standard error of the mean). Statistical analysis was performed using ordinary 1-way ANOVA with Holm–Sidak's multiple comparison test. ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; O.D., optical densitiy; Sig, Siglec.
