Heparin-induced thrombocytopenia: detection of antiheparin/PF4 antibodies by means of heparin/FP4-coated beads and flow cytometry

A total of 70 serum samples from heparin-induced thrombocytopenia (HIT II) patients, non-HIT patients and healthy subjects, respectively, were studied for the presence of antiheparin/PF4 antibodies. Two enzyme-linked immunosorbent assay (ELISA) assays were compared with the particle gel immunoassay (PaGIA). Beads of the PaGIA kit were also used to evaluate the feasibility of flow cytometric detection of antiheparin/PF4 antibodies in patient samples. Experiments have shown that all samples found positive by ELISA and PaGIA, were also positive when analysed by flow cytometry by an indirect test using the high-density particles coated with heparin/PF4 complexes and a second step fluorescein isothiocyanate (FITC) antihuman immunoglobulin (Ig)G reagent. The procedure was easy to perform, repetitive and beads were promptly visualized by physical parameters, with a very low background. In conclusion, the results of this study suggest that flow cytometry is a reliable method for the detection of antiheparin/PF4 antibodies.