Randomized trial of red cell washing for the prevention of transfusion-associated organ injury in cardiac surgery M.J. Woźniak, N. Sullo, S. Qureshi,

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Randomized trial of red cell washing for the prevention of transfusion-associated organ injury in cardiac surgery M.J. Woźniak, N. Sullo, S. Qureshi, W. Dott, R. Cardigan, M. Wiltshire, T. Morris, M. Nath, N. Bittar, S.K. Bhudia, T. Kumar, A.H. Goodall, G.J. Murphy British Journal of Anaesthesia Volume 118, Issue 5, Pages 689-698 (May 2017) DOI: 10.1093/bja/aex083 Copyright © 2017 The Author(s) Terms and Conditions

Fig 1 (A) CONSORT diagram of REDWASH trial. (B) Haemoglobin concentrations [mean (sd)] in samples collected from REWASH participants. (C) Number of red blood cell (RBC) units transfused during the trial, during cardiac surgery, and after surgery. (D) Histogram of the transfused RBC unit age distribution. British Journal of Anaesthesia 2017 118, 689-698DOI: (10.1093/bja/aex083) Copyright © 2017 The Author(s) Terms and Conditions

Fig 2 Inflammation and organ injury. (A) Serum interleukin-8 (IL-8). (B) Multiple organ dysfunction scores (MODS). (C) Arterial partial pressure of oxygen/inspired oxygen fraction ( P a O 2 / F I O 2 ) ratio. (D) Serum troponin. (E) Urine neutrophil gelatinase-associated lipocalcin (NGAL). (F) Serum creatinine clearance. Values represent the mean (sd). Findings were not different in prespecified per protocol and safety analyses. British Journal of Anaesthesia 2017 118, 689-698DOI: (10.1093/bja/aex083) Copyright © 2017 The Author(s) Terms and Conditions

Fig 3 Effects of washing on the red blood cell (RBC) storage lesion in vitro. (A) Fractional changes in CD235a/annexin V (AV)-positive microparticles during storage and after washing; eight RBC units were analysed. (B) Fractional changes in RBC-derived microparticles in 21-day-stored RBC bags in response to washing (6 units analysed before and 3 units after washing). (C) Cell-free haemoglobin (Hgb) in washed RBC units. (D) Haemoglobin concentrations after washing at day 21. (E) Adenosine triphosphate (ATP) concentrations in 21-day-old RBC bags before and after washing (6 units analysed before and 3 units after washing). (F) Osmotic fragility of 21-day-old RBCs (6 units analysed). Values represent means (sd). British Journal of Anaesthesia 2017 118, 689-698DOI: (10.1093/bja/aex083) Copyright © 2017 The Author(s) Terms and Conditions

Fig 4 Effects of red blood cell (RBC) washing on microparticle concentrations and platelet and leucocyte activation. (A) Microparticle concentration in collected plasma samples. (B and C) Fractional content of annexin V- (AV) and CD235a-positive microparticles in plasma samples. (D) Activation of monocytes measured by expression of CD64 on monocytes (CD163). (E) Expression of integrin αIIb/β3 (PAC-1) on activated platelets. (F) Expression of CD62P on platelets (CD41). Values represent means (sd). British Journal of Anaesthesia 2017 118, 689-698DOI: (10.1093/bja/aex083) Copyright © 2017 The Author(s) Terms and Conditions

Fig 5 Effects of washing on oxidative stress markers in REDWASH subjects. (A) Plasma cell-free haemoglobin. (B) Plasma total iron concentrations. (C) Serial haematocrit. (D) Total bilirubin. (E) Non-transferrin-bound iron. (F) Serum hepcidin. (G) Nitric oxide bioavailability. (H) Serum intercellular adhesion molecule (ICAM)-1. (I) Endothelial (CD144) microparticle concentrations in plasma. (J) Oxidative potential of cell-free haemoglobin (Hgb) derived from microparticle-free storage supernatant from stored RBCs (HS Sup) or derived from osmotically lysed stored RBCs or lysed fresh RBCs. ROS, reactive oxygen species. (K) Serum protein carbonylation. (L) Serum thiobarbituric acid reactive substances (TBARS, lipid peroxidation). Values represent means (sd). British Journal of Anaesthesia 2017 118, 689-698DOI: (10.1093/bja/aex083) Copyright © 2017 The Author(s) Terms and Conditions