Heparin-induced thrombocytopenia (HIT), vaccine-induced immune thrombocytopenia and thrombosis (VITT), and VITT-like disorders are caused by anti-platelet factor 4 (PF4)/polyanion and/or anti-PF4 IgG antibodies. The rapid anti-PF4/polyanion and anti-PF4 chemiluminescence assays differentiate between typical HIT (anti-PF4/heparin) and VITT antibodies (anti-PF4 antibodies).To characterize monoclonal antibodies(moAbs) and recombinant anti-PF4 antibodies in the two chemiluminescence assays specific for anti-PF4/polyanion and anti-PF4 antibodies, respectively; and to characterize the anti-PF4(/polyanion) antibodies from patients whose sera tested positive in both assays.Anti-PF4/heparin and anti-PF4 antibodies: The chimeric moAbs 5B9, 1C12, and 1E12 were generated by standard technology. Recombinant antibodies from VITT and VITT-like patients were generated based on the amino acid sequence of affinity purified patient anti-PF4 antibodies. KKO was humanized (hKKO) by cloning its Fab sequences and expressing it as chimeric recombinant human IgG1 antibody. Anti-PF4 antibodies from patient sera testing positive in both chemiluminescence assays were affinity purified using PF4/polyanion and PF4 beads, respectively.In chemiluminescence assays, 5B9, hKKO, 1C12 and 1E12 bound to PF4/polyanion complexes but differed largely in their PF4-reactivity. VITT(-like) recombinant antibodies bound only to PF4. Some patients' sera contain antibodies that showed similar reactivity in both assays, regardless, whether purified by PF4/polyanion or PF4 beads, suggesting cross-reactive antibodies, while sera of other patients contained both anti-PF4/heparin and anti-PF4 antibodies.The different reactivities of monoclonal anti-PF4/heparin antibodies need to be considered when planning experiments and interpreting results. Patients may have very different antibody reactivities despite giving the same results in the two chemiluminescence assays.