Cryobiology of platelets: II. Effects of freezing and storage at low temperatures on the survival of isolated human blood platelets as measured by assays for aminopeptidases

Abstract

The effects of freezing and storage at low temperatures on isolated platelets suspended in potassium phosphate buffer only, phosphate buffer plus dimethyl sulfoxide, phosphate buffer plus sodium glycerophosphate, or combinations of the foregoing were studied. The effects of freezing on platelets suspended in 0.25 m phosphate buffer at different hydrogen ion concentrations were investigated also. The least disruption or destruction of platelets after freezing and thawing occurred at pH 7.4, a loss of 29%. No losses in the activities of aminopeptidases, as a measure of platelet intactness, were observed in preparations of platelets suspended in potassium phosphate buffer plus 0.50 to 1.00 m dimethyl sulfoxide and cooled at 1 °C per min to a terminal temperature of −70 °C; preparations of platelets suspended in 0.25 to 1.00 m sodium β-glycerophosphate and treated in a similar manner lost 32% of their numbers. A synergistic effect was observed when platelets were suspended in a combination of 0.05 m dimethyl sulfoxide and 0.25 m sodium β-glycerophosphate. Platelet numbers in preparations containing 1 m dimethyl sulfoxide and stored at −70 °C for 8 days were not significantly different from those of the control suspensions; a 6% decrease in the numbers of platelets was found after 16 days of storage. Platelets suspended in 0.05 m dimethyl sulfoxide plus 0.25 m sodium β-glycerophosphate in 0.25 m potassium phosphate buffer, pH 7.4, did not decline significantly in numbers after 4 days of storage at −70 °C. A decline in the number of platelets occurred after the 4th day of storage at −70 °C, and by the 16th day of storage, these preparations had lost 19% of the number present before freezing and storing. A mixture of extremely low concentrations of dimethyl sulfoxide plus sodium β-glycerophosphate in potassium phosphate buffer appears, therefore, to be suitable menstruum for the freezing and storage at low temperatures of platelets, with minimal losses of activities as measured by the levels of activities of aminopeptidases.