Cryopreservation of human platelets with 1.4m glycerol at −196°C

Abstract

The aim of this study was to develop a method for the cryopreservation of human platelets that would be more effective than previous methods using glycerol. The problem of defining the concentration of glycerol that would be required and of introducing and removing that concentration without serious osmotic disturbance was approached fundamentally by proposing, on the basis of previously published evidence, the limiting salt concentration that should not be exceeded during the freezing process, and the limits of platelet volume that should not be transgressed during addition and removal of the cryoprotectant. A method was developed to satisfy these requirements and its effectiveness was assessed by measuring the numerical recovery of cryopreserved platelets, their ability to osmoregulate in the hypotonic stress test and their aggregation response to ADP. The physical manipulations of dilution, centrifugation and resuspension etc, and the addition and removal of 1.4 molar glycerol without freezing had, respectively, a moderate effect on the aggregation response and a lesser effect on the hypotonic stress response. Although freezing and thawing produced additional decrements in all the assays, the hypotonic stress response was better by a factor of 3.5 than that previously obtained in a cryopreservation method using 0.5 molar glycerol. The method is technically straightforward and should be adaptable to the requirements of blood banks.