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

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.