Developing an assay to distinguish between HIT and VITT antibodies

Introduction Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare, but severe side effect after Covid-19 and other vaccinations. First cases of VITT-mimicking antibodies in unvaccinated patients with recurrent thrombosis have been described. Differentiation between heparin-induced thrombocytopenia (HIT) and VITT is difficult in some patients. Widely used enzyme-linked immunoassays (EIA) cannot differentiate between the two, some of them even fail to detect VITT antibodies. So far, differentiation between HIT-like and VITT-like anti-PF4 antibodies can only be performed in specialized laboratories by functional tests using the heparin-induced platelet activation (HIPA) or PF4-induced platelet activation (PIPA) test. We have developed an assay, which can distinguish between HIT and VITT antibodies and can be used in any hospital laboratory.

Method Confirming platelet-activation assays (HIPA and PIPA) were performed as described.[1] We defined 3 cohorts: 1) Negative controls (n=112, including 35 healthy donors from before 2020, 46 clinical patients suspected for HIT but with negative EIA and HIPA and 31 non-thrombotic patients); 2) classical HIT-patients with positive EIA and HIPA (n=121); 3) typical VITT patients (n=63; presenting after vaccination with adenoviral vector-based Covid-19 vaccine and positive EIA and PIPA). Samples were analyzed by an automated coagulation analyzer ACL AcuStar (Werfen / IL Inc., Bedford, MA, USA) using HemosIL AcuStar HIT-IgG(PF4-H) and a prototype of VITT-IgG(PF4) assay according to the manufacturer’s protocol. For both assays, raw data was analyzed as relative light units (RLU).

Results All VITT samples were positive in the prototype VITT-assay ([Fig. 1]); only a few (n=9; 14.3%) also showed weakly positive results in the HIT-assay. On the other hand, most of the HIT samples showed positive results in the HIT-assay (113; 93.4%), 34 of them (30.1%) also reacted positive in the prototype VITT-assay (12 of them strongly; 10.6%), and three demonstrated an antibody pattern like autoimmune VITT. Negative control samples where all non-reactive in the HIT-assay and served to adjust the cutoff for the prototype VITT-assay.

Fig. 1 Results of the HemosIL AcuStar HIT-IgG(PF4-H) and of the prototype VITT assay; Results of the HemosIL AcuStar HIT-IgG(PF4-H) are given on the x-axis (cutoff: dashed vertical line). Results of the prototype VITT assay are given on the y-axis (cutoff: dashed horizontal line). HIT sera are shown in red, VITT sera in blue, and controls in grey symbols.

Conclusion The different reaction pattern of samples of HIT and VITT patients using HemosIL AcuStar HIT-IgG(PF4-H) and a VITT prototype assay was able to distinguish between the two antibody entities for the first time. The combination of assays can facilitate a rapid decision whether heparin may be used for treatment and also identify patients with autoimmune-VITT as a cause of recurrent thrombosis.

Publication History

Article published online:
20 February 2023

• References

• 1 Greinacher A. et al Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination. N Engl J Med 2021; 384: 2092-2101