Pathogen reduction of platelets: Experience of a single blood bank

Pathogen reduction technology (PRT) has the potential to prevent pathogen transfusion transmission from blood donor to patient by impeding the replication of bacteria, viruses and parasites in blood components. Additionally, PRT can help to guarantee blood safety in challenging situations for blood supply, as in the Ebola or Chikungunya epidemics, or in a scenario full of uncertainties such as the current SARS-CoV-2 pandemic. The Balearic Islands Blood Bank (BIBB) is one of the few blood establishments worldwide with more than 10 years of experience in the routine use of amotosalen/UVA (Intercept Blood System) and riboflavin/UVA-UVB (Mirasol PRT system) for platelets (PLTs), the use of riboflavin/UVA-UVB for plasma and with research experience in riboflavin/UVA-UVB applied to whole blood. Over the years, we have had the opportunity to evaluate PRT from different perspectives, such as clinical and hemovigilance research in adults and children, in vitro studies on PRT effects on PLTs and assessing the financial impact of PRT implementation. PRT methods offer remarkable benefits but also have certain limitations, which are important to bear in mind during the decision-making process for PRT implementation. The purpose of this study is to review the current knowledge on PRT for PLTs drawing on our experience acquired over the last decade.Copyright © 2021 AME Publishing Company.

Platelet Function at the Intersection of the COVID-19 “Cytokine Storm” and Mechanical Circulatory Support

Purpose: Release and circulation of pro-inflammatory cytokines or “cytokine storm,” a pathophysiologic component of severe COVID-19, is associated with thrombosis and clot embolization. Compromised patients often require extracorporeal oxygenation and mechanical circulatory support (MCS), imparting blood flow disturbances and exogenous shear stress, further amplifying thrombotic potential. Central in these processes is the platelet. The dynamic interaction of MCS flow/shear and inflammatory cytokines and their propensity for altering platelet function remains unknown. We hypothesized that platelet function is modified in an MCS + pro-inflammatory cytokine environment. We examined platelet aggregation as a function of time, exposing platelets to COVID-19-associated cytokines under MCS flow in vitro. Methods: An Impella5.5® was affixed in a closed loop and positioned with outflow cannula in a 1-inch tube region, maintained at differential 60mmHg pressure. Alternatively, a CentriMag® was affixed in series with a similar closed loop. Porcine PRP, obtained via centrifugation of fresh, ACD-A anticoagulated whole blood was used as circulating fluid. A cytokine “COVID cocktail” of porcine IL-6 (4.5 ng/mL), IL-1β (0.5 ng/mL), IL-8 (2.7 ng/mL), and TNFα (1 ng/mL) was added to PRP and circulated at 5 L/ min. After 5, 60 and 240min of circulation, platelet samples were taken and measured for aggregation with ADP (20uM), and expression of activation markers (CD62P, AnnV) via flow cytometry. Samples were measured in duplicate from N ≥ 2 pigs per experiment. Results: The addition of COVID Cocktail cytokines led to an increase in overall aggregability of platelets over time. In contrast, the addition of shear via MCS devices led to a decrease in platelet aggregability despite Cytokine addition (Fig 1). Notably, platelet aggregability was more greatly reduced with CentriMag (85% reduction) than with Impella (65% reduction). There was no significant difference in platelet activation (AnnV binding, CD62P exposure) between CentriMag and Impella 5.5 in the cytokine environment. (Figure Presented).

ASAIO 2022 Annual Meeting Abstracts

The proceedings contain 226 papers. The topics discussed include: identification of biomarkers sensitive to pulsatile and continuous flow for identification of promising continuous flow VAD modulation protocols to mitigate non-surgical bleeding events;comprehensive machine learning analysis of pre-implantation risk factors for right heart failure after LVAD implantation;combining VA-ECMO And Impella (EC-Pella) before reperfusion mitigates left ventricular loading and injury due to VA-ECMO in acute myocardial infarction;platelet function at the intersection of the COVID-19 'cytokine storm' and mechanical circulatory support;a dialysate free portable artificial kidney device;durable right heart mechanical support system: a multi-day proof-of-concept study in pulmonary hypertension sheep;a dual-action nitric oxide-releasing slippery surface coating for extracorporeal organ support: first evaluation at clinically relevant blood flow rate for partial lung support;cannula add-on for pressure and flow measurement in VADs;and comparison of interlaboratory CFD simulations of the FDA benchmark blood pump model.

Platelets genetically modified with mRNA-lipid nanoparticles are functionally similar to clinically transfused platelets in vitro

Background: Platelet transfusions are an essential treatment for attenuating bleeding but are often ineffective in cases of intractable haemorrhage. Although anucleate, mature platelets synthesize protein de novo, making them amenable to mRNA gene therapy;however, there remains to be an effective transfection technique. Advancements in lipid nanoparticle technology has enabled leading COVID vaccines and is an efficient method to deliver nucleic acids into target cells. Recently, we developed a LNP approach to successfully express exogenous protein in platelets [unpublished data], a first step towards demonstrating that donor platelet coagulability can be engineered. However, the effects of LNP treatment on platelet function has yet to be investigated. Aims: To determine whether LNP-treated donor platelets are functionally similar, or better, in vitro, than platelets currently transfused clinically as a next step to establish LNP engineered platelets as a new cell therapy. Methods: The hemostatic profiles of LNP-treated and clinical donor platelets were assessed using an adapted rotational thromboelastometry model of dilutional coagulopathy. LNP-treated platelets were also stimulated with conventional platelet agonists to test if responsiveness is similar, or better than clinical platelets using flow cytometry. Results: LNP-treated platelets have a comparable hemostatic profile to clinically transfused platelets and significantly improved clotting dynamics when spiked into hemodiluted whole blood in an in vitro transfusion simulation. LNP-treated platelets also respond comparably, and in some cases more potently to agonist simulation compared to untreated platelets as indicated by similar p-selectin surface presentation. Summary/Conclusions: LNPs are an effective way to deliver exogenous nucleic acids into platelets;they do not significantly change platelet coagulability or responsiveness to agonists. LNP platelet engineering is a promising new approach to load platelets with procoagulant protein to enhance their function.

In vitro ACE2 Enzyme Inhibitory Activity Evaluation of Different Salvia Essential Oils

In this present study, commertially available Salvia triloba L., S.officinalis L., and S. sclarea L. essential oils were evaluated for their in vitro angiotensin converting enzyme 2 (ACE2) inhibitory activity. The Salvia essential oils compositions were confirmed both by GC-FID and GC/MS. Main components of the S. triloba essential oil was characterized as 1,8-cineole (22.8%), camphor (17.2%), α-thujone (15.2 %), β-caryophyllene (11.4 %), and α-humulene (3%). Major constiutents were identified as α-thujone (28.5 %), camphor (20.6 %), 1,8-cineole (10.9%), α-humulene (5%), and camphene (4.9 %) in S. officinalis essential oil. Whereas, linalylacetate (56.8 %), linalool (21.1%), α-terpineol (6.1%), geraniol (5%), and β-caryophyllene (3.4%) were the major components of S. sclarea essential oil. The essential oils were evaluated using a fluorometric multiplate based enzyme inhibition kit, where the ACE2 inhi-bitions of S. triloba, S. officinalis, and S. sclarea essential oils were 50.1%, 60.5%, and 72.1% at a concentration of 20 µg/mL, respectively. As a result, further tests of Salvia essential oils supported by in vivo studies may have antiviral potential ap-plications against coronaviruses due to ACE2 enzyme inhibitions.

Vaccine-induced thrombotic thrombocytopenia: Novelty testing in the diagnostic workup?

COVID-19 vaccination campagnies with several vaccines types are currently undeway. Recently, the ASTRA ZENECA vaccine has raised public alarm with concerns regarding the development of thrombotic events known as vaccine-induced thrombotic thrombocytopenia (VITT). Early and limited studies have implicated an antibody-mediated platelet activation as the mechanism of the clotting events. Aim of this study was to investigate the platelet and coagulation activation using specialized tests. In this study we enrolled 60 patients (40 men, 20 women;mean age 55±10 years) without cardiovascular risk factors or a history of thrombosis who reported having poplitea deep vein thrombosis (35/60) and pulmonary embolism (25/60) revealed with lower-limb ultrasonography and computed tomography (CT) angiography, respectively, 7 days after vaccination with ASTRA ZENECA. All patients were evaluated for initial testing such as platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib) and D-dimer (DD). Platelets were measured by automated analyzer, PT and APTT by coagulometric test, Fib using Clauss method, and DD using ELISA. Complete blood hemostasis was studied by platelet function assay (PFA-100) on Collagen/ADP (CT-ADP) and Collagen/Epinephrine (CT-EPI) cartridges and Thromboelastometry method on Clotting Time (CT), Clotting Formation Time (CFT), Maximum Clot Firmness (MCF), and clot lysis at 30 minutes (LY-30). All patients had thrombocytopenia (60±5x109/L), longer PT (28±10 s) and PTT (50±10 s), lower Fib (80±20 mg/dl), higher DD ((550±100 mg/l). All patients had shorter C/ADP and C/EPI (C/ADP, n.v. 68-121 s (42±10 s) and C/EPI n.v. 84-160 s (38±5 s) and shorter CT (CT, unit: s. n.v. 100-240 s) (INTEM 30±20 s, EXTEM 18±10 s), shorter CFT (CFT, unit: s, n.v. 30-160 s (INTEM 11±10 s, EXTEM 19±10 s), longer MCF (MCF, unit: mm, n.v. 50-72 mm (INTEM 128±10 mm, EXTEM 110±10 mm), and lower LY-30 (LY-30, %: v.n. 15% (INTEM 0.8%, EXTEM 0.7%). These interesting findings may be the novelty in the diagnostic work-up of the VITT. If these tests may aid in the diagnosis of VITT deserve to be confirmed and need reproducing in other studies.

Platelets contribute to disease severity in covid-19

Objective: Heightened inflammation, dysregulated immunity, and thrombotic events are characteristic of hospitalized COVID-19 patients. Platelets are key regulators of thrombosis, inflammation, immunity and are prime candidates for a role in the pathogenesis of COVID-19. The objective of this study was to analyze the platelet phenotype in COVID-19. Approach and Results: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is detected in human megakaryocytes and platelets in COVID-19 patients and following incubation with the virus in vitro. We show a direct interaction between SARS-CoV-2 and megakaryocytes that alters the platelet transcriptome and platelet activity. COVID-19 platelets are hyperreactive and have a distinct transcriptomic profile characteristic of prothrombotic large and immature platelets. We find transcriptomic changes mediated by SARS-CoV-2 do not occur following exposure of megakaryocytes with a coronavirus responsible for the common cold, CoV-OC43. In a cohort of 3,915 hospitalized COVID-19 patients, we analyzed blood platelet indices collected at hospital admission. Following adjustment for demographics, clinical risk factors, medication use, and biomarkers of inflammation and thrombosis, platelet count, size, and immaturity are each associated with increased critical illness and all-cause mortality. Conclusions: Our findings demonstrate that SARS-CoV-2 virions invade megakaryocytes and platelets, inducing alterations to the platelet transcriptome and activation profile, which correlate with critical illness and mortality in hospitalized COVID-19 patients.

Flow Cytometric Detection of Procoagulant Properties of Plasma from Patients with Clinically Confirmed Vaccine-Induced Immune Thrombotic Thrombocytopenia

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines including ChAdOx1 nCoV-19 (AstraZeneca) vaccine. The putative mechanism involves formation of pathological anti-PF4 antibodies that activate platelets via the FcγRIIa receptor to drive thrombosis and the associated thrombocytopenia. Functional assays are important in the VITT diagnostic pathway as not all detectable PF4 antibodies are pathogenic. Detection of procoagulant platelets (platelets supporting thrombin generation) in presence of PF4 has been proposed as a diagnostic assay for VITT (Althaus et al). Procoagulant platelets are not typically generated in response to low level agonist stimulation;however, combination of ligand binding of G-protein coupled receptors (GPCR) (eg. PAR1) and ITAM linked receptors (eg. GPVI, CLEC2 and FcγRIIa) synergistically induce procoagulant platelet formation. Here, we describe an alternative flow cytometric assay to diagnose VITT. We hypothesized that priming of platelets with a PAR1 agonist at a level sufficient to release PF4, but insufficient to generate a significant procoagulant response in donor platelets, would provide a platform in which procoagulant response would be dependent on presence of FcγRIIa dependent procoagulant antibodies in patient plasma, without requirement for additional PF4. Methods: Our previously established flow cytometry-based procoagulant platelet assay (using cell death marker GSAO and P-selectin) was modified to incorporate exogenous patient plasma and performed on whole blood from healthy donors screened for FcγRIIa responsiveness (aggregation response to anti-CD9 antibody, ALB6), primed with 5 μM SFLLRN. The assay was performed on Australian patients referred for confirmatory VITT testing with probable VITT (confirmed thrombosis within 4-42 days of ChAdOx1 nCov-19 vaccination, D-Dimer > 5x ULN, platelets < 150 x 10 9/L or falling platelet count) after screening on PF4/heparin ELISA (Asserachrom HPIA IgG Assay, Stago Diagnostics). Procoagulant response was also measured in presence of 0.5 U/mL and 100 U/mL heparin, monoclonal FcγRIIa blocking antibody, IV.3, and intravenous immunoglobulin, IVIg. Some plasmas were incubated with ChAdOx1 nCoV-19 or SARS-CoV-2 spike protein. Flow cytometry positive patients were also tested by serotonin release assay (SRA) and multiplate aggregometry. Clinical correlation was obtained. Results: Citrated plasma from 49 unique patients with suspected VITT are reported. Plasma from ELISA+ve patients with clinical picture consistent with VITT (n=31), significantly increased the procoagulant platelet proportions in healthy donors in presence of 5 μM SFLLRN (p<0.0001, Figure 1A). This increase was not seen with plasma from healthy donors (n=14);or individuals exposed to ChAdOx1 nCov-19 vaccine without VITT: thrombocytopenic thrombosis patients who were ELISA-ve and SRA-ve (n=14);or low-level ELISA+ve patients without thrombocytopenia who were negative by either multiplate or SRA (n=4). The procoagulant platelet response induced by VITT positive plasma was reduced with low dose heparin (0.5 U/mL, p<0.01) except for 3 patients who showed a heparin-enhancing effect (Figure 1B). High dose heparin (100 U/mL, p<0.0001), IV.3 (10 µg/mL, p<0.0001) or IVIg (10 mg/mL, p<0.0001) abolished the procoagulant response (Figures 1C-D). The in vitro effect of IVIg was predictive of the in vivo response to IVIg therapy (Figure 1E). Addition of SARS-CoV-2 spike protein had no effect on the procoagulant platelet response. ChAdOx1 nCov-19 had an inconsistent effect on procoagulant platelet formation in presence of VITT plasma. Use of donors without a robust aggregation response to ALB6 resulted in false negative results. Conclusion: Induction of FcγRIIa dependent procoagulant response by patient plasma, suppressible by high dose heparin and IVIg, is highly indicative of VITT in the correct clinical circumstance. This assay modification of priming donor platelets from known FcγRIIa responsive donors ith a GPCR agonist to potentiate the ITAM signaling from platelet activating immune complexes, results in a sensitive and specific assay. This may represent a functional platform that can be adopted into diagnostic laboratories to identify patients with platelet-activating antibodies and potentially predict treatment responses. [Formula presented] Disclosures: No relevant conflicts of interest to declare.