The effects of freeze‐preservation on the metabolic integrity of human erythrocytes are shown to be largely dependent on the length of time they are stored in ACD at 4 C prior to being frozen. In vitro evaluation involving incubation for 12 hours at 37 C with adenine, inosine and buffering to pH 7.3 showed that when stored for ten days, red blood cells could be preserved by the low‐glycerol‐rapid‐freeze process with no deleterious effects on their capability to produce ATP and 2,3 DPG and to restore the oxygen dissociation curve and sodium ‐potassium gradient to normal. Storage for 20 days, however, resulted in changes in the metabolic response of red blood cells to the incubation so that ATP production was increased while that of 2,3 DPG was decreased and freeze‐preservation lowered production of both compounds. The findings indicate that red blood cells can be stored in ACD at 4 C for up to ten days prior to being freeze‐preserved and still be capable of resuming normal metabolic function. This, in view of previous evidence showing that the oxygen affinity and metabolic status of red blood cells remain unaffected by the low‐glycerol‐rapid‐freeze process, indicates that if the cells are frozen immediately after being drawn, preservation of their respiratory function will be optimal.

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