Laboratory approach for vaccine-induced thrombotic thrombocytopenia (VITT) diagnostics: A retrospective study of clinically suspected cases in the Netherlands

ABSTRACT

Background: Vaccines have been an important strategy to control the SARS-CoV-2 pandemic. In The Netherlands two mRNA vaccines (BTN162b2, Pfizer-BioNTech;mRNA-1273, Moderna), and two adenovirus vector-based vaccines (ChAdOx1 nCoV-19, AstraZeneca;Ad26. COV2.S, Johnson & Johnson/Janssen) have been administered. In 2021, vaccination with the ChAdOx1 nCoV-19 was ceased in The Netherlands and other European countries due to the occurrence of thrombocytopenia and thromboembolic events. This new phenomenon was termed vaccine-induced thrombotic thrombocytopenia (VITT) and was clinically associated with thrombocytopenia and thrombosis at unusual sites, in particular cerebral venous sinus thrombosis (CVST). In addition, VITT was characterized by the presence of IgG-antibodies directed against platelet factor 4 (PF4). As PF4 appears to play a central role in the pathophysiology of VITT, it is recommended that the role of PF4 should be taken into account for VITT diagnostic testing. Aims: To characterize and define the patient population of clinically suspected VITT cases in The Netherlands from a diagnostics perspective. Methods: We describe a cohort of 283 clinically suspected VITT patients. We assessed these patients based on their clinical presentation and using an anti-PF4 IgG ELISA and a functional PF4-dependent platelet activation assay. Results: We found that when the 283 clinically suspected VITT patients were analysed in the anti-PF4 IgG ELISA 24 (8.5%) tested positive, 248 (87.6%) tested negative, and 11 (3.9%) tested weak positive. Out of the 24 patients that tested positive in the anti-PF4 IgG ELISA, 19 (79.2%) patients also demonstrated increased PF4-dependent platelet activation. Furthermore, we observed that platelet activation was inhibited by excess levels of heparin and by a FcγRIIa-blocking antibody, indicating a role for platelet-FcγRIIa in the pathophysiology of VITT. Remarkably, we found that patients vaccinated with mRNA vaccines BTN162b2 (N = 84) and mRNA-1273 (N = 36), did not test positive in the anti-PF4 IgG ELISA. Based on these test results 19 patients (11 women) were eventually diagnosed with probable VITT, of which 13 presented with both thrombocytopenia and thrombosis and three suffered from CVST. Strikingly, discrepancies in test results were also present, including nine patients with low levels of anti-PF4 IgG but with increased PF4-dependent platelet activation, and two patients with high levels of anti-PF4 IgG but without any PF4-dependent platelet activation. Summary/Conclusions: VITT is a rare but serious complication of SARS-CoV-2 virus vaccination, particularly due to adenovirus vectorbased vaccines. Our results underline the importance of using the clinical presentation, in combination with the anti-PF4 IgG ELISA and the PF4-dependent platelet activation assay for VITT diagnostics. Discrepancies in test results, however, proved it difficult to unequivocally diagnose VITT. Therefore, it will be essential to obtain more insights into the pathophysiology of VITT in order to improve the diagnostic accuracy and identify preventive and therapeutic approaches.