Component Development Laboratory, NHS Blood and Transplant, Cambridge, United Kingdom.
Manufacturing & Development, NHS Blood and Transplant, Bristol, United Kingdom.
Transfusion. 2019 Sep;59(9):2952-2963. doi: 10.1111/trf.15426. Epub 2019 Jul 11.
Rejuvenation of stored red blood cells (RBCs) increases levels of adenosine 5'-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.
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 ± 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.
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.
The validated rejuvenation process operates within defined quality limits, preserving RBC immunophenotypes, enabling manufacture for clinical trials.
储存的红细胞 (RBC) 的复壮会将三磷酸腺苷 (ATP) 和 2,3-二磷酸甘油酸 (2,3-DPG) 的水平提高到新鲜细胞的水平。本研究旨在针对英国环境对已获美国批准的复壮工艺进行优化和验证，以便在心脏手术的多中心随机对照临床试验中制造和发放复壮 RBC。
将含有丙酮酸盐、肌苷、磷酸盐和腺嘌呤的溶液添加到白细胞减少的 RBC 单位中（Rejuvesol，Zimmer Biomet），在 37°C 下孵育 60 分钟，然后用生理盐水腺嘌呤葡萄糖甘露醇溶液进行“手动”洗涤。在捐献后第二天对六个 ABO/D 匹配的单位池进行了实验室研究。在 4°C ± 2°C 储存的第 7、21 和 28 天，每个池中的一个单位进行复壮，并在 96 小时内测量体积、血细胞比容、溶血、ATP、2,3-DPG、上清液钾、乳酸和复壮过程中添加的（肌苷）或产生的（次黄嘌呤）嘌呤。随后进行了操作验证（两个 32 个单位的阶段），第一阶段的结果为第二阶段提供了试验组件规范的信息。复壮效果也在交叉配血反应性和 RBC 抗原谱上进行了测试。
复壮将 2,3-DPG 提高到并超过新鲜细胞的 ATP 水平。最终成分的钾和溶血值分别低于标准储存第 7 天和第 21 天的水平，分别含有 1.2%外源性肌苷和 500 至 1900 μmol/单位的次黄嘌呤。第二个操作验证符合试验组件规范。复壮不会对交叉配血反应性或 RBC 抗原谱产生不利影响。
经过验证的复壮工艺在规定的质量范围内运行，保持 RBC 免疫表型，为临床试验制造提供了可能。
