The transfusion of red blood cells (RBC) is associated with adverse outcomes in critically ill patients. One hypothesis explaining this observation involves the accumulation of potentially inflammatory/prothrombotic microparticles (MPs), released from cellular membranes during unit processing and storage. Despite their implication, the exact nature of MPs within RBC units remains incompletely defined. Here, we characterized the MP phenotypes present within stored RBC units and quantified the various populations. We directly sampled 11 O-positive non-leukoreduced RBC unit bags and isolated MPs by sequential centrifugation. We used a two-step process to detect MPs by flow cytometry. First, we used commercially available Megamix beads to define MPs according to their size during flow cytometric analysis and Trucount beads to quantify MP counts/mL (reported as median [IQR]). MPs ≤1 μm in size were subsequently classified into the following populations: total annexin V-positive, tissue factor-positive (CD142), and those staining for RBC- (intra/extracellular domain-specific glycophorin A, and band-3), leukocyte- (CD45), platelet- (CD42b), and endothelial-specific (CD144) markers. The total concentration of MPs was 1.52×109 MPs/mL [1.39×109]; however, only 4.3% were Annexin V-positive (5.16×107 MPs/mL [7.27×107]), and 11.7% were tissue factor-positive (4.24×107 MPs/mL [1.11×108]). Likewise, MPs with leukocyte-, platelet-, and endothelial-specific markers accounted for only 0.05% (4.44×105 MPs/mL [7.83×105]), 0.17% (1.99×106 MPs/mL [3.11×106]), and 0.06% (7.58×105 MPs/mL [8.04×105]), respectively (Figure 1). The amount of RBC-derived MPs varied depending on the antibody used. While only 4.2% of MPs (3.40×107 MPs/mL [2.91×107]) stained positive for the extracellular domain of glycophorin A, 41.3% (8.20×108 MPs/mL [4.06×108]) and 20.6% MPs (3.03×108 MPs/mL [3.53×108]) stained positive for the cytoplasmic domain of glycophorin A and band-3 respectively (Figure 1). The concentration of MPs positive for the cytoplasmic domain of glycophorin A was significantly greater their extracellular domain-specific counterparts (p<0.01). The results indicate that MPs in stored RBC units are primarily derived from RBCs, but also from other blood cells and vascular endothelial cells. A strong stain for the cytoplasmic domains of two RBC markers (cytoplasmic domain-specific glycophorin A and band 3) suggest the presence of a unique inside-out MP phenotype, potentially derived by RBC membrane “flipping” during microvesiculation. Ongoing studies are required to confirm these results and focus on 1) the mechanism of MP formation and (patho)physiological significance of inside-out RBC MPs and 2), the association between these MPs and the adverse effects seen in patients receiving RBC transfusions.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.