Evaluation of Four Different Methods for Platelet Freezing: In vitro and in vivo Studies

Abstract

This report describes our experience with various techniques for the freezing of platelet-rich plasma, removed from the final product after leukapheresis procedures performed on 14 hematological patients. A total of 194 platelet units were frozen for subsequent autologous transfusion, by the following four methods: (1) 6% dimethyl sulfoxide (DMSO); (2) a combination of 5% DMSO/6% hydroxyethyl starch; (3) 3% glycerol; (4) 5% glycerol/4% glucose. Each technique was evaluated by measuring the percentage of platelet recovery, malondialdehyde (MDA) production, and lactate dehydrogenase release. To investigate the safety and therapeutic effectiveness of the previously frozen platelets, in vivo comparison of four platelet freezing methods was made in 8 thrombocytopenic patients, using corrected platelet increment (CCI), determined at 24 h. Our in vitro results indicate that the cryopreservation with 6% DMSO, without controlled cooling rate, provides significantly (p less than 0.05) greater platelet recovery (75%) as compared to other systems. The decrease of MDA production and the increase in plasma lactate measured after the thawing process was less in the DMSO-frozen units than in the other platelet units. When platelets, cryopreserved by this method, were subsequently transfused into patients, a significantly better CCI (greater than 5,000/microliters) was obtained. In our series, 6 patients were entirely supported with frozen autologous platelets. It appears from this study that a better understanding of the physical and biochemical events occurring during the freezing process will improve platelet cryopreservation, allowing a more systematic use of frozen platelets in the support of thrombocytopenic patients.

Effects of the addition of second‐messenger effectors to platelet concentrates separated from whole‐blood donations and stored at 4°C or –80°C

“…In the second set of experiments, PCs separated from whole‐blood donations by the buffy coat method were cryo‐preserved with 6‐percent DMSO or TC. Platelet recovery was similar with both methods (>70 %), which is in agreement with previous reports in PCs separated from whole‐blood donations by the platelet‐rich plasma method 23 …”

PAS‐G supports platelet reconstitution after cryopreservation in the absence of plasma

“…However, dimethyl sulfoxide (DMSO) at a concentration of 5% to 6% is currently considered the best cryoprotectant for PLTs. 5,6 The most commonly used method for PLT cryopreservation involves the addition of prediluted DMSO to a PLT unit, followed by centrifugation to concentrate the PLTs and removal of the majority of the supernatant, to minimize residual DMSO. As such, upon thawing, PLTs need to be resuspended in solution before transfusion.…”

Platelet cryopreservation using a trehalose and phosphate formulation

“…Me 2 SO 5%-6% has been reported to be the most effective cryoprotectant for platelet cryopreservation (Angelini et al, 1992;Kotelba-Witkowska and Schiffer, 1982;Raymond et al, 1975;Taylor, 1981). Since Me 2 SO is a penetrating cryoprotectant and trehalose is nonpenetrating, and these protectants may utilize different mechanisms to protect biological structures, we utilized the annexin V binding assay to investigate whether the addition of Me 2 SO to our optimal trehalose plus phosphate formulation would further improve the recovery of intact, nonactivated platelets (Fig.…”

“…The effectiveness of nearly every type of cryoprotectant, including glycerol, hydroxyethyl starch, dextran (Taylor, 1981), propane-1,2-diol (Arnaud and Pegg, 1990), glycerol-glucose (Balduini et al, 1993;KotelbaWitkowska and Schiffer, 1982), dimethyl sulfoxide (Me 2 SO) (Balduini et al, 1993;Taylor, 1981;Valeri et al, 1974), Me 2 SO in the presence of second messenger effectors (ThromboSol 1 ) (Lozano et al, 1999), Me 2 SO in the presence of epinephrine (Xiao et al, 2000), has been tested. To date, 5%-6% Me 2 SO (Angelini et al, 1992;KotelbaWitkowska and Schiffer, 1982;Raymond et al, 1975;Taylor, 1981) or 2% Me 2 SO with ThromboSol (Borzini et al, 2000) are considered to be the most effective cryoprotectants for platelets. In fact, Me 2 SO preserved platelets have been used clinically (Herve, 1982;Schiffer et al, 1982).…”