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Antibody-mediated procoagulant platelets in VITT
Introduction
Testing for anti-PF4/heparin antibodies
A commercially available EIA was used in accordance with the manufacturer’s instructions (Hyphen Biomed, Neuville-sur-Oise, France). The ability of sera to activate platelets was tested using the functional assay heparin-induced platelet aggregation assay (HIPA) as previously described.6 For more details, see the Online Supplementary Appendix.
Serological characterization of PF4 antibody
Antibody binding to PF4 and the receptor binding domain of Spike protein (Spike-RBD and S2 domain) was analyzed using an in-house EIA.
Assessment of antibody-mediated procoagulant platelets
Patients’ sera were incubated with washed platelets (7.5x106) for 1.5 h* under different conditions at room temperature. Platelets were then stained with Annexin V-FITC and CD62-APC (Immunotools, Friesoythe Germany) and directly analyzed by flow cytometry (FC). For more details see the Online Supplementary Appendix.
Ethics statement
The study was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the Institutional Review Board of the University of Tuebingen (236/2021BO2, 224/2021BO2) and analysis of sera from ChAdOx1 nCoV-19 vaccinated individuals were performed at the University of Ulm (99/21).
Statistical analyses
The statistical analysis was performed using GraphPad Prism, Version 7.0 (GraphPad, La Jolla, USA). Since potential daily vari- ations in FC measurements might result in bias in data analysis, test results were normalized to two healthy donors tested in par- allel at the same time point. Data in the text are presented as median (range), mean ± standard deviation (SD) or numbers (n in %).
Data sharing statement
Data may be requested for academic collaboration from the corresponding author.
Results
Clinical and laboratory features of vaccine-induced immune thrombotic thrombocytopenia
Eight patients (five females, three males) with a median age of 41.5 years (range, 24-53) were referred with sus- pected thrombotic complications after ChAdOx1 nCoV- 19 vaccination. Demographic data are summarized in Table 1. The patients were admitted to hospital 6 to 20 days after ChAdOx1 nCoV-19 vaccination. All patients had thrombocytopenia at admission with a median platelet count of 46.5x109/L (range, 8-92). D-dimer was available in five patients, which was 9 μg/mL or higher. Thrombosis was detected in six patients at admission and two developed thrombosis during hospitalization (Figure 1). Thrombotic events included cerebral venous sinus thrombosis (five patients), pulmonary embolism (four patients), deep vein thrombosis (one patient), and throm- bosis in other organs (three patients). Three of eight patients had more than one thrombotic event. Three patients presented initially with bleeding signs with easy
COVID-19 infection has resulted in considerable mor- bidity and mortality in the last 15 months.1 Within an exceptionally short time, several SARS-CoV-2 vaccines have been licensed and used worldwide.2 Safety signals have been, however, noted. Center for Disease Control and Prevention (CDC) in the US reported in the begin- ning of March 26 cases of venous thromboembolism, 20 cases of thrombosis and 41 ischemic strokes in individu- als vaccinated with mRNA vaccines in the US. More than 200 cases with thrombosis among 34 million persons vac- cinated with ChAdOx1 nCoV-19 have been reported to the European database of suspected adverse reactions, EudraVigilance. After the investigation of reported cases, European Medical Association (EMA) found a link between ChAdOx1 nCoV-19 and unusual thrombotic events and concomitant thrombocytopenia. Although the World Health Organization (WHO) and EMA concluded that the benefit of vaccination with ChAdOx1 nCoV-19 outweighs the risks associated with thrombosis and thrombocytopenia, several countries instituted restric- tions on the use of ChAdOx1 nCoV-19. The unusual clin- ical constellation of cerebral venous sinus thrombosis (CVST) and thrombocytopenia is called vaccine-induced immune thrombotic thrombocytopenia (VITT). We stud- ied eight cases with thrombocytopenia and primarily with suspected CVST but also other thromboembolic complications to better understand the pathophysiology of VITT. In this study, we identified antibody-mediated procoagulant platelets as a novel mechanism associated with VITT.
Methods
Study cohort and evaluation of the clinical data
Eight patients were referred to different university hospitals with neurological or hematological symptoms after vaccination with ChAdOx1 nCoV-19 (AstraZeneca, London, UK) between February 1st and April 6th 2021. Six patients admitted to university hospitals and two patients, who were initially admitted to local hospitals, were later transferred to university hospitals. Medical records were used to collect treatments and outcome. Diagnosis of thromboembolic complications was made when indicated by clinical or laboratory findings and/or based on computed tomog- raphy, ultrasound imaging or in case of death by autopsy.
Blood samples were collected to exclude heparin-induced thrombocytopenia (HIT). Blood samples from non-vaccinated healthy blood donors (n=24, 17 females, mean age 36.1±13.7 years) and from healthy vaccinated before and after the first vac- cination with ChAdOx1 nCoV-19 (n=41, 29 females, mean age 37.3±10.9 years) served as healthy controls.
In addition, sera from 29 COVID-19 patients who had serial HIT immunoglobulin G (IgG)-enzyme immune assay (EIA) measurements during hospitalization were also included in the study (seven females, mean age 65.3±14.1 years). Clinical data from the ICU COVID-19 patients and a VITT-patient (case #7) were reported in previous studies.3,4
Bead-based multiplex assay for detection of COVID-19 antibodies
COVID-19 antibodies were measured with a multiplex assay (NMI, Reutlingen, Germany) with the FLEXMAP 3D® system (Luminex Corporation, Austin, USA).5
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