Measurements of intracellular potassium and mean corpuscular volume (MCV) were used to evaluate the effects of washing human red cells by either a continuous wash technic with ionic solutions, or a dilution technic with non‐electrolyte solutions and recovery by agglomeration. The red cells were either ACD‐stored, or preserved in glycerol with or without slow freezing. Significant reduction in cellular potassium was observed when agglomeration was used to recover the red cells, whereas no significant change was observed with washing using continuous centrifugation with ionic solutions. No significant change in mean corpuscular volume of red cells was observed with either technic. The effect of hypertonic ionic and non‐ionic washing solutions on intracellular potassium was also evaluated. The data suggest that the removal of glycerol from previously frozen red cells should be accomplished by hypertonic ionic solutions.

Human red blood cells preserved with approximately 45 per cent w/v glycerol, frozen by the slow freeze‐thaw technic, and stored at ‐80 C for up to six years were evaluated by measurements of the posttransfusion survival of autologous 51chromium‐labeled red blood cells, the per cent recovery of the preserved red blood cells, supernatant hemoglobin in the unit, and intracellular potassium levels. The glycerolized red blood cells were washed either with electrolyte solutions using continuous centrifugation, or by dilution with nonelectrolyte solutions and recovery of the red blood cells by agglomeration (Huggins technic). Glycerolized red blood cells stored frozen for more than two years and washed by the Huggins technic had significantly decreased postthaw stability when the de‐glycerolized red blood cells were kept after thawing for longer than four hours at 4 C. Glycerolized red blood cells stored frozen for up to six years and washed by continuous centrifugation with electrolyte solutions showed clinically acceptable post‐transfusion survival after postthaw storage at 4 C for up to 24 hours. The results of washing glycerolized red blood cells by continuous centrifugation showed that prior dilution of the thawed cells with a ten per cent glycerol solution was vitally important. The method used in washing glycerolized red blood cells may significantly limit the length of time that red blood cells may be stored at 4 C after having been frozen.

Cohn‐processed red blood cells that had been stored for as long as seven years at ‐80 C., washed by the ADL procedure and then stored at 4 C for up to 48 hours, showed approximately 90 per cent 24‐hour recovery in vivo by an automated differential agglutination (ADA) technic, recovery in vitro of approximately 90 per cent, and an index of therapeutic effectiveness of approximately 80 per cent. Washing Huggins‐preserved red blood cells with EDTA by the Huggins process produced a significant deterioration (decreased 24‐hour posttransfusion survival and decreased recovery in vitro) following storage at ‐80 C for as long as three years. In two of seven patients studied the Huggins‐processed red blood cells that had been stored at ‐80 C for 1.8 years and longer and washed by the Huggins procedure showed intravascular destruction of the compatible nonviable red blood cells. Huggins‐preserved red blood cells with EDTA that had been stored at ‐80 C up to 1.6 years showed, following washing with an electrolyte solution in the ADL bowl, a somewhat better 24‐hour ADA survival, better recovery of the preserved red blood cells, lower supernatant hemoglobin concentrations, and higher intracellular potassium levels on the day of washing and resuspension. These findings suggest that Hugginspreserved red blood cells following storage at ‐80 C for one and one half years or more should not be washed by the Huggins dilution/agglomeration procedure.

Washing of either nonfrozen or previously frozen red blood cells with nonelectrolyte solutions caused a significant reduction in intracellular potassium concentration. Storage of the nonfrozen or previously frozen washed red blood cells at 4 C for up to 48 hours produced further reductions in intracellular potassium levels. The loss of cellular potassium after the washing was much greater in previously frozen red blood cells than in the non‐frozen cells. Washing with 4.5 per cent glucose and 4.5 per cent fructose, instead of 8 per cent glucose and 1 per cent fructose, decreased MCV and osmotic fragility, and increased MCHC and cellular density, which suggests that blood so treated was not acceptable for clinical use. Thus, the volume and composition of the electrolyte solutions used to disaggregate red blood cells determined the magnitude of the changes in the washed red blood cells during postthaw storage. Significant correlations relating changes in red blood cell indices, osmotic fragility, intracellular electrolyte levels (K+, Na+, Cl‐), and the density distribution of the red blood cells provided useful guidelines for determining the environmental conditions for the washing of both nonfrozen and previously frozen red blood cells with nonelectrolyte solutions under which the restored red blood cells will have acceptable physical and structural characteristics.

Freeze‐preservation of human red blood cells with a low concentration of glycerol (approximately 20 per cent w/v), liquid‐nitrogen refrigeration, and a stainless steel container was evaluated by measurements of the posttransfusion survival of 10‐ml samples of 51chromium‐labeled autologous red blood cells, together with several measurements in vitro. Removal of glycerol prior to transfusion (the major technologic problem) was carried out both by serial centrifugation (batch washing) and by continuous centrifugation, using either predilution or sequence wash cycles. A biologic product of glycerolized red blood cells was prepared to the following specifications: the recovery in vitro of at least 90 per cent of the red blood cells after thawing and washing was achieved, together with 24‐hour posttransfusion survival in vivo of approximately 85 per cent, a total amount of supernatant hemoglobin in the unit on the day of washing of approximately 450 mg, a preparation time of approximately 30 minutes, and a total volume of wash solution of less than 3.0 liters. The present urgent need in frozen blood preservation is for systems which use disposable software in a completely automated wash cycle.