Rejuvenation of Aged Human Red Cells Improves Their in Vivo Recovery in a Mouse Model
Authors: GelderMan-Fuhrmann Monique , Vostal Jaroslav G. 
Published: October 2019

Evaluation of novel storage or processing technology for human red blood cells (RBCs) involves in vitro tests on the red cells to determine biochemical changes and in vivo studies in healthy human volunteers with radiolabeled red cells to determine in vivo recovery 24 hours post infusion. In vivo studies are needed because our understanding of red cell storage lesions is not sufficient to identify an in vitro test(s) that would adequately predict red cell performance in vivo. The clinical studies with radiolabeled cells are used as the gold standard for evaluation prior to approval of a novel technology by the FDA. However, in vivo studies require time and funds and can be a significant hurdle in the development of new products. An animal model that could predict performance of human red cells in vivo would be useful in the development process. We previously reported that severe combined immunodeficient (SCID) mice could be used as a model to identify damaged human platelets (Transfusion. 47(8):1540–9, 2007). In the current study, we investigated if this murine model could be used to distinguish between the recovery of fresh and aged human RBCs, non-rejuvenated and rejuvenated aged RBCs, gamma-irradiated (25 Gy) fresh RBCs and irradiated fresh RBCs and stored for 28 days. “Fresh” RBCs were processed from whole blood within 24 hrs of collection and the “aged” RBCs were either RBC products stored for 42 or 100 days in an additive solution at 4°C. For in vivo recovery, approximately 1x109 human RBCs were injected into the tail vein of SCID mice (n=5 or 7 per condition) and serial blood samples were collected. Human RBCs were detected in mouse whole blood by flow cytometry using an anti-human glycophorin A mAb (clone CLB-ery-1). Recovery was defined as percent of human RBCs in the mouse circulation at 2 hours post infusion. Rejuvenation of cells was accomplished by incubating RBCs for 1 hour with Rejuvesol solution (Table 1).

2,3-DPG Levels (mM/L) Pre- and Post-Rejuvenation Fresh RBCs Aged for 42 Days Aged for 100 Days Control 3.25±0.40 0.17±0.04 0.38 ±0.06 Rejuvenated 8.58±0.82 4.56±0.17 2.31±0.13

Fresh red cells exhibited recovery of 58.4±4.4 % of total cells injected. Aged RBCs showed a reduced in vivo recovery of 35.7±7.3 % and 5.7±1.6 % of total cells injected for 42 and 100 day old RBC, respectively. Gamma-irradiated fresh RBCs and irradiated fresh RBCs stored for 28 days showed a recovery of 66.7±8.6 % and 55±13.2 % respectively, whereas the recovery of control fresh RBCs and control fresh RBCs stored for 28 days showed a recovery of 58.4±4.4 % and 49.1±7.0 % (p=0.44) respectively (Table 2).

In VivoRecovery Fresh RBCs Stored for 28 days Aged for 42 Days Aged for 100 Days nd - not determined Control 58.4±4.5 49.1±7.0 35.7±7.3 5.17±1.6 Rejuvenated 52.5±11.5 nd 55.4±7.1 21.3±5.0 Irradiated (25Gy) 66.7±8.6 55±13.2 nd nd

Our data indicate that the SCID mouse model can distinguish between fresh and aged red cells and that rejuvenation of the red cells increases intracellular 2,3-DPG levels and in vivo recovery of aged red cells. The SCID mouse model could be used to develop or improve existing methods of red cell storage and processing. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.