Distribution Dynamics of Biologically Aged RBC Subpopulations: Impact of Blood Component Manufacturing Methods, Cryopreservation, and Irradiation
Creators: Hemmatibardehshahi, Sanaz
Publisher: name: University of Alberta Library
Year: 2024

<p>The quality of red blood cell concentrates (RCCs) is influenced by storage duration, donor variability, and processing methods. RCCs contain heterogeneous subpopulations of red blood cells (RBCs), ranging from young (Y-RBC) to old (O-RBC), each with distinct structural and biochemical characteristics. A higher proportion of O-RBCs contributes to storage lesions and decreased post-transfusion survival rates. Understanding how manufacturing methods impact the distribution of RBC subpopulations is crucial in estimating the quality of RCCs. Additionally, cryopreservation processes result in approximately 15% loss of vulnerable RBCs due to osmotic and freezing stress. Investigating how different RBC subpopulations respond to these stresses can provide insights into RBC variability in response to cryopreservation techniques and minimize RBC loss. Gamma irradiation, a common practice in transfusion medicine, has been linked to increased hemolysis and supernatant potassium (K+) levels. Studying RBC subpopulations&#39; responses to irradiation can help develop strategies to minimize irradiation-induced lesions by achieving a favorable ratio of Y- to O-RBCs in units undergoing irradiation. This thesis explored how manufacturing methods, cryopreservation, and irradiation affect various biologically aged RBC subpopulations. It compared the density-based distribution of RBCs, osmotic parameters, irradiation-induced lesions, and recovery rates of Y- and O-RBCs after thawing and irradiation during hypothermic storage.<br>
Density-based Percoll® separation was employed to evaluate the impact of manufacturing methods on the distribution of RBCs in RCCs produced by whole blood filtration (WBF) and red cell filtration (RCF) methods. The membrane water (Lp) and solute permeability (Ps), osmotic fragility, and osmoscan parameters of Y- and O-RBC subpopulations were measured using stopped-flow and ektacytometry. The biotinylation technique was used to assess the recovery rate of spiked young and old bio-labeled RBCs (Y- and O-BioRBCs) in RCC units post-thaw. The impact of gamma irradiation on Y- and O-RBC subpopulations was assessed by measuring oxidative hemolysis, supernatant K+, and p50 before and after irradiation during hypothermic storage. The biotinylation approach was implied to evaluate the recovery of Y- and O-RBCs after irradiation. <br>
The comparative analysis of RCCs processed using RCF and WBF methods revealed that RCCs processed with the WBF method exhibited higher RBC counts, hemoglobin (Hb), hematocrit (HCT), and volume. Despite variations observed in individual units, the overall ratio of Y- to O-RBCs remained consistent across all units regardless of the processing method employed. The stopped-flow experiment revealed that O-RBCs have higher Lp values followed by Y-RBCs, with unseparated RBCs (U-RBCs) showing the lowest Lp values across all NaCl solutions at 4 and 20 °C (p&lt;0.0001). O-RBCs displayed a higher Ps value than Y-RBCs during deglycerolization, indicating faster glycerol efflux (p&lt;0.0001). Furthermore, Y-RBCs exhibited higher Ohyper, EImax, and lower rigidity compared to O-RBCs, suggesting greater shear stress tolerance (p&lt;0.0001). Despite these findings, no advantages in Y-RBC post-deglycerolization survival were observed. The irradiation results show that hemolysis levels and supernatant K+ significantly increased after gamma irradiation during hypothermic storage for all RBC subpopulations, with Y- and O-RBCs consistently exhibiting higher hemolysis levels than U-RBCs. Oxidative hemolysis increased across all subpopulations (p&lt;0.0001), with O-RBCs showing a more pronounced trend (p=0.0110). p50 values were decreased across all RBC subpopulations following irradiation. The number of Y- and O-BioRBCs decreased during hypothermic storage, with no significant differences observed between RBC subpopulations.<br>
In conclusion, although WBF-derived RCCs had higher levels of HCT, Hb, RBC counts, and volume, the distribution of Y- and O-RBCs in RCCs was not significantly influenced by manufacturing methods. Variations observed in the distribution of RBCs in RCCs are likely attributed to donor factors rather than the manufacturing method. This highlights the importance of considering donor-related factors when assessing RCC composition. Despite Y-RBCs exhibiting lower membrane permeability and superior osmoscan parameters, no advantage in their post-deglycerolization survival was observed. The hemolysis and supernatant K+ levels increased in Y- and O-RBCs following irradiation, suggesting comparable membrane damage. The impaired antioxidant capacity of O-RBCs leads to a more pronounced increase in oxidative hemolysis post-gamma irradiation. Despite these differences, no significant variations in RBC survival were observed across RBC subpopulations. Further research is needed to elucidate the complex factors influencing RBC behavior post-irradiation and during hypothermic storage. This research enriches our understanding of how processing methods may impact RBC subpopulations in RCCs, providing valuable insights into optimizing methodologies, considering the diverse subpopulations within RCCs.</p>