Estimated median density identifies donor age and sex differences in red blood cell biological age.

BACKGROUND: Donors possess heterogeneous red cell concentrates (RCCs) in terms of the biological age of their red blood cells (RBCs) as a direct result of various donor-dependent factors influencing rates of erythropoiesis. This study aimed to estimate the median biological age of RBCs in RCCs based on donor age and sex to investigate inherent differences in blood products' biological ages over hypothermic storage using estimated median densities (EMDs). STUDY DESIGN: Sixty RCCs were collected from four donor groups; male and female teenagers (17-19 years old) and seniors (75+ years old). A Percoll density-based separation approach was used to quantify the EMDs indicative of biological age. EMD and mean corpuscular hemoglobin (MCHC) were compared by correlation analyses. RESULTS: Differences in the median biological age of RCC units were observed with male donors having significantly higher EMDs compared to females (p < .001). Teen male donors possessed the highest EMDs with significantly elevated levels of biologically aged RBCs compared to both female donor groups, regardless of storage duration (p < .05). Throughout most of the 42-day storage period, senior donors, particularly senior females, demonstrated the strongest correlation between EMD and MCHC (R2 > 0.5). CONCLUSIONS: This study provides further evidence that there are inherent differences between the biological age profiles of RBCs between blood donors of different sex and age. Our findings further highlight that biological age may contribute to RBC quality during storage and that donor characteristics need to be considered when evaluating transfusion safety and efficacy.

Functional effects of an African glucose-6-phosphate dehydrogenase (G6PD) polymorphism (Val68Met) on red blood cell hemolytic propensity and post-transfusion recovery.

BACKGROUND: Donor genetic variation is associated with red blood cell (RBC) storage integrity and post-transfusion recovery. Our previous large-scale genome-wide association study demonstrated that the African G6PD deficient A- variant (rs1050828, Val68Met) is associated with higher oxidative hemolysis after cold storage. Despite a high prevalence of X-linked G6PD mutation in African American population (>10%), blood donors are not routinely screened for G6PD status and its importance in transfusion medicine is relatively understudied. STUDY DESIGN AND METHODS: To further evaluate the functional effects of the G6PD A- mutation, we created a novel mouse model carrying this genetic variant using CRISPR-Cas9. We hypothesize that this humanized G6PD A- variant is associated with reduced G6PD activity with a consequent effect on RBC hemolytic propensity and post-transfusion recovery. RESULTS: G6PD A- RBCs had reduced G6PD protein with ~5% residual enzymatic activity. Significantly increased in vitro hemolysis induced by oxidative stressors was observed in fresh and stored G6PD A- RBCs, along with a lower GSH:GSSG ratio. However, no differences were observed in storage hemolysis, osmotic fragility, mechanical fragility, reticulocytes, and post-transfusion recovery. Interestingly, a 14% reduction of 24-h survival following irradiation was observed in G6PD A- RBCs compared to WT RBCs. Metabolomic assessment of stored G6PD A- RBCs revealed an impaired pentose phosphate pathway (PPP) with increased glycolytic flux, decreasing cellular antioxidant capacity. DISCUSSION: This novel mouse model of the common G6PD A- variant has impaired antioxidant capacity like humans and low G6PD activity may reduce survival of transfused RBCs when irradiation is performed.

Red cell concentrates from teen male donors contain poor-quality biologically older cells.

BACKGROUND AND OBJECTIVES: Donor factors influence the quality characteristics of red cell concentrates (RCCs) and the lesions that develop in these heterogeneous blood products during hypothermic storage. Teen male donors' RCCs contain elevated levels of biologically old red blood cells (RBCs). The aim of this study was to interrogate the quality of units of different donor ages and sexes to unravel the complex interplay between donor characteristics, long-term cold storage and, for the first time, RBC biological age. MATERIALS AND METHODS: RCCs from teen males, teen females, senior males and senior females were density-separated into less-dense/young (Y-RBCs) and dense/old RBCs (O-RBCs) throughout hypothermic storage for testing. The unseparated and density-separated cells were tested for haematological parameters, stress (oxidative and osmotic) haemolysis and oxygen affinity (p50). RESULTS: The O-RBCs obtained from teen donor samples, particularly males, had smaller mean corpuscular volumes and higher mean corpuscular haemoglobin concentrations. While biological age did not significantly affect oxygen affinity, biologically aged O-RBCs from stored RCCs exhibited increased oxidative haemolysis and decreased osmotic fragility, with teenage male RCCs exhibiting the highest propensity to haemolyse. CONCLUSION: Previously, donor age and sex were shown to have an impact on the biological age distribution of RBCs within RCCs. Herein, we demonstrated that RBC biological age, particularly O-RBCs, which are found more prevalently in male teens, to be a driving factor of several aspects of poor blood product quality. This study emphasizes that donor factors should continue to be considered for their potential impacts on transfusion outcomes.

Quality assessment of red blood cell concentrates from blood donors at the extremes of the age spectrum: The BEST collaborative study.

BACKGROUND: Blood donors at the extremes of the age spectrum (16-19 years vs. ≥75 years) are characterized by increased risks of iron deficiency and anemia, and are often underrepresented in studies evaluating the effects of donor characteristics on red blood cells (RBC) transfusion effectiveness. The aim of this study was to conduct quality assessments of RBC concentrates from these unique age groups. STUDY DESIGN: We characterized 150 leukocyte-reduced (LR)-RBCs units from 75 teenage donors, who were matched by sex, and ethnicity with 75 older donors. LR-RBC units were manufactured at three large blood collection centers in the USA and Canada. Quality assessments included storage hemolysis, osmotic hemolysis, oxidative hemolysis, osmotic gradient ektacytometry, hematological indices, and RBC bioactivity. RESULTS: RBC concentrates from teenage donors had smaller (9%) mean corpuscular volume and higher (5%) RBC concentration compared with older donors counterparts. Stored RBCs from teenage donors exhibited increased susceptibility to oxidative hemolysis (>2-fold) compared with RBCs from older donors. This was observed at all testing centers independent of sex, storage duration, or the type of additive solution. RBCs from teenage male donors had increased cytoplasmatic viscosity and lower hydration compared with older donor RBCs. Evaluations of RBC supernatant bioactivity suggested that donor age was not associated with altered expression of inflammatory markers (CD31, CD54, and IL-6) on endothelial cells. CONCLUSIONS: The reported findings are likely intrinsic to RBCs and reflect age-specific changes in RBC antioxidant capacity and physical characteristics that may impact RBC survival during cold storage and after transfusion.

Optimizing interpretation of survival studies of fresh and aged transfused biotin-labeled RBCs.

BACKGROUND: Ex vivo labeling with 51 chromium represents the standard method to determine red blood cell (RBC) survival after transfusion. Limitations and safety concerns spurred the development of alternative methods, including biotinylated red blood cells (BioRBC). STUDY DESIGN AND METHODS: Autologous units of whole blood were divided equally into two bags and stored under standard blood bank conditions at 2 to 6°C (N = 4 healthy adult volunteers). One bag was biotinylated (15 µg/ml) on storage days 5 to 7 (fresh) and the other was biotinylated (3 µg/ml) on days 35 to 42 (aged). The proportion of circulating BioRBC was measured serially, and cell-surface biotin was quantified with reference to molecules of equivalent soluble fluorochrome. Clearance kinetics were modeled by RBC age distribution at infusion (Gaussian vs. uniform) and decay over time (constant vs. exponential). RESULTS: Data were consistent with biphasic exponential clearance of cells of uniform age. Our best estimate of BioRBC clearance (half-life [T1/2 ]) was 49.7 ± 1.2 days initially, followed by more rapid clearance 82 days after transfusion (T1/2  = 15.6 ± 0.6 days). As BioRBC aged in vivo, molecules of equivalent soluble fluorochrome declined with a T1/2 of 122 ± 9 days, suggesting gradual biotin cleavage. There were no significant differences between the clearance of fresh and aged BioRBC. CONCLUSION: Similar clearance kinetics of fresh and aged BioRBC may be due to the extensive washing required during biotinylation. Survival kinetics consistent with cells with uniform rather than Gaussian or other non-uniform age distributions suggest that washing, and potentially RBC culling, may extend the storage life of RBC products.

Erythrocyte mitogen-activated protein kinases mediate hemolytic events under osmotic and oxidative stress and in hemolytic diseases.

p38 MAPKs are key regulators of cellular adaptation to various stress stimuli, however, their role in mediating erythrocyte cell death and hemolysis is largely unknown. We hypothesized that activation of erythrocyte p38 MAPK is a common event in the stimulation of hemolysis, and that inhibition of p38 MAPK pathways could mitigate hemolysis in hemoglobinopathies. We exposed human erythrocytes to diamide-induced oxidative stress or to hypoosmotic shock in the presence or absence of p38 MAPK inhibitors (SCIO469, SB203580, CMPD1) and used immunoblotting to determine MAPK activity and to identify possible downstream effectors of p38 MAPK. We also evaluated the impact of p38 MAPK inhibitors on stress-induced hemolysis or hypoxia-induced sickling in erythrocytes from mouse models of sickle cell disease. We found that human erythrocytes express conventional MAPKs (MKK3, p38 MAPK, MAPKAPK2) and identified differential MAPK activation pathways in each stress condition. Specifically, p38 MAPK inhibition in diamide-treated erythrocytes was associated with decreased phosphorylation of Src tyrosine kinases and Band 3 protein. Conversely, hypoosmotic shock induced MAPKAPK2 and RSK2 phosphorylation, which was inhibited by SCIO469 or CMPD1. Relevant to hemoglobinopathies, sickle cell disease was associated with increased erythrocyte MKK3, p38 MAPK, and MAPKAPK2 expression and phosphorylation as compared with erythrocytes from healthy individuals. Furthermore, p38 MAPK inhibition was associated with decreased hemolysis in response to diamide treatments or osmotic shock, and with decreased erythrocyte sickling under experimental hypoxia. These findings provided insights into MAPK-mediated signaling pathways that regulate erythrocyte function and hemolysis in response to extracellular stressors or human diseases.

Sex-specific genetic modifiers identified susceptibility of cold stored red blood cells to osmotic hemolysis.

BACKGROUND: Genetic variants have been found to influence red blood cell (RBC) susceptibility to hemolytic stress and affect transfusion outcomes and the severity of blood diseases. Males have a higher susceptibility to hemolysis than females, but little is known about the genetic mechanism contributing to the difference. RESULTS: To investigate the sex differences in RBC susceptibility to hemolysis, we conducted a sex-stratified genome-wide association study and a genome-wide gene-by-sex interaction scan in a multi-ethnic dataset with 12,231 blood donors who have in vitro osmotic hemolysis measurements during routine blood storage. The estimated SNP-based heritability for osmotic hemolysis was found to be significantly higher in males than in females (0.46 vs. 0.41). We identified SNPs associated with sex-specific susceptibility to osmotic hemolysis in five loci (SPTA1, KCNA6, SLC4A1, SUMO1P1, and PAX8) that impact RBC function and hemolysis. CONCLUSION: Our study established a best practice to identify sex-specific genetic modifiers for sexually dimorphic traits in datasets with mixed ancestries, providing evidence of different genetic regulations of RBC susceptibility to hemolysis between sexes. These and other variants may help explain observed sex differences in the severity of hemolytic diseases, such as sickle cell and malaria, as well as the viability of red cell storage and recovery.

Donor genetic and nongenetic factors affecting red blood cell transfusion effectiveness.

BACKGROUNDRBC transfusion effectiveness varies due to donor, component, and recipient factors. Prior studies identified characteristics associated with variation in hemoglobin increments following transfusion. We extended these observations, examining donor genetic and nongenetic factors affecting transfusion effectiveness.METHODSThis is a multicenter retrospective study of 46,705 patients and 102,043 evaluable RBC transfusions from 2013 to 2016 across 12 hospitals. Transfusion effectiveness was defined as hemoglobin, bilirubin, or creatinine increments following single RBC unit transfusion. Models incorporated a subset of donors with data on single nucleotide polymorphisms associated with osmotic and oxidative hemolysis in vitro. Mixed modeling accounting for repeated transfusion episodes identified predictors of transfusion effectiveness.RESULTSBlood donor (sex, Rh status, fingerstick hemoglobin, smoking), component (storage duration, γ irradiation, leukoreduction, apheresis collection, storage solution), and recipient (sex, BMI, race and ethnicity, age) characteristics were associated with hemoglobin and bilirubin, but not creatinine, increments following RBC transfusions. Increased storage duration was associated with increased bilirubin and decreased hemoglobin increments, suggestive of in vivo hemolysis following transfusion. Donor G6PD deficiency and polymorphisms in SEC14L4, HBA2, and MYO9B genes were associated with decreased hemoglobin increments. Donor G6PD deficiency and polymorphisms in SEC14L4 were associated with increased transfusion requirements in the subsequent 48 hours.CONCLUSIONDonor genetic and other factors, such as RBC storage duration, affect transfusion effectiveness as defined by decreased hemoglobin or increased bilirubin increments. Addressing these factors will provide a precision medicine approach to improve patient outcomes, particularly for chronically transfused RBC recipients, who would most benefit from more effective transfusion products.FUNDINGFunding was provided by HHSN 75N92019D00032, HHSN 75N92019D00034, 75N92019D00035, HHSN 75N92019D00036, and HHSN 75N92019D00037; R01HL126130; and the National Institute of Child Health and Human Development (NICHD).

Toxic masculinity in red blood cell units? Testosterone therapy in blood donors revisited.

BACKGROUND: FDA guidelines limit the use of blood from donors taking testosterone replacement therapy (TRT) to red blood cell (RBC) concentrates, whereas plasma and platelets are discarded. The purpose of this study is to bring awareness to above-average free testosterone concentrations in RBC units from TRT donors. STUDY DESIGN: We quantified the concentrations of free (bioavailable; pg/ml) and total (protein bound and free; ng/dl) testosterone in plasma (frozen within 24 h) and supernatants from 42-day stored leukocyte-reduced RBC units from 17 TRT male donors and 17 matched controls (no TRT). Total testosterone concentrations were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Free testosterone concentrations were quantified in the same samples using equilibrium dialysis/LC-MS/MS. RESULTS: Plasma free and total testosterone concentrations in TRT donors were 2.9 and 1.8 times higher than that of controls. Total testosterone concentrations in RBC supernatants were about 30% of that of plasma. In contrast, free testosterone concentrations in RBC supernatants were 80%-100% of that of plasma and were significantly (p = .005) higher in TRT compared with controls (252.3 ± 245.3 vs. 103.4 ± 88.2 pg/ml). Supraphysiological free testosterone concentrations (>244 pg/ml) in RBC supernatants were observed in five TRT donors and two control donors. CONCLUSIONS: RBC units from TRT donors may contain supraphysiological concentrations of free testosterone. This may be resolved by avoiding blood collections soon after testosterone dosing and by enhanced screening of TRT donors. These data establish a rationale for new studies and reexamination of the current guidelines concerning the utilization of blood components from TRT donors.

Multiple-ancestry genome-wide association study identifies 27 loci associated with measures of hemolysis following blood storage.

BackgroundThe evolutionary pressure of endemic malaria and other erythrocytic pathogens has shaped variation in genes encoding erythrocyte structural and functional proteins, influencing responses to hemolytic stress during transfusion and disease.MethodsWe sought to identify such genetic variants in blood donors by conducting a genome-wide association study (GWAS) of 12,353 volunteer donors, including 1,406 African Americans, 1,306 Asians, and 945 Hispanics, whose stored erythrocytes were characterized by quantitative assays of in vitro osmotic, oxidative, and cold-storage hemolysis.ResultsGWAS revealed 27 significant loci (P < 5 × 10-8), many in candidate genes known to modulate erythrocyte structure, metabolism, and ion channels, including SPTA1, ALDH2, ANK1, HK1, MAPKAPK5, AQP1, PIEZO1, and SLC4A1/band 3. GWAS of oxidative hemolysis identified variants in genes encoding antioxidant enzymes, including GLRX, GPX4, G6PD, and SEC14L4 (Golgi-transport protein). Genome-wide significant loci were also tested for association with the severity of steady-state (baseline) in vivo hemolytic anemia in patients with sickle cell disease, with confirmation of identified SNPs in HBA2, G6PD, PIEZO1, AQP1, and SEC14L4.ConclusionsMany of the identified variants, such as those in G6PD, have previously been shown to impair erythrocyte recovery after transfusion, associate with anemia, or cause rare Mendelian human hemolytic diseases. Candidate SNPs in these genes, especially in polygenic combinations, may affect RBC recovery after transfusion and modulate disease severity in hemolytic diseases, such as sickle cell disease and malaria.

Testosterone replacement therapy in blood donors modulates erythrocyte metabolism and susceptibility to hemolysis in cold storage.

BACKGROUND: Red blood cells (RBCs) derived from patients who receive testosterone replacement therapy (TRT) may be considered eligible for component production and transfusion. The aim of this study was to identify testosterone-dependent changes in RBC metabolism and to evaluate its impact on susceptibility to hemolysis during cold storage. STUDY DESIGN AND METHODS: We characterized stored RBCs from two cohorts of TRT patients who were matched with control donors (no TRT) based upon sex, age, and ethnicity. We further evaluated the impact of testosterone deficiency (orchiectomy) on RBC metabolism in FVB/NJ mice. RBC metabolites were quantified by ultra-high-pressure liquid chromatography-mass spectrometry. RBC storage stability was determined in RBC units from TRT and controls by quantifying storage, osmotic, and oxidative hemolysis. RESULTS: Orchiectomy in mice was associated with significant (P < 0.05) changes in RBC metabolism as compared with intact males including increased levels of acyl-carnitines, long-chain fatty acids (eg, docosapentaenoic acids), arginine, and dopamine. Stored RBCs from TRT patients exhibited higher levels of pentose phosphate pathway metabolites, glutathione, and oxidized purines (eg, hypoxanthine), suggestive of increased activation of antioxidant pathways in this group. Further analyses indicated significant changes in free fatty acids and acyl-carnitines in response to testosterone therapies. With regard to hemolysis, TRT was associated with enhanced susceptibility to osmotic hemolysis. Correlation analyses identified acyl-carnitines as significant modifiers of RBC predisposition to osmotic and oxidative hemolysis. CONCLUSIONS: These observations provide new insights into testosterone-mediated changes in RBC metabolome and biology that may impact the storage capacity and posttransfusion efficacy of RBCs from TRT donors.

Donor sex, age and ethnicity impact stored red blood cell antioxidant metabolism through mechanisms in part explained by glucose 6-phosphate dehydrogenase levels and activity.

Red blood cell storage in the blood bank promotes the progressive accumulation of metabolic alterations that may ultimately impact the erythrocyte capacity to cope with oxidant stressors. However, the metabolic underpinnings of the capacity of RBCs to resist oxidant stress and the potential impact of donor biology on this phenotype are not known. Within the framework of the REDS-III RBC-Omics study, RBCs from 8,502 healthy blood donors were stored for 42 days and tested for their propensity to hemolyze following oxidant stress. A subset of extreme hemolyzers donated a second unit of blood, which was stored for 10, 23, and 42 days and profiled again for oxidative hemolysis and metabolomics (599 samples). Alterations of RBC energy and redox homeostasis were noted in donors with high oxidative hemolysis. RBCs from females, donors over 60 years old, donors of Asian/South Asian race-ethnicity, and RBCs stored in additive solution-3 were each independently characterized by improved antioxidant metabolism compared to, respectively, males, donors under 30 years old, Hispanic and African American race ethnicity donors, and RBCs stored in additive solution-1. Merging metabolomics data with results from an independent GWAS study on the same cohort, we identified metabolic markers of hemolysis and G6PD-deficiency, which were associated with extremes in oxidative hemolysis and dysregulation in NADPH and glutathione-dependent detoxification pathways of oxidized lipids. Donor sex, age, ethnicity, additive solution and G6PD status impact the metabolism of the stored erythrocyte and its susceptibility to hemolysis following oxidative insults.

Blood donor obesity is associated with changes in red blood cell metabolism and susceptibility to hemolysis in cold storage and in response to osmotic and oxidative stress.

BACKGROUND: Obesity is a global pandemic characterized by multiple comorbidities, including cardiovascular and metabolic diseases. The aim of this study was to define the associations between blood donor body mass index (BMI) and RBC measurements of metabolic stress and hemolysis. STUDY DESIGN AND METHODS: The associations between donor BMI (<25 kg/m2 , normal weight; 25-29.9 kg/m2 , overweight; and ≥30 kg/m2 , obese) and hemolysis (storage, osmotic, and oxidative; n = 18 donors) or posttransfusion recovery (n = 14 donors) in immunodeficient mice were determined in stored leukocyte-reduced RBC units. Further evaluations were conducted using the National Heart, Lung, and Blood Institute RBC-Omics blood donor databases of hemolysis (n = 13 317) and metabolomics (n = 203). RESULTS: Evaluations in 18 donors revealed that BMI was significantly (P < 0.05) and positively associated with storage and osmotic hemolysis. A BMI of 30 kg/m2 or greater was also associated with lower posttransfusion recovery in mice 10 minutes after transfusion (P = 0.026). Multivariable linear regression analyses in RBC-Omics revealed that BMI was a significant modifier for all hemolysis measurements, explaining 4.5%, 4.2%, and 0.2% of the variance in osmotic, oxidative, and storage hemolysis, respectively. In this cohort, obesity was positively associated (P < 0.001) with plasma ferritin (inflammation marker). Metabolomic analyses on RBCs from obese donors (44.1 ± 5.1 kg/m2 ) had altered membrane lipid composition, dysregulation of antioxidant pathways (eg, increased oxidized lipids, methionine sulfoxide, and xanthine), and dysregulation of nitric oxide metabolism, as compared to RBCs from nonobese (20.5 ± 1.0 kg/m2 ) donors. CONCLUSIONS: Obesity is associated with significant changes in RBC metabolism and increased susceptibility to hemolysis under routine storage of RBC units. The impact on transfusion efficacy warrants further evaluation.

Immunodeficient mice are better for modeling the transfusion of human blood components than wild-type mice.

Animal models are vital to the study of transfusion and development of new blood products. Post-transfusion recovery of human blood components can be studied in mice, however, there is a need to identify strains that can best tolerate xenogeneic transfusions, as well as to optimize such protocols. Specifically, the importance of using immunodeficient mice, such as NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, to study human transfusion has been questioned. In this study, strains of wild-type and NSG mice were compared as hosts for human transfusions with outcomes quantified by flow cytometric analyses of CD235a+ erythrocytes, CD45+ leukocytes, and CD41+CD42b+ platelets. Complete blood counts were evaluated as well as serum cytokines by multiplexing methods. Circulating human blood cells were maintained better in NSG than in wild-type mice. Lethargy and hemoglobinuria were observed in the first hours in wild-type mice along with increased pro-inflammatory cytokines/chemokines such as monocyte chemoattractant protein-1, tumor necrosis factor α, keratinocyte-derived chemokine (KC or CXCL1), and interleukin-6, whereas NSG mice were less severely affected. Whole blood transfusion resulted in rapid sequestration and then release of human cells back into the circulation within several hours. This rebound effect diminished when only erythrocytes were transfused. Nonetheless, human erythrocytes were found in excess of mouse erythrocytes in the liver and lungs and had a shorter half-life in circulation. Variables affecting the outcomes of transfused erythrocytes were cell dose and mouse weight; recipient sex did not affect outcomes. The sensitivity and utility of this xenogeneic model were shown by measuring the effects of erythrocyte damage due to exposure to the oxidizer diamide on post-transfusion recovery. Overall, immunodeficient mice are superior models for xenotransfusion as they maintain improved post-transfusion recovery with negligible immune-associated side effects.

Additive effects of blood donor smoking and gamma irradiation on outcome measures of red blood cell transfusion.

BACKGROUND: Recent publications have reported conflicting results regarding the role of blood donor tobacco use on hemoglobin (Hb) levels in patients after red blood cell (RBC) transfusion. We examined associations and interactions between donor, component, and recipient factors to better understand the impact of donor smoking on transfusion outcomes. STUDY DESIGN AND METHODS: We linked blood donor and component manufacturing data, including self-reported cigarette smoking, with a cohort of patients transfused RBCs between 2013 and 2016. Using multivariable regression, we examined Hb increments and subsequent transfusion requirements after single-unit RBC transfusion episodes, adjusting for donor, component, and recipient factors. RESULTS: We linked data on 4038 transfusion recipients who received one or more single-unit RBC transfusions (n = 5086 units) to donor demographic and component manufacturing characteristics. Among RBC units from smokers (n = 326), Hb increments were reduced after transfusion of gamma-irradiated units (0.76 g/dL; p = 0.033) but not unirradiated units (1.04 g/dL; p = 0.54) compared to those from nonsmokers (1.01 g/dL; n = 4760). In parallel with changes in Hb levels, donor smoking was associated with the receipt of additional RBC transfusions for irradiated (odds ratio [OR], 2.49; p = 0.01) but not unirradiated RBC units (OR, 1.10; p = 0.52). CONCLUSION: Donor smoking was associated with reduced Hb increments and the need for additional transfusions in recipients of gamma-irradiated RBC units. Additional research is needed to better understand interactions between donor, component, and recipient factors on efficacy measures of RBC transfusion.

Stored RBC metabolism as a function of caffeine levels.

BACKGROUND: Coffee consumption is extremely common in the United States. Coffee is rich with caffeine, a psychoactive, purinergic antagonist of adenosine receptors, which regulate red blood cell energy and redox metabolism. Since red blood cell (purine) metabolism is a critical component to the red cell storage lesion, here we set out to investigate whether caffeine levels correlated with alterations of energy and redox metabolism in stored red blood cells. STUDY DESIGN AND METHODS: We measured the levels of caffeine and its main metabolites in 599 samples from the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study via ultra-high-pressure-liquid chromatography coupled to high-resolution mass spectrometry and correlated them to global metabolomic and lipidomic analyses of RBCs stored for 10, 23, and 42 days. RESULTS: Caffeine levels positively correlated with increased levels of the main red cell antioxidant, glutathione, and its metabolic intermediates in glutathione-dependent detoxification pathways of oxidized lipids and sugar aldehydes. Caffeine levels were positively correlated with transamination products and substrates, tryptophan, and indole metabolites. Expectedly, since caffeine and its metabolites belong to the family of xanthine purines, all xanthine metabolites were significantly increased in the subjects with the highest levels of caffeine. However, high-energy phosphate compounds ATP and DPG were not affected by caffeine levels, despite decreases in glucose oxidation products-both via glycolysis and the pentose phosphate pathway. CONCLUSION: Though preliminary, this study is suggestive of a beneficial correlation between the caffeine levels and improved antioxidant capacity of stored red cells.

Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers.

BACKGROUND: Cigarette smoking is a frequent habit across blood donors (approx. 13% of the donor population), that could compound biologic factors and exacerbate oxidant stress to stored red blood cells (RBCs). STUDY DESIGN AND METHODS: As part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, a total of 599 samples were sterilely drawn from RBC units stored under blood bank conditions at Storage Days 10, 23, and 42 days, before testing for hemolysis parameters and metabolomics. Quantitative measurements of nicotine and its metabolites cotinine and cotinine oxide were performed against deuterium-labeled internal standards. RESULTS: Donors whose blood cotinine levels exceeded 10 ng/mL (14% of the tested donors) were characterized by higher levels of early glycolytic intermediates, pentose phosphate pathway metabolites, and pyruvate-to-lactate ratios, all markers of increased basal oxidant stress. Consistently, increased glutathionylation of oxidized triose sugars and lipid aldehydes was observed in RBCs donated by nicotine-exposed donors, which were also characterized by increased fatty acid desaturation, purine salvage, and methionine oxidation and consumption via pathways involved in oxidative stress-triggered protein damage-repair mechanisms. CONCLUSION: RBCs from donors with high levels of nicotine exposure are characterized by increases in basal oxidant stress and decreases in osmotic hemolysis. These findings indicate the need for future clinical studies aimed at addressing the impact of smoking and other sources of nicotine (e.g., nicotine patches, snuff, vaping, secondhand tobacco smoke) on RBC storage quality and transfusion efficacy.

Impact of taurine on red blood cell metabolism and implications for blood storage.

BACKGROUND: Taurine is an antioxidant that is abundant in some common energy drinks. Here we hypothesized that the antioxidant activity of taurine in red blood cells (RBCs) could be leveraged to counteract storage-induced oxidant stress. STUDY DESIGN AND METHODS: Metabolomics analyses were performed on plasma and RBCs from healthy volunteers (n = 4) at baseline and after consumption of a whole can of a common, taurine-rich (1000 mg/serving) energy drink. Reductionistic studies were also performed by incubating human RBCs with taurine ex vivo (unlabeled or 13 C15 N-labeled) at increasing doses (0, 100, 500, and 1000 µmol/L) at 37°C for up to 16 hours, with and without oxidant stress challenge with hydrogen peroxide (0.1% or 0.5%). Finally, we stored human and murine RBCs under blood bank conditions in additives supplemented with 500 µmol/L taurine, before metabolomics and posttransfusion recovery studies. RESULTS: Consumption of energy drinks increased plasma and RBC levels of taurine, which was paralleled by increases in glycolysis and glutathione (GSH) metabolism in the RBC. These observations were recapitulated ex vivo after incubation with taurine and hydrogen peroxide. Taurine levels in the RBCs from the REDS-III RBC-Omics donor biobank were directly proportional to the total levels of GSH and glutathionylated metabolites and inversely correlated to oxidative hemolysis measurements. Storage of human RBCs in the presence of taurine improved energy and redox markers of storage quality and increased posttransfusion recoveries in FVB mice. CONCLUSION: Taurine modulates RBC antioxidant metabolism in vivo and ex vivo, an observation of potential relevance to transfusion medicine.

The prevalence and demographic determinants of blood donors receiving testosterone replacement therapy at a large USA blood service organization.

BACKGROUND: Blood donors receiving testosterone replacement therapy (TRT) often require therapeutic phlebotomy due to erythrocytosis. Red blood cells (RBCs) donated by eligible TRT donors are approved for collection and transfusion. This study was aimed at defining the prevalence and demographic determinants of TRT donors at a large USA blood service organization. STUDY DESIGN: Donation data from TRT donors and matched controls was collected from a de-identified electronic donor database across 16 blood centers in 2017-2018. Demographic determinants included race, sex, age, hemoglobin (Hb), body mass index (BMI), mean arterial pressure (MAP), and the frequency of donations in the 2-year period. RESULTS: TRT donors comprised 1.6% of the donor population and produced 2.2% of RBC units during 2018. TRT donors were likely to be middle-aged white or Hispanic men, with high prevalence of obesity (50.8% of TRT donors had BMI ≥30 kg/m2 compared with 36.2% in controls) and intensive donation frequency (1 to 29 donations in 2 years vs. 1 to 12 in controls). TRT donors had significantly (p < 0.0001) higher MAP and Hb compared with controls (MAP 99.9 ± 9.81 vs. 96.5 ± 10.1 mmHg; Hb 17.8 ± 1.44 vs. 15.6 ± 1.37 g/dL). One year of donations was associated with significant decreases in MAP and Hb for TRT donors. CONCLUSIONS: TRT is associated with high prevalence of erythrocytosis and obesity that may explain the intensive donation frequency, high MAP, and Hb. Frequent phlebotomies had a moderately positive effect on blood pressure and Hb levels. Potential implications of TRT on the quality of the RBC products require further evaluation.

Sex hormone intake in female blood donors: impact on haemolysis during cold storage and regulation of erythrocyte calcium influx by progesterone.

BACKGROUND: Sex hormone intake in blood donors may affect the quality of red blood cell (RBC) products via modulation of RBC function and predisposition to haemolysis during cold storage. The aims of this study were to evaluate the association between female sex hormone intake and RBC storage outcomes, and to examine possible mechanisms by which sex hormones interact with RBCs. MATERIALS AND METHODS: Sex hormone intake by race/ethnicity and menopausal status, and association analyses between hormone intake and donor scores of storage, osmotic or oxidative haemolysis, were evaluated in 6,636 female donors who participated in the National Heart, Lung and Blood Institute's RBC-Omics study. A calcium fluorophore, Fluo-3AM, was used to define RBC calcium influx in response to exogenous sex hormones or transient receptor potential cation (TRPC) channel drugs. RESULTS: Sex hormone intake was more prevalent in premenopausal women from all racial groups (18-31%) than in postmenopausal women (4-8%). Hormone intake was significantly (p<0.0001) associated with reduced storage haemolysis in all females, reduced osmotic haemolysis in postmenopausal donors (23.1±10.2% vs 26.8±12.0% in controls, p<0.001), and enhanced susceptibility to oxidative haemolysis in premenopausal women. In vitro, supraphysiological levels of progesterone (10 µmol/L), but not 17ß-oestradiol or testosterone, inhibited calcium influx into RBC and was associated with lower spontaneous haemolysis after 30 days of cold storage (0.95±0.18% vs 1.85±0.35% in controls, p<0.0001) or in response to a TRPC6 activator. CONCLUSIONS: Sex hormone intake in female donors is associated with changes in RBC predisposition to haemolysis. Menstrual status and the type of hormone preparation may contribute to differences in haemolytic responses of female RBCs to osmotic and oxidative stress. Progesterone modulates calcium influx into RBC via a mechanism that may involve interactions with membrane TRPC6 channels.