# RBC Storage Lesion Studies in Humans and Experimental Models of Shock
## 4 Effects of Longer-Stored versus Fresher Blood Transfusions  
When septic canines underwent RBC transfusion with longer-stored RBCs, there was increased mortality (\(p=0.0005\)), worsening of lung injury as measured by the arterial-alveolar oxygen gradient (\(p\leq 0.01\)) (Figure 2), and more histologically quantifiable lung damage (\(p=0.03\)). After transfusion, animals receiving longer-stored RBCs had evidence of increased in vivo hemolysis with higher levels of both free iron and cell-free hemoglobin (CFH) (\(p\leq 0.03\)) [9]. These animals also had elevations in both the mean systemic and pulmonary pressures (all \(p\leq 0.02\)) which mechanistically fit with the known vasoconstretric effect of CFH. This vasoconstriction results from CFH scavenging nitric oxide  
Figure 1: Meta-analysis of the effects of transfusion of old vs. new blood on mortality. The data marker size is proportional to the inverse variance of each point estimate. Meta-analysis of 21 studies revealed a highly statistically significant difference in mortality favoring transfusion of new blood vs. old blood with respect to mortality [odds ratio 1.16, 95% confidence interval (CI) 1.07–1.24, \(p=0.0001\)]. From: [PERSON]; [PERSON]; [PERSON]; [PERSON]; [PERSON] Transfusion of older stored blood and risk of death: a meta-analysis. Transfusion 2012, 52, 1184-1195, doi:10.1111/j.1537-2995.2011.03466.x.  
(NO), a vasodilator. In the animals that did not survive, there was extensive pulmonary necrosis associated with the mortality. This represented one of the first randomized blinded large animal trials demonstrating that mortality increases when blood is transfused at the end of the storage period.  
Mechanisms that Increase Risk after Transfusion of Longer-Stored Red Blood Cells (RBCs) during Infection  
This data suggests two main pathways underlying the reported adverse effects of longer-stored RBCs during infection. In the case of both canine and human RBCs, when donor cells are obtained for transfusion they are at different stages in the life span of an RBC. As these donor RBCs age during cold storage, the oldest RBCs likely become even more fragile and prone to in vivo hemolysis after transfusion, resulting in increased release of plasma CFH and iron. Both CFH and iron have well described toxicities that can potentially result in worse outcomes in transfusion settings [10; 11; 12]. CFH is well known to cause vasoconstriction through its ability to scavenge NO, an endogenous potent vasodilator. This CFH-induced vasoconstriction can cause ischemia and vascular endothelial injury at sites of infection. Iron serves as a critical nutrient for bacterial growth, potentially worsening infection.  
Bacteria associated with human infection commonly produce hemolysis, a virulence factor which can lyse RBCs. Haptoglobin is a natural plasma protein that binds and complexes to CFH. These CFH and haptoglobin complexes, once formed, are sequestered in the intravascular space because of size and then bind to specific receptors on macrophages in the liver and are cleared from the circulation. With excess hemolysis caused both by transfusing longer-stored RBCs and bacteria producing hemolysis, the haptoglobin proteins during infection can become saturated and the reticuloendothelial system is unable to perform the normal function of clearing CFH. This results in excess circulating CFH as well as free and protein-bound iron from RBCs, resulting in increased toxicity during infection.