Characterization of monoclonal and patient-derived antiplatelet factor 4 antibodies in platelet factor 4 and platelet factor 4/polyanion chemiluminescence assays

BACKGROUND: Heparin-induced thrombocytopenia, vaccine-induced immune thrombocytopenia and thrombosis (VITT), and VITT-like disorders are caused by antiplatelet factor 4 (PF4)/polyanion and/or anti-PF4 immunoglobulin G antibodies. The rapid anti-PF4/polyanion and anti-PF4 chemiluminescence assays differentiate between typical heparin-induced thrombocytopenia (anti-PF4/heparin) and VITT antibodies (anti-PF4). OBJECTIVES: To characterize monoclonal antibodies (mAbs) and recombinant anti-PF4 antibodies in the 2 chemiluminescence assays specific for anti-PF4/polyanion and anti-PF4 antibodies, respectively, and to characterize anti-PF4/polyanion antibodies in patients whose sera tested positive in both assays. METHODS: For anti-PF4/heparin and anti-PF4 antibodies, the chimeric mAbs 5B9, 1C12, and 1E12 were generated using standard technology. Recombinant antibodies from VITT and VITT-like patients were generated based on the amino acid sequences of affinity-purified patient anti-PF4 antibodies. KKO was humanized by cloning its Fab sequences and expressing them as a chimeric recombinant human immunoglobulin G1 antibody. Anti-PF4 antibodies from patient sera testing positive in both chemiluminescence assays were affinity-purified using PF4/polyanion and PF4 beads. RESULTS: In chemiluminescence assays, 5B9, humanized KKO, 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 contained antibodies that showed similar reactivity in both assays, regardless of whether purified with PF4/polyanion or PF4 beads, suggesting cross-reactive antibodies, while sera from other patients contained both anti-PF4/heparin and anti-PF4 antibodies. CONCLUSION: The different reactivities of anti-PF4 mAbs/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 2 chemiluminescence assays.