Prolonged post-thaw shelf life of red cells frozen without prefreeze removal of excess glycerol

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Background and Objectives

The use of a functionally closed system (ACP215, Haemonetics) for (de)glycerolization of RBCs allows for prolonged post-thaw storage. Currently, glycerolization is followed by supernatant glycerol reduction before freezing. The aim of this study was to investigate the influence of supernatant glycerol reduction before freezing on the stability of thawed, deglycerolized RBCs during subsequent cold storage.

Materials and Methods

Leucoreduced RBCs were stored for 6 days at 2–6°C before glycerolization. The RBCs were pooled and split, and glycerol was added using the ACP215 to a final concentration of 40%. Units were either frozen as such (n = 4) or supernatant reduced before freezing (n = 4). After storage at −80°C, the units were thawed, deglycerolized and resuspended in SAGM. An additional sixteen units, frozen without supernatant reduction, were resuspended in either AS-3 (n = 8) or SAGM (n = 8) after deglycerolization. During cold storage (2–6°C), the red cells were analysed for their stability and in vitro quality.


The freeze-thaw-wash recovery was comparable for both volume reduced and non-reduced units. During post-thaw storage in SAGM, non-glycerol reduced units showed significantly less potassium leakage and haemolysis and higher ATP levels. AS-3 strongly reduced haemolysis during post-thaw storage of non-glycerol reduced units: haemolysis remained below 0·8% for up to 28 days of storage.


Omitting glycerol supernatant reduction before freezing simplifies the cryopreservation procedure and increases the stability and therefore the outdating period of thawed RBCs. This increases the practical applicability of cryopreserved RBCs in both civil (rare blood) and military blood transfusion practice.

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