BACKGROUND: Rejuvenation of stored red blood cells (RBCs) increases levels of adenosine 5 0 -triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) to those of fresh cells. This study aimed to optimize and validate the US-approved process to a UK setting for manufacture and issue of rejuvenated RBCs for a multicenter randomized controlled clinical trial in cardiac surgery.STUDY DESIGN AND METHODS: Rejuvenation of leukoreduced RBC units involved adding a solution containing pyruvate, inosine, phosphate, and adenine (Rejuvesol, Zimmer Biomet), warming at 37 C for 60 minutes, then "manual" washing with saline adenine glucose mannitol solution. A laboratory study was conducted on six pools of ABO/D-matched units made the day after donation. On Days 7, 21, and 28 of 4 AE 2 C storage, one unit per pool was rejuvenated and measured over 96 hours for volume, hematocrit, hemolysis, ATP, 2,3-DPG, supernatant potassium, lactate, and purines added (inosine) or produced (hypoxanthine) by rejuvenation. Subsequently, an operational validation (two phases of 32 units each) was undertaken, with results from the first informing a trial component specification applied to the second. Rejuvenation effects were also tested on crossmatch reactivity and RBC antigen profiles. RESULTS:Rejuvenation raised 2,3-DPG to, and ATP above, levels of fresh cells. The final component had potassium and hemolysis values below those of standard storage Days 7 and 21, respectively, containing 1.2% exogenous inosine and 500 to 1900 μmoles/unit of hypoxanthine. The second operational validation met compliance to the trial component specification. Rejuvenation did not adversely affect crossmatch reactivity or RBC antigen profiles. CONCLUSION:The validated rejuvenation process operates within defined quality limits, preserving RBC immunophenotypes, enabling manufacture for clinical trials. Abbreviations: AHG = anti-human globulin; 2,3-DPG = 2,3-diphosphoglycerate; ATP = adenosine 5 0 -triphosphate; Hb = hemoglobin; HCT = hematocrit; NHSBT = National Health Service Blood and Transplant; RCMV = RBC microvesicle; SAGM = saline adenine glucose mannitol solution; XM = serologic tube technique without anti-human globulin.