EV relative size, absolute count, and composition in stored RBC units. (A) Day 0 isolated EVs were subject to flow cytometry, and FSC-SSC data were acquired on log mode. The same settings were used for data acquisition using 0.2-, 1.0-, and 3.2-μm polystyrene beads, and the plots show the overlay of EVs (gray) and beads (black), representative of 3 independent experiments. (B) Electron micrograph of day 0 EVs prepared by positive staining, representative of 2 independent experiments (bar represents 200 nm). (C) Six RBC unit aliquots were tested by flow cytometry on day 0, 21, and 42 of storage at 4°C for absolute count of EVs (*P < .05). (D) EVs binding lactadherin-FITC (4 samples), measured by flow cytometry. (E) The following markers were used for characterization of EVs: RBC: CD235a; platelet: CD41a; T cell: CD3; B cell: CD19; natural killer cell: CD16; monocyte: CD14; and endothelial cell: CD142. (F) The increase of platelet- and RBC-derived EVs was significant at day 21. At least 50 000 EV events were acquired at each time point, and 4 independent experiments were performed for each condition (*P < .05). (G) A total of 6 EV samples (day 0, 21, 42) were fractionated into small or large particles using 0.22-μm centrifugal filters. Beads (6 µm) were coated with EVs and blocked using bovine serum albumin. The negative control was beads incubated with only bovine serum albumin and no EVs. Immunostaining was performed with the exosome marker CD63 on the small EV fraction (<200 nm, exosomes) and the large EV fraction (>200 nm, microvesicles). The negative control is shown as a gray line and EVs as a dark line (*P < .05).