Combined use of the high heparin step and optical density to optimize diagnostic sensitivity and specificity of an anti-PF4/heparin enzyme-immunoassay

Background: IgG-specific anti-PF4/heparin enzyme-immunoassays (EIAs) are sensitive but not specific for platelet-activating antibodies, the cause of heparin-induced thrombocytopenia (HIT). Two features of EIA reactivity predict for presence of HIT antibodies - the magnitude of a positive result (in optical density [OD] units) and the inhibition of reactivity at high heparin concentrations - but their combined utility remains uncertain. Objective: To determine for an IgG-specific EIA how the OD values of a positive reaction and its inhibition by high heparin can be optimally combined. Methods: We screened 1,000 consecutive patients with suspected HIT using an IgG-specific PF4/heparin in-house EIA with and without high heparin (100 IU/mL); and by the heparin-induced platelet activation test. Results: Platelet-activating antibodies were rarely detected (<0.2%) when the IgG-specific EIA was negative at the conventional cut-off (OD, 0.5). However, an OD cut-off of 1.0 resulted in an unacceptable loss of sensitivity (14/83 = 17%) for detecting platelet-activating antibodies. The high heparin step increased specificity for platelet-activating antibodies from 72% to 89% without loss of sensitivity when applied to weak-positive sera (OD <= 1.0). However, decreased sensitivity was observed with strong-positive sera (OD > 1.0): 11/69 such sera (16%) that did not show >40% inhibition by high heparin nevertheless contained platelet-activating antibodies. Conclusion: Specificity of an IgG-specific EIA for detecting platelet-activating antibodies can be optimized by applying the high heparin inhibition step to weak-positive reactions (0.5- <= 1.0 OD). However, applying the high heparin inhibition step to strong-positive reactions (>1.0 OD) in our in-house assay risks falsely classifying a serum as negative for platelet-activating antibodies. (C) 2011 Elsevier Ltd. All rights reserved.