The Pro-Inflammatory Activity of Microparticles in Red Blood Cell Concentrates (RBC)

RBC's were separated from the whole blood of 8 healthy donors and stored in AS-5 via current U.S. industry standards. The first 5 units were split into two equal aliquots (weight) with 50% undergoing standard leukoreuction (LR) and the remaining was left as an unmodified control (NLR). The other three units were processed as standard LR-RBCs. RBC samples were taken at days (D) 1 and 42 and cell-free supernatants were separated by centrifugation and stored (-80°C). The supernatant was centrifuged at 100,000g – 120 minutes into MP and MP free (MPF) portions and MP's resuspended in equal volume of 1.25 % HSA (MP-HSA) or cell-free plasma (MP-FFP). MP fractions were incubated with specific antibodies to RBCs (CD235), WBCs (CD45), and PLTs (CD41a) and analyzed by flow cytometry employing commercially obtained counting beads. Isolated neutrophils (PMNs) were incubated with the MP and MPF fractions [10%] _FINAL for 5 min at 37°C followed by activation of the NADPH oxidase with fMLF. The maximal rate of O₂⁻ production was measured as the SOD-inhibitable reduction of cytochrome c. Results were analyzed as an ANOVA with a post-hoc Newman Keuls test for multiple comparisons.

The total number of MP's increased during storage in all units irrespective of LR. The predominant MPs came from RBCs with PLTs representing the least (Table 1). Although the total number of MPs increased in all units, the number of MPs that specifically marked for precursor cell types decreased over storage (Table 1). The MPF fractions from stored (D.42) NLR-RBCs and LR-RBCs caused significant priming of the PMN oxidase vs. both the MPF from D.1 and the fMLF controls (Table 1). The MP fraction from NLR-RBCs did not evidence priming activity (data not shown); however, the LR-MP-HSA fractions from both D.1 and D.42 caused significant priming of the oxidase. Resuspension in plasma (LR-MP-FFP) inhibited this priming activity (Table 1). We conclude that during storage of NLR- and LR-RBCs, MPs increase; however the number of MPs associated with a specific cell type decrease but not significantly. This loss of cellular markers may be due to non-specific loss, antigen capping, internalization or proteolysis because proteases are released and increase during RBC storage. The presence of MPs from leukocytes and platelets are likely due to filtration, which is known to cause release of WBC- and PLT-derived proteins. While the majority of the PMN priming activity is in the acellular MPF supernatant in all NLR- and LR-RBC units, there is a modest amount of priming activity in the MP fraction following LR (LR-MP-HSA), which is likely a function of LR and may be inhibited by resuspension of the MPs in plasma.

Silliman:Pall Corporation: Honoraria, Research Funding.