Identification and quantification of a subpopulation of SMEs in a mouse model. (A) Posttransfusion recovery of long-stored RBCs (14 days of storage; lavender line; n = 7) is decreased at 2 and 24 hours after transfusion to control recipients compared with short-stored RBCs (1 day of storage; red line; n = 7). (B) Normalized frequency plots of projected surface area for fresh (purple line; n = 11) and short-stored RBCs (red line; n = 9) show similar patterns. (C) Long-stored RBCs (lavender line; n = 9) have a reduced projected surface area compared with short-stored RBCs (red line). (D) Quantification of projected surface area of front views of focused RBCs obtained by imaging flow cytometry shows a significant decrease in long-stored RBCs. (E) Representative analysis of brightfield images of long-stored RBCs shows that morphologically altered RBCs (dashed red line) have a reduced projected surface area compared with RBCs with normal morphology (blue line). Morphologically altered RBCs defined as type III echinocytes, spheroechinocytes, spherostomatocytes, and spherocytes, whereas normal RBCs comprised discocytes and type I and II echinocytes. (F) SMEs in short- and long-stored RBCs accumulate during storage. Data are presented as means ± standard errors of the mean. ***P < .001, ****P < .0001 by Sidak multiple comparisons test comparing, at each time point, recovery of short- vs long-stored RBCs (A), by Tukey multiple comparisons test comparing projected surface area for each condition (D), or by Student t test comparing proportion of SMEs in short-stored vs long-stored RBC (F). ns, not significant.