Background Intravascular red cell hemolysis impairs NO-redox homeostasis, producing endothelial dysfunction, platelet activation and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of exocytic microvesicles or “microparticles” and hemolysis, which we hypothesized could impair vascular function and contribute to the putative “storage lesion” of banked blood. Methods and Results We now find that storage of human red blood cells under standard blood banking conditions results in the accumulation of cell free and microparticle-encapsulated hemoglobin which, despite 39 days of storage, remains in the reduced ferrous oxyhemoglobin redox state and stoichiometrically reacts with and scavenges the vasodilator nitric oxide (NO). Using stopped-flow spectroscopy and laser triggered NO release from a caged NO compound we found that both free hemoglobin and microparticles react with NO about 1000 times faster than with intact erythrocytes. In complementary in vivo studies we show that hemoglobin, even at concentrations below 10 μM (in heme), produces potent vasoconstriction when infused into the rat circulation, while controlled infusions of methemoglobin and cyanomethemoglobin, which do not consume NO, have substantially reduced vasoconstrictor effects. Infusion of the plasma from stored human red cell units into the rat circulation produces significant vasoconstriction related to the magnitude of storage related hemolysis. Conclusions The results of these studies suggest new mechanisms for endothelial injury and impaired vascular function associated with the most fundamental of storage lesions, hemolysis.

Genetic polymorphisms in blood donors may contribute to donor-specific differences in the survival of red blood cells (RBCs) during cold storage and after transfusion. Genetic variability is anticipated to be high in donors with racial admixture from malaria endemic regions such as Africa and Asia. The purpose of this study was to test the hypothesis that donor genetic background, reflected by sex and self-reported ethnicity, significantly modulates RBC phenotypes in storage. High throughput hemolysis assays were developed and used to evaluate stored RBC samples from 11 115 African American, Asian, white, and Hispanic blood donors from 4 geographically diverse regions in the United States. Leukocyte-reduced RBC concentrate-derived samples were stored for 39 to 42 days (1–6°C) and then evaluated for storage, osmotic, and oxidative hemolysis. Male sex was strongly associated with increased susceptibility to all 3 hemolysis measures (P < .0001). African American background was associated with resistance to osmotic hemolysis compared with other racial groups (adjusted P < .0001). Donor race/ethnicity was also associated with extreme (>1%) levels of storage hemolysis exceeding US Food and Drug Administration regulations for transfusion (hemolysis >1% was observed in 3.51% of Asian and 2.47% of African American donors vs 1.67% of white donors). These findings highlight the impact of donor genetic traits on measures of RBC hemolysis during routine cold storage, and they support current plans for genome-wide association studies, which may help identify hereditable variants with substantive effects on RBC storage stability and possibly posttransfusion outcomes.

Key Points• In canine S aureus pneumonia, first randomized blinded trial showing blood transfused at end of storage period increases mortality.• Increased in vivo hemolysis, cell-free hemoglobin, pulmonary hypertension, tissue damage, and gas exchange abnormalities each contributed.Two-year-old purpose-bred beagles (n ‫؍‬ 24) infected with Staphylococcus aureus pneumonia were randomized in a blinded fashion for exchange transfusion with either 7-or 42-day-old canine universal donor blood (80 mL/kg in 4 divided doses). Older blood increased mortality (P ‫؍‬ .0005), the arterial alveolar oxygen gradient (24-48 hours after infection; P < .01), systemic and pulmonary pressures during transfusion (4-16 hours) and pulmonary pressures for ϳ 10 hours afterward (all P < .02). Further, older blood caused more severe lung damage, evidenced by increased necrosis, hemorrhage, and thrombosis (P ‫؍‬ .03) noted at the infection site postmortem. Plasma cell-free hemoglobin and nitric oxide (NO) consumption capability were elevated and haptoglobin levels were decreased with older blood during and for 32 hours after transfusion (all P < .03). The low haptoglobin (r ‫؍‬ 0.61; P ‫؍‬ .003) and high NO consumption levels at 24 hours (r ‫؍‬ ؊0.76; P < .0001) were associated with poor survival. Plasma nontransferrin-bound and labile iron were significantly elevated only during transfusion (both P ‫؍‬ .03) and not associated with survival (P ‫؍‬ NS). These data from canines indicate that older blood after transfusion has a propensity to hemolyze in vivo, releases vasoconstrictive cell-free hemoglobin over days, worsens pulmonary hypertension, gas exchange, and ischemic vascular damage in the infected lung, and thereby increases the risk of death from transfusion. (Blood. 2013;121(9):1663-1672) IntroductionTransfusion of red blood cells (RBCs) is one of the most commonly used, potentially lifesaving medical therapies. Each year, some 80.7 million units of blood are collected in 167 countries worldwide, and approximately 15 million units are collected and transfused in the United States alone. 1,2 RBCs can be stored for up to 42 days to meet inventory needs, and by standard practice the oldest blood is usually used first ("first in, first out"). Food and Drug Administration (FDA) regulations only stipulate that at the end of the storage period 75% of the cells remain in the circulation at 24 hours after transfusion and that hemolysis in the storage bag does not exceed 1%, 3 no other product specification of quality is required. Although 6-week-old stored blood meets current FDA standards, laboratory and clinical studies have raised concerns that "older" blood may not be as safe as blood stored for a shorter duration. [4][5][6][7][8] Refrigerated storage of blood results in a "storage lesion" characterized by rheologic changes, metabolic derangements, changes in oxygen affinity and delivery, oxidative injury to lipids and proteins, RBC shape change, loss of membrane carbohydrates, and reduced RBC lifespan. [8][9][10] The storage lesion resu...

464haematologica | 2013; 98(3)The intensity of hemolytic anemia has been proposed as an independent risk factor for the development of certain clinical complications of sickle cell disease, such as pulmonary hypertension, hypoxemia and cutaneous leg ulceration. A composite variable derived from several individual markers of hemolysis could facilitate studies of the underlying mechanisms of hemolysis. In this study, we assessed the association of hemolysis with outcomes in sickle cell anemia. A hemolytic component was calculated by principal component analysis from reticulocyte count, serum lactate dehydrogenase, aspartate aminotransferase and total bilirubin concentrations in 415 hemoglobin SS patients. Association of this component with direct markers of hemolysis and clinical outcomes was assessed. As primary validation, both plasma red blood cell microparticles and cell-free hemoglobin concentration were higher in the highest hemolytic component quartile compared to the lowest quartile (P≤0.0001 for both analyses). The hemolytic component was lower with hydroxyurea therapy, higher hemoglobin F, and alpha-thalassemia (P≤0.0005); it was higher with higher systemic pulse pressure, lower oxygen saturation, and greater values for tricuspid regurgitation velocity, left ventricular diastolic dimension and left ventricular mass (all P<0.0001). Two-year follow-up analysis showed that a high hemolytic component was associated with an increased risk of death (hazard ratio, HR 3.44; 95% confidence interval, CI: 1.2-9.5; P=0.02). The hemolytic component reflects direct markers of intravascular hemolysis in patients with sickle cell disease and allows for adjusted analysis of associations between hemolytic severity and clinical outcomes. These results confirm associations between hemolytic rate and pulse pressure, oxygen saturation, increases in Doppler-estimated pulmonary systolic pressures and mortality (Clinicaltrials.gov identifier: NCT00492531). The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe

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