(P-CB-5) Biologically Old RBCs Are More Susceptible to Oxidative Stress Induced by Gamma Irradiation

Gamma irradiation of red cell concentrates (RCCs) prevents transfusion-associated graft-versus-host disease by preventing donor T-lymphocyte DNA replication; however, it can induce the generation of reactive oxygen species, leading to protein and lipid peroxidation. RCCs consist of a heterogeneous population of biologically aged red blood cells (RBCs), including young (Y-) and old (O-) RBCs. The ability of RBCs to manage oxidative stress declines with biological age. This study investigates the impact of gamma irradiation and subsequent hypothermic storage on biological Y- and O-RBC. Biotinylation was utilized to evaluate the post-irradiation recovery of Y- and O- biotin-labeled RBCs (BioRBCs).

Study

Design/Methods: Six RCCs were pooled, and their density profiles were determined using a Percoll-based method. Y- and O-RBCs (20 mL) were isolated, with Y-RBCs comprising 13.8% ± 0.07% and O-RBCs 18.5% ± 0.07% of the density spectrum. Aliquots of Y- and O-RBCs were labeled with two biotin concentrations (15 µg/mL and 48 µg/mL, respectively) and spiked back into the pooled unit. The pooled unit was divided into 5 RCCs. Aliquots of unseparated (U-), Y-, and O-RBCs along with RCC units, were irradiated with a minimum dose of 25 Gy. Hemolysis levels, supernatant potassium (K+), oxidative hemolysis, and p50 value were measured in RBC subpopulations before and at days 1, 7, and 14 post-gamma irradiation. Additionally, BioRBCs in RCCs were quantified using flow cytometry at the same intervals.

Results/Findings:

Hemolysis levels increased significantly across all RBC subpopulations post-gamma irradiation (p< 0.0001), with Y- and O-RBCs consistently showing higher levels than U-RBCs. Oxidative hemolysis increased across all subpopulations (p< 0.0001), with O-RBCs having a more pronounced trend (p=0.0110). Supernatant K+ levels correlated significantly with irradiation in Y-RBCs and U-RBCs (p< 0.0001) but not in O-RBCs during hypothermic storage. p50 values significantly decreased across all RBC subpopulations by day 14 (p< 0.0001), with Y-RBCs having the highest initial values and O-RBCs the lowest. No significant differences in p50 values were observed between irradiated and non-irradiated RBC subpopulations, suggesting hypothermic storage has a greater impact on p50 levels than irradiation. The number of Y- and O-BioRBCs decreased during hypothermic storage post-gamma irradiation with no significant differences between them.

Conclusions: Following irradiation, Y- and O-RBCs experienced increased hemolysis and supernatant K+, indicating similar membrane damage in both groups. The impaired antioxidant capacity of O-RBCs led to a more pronounced increase in oxidative hemolysis post-gamma irradiation. No significant variations in RBC survival across the subpopulations, emphasizing the need for further research to understand the complex factors affecting RBC behavior after irradiation and during hypothermic storage