Sanne de Bruin

165 Storage of RBCs in PAGGGM improves metabolism after transfusion but has no effect on PTR the PTR up to six hours after transfusion, also no difference in PTR between 2 and 35 days stored RBCs was found 30 . Here we show that, in concordance with previous published work, storage in PAGGGM resulted in better preservation of glycolysis, redox metabolism and purine metabo- lism 10,18 . However, that this did not result in an increased PTRwas unexpected, especially in light of recent double-blind randomized studies showing that improvements in met- abolic phenotypes correlate to improved PTR 31 . Analogously, boosting RBCmetabolism through storage in additive solution 7 (AS-7), which is a similar alkaline additive solution, showed an increased PTR 19,32 . The main difference between these additive solutions is that AS-7 contains bicarbonate instead of guanosine and gluconate. How this may affect RBC clearance remains unclear. After transfusion, the RBCs metabolic profile improved. This recovery can be explained by a technical bias of the study design, cells that are amenable to recover from storage, biotinylation and transfusion are those that survived intra- and extra-vascular haemoly- sis via splenic sequestration and phagocytosis. Recent studies have indeed shown that RBCs with the highest degree of morphological alterations (small microcytic erythro- cytes) are the most likely to be removed rapidly from circulation upon transfusion in humans and mouse models 33 . Acknowledged the potential survival bias of this study, we can also speculate on the effect of exposure to the physiological extracellular pH in the blood circulation after transfusion. After 35 days of storage the pH of RBC con- centrates decreased to approximately 6.5 18 while the pH in the circulation is 7.45 under physiological circumstances. By infusing the RBCs in the circulation, the PFK and G6PD activity may normalize and thereby restore the glucose flux through glycolysis and PPP, provided the enzymatic activities are not irreversibly compromised by oxidant stress-induced fragmentation of metabolism regulatory elements (e.g., N-terminus of band 3) or post-translational modifications, such as beta-elimination of thiols to de- hydroalanine or deamidation of asparagines – as reported for all glycolytic enzymes in end of storage units 34–39 . To investigate factors involved in RBC-clearance we assessed several eat me and don’t eat me signals. An increased C3 deposition was found in 35 days stored PAGGGM RBCs. It is unclear whether this concerns active C3, since also the deposition of the non-ac- tive component iC3b was increased. Furthermore, none of the other assessed markers differed significantly. This may be explained by the chosen timeframe in which we mea- sured thesemarkers. The phenotype of the donor RBCwas assessed up to six hours after transfusion, while in this timeframe the RBC clearance was limited. Another possibility 6