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.

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.

Current good manufacturing practices-compliant manufacture and measurement of biotin-labeled red blood cells.

BACKGROUND: Red blood cells (RBCs) can be labeled with N-hydroxysuccinimidobiotin (sulfo-NHS-biotin), which binds to cell surface proteins under aqueous conditions. Biotinylated RBCs can be safely infused and detected in peripheral blood samples using flow cytometry, using a fluorochrome-conjugated streptavidin (SA) detection reagent. Biotinylated RBCs have been used to track survival of transfused RBCs, and have applications in optimizing RBC storage and in understanding donor genetic, environmental and disease factors affecting RBC products. METHODS: We have developed a closed-system, current good manufacturing practices (cGMP)-compliant procedure for biotinylation of RBCs and a quantitative flow cytometric assay to estimate the dose of cell-bound biotin delivered to the patient. Resulting products were characterized for variability, sterility, endotoxin, hemolysis, total dose of cell-bound biotin and stability. RESULTS: The density of biotin-labeling increased as a log-linear function of sulfo-NHS-biotin-labeling concentration, with greater variability at lower concentrations. The upper estimates of biotin doses in the average product (mean RBC content = 5.55 × 1011) were 9.8 and 73.0 µg for products labeled at 3 and 15 µg sulfo-NHS-biotin/mL of total reaction mixture (27 and 135 nmol/mL packed RBCs), respectively. All products were negative for bacterial and fungal growth at 14 days and were below the limit of endotoxin detection. Biotinylated RBCs were stable in vitro for up to 50 days after labeling. DISCUSSION: We have validated a closed-system procedure for biotinylating RBCs for investigational use. A standard operating procedure is presented in sufficient detail for implementation in a cGMP-compliant cell-processing facility.

Piloting and implementation of quality assessment and quality control procedures in RBC-Omics: a large multi-center study of red blood cell hemolysis during storage.

BACKGROUND: The major aims of the RBC-Omics study were to evaluate the genomic and metabolomic determinants of spontaneous and stress-induced hemolysis during RBC storage. This study was unique in scale and design to allow evaluation of RBC donations from a sufficient number of donors across the spectrum of race, ethnicity, sex, and donation intensity. Study procedures were carefully piloted, optimized, and controlled to enable high-quality data collection. METHODS: The enrollment goal of 14,000 RBC donors across four centers, with characterization of RBC hemolysis across two testing laboratories, required rigorous piloting and optimization and establishment of a quality assurance (QA) and quality control (QC) program. Optimization of WBC elution from leukoreduction (LR) filters, development and validation of small-volume transfer bags, impact of manufacturing and sample-handling procedures on hemolysis parameters, and testing consistency across laboratories and technicians and over time were part of this quality assurance/quality control program. RESULTS: LR filter elution procedures were optimized for obtaining DNA for analysis. Significant differences between standard and pediatric storage bags led to use of an alternative LR-RBC transfer bag. The impact of sample preparation and freezing methods on metabolomics analyses was evaluated. Proficiency testing monitored and documented testing consistency across laboratories and technicians. CONCLUSION: Piloting and optimization, and establishment of a robust quality assurance/quality control program documented process consistency throughout the study and was essential in executing this large-scale multicenter study. This program supports the validity of the RBC-Omics study results and a sample repository that can be used in future studies.

Washing older blood units before transfusion reduces plasma iron and improves outcomes in experimental canine pneumonia.

In a randomized controlled blinded trial, 2-year-old purpose-bred beagles (n = 24), with Staphylococcus aureus pneumonia, were exchanged-transfused with either 7- or 42-day-old washed or unwashed canine universal donor blood (80 mL/kg in 4 divided doses). Washing red cells (RBC) before transfusion had a significantly different effect on canine survival, multiple organ injury, plasma iron, and cell-free hemoglobin (CFH) levels depending on the age of stored blood (all, P < .05 for interactions). Washing older units of blood improved survival rates, shock score, lung injury, cardiac performance and liver function, and reduced levels of non-transferrin bound iron and plasma labile iron. In contrast, washing fresh blood worsened all these same clinical parameters and increased CFH levels. Our data indicate that transfusion of fresh blood, which results in less hemolysis, CFH, and iron release, is less toxic than transfusion of older blood in critically ill infected subjects. However, washing older blood prevented elevations in plasma circulating iron and improved survival and multiple organ injury in animals with an established pulmonary infection. Our data suggest that fresh blood should not be washed routinely because, in a setting of established infection, washed RBC are prone to release CFH and result in worsened clinical outcomes.

Testosterone-dependent sex differences in red blood cell hemolysis in storage, stress, and disease.

BACKGROUND: Red blood cell (RBC) hemolysis represents an intrinsic mechanism for human vascular disease. Intravascular hemolysis releases hemoglobin and other metabolites that inhibit nitric oxide signaling and drive oxidative and inflammatory stress. Although these pathways are important in disease pathogenesis, genetic and population modifiers of hemolysis, including sex, have not been established. STUDY DESIGN AND METHODS: We studied sex differences in storage or stress-induced hemolysis in RBC units from the United States and Canada in 22 inbred mouse strains and in patients with sickle cell disease (SCD) using measures of hemolysis in 315 patients who had homozygous SS hemoglobin from the Walk-PHASST cohort. A mouse model also was used to evaluate posttransfusion recovery of stored RBCs, and gonadectomy was used to determine the mechanisms related to sex hormones. RESULTS: An analysis of predisposition to hemolysis based on sex revealed that male RBCs consistently exhibit increased susceptibility to hemolysis compared with females in response to routine cold storage, under osmotic or oxidative stress, after transfusion in mice, and in patients with SCD. The sex difference is intrinsic to the RBC and is not mediated by plasmatic factors or female sex hormones. Importantly, orchiectomy in mice improves RBC storage stability and posttransfusion recovery, whereas testosterone repletion therapy exacerbates hemolytic response to osmotic or oxidative stress. CONCLUSION: Our findings suggest that testosterone increases susceptibility to hemolysis across human diseases, suggesting that male sex may modulate clinical outcomes in blood storage and SCD and establishing a role for donor genetic variables in the viability of stored RBCs and in human hemolytic diseases.

In a canine pneumonia model of exchange transfusion, altering the age but not the volume of older red blood cells markedly alters outcome.

BACKGROUND: Massive exchange transfusion of 42-day-old red blood cells (RBCs) in a canine model of Staphylococcus aureus pneumonia resulted in in vivo hemolysis with increases in cell-free hemoglobin (CFH), transferrin-bound iron (TBI), non-transferrin-bound iron (NTBI), and mortality. We have previously shown that washing 42-day-old RBCs before transfusion significantly decreased NTBI levels and mortality, but washing 7-day-old RBCs increased mortality and CFH levels. We now report the results of altering volume, washing, and age of RBCs. STUDY DESIGN AND METHODS: Two-year-old purpose-bred infected beagles were transfused with increasing volumes (5-10, 20-40, or 60-80 mL/kg) of either 42- or 7-day-old RBCs (n = 36) or 80 mL/kg of either unwashed or washed RBCs with increasing storage age (14, 21, 28, or 35 days; n = 40). RESULTS: All volumes transfused (5-80 mL/kg) of 42-day-old RBCs resulted in alike (i.e., not significantly different) increases in TBI during transfusion as well as in CFH, lung injury, and mortality rates after transfusion. Transfusion of 80 mL/kg RBCs stored for 14, 21, 28, and 35 days resulted in increased CFH and NTBI in between levels found at 7 and 42 days of storage. However, washing RBCs of intermediate ages (14-35 days) does not alter NTBI and CFH levels or mortality rates. CONCLUSIONS: Preclinical data suggest that any volume of 42-day-old blood potentially increases risks during established infection. In contrast, even massive volumes of 7-day-old blood result in minimal CFH and NTBI levels and risks. In contrast to the extremes of storage, washing blood stored for intermediate ages does not alter risks of transfusion or NTBI and CFH clearance.

Transfused older stored red blood cells improve the clinical course and outcome in a canine lethal hemorrhage and reperfusion model.

BACKGROUND: In canine models, transfused older stored red blood cells (RBCs) hemolyze in vivo resulting in significantly increased intravascular cell-free hemoglobin (CFH) and non-transferrin-bound iron (NTBI). During canine bacterial pneumonia with septic shock, but not in controls, older stored RBCs were associated with significantly increased lung injury and mortality. It is unknown if in shock without infection transfusion of older RBCs will result in similar adverse effects. STUDY DESIGN AND METHODS: Two-year-old purpose-bred beagles (n = 12) were transfused similar quantities of either older (42-day) or fresher (7-day) stored universal donor canine RBCs 2.5 hours after undergoing controlled hemorrhage (55 mL/kg). RESULTS: With older transfused RBCs, CFH (p < 0.0001) and NTBI (p = 0.004) levels increased, but lung injury (p = 0.01) and C-reactive protein levels (p = 0.002) declined and there was a trend toward lower mortality (18% vs. 50%). All three deaths after transfused fresher RBCs resulted from hepatic fractures. Lowered exogenous norepinephrine requirements (p < 0.05) and cardiac outputs (p < 0.05) after older transfused RBCs were associated with increased CFH levels that have known vasoconstrictive nitric oxide scavenging capability. CONCLUSIONS: In hemorrhagic shock, older RBCs altered resuscitation physiology but did not worsen clinical outcomes. Elevated CFH may lower norepinephrine requirements and cardiac outputs ameliorating reperfusion injuries. With hemorrhagic shock, NTBI levels persist in contrast to the increased clearance, lung injury, and mortality in the previously reported infection model. These preclinical data suggest that whereas iron derived from older RBCs promotes bacterial growth, worsening septic shock mortality during infection, release of CFH and NTBI during hemorrhagic shock is not necessarily harmful.

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 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.

Sickle Cell Trait Increases Red Blood Cell Storage Hemolysis and Post-Transfusion Clearance in Mice.

BACKGROUND: Transfusion of blood at the limits of approved storage time is associated with lower red blood cell (RBC) post-transfusion recovery and hemolysis, which increases plasma cell-free hemoglobin and iron, proposed to induce endothelial dysfunction and impair host defense. There is noted variability among donors in the intrinsic rate of storage changes and RBC post-transfusion recovery, yet genetic determinants that modulate this process are unclear. METHODS: We explore RBC storage stability and post-transfusion recovery in murine models of allogeneic and xenogeneic transfusion using blood from humanized transgenic sickle cell hemizygous mice (Hbatm1PazHbbtm1TowTg(HBA-HBBs)41Paz/J) and human donors with a common genetic mutation sickle cell trait (HbAS). FINDINGS: Human and transgenic HbAS RBCs demonstrate accelerated storage time-dependent hemolysis and reduced post-transfusion recovery in mice. The rapid post-transfusion clearance of stored HbAS RBC is unrelated to macrophage-mediated uptake or intravascular hemolysis, but by enhanced sequestration in the spleen, kidney and liver. HbAS RBCs are intrinsically different from HbAA RBCs, with reduced membrane deformability as cells age in cold storage, leading to accelerated clearance of transfused HbAS RBCs by entrapment in organ microcirculation. INTERPRETATION: The common genetic variant HbAS enhances RBC storage dysfunction and raises provocative questions about the use of HbAS RBCs at the limits of approved storage.

Towards microfluidic-based depletion of stiff and fragile human red cells that accumulate during blood storage.

In this study, the effects of prolonged storage on several biophysical properties of red blood cells (RBCs) were investigated. Single cell deformability was used as an important criterion in determining subgroups of RBCs evolved during storage lesion. A deformability-based microfluidic cell sorting technology was applied, which demonstrates the ability to enrich and separate the less deformable subpopulations of stored blood. These less deformable RBC subpopulations were then associated with other important markers such as osmotic fragility indicating cell integrity as well as microparticle content. This work demonstrates a systematic methodology to both monitor and improve banked blood quality, thereby reducing risks related to blood transfusion.