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.

Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I–deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I–deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. However, the genetic underpinnings of RBC metabolic heterogeneity and extravascular hemolysis at population scale are incompletely understood. On the basis of the breeding of 8 founder strains with extreme genetic diversity, the Jackson Laboratory diversity outbred population can capture the impact of genetic heterogeneity in like manner to population-based studies. RBCs from 350 outbred mice, either fresh or stored for 7 days, were tested for posttransfusion recovery, as well as metabolomics and lipidomics analyses. Metabolite and lipid quantitative trait loci (QTL) mapped >400 gene-metabolite associations, which we collated into an online interactive portal. Relevant to RBC storage, we identified a QTL hotspot on chromosome 1, mapping on the region coding for the ferrireductase 6-transmembrane epithelial antigen of the prostate 3 (Steap3), a transcriptional target to p53. Steap3 regulated posttransfusion recovery, contributing to a ferroptosis-like process of lipid peroxidation, as validated via genetic manipulation in mice. Translational validation of murine findings in humans, STEAP3 polymorphisms were associated with RBC iron content, lipid peroxidation, and in vitro hemolysis in 13 091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. QTL analyses in humans identified a network of gene products (fatty acid desaturases 1 and 2, epoxide hydrolase 2, lysophosphatidylcholine acetyl-transferase 3, solute carrier family 22 member 16, glucose 6-phosphate dehydrogenase, very long chain fatty acid elongase, and phospholipase A2 group VI) associated with altered levels of oxylipins. These polymorphisms were prevalent in donors of African descent and were linked to allele frequency of hemolysis-linked polymorphisms for Steap3 or p53. These genetic variants were also associated with lower hemoglobin increments in thousands of single-unit transfusion recipients from the vein-to-vein database.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.

Red blood cell (RBC) membrane lipid peroxidation during blood bank storage profoundly impacts transfusion efficacy; however, the genetic determinants underlying RBC resilience remain incompletely defined. Here, we identify a critical role for glutathione peroxidase 4 (GPX4) - a pivotal enzyme protecting against iron-dependent lipid peroxidation (ferroptosis) - in regulating RBC storage quality and post-transfusion survival. Conditional erythroid-specific deletion of Gpx4 in mice exacerbated lipid hydroperoxide accumulation, oxidation and ubiquitination of membrane proteins, and reduced RBC recovery after transfusion. Multi-omics analyses in 13,091 human blood donors from the REDS RBC Omics cohort identified regulatory intergenic (rs8178962), intronic and missense genetic variants in GPX4 (rs73507255, rs8178967), particularly prevalent among donors of African descent, that were linked to increased lipid peroxidation and compromised post-transfusion hemoglobin increments. Single protein- and metabolome-wide association studies (pQTL/mQTL) highlighted genetic variants associated with enhanced (rs8178962) or impaired GPX4 expression, disrupted glutathione homeostasis, lipid hydroperoxide accumulation, accelerated membrane damage, and activation of ferroptotic signatures during RBC storage. These effects were exacerbated by genetic traits impairing redox homeostasis, including glucose 6-phosphate dehydrogenase (G6PD) deficiency (African variant rs1050828 V68M/N126D). Storage of murine RBCs in presence of the ferroptosis inhibitor ferrostatin-1 prevented storage-induced lipid peroxidation and boosted post-transfusion recovery, a beneficial effect in part phenocopied by supplementation of lipophilic antioxidants vitamin E and Lands cycle fueling via L-carnitine, and in part ablated by GPX4 inhibition via the covalent inhibitor ML210. This study offers mechanistic insights into RBC ferroptosis and positions GPX4 genetic status as a promising biomarker for precision transfusion medicine.

Administration of anti-RhD immunoglobulin (Ig) to decrease maternal alloimmunization (antibody-mediated immune suppression [AMIS]) was a landmark clinical development. However, IgG has potent immune-stimulatory effects in other settings (antibody-mediated immune enhancement [AMIE]). The dominant thinking has been that IgG causes AMIS for antigens on RBCs but AMIE for soluble antigens. However, we have recently reported that IgG against RBC antigens can cause either AMIS or AMIE as a function of an IgG subclass. Recent advances in mechanistic understanding have demonstrated that RBC alloimmunization requires the IFN-α/-β receptor (IFNAR) and is inhibited by the complement C3 protein. Here, we demonstrate the opposite for AMIE of an RBC alloantigen (IFNAR is not required and C3 enhances). RBC clearance, C3 deposition, and antigen modulation all preceded AMIE, and both CD4 ⁺ T cells and marginal zone B cells were required. We detected no significant increase in antigen-specific germinal center B cells, consistent with other studies of RBC alloimmunization that show extrafollicular-like responses. To the best of our knowledge, these findings provide the first evidence of an RBC alloimmunization pathway which is IFNAR independent and C3 dependent, thus further advancing our understanding of RBCs as an immunogen and AMIE as a phenomenon.