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The relationship between red blood cell hematocrit and hemolysis during cryopreservation has been examined. Cells were frozen with glycerol, thawed, and deglycerolized in a model system based on the protocols used in transfusion medicine. Analysis included determination of hemolysis following thaw (Thaw) and deglycerolization (Overall) and osmotic fragility of the final cell suspensions. Results demonstrate that thaw hemolysis decreased with increasing hematocrit at all glycerol levels tested. Overall hemolysis increased with increasing hematocrit at low (15% w/v) glycerol and decreased with increasing hematocrit at high (40% w/v) glycerol levels. These results were paralleled by changes in the fragility index. Furthermore, these results indicate a distinction between freeze/thaw lysis and damage which leads to lysis during postthaw processing. To examine this further, a biochemical stabilizing solution, having no cryoprotective effects itself, was added to suboptimal glycerol concentrations. This addition resulted in hemolysis levels and fragility indices comparable to those using high (40% w/v) glycerol levels. Thus, the damage observed with increasing hematocrit is not necessarily a function of the packing on the volume of the ice-free zone, but rather an expression of cell damage. Furthermore, this damage is, in part, biochemical in nature and may be protected against through specific cellular stabilization prior to cryopreservation.