Platelet preservation

Abstract

This thesis deals with the cryopreservation of human platelets. Chapter 1 briefly summarizes the properties and clinical applications of platelets. The importance of well working preservation methods is pointed out. The purpose and the design of the investigations are explained. Chapter 2 reviews the literature on the preparation and preservation of platelet suspensions, and on in vitro quality criteria for platelets. Viability and function, both of essential importance for the success of platelet transfusion, can be differently influenced by preservation. Criteria based on the uptake of serotonin and on the response to a hypotonic stress, appear to be valuable assays to estimate the viability of platelets. In chapter 3 a comparative investigation is presented between electronic and light microscopic counting of platelets. No significant differences are observed for samples of fresh blood from healthy individuals. In chapter 4 it is shown that cryopreservation causes a decrease in light absorbance of platelet suspensions when the conditions are not optimal. This phenomenon is used as a screening method for the rapid evaluation of a number of cryoprotectants and cooling conditions. Best results are obtained for dimethylsulfoxide (DMSO) and N,N dimethylacetamide (DMAC). Based on other more detailed investigations the use of DMSO appears to be more favourable. Electron microscopic observations show after cryopreservation with 5% (v/v) DMSO intact cells and cells which have lost their normal ultrastructure. Therefore it is concluded that cryopreservation under these conditions is an 'all or none' process: there are cells which survive the procedure and there are cells which do not. In chapter 5 the response to hypotonic stress is investigated. A parameter is found which is independent of platelet concentration and, within a certain range, decrease in osmolarity. Cryoprotectants disturb the assay. In chapter 6 the use of DMSO is investigated more closely. Serotonin uptake velocity and, in some cases, response to hypotonic stress are used as criteria. Cryopreservation in the presence of 5% (v/v) DMSO causes a considerable decrease in activity. Best results are obtained for cooling rates from 2 to 10°C/min. In chapter 7 sedimentation behaviour of cells in whole blood is studied in the IBM 2991 Blood Cell Processor, a centrifuge originally developed for the deglycerolization of erythrocytes. Using this centrifuge, platelet concentrates can be prepared in one centrifugation step in a relatively short period of time. In chapter 8 two methods are compared for the preparation of platelet concentrates: a method in one centrifugation step as described in chapter 7, and the conventional 'two step' method. Based on in vitro criteria and on the manageability of both methods, it is concluded that the 'two step' method is preferable. In chapter 9 the recovery of the cryopreservation procedure is determined with in vivo survival experiments. Furthermore, it is investigated whether the data obtained from the in vivo experiments correlate with the results of the serotonin uptake velocity and the response to hypotonic stress. It is concluded that both assays are useful for the estimation of changes in viability of platelets, caused by preservation, but that they are only of limited value for the determination of the quality of single platelet suspensions. In chapter 10 it is shown that the volume of platelets and their degree of labeling with 51Cr or 14C serotonin is dependent on their density. Density centrifugation is not suitable for separating the morphologically intact and morphologically damaged platelets which are present after cryopreservation. Although this is not proved, it is likely that the morphologically damaged platelets do not take up serotonin anymore, but that they can be labeled with 51Cr.