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An ektacytometer was used to measure red blood cell deformability during blood storage for 42 days at different degrees of cell packing in citrate-phosphate-dextrose preservative. Observed changes in deformability were studied in relationship to red blood cell adenosine triphosphate (ATP) levels, lipid content, osmotic fragility, and hematocrit during storage. Decrease in whole cell deformability as measured in isotonic medium did not occur until cellular ATP levels were reduced to less than 30 percent of initial values. The rate of deformability loss during storage increased with increased degrees of cell packing because of more rapid substrate depletion. The loss in isotonic deformability was further magnified when deformability measurements were carried out in hypotonic media, suggesting that a reduction in surface area-to-volume ratio was the dominant cause of the reduced deformability of stored red blood cells. Marked spherocytosis, decreased membrane lipid content and increased osmotic fragility confirmed loss of membrane surface area during storage. The significantly higher rate of deformability loss observed in packed red blood cells suggests that careful control of storage hematocrit may be necessary to avoid loss of cellular deformability and possibly posttransfusion viability.