Background/Case Studies: Red blood cell (RBC) cryopreservation using the high glycerol method is an accepted approach for long-term storage of red cell concentrates (RCCs). This process is commonly used to maintain frozen RCC units for rare blood programs. However, small-scale (SS) cryopreservation is advantageous for research and development applications where functional and intact RBCs are required, but large-scale processing is not feasible or practical. Cryopreservation protocols for SS RBC samples were previously developed. This work aimed to characterize the inherent variability of these methods.
Study
Design/Methods: Two glycerolization methods were evaluated: (G1) Glycerolyte 57 (Fenwal) was added dropwise to an aliquot of RBCs from an RCC, and (G2) the RCC aliquot was centrifuged (2200 x g, 10 min, RT) for supernatant reduction, glycerolized, then centrifuged for glycerol reduction. For both methods, 1 mL of glycerolized RCCs was aliquoted into vials and frozen at 1°C/min to -80°C. Samples were stored at ≤ -65°C, thawed, and deglycerolized using a 3-step wash method (12% NaCl, 1.6% NaCl, and 0.9% NaCl/0.2% dextrose). RBCs were suspended in AS-3 post-deglycerolization and tested for hemolysis and hematocrit at < 1 h and 24 h post-deglycerolization (PD). Within lot (WL) repeatability was evaluated by glycerolizing an RCC sample and splitting it into multiple vials (n=10/method). Across lot (AL) repeatability was evaluated for both methods by glycerolizing 10 separate RCC samples. Three technicians glycerolized RCCs (n=6) using G1 and G2 methods to determine technician variability.
Results/Findings: WL and AL results (Table 1) demonstrated that the SS cryopreservation process using either glycerolization method is highly repeatable and produces deglycerolized RBCs with < 0.8% hemolysis after 24 h of storage. The largest increase in variability was observed for hemolysis at the 24-h time point. When comparing across technicians, the bias between techs varied from -0.02 to 0.05 and -0.09 to 0.17 for < 1 h PD hemolysis results for G1 and G2, respectively. Conclusions: This work demonstrated that the SS method for cryopreservation of RCCs was highly repeatable even when performed by multiple trained technicians. A source of variability within the method is the rate of addition of deglycerolization solutions, which could be improved with automated addition and mixing systems.
Importance of research: Repeatable and practicable small scale cryopreservation methods for red cell concentrates used for research and development applications requiring intact red blood cells for novel research studies or as control cells.
