A deep eutectic solvent is an effective cryoprotective agent for platelets.

The most widely used method of platelet cryopreservation requires the addition of dimethyl sulfoxide (DMSO; Me2SO) as a cryoprotective agent (CPA) and pre-freeze removal of Me2SO before freezing to mitigate toxicity. However, alternative CPAs such as deep eutectic solvents (DES), which are less toxic could simplify this process. The aim of this study was to determine the effectiveness of a Proline-Glycerol (Prol-Gly 1:3) DES as a platelet CPA. Platelets were cryopreserved at -80 °C using 10 % Prol-Gly 1:3 (DES; n = 6), or in the absence of a cryoprotectant (no CPA; n = 6). Platelets were also cryopreserved according to the gold-standard blood-banking method using 5.5 % Me2SO (n = 6), with centrifugation and pre-freeze removal of the excess Me2SO. Platelet quality was assessed by flow cytometry and thromboelastography (TEG). Post-thaw recovery was similar between the three groups. The abundance of labile platelet glycoproteins GPIbα and GPVI were highest in the DES group, however, markers of activation (CD62P and annexin-V) were also higher in this group. In terms of function, the strength of the clot (maximum amplitude; TEG) and extent of clot retraction was better with DES platelets compared to no CPA, but lower than Me2SO platelets. DES provides a cryoprotective advantage to platelets when compared to no CPA. Importantly, when compared to Me2SO platelets, most quality parameters were similar in DES platelets. The major advantage with using a DES is biocompatibility, therefore it does not need to be removed prior to transfusion. This greatly simplifies the freezing and thawing process, avoiding the toxic effects of Me2SO.

The role of sodium citrate during extended cold storage of platelets in platelet additive solutions.

BACKGROUND: Cold-stored platelets are increasingly being used to treat bleeding. Differences in manufacturing processes and storage solutions can affect platelet quality and may influence the shelf life of cold-stored platelets. PAS-E and PAS-F are approved platelet additive solutions (PAS) in Europe and Australia, or the United States respectively. Comparative data are required to facilitate international transferability of laboratory and clinical data. STUDY DESIGN AND METHODS: Single apheresis platelets from matched donors (n = 8) were collected using the Trima apheresis platform and resuspended in either 40% plasma/60% PAS-E or 40% plasma/60% PAS-F. In a secondary study, platelets in PAS-F were supplemented with sodium citrate, to match the concentration in PAS-E. Components were refrigerated (2-6°C) and tested over 21 days. RESULTS: Cold-stored platelets in PAS-F had a lower pH, a greater propensity to form visible (and micro-) aggregates, and higher activation markers compared to PAS-E. These differences were most pronounced during extended storage (14-21 days). While the functional capacity of cold-stored platelets was similar, the PAS-F group displayed minor improvements in ADP-induced aggregation and TEG parameters (R-time, angle). Supplementation of PAS-F with 11 mM sodium citrate improved the platelet content, maintained the pH above specifications and prevented aggregate formation. DISCUSSION: In vitro parameters were similar during short-term cold storage of platelets in PAS-E and PAS-F. Storage in PAS-F beyond 14 days resulted in poorer metabolic and activation parameters. However, the functional capacity was maintained, or even enhanced. The presence of sodium citrate may be an important constituent in PAS for extended cold storage of platelets.

Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry.

Cryopreservation of platelets, at - 80 °C with 5-6% DMSO, results in externalisation of phosphatidylserine and the formation of extracellular vesicles (EVs), which may mediate their procoagulant function. The phenotypic features of procoagulant platelets overlap with other platelet subpopulations. The aim of this study was to define the phenotype of in vitro generated platelet subpopulations, and subsequently identify the subpopulations present in cryopreserved components. Fresh platelet components (n = 6 in each group) were either unstimulated as a source of resting platelets; or stimulated with thrombin and collagen to generate a mixture of aggregatory and procoagulant platelets; calcium ionophore (A23187) to generate procoagulant platelets; or ABT-737 to generate apoptotic platelets. Platelet components (n = 6) were cryopreserved with DMSO, thawed and resuspended in a unit of thawed plasma. Multi-colour panels of fluorescent antibodies and dyes were used to identify the features of subpopulations by imaging flow cytometry. A combination of annexin-V (AnnV), CD42b, and either PAC1 or CD62P was able to distinguish the four subpopulations. Cryopreserved platelets contained procoagulant platelets (AnnV+/PAC1-/CD42b+/CD62P+) and a novel population (AnnV+/PAC1-/CD42b+/CD62P-) that did not align with the phenotype of aggregatory (AnnV-/PAC1+/CD42b+/CD62P+) or apoptotic (AnnV+/PAC1-/CD42b-/CD62P-) subpopulations. These data suggests that the enhanced haemostatic potential of cryopreserved platelets may be due to the cryo-induced development of procoagulant platelets, and that additional subpopulations may exist.