Accuracy of the hyphen Zymutest IgG ELISA assay for anti-platelet factor 4 antibody detection after ChAdOx1 nCov-19 vaccination in diagnosing vaccine-induced immune thrombotic thrombocytopenia (VITT)

ABSTRACT

Background: Vaccine-induced Immune Thrombotic Thrombocytopenia (VITT) is a rare, anti-platelet factor 4 (PF4) antibody-mediated prothrombotic syndrome associated with the AstraZeneca (AZ) Covid-19 vaccination. Diagnostic criteria include thrombosis, thrombocytopenia, appropriate timing post AZ vaccine, and demonstration of an anti-PF4 antibody by enzyme-linked (Table Presented) immunosorbent assay (ELISA). ELISA methods have varying sensitivity and specificity for detecting anti-PF4 antibodies, therefore, functional assays (modified serotonin release and flow cytometry) assist in diagnosis. In Australia, the primary assay for detection of anti-PF4 antibodies is the Asserachrom HPIA IgG ELISA. Aim(s) To assess the accuracy of an alternate ELISA method for suspected VITT compared to the gold standard (clinicopathological diagnosis). Method(s) 96 stored frozen samples from patients with suspected VITT, previously analysed by Asserachrom HPIA IgG ELISA (Stago, Melbourne, Australia), were tested using the Hyphen Biomed Zymutest HIA IgG ELISA (Haematex Research, Sydney, Australia). These patients had confirmed thrombosis within 42 days post first dose of AZ vaccine. Cases were classified as 'serologically confirmed VITT' if clinicopathological criteria were met, and initial ELISA testing and/or functional assays were positive. Results were interpreted as positive or negative for anti-PF4 antibodies using the manufacturer's cut-offs derived for the diagnosis of Heparin-Induced Thrombocytopenia. Result(s) 96 patient samples were tested, including 35 classified as VITT (Table 1). The Hyphen assay had a moderate sensitivity (82%), including 2 probable false negatives on initial testing which were strongly positive by Hyphen assay. The specificity was 93%, including 5 probable false positives on initial ELISA which were negative by Hyphen assay (Table 2). Conclusion(s) The Hyphen ELISA is suitably accurate for the diagnosis of VITT. These data highlight the limitations of using a single ELISA testing method for anti-PF4 antibody detection. In cases with high clinical suspicion, a second ELISA method or functional assay should be considered.