Detection of Platelet-Activating Antibodies Necessary to Diagnose HIT in Patients With COVID-19

While high titer anti-PF4/heparin antibodies can often confirm a diagnosis of heparin-induced thrombocytopenia (HIT) and predict a positive platelet activation test in most patients, this does not appear to be the case in patients with COVID-19, according to a new study. Findings from this report, which was published in the Journal of Thrombosis and Haemostasis, indicate that clinicians should confirm HIT suspicion in patients with COVID-19 who present with strong reactivity in PF4/heparin antigen tests without platelet-activating antibodies, as this could help avoid overtreatment with non-heparin anticoagulants.

Heparin represents a cornerstone of treatment in critically ill patients with COVID-19 who enter a prothrombotic state, explained the authors, led by Justine Brodard, MSc, from Bern University Hospital in Switzerland. Previous reports indicate HIT usually occurs between day five and 14 of treatment with heparin, but diagnosis of this severe adverse reaction is often challenging to establish in critically ill patients. The diagnosis of HIT is typically based on both clinical criteria and laboratory tests, but current knowledge of how this diagnostic approach generalizes to severely ill patients with COVID-19 remains unknown.

With this study, the researchers tested serum samples from 12 patients with COVID-19 and suspected HIT for anti-PF4/heparin antibodies. They also performed heparin-induced platelet activation (HIPA) tests, during which washed platelets of healthy donors were incubated within patient serum in the presence of buffer, low-molecular-weight heparin, reviparin 0.2 aFXaU, and unfractionated heparin 100 units. The investigators visually assessed platelet aggregation every five minutes.

In addition, the serum samples of the patients and a healthy control were spiked with monoclonal antibody 5B9 before being assessed in the HIPA test. The antibody features a human Fc fragment that recognizes PF4/heparin complexes and activates platelets in the presence of low heparin/reviparin concentrations. This action mimics a typical human HIT antibody.

The observation period lasted from March to April 2020. A total of three samples tested negative in all tests, while nine samples tested positive by the antigen test. Only three of the nine positive samples returned a positive result with the functional test.

HIT prevalence could be higher in severely ill patients with COVID-19 receiving unfractionated heparin versus other patients in the ICU.

Next, the investigators purified the IgG fraction of the serum samples that were strongly positive with the antigen tests but negative with the HIPA test. They found very weak to no reactivity of the IgG fraction at up to 30 minutes in the four test cells per sample when tested with the HIPA. “We did not consider these reactions as reflecting the presence of typical HIT antibodies as purified IgG fractions may contain some aggregated IgG, which can alter the HIPA test,” the authors noted.

The researchers suggest that these findings indicate patients with COVID-19 have a different reactivity pattern in HIT tests compared with patients without COVID-19. While patients without COVID-19 may demonstrate platelet-activating anti-PF4/heparin antibodies, those with COVID-19 do not, even if they have strongly positive antigen tests.

“It is unlikely that this is caused by an inhibitory factor present in the serum of COVID-19 patients,” they remarked. “An alternative explanation might be that sera which test only positive in the functional test after addition of PF4 are prevalent in COVID-19 patients at an unusually high rate. Although we have not tested this, we regard this explanation as very unlikely.”

Limitations of this study included its small sample size, observational nature, and narrow enrollment window.

In addition, the investigators suggest that HIT prevalence could be higher in severely ill patients with COVID-19 receiving unfractionated heparin versus other patients in the intensive care unit. Therefore, they added, HIT should “be considered as an important differential diagnosis in patients with a rapid decrease in platelet count associated with new thrombotic complications.”

Study authors report no relevant conflicts of interest.


Brodard J, Kremer Hovinga JA, Fontana P, et al. COVID-19 patients often show high-titer non-platelet-activating anti-PF4/heparin IgG antibodies. J Thromb Haemost. 2021;19(5):1294-1298.