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

Blood Adv. 2022 Jun 14;6(11):3494-3506. doi: 10.1182/bloodadvances.2021006698.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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


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