Sanne de Bruin

147 Storage of RBCs in PAGGGM improves metabolism after transfusion but has no effect on PTR The study was approved by the Institutional Review Board of the Academic Medical Center, Amsterdam the Netherlands and registered in the Dutch trial register (NTR6492). Written informed consent was obtained from each subject prior to screening. Blood donation Twenty subjects donated 500 mL and 200 mL of whole blood in citrate-phosphate-dex- trose at the national blood bank 35 and 2 days prior to the transfusion, respectively. After overnight storage at 20 – 24°C, the units whole blood were processed according to the Dutch Blood Bank standards: separated into plasma, buffycoat and leukoreduced RBCs. RBC storage was randomly allocated to saline-adenine-glucose-mannitol (SAGM, 150 mmol/L NaCl, 1.25 mmol/L adenine, 50 mmol/L glucose, 29 mmol/L mannitol, pH 5-6, Fresenius Kabi) or the experimental additive solution (PAGGGM, 1.44 mmol/L ade- nine, 1.44 mmol/L guanosine, 47.5 mmol/L glucose, 40 mmol/L Na-gluconate, 8 mmol/ NaH 2 PO 4 ·2H 2 O, 8 mmol Na 2 HPO 4 ·2H 2 O, 55 mmol/L mannitol, pH 8-9). Blood was stored according to standard blood bank practice until biotinylation. Biotinylation of red blood cells One day prior to transfusion, 25 ml of each donation was labelled with 15 µg /ml (low density) or with 48ug/ml (high density) biotin. Directly after labelling, the biotinyla- tion was checked using flow cytometry, to ensure that the different populations were distinguishable. To exclude any effect of biotin label concentration, half of each group received fresh RBCs labelled with a low biotin concentration and stored RBCs labelled with a high concentration. The other half received fresh RBCs labelled with a high con- centration biotin and stored RBCs labelled with a low concentration of biotin. All units were biotinylated in a closed system according to methods described previously 20 . In short, biotin (EZ-link Sulfo-NHS-LC-Biotin, 100mg; Thermo scientific) was diluted in SAGM (Fresenius Kabi) in two different concentrations: 48 µg /ml and 15 µg /ml, and sterilized using a 0.22 µ filter (Fresenius HemoCare, Fresenius Kabi) in a closed system using a sterile connection device (Sterile TurbingWelder-Terumo BCT, TSCD II). A portion of 25 mL red cell concentrate from each donation was transferred to a sample bag in a closed system and washed once with 125 mL SAGM (2000 RPM with 5.00X10 7 ACE) to avoid binding of the biotin to plasma proteins. Supernatant was removed with a plasma clamp, after which the cell pellet was resuspended in biotin diluted in SAGM and incu- bated for 60 minutes at 22˚C. The biotinylated RBCs (bioRBCs) were washed twice with 125 mL SAGM (2000 RPM with 5.00X10 7 ACE). After removing the supernatant, the cell pellet of the 2 days stored RBCs was resuspended in SAGM to its original haematocrit. 6