Heparin-induced thrombocytopenia (HIT) is a dangerous complication of heparin therapy. HIT diagnosis is established by recognizing thrombocytopenia and/or thrombosis in an affected patient and from the results of serological tests such as the platelet factor 4 (PF4)/heparin immunoassay (PF4 ELISA) and serotonin release assay (SRA). Recent studies suggest that HIT antibodies activate platelets by recognizing PF4 in a complex with platelet glycosaminoglycans (and/or polyphosphates) and that an assay based on this principle, the PF4-dependent P-selectin expression assay (PEA), may be even more accurate than the SRA for HIT diagnosis. Here, we demonstrate that the PEA detected pathogenic antibodies before the SRA became positive in two patients with HIT studied serially, in one case even before seropositivity in the PF4 ELISA. In one of the patients treated with plasma exchange, persistent dissociation between the PEA and SRA test results was observed. These results support a role for the PEA in early HIT diagnosis.