Given the delicate redox balance that must be maintained in biologic systems, alterations in blood redox state may also directly affect the overall health and viability of banked blood, contributing to storage lesions due to increases in overall oxidative stress (13), potentiation of red cell lysis in oxidized states (14), and impact on clot formation and contraction (15). This also includes decreases in red cell deformability that can be present in states of increased oxidative stress (16). As a result, these alterations can contribute direct effects on systemic coagulopathy, as well as impairments in the ability of red cells to traverse the microcirculation and provide effective systemic oxygen delivery. Despite the seeming importance of making direct assessments of redox state to assess the viability of banked blood, there are currently few, if any, means by which to make these measurements in the clinical setting, and none by which to make them at the point of care (POC), immediately upon sample collection where these measurements could make the most impact. However, with the use of novel nanoporous (np) gold electrodes our group has described previously (10, 11), we can now make direct measurements of the redox state, via RP, at the point of care that could provide new insight into overall oxidative stress, and the degree of oxidative injury, present in RBC units and in circulating blood. The value measured reflects the overall (ambient) redox balance (RP) arising from the sum of metabolically active oxidant and reductant species contributing to the signal. The more positive the RP, the more oxidized the sample, and the more negative the RP, the more reduced (anti-oxidized) the sample. Having a direct measure of the oxidative state of RBCs available during storage and prior to transfusion could enable more advanced monitoring and utilization of banked RBCs.
Although RBC transfusions are vital in cases of trauma and hemorrhagic shock, multiple studies indicate that the administration of older blood units, as much as 14 days and older, can increase the risk of transfusion related lung injury (17) and increase the risk of adverse clinical outcomes and mortality in critically ill patients, especially if multiple transfusions of RBCs are required (18, 19). These effects are attributed to storage lesions that accumulate over time as RBCs are stored, and include altered redox states (increased oxidation/oxidative injury), with multiple reports describing oxidative injury and changes in redox state as a major contributing factor in red cell storage lesions (2–4). Also, while a previous study of oxidation-reduction potential in stored RBCs did show an increase in oxidation during storage, a small number of RBC units were tested and only units from day 1 and day 42 were included, with no evaluation of units between these extremes of age (20). In addition, redox potential was only measured in the supernatant, which may not provide the best measurement for assessing the RBC unit as a whole, as we have noted dampened/altered RP signals measured from plasma vs whole blood samples taken simultaneously from the same blood draw in previous studies (11).
Indeed, the data presented here support the concept as the overall RP of banked blood increases across the units tested reflecting an overall increase in oxidation present, and thus oxidative stress, as they age. When compared to the circulating redox state of healthy volunteers (representing fresh blood from healthy donors), all RBC units tested were found to have a median RP that was more positive (more oxidated), even when compared to RBCs on day 5 of storage. While time of storage appears to contribute, the discrepancy between healthy volunteers and day 5 of storage may also be related to the way in which the RBCs are processed and stored, producing a more oxidized environment from the onset of collection and processing. Variables contributing to this include temperature and pH variations, as well as the anticoagulant utilized in collection, and could result in an increase in the initial level of oxidation present at the onset of RBC storage (21).
However, age alone does not accurately predict the RP of any individual unit of RBCs. While data presented here demonstrates oxidation increases over time as RBCs are stored, there can also be significant variation in the RP status of any given donor due to multiple variables including age, state of health, comorbid conditions, and medications the subject may be taking at the time of donation. One study of 15 donors noted a smaller increase in malondialdehyde (MDA) and less decline in antioxidant capacity in blood taken from the same donors after receiving a 10 day regimen of antioxidant supplementation vs blood collected from these individuals prior to initiating the antioxidant regimen (22). Baseline variation in the RP values of peripheral blood taken from the healthy volunteers in our study also provides evidence to indicate that individual variations in baseline RP status, or oxidative state, exist throughout the population. This may have implications for donor screening and allow for better characterization of blood throughout initial processing and storage.
It should be noted that length of storage of RBCs is not itself necessarily a predictor of adverse clinical outcomes of transfusion. Randomized clinical trials in the settings of critically ill adults (23), cardiac surgery (24), and adult hospitalized patients (25), in which subjects were assigned to transfusion of either short duration storage RBCs or prolonged duration stored RBCs, have not demonstrated differences in mortality or secondary outcomes such as length of stay, or transfusion reactions. The generalizability of these trials to the treatment of specific patients has been questioned on the basis of a number of clinical and methodologic issues, including inconsistency in the definition of short, standard, and prolonged storage; heterogeneity of case mix; multiple transfusions; and dichotomization of the continuous variable of storage duration (26). Notably, none of these trials utilized any direct measure of RBC unit quality. However, a recent trauma study analyzing data from the Pragmatic Randomized Optimal Platelet and Plasma Ratios (PROPPR) trial reported an increased likelihood of 24-hour mortality in patients receiving massive transfusions (> 10 units) of RBCs if older than 22 days (27). In addition, a second study analyzing PROPPR data used a scalar metric (Scalar Age of Blood Index, SBI) that accounted for the distribution of the blood ages of all transfusions received by each patient and found that a higher, more positive SBI (indicating older RBC units were used) was associated with both 24-hour and 30 day mortality, despite adjustments for total units received and clinical covariates (28). Therefore, an open question remains whether there are critical aspects of the RBC storage lesion that can adversely impact clinical outcomes for specific patients.
While the data presented here provides further evidence that progressive oxidation occurs over time when RBCs are stored, it also suggests that RP screening of banked blood could be important independent of age and may serve as a precision medicine marker of RBC quality and blood oxidation state allowing for more selective and efficient use of RBC utilization. The ability to determine the RP status of RBC units in the blood bank, or at the point of care, could provide a useful blood “vital sign” to evaluate the health and oxidative state of RBCs. With a direct measure of the redox status, and oxidative stress of any given RBC unit planned for transfusion, therapeutic interventions could be delivered to improve the redox state, such as antioxidant therapies (e.g. Vitamin C, N-acetylcysteine, and others) that have been shown to reduce oxidative stress in RBCs (5, 6), reduce cell damage by free radicals (22), and preserve red cell energy and overall RBC quality (7). Although evaluating a limited number of single pRBC units over time, our colleagues at Virginia Commonwealth University have also reported evidence that the addition of Vitamin C may help to stabilize the redox state over the duration of pRBC storage and that progressive oxidation occurs in these units (29). Furthermore, RP values may also serve as a gauge for providing systemic antioxidant therapy in patients receiving large transfusions if their systemic RP values increase significantly after resuscitation with RBCs. In fact, traumatic injury itself can produce negative effects on the health and viability of circulating blood by causing changes in circulating redox state, as well as the induction of platelet activation that can stimulate the production of reactive oxygen species (ROS) that alter the systemic redox state and promote the adhesion and activation of additional neutrophils, platelets, and endothelial cells, stimulating the extrinsic coagulation pathway and ultimately leading to dysregulation of the coagulation cascade (30). Given that 33% of patients suffering from trauma and hemorrhage present with coagulopathy on hospital admission (31), if the redox state of banked blood units given to these patients is also altered, the negative effects of storage lesions could be amplified, worsening systemic oxidation and oxidative stress. Therefore, having bedside measurements of RP at the point of care could add a new dimension to patient monitoring that could improve both banked blood viability, its effectiveness when given to those in need of transfusion, and the overall health and function of circulating blood in these patients.
There are a number of important limitations to this study. Overall, the total number of units sampled was relatively small. Sampling was done from the RBC segments and not directly from the blood bag itself where RP may have been different. Current studies are underway that include direct sampling from the blood bag itself. We performed only single RP measures on each sample, however our previous work has demonstrated excellent reproducibility of measurement (10, 11). Given this and the small sample size of the segments we did not feel it necessary to perform duplicate measures. We did not measure RP of RBCs at day zero, although they were not available due to the time required for processing and subsequent delivery to the blood bank after initial collection from blood donors. While we did measure RP in fresh whole blood of non-acutely ill/injured volunteers, this blood was not processed in the same way that occurs with blood donation. As mentioned earlier, such processing could change RP, and we decided to make direct measures of RP from these samples as the result would more likely reflect circulating RP values of patients receiving transfusions. Lastly, we made no additional measures of oxidative stress or RBC damage such as fragility-deformability or oxygen carrying capacity (p50). Thus, it is not possible to know with certainly the extent of storage lesions present at RP values measured in this study.