Red Blood Cells Stored in Hypoxic State Show Lower Adhesion Levels Vs Normoxia

Erdem Kucukal, PhD

Red blood cells (RBCs) processed and stored in a hypoxic state maintain their integrity, which may reduce the risk of developing vaso-occlusion (VOC) after RBC transfusion.

Data released at the 2021 ASH Annual Meeting showed that storing RBCs with the Hemanext RBC Processing and Storage System resulted in amelioration of storage-mediated RBC adhesion to heme-activated human umbilical vein endothelial cells (HUVECs).¹

RBCs stored in a hypoxic environment are exposed to much lower oxidative stress during storage. “This study showed a decrease in the adhesion of hypoxic RBCs to heme-activated HUVECs when compared to conventionally stored RBCs for transfusion,” said lead investigator Erdem Kucukal, PhD, of BioChip Labs, Cleveland, in a poster presentation of the results. “Based on our findings, we postulate that hypoxic RBCs may reduce the risk of developing vaso-occlusive crises after transfusion in patients such as sickle cell disease.”

Current regulations require RBCs to be stored at 4° C not more than 42 days before a transfusion. “RBCs undergo extensive rheological changes during this time and may contribute to complications associated with transfusion,” he said. “Hemanext has recently introduced an innovative hypoxic system to ameliorate such storage lesions.”

His group studied the changes in adhesion properties of stored RBCs to human endothelial cells following a 42-day storage in a normoxic condition vs hypoxic, using a standardized endothelialized microfluidic platform (Endothelium-on-a Chip).

HUVECs were first cultured within microfluidic channels under flow for at least 48 hours, and then treated with 40 µM heme for 4 hours at 37° C. RBCs were centrifuged to remove the storage buffer and resuspended in basal cell culture medium A 15 µl RBC solution, and was then injected over heme-activated HUVECs. Control experiments were conducted with nonactivated HUVECs.

RBCs that were stored under conventional normoxic condition displayed higher adhesion to heme-activated HUVECs than hypoxic condition. At baseline (week 1), adhesion levels to nonactivated HUVECs (control) were negligible (normoxia: 13 ± 6; hypoxia, 18 ± 10, P > .05) while both cell lines had higher adhesion levels to nonactivated HUVECs at week 6 (normoxia: 132 ± 64; hypoxia, 292 ± 31). “While the P values were greater than 0.05 when the week 1 and week 6 results were compared for both normal and hypoxic storage conditions, we expect to see a statistically significant difference as the sample size increases,” the researchers wrote in their poster.

Following 6-week storage, adhesion of erythrocytes stored in normoxia to heme-activated HUVECs was higher compared with those stored in hypoxic conditions (1333 ± 407 vs 544 ± 149; P = .09). Overall, erythrocyte adhesion to heme-activated HUVECs was significantly greater compared with nonactivated HUVECs when combining data at weeks 1 and 6 (heme: 681 ± 150; nonactivated: 81 ± 33).

At week 6, stored RBCs adhesion to heme-activated HUVECs vary from sample to sample: 4 of 5 samples tested displayed lower adhesion when stored in hypoxia.

“These results suggest that storage-mediated RBC adhesion to heme-activated HUVECs may be ameliorated by the novel hypoxic-storage condition,” the researchers concluded. “This study showed a decrease in the adhesion of hypoxic RBCs to heme-activated HUVECs when compared to conventionally stored RBCs for transfusion. This result suggests that hypoxic RBCs may reduce the risk of developing VOC after RBC transfusion in patients, such as in sickle cell disease, where adhesion to heme-activated HUVECs has been implicated in the pathogenesis of VOC.”

Reference

1. Kucukal E, Wolfe A, Kocevar R, et al. Hypoxic storage of red blood cells: assessment of adhesion properties using a standardized endothelium-on-a-chip microfluidic platform. Presented at: 2021 ASH Annual Meeting; December 11-14, 2021; Atlanta GA. Abstract 1072.