Sanne de Bruin

151 Storage of RBCs in PAGGGM improves metabolism after transfusion but has no effect on PTR themagnetic field. This cell suspension, containing bioRBCs with two different densities biotin, was sorted using flowcytometry. After flow cytometry, the cell suspension was centrifuged (2500 RCF, 5 minutes, 4˚C), removing the supernatant. The cell pellet was stored at -80˚C until further analysis. Liquid chromatography mass spectrometry Metabolomics analyses were performed as extensively described in previous studies 22–24 . A volume of 50μl of frozen RBC aliquots was extracted in 450μl of methanol:acetoni- trile:water (5:3:2, v/v/v ). After vortexing at 4°C for 30min, extracts were separated from the protein pellet by centrifugation for 10min at 10,000g at 4°C and stored at −80°C until analysis. Ultra-High-Pressure Liquid Chromatography-Mass Spectrometry analyses were performed using a Vanquish UHPLC coupled online to a Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were analyzed using a 5-minute gradient as described. Solvents were supplemented with 0.1% formic acid for positivemode runs and 1 mM ammonium acetate for negativemode runs. MS acquisition, data analysis and elaboration was performed as described 25–27 . Statistical analysis Data was visually checked for distribution. Normally distributed and non- normally distributed data was reported as mean (standard deviation) or as median [first quar- tile-third quartile], respectively. Normally distributed data were analysed using the stu- dents t test or ANOVA analysis and non-parametric data was first ranked prior to analysis or analysed with the Wilcoxon-sum rank or Kruskal-Wallis test. Significance between two groups (SAGM versus PAGGGM and 2 days versus 35 days stored) was evaluated using a two-way ANOVA with grouping variables additive solution or storage time and time after transfusion. To assess the effect of additive solution, only the 35 days stored RBCs were included for analysis. P< 0.05 was considered to be statistically significant. Metabolomics analysis was considered to be exploratory, therefore no Bonferroni cor- rection was applied. As predefined in the trial registry (NTR6492), only glycolysis, pen- tose phosphate pathway and redox metabolism related metabolites were analysed. 35 days stored SAGM RBCs were compared with 35 days stored PAGGGM RBCs. Two days stored SAGM RBCs were compared with 35 days stored SAGM RBCs. Data was analysed using computer software (R studio version, R version 3.5.2). 6