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

Research output: Contribution to journalArticlepeer-review


Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti-PF4 antibodies that activate platelets via the FcγRIIa receptor 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 (PAR1) and ITAM-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 presence of a PAR1 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 ChAdOx1 nCov-19 vaccine without VITT (n=24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified by clinic-pathological adjudication, procoagulant flow cytometry identified 93% of VITT cases including ELISA-negative and SRA-negative patients. The in vitro effect of 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.

Original languageEnglish
JournalBlood Advances
Publication statusE-pub ahead of print - 31 Mar 2022


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