M W Kent1, M R Kelher, F B West, C C Silliman. 1. Research Laboratory, Bonfils Blood Center, Denver, Colorado, USA; Department of Pediatrics, University of Colorado, Denver, Colorado, USA.
Abstract
BACKGROUND: Microparticles (MPs) are submicron size cell fragments that are released from cells. OBJECTIVES: We hypothesise that MPs increase during red blood cell (RBC) storage and are part of the pro-inflammatory activity, which accumulates in the RBC supernatant. METHODS/MATERIALS: RBC units were separated from whole blood of eight healthy donors: 5 U were split, with 50% undergoing leucoreduction (LR) and the remaining left as unmodified controls. The remaining 3 U were leucoreduced. Samples were obtained at days (D) 1 and 42 and cell-free supernatants separated and stored. The supernatants were centrifuged at 17 000 × g (60 min) or 100 000 × g (120 min) into microparticle-rich (MPR) and microparticle-poor (MPP) portions, resuspended in albumin, incubated with antibodies to CD235 (RBCs), CD45 [white blood cells (WBCs)] and CD41a [platelets (Plts)], and analysed by flow cytometry. Isolated neutrophils were incubated with these samples, and priming activity measured. RESULTS: Total MPs increased during storage; however, MPs that marked for precursor cell types did not. Significant priming accumulated in the MPP fraction during storage with some activity present in the MPR fraction from D1 and D42 LR-RBCs. CONCLUSION: Most of the pro-inflammatory priming activity from stored RBCs resides in the MPP supernatant, although the MPR fraction from D42 LR-RBCs does contain some priming activity.
BACKGROUND: Microparticles (MPs) are submicron size cell fragments that are released from cells. OBJECTIVES: We hypothesise that MPs increase during red blood cell (RBC) storage and are part of the pro-inflammatory activity, which accumulates in the RBC supernatant. METHODS/MATERIALS: RBC units were separated from whole blood of eight healthy donors: 5 U were split, with 50% undergoing leucoreduction (LR) and the remaining left as unmodified controls. The remaining 3 U were leucoreduced. Samples were obtained at days (D) 1 and 42 and cell-free supernatants separated and stored. The supernatants were centrifuged at 17 000 × g (60 min) or 100 000 × g (120 min) into microparticle-rich (MPR) and microparticle-poor (MPP) portions, resuspended in albumin, incubated with antibodies to CD235 (RBCs), CD45 [white blood cells (WBCs)] and CD41a [platelets (Plts)], and analysed by flow cytometry. Isolated neutrophils were incubated with these samples, and priming activity measured. RESULTS: Total MPs increased during storage; however, MPs that marked for precursor cell types did not. Significant priming accumulated in the MPP fraction during storage with some activity present in the MPR fraction from D1 and D42 LR-RBCs. CONCLUSION: Most of the pro-inflammatory priming activity from stored RBCs resides in the MPP supernatant, although the MPR fraction from D42 LR-RBCs does contain some priming activity.
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