Sanne de Bruin

154 Chapter 6 a higher rate compared to six subjects in whom the 35 days stored RBCs were cleared at a higher rate. However, for the group as a whole there was no statistically significant difference between the ratio of fresh and stored RBCs before and after transfusion. To exclude any effect of the differential biotinylation on our results, half of each group received 35 days stored RBCs with the high-density biotin and the 2 days stored RBCs low density of biotin. The other half of the participants received RBCs that were labelled in the opposite manner. Our data revealed no effect of the density of biotin on the PTR (Figure 1D). Expression of “eat me” and “don’t eat me” signals after transfusion In addition to the analysis of the PTR, attempts were made to identify the occurrence of changes of the membrane of the transfused RBCs in time. Transfusion of stored RBCs offers a window in which a relatively large proportion of the RBC is cleared within a short time, thus allowing to detect changes on the RBC that precede their clearance, which may be linked to the clearance. In particular, the expression of several “eat me” and “don’t eat me” signals, which have been linked to increased phagocytosis of the RBCs in vitro, were tested. Among these were phosphatidylserine exposure, IgG binding, complement deposition and CD47 expression28. No changes in the expression of poten- tial “eat me” or “don’t eat me” signals were found, except for complement deposition on RBCs stored in PAGGGM. PAGGGM-RBCs showed significantly higher C3 deposition 10 minutes, 30 minutes, one hour and two hours after transfusion (supplement Figure S5A). However, also higher iC3b deposition was found on these time points (supplement Figure S5B). Increased glycolytic activity in PAGGGM RBCs Besides the PTR and the expression of several membrane markers related to RBC clear- ance, the metabolic profile of the donor RBC was also determined prior and after the transfusion. To this end, donor RBCs were isolated from whole blood samples after transfusion by a protocol in which the biotinylated RBCs were first enriched for by mag- netic bead isolation and then subjected to cell sorting by FACS. Using this protocol, pure populations of high and low biotinylated RBCs were extracted from blood samples of the healthy volunteers (Supplementary figure 1). After their sorting and processing, metabolomics was performed on the different cell fractions, and the metabolic profile of the RBCs was determined in time. As described before, glycolysis was more active during storage in PAGGGM (Figure 2).