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The laboratory diagnosis of heparin-induced thrombocytopenia (HIT) is based on an enzyme immunoassay combined with a functional test, and serotonin release assay (SRA) is the gold standard for detecting activating HIT antibodies. However, a recent atypical history of HIT prompted us to evaluate whether addition of platelet factor 4 (PF4) during SRA could improve its ability to detect pathogenic HIT antibodies. Using 5B9, a monoclonal antibody to PF4/H with a human Fc fragment, we first defined the optimal PF4 concentration for detecting low amounts of platelet-activating IgG with SRA. Plasma samples from 50 patients with suspected HIT were then studied, and SRA was positive in 17 cases (Group SRA pos ), with relatively high levels of PF4-specific IgG (median optical density = 2·66). SRA was also systematically performed after adding 10 μg/ml of PF4 in the reaction mixture, and significant serotonin release was measured with samples from 9 additional patients (Group PF4-SRA pos ). Importantly, levels of PF4-specific IgG were similar in these samples and those from the 24 persistently SRA negative patients. Moreover, the pre-test probability of HIT was intermediate/high in all 'SRA pos ' or 'SRA-PF4 pos ' patients. In conclusion, addition of exogenous PF4 might improve the detection of pathogenic HIT antibodies by SRA., (© 2017 John Wiley & Sons Ltd.)