Assays for phenotypic and functional characterization of cryopreserved platelets

Platelet concentrates for transfusion have a limited shelf-life, and cryopreservation offers a means of extending platelet shelf-life for at least 2 years. Cryopreservation, however, has some disadvantages, as platelets can be damaged during the freezing and thawing process. Consequently the phenotype of cryopreserved platelets is very different to that of freshly collected, liquid stored platelets. To obtain a reliable overview of cryopreserved platelet quality and function, specific testing methodologies should be considered. Whilst light transmission aggregometry (LTA) is the gold standard for measuring in vitro platelet reactivity in liquid stored platelets, it is not suited for cryopreserved platelets, as they retain little response to typical platelet agonists, even at high doses. Instead, phenotypic characterization by flow cytometry in combination with global assays of coagulation provides a clearer delineation of the phenotype and function of cryopreserved platelets. With increasing international uptake of platelet cryopreservation, it is important to recognize the need for appropriate measures of platelet quality and function

Ultraviolet-Based Pathogen Inactivation Systems: Untangling the Molecular Targets Activated in Platelets

Transfusions of platelets are an important cornerstone of medicine; however, recipients may be subject to risk of adverse events associated with the potential transmission of pathogens, especially bacteria. Pathogen inactivation (PI) technologies based on ultraviolet illumination have been developed in the last decades to mitigate this risk. This review discusses studies of platelet concentrates treated with the current generation of PI technologies to assess their impact on quality, PI capacity, safety, and clinical efficacy. Improved safety seems to come with the cost of reduced platelet functionality, and hence transfusion efficacy. In order to understand these negative impacts in more detail, several molecular analyses have identified signaling pathways linked to platelet function that are altered by PI. Because some of these biochemical alterations are similar to those seen arising in the context of routine platelet storage lesion development occurring during blood bank storage, we lack a complete picture of the contribution of PI treatment to impaired platelet functionality. A model generated using data from currently available publications places the signaling protein kinase p38 as a central player regulating a variety of mechanisms triggered in platelets by PI systems

Not all (N)SAID and done: Effects of nonsteroidal anti‐inflammatory drugs and paracetamol intake on platelets

Abstract Platelets are key mediators of hemostasis and thrombosis and can be inhibited by nonsteroidal anti‐inflammatory drugs (NSAIDs). As a result, platelet donors are temporarily deferred from donating if they have recently taken NSAIDs such as aspirin or ibuprofen. Despite these measures, a proportion of platelet donations show exposure to these drugs; however, little is known about the effect of NSAIDs and their metabolites on platelet quality in vivo and during storage. In this review, the effect of NSAIDs on platelet function is summarized, with a focus on the widely consumed over‐the‐counter (OTC) medications aspirin, ibuprofen, and the non‐NSAID paracetamol. Aspirin and ibuprofen have well‐defined antiplatelet effects. In comparison, studies regarding the effect of paracetamol on platelets report variable findings. The timing and order of NSAID intake is important, as concurrent NSAID use can inhibit or potentiate platelet activation depending on the drug taken. NSAID deferral periods and maximum platelet shelf‐life is set by each country and are revised regularly. Reduced donor deferral periods and longer platelet storage times may affect the quality of platelet products, and it is therefore important to identify the possible impact of NSAID intake on platelet quality before and after storage

The hemostatic activity of cryopreserved platelets is mediated by phosphatidylserine-expressing platelets and platelet microparticles

BACKGROUND: Cryopreservation of platelets (PLTs) at -80 degrees C with dimethyl sulfoxide (DMSO) can extend the shelf life from 5 days to 2 years. Cryopreserved PLTs are reported to have a greater in vivo hemostatic effect than liquid-stored PLTs. As such, the aim of this study was to understand the mechanisms responsible for the hemostatic potential of cryopreserved PLTs and the contribution of the reconstitution solution to this activity. STUDY DESIGN AND METHODS: DMSO (5% final concentration) was added to buffy coat-derived PLTs, followed by prefreeze removal of DMSO and storage at -80 degrees C. Cryopreserved PLTs (n = 8 per group) were thawed at 37 degrees C, reconstituted with either 1 unit of thawed frozen plasma or PLT additive solution (PAS-G). In vitro assays were performed before freezing and after thawing to assess the hemostatic activity of PLTs. RESULTS: Cryopreserved PLTs expressed high levels of phosphatidylserine and contained significantly more phosphatidylserine-positive PLT microparticles than liquid-stored PLTs. This was accompanied by a significant decrease in the time to clot formation and clot strength, as measured by thromboelastography. The supernatant from cryopreserved PLTs was sufficient to reduce the phosphatidylserine-dependent clotting time and increase the thrombin generation potential. Overall, plasma-reconstituted cryopreserved PLTs were more procoagulant than those reconstituted in PAS-G. CONCLUSION: PLT cryopreservation results in the generation of phosphatidylserine-expressing PLT microparticles which contribute to the hemostatic activity. Understanding the hemostatic activity of these components may assist in extending the use of these specialized components beyond military applications

Propensity of red blood cells to undergo P2X7 receptor-mediated phosphatidylserine exposure does not alter during in vivo or ex vivo aging

Phosphatidylserine (PS) exposure facilitates the removal of red blood cells (RBCs) from the circulation, potentially contributing to the loss of stored RBCs after transfusion, as well as senescent RBCs. Activation of the P2X7 receptor by extracellular adenosine 5′-triphosphate (ATP) can induce PS exposure on freshly isolated human RBCs, but whether this process occurs in stored RBCs or changes during RBC aging is unknown. STUDY DESIGN AND METHODS RBCs were processed and stored according to Australian blood banking guidelines. PS exposure was determined by annexin V binding and flow cytometry. Efficacy of P2X antagonists was assessed by flow cytometric measurements of ATP-induced ethidium+ uptake in RPMI 8226 cells. Osmotic fragility was assessed by lysis in hypotonic saline. RBCs were fractionated by discontinuous density centrifugation. RESULTS ATP (1 mmol/L) induced PS exposure on RBCs stored for less than 1 week. This process was near-completely inhibited by the P2X7 antagonists A438079 and AZ10606120 and the P2X1/P2X7 antagonist MRS2159 but not the P2X1 antagonist NF499. ATP-induced PS exposure on RBCs was not dependent on K+, Na+, or Cl− fluxes. ATP did not alter the osmotic fragility of stored RBCs. ATP-induced PS exposure was similar between RBCs of different densities. ATP-induced PS exposure was also similar between RBCs stored for less than 1 week or for 6 weeks. CONCLUSION The propensity of RBCs to undergo P2X7-mediated PS exposure does not alter during in vivo and ex vivo aging. Thus, P2X7 activation is unlikely to be involved in the removal of senescent RBCs or stored RBCs after transfusion

The phenotype of cryopreserved platelets influences the formation of platelet-leukocyte aggregates in an in vitro model

Cryopreservation significantly alters the phenotype of platelets; generating distinct subpopulations, which may influence the formation of platelet leukocyte aggregates (PLA). PLAs are immunomodulatory and have been associated with transfusion-associated adverse events. As such, the aim of this study was to examine the effect of cryopreservation on the ability of platelets to form PLAs, using a monocyte-like cell line (THP-1). Platelets were tested pre-freeze, post-thaw and following stimulation with TRAP-6 or A23187, both alone and following co-culture with THP-1 cells for 1 and 24 hours (n = 6). Platelet subpopulations and platelet-THP-1 cell aggregates were analyzed using multi-color imaging flow cytometry using Apotracker Green (ApoT), CD42b, CD62P, CD61, and CD45. Cryopreservation resulted in the generation of activated (ApoT-/CD42b+/CD62P+), procoagulant (ApoT+/CD42b+/CD62P+) and a novel (ApoT+/CD42b+/CD62P-) platelet subpopulation. Co-incubation of cryopreserved platelets with THP-1 cells increased PLA formation compared to pre-freeze but not TRAP-6 or A23187 stimulated platelets. P-selectin on the surface membrane was correlated with increased PLA formation. Our findings demonstrate that cryopreservation increases the interaction between platelets and THP-1 cells, largely due to an increase in procoagulant platelets. Further investigation is required to determine the immunological consequences of this interaction

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

Abstract 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