Genetic polymorphisms and expression of Rhesus blood group RHCE are associated with 2,3-bisphosphoglycerate in humans at high altitude.

Red blood cell (RBC) metabolic reprogramming upon exposure to high altitude contributes to physiological human adaptations to hypoxia, a multifaceted process critical to health and disease. To delve into the molecular underpinnings of this phenomenon, first, we performed a multi-omics analysis of RBCs from six lowlanders after exposure to high-altitude hypoxia, with longitudinal sampling at baseline, upon ascent to 5,100 m and descent to sea level. Results highlighted an association between erythrocyte levels of 2,3-bisphosphoglycerate (BPG), an allosteric regulator of hemoglobin that favors oxygen off-loading in the face of hypoxia, and expression levels of the Rhesus blood group RHCE protein. We then expanded on these findings by measuring BPG in RBCs from 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. These data informed a genome-wide association study using BPG levels as a quantitative trait, which identified genetic polymorphisms in the region coding for the Rhesus blood group RHCE as critical determinants of BPG levels in erythrocytes from healthy human volunteers. Mechanistically, we suggest that the Rh group complex, which participates in the exchange of ammonium with the extracellular compartment, may contribute to intracellular alkalinization, thus favoring BPG mutase activity.

Genetic regulation of carnitine metabolism controls lipid damage repair and aging RBC hemolysis in vivo and in vitro.

ABSTRACT: Recent large-scale multiomics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on 2 separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured l-carnitine and acyl-carnitines in 13 091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation study. Genome-wide association studies against 879 000 polymorphisms identified critical genetic factors contributing to interdonor heterogeneity in end-of-storage carnitine levels, including common nonsynonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, and SLC16A9); carnitine synthesis (FLVCR1 and MTDH) and metabolism (CPT1A, CPT2, CRAT, and ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying 2 alleles of the rs12210538 SLC22A16 single-nucleotide polymorphism exhibited the lowest l-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice, and Percoll density gradients of human RBCs, showed age-dependent depletions of l-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process after chemically induced membrane lipid damage. Supplementation of stored murine RBCs with l-carnitine boosted posttransfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.

Patient and Immunological Factors Associated With Delayed Clearance of Mucosal Severe Acute Respiratory Syndrome Coronavirus 2 RNA and Symptom Persistence.

Serial blood and mucosal samples were characterized for 102 participants enrolled a median of 7.0 days after coronavirus disease 2019 diagnosis. Mucosal RNA was detectable for a median of 31.5 (95% confidence interval [CI], 20.5-63.5) days, with persistence ≥1 month associated with obesity (body mass index [BMI] ≥30 kg/m2; odds ratio [OR], 3.9 [95% CI, 1.2-13.8]) but not age, sex, or chronic conditions. Fifteen participants had likely reinfection; lower serum anti-spike IgG levels were associated with reinfection risk. Nearly half of participants (47%) reported symptoms lasting ≥2-3 months; persistence ≥3 months was associated with BMI ≥30 kg/m2 (OR, 4.2 [95% CI, 1.1-12.8]) and peak anti-spike and anti-nucleocapsid antibody levels.

Detection of Nucleocapsid Antibodies Associated with Primary SARS-CoV-2 Infection in Unvaccinated and Vaccinated Blood Donors.

Nucleocapsid antibody assays can be used to estimate SARS-CoV-2 infection prevalence in regions implementing spike-based COVID-19 vaccines. However, poor sensitivity of nucleocapsid antibody assays in detecting infection after vaccination has been reported. We derived a lower cutoff for identifying previous infections in a large blood donor cohort (N = 142,599) by using the Ortho VITROS Anti-SARS-CoV-2 Total-N Antibody assay, improving sensitivity while maintaining specificity >98%. We validated sensitivity in samples donated after self-reported swab-confirmed infections diagnoses. Sensitivity for first infections in unvaccinated donors was 98.1% (95% CI 98.0-98.2) and for infection after vaccination was 95.6% (95% CI 95.6-95.7) based on the standard cutoff. Regression analysis showed sensitivity was reduced in the Delta compared with Omicron period, in older donors, in asymptomatic infections, <30 days after infection, and for infection after vaccination. The standard Ortho N antibody threshold demonstrated good sensitivity, which was modestly improved with the revised cutoff.

Higher intraindividual variability of body mass index is associated with elevated risk of COVID-19 related hospitalization and post-COVID conditions.

BACKGROUND: Cardiometabolic diseases are risk factors for COVID-19 severity. The extent that cardiometabolic health represents a modifiable factor to mitigate the short- and long-term consequences from SARS-CoV-2 remains unclear. Our objective was to evaluate the associations between intraindividual variability of cardiometabolic health indicators and COVID-19 related hospitalizations and post-COVID conditions (PCC) among a relatively healthy population. METHODS: This retrospective, multi-site cohort study was a post-hoc analysis among individuals with cardiometabolic health data collected during routine blood donation visits in 24 US states (2009-2018) and who responded to COVID-19 questionnaires (2021-2023). Intraindividual variability of blood pressure (systolic, diastolic), total circulating cholesterol, and body mass index (BMI) were defined as the coefficient of variation (CV) across all available donation timepoints (ranging from 3 to 74); participants were categorized into CV quartiles. Associations were evaluated by multivariable binomial regressions. RESULTS: Overall, 3344 participants provided 42,090 donations (median 9 [IQR 5, 17]). The median age was 48 years (38, 56) at the first study donation. 1.2% (N = 40) were hospitalized due to COVID-19 and 15.5% (N = 519) had PCC. Higher BMI variability was associated with greater risk of COVID-19 hospitalization (4th quartile aRR 4.15 [95% CI 1.31, 13.11], p = 0.02; 3rd quartile aRR 3.41 [95% CI 1.09, 10.69], p = 0.04). Participants with higher variability of BMI had greater risk of PCC (4th quartile aRR 1.29 [95% CI 1.02, 1.64]; p = 0.04). Intraindividual variability of blood pressure (systolic, diastolic) and total circulating cholesterol were not associated with COVID-19 hospitalization or PCC risk (all p > 0.05). From causal mediation analysis, the association between the highest quartiles of BMI variability and PCC was not mediated by hospitalization (p > 0.05). CONCLUSIONS: Higher intraindividual variability of BMI was associated with COVID-19 hospitalization and PCC risk. Our findings underscore the need for further elucidating mechanisms that explain these associations and importance for consistent maintenance of body weight.

SARS-CoV-2 antibody levels and long COVID occurrence in blood donors.

BACKGROUND: Long COVID is a common condition lacking consensus definition; determinants remain incompletely understood. Characterizing immune profiles associated with long COVID could support the development of preventive and therapeutic strategies. METHODS: We used a survey to investigate blood donors' infection/vaccination history and acute/persistent symptoms following COVID-19. The prevalence of long COVID was evaluated using self-report and an adapted definition from the RECOVER study. We evaluated factors associated with long COVID, focusing on anti-spike and anti-nucleocapsid SARS-CoV-2 antibodies. Lastly, we investigated long COVID clinical subphenotypes using hierarchical clustering. RESULTS: Of 33,610 participants, 16,003 (48%) reported having had COVID-19; 1853 (12%) had self-reported long COVID, 685 (4%) met an adapted RECOVER definition, and 2050 (13%) met at least one definition. Higher anti-nucleocapsid levels measured 12-24 weeks post-infection were associated with higher risk of self-reported and RECOVER long COVID. Higher anti-spike IgG levels measured 12-24 weeks post-infection were associated with lower risk of self-reported long COVID. Higher total anti-spike measured 24-48 weeks post-infection was associated with lower risk of RECOVER long COVID. Cluster analysis identified four clinical subphenotypes; patterns included neurological and psychiatric for cluster 1; neurological and respiratory for cluster 2; multi-systemic for cluster 3; and neurological for cluster 4. DISCUSSION: Long COVID prevalence in blood donors varies depending on the adopted definition. Anti-SARS-CoV-2 antibodies were time-dependently associated with long COVID; higher anti-nucleocapsid levels were associated with higher risk; and higher anti-spike levels were associated with lower risk of long COVID. Different underlying pathophysiologic mechanisms may be associated with distinct clinical subphenotypes.

SARS-CoV-2 immunoassays in a predominantly vaccinated population: Performances and qualitative agreements obtained with two analytical approaches and four immunoassays.

BACKGROUND AND OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurveys are typically analysed by applying a fixed threshold for seropositivity ('conventional approach'). However, this approach underestimates the seroprevalence of anti-nucleocapsid (N) in vaccinated individuals-who often exhibit a difficult-to-detect anti-N response. This limitation is compounded by delays between the onset of infection and sample collection. To address this issue, we compared the performance of four immunoassays using a new analytical approach ('ratio-based approach'), which determines seropositivity based on an increase in anti-N levels. MATERIALS AND METHODS: Two groups of plasma donors and four immunoassays (Elecsys total anti-N, VITROS total anti-N, Architect anti-N Immunoglobulin G (IgG) and in-house total anti-N) were evaluated. First-group donors (N = 145) had one positive SARS-CoV-2 polymerase chain reaction (PCR) test result and had made two plasma donations, including one before and one after the PCR test (median = 27 days post-PCR). Second-group donors (N = 100) had made two plasma donations early in the Omicron wave. RESULTS: Among first-group donors (97.9% vaccinated), sensitivity estimates ranged from 60.0% to 89.0% with the conventional approach, compared with 94.5% to 98.6% with the ratio-based approach. Among second-group donors, Fleiss's κ ranged from 0.56 to 0.83 with the conventional approach, compared with 0.90 to 1.00 with the ratio-based approach. CONCLUSION: With the conventional approach, the sensitivity of four immunoassays-measured in a predominantly vaccinated population based on samples collected ~1 month after a positive test result-fell below regulatory agencies requirement of ≥95%. The ratio-based approach significantly improved the sensitivities and qualitative agreement among immunoassays, to the point where all would meet this requirement.

The International Society of Blood Transfusion (ISBT) Public Health Research Toolkit: A report from the Surveillance, Risk Assessment and Policy Sub-group of the ISBT Transfusion Transmitted Infectious Diseases Working Party.

BACKGROUND AND OBJECTIVES: Data provided from blood donors have contributed to the understanding of public health epidemiology and policy decisions. A recent example was during the severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) pandemic when blood services monitored the seroprevalence in blood donors. Based on this experience, blood services have the opportunity to expand their role and participate in public health surveillance and research. The aim of this report is to share available resources to assist blood services in this area. MATERIALS AND METHODS: The Surveillance, Risk Assessment and Policy (SRAP) Sub-group of the International Society of Blood Transfusion (ISBT) Transfusion Transmitted Infectious Diseases (TTID) Working Party developed a Public Health Research Toolkit to assist blood services and researchers interested in expanding their role in public health research. RESULTS: The ISBT Public Health Research Toolkit provides resources for what blood services can offer to public health, examples of donor research studies, the utility of donor data and website links to public health agencies. The toolkit includes a customizable template for those interested in establishing and managing a biobank. CONCLUSION: The ISBT Public Health Research Toolkit includes resources to increase the recognition of the role blood donors can play in public health and to help blood services gain commitment and funding from various agencies for new research and surveillance.

End of selection criteria based on sexual orientation: An international symposium on alternatives to donation deferral.

BACKGROUND AND OBJECTIVES: Until recently, gay, bisexual and other men who have sex with men (MSM) were deferred from donating blood for 3-12 months since the last male-to-male sexual contact. This MSM deferral has been discontinued by several high-income countries (HIC) that now perform gender-neutral donor selection. MATERIALS AND METHODS: An international symposium (held on 20-04-2023) gathered experts from seven HICs to (1) discuss how this paradigm shift might affect the mitigation strategies for transfusion-transmitted infections and (2) address the challenges related to gender-neutral donor selection. RESULTS: Most countries employed a similar approach for implementing a gender-neutral donor selection policy: key stakeholders were consulted; the transition was bridged by time-limited deferrals; donor compliance was monitored; and questions or remarks on anal sex and the number and/or type of sexual partners were often added. Many countries have now adopted a gender-neutral approach in which questions on pre- and post-exposure prophylaxis for human immunodeficiency virus (HIV) have been added (or retained, when already in place). Other countries used mitigation strategies, such as plasma quarantine or pathogen reduction technologies for plasma and/or platelets. CONCLUSION: The experience with gender-neutral donor selection has been largely positive among the countries covered herein and seems to be acceptable to stakeholders, donors and staff. The post-implementation surveillance data collected so far appear reassuring with regards to safety, although longer observation periods are necessary. The putative risks associated with HIV antiretrovirals should be further investigated.

Using routine health-care data to search for unknown transfusion-transmitted disease: a nationwide, agnostic retrospective cohort study.

BACKGROUND: Identification and prevention of transfusion-transmitted disease is essential for blood transfusion safety. However, current surveillance systems are largely driven by reports of sentinel events, which is an approach that might be inadequate for identifying transmission of pathogens not known to be transmissible or pathogens with long incubation periods. Using a combination of health-data registers and blood-bank databases, we aimed to perform an agnostic search for potential transfusion-transmitted diseases and to identify unknown threats to the blood supply. METHODS: In this nationwide, agnostic retrospective cohort study, we developed a systematic algorithm for performing a phenome-wide search for transfusion-transmitted disease without consideration of any a-priori suspicion of blood-borne transmissibility. We applied this algorithm to a nationwide Swedish transfusion database (SCANDAT-3S) to test for possible transmission of 1155 disease entities based on all relevant diagnostic coding systems in use during the period. We ascertained health outcomes of blood donors and transfusion recipients from the Swedish National Inpatient Register, Swedish Cause of Death Register, and Swedish Cancer Register. Analyses were two-pronged, studying both disease diagnosis concordance between donors and recipients and a possible shared increased disease risk among all recipients of a given donor. For both approaches, we used Cox proportional hazards regression models with time-dependent covariates. Adjustment for multiple comparisons was done using a false discovery rate method. FINDINGS: The analyses included data on 1·72 million patients who had received 18·97 million transfusions (red blood cell, plasma, platelet, or whole blood units) between Jan 1, 1968, and Dec 31, 2017, from 1·04 million blood donors. The median follow-up was 4·5 (IQR 0·9-11·4) years for recipients and 18·5 (8·3-26·2) years for donors. We found evidence of transfusion-transmission for 15 diseases, of which 13 were validated using a second conceptually different approach. We identified transmission of viral hepatitis and its complications (eg, oesophageal varices) but also transmission of other conditions (eg, pneumonia of unknown origin). The diseases that could not be validated in this second approach, HIV and abnormal findings in specimens from male genital organs, were not statistically significant after adjustment for multiple testing. The effect sizes were small (close to 1) for other conditions. INTERPRETATION: We find no strong evidence of unexpected, widespread transfusion-transmitted disease. This novel approach serves as a proof-of-concept for agnostic, data-driven surveillance for transfusion-transmitted disease using routinely collected blood-bank and health-care data. FUNDING: Department of Health and Human Services, US National Heart, Lung, and Blood Institute, US National Institutes of Health, Swedish Research Council and Region Stockholm.

Performance of a rapid recency assay for detection of early HIV infection.

The Asanté HIV-1 Rapid Recency assay's 'verification' line detected HIV infection a median of 18 days later than a nucleic acid detection assay and performed similarly to 19 other existing rapid HIV antibody tests. Pending regulatory approval, the assay could be an option with other rapid tests in national HIV-1 testing algorithms, which would allow collection of HIV recency data as part of a national screening program without requiring additional testing.

Associations of Temporal Cardiometabolic Patterns and Incident SARS-CoV-2 Infection Among U.S. Blood Donors With Serologic Evidence of Vaccination.

Introduction: Cardiometabolic diseases are associated with greater COVID-19 severity; however, the influences of cardiometabolic health on SARS-CoV-2 infections after vaccination remain unclear. Our objective was to investigate the associations between temporal blood pressure and total cholesterol patterns and incident SARS-CoV-2 infections among those with serologic evidence of vaccination. Methods: In this prospective cohort of blood donors, blood samples were collected in 2020-2021 and assayed for binding antibodies of SARS-CoV-2 nucleocapsid protein antibody seropositivity. We categorized participants into intraindividual pattern subgroups of blood pressure and total cholesterol (persistently, intermittently, or not elevated [systolic blood pressure <130 mmHg, diastolic blood pressure <80 mmHg, total cholesterol <200 mg/dL]) across the study time points. Results: Among 13,930 donors with 39,736 donations representing 1,127,071 person-days, there were 221 incident SARS-CoV-2 infections among those with serologic evidence of vaccination (1.6%). Intermittent hypertension was associated with greater SARS-CoV-2 infections among those with serologic evidence of vaccination risk (adjusted incidence rate ratio=2.07; 95% CI=1.44, 2.96; p<0.01) than among participants with consistent normotension on the basis of a multivariable Poisson regression. Among men, intermittently elevated total cholesterol (adjusted incidence rate ratio=1.90; 95% CI=1.32, 2.74; p<0.01) and higher BMI at baseline (adjusted hazard ratio=1.44; 95% CI=1.07, 1.93; p=0.01; per 10 units) were associated with greater SARS-CoV-2 infections among those with serologic evidence of vaccination probability; these associations were null among women (both p>0.05). Conclusions: Our findings underscore that the benefits of cardiometabolic health, particularly blood pressure, include a lower risk of SARS-CoV-2 infection after vaccination.

Comparative Evaluation of Select Serological Assays for Zika Virus Using Blinded Reference Panels.

In response to the 2015 Zika virus (ZIKV) epidemic that occurred in Brazil, numerous commercial serological assays have been developed for clinical and research applications. Diagnosis of recent infection in pregnant women remains challenging. Having standardized, comparative studies of ZIKV tests is important for implementing optimal diagnostic testing and disease surveillance. This is especially important for serology tests used to detect ZIKV infection given that antibodies against ZIKV can cross-react with other arboviruses in the same virus family, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). We looked at the sensitivity and specificity of tests detecting ZIKV antibodies (IgM, IgG) from multiple manufacturers using panels of samples previously collected with known exposure to ZIKV and other arboviruses. We found that performance of the IgM tests was highly variable, with only one test (Inbios 2.0 IgM capture ELISA) having both high sensitivity and specificity. All IgG tests showed good sensitivity; however, specificity was highly variable, with some assays giving false-positive results on samples infected by another flavivirus. Overall, the results confirmed that accurate ZIKV antibody testing is challenging, especially in specimens from regions endemic for multiple other flaviviruses, and highlight the importance of available and suitable reference samples to evaluate ZIKV diagnostics.

HIV Subtypes and Drug-resistance-associated Mutations in US Blood Donors, 2015-2020.

Background: Monitoring genotypes of HIV infections in blood donors may provide insights into infection trends in the general population. Methods: HIV RNA was extracted from plasma samples of blood donors confirmed as HIV positive by blood screening nucleic acid and antibody tests. HIV genome target regions were amplified using nested real time-polymerase chain reaction followed by next-generation sequencing. Sequences were compared to those in the Los Alamos National Laboratory (LANL) database. Sequences were also assessed for drug resistance mutations (DRM) using the Stanford HIV DRM Database. Results: From available HIV-positive donations collected between 1 September 2015 and 31 December 2020, 563 of 743 (75.8%) were successfully sequenced; 4 were subtype A, 543 subtype B, 5 subtype C, 1 subtype G, 5 circulating recombinant forms (CRF), and 2 were subtype B and D recombinants. Overall, no significant differences between blood donor and available LANL genotypes were found, and the genotypes of newly acquired versus prevalent HIV infections in donors were similar. The proportion of non-B subtypes and CRF remained a small fraction, with no other subtype or CRF representing more than 1% of the total. DRM were identified in 122 (21.6%) samples with protease inhibitor, nucleoside reverse transcriptase inhibitor and non-nucleoside reverse transcriptase inhibitor DRMs identified in 4.9%, 4.6% and 14.0% of samples, respectively. Conclusions: HIV genetic diversity and DRM in blood donors appear representative of circulating HIV infections in the US general population and may provide more information on infection diversity than sequences reported to LANL, particularly for recently transmitted infections.

Ferroptosis regulates hemolysis in stored murine and human red blood cells.

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. Here, we leveraged a diversity outbred mouse population to map the genetic drivers of fresh/stored RBC metabolism and extravascular hemolysis upon storage and transfusion in 350 mice. We identify the ferrireductase Steap3 as a critical regulator of a ferroptosis-like process of lipid peroxidation. Steap3 polymorphisms were associated with RBC iron content, in vitro hemolysis, and in vivo extravascular hemolysis both in mice and 13,091 blood donors from the Recipient Epidemiology and Donor evaluation Study. Using metabolite Quantitative Trait Loci analyses, we identified a network of gene products (FADS1/2, EPHX2 and LPCAT3) - enriched in donors of African descent - associated with oxylipin metabolism in stored human RBCs and related to Steap3 or its transcriptional regulator, the tumor protein TP53. Genetic variants were associated with lower in vivo hemolysis in thousands of single-unit transfusion recipients. Highlights: Steap3 regulates lipid peroxidation and extravascular hemolysis in 350 diversity outbred miceSteap3 SNPs are linked to RBC iron, hemolysis, vesiculation in 13,091 blood donorsmQTL analyses of oxylipins identified ferroptosis-related gene products FADS1/2, EPHX2, LPCAT3Ferroptosis markers are linked to hemoglobin increments in transfusion recipients.

Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes.

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in the blood bank. Here we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify an association between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs), hexokinase 1, ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice, and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine levels - and the genetic traits linked to them - were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes. Highlights: Blood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers;Ancestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis;Modeled PFKP effects relate to preventing loss of the total AXP pool in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

Biological and genetic determinants of glycolysis: Phosphofructokinase isoforms boost energy status of stored red blood cells and transfusion outcomes.

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes.