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

Christine Shu Mei 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

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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 FcgRIIa 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 (FcgRIIa) 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 5 23), but not healthy donors (n 5 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n 5 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 FcgRIIa-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.

Original languageEnglish
Pages (from-to)3494-3506
Number of pages13
JournalBlood Advances
Volume6
Issue number11
Early online date10 Jun 2022
DOIs
Publication statusPublished - 14 Jun 2022

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