Red blood cell stabilization reduces the effect of cell density on recovery following cryopreservation

The relationship between red blood cell hematocrit and hemolysis during cry opreservation has been examined. Cells were frozen with glycerol, thawed, a nd deglycerolized in a model system based on the protocols used in transfus ion medicine. Analysis included determination of hemolysis following thaw ( Thaw) and deglycerolization (Overall) and osmotic fragility of the final ce ll suspensions. Results demonstrate that thaw hemolysis decreased with incr easing hematocrit at all glycerol levels tested. Overall hemolysis increase d with increasing hematocrit at low (15% w/v) glycerol and decreased with i ncreasing hematocrit at high (40% w/v) glycerol levels. These results were paralleled by changes in the fragility index. Furthermore, these results in dicate a distinction between freeze/thaw lysis and damage which leads to ly sis during postthaw processing. To examine this further, a biochemical stab ilizing solution, having no cryoprotective effects itself, was added to sub optimal glycerol concentrations. This addition resulted in hemolysis levels and fragility indices comparable to those using high (40% w/v) glycerol le vels. Thus, the damage observed with increasing hematocrit is not necessari ly 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, bioche mical in nature and may be protected against through specific cellular stab ilization prior to cryopreservation. (C) 2000 Academic Press.