Cryopreservation of Red Cells by Freezing and Vitrification – Some Recollections and Predictions

Abstract

A science that originated over 50 years ago, cryobiology, has matured to become an indispensable adjunct to blood transfusion medicine. Before we can look to the future of cryobiology and blood preservation, it is essential to understand what has gone before, what is the current state of the art, and finally where do we go from here. This paper attempts to meet this challenge. Red cells can be cryopreserved successfully using one of two approaches: colligative or kinetic. The colligative approach utilizes glycerol in high concentration and slow cooling at uncontrolled rates. Kinetic approaches utilize either glycerol at low concentration and rapid controlled cooling rates, or extracellular additives such as polyvinylpyrrolidone (PVP), hydroxyethyl starch (HES), polyethylene oxide (PEO), etc. and high rates of cooling usually in liquid nitrogen at –196 °C. Current data with all methods of red cell cryopreservation indicate excellent red cell recovery (>90%) and clinically acceptable in vivo survival times. Of particular interest is the innovative HES cryopreservation technique and results whereby the additive HES does not have to be removed prior to transfusion of the thawed red cells. Clinical results are most promising and more clinical trials are warranted. The future for cryopreservation of red cells in glycerol as well as in HES looks bright based on extensive clinical data. New developments in automation and sterile closed systems can now extend postthaw storage. Recent data show that thawed deglycerolized blood, processed in an apparatus with sterile connectors, can be stored safely at 4 °C for 15 days or more with acceptable 24- hour survival (⁵¹Cr) values. Current regulations need to be reviewed and revised to remove the 24-hour outdating restriction on thawed blood to allow it to reach its full potential. Experimental data also support extending the storage life of blood in the frozen state from its current limit of 10 years to at least 25 years or longer.

Utilization and quality of cryopreserved red blood cells in transfusion medicine

“…The permeating additive glycerol has been used to freeze RBCs since the early fifties. The concentration of glycerol that is necessary to protect the RBCs is dependent on the cooling rate and the storage temperature . At slow cooling rates, ice crystals will form extracellular.…”

Cryopreservation of Erythrocytes, Thrombocytes, and Lymphocytes

“…In principle, three different methods have been established for routine clinical use of frozen red cells (RBC) [4]; these are: i) The Huggins [5] technique utilizes ~40% [wt/v] glycerol as the cryoprotective additive. Prior to transfusion the glycerol is removed by reversible agglomeration of the RBC using nonionic sugar solutions.…”

Frozen Red Blood Cells in Transfusion

“…Infusion of incompletely deglycerolized RBC has negligible effect except for shift in intracellular fluid volume [4]. Polyvinylpyrrolidone, hydroxyethyl starch (HES), polyethylene oxide are non penetrating cryoprotecting agents as they require high rates of cooling in liquid nitrogen at -196°C [6]. Non penetrating cryoprotecting agents protect cells by a process called 'vitrification', where they form glassy shell around the cell.…”

“…Non penetrating cryoprotecting agents protect cells by a process called 'vitrification', where they form glassy shell around the cell. HES is one of the promising cryoprotecting agent for cryopreservation of RBC, since its removal from thawed RBC prior to transfusion is not required [4][5][6].…”