PB1395 - Synergistic combination of polyampholyte with DMSO/trehalose improves cryopreservation of red blood cells and reduces preparation time

Background: Storing red blood cells (RBCs) long-term is crucial in modern medicine, for the storage of rare blood types and for treating patients for blood loss due to surgery and injury. Traditional glycerol-based methods are effective but involve time-consuming deglycerolization, leading to RBC loss. This study investigates PaDT, a mixed cryoprotectant system with polyampholyte, trehalose, and DMSO, as a faster and more efficient alternative for RBC cryopreservation.

Aims: Compare the effectiveness of PaDT and glycerol methods in RBC cryopreservation

Methods: Isolated human RBCs (n=4) were suspended in glycerol and PaDT solutions at 30% haematocrit and frozen at -80°C or in liquid nitrogen. Samples were thawed at 37oC and washed with recovered cells assessed post-thaw and post washout. Viability was assessed by measuring RBC lysis using spectrophotometry and microscopic imaging. Effects of freezing method, haematocrit and washout were explored on RBC recovery. Morphological evaluations were conducted post-thaw and post-washout.

Results: Freezing RBCs in glycerol at -80°C or liquid nitrogen resulted in 98±0.5% recovery post-thaw, while PaDT only demonstrated efficacy with liquid nitrogen, retaining 97±1% of RBCs post-thaw. Glycerol maintained consistent recovery at haematocrit levels up to 50%, whereas PaDT exhibited a 4% and 10% decrease in recovery at 40% and 50% haematocrit, respectively (p=0.0006 and p< 0.0001). Incubation with PaDT resulted in lower pre-freeze RBC lysis (0.4±0.6%) compared to glycerol (0.7±0.2%, p< 0.0001). Post-thaw and washout recovery rates were higher with PaDT (97±2% and 82±4%) compared to glycerol (95±4% and 76±3%, p=0.03). PaDT washout process was more efficient (45 mins) than glycerol (more than 70 mins), and morphologically, RBCs appeared healthier with PaDT post-thaw and post-washout compared to glycerol.

Conclusion(s): The PaDT cryopreservation method demonstrates superior RBC retention, shorter washout times, and improved morphological characteristics compared to the conventional glycerol method. These findings highlight the potential of PaDT as a more efficient RBC cryopreservation method.