title: Combined use of the high heparin step and optical density to optimize diagnostic sensitivity and specificity of an anti-PF4/heparin enzyme-immunoassay.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Ernst-Moritz-Arndt Universität Greifswald, Germany.
authors.forename: Karina
authors.initials: K
authors.lastname: Althaus
authors.forename: Ulrike
authors.initials: U
authors.lastname: Strobel
authors.forename: Theodore E
authors.initials: TE
authors.lastname: Warkentin
authors.forename: Andreas
authors.initials: A
authors.lastname: Greinacher
mesh_terms: D000918:Antibody Specificity; D003720:Densitometry / Q000379:methods; D006493:Heparin / Q000097:blood / Q000276:immunology*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods* / Q000592:standards; D007074:Immunoglobulin G / Q000097:blood* / Q000276:immunology; D010865:Pilot Projects; D010978:Platelet Factor 4 / Q000097:blood / Q000276:immunology*; D012680:Sensitivity and Specificity; D013921:Thrombocytopenia / Q000097:blood / Q000175:diagnosis* / Q000276:immunology
chemical_list: D007074:Immunoglobulin G; D010978:Platelet Factor 4; D006493:Heparin