Red blood cells stored for increasing periods produce progressive impairments in nitric oxide-mediated vasodilation.

Blood transfusion is the most commonly employed procedure for hospitalized patients in the United States, based on discharge codes.1 Optimal functioning of this system depends on the ability to store blood for up to 42 days before transfusion. Studies used to support 42-day post-donation storage include biochemical measurements (2,3-diphosphoglycerate acid [2,3-DPG] and adenosine triphosphate [ATP]), measures of red blood cell (RBC) integrity (plasma free hemoglobin [Hb]), and quantification of survival of stored RBCs in autologous transfusion recipients at 24 hours after transfusion.2,3 However, there are no specific measurements performed to show that RBCs stored up to 42 days achieve minimal standards of efficacy or produce acceptably low rates of adverse events in transfusion recipients. Despite the undeniable therapeutic benefits of blood transfusion, numerous studies have demonstrated significant biochemical, structural, and morphologic changes in RBCs during pretransfusion storage.4-6 These changes (the “RBC storage lesion”) may be of negligible impact after short storage periods (“fresh RBCs”), but longer-term storage approaching 42 days (“storage-aged RBCs”; saRBCs) may have deleterious effects on the recipient. Tinmouth and coworkers7 and Wang and coworkers8 have performed systematic reviews of dozens of studies that investigated the relationship between blood storage and adverse transfusion events. Meta-analyses showed worse recipient outcomes after transfusion of saRBCs. Since the largest clinical studies included in these reviews were retrospective, further elucidation of the possible adverse effects of saRBCs may be provided by prospective randomized trials. The largest to be published to date, ARIPI,9 compared fresh blood (stored up to 7 days; median, 5 days) with standard of care (stored up to 42 days; median, 13 days) in low-birthweight neonates. Although the outcomes showed no difference between study arms, the relatively short storage times in the standard-of-care arm do not allow an assessment of the efficacy of saRBCs stored for long periods (21-42 days). This issue may be better addressed in the ongoing RECESS,10 ABLE,11 and Red Cell Storage Duration and Outcomes in Cardiac Surgery studies.12 As an adjunct to biochemical, molecular, and clinical outcomes studies, investigations of the acute physiologic effects of saRBCs may identify potential mechanisms by which saRBC transfusions could cause adverse outcomes. In addition to oxygen and carbon dioxide transport, another significant physiologic role of RBCs is in the process of hypoxic vasodilation. This activity, which regulates local blood flow to preferentially perfuse and provide oxygen for the most hypoxic tissues, involves combined activities of RBCs and endothelial cells to regulate arteriolar smooth muscle tone.13 Disruption of hypoxic vasodilation by saRBCs represents a viable physiologic hypothesis to explain an association between blood storage and adverse transfusion events.14 Although hypoxic vasodilation is not yet fully understood at a biochemical level, it is likely to involve regulation of nitric oxide (NO) signaling. Theoretically, if saRBCs did not produce or stimulate sufficient NO, then replacement of a patient’s normal RBCs with transfused cells could result in an NO synthesis defect. There is some evidence for this possibility. For example, studies show that S-nitrosylation of Hb (SNO-Hb) may produce a storage pool of NO within RBCs that can be released in hypoxic environments,15-18 and S-nitrosylation of Hb has been shown to decline with RBC storage.19 Other investigators have shown that nitrite (which is present at high levels in plasma) can be converted to NO by Hb.20-23 In this model, deoxygenated Hb, which is at high concentrations in hypoxic tissues, binds nitrite and a proton to generate NO and methemoglobin. If blood storage altered the nitrite reductase activity of Hb, this NO source would be disrupted. With either of these models, NO must diffuse from the RBC to the smooth muscle to produce vasodilation.24,25 In a third model that could potentially explain reduced NO production with saRBCs, ATP release by RBCs (a robust mechanism for stimulating endothelial NO production26-29) may be impaired by storage.30 As an alternative mechanism that could also underlie reduced hypoxic vasodilation after transfusion, infused saRBCs could produce an inhibitory factor that interrupts normal NO signaling between endogenous RBCs, endothelium, and the underlying smooth muscle. This mechanism, which may function in conjunction with (or instead of) the synthesis defects described above, is attractive because it could allow a small volume of transfused saRBCs to broadly impair hypoxic vasodilation. Disruption of NO signaling by saRBCs could be mediated by free Hb (or other RBC constituents) released into the plasma as a consequence of RBC hemolysis or by Hb or other factors encapsulated in RBC-derived microparticles.4,24,25 The free Hb mechanism is supported by clinical and experimental studies showing that plasma free Hb can scavenge NO, reducing its bioavailability and causing vascular sequelae.31-33 Microparticles may be important because they can flow closer to the endothelium than intact RBCs, bringing Hb close to the sites of NO synthesis, which may further accentuate NO scavenging after transfusion.34 Although intact saRBCs could also potentially interfere with NO-mediated vasodilation, there is currently little evidence for this mechanism. In these studies, we used an ex vivo system where endothelium-dependent, NO-mediated vasodilation can be experimentally controlled and sensitively monitored to assess the impact of fresh blood versus saRBCs on vasodilation. This system has been previously employed to probe interactions between RBCs and the vasculature and to demonstrate how RBC-derived ATP and deoxyhemoglobin-catalyzed NO production can stimulate vasodilation.14 Using this model, we have instead focused on determining whether saRBCs can interfere with normal NO-mediated vasodilatory activities and to what extent the inhibitory effect is mediated by substances, such as Hb, released from saRBCs, or by an alternate mechanism.