Cryopreserving Small Volumes of Red Blood Cells Using the High Glycerol Method

Cryopreservation of red blood cells (RBCs) plays a critical role in blood inventory management by enabling long-term frozen storage of rare blood types. This process requires the addition of glycerol, a cryoprotectant that mitigates cellular damage caused by ice crystal formation during freezing and thawing. A high glycerol method (HGM) is commonly used, which requires an approximate concentration of 40% glycerol and enables slow freezing rates (1-3°C/min) and extended storage in low-temperature freezers (≤-65°C). While automated cell processing systems have been developed for the glycerolization and deglycerolization of full-size RBC units for transfusion, there is no such technology for the small-scale cryopreservation of RBC aliquots. This chapter presents a high-recovery, small-scale model that mirrors current automated cell processing systems that employ the HGM for cryopreservation of RBCs. This technique is valuable for (1) research studies, particularly for investigations on cryopreservation of rare donor phenotypes or investigating novel cryoprotectants, (2) to provide control or reagent cells for antibody identification or compatibility testing, or (3) any other applications requiring small volumes of functional and intact RBCs.