Microvesicle (MV) analysis in erythrocyte bags. (A) Annexin V–positive MVs in aging porcine blood bags (minimum of 3 units analyzed). (B) Fractional changes in microvesicle subtypes during storage and after washing and rejuvenation; 8 units were analyzed with indicated antibodies. (C) Schematics depicting a flow system used to analyze leukocytes and platelets in real time as described in Materials and Methods. (D) Effect of stored blood and MV fractions on leukocyte adhesion under flow in vitro (at least three independent repeats). Donor blood was mixed with phosphate-buffered saline (N/T), samples from whole red cell units that were stored for 35 days and washed (Wash), MV fraction (Supplemental Digital Content, https://links.lww.com/ALN/B560), or the high-speed centrifugation supernatant fraction (HS; Supplemental Digital Content, https://links.lww.com/ALN/B560) at 5:1 ratio. The microvesicle fraction was mixed with the donor blood at 1:10 ratio. (E) Effect of annexin V (AV) blocking on leukocyte adhesion. The values represent means ± SD. CTRL = control; pos = positive; Rej. = rejuvenation.