Sanne de Bruin

148 Chapter 6 35-days stored bioRBCs were resuspended in their allocated additive solution (SAGM or PAGGGM). Study day Subjects were admitted for one day at the research unit of the Department of Intensive Care. Prior to the study procedures, a research physician medically checked the sub- jects and blood was cross matched as additional safety procedure. Fresh and stored bioRBCs were mixed prior to infusion in a closed system. 50 mL mixed bioRBC sample was infused within ten minutes. The infusion system was flushed with 100 mL 0.9% saline (Baxter, Viaflo). Ten minutes, 30 minutes, 1, 2, 4, 6 and 8 hours after infusion blood samples were drawn from an indwelling venous cannula and stored in the dark at 4˚C until further analysis. Temperature, blood pressure, heart rate and respiration rate were monitored up to eight hours after transfusion. Whole blood samples were drawn using EDTA tubes (BD Plymouth, United Kingdom). In the hospital’s general laboratory of clinical biochemistry, total bilirubin, lactate dehy- drogenase (LDH), aspartate aminotransferase (ASAT) and haptoglobin were measured with the Roche cobas c702 chemical analyser (Roche Diagnostics, Indianapolis, USA). Follow up: All study subjects were followed for 90 days after transfusion: blood was sampled 1, 2, 7, 30 and 90 days after transfusion. On return visits, blood samples were collected using venipunctures. All samples were stored in the dark at 4˚C until further analysis. Antibodies The following antibodies were used in our study: CD44 (conjugate FITC, Diaclone, prod- uct code: 852601010, 1:5), CD47 (2D3) (conjugate FITC, eBioscience, San Diego, USA, product code: 11-0478-42, 1:5), CD47 (B6H12) (Santa Cruz Biotechnology,sc-12730, 1:250), CD55 (conjugate FITC, Sanquin, Amsterdam, The Netherlands, product code: M2192, 1:5), CD59 (conjugate FITC, Diaclone, Besançon, France, product code: 954361010, 1:5), CD147 (conjugate PE, eBioscience, San Diego, USA, product code: 12-1472-42, 1:10), ICAM-4 (Abnova, Taipei, Taiwan, product code: H00003386-B01P, 1:20), Band 3 (EMA staining) (conjugate FITC/PE, Biotum, Fremont, USA, product code: 92013, 25 µl (undiluted)), Annexin V (conjugate FITC, VPS diagnostics, Hoeven, The Netherlands, product code: A705, 1:200), Lactadherin (conjugate FITC, Bio Connect, Huissen, The Netherlands, prod- uct code: BLAC-FITC, 1:100), Anti-lactadherin (conjugate PE, R&D systems, Minneapolis,