Cold-stored platelets: revisiting assumptions and addressing variability to support implementation

Platelet transfusion has become essential therapy in modern medicine. Although the clinical advantage of platelet transfusion has been well established, adverse reactions upon transfusion, especially transmission of bacterial infection, still represent a major challenge. While bacterial contamination is favored by the storage of platelets at room temperature, cold storage may represent a solution for this important clinical issue. In this study, we aimed to clarify whether plasma has protective or detrimental effects on cold-stored platelets. We investigated the impact of different residual plasma contents in apheresis-derived platelet concentrates, stored at 4°C or room temperature, on platelet function and survival. We found that platelets stored at 4°C have higher expression of apoptosis marker compared to platelets stored at room temperature, leading to accelerated clearance from the circulation in a humanized animal model. While cold-induced apoptosis was independent of the residual plasma concentration, cold storage was associated with better adhesive properties and higher response to activators. Interestingly, delta (δ) granule-related functions, such as ADP-mediated aggregation and CD63 release, were better preserved at 4°C, especially in 100% plasma. An extended study to assess cold-stored platelet concentrates produced under standard care Good Manufacturing Practice conditions showed that platelet function, metabolism and integrity were better compared to those stored at room temperature. Taken together, our results show that residual plasma concentration does not have a cardinal impact on the cold storage lesions of apheresis-derived platelet concentrates and indicate that the current generation of additive solutions represent suitable substitutes for plasma to store platelets at 4°C.

Conventional platelet (PLT) storage at room temperature under continuous agitation results in a limited shelf life (5 days) and an increased risk of bacterial contamination. However, both of these aspects can be ameliorated by cold storage. Preliminary work has suggested that PLTs can be cold stored for up to 3 weeks, while preserving their metabolic activity longer than in PLTs stored at room temperature. As such, in the present study, we hypothesized that the metabolic phenotypes of PLTs stored at 4°C for 3 weeks could be comparable to that of room temperature-stored PLTs at 22°C for 5 days. Metabolomics analyses were performed on nine apheresis PLT concentrates stored either at room temperature (22°C) for 5 days or refrigerated conditions (4°C) for up to 3 weeks. Refrigeration did not impact the rate of decline in glutamine or the intracellular levels of Krebs cycle metabolites upstream to fumarate and malate. It did, however, decrease oxidant stress (to glutathione and purines) and slowed down the activation of the pentose phosphate pathway, glycolysis, and fatty acid metabolism (acyl-carnitines). The overall metabolic phenotypes of 4°C PLTs at Storage Day 10 are comparable to PLTs stored at 22°C at the end of their 5-day shelf life, while additional changes in glycolysis, purine, and fatty acid metabolism are noted by Day 21.

Refrigeration and cryopreservation are attractive alternatives that could support an extension of the current room temperature shelf life of platelets. As transfusion of cold-stored platelets (CSP) is currently limited to bleeding patients, multiple platelet inventories will be required, which may lead to expiry and wastage. To avoid this, it may be beneficial to freeze CSP to further extend their shelf life. As such, the aim of this study was to determine whether cryopreservation of expiring CSP adversely affects in vitro platelet quality. On Day 1 following collection, double-dose single-donor plateletsharvested apheresis (60% PAS-E/40% plasma; n = 10) were frozen (-80°C with 5%-6% dimethylsulfoxide) or refrigerated (2-6°C without agitation) for 14 days. The CSP was then frozen. In vitro platelet quality was measured prior to freezing, and after thawing and reconstitution in plasma. Platelet recovery was lower in the thawed CSP (70 ± 5%), compared to standard thawed components (77 ± 8%; p = .0340). However, the recovery of all thawed platelets was above the minimum specification of 50%. Platelet glycoproteins (GPIIIa, GPIbα, and GPVI) were equivalent between thawed groups. Thawed CSP had higher P-selectin exposure, but less externalized phosphatidylserine and extracellular vesicle release, compared to standard thawed platelets. Functional parameters, including ADP-induced PAC-1 activation and thromboelastography were similar after thawing, regardless of pre-freeze storage conditions. These data suggest that cryopreservation may be a suitable way to further extend the shelf life of CSP, thus offering a strategy to reduce wastage and enhance inventory management.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.

Cold-stored platelets are making a comeback. They were abandoned in the late 1960s in favor of room-temperature stored platelets due to the need for longer post-transfusion platelet recoverability and survivability in patients with chronic thrombocytopenia. However, the current needs for platelet transfusions are rapidly changing. Today, more platelets are given to patients who are actively bleeding, such as ones receiving cardiac surgeries. It has been established that cold-stored platelets are more hemostatically effective, have reduced bacterial growth, and have longer potential shelf lives. These compelling characteristics led to the recent interest in bringing back cold-stored platelets to the blood systems. However, before reinstating cold-stored platelets in the clinics again, a thorough investigation of in vitro storage characteristics and in vivo transfusion effects is required. This review aims to provide an update on the recent research efforts into the storage characteristics and functions of cold-stored platelets using modern investigative tools. We will also discuss efforts made to improve cold-stored platelets to be a better and safer product. Finally, we will finish off with discussing the relevance of in vitro data to in vivo transfusion results and provide insights and directions for future investigations of cold-stored platelets.

Currently, platelets for transfusion are stored at room temperature (RT) for 5-7days with gentle agitation, but this is less than optimal because of loss of function and risk of bacterial contamination. We have previously demonstrated that cold (4°C) storage is an attractive alternative because it preserves platelet metabolic reserves, invitro responses to agonists of activation, aggregation and physiological inhibitors, as well as adhesion to thrombogenic surfaces better than RT storage. Recently, the US Food and Drug Administration clarified that apheresis platelets stored at 4°C for up to 72h may be used for treating active haemorrhage. In this work, we tested the hypothesis that cold-stored platelets contribute to generating clots with superior mechanical properties compared to RT-stored platelets. Rheological studies demonstrate that the clots formed from platelets stored at 4°C for 5days are significantly stiffer (higher elastic modulus) and stronger (higher critical stress) than those formed from RT-stored platelets. Morphological analysis shows that clot fibres from cold-stored platelets were denser, thinner, straighter and with more branch points or crosslinks than those from RT-stored platelets. Our results also show that the enhanced clot strength and packed structure is due to cold-induced plasma factor XIII binding to platelet surfaces, and the consequent increase in crosslinking.

Cryopreservation of platelets with dimethylsulfoxide (DMSO) at -80°C increases their shelf life from days to years. Once thawed, platelets are stored at room temperature (RT), and the shelf life is limited to 4-6 hours. However, refrigeration (cold storage) may facilitate a prolongation of the shelf life of thawed platelets. ABO-matched buffy coat-derived platelets (30% plasma/70% SSP+) were cryopreserved at -80°C in 5%-6% DMSO. Paired cryopreserved platelet components were thawed, resuspended in 30% plasma/70% SSP+, and then stored at either 20°C-24°C with agitation (RT) or at 2°C-6°C (cold). In vitro platelet quality was assessed over 10 days of postthaw storage. During postthaw storage, the platelet concentration of RT-stored components decreased significantly more than components in cold storage (Day 10 RT 58 ± 10 × 109 /unit vs Day 10 cold 142 ± 16 × 109 /unit; P < .0001). Cold storage reduced the metabolic rate of thawed platelets. During storage, the surface glycoprotein ([GP] Ibα, GPVI, GPIIb, GPIIIa) and activation marker (P-selectin and phosphatidylserine) profile of cold platelets was closer to freshly thawed platelets (Day 0) than those stored at RT. Thromboelastography (reaction time) demonstrated that the procoagulant nature of cryopreserved platelets was preserved during 10 days of cold storage, while RT-stored thawed platelets displayed a gradual prolongation of the time taken to initiate clot formation. Cold storage of thawed platelets preserves the platelet phenotype and function for up to 10 days, compared to thawed platelets stored at RT. Thus, cold storage of thawed platelets may represent a simple approach to extend the postthaw shelf life.

Ethylene is an essential indicator of fruit ripening and climacteric or non-climacteric nature. This study investigated the postharvest behavior of five Chinese bayberry cultivars ‘Biqi’, ‘Dongkui’, ‘Fenhong’, ‘Xiazhihong’, and ‘Shuijing’. The fruits were harvested mature and stored at room temperature (25 °C) and under cold storage conditions (4 °C) to investigate the dynamics of ethylene production, firmness, anthocyanin content, and cell wall polysaccharide composition, as well as basic fruit physicochemical characteristics. The results show that Chinese bayberry is a climacteric fruit with ethylene production peaking shortly after harvest, especially at room temperature. Fruit color intensified over time due to anthocyanin accumulation, particularly in the flesh core. Darker cultivars produced more ethylene, which correlated with higher anthocyanin levels. At room temperature, ‘Biqi’ (black) had the highest ethylene production (4.03 µL·kg−1·h−1) and anthocyanin content (0.91 mg/g FW), while ‘Shuijing’, the white cultivar, had the lowest ethylene levels (1.9 µL·kg−1·h−1) and anthocyanin content (0.03 mg/g FW). Firmness significantly decreased at room temperature due to the degradation of hemicellulose and insoluble pectin, whereas cold storage mitigates this effect. After 3 days at room temperature, the average of firmness decreased by 23.7% in the five cultivars, compared to 12.7% under cold storage. Total soluble solids increase during storage, enhancing sweetness, especially at room temperature, with ‘Biqi’ increasing from 9.2 to 10.9% at 4 °C. Titratable acidity slightly decreased over time: the value for ‘Biqi’ decreased from 1.2% to 0.95% at room temperature and 1.1% at 4 °C. Citric, malic, and tartaric acid generally declined at room temperature but stabilized under cold storage. Sucrose, fructose, and glucose increased or remained stable, with significant varietal differences. Our results indicate that storing Chinese bayberry at 4 °C effectively preserves its quality and extends postharvest life. These findings underscore ethylene’s key role in regulating ripening, postharvest quality, and shelf life by influencing fruit color, firmness, and overall consumer appeal.

There and back again: the once and current developments in donor-derived platelet products for hemostatic therapy

Natural products chemists, microbiologists, and ethnobotanists, are actively investigating plant-derived compounds and natural fungicide alternatives from around the world to improve post-harvest management of vegetables and fruits, reducing reliance on synthetic chemicals. Although 25 to 50% of modern medicines are originate from plants, not a single one currently utilized as anti-microbials. Conventional farmers have historically used plants to prevent and treat infections, and Western medicine is now aiming to replicate these effective practices. Plants comprise a diverse secondary metabolite such as flavonoids, alkaloids, tannins, and terpenoids, which demonstrated antimicrobial properties in vitro. This study aimed to assess the effectiveness of turmeric and neem plant extracts in extending the mango fruits shelf life (cv. Gulab Khas) under two different storage environments: cold storage and ambient temperature. The fruits were subjected to four different concentrations (5%, 10%, 15%, and 20%) of each plant extract and stored in perforated polyethylene bags. They were then stored under the specified conditions of cold and ambient storage. The combination of neem leaf extract treatment with cold storage yielded promising results. Our results showed that 20% neem leaf extract treatment in cold storage effectively inhibited pathogens, preventing any spoilage. This combination also demonstrated minimal physiological weight loss (6.2%), low spoilage rates, and maximum ascorbic acid content (28.5 mg per 100 g of pulp). Additionally, it exhibited minimal acidity (0.12%), maximum total soluble solids (20.1ºB), and the highest levels of total sugars (12.50%), reducing sugars (3.5%), and non-reducing sugars (8.55%). The organoleptic score was also notably high at 7.3 out of 10. The repressive influence of neem leaf extract is attributed owing to the active compound azadirachtin. This study emphasizes the capability of neem leaf extract as an efficient and natural alternative for extending the shelf life of mango fruits during storage. Therefore, the successful application of neem extract provides a promising avenue for future research and practical implementation in post-harvest technology.

Targeting Hemorrhage: Alternative Storage of Platelets for Hemostatic Transfusion

Platelets stored at room temperature (22-24°C) for transfusion purposes have a shelf life of 5-7 days, or 72 h when stored refrigerated (1-6°C). The limited shelf life of platelet products severely compromises platelet inventory. We hypothesized that cold storage of platelets in 100% plasma using xenon gas under high pressure would extend shelf life to 14 days. Double apheresis platelet units were collected and split equally between two bags. One unit was placed in a hyperbaric chamber, pressurized to 4 bars with a xenon/oxygen gas mixture, and placed in a refrigerator for 14 days (Xe). The remaining unit was aliquoted into mini-bags (10 ml) for storage at room temperature (RTP) or in cold (CSP). Samples were assayed on days 5 (RTP) or 14 (Xe and CSP) for count, metabolism, clot strength, platelet aggregation, and activation markers. The platelet count in Xe samples was lower than that of RTP but significantly higher than CSP. Despite similar levels of glucose and lactate, the pH of Xe samples was significantly lower than CSP. Glycoprotein expression was better preserved by Xe storage compared to CSP, but no differences in activation were observed. Thromboelastography and aggregometry results were comparable between all groups. Cold storage of platelets in plasma with hyperbaric xenon provides no significant improvement in platelet function over cold storage alone. The use of a hyperbaric chamber and the slow off-gassing of Xe-stored units complicate platelet storage and delivery logistics.

Cofilin-1 Activation in Stored Platelets

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Ciba-Geigy renames colour range