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Impact of donor biology on stored RBC metabolism
available for transfusion every year worldwide, the process comes at a significant cost in terms of RBC struc- tural2–4 and biochemical homeostasis.1 Some of these “storage lesions” are inevitable, since refrigeration tem- peratures negatively impact the activity of key enzymes regulating red cell energy and ion pump homeostasis.5 In small scale studies, the metabolic lesion has been repro- ducibly assessed to a quantifiable extent, which allowed the definition of metabolic markers of the so-called “metabolic age” of stored RBC.2,6 Despite the consistency of these laboratory observations, it is still a matter of debate whether the (metabolic) storage lesion could rep- resent an etiological contributor to (or a reliable predictor of) transfusion outcomes in the recipient.7 Indeed, stor- age-induced impairments in the homeostasis of high energy phosphate compounds adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) should nega- tively impact RBC capacity to bind and off-load oxygen upon transfusion.8 Depletion of ATP and DPG could rep- resent a concern in massively transfused patients, since the rate at which these compounds are replenished with- in the first 72 hours (h) upon transfusion may not be suf- ficient to meet the oxygen metabolic demands in severely hypoxic recipients.9,10 Studies in animal models and humans have shown that some small molecule metabo- lites could represent reliable correlates to Food and Drug Administration gold standards for stored blood quality, i.e., storage hemolysis11,12 and post-transfusion recov- ery.13,14 For example, metabolites like hypoxanthine, an ATP-breakdown and oxidation product, have been corre- lated to hemolysis and post-transfusion recoveries in mice and humans.13 Similar correlations have been report- ed for lipid oxidation products.14
Despite the overwhelming evidence from in vitro stud- ies, randomized clinical trials15 have hitherto failed to capture any signal associated with poorer outcomes when comparing transfusion of the freshest available units versus the standard of practice. On the other hand, a recent analysis of a linked donor and recipient database indicated that transfusion of RBC units less than 35 days old was associated with a higher recipient hemoglobin increment compared to transfusion of 35- to 42-day old RBC units.16 The apparent inconsistencies among the studies on the age of blood in the literature could be rec- onciled by the appreciation of the fact that RBC, like people, do not always age the same.17 In other terms, the molecular age of blood may be a distinct parameter from the storage age calculated in days since the time of dona- tion.18 Biological variability in donors19 and different RBC component processing strategies20 may for example impact hemoglobin oxygen saturation across donors/components,21 which in turn affects RBC suscep- tibility to oxidative stress during storage.13,22,23 Small-scale laboratory studies corroborated the hypothesis that RBC antioxidant capacity24 and storage-induced susceptibility to oxidative stress may indeed be donor-dependent.25 This statement holds true when considering some cate- gories of routinely accepted donors who are more sus- ceptible to storage-induced oxidative stress owing to common enzymopathies. For example, deficiency of glu- cose 6-phosphate dehydrogenase (G6PD) activity affects ~400 million people worldwide, including ~10% of the African American donor population in some metropoli- tan areas.26 These subjects are characterized by a decreased capacity to activate the pentose phosphate
pathway (PPP) and thus to generate the nicotinamide adenine dinucleotide phosphate (NADPH) necessary to reduce oxidized glutathione (GSH) and NADPH-depen- dent antioxidant enzymes.26 RBC from G6PD deficient donors are characterized by altered energy and redox metabolism,27,28 a feature that has been preliminarily associated with poorer capacity to circulate upon trans- fusion to sickle cell recipients29 and poorer post-transfu- sion recoveries in autologous volunteers.30 As such, pop- ulation screening in regions where the prevalence of G6PD deficiency is 3–5% or greater (in males) is recom- mend by the World Health Organization,31 but no specif- ic screening for G6PD activity is routinely in place for blood donors in the United States.
In the past few years, large scale studies have been designed to focus on the impact of donor biology on stor- age quality and transfusion outcomes. Within the frame- work of the National Heart Lung and Blood Institute (NHLBI, NIH) Recipient Epidemiology and Donor Evaluation Study (REDS)-III RBC-Omics study, four blood centers across the United States enrolled ~13,800 healthy donor volunteers of different ages, sex and eth- nicities. Preliminary analyses of the data obtained from this cohort allowed us to conclude that (i) donor sex (and testosterone levels32), age and ethnicity impact the hemolytic propensity of stored RBC;33,34 (ii) stored RBC from multiple units donated by the same donors have a similar propensity to hemolyze following pro-oxidant or osmotic insults;35 and (iii) the storage duration contributes to explain ~13% of the total metabolic heterogeneity of stored RBC, a percentage similar to the impact noted for storage additives in a subgroup of recalled donors from the original RBC-Omics cohort.36
In the light of this background, the continued character- ization of the impact of donor biology on storability and transfusion outcomes is a critical step towards the estab- lishment of personalized transfusion medicine practices.
Methods
REDS-III RBC-Omics study participants and samples
Donor selection and recruitment for the RBC-Omics study under approved protocols (BioLINCC Study: HLB02071919a) were previously detailed.35,37,38 Donors were enrolled at the four participating REDS-III US blood centers. Overall, 13,758 whole blood donors were enrolled and 13,403 (97%) age 18+ provided informed consent to participate in the study; of these, 8,502 were evaluated for oxidative hemolysis on RBC stored for ~39- 42 days. Extreme hemolysers (5th and 95th percentile) from the donors tested for end of storage oxidative hemolysis were asked to donate a second unit of blood. These units were sterilely sam- pled at storage day 10, 23 and 42 for oxidative hemolysis and metabolomics analyses (599 samples in total). Blood collection, sample processing and other aspects of the screening and recall phases of the RBC-Omics study have been extensively described.33,34
Oxidative hemolysis
Oxidative hemolysis was determined at the University of Pittsburgh and Vitalant Research Institute as reported and fur- ther detailed in references. Briefly, RBC were incubated with 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH, 150 mmoL) to determine susceptibility to oxidative hemolysis, as extensively described.33
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