Cell Biology, Immunology and Biochemistry (basic and preclinical research)
Tatsuro Yoshida
Hemanext
Lexington, Massachusetts, United States
Processing and storing red blood cells (RBCs) under O2/CO2-reduced conditions using HemanextONE protects RBCs from oxidative stress, enhances metabolic status and reduces storage lesion markers, as demonstrated in extensive in vitrostudies and enhanced 24-hr post-transfusion recovery in vivo.
We developed an experimental miniaturized O2/CO2-reduction blood storage platform capable of processing and storing small blood volumes (1-50mL) to enable blood cell storage under O2/CO2-controlled conditions for studies using umbilical cord blood and small animal models.
The basic construct of the HemanextOne (Hemanext Inc.) was scaled down and optimized to efficiently process the necessary small volumes. For the O2 reduction bag (ORB) used for pre-storage processing, a mini-ORB (mORB) was fabricated using a reduced amount of the same iron-based O2/CO2 sorbent and a heat-sealable microporous gas-permeable membrane (53cm2 UPE-0.04M, Cobetter Industries). For the hypoxic storage of processed blood in ambient air, a mini-hypoxic storagebag (15mL) was fabricated with transparent gas barrier film (polyethylene contact layer, Clearfoil Z, PAXXUS) (Figure A).
Fresh CPDA1 whole blood (WB) was procured and 8.5mL were transferred into mORBs and then continuously mixed by a rotating agitator (Mini tube rotator, Fischer Scientific) at 10 - 40 RPM settings for 2 hours. SO2, pH, pO2 and pCO2were measured every 30 min with ABL90 (Radiometer). Percent hemolysis was determined at the end of the mORB processing.
Longer process times and faster rotation settings yielded higher SO2 reduction rates but resulted in increased hemolysis. SO2 reduction kinetics followed a simple first order process: SO2 (t) = SO2 (t=0)* e-k*t, where k is rate constant (min-1) and t is time (min). Rotation over 20 RPM resulted in excessive hemolysis, and thus only 10 and 20 RPM were examined further. Additional mixing (drawing and expressing half of the volume) using a syringe every 30 minutes during rotation significantly optimized the rate of O2 reduction while minimizing hemolysis. There were no significant differences between 10 and 20 RPM results. A finalized design targeted for a 10mL processing volume, rotated on its short axis at 10-20 RPM, yielded optimal performance with an hourly SO2 reduction rate of 24% (95% CI:23-25, n=8). Starting with fully oxygenated WB (10g/dL tHb), SO2 was reduced to 6% with hemolysis of 1.2% (95% CI:1.0,1.2) during a 2-hour process.
Small volumes of blood, such as those used for studies with umbilical cord blood and small animal models, can be successfully processed for O2/CO2-reduced storage, yielding blood with similar properties to the full-scale HemanextONE RBCs.