Beneficial effect of exogenous platelet factor 4 for detecting pathogenic heparin-induced thrombocytopenia antibodies

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.