Cryopreserved Platelet Research Articles

Platelet transfusion.

Platelet transfusions, used as prophylaxis or treatment for bleeding, are potentially life-saving. In many countries, demand for platelet transfusion is rising. Platelets are a limited and costly resource, and it is vital that they are used appropriately. This study will explore the evidence behind platelet transfusions in different contexts, in particular recent and important research in this area. Recent randomized clinical trials demonstrate the efficacy of platelet transfusions in some contexts but potential detrimental effects in others. Platelet transfusions also carry risk of transfusion reactions, bacterial contamination and platelet transfusion refractoriness. Observational and clinical studies, which highlight approaches to mitigate these risks, will be discussed. There is growing interest in cold-stored or cryopreserved platelet units, which may improve platelet function and availability. Clinical trials also highlight the efficacy of other supportive measures such as tranexamic acid or thrombopoietin receptor agonists in patients with bleeding. Although platelet transfusions are beneficial in many patients, there remain many settings in which the optimal use of platelet transfusions is unclear, and some situations in which they may have detrimental effects. Future clinical trials are needed to determine optimal use of platelet transfusions in different patient populations.

A Thrombospondin-1 Release Assay (TRA) Coupled to PF4-Treated Cryopreserved Platelets for the Detection of Pathogenic HIT and VITT Antibodies

Background: Platelet-activating anti-PF4 antibodies that cause thrombocytopenia and thrombosis are implicated in Heparin-induced thrombocytopenia (HIT) and, more recently, in a newly recognized entity, vaccine-induced immune thrombotic thrombocytopenia (VITT). Current frontline PF4-Polyanion ELISAs utilize simple techniques but are highly non-specific. Functional gold-standard assays such as the serotonin-release assay (SRA) and PF4-dependent P-selectin expression assay (PEA) have limited availability due to technical complexity and the requirement for freshly-drawn normal donor platelets which results in significant delays in diagnosis. Thus, there is an unmet need for near-patient, functional assays for HIT/VITT diagnosis.Aim: To develop a highly sensitive and specific functional assay to detect platelet-activating antibodies associated with HIT and VITT using long-term stored platelets. The assay should be technically simple, avoiding current functional HIT testing endpoints such as aggregometry, radioactivity measurement or flow cytometry.Methods: Platelets were subjected to controlled-rate freezing in a trehalose-based buffer. Cryopreserved platelets were then thawed after variable storage periods, incubated with platelet factor 4 (PF4), and sera from HIT or VITT suspected patients. Thrombospondin-1 (TSP1), a protein highly expressed in platelet α-granules, was quantified from activated platelet supernatants in an ELISA-based assay as a measure of platelet activation.Results: In initial studies, p-selectin expression was induced by platelet-activating HIT antibodies using cryopreserved platelets and the TSP1-release assay (TRA) detected HIT antibody-mediated PF4-dependent cryopreserved platelet activation across 20 different platelet lots (data not shown). A representative study using platelets stored for 11 months is shown in Figure 1A. A cohort of thirty-six HIT-suspected patient sera, half with PF4-dependent platelet-activating antibodies (PEA+) and without (PEA-), were used to evaluate the diagnostic accuracy of the TRA (Figure 1B). All 18 PEA+ samples activated platelets strongly with a mean TSP1 concentration of 147 µg/mL measured in the supernatant compared to a mean of 43 µg/mL obtained with non-activating samples. TSP1 release in a subgroup of 7 PEA+/SRA+/ELISA+ and 4 PEA-/SRA-/ELISA+ samples that were matched for ELISA Optical Density was examined (Figure 1C). Results demonstrated that even strong ELISA+ samples that were non-activating (PEA-/SRA-) did not induce platelet activation in the TRA, confirming high specificity of the assay. The TRA was tested for its ability to detect platelet-activating antibodies in four patients who developed VITT after the Janssen vaccine (Ad26.COV2.S). All samples induced high levels of TSP1 release from cryopreserved platelets (Figure 1D).Conclusions: Our results demonstrate that cryopreserved platelets stored for several months are viable and capable of degranulation upon activation. Further, we demonstrate that measurement of TSP-1 released from cryopreserved, PF4-treated platelets is highly accurate for detecting platelet-activating HIT and VITT anti-PF4 antibodies. Specificity and sensitivity were both 100% even with samples that were strongly false-positive in the HIT ELISA. Coupling cryopreserved platelet activation to a simple ELISA endpoint, as demonstrated here, precludes the need for fresh platelets and complex testing techniques. Thus, the TRA has the potential to transform the diagnostic testing paradigm in HIT and VITT by making near-patient in-hospital functional testing available for rapid and accurate diagnosis.Figure Legend:Figure 1. TSP-1 quantification of (A) 3 HIT patient samples (closed circles) relative to 3 normal control samples (open circles) after 11-months of frozen platelet storage. (B) Thirty-six HIT-suspected patient samples: 18 PEA+ samples (closed circles), and 18 PEA- samples (open squares). Data are compared using two-tailed, unpaired Student's t test. **** P<0.0001. (C) Eleven high optical density matched HIT ELISA patient sera: 7 PEA+/SRA+ samples (closed circles) and 4 PEA-/SRA- samples (open squares). (D) TSP-1 quantification of VITT patient sera. NC-normal control. [Display omitted] DisclosuresPruthi: Instrumentation Laboratory: Honoraria; HEMA Biologics: Honoraria; Bayer Healthcare AG: Honoraria; Genentech: Honoraria; CSL Behring: Honoraria; Merck: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Frozen Platelets-Development and Future Directions.

Storage requirements and outdating of platelets represent a continued challenge for blood banks. These hurdles are confounded for rural area hospitals or in military deployments. Over 60 years of research and development into frozen platelets have generated a stable and reproducible product. Valeri's method to freeze platelets in 6% dimethyl sulfoxide (DMSO) and storage at -80°C allows for long-term storage alleviating burdens placed on blood banks. Clinical studies show that frozen platelet transfusions are safe with no related thrombotic or other serious adverse events. There are ongoing efforts to demonstrate cryopreserved platelet (CPP) superiority in efficacy studies designed in trauma or cardiac surgery patients. Technical advances in CPP manufacturing including closed system manufacturing, applications of pathogen reduction technology and potency standard characterization add to the appeal of CPP as an alternative to traditional liquid-stored platelets (LP) in settings of supply shortages, mass casualty, active bleeding, rapid provision of HLA-compatible platelets, and remote care.

The use of cryopreserved platelets in a trauma-induced hemorrhage model.

BackgroundCryopreserved platelet products can be stored for years and are mainly used in military settings. Following thawing, cryopreserved platelets are activated, resulting in faster clot formation but reduced aggregation in vitro, rendering their efficacy in bleeding unknown. Also, concerns remain on the safety of these products. The aim was to investigate the efficacy and safety of cryopreserved platelets in a rat model of traumatic hemorrhage.Study Design and MethodsAfter 1 hour of shock, rats (n = 13/group) were randomized to receive a balanced transfusion pack (1:1:1 red blood cell:plasma:platelet) made from syngeneic rat blood, containing either liquid stored platelets or cryopreserved platelets. Primary outcome was the transfusion volume required to obtain a mean arterial pressure (MAP) of 60 mmHg. Secondary outcomes were coagulation as assessed by thromboelastometry (ROTEM®) and organ failure as assessed by biochemistry and histopathology.ResultsThe transfusion volume to obtain a MAP of 60 mmHg was lower in animals receiving cryopreserved platelets (5.4 [4.1‐7.1] mL/kg) compared to those receiving liquid stored platelets (7.5 [6.4‐8.5] mL/kg, p < 0.05). ROTEM® clotting times were shorter (45 [41‐48] vs. 49 [45‐53]sec, p < 0.05), while maximum clot firmness was slightly lower (68 [67‐68] vs. 69 [69‐71]mm, p < 0.01). Organ failure was similar in both groups.ConclusionsUse of cryopreserved platelets required less transfusion volume to reach a targeted MAP compared to liquid stored platelets, while organ injury was similar. These results provide a rationale for clinical trials with cryopreserved platelets in (traumatic) bleeding.

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.

Transfusion of cryopreserved platelets exacerbates inflammatory liver and lung injury in a mice model of hemorrhage.

Platelets are essential for primary hemostasis and also play an important role in inflammatory reactions. The hemostatic property of cryopreserved platelets (CPPs) has been confirmed in the treatment of bleeding casualties, but inflammatory injury induced by CPP transfusion is relatively unclear. We aim to investigate the effects of CPP transfusion on inflammatory organ injury in mice after hemorrhage. Mice were subjected to a volume-controlled hemorrhage over 1 hour, and then were transfused with fresh platelets (FPs), Liquid-stored platelets (LPPs), CPPs, or fresh frozen plasma (FFP, control). At 6 hours posttransfusion, mice were sacrificed, and blood and tissues were sampled. Tissue sections were examined histologically and by immunohistochemical staining of neutrophils and macrophages. Plasma alanine aminotransferase, hepatic myeloperoxidase activity and inflammatory cytokine levels were measured. Transfusion of stored platelets (LPPs and CPPs) caused more serious histological injury in liver and lung compared with FPs and FFP (p < 0.05). However, kidney histological injury was similar among groups. Significantly higher numbers of Ly-6G-positive neutrophils were detected in liver and of F4/80-positive macrophages in liver and lung of mice transfused with LPPs or CPPs compared with FPs or FFP (p < 0.05). Transfusion of CPPs caused the most severe inflammatory liver injury, as reflected by alanine aminotransferase levels, hepatic macrophage infiltration, and hepatic myeloperoxidase activity and inflammatory cytokine levels (macrophage inflammatory protein-2, tumor necrosis factor-α, and interleukin-1β). Cryopreserved platelet transfusion is more likely to aggravate hemorrhage-induced liver and lung injury by activating macrophage and facilitating neutrophil infiltration into hepatic tissues.

Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets

BackgroundFreezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs.MethodsCPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test.ResultsSEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing–thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a+ PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent.ConclusionsMajor portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing–thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant contribution to PCA of CPPs.

In vitro evaluation of the hemostatic effectiveness of cryopreserved platelets.

Cryopreserved platelet (CPP) concentrates exhibit a variety of morphologic and functional alterations that may affect the action of CPP with accelerated platelet (PLT) response and clotting. The objective of this study was to compare the in vitro hemostatic effect of CPP with fresh whole blood (WB) and standard 5-day PLT concentrates (PCs). WB collected from eight healthy donors was used to prepare fresh WB, PLT-depleted WB (TPN), and PLT-restored TPN using CPP (TPN-CPP) or PC (TPN-PC). Clot properties were evaluated with thromboelastometry (ROTEM); adhesion and aggregate formation under high shear (Impact-R); and PLT adhesion, aggregate formation, fibrin formation, and prothrombin activation under medium shear in a perfusion system. TPN-CPP had faster clot initiation (ROTEM clot time--TPN-CPP 115 sec, WB 194 sec, TPN-PC 161 sec), and CPP contributes to a strong clot with PLT involvement (maximum clot firmness--TPN-CPP 32 mm, WB 62 mm, TPN-PC 59 mm). The Impact-R PLT-covered area with TPN-CPP was less than those of WB and PCs, but aggregate size was the same as WB. PLT coverage in perfusion studies was observed with TPN-CPP, although generally less than both WB and PC. Fibrin was deposited with CPP-restored samples, but did not exceed the level of WB. CPPs present a phenotype supporting a moderate increase in the rate of clot formation, form stable PLT clots, and do not present a hypercoagulable phenotype during in vitro functional tests.

Comparison of the effects of transfusions of cryopreserved and liquid-preserved platelets on hemostasis and blood loss after cardiopulmonary bypass

Objective: The aim of the study was to compare the clinical effects and hemostatic efficiency of transfusions of platelets preserved in the frozen state for as long as 2 years with transfusions of platelets preserved in the conventional manner for as long as 5 days in patients undergoing cardiopulmonary bypass. Methods: Seventy-three patients were prospectively randomly assigned to receive transfusions of cryopreserved or liquid-preserved platelets. Nonsurgical blood loss was measured during and after the operation. Bleeding time, hematologic variables, and the bleeding time site shed blood were assayed before cardiopulmonary bypass and at 30 minutes and 2, 4, and 24 hours after transfusion. In vitro platelet function tests were conducted on platelets obtained from healthy volunteers. Results: No adverse sequelae of the transfusions were observed. Blood loss and the need for postoperative blood product transfusions were lower in the group receiving cryopreserved platelets. Lower posttransfusion platelet increments and a tendency toward decreased platelet survival were observed in patients receiving cryopreserved platelets. Hematocrit and plasma fibrinogen were significantly higher in this group, and the duration of intubation was shorter. In vitro, cryopreserved platelets demonstrated less aggregation, lower pH, and decreased response to hypotonic stress but generated more procoagulant activity and thromboxane. Conclusions: (1) Cryopreserved platelet transfusions are superior to liquid-preserved platelets in reducing blood loss and the need for blood product transfusions after cardiopulmonary bypass. (2) The reduction in blood loss in the patients receiving cryopreserved platelet transfusions after cardiopulmonary bypass probably reflects improved in vivo hemostatic function of cryopreserved platelets. (3) Some in vitro measures of platelet quality (aggregation, pH, hypotonic stress) may not reflect in vivo quality of platelet transfusions after cardiopulmonary bypass, whereas other in vitro measures (platelet procoagulant activity and thromboxane) do. (J Thorac Cardiovasc Surg 1999;117:172-84)