Multicomponent Cryopreservation Agent Could Speed RBC Availability

Researchers at the University of Leeds have developed a multi-component cryopreservation agent that appears to improve the speed of freezing, thawing and washing red blood cells (RBCs) compared with glycerol, the currently used agent. The findings were published this month in Cryobiology.

The availability of RBCs has long been affected by their short shelf-life. While cryopreservation can extend that availability, the process of cryopreserving, freezing, thawing and washing is time-consuming.

“Our goal was to create a system that allows blood to be frozen and then used almost on demand, with PaDT, we’ve achieved that. It’s faster, simpler and results in better recovery of healthy, functional red blood cells,” primary investigator Fraser Macrae, PhD, of the University of Leeds in the United Kingdom, said in a press statement.

According to the authors, the breakthrough has the potential to revolutionize RBC availability. “The multicomponent cryoprotectant can be used to freeze large volumes of RBCs and facilitate faster cryopreserved RBC preparation. This study emphasizes that with faster preparation times, the routine use of cryopreserved red blood cells in clinical settings could become a game-changing solution to address red blood cell shortages in emergency situations and reduce the need for walking donors, and associated challenges of blood group matching,” the researchers wrote.

The new cryopreservation mixture, called PaDT, includes polyampholyte, dimethyl sulfoxide (DMSO) and trehalose. Polyampholytes, a class of polymers, are potent cryopreservation enhancers, thought to work by stabilizing the cell membrane and improving the vitrification process during cryopreservation. DMSO is the most widely used cryprotectant and is highly effective. It works by quickly permeating cells and reducing the likelihood of ice crystal formation. Trehalose is a natural sugar produced by “extremophile” species, such as tardigrades (water bears), which are able to survive a range of extreme conditions, including extreme temperatures, pressures, radiation, dehydration and starvation. They’ve even survived in outer space. Trehalose is a cryoprotectant that works primarily outside of cells.

The researchers compared the quantity, quality, and functionality of RBCs cryopreserved using   PaDT to those preserved with glycerol. They started by optimizing the factors involved in RBC yield and function for each technique.

They then compared washout time, RBC quantity, viability, morphological integrity and function with 40% glycerol. PaDT washout could be accomplished in 30 minutes, roughly three times faster than the 40% glycerol washout process, which takes 30–60 minutes per unit using an automated system.

The researchers used several techniques to assess cell integrity, including flow cytometry, the cell counter, adenosine triphosphate assays and microscopy. These revealed that RBCs were “intact with normal metabolic health and no differences in cell morphology between PaDT and glycerol preserved cells, demonstrating that with both methods revived cells were structurally normal.”

They also noted that differences in osmotic fragility with cells preserved in PaDT demonstrated a small reduction in propensity to rupture compared to fresh and glycerol preserved RBCs. It’s unclear what the mechanism is. However, RBCs with reduced fragility could result in more functional RBCs after transfusion, while reducing complications from hemolysis — essentially making transfusions more effective against anemia or other blood disorders.

The researchers also looked preliminarily at optimizing PaDT for use with larger volumes of RBCs. They demonstrated that “our mixed cryoprotectant can be used to successfully cryopreserve RBCs in large volumes, using slower freezing rates, with a more rapid washout while recovering a comparable number of RBCs to a standard glycerol protocol.”