A modification of previously reported methods for freezing red blood cells with a high concentration of glycerol is described. The limits of osmotic tolerance of human red blood cells range from one‐half to four times isotonic. The introduction and removal of glycerol creates transient osmotic gradients that can exceed these limits. It is shown that much of the cell lysis following thawing and during washing can be related to hypertonic injury incurred during the glycerolizing procedure. Refinements of this portion of the process produce significant gains in recovery. Problems associated with washing out a high concentration of glycerol by centrifugal means can be alleviated by a preliminary dilution with hypertonic sodium chloride which serves also to reduce cell volume and increase cell specific gravity improving sedimentation characteristics and affording more latitude for the hypotonic stresses associated with glycerol removal. Careful attenion to the osmotic limits during both glycerolizing and washing results in a process in which hemolysis from freezing, thawing and washing can be reduced to less than three per cent.

One of the events associated with red cell storage at 4 degrees C is the development of an increasing proportion of echinocytes. Vesicles also may bud off the spicules, presumably leading to a decreased surface-to-volume ratio and decreased deformability. Pursuing the hypothesis that increasing the surface tension of the cells by increasing their volume might reduce the tendency toward echinocytosis and extend refrigerated storage time, packed red cells were resuspended in a solution hypotonic (210 mOsm) with respect to solutes that do not penetrate the cell. Since a reduced ionic concentration results in increased membrane permeability for cations, normal ionic concentration was maintained by the addition of NH4C1, which readily penetrates red cells and therefore contributes no osmotic support. Adenine, glucose, mannitol, citrate, and phosphate also were included. Unexpectedly, the predominant effect of red cell storage in this solution was a remarkable elevation of adenosine triphosphate (ATP). At 4, 8, and 10 weeks, (ATP) levels averaged 165, 135, and 110 percent of initial values, respectively. At 16 weeks, ATP still averaged 50 percent of initial values. Twenty-four-hour in vivo survival of red cells measured at 12 to 18 weeks ranged between 70 and 80 percent, and hemolysis ranged from 0.3 to 7.1 percent. Both the hypotonicity and the ammonium salt appear to be necessary for the high ATP.

Red cells washed and stored in a citrate-phosphate-glucose-adenine solution at pH 7.4-7.6 demonstrate excellent maintenance of adenosine triphosphate, elevation of 2,3-diphosphoglycerate well above normal levels for more than 6 weeks, reduced hemolysis and 24-hour in vivo survival comparable to that of cells stored in ADSOL. These results can be attributed in part to a chloride shift in which the washout of intracellular chloride is associated with an influx of OH-, which increases intracellular pH and thereby increases the rate of glycolysis. The phosphate functions primarily as a buffer to maintain both extra- and intracellular pH. Reducing the effective osmolality of the storage solution reduces hemolysis and improves cell morphology.

Although the high (40 to 50 per cent) glycerol method of freezing red blood cells has many advantages, no feasible procedure for deglycerolizing with only a clinical centrifuge has been available. Thus this method has been restricted to blood centers that distribute a sufficient number of frozen cells to warrant the installation of automated cell washing equipment. However, by sedimenting the glycerolized cells prior to freezing and discarding the excess glycerol solution, postthaw deglycerolizing can be simplified to consist of an initial double dilution with hypertonic NaCl and isotonic saline-glucose followed by two cycles of sedimentation and resuspension in isotonic saline-glucose. The method is applicable to units frozen either at -80 C or in liquid nitrogen vapor. Units frozen in this way can also be deglycerolized in commercial automated cell washers using suitably modified protocols.

The most commonly used methods for depleting red cells of leukocytes are the inverted spin procedure, which results in excessive red cell loss and poor leukocyte depletion, and washing, which is expensive and yields a product with a 24-hour shelf life. Clinical data now available suggest that nonhemolytic febrile transfusion reactions can be prevented in most clinical situations by microaggregate filtration of red cells during transfusion using the spin-cool-filter procedure with a screen filter. This procedure holds advantages for leukocyte removal, red cell recovery, cost, normal shelf-life, and simplicity over other washing and buffy coat removal procedures currently used. For highly alloimmunized patients requiring virtually total leukocyte removal and where alloimmunization is to be avoided at any cost, adhesion filtration or freezing and deglycerolization should be used.

Sickle-trait red blood cells respond to freezing and thawing like normal cells, but during deglycerolization by centrifugal methods, they pack and hemolyze. This appears to be the result of changes in surface characteristics with suspension in hyperosmotic saline and even modest increases in osmolality result in packing with centrifugation. Modifications to permit deglycerolization of sickle-trait cells have been designed for use with both the Haemonetics and the IBM cell washing systems. Sickle-trait cells do not agglomerate satisfactorily but we were unable to devise a modification of this deglycerolizing system that would accommodate sickle-trait cells.

The effect of short-term cryopreservation on metabolic, functional, and survival characteristics of erythrocytes from patients with sickle cell disease was examined. Post-thaw hemolysis of glycerolized sickle (SS) erythrocytes was greater (mean 12.9%) than in hemoglobin-AA cells (mean 4.7%). Freeze preservation had no apparent effect on red cell morphology, percent irreversibly sickled cells, and fetal hemoglobin content. There were modest reductions in ATP and 2,3-diphosphoglycerate in thawed, washed sickle erythrocytes (12.7% and 29.7%, respectively). However, the autologous survival of 51Cr-labelled SS red cells was not shortened by cryopreservation. The safety and efficacy of autotransfusion of cryopreserved red cells in alloimmunized sickle cell disease patients with anemic episodes unrelated to sickling need to be determined.

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