Regional
Functional assessment of alternatively stored platelet components
Christopher Roan
Research Assistant at Australian Red Cross Lifeblood, Australia
Lacey Johnson
Principal Research Scientist at the Australian Red Cross Lifeblood, Australia
Platelets for transfusion are stored at room temperature (RT; 20-24°C) with constant agitation for up to 7 days. This short shelf-life is due to the risk of continued bacterial proliferation and the progression of the platelet storage lesion, leading to reduced platelet functionality. Alternative platelet storage conditions include refrigeration (cold-stored; 2-6°C) and cryopreservation (-80°C). Cold-stored platelets are currently approved for storage for 14 days have the potential to be stored for up to 21 days and do not require agitation. Cryopreserved platelets have a shelf-life of at least 2 years but must be transfused within 4-6 hours of thawing and reconstitution. Cold-stored and cryopreserved platelets provide logistical advantages over RT storage but appear functionally different to RT-stored platelets.
Measurement of platelet function by in vitro assays is influenced by the storage temperature, platelet collection method, storage solution (plasma versus platelet additive solution [PAS]), irradiation and/or pathogen inactivation. The traditional in vitro assays of light transmission aggregometry (LTA) and viscoelastic testing assess aggregation and clot formation, respectively. These assays indicate that aspects of platelet function are differentially affected by alternative storage conditions. However, both assays have limitations for testing stored platelets, and neither assay correlates directly with platelet function following transfusion. As such, a more extensive assay panel may provide a better understanding of the functional potential of alternatively stored platelets.
LTA is considered the gold standard of platelet function testing, particularly for investigating platelet defects and effects of anti-platelet drugs in freshly collected patient samples. LTA measures the aggregatory response of platelets to a range of agonists including collagen, adenosine diphosphate (ADP), arachidonic acid and epinephrine. Platelets stored at RT aggregate in response to these agonists, however a gradual decline in aggregation response occurs during storage. Cold-stored platelets maintain a stronger aggregation response to collagen and ADP compared to RT-stored platelets over storage1, although this varies depending on the storage solution. Cryopreserved platelets have significantly reduced aggregation responses regardless of agonist type or concentration2. Viscoelastic testing using thromboelastography (TEG) or rotational thromboelastometry (ROTEM) measures overall clot formation and lysis. The interaction between coagulation factors and platelet function can be assessed by activation with calcium chloride and an activator (e.g. kaolin). Compared to RT-stored platelets, cold-stored platelets clot slightly faster but have similar clot strength1. Cryopreserved platelets demonstrate even faster clotting times, but decreased clot strength compared to RT- and cold-stored platelets1,3,4. Faster clotting times have been attributed to the increased number of microparticles in cold-stored and cryopreserved platelets. Plasma coagulation factors also influence viscoelastic readouts, so the use of PAS may result in prolongation of clotting times and reduced clot strength3,5.
Additional platelet function assays, such as clot retraction and thrombin generation assay (TGA), provide measures of platelet function not captured by traditional assays. TGA measures the overall contribution of thrombin formation and inhibition and provides insight into the procoagulant/anticoagulant activity of platelet components. Supernatants from cold-stored platelets generate more thrombin at a faster rate than RT-stored platelets6. Cryopreserved platelets have been found to elicit a stronger and faster thrombin generation response than both RT and cold-stored platelets2,3,4,6, likely due to the greater phosphatidylserine exposure and microparticle numbers present in these components.
Figure 1. Clot retraction of stored platelets.
Clot retraction (contraction) is another physiologically important platelet function whereby the clot volume decreases following cessation of bleeding to prevent vessel occlusion and promote wound closure. Assays to measure clot retraction have only recently been used to measure stored platelets, quantifying contractile stress by rheometry, changes in the clot size by imaging, or the clot mass by weight. Cold-stored platelets stored in plasma maintain a consistent capacity for clot retraction over 21 days, while RT-stored platelets lose contractile function after day 57. When stored in PAS, clot contractility of RT-stored platelets is better preserved over storage7,8, resulting in similar size clots at expiry (Figure 1). There is currently no published literature describing the impact of cryopreservation on clot retraction capabilities, although we are actively investigating this aspect of platelet function. The assessment of other platelet functions, such as fibrinolysis, platelet spreading, and platelet function under shear flow may also provide valuable information as to the overall contribution of platelets to haemostasis. However, specialised equipment and expertise is required to perform these assays.
Due to differences in the function of cold-stored and cryopreserved platelets compared to RT-stored platelets, careful interpretation of in vitro results must be made, with acknowledgement of assay limitations. A comprehensive panel of assays is therefore required to understand the effects of alternative storage conditions on the multiple roles that platelets play in mediating haemostasis. Comparisons of these in vitro assays alongside clinical data such as that being collected from ongoing clinical trials (NCT04834414 and NCT03991481) may determine which assay(s) provide the best indicator of platelet function for each component type.
References
1. Johnson L, Tan S, Wood B, Davis A, Marks DC. Refrigeration and cryopreservation of platelets differentially affect platelet metabolism and function: a comparison with conventional platelet storage conditions. Transfusion. 2016;56:1807-18.
2. Marks DC, Johnson L. Assays for phenotypic and functional characterization of cryopreserved platelets. Platelets. 2019;30:48-55.
3. Johnson L, Coorey CP, Marks DC. The hemostatic activity of cryopreserved platelets is mediated by phosphatidylserine-expressing platelets and platelet microparticles. Transfusion. 2014;54:1917-26.
4. Johnson L, Reade MC, Hyland RA, Tan S, Marks DC. In vitro comparison of cryopreserved and liquid platelets: potential clinical implications. Transfusion. 2015;55:838-47.
5. Reddoch-Cardenas KM, Sharma U, Salgado CL, Montgomery RK, Cantu C, Cingoz N, et al. An in vitro pilot study of apheresis platelets collected on Trima Accel system and stored in T-PAS+ solution at refrigeration temperature (1-6°C). Transfusion. 2019;59:1789-1798.
6. Johnson L, Tan S, Jenkins E, Wood B, Marks DC. Characterization of biologic response modifiers in the supernatant of conventional, refrigerated, and cryopreserved platelets. Transfusion. 2018;58:927-937.
7. Nair PM, Meledeo MA, Wells AR, Wu X, Bynum JA, Leung KP, et al. Cold-stored platelets have better preserved contractile function in comparison with room temperature-stored platelets over 21 days. Transfusion. 2021;61:S68-S79.
8. Muraoka WT, Nair PM, Darlington DN, Wu X, Bynum JA, Cap AP. A novel, quantitative clot retraction assay to evaluate platelet function. Platelets. 2023;34:1-9.
