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

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

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9 Citations (Scopus)


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
Issue number11
Publication statusPublished - 14 Jun 2022

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