Fractionation of Aged RBCs Based on Hemoglobin Content

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Red blood cell (RBC) transfusion is clinically used to treat hemodynamic instability and O2 carrying deficits in patients with acute blood loss, and patients with chronic anemia caused by bone marrow failure/suppression (1-4). Currently, cold storage of human RBCs (hRBCs) can preserve hRBCs for a maximum of six weeks (i.e. 42 days) (5). This relatively short ex vivo storage length has been set by the United States Food and Drug Administration (US FDA) based on the post transfusion viability (PTV) of stored hRBCs at 24 hours, which must be greater than or equal to 75 ± 9% (7) and the percent hemolysis of stored hRBCs which must be less than 1% (8). Despite widespread clinical use, stored RBCs face two major problems, namely: the steadily decreasing supply of RBC units (1, 9, 10), and the questionable clinical safety of RBCs stored for extended periods of time (11-17). The supply of RBCs is expected to diminish as the population base ages and demand increases (1, 9, 10, 18). As stored RBCs age, they undergo biochemical and biophysical changes that are often referred to as the storage lesion (8, 19-24). It is well known that upon transfusion of stored RBCs, there is a population of RBCs (i.e. healthy RBCs) that circulate for more than 24 hours, and another smaller population (i.e. damaged RBCs) that are cleared within 24 hours post transfusion (54). This population of cells destined to be cleared quickly can be higher than 25% in units stored for a mean of 30 days. Therefore, it could be clinically beneficial if the damaged RBCs in any unit of RBCs could be separated leaving a population of only healthy RBCs behind for transfusion. When a recipient is transfused with a dose of RBCs that overwhelms their circulatory system's ability to compensate for the increased intravascular volume, heart failure can ensue. This condition is known as Transfusion Associated Circulatory Overload (TACO). It is the second leading cause of death related to transfusion reported to the FDA (106). We hypothesize that reducing the amount of soon to be cleared RBCs from older units by 25% and fresher units by 15% will reduce the volume of transfused product, thereby reducing the risk of death from TACO. In fact, as the incidence of TACO is usually quite underestimated as it is often not appreciated as a transfusion reaction, and as the transfusion community has undertaken several large scale initiatives to reduce the incidence of the most common cause of death as reported to the FDA (transfusion related acute lung injury, TRALI), TACO will likely surpass TRALI as the leading cause of transfusion related mortality (52). Thus, taking steps to reduce the incidence of TACO will have far reaching benefits for many recipients of RBCs. Utilizing technology that exploits the intrinsic magnetization of the deoxygenated form of Hb inside RBC's in an applied magnetic field, the Yazer, Chalmers and Zborowski laboratories (6) demonstrated that RBCs lose magnetization during storage, which indicates that the aged RBCs lose Hb during storage. Furthermore, Chalmers and Zborowski have demonstrated technology capable of exploiting the intrinsic magnetization of deoxygenated Hb to perform a bulk separation of RBCs based on Hb content (55, 56). In this application, we hypothesize that RBCs with higher Hb content (i.e. healthy RBCs) correlate with higher deformability and PTV versus RBCs with lower Hb content (i.e. damaged RBCs). To test this hypothesis, we propose to further develop and optimize our magnetic separation technology to fractionate aged RBCs into different sub-fractions based on Hb content and Hb magnetic susceptibility, Characterize the biophysical and biochemical properties of these different sub-fractions of stored RBCs during storage and transfuse these different sub-fractions into an animal model with antioxidant status similar to humans to assess their ability to reduce/prevent systemic hypertension and oxidative tissue injury, both side-effects of exposure to cell-free Hb. This work is significant, since new technology will be developed to facilitate bulk separation of RBCs without labelling the cells to make the resulting RBC unit safer for clinical use.