A novel flow cytometry procoagulant assay for diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Abstract

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti-platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcγRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein-coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine-based activation motif-linked receptors (FcγRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry-based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n = 23), but not healthy donors (n = 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n = 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay-negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcγRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.

• Authors: Christine S M Lee, Hai Po Helena Liang, David E Connor, Agnibesh Dey, Ibrahim Tohidi-Esfahani, Heather Campbell, Shane Whittaker, David Capraro, Emmanuel J Favaloro, Dea Donikian, Mayuko Kondo, Sarah M Hicks, Philip Y-I Choi, Elizabeth E Gardiner, Lisa Joanne Clarke, Huyen Tran, Freda H Passam, Timothy Andrew Brighton, Vivien M Chen
• Journal: Blood advances (Blood Adv) Vol. 6 Issue 11 Pg. 3494-3506 (06 14 2022) ISSN: 2473-9537 [Electronic] United States
• PMID: 35359002 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

Chemical References

• Immunoglobulins, Intravenous
• Receptors, Proteinase-Activated
• Platelet Factor 4
• Heparin
• ChAdOx1 nCoV-19

Topics

• ChAdOx1 nCoV-19
• Flow Cytometry
• Heparin (therapeutic use)
• Humans
• Immunoglobulins, Intravenous (adverse effects)
• Platelet Factor 4
• Purpura, Thrombocytopenic, Idiopathic (drug therapy)
• Receptors, Proteinase-Activated (therapeutic use)
• Thrombocytopenia (diagnosis)
• Thrombosis (drug therapy)