Dilution‐Endpoint Comparison of a Flow Cytometric Platelet Activation Assay With <scp>PF4</scp> /Heparin <scp>ELISA</scp> and a Lateral Flow Immunoassay in Suspected <scp>HIT</scp>

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is a rare but potentially life-threatening, immune-mediated adverse reaction to heparin. PF4-dependent immunoassays are sensitive screening tests, but they detect both platelet-activating and nonactivating antibodies; therefore, positive results require interpretation in clinical context and, when available, confirmation with a functional platelet activation assay. METHODS: We compared dilution endpoints of an in-house flow cytometric platelet activation assay with an IgG-specific PF4/polyanion ELISA and a PF4-dependent lateral flow immunoassay (LFA). Six consecutive ELISA-positive patient sera and one external ELISA-positive control serum were tested neat and in serial twofold dilutions (up to 1:2048) in parallel with all three methods. Platelet activation was assessed using platelet-rich plasma from four healthy donors in the presence of low and high doses of heparin and quantified by CD62P expression on CD61-positive platelets. RESULTS: Five patient sera and the external control showed heparin-dependent platelet activation, whereas one ELISA-positive serum was nonactivating. All three assays were concordantly positive through dilutions up to 1:32 in platelet-activating samples. At higher dilutions, the LFA remained positive more often than the ELISA or the flow cytometric assay. Across 72 dilution points from platelet-activating sera and the external control, the ELISA was positive in 47/72, the flow assay in 51/72, and the LFA in 67/72. In several sera, mild dilution increased the ELISA signal and/or platelet activation, consistent with a prozone-like stoichiometric effect. CONCLUSION: In this pilot dilution-series study, the in-house flow cytometric assay showed dilution endpoints broadly comparable to the IgG-specific ELISA for platelet-activating anti-PF4/heparin antibodies. The LFA remained positive at lower apparent antibody levels than either the ELISA or the functional assay, but also gave a positive result in the serum that did not contain platelet-activating anti-PF4/heparin antibodies.