Platelet vesicles are potent inflammatory mediators in red blood cell products and washing reduces the inflammatory phenotype.

BACKGROUND: Studies suggest that washing red cell concentrates (RCCs) to remove soluble mediators and/or inflammatory components, such as extracellular vesicles (EVs), may lead to better clinical outcomes. This study tested the hypothesis that non-red blood cell (RBC) generated vesicles in RCC are potent inflammatory mediators in vitro and washing RCCs can reduce these vesicles and subsequently decrease the inflammatory activity of RCCs. STUDY DESIGN AND METHODS: Sixteen RCCs were pooled and split into four groups based on pre-wash storage time (Day 2 or 14; n = 4/group). Each group was tested 24 hours and 7 days post-wash. Characteristics of RBCs and EVs, cytokines released by monocytes, and expression of human umbilical vein endothelial cells (HUVECs) adhesion molecules were assessed. RESULTS: All RCCs meet quality standards for hemolysis, hematocrit, and hemoglobin. Washing did not remove residual platelets from RCCs but led to a significant reduction in platelet-EV count regardless of the group. Supernatant of RCCs washed on Day 14 and stored for 24 hours had significantly lower concentrations of RBC-EVs and white blood cell EVs compared to unwashed controls. Supernatant of unwashed RCCs showed higher production of inflammatory cytokines/chemokines MCP-1, IL-8, and TNF-α, and heightened expression of HUVEC VCAM-1, which were significantly reduced by washing. Spiking washed RCC supernatants with platelet-EVs showed significant increase in IL-8, MCP-1, VCAM-1, and E-selection in groups washed on Day 14. CONCLUSIONS: Platelet-EVs in RCCs are associated with pro-inflammatory activity. As washing significantly reduced RCC immunomodulatory activity, implementation of this process may improve transfusion outcomes.

Evaluation of the functional properties of cryopreserved buffy coat-derived monocytes for monocyte monolayer assay.

BACKGROUND: Monocyte monolayer assay (MMA) is a compatibility testing method for evaluating the clinical significance of red blood cell (RBC) alloantibodies. Time-consuming monocyte isolation procedures and requirement for fresh monocytes have limited application of the MMA. The aim of this study was to develop and assess the utility and efficacy of cryopreserved buffy coat (BC)-derived monocytes for MMA application. STUDY DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMNCs) were isolated from BC or peripheral blood (PB) and pooled and BC PBMNCs were cryopreserved. Monocytes from pooled PBMNCs were incubated with anti-D-sensitized, anti-Scianna2 (Sc2)-sensitized, anti-AnWj-sensitized, or anti-Jra -sensitized RBCs or lipopolysaccharide (LPS). MMA phagocytic index (PI) and membrane integrity were determined microscopically, and cytokine release was measured by Luminex technology. RESULTS: PBMNC isolation rates from fresh BC and PB were not comparable (67.4 ± 6.3 and 75.8 ± 7.7% respectively, p = 0.024). There was no significant difference in PBMNC membrane integrity (fresh PB, 100%; fresh BC, 100%; cryopreserved BC, 95.2 ± 1.2%), postwash recovery (fresh PB, 85.9 ± 3.1; fresh BC, 86.9 ± 6.7; cryopreserved BC, 84.8 ± 5.1), or monocyte PI (fresh PB, 82 ± 10; fresh BC, 77 ± 11; cryopreserved BC = 80 ± 6). Monocytes from pooled cryopreserved BC PBMNCs reacted with RBCs sensitized with anti-D and RBC alloantibodies, including anti-Sc2, anti-Jra , and anti-AnWj. CONCLUSIONS: Monocytes from pooled cryopreserved BC PBMNCs can be used reliably to evaluate phagocytic responses of sensitized RBCs and to assess clinical significance of RBC alloantibodies.