Characterization of P-RBC complex clearance from the bloodstream. (A) Platelets and RBCs were separately isolated from donor mice, labeled with CFSE and atto633, respectively, coincubated to generate P-RBC complexes (inset image; platelet, green; RBC, red), and then transfused into recipient mice, either untreated, pretreated with clodronate or phosphate-buffered saline (PBS) liposomes (phagocyte depletion), or splenectomized. Blood samples collected at indicated times after transfusion (hours posttransfusion [h.p.]) were analyzed by flow cytometry using the method depicted. Total (recipient plus donor) populations of RBCs (ter119⁺), platelets (CD41⁺), and P-RBC complexes (ter119⁺ CD41⁺) were gated and then separately analyzed for proportions of respective donor cells (atto633⁺, CFSE⁺, or atto633⁺ and CFSE⁺). The panels show the same mouse analyzed 0.17 (10 minutes) and 18 hp. (B) Proportions of transfused RBCs, platelets, and P-RBC complexes in the bloodstream of recipient mice measured up to 24 h.p. transfusion (left) and the clearance rates derived from these data (right). Levels and clearance rates of P-RBC complexes were significantly lower than those of platelets and RBCs (P < .0001), and platelet levels were significantly lower than RBC numbers at indicated time points (P < .001). Nine mice and 3 independent experiments. (C) Proportions of transfused RBCs, platelets, and P-RBC complexes in recipient mice treated 24 hours prior with clodronate or PBS (control) liposomes (left) and clearance rates derived from these data (right). Levels and clearance rates of P-RBC complexes were significantly higher in clodronate vs control mice (P < .001 and .0001, respectively); 10 mice and 2 experiments. (D) Proportions of transfused RBC, platelet, and P-RBC complex levels in recipient mice previously splenectomized (splenec.) compared with nonsplenectomized (intact) mice (left), clearance rates derived from these data (right), and (E) relative proportions of recipient (unlabeled) RBCs, platelets, and complexes; 3 and 4 mice, respectively. (B-D) Dashed lines indicate decay curves fitted using nonlinear regression. (B-E) Symbols and bars indicate means and SEM or individuals; comparisons shown using ANOVA. (F) P-RBC complexes in 20 population-matched spleen-intact individuals (healthy) and 16 patients measured before (pre) and 60 and 360 days (6 of 16 patients) after surgical removal of their spleen (mean, 95% confidence interval, and minimum and maximum values; comparisons shown using ANOVA), and (G) the P-RBC:platelet ratio for the same patients plotted longitudinally. Comparisons shown using Kruskal-Wallis test. Plt, platelet.