In vitro Hemostatic Functions of Cold-Stored Platelets.

Background: Transfusion of platelets is a life-saving medical strategy used worldwide to treat patients with thrombocytopenia as well as platelet function disorders. Summary: Until the end of 1960s, platelets were stored in the cold because of their superior hemostatic functionality. Cold storage of platelets was then abandoned due to better posttransfusion recovery and survival of room temperature (RT)-stored platelets, demonstrated by radioactive labeling studies. Based on these findings, RT became the standard condition to store platelets for clinical applications. Evidence shows that RT storage increases the risk of septic transfusion reactions associated with bacterial contamination. Therefore, the storage time is currently limited to 4-7 days, according to the national guidelines, causing a constant challenge to cover the clinical request. Despite the enormous efforts made to optimize storage conditions of platelets, the quality and efficacy of platelets still decrease during the short storage time at RT. In this context, during the last years, cold storage has seen a renaissance due to the better hemostatic functionality, reduced risk of bacterial contamination, and potentially longer storage time. Key Messages: In this review, we will focus on the impact of cold storage on the in vitro platelet functions as promising alternative storage temperature for future medical applications.

Characteristics Associated with COVID-19 Breakthrough Infections after Booster Vaccinations in Healthcare Workers: Insights from the TüSeRe:exact Study.

Background: The prevalence of COVID-19 breakthrough infections in healthcare workers (HCWs) remains an issue of concern. This study examines the different characteristics associated with breakthrough infections in HCWs. Methods: From the total participants in the TüSeRe:exact study (n = 1046), we specifically included study participants who had received three vaccinations and were not infected prior to the third vaccination. Participants were invited to complete an online questionnaire, which included inquiries about any breakthrough infections they might have experienced. Univariate Cox regression analysis was used to investigate the association between participant characteristics and breakthrough infections. Results: Among 629 HCWs (497 female and 132 male), 241 (38%) experienced breakthrough infections during the follow-up period. The frequency of breakthrough infections was 39.2% (195/497) among female participants and 34.8% (46/132) among male participants (p = 0.357). The Cox regression model adjusted for age and sex showed that participants with cardiovascular disease (hazard ratio (95%CI) = 0.621 (0.392-0.985); p = 0.043) and those taking antihypertensives (hazard ratio (95%CI) = 0.551 (0.331-0.915); p = 0.021) had a significantly lower hazard ratio for breakthrough infections. The use of analgesics after the first vaccine (hazard ratio (95%CI) = 1.343 (1.025-1.759); p = 0.032) was associated with an increased risk of breakthrough infections. Conclusions: These findings can inform targeted preventive measures and risk management strategies to protect frontline workers and maintain a resilient healthcare system during the ongoing pandemic.

Results of an international survey of opinions on the definitions and treatments for heparin-induced thrombocytopenia: communication from the ISTH SSC Subcommittee on Platelet Immunology.

Heparin-induced thrombocytopenia (HIT) is rare, affecting fewer than 1 in 1500 hospital admissions. Despite the increasing adoption of new therapies in HIT, such as direct oral anticoagulants and pooled immunoglobulins, there is limited high-quality evidence to guide clinicians. Numerous uncommon presentations of HIT and HIT-like entities have recently been recognized, and a harmonized approach to their classification is required to study them better. We present the results of an international survey of opinions from experts and practitioners in the field of platelet immunology regarding the role of direct oral anticoagulants in HIT, novel definitions of subclassifications of HIT-like platelet factor 4 immune conditions (spontaneous autoimmune HIT, persistent autoimmune HIT, and treatment-refractory HIT), and the role for intravenous immunoglobulins in the treatment paradigm of HIT and these HIT-like conditions. From 102 survey responses, there was broad acceptance of rivaroxaban (74.5%) and apixaban (73.5%) even before platelet recovery, as well as for intravenous immunoglobulin in the management of spontaneous (85.6%), persistent (83.7%), and treatment-refractory HIT (87.4%). With this mandate for harmonizing terminologies and treatment approaches in special situations without robust clinical data owing to their rarity, we plan to conduct a robust survey, establish international consensus, and draft management guidelines for HIT and platelet factor 4 immune diseases in the near future.

Heparin-activated procoagulant platelet assay: a flow cytometry-based functional test for heparin-induced thrombocytopenia.

BACKGROUND: Functional platelet activation assays are required for the diagnosis of heparin-induced thrombocytopenia (HIT). Due to their sophisticated methodology, they are only available in reference centers. OBJECTIVES: To evaluate the diagnostic accuracy of the flow cytometry-based heparin-activated procoagulant platelet (HAPP) assay in the laboratory diagnosis of HIT. METHODS: Procoagulant platelets (PCP), defined by the expression of phosphatidylserine and CD62-P, were evaluated by flow cytometry in platelet-rich plasma from healthy donors after incubation with patient sera in the absence and presence of heparin. A sample was considered positive in HAPP assay, if the following 3 criteria were met: 1) the percentage of PCPs was ≥10.3% after incubation with 0.2 IU/mL heparin, 2) the fold increase in presence of 0.2 IU/mL heparin compared with buffer was ≥1.5, and 3) 100 IU/mL of heparin resulted in ≥50% inhibition of PCP. HAPP assay was validated in a prospective cohort (n = 202) of consecutive specimens submitted to our laboratory for serologic diagnosis of HIT. Heparin-induced platelet activation (HIPA) assay was used as the reference standard. RESULTS: HIT-positive sera induced PCPs in the presence of 0.2 IU/mL heparin, which was inhibited with 100 IU/mL of heparin. In the prospective validation cohort, there were 15 HIPA+ and 187 HIPA- sera. HAPP was positive in 20 samples in this cohort. Using optimized cut-offs, HAPP assay had a sensitivity of 93.3% and specificity of 96.8%. CONCLUSION: HAPP assay is promising as a simple and reliable functional assay for HIT; however, further studies are needed to confirm our results in larger cohorts.

Cerebral venous sinus thrombosis and thrombocytopenia due to heparin-independent anti-PF4 antibodies after adenovirus infection.

Not available.

Platelet-neutrophil interaction in COVID-19 and vaccine-induced thrombotic thrombocytopenia.

Coronavirus disease 2019 (COVID-19) is known to commonly induce a thrombotic diathesis, particularly in severely affected individuals. So far, this COVID-19-associated coagulopathy (CAC) has been partially explained by hyperactivated platelets as well as by the prothrombotic effects of neutrophil extracellular traps (NETs) released from neutrophils. However, precise insight into the bidirectional relationship between platelets and neutrophils in the pathophysiology of CAC still lags behind. Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare autoimmune disorder caused by auto-antibody formation in response to immunization with adenoviral vector vaccines. VITT is associated with life-threatening thromboembolic events and thus, high fatality rates. Our concept of the thrombophilia observed in VITT is relatively new, hence a better understanding could help in the management of such patients with the potential to also prevent VITT. In this review we aim to summarize the current knowledge on platelet-neutrophil interplay in COVID-19 and VITT.

Correlation between Clinical Characteristics and Antibody Levels in COVID-19 Convalescent Plasma Donor Candidates.

COVID-19 convalescent plasma (CCP) with high neutralizing antibodies has been suggested in preventing disease progression in COVID-19. In this study, we investigated the relationship between clinical donor characteristics and neutralizing anti-SARS-CoV-2 antibodies in CCP donors. COVID-19 convalescent plasma donors were included into the study. Clinical parameters were recorded and anti-SARS-CoV-2 antibody levels (Spike Trimer, Receptor Binding Domain (RBD), S1, S2 and nucleocapsid protein) as well as ACE2 binding inhibition were measured. An ACE2 binding inhibition < 20% was defined as an inadequate neutralization capacity. Univariate and multivariable logistic regression analysis was used to detect the predictors of inadequate neutralization capacity. Ninety-one CCP donors (56 female; 61%) were analyzed. A robust correlation between all SARS-CoV-2 IgG antibodies and ACE2 binding inhibition, as well as a positive correlation between donor age, body mass index, and a negative correlation between time since symptom onset and antibody levels were found. We identified time since symptom onset, normal body mass index (BMI), and the absence of high fever as independent predictors of inadequate neutralization capacity. Gender, duration of symptoms, and number of symptoms were not associated with SARS-CoV-2 IgG antibody levels or neutralization. Neutralizing capacity was correlated with SARS-CoV-2 IgG antibodies and associated with time since symptom onset, BMI, and fever. These clinical parameters can be easily incorporated into the preselection of CCP donors.

Platelet phosphatidylserine is the critical mediator of thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies (Ab) reactive to complexes of platelet factor 4 and heparin. Platelets (PLT) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations in this prothrombotic environment remain poorly understood. In this study, we observed that HIT patient Ab induce a new PLT population that is characterized by increased P-selectin expression and phosphatidylserine (PS) externalization. Formation of this procoagulant PLT subpopulation was dependent on engagement of PLT Fc-γ-RIIA by HIT Ab and resulted in a significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLT propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLT intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of P-selectin and PS was dissected. While inhibition of P-selectin did not affect thrombus formation, the specific blockade of PS prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLT are critical mediators of prothrombotic conditions in HIT. Specific PS targeting could be a promising therapeutic approach to prevent thromboembolic events in HIT patients.

Diagnostic Performance of a Particle Gel Immunoassay in Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious complication of adenoviral vector-based COVID-19 vaccines. Similar to heparin-induced thrombocytopenia (HIT), antibodies reacting to platelet factor 4 (PF4) are responsible for platelet activation in VITT. The diagnosis of VITT includes the detection of anti-PF4 antibodies. Particle gel immunoassay (PaGIA) is one of the rapid immunoassays that is commonly used in the diagnosis of HIT to detect anti-PF4 antibodies. The aim of this study was to investigate the diagnostic performance of PaGIA in patients suspected of VITT. In this retrospective, single-center study, the correlation between PaGIA, enzyme immunoassay (EIA), and modified heparin-induced platelet aggregation assay (HIPA) in patients with findings suggestive of VITT was investigated. A commercially available PF4 rapid immunoassay (ID PaGIA H/PF4, Bio-Rad-DiaMed GmbH, Switzerland) and an anti-PF4/heparin EIA (ZYMUTEST HIA IgG, Hyphen Biomed) were used according to manufacturer's instructions. Modified HIPA was accepted as the gold standard test. Between March 8 and November 19, 2021, a total of 34 samples from clinically well-characterized patients (14 males, 20 females, mean age: 48.2 ± 18.2 years) were analyzed with PaGIA, EIA, and modified HIPA. VITT was diagnosed in 15 patients. Sensitivity and specificity of PaGIA were 54 and 67%, respectively. Anti-PF4/heparin optical density values were not significantly different between PaGIA positive and negative samples (p = 0.586). The sensitivity and specificity of EIA, on the other hand, were 87 and 100%, respectively. In conclusion, PaGIA is not reliable in the diagnosis of VITT because of its low sensitivity and specificity.

Antibody Binding and Angiotensin-Converting Enzyme 2 Binding Inhibition Is Significantly Reduced for Both the BA.1 and BA.2 Omicron Variants.

BACKGROUND: The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.2), which currently represent the majority of global infections. Initial studies of the neutralizing response toward BA.1 in convalescent and vaccinated individuals showed a substantial reduction. METHODS: We assessed antibody (immunoglobulin G [IgG]) binding, ACE2 (angiotensin-converting enzyme 2) binding inhibition, and IgG binding dynamics for the Omicron BA.1 and BA.2 variants compared to a panel of VOCs/variants of interest, in a large cohort (N = 352) of convalescent, vaccinated, and infected and subsequently vaccinated individuals. RESULTS: While Omicron was capable of efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to wild type. Whereas BA.1 exhibited less IgG binding compared to BA.2, BA.2 showed reduced inhibition of ACE2 binding. Among vaccinated samples, antibody binding to Omicron only improved after administration of a third dose. CONCLUSIONS: Omicron BA.1 and BA.2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind to Omicron. The extent of the mutations within both variants prevents a strong inhibitory binding response. As a result, both Omicron variants are able to evade control by preexisting antibodies.

Platelets and Sera from Donors of Convalescent Plasma after Mild COVID-19 Show No Procoagulant Phenotype.

Coronavirus disease-2019 (COVID-19) is associated with increased thromboembolic complications. Long-term alteration in the coagulation system after acute COVID-19 infection is still a subject of research. Furthermore, the effect of sera from convalescent subjects on platelets is not known. In this study, we investigated platelet phenotype, coagulation, and fibrinolysis in COVID-19 convalescent plasma (CCP) donors and analyzed convalescent sera-induced effects on platelets. We investigated CCP donors who had a history of mild COVID-19 infection and donors who did not have COVID-19 were used as controls. We analyzed phosphatidylserine (PS) externalization, CD62p expression, and glycoprotein VI (GPVI) shedding both in platelet-rich plasma (PRP) and after incubation of washed healthy platelets with donors' sera using flow cytometry. Coagulation and fibrinolysis systems were assessed with thromboelastometry. Forty-seven CCP donors (22 males, 25 females; mean age (±SD): 41.4 ± 13.7 years) with a history of mild COVID-19 infection were included. Median duration after acute COVID-19 infection was 97 days (range, 34-401). We did not find an increased PS externalization, CD62p expression, or GPVI shedding in platelets from CCP donors. Sera from CCP donors did not induce PS externalization or GPVI shedding in healthy platelets. Sera-induced CD62p expression was slightly, albeit statistically significantly, lower in CCP donors than in plasma donors without a history of COVID-19. One patient showed increased maximum clot firmness and prolonged lysis time in thromboelastometry. Our findings suggest that procoagulant platelet phenotype is not present after mild COVID-19. Furthermore, CCP sera do not affect the activation status of platelets.

Increased plasma level of soluble P-selectin in non-hospitalized COVID-19 convalescent donors.

BACKGROUND: The coronavirus disease-2019 (COVID-19) is a systemic disease with severe implications on the vascular and coagulation system. A procoagulant platelet phenotype has been reported at least in the acute disease phase. Soluble P-selectin (sP-sel) in the plasma is a surrogate biomarker of platelet activation. Increased plasma levels of sP-sel have been reported in hospitalized COVID-19 patients associated with disease severity. Here, we evaluated in a longitudinal study the sP-sel plasma concentration in blood donors who previously suffered from moderate COVID-19. METHODS: 154 COVID-19 convalescent and 111 non-infected control donors were recruited for plasma donation and for participation in the CORE research trial. First donation (T1) was performed 43-378 days after COVID-19 diagnosis. From most of the donors the second (T2) plasma donation including blood sampling was obtained after a time period of 21-74 days and the third (T3) donation after additional 22-78 days. Baseline characteristics including COVID-19 symptoms of the donors were recorded based on a questionnaire. Platelet function was measured at T1 by flow cytometry and light transmission aggregometry in a representative subgroup of 25 COVID-19 convalescent and 28 control donors. The sP-sel plasma concentration was determined in a total of 704 samples by using a commercial ELISA. RESULTS: In vitro platelet function was comparable in COVID-19 convalescent and control donors at T1. Plasma samples from COVID-19 convalescent donors revealed a significantly higher sP-sel level compared to controls at T1 (1.05 ± 0.42 ng/mL vs. 0.81 ± 0.30 ng/mL; p < 0.0001) and T2 (0.96 ± 0.39 ng/mL vs. 0.83 ± 0.38 ng/mL; p = 0.0098). At T3 the sP-sel plasma level was comparable in both study groups. Most of the COVID-19 convalescent donors showed a continuous decrease of sP-sel from T1 to T3. CONCLUSION: Increased sP-sel plasma concentration as a marker for platelet or endothelial activation could be demonstrated even weeks after moderate COVID-19, whereas, in vitro platelet function was comparable with non-infected controls. We conclude that COVID-19 and additional individual factors could lead to an increase of the sP-sel plasma level.

Treatment of drug-induced immune thrombocytopenias.

Several therapeutic agents can cause thrombocytopenia by either immune-mediated or non-immune-mediated mechanisms. Non-immune-mediated thrombocytopenia is due to direct toxicity of drug molecules to platelets or megakaryocytes. Immune-mediated thrombocytopenia, on the other hand, involves the formation of antibodies that react to platelet-specific glycoprotein complexes, as in classic drug-induced immune thrombocytopenia (DITP), or to platelet factor 4, as in heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT). Clinical signs include a rapid drop in platelet count, bleeding or thrombosis. Since the patient's condition can deteriorate rapidly, prompt diagnosis and management are critical. However, the necessary diagnostic tests are only available in specialized laboratories. Therefore, the most demanding step in treatment is to identify the agent responsible for thrombocytopenia, which often proves difficult because many patients are taking multiple medications and have comorbidities that can themselves also cause thrombocytopenia. While DITP is commonly associated with an increased risk of bleeding, HIT and VITT have a high mortality rate due to the high incidence of thromboembolic complications. A structured approach to drug-associated thrombocytopenia/thrombosis can lead to successful treatment and a lower mortality rate. In addition to describing the treatment of DITP, HIT, VITT, and vaccine-associated immune thrombocytopenia, this review also provides the pathophysiological and clinical information necessary for correct patient management.

The interaction between anti-PF4 antibodies and anticoagulants in vaccine-induced thrombotic thrombocytopenia.

Life-threatening thrombotic events at unusual sites have been reported after vector-based vaccinations against severe acute respiratory syndrome coronavirus 2. This phenomenon is now termed vaccine-induced immune thrombotic thrombocytopenia (VITT). The pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT) and is associated with platelet-activating antibodies (Abs) against platelet factor 4 (PF4). Therefore, current guidelines suggest nonheparin anticoagulants to treat VITT patients. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux, and argatroban with VITT-Ab/PF4 complexes using an ex vivo model for thrombus formation as well as in vitro assays to analyze Ab binding and platelet activation. We found that immunoglobulin Gs (IgGs) from VITT patients induce increased adherent platelets/thrombus formation in comparison with IgGs from healthy controls. In this ex vivo flow-based model, the procoagulant activity of VITT IgGs was effectively inhibited with danaparoid and argatroban but also by heparin. Interestingly, heparin and danaparoid not only inhibited IgG binding to PF4 but were also able to effectively dissociate the preformed PF4/IgG complexes. Fondaparinux reduced the in vitro generation of procoagulant platelets and thrombus formation; however, it did not affect platelet aggregation. In contrast, argatroban showed no effect on procoagulant platelets and aggregation but significantly inhibited VITT-mediated thrombus formation. Taken together, our data indicate that negatively charged anticoagulants can disrupt VITT-Ab/PF4 interactions, which might serve as an approach to reduce Ab-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulants in VITT patients could be made.

Assessment and Monitoring of Coagulation in Patients with COVID-19: A Review of Current Literature.

Coagulation abnormalities are common in patients with COVID-19 and associated with high morbidity and mortality. It became a daily challenge to navigate through these abnormal laboratory findings and deliver the best possible treatment to the patients. The unique character of COVID-19-induced coagulopathy necessitates not only a dynamic follow-up of the patients in terms of hemostatic findings but also the introduction of new diagnostic methods to determine the overall function of the coagulation system in real time. After the recognition of the high risk of thromboembolism in COVID-19, several professional societies published their recommendations regarding anticoagulation in patients with COVID-19. This review summarizes common hemostatic findings in COVID-19 patients and presents the societal recommendations regarding the use of coagulation laboratory findings in clinical decision-making. Although several studies have investigated coagulation parameters in patients with COVID-19, the methodological shortcomings of published studies as well as the differences in employed anticoagulation regimens that have changed over time, depending on national and international guidelines, limit the applicability of these findings in other clinical settings. Accordingly, evidence-based recommendations for diagnostics during acute COVID-19 infection are still lacking. Future studies should verify the role of coagulation parameters as well as viscoelastic methods in the management of patients with COVID-19.

Immune-Mediated Platelet Activation in COVID-19 and Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Both qualitative and quantitative platelet abnormalities are common in patients with coronavirus disease 2019 (COVID-19) and they correlate with clinical severity and mortality. Activated platelets contribute to the prothrombotic state in COVID-19 patients. Several groups have shown immune-mediated activation of platelets in critically ill COVID-19 patients. Vaccine-induced immune thrombotic thrombocytopenia is an autoimmune condition characterized by thrombocytopenia and life-threatening thrombotic events in the arterial and venous circulation. Although the initial trigger has yet to be determined, activation of platelets by immune complexes through Fc gamma RIIA results in platelet consumption and thrombosis. A better understanding of platelet activation in COVID-19 as well as in vaccine-induced thrombotic complications will have therapeutic implications. In this review, we focused on the role of immune-mediated platelet activation in thrombotic complications during COVID-19 infection and vaccine-induced immune thrombotic thrombocytopenia.

Diagnosis of Platelet Function Disorders: A Challenge for Laboratories.

In patients with normal plasmatic coagulation and bleeding tendency, platelet function defect can be assumed. Congenital platelet function defects are rare. Much more commonly they are acquired. The clinical bleeding tendency of platelet function defects is heterogeneous, which makes diagnostic approaches difficult. During the years, a large variety of tests for morphological phenotyping and functional analysis have been developed. The diagnosis of platelet function defects is based on standardized bleeding assessment tools followed by a profound morphological evaluation of the platelets. Platelet function assays like light transmission aggregation, luminoaggregometry, and impedance aggregometry followed by flow cytometry are commonly used to establish the diagnosis in these patients. Nevertheless, despite great efforts, standardization of these tests is poor and in most cases, quality control is lacking. In addition, these tests are still limited to specialized laboratories. This review summarizes the approaches to morphologic phenotyping and platelet testing in patients with suspected platelet dysfunction, beginning with a standardized bleeding score and ending with flow cytometry testing. The diagnosis of a functional defect requires a good collaboration between the laboratory and the clinician.

Vaccine Side Effects in Health Care Workers after Vaccination against SARS-CoV-2: Data from TüSeRe:exact Study.

As the Corona Disease 2019 (COVID-19) caused by SARS-CoV-2 persists, vaccination is one of the key measures to contain the spread. Side effects (SE) from vaccination are one of the reasons for reluctance to vaccinate. We systematically investigated self-reported SE after the first, second, and booster vaccinations. The data were collected during the TüSeRe: exact study (Tübinger Monitoring Studie zur exakten Analyse der Immunantwort nach Vakzinierung). Employees of health and research institutions were invited to participate. Study participants were asked to fill out an online questionnaire and report their SE after each dose of SARS-CoV-2 vaccination. A total of 1046 participants (mean age: 44 ± 12.9 years; female, n = 815 (78%); male, n = 231 (22%)) were included in the analysis. Local and systemic SE were more frequent after receiving the vector-based vaccine ChAdOx1 nCoV-19 in the first vaccination. However, local and systemic SE were more common after receiving mRNA vaccines (BNT162b2, mRNA-1273) in the second vaccination. Compared to the BNT162b2 vaccine, more SE have been observed after receiving the mRNA-1273 vaccine in the booster vaccination. In multivariate analysis, local and systemic side effects were associated with vaccine type, age and gender. Local and systemic SE are common after SARS-CoV-2 vaccines. The frequency of self-reported local and systemic SE differ significantly between mRNA and vector-based vaccines.