title: New insights in heparin-induced thrombocytopenia by the use of fluid-phase assays to detect specifically platelet factor 4/heparin complex antibodies and antibody-secreting cells.
abstract: BACKGROUND
  The key feature of heparin-induced thrombocytopenia (HIT) is the production of antibodies (Ab) against the platelet factor 4 (PF4)/heparin complex. These Ab are directed against neoepitopes of the PF4 tetramer, which are induced by the complex formation with heparin. To study this humoral immune response in greater detail, either in a murine immunization model or in human blood samples, reliable and specific immune assays to detect specifically Ab against the PF4/heparin complexes, but not PF4 alone are required.
  
  
  METHODS
  We established fluid-phase enzyme-immunoassays in which the soluble biotinylated antigen, PF4/heparin, is firstly captured by specific Ab, and secondly directly detected with enzyme-conjugated streptavidin.
  
  
  RESULTS
  The use of this fluid-phase principle allowed a higher specificity than the traditional solid-phase enzyme-immunoassays in terms of Ab binding to murine PF4/heparin compared to murine PF4 alone. This fluid-phase approach applied to the detection of specific murine PF4/heparin Ab-secreting cells (ASC) identified the spleen as the main lymphatic organ that contributes to the PF4/heparin Ab response in mice. IgG ASC specific for PF4/heparin are very transiently detectable in mice, which might explain why anti-PF4/heparin IgG Ab typically disappear within 100 days in humans. Furthermore, this fluid-phase approach was successfully transferred to detect human PF4/heparin-specific Ab.
  
  
  CONCLUSIONS
  The fluid-phase principle for the specific detection of anti-PF4/heparin IgG and IgM Ab enables new and improved assays for HIT research in men and mice. At least in mice PF4/heparin antibodies are produced by transient B cells.
authors.affiliation: Center for Innovation Competence - Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Schulze
authors.affiliation: Center for Innovation Competence - Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald, Germany.
authors.forename: Inga
authors.initials: I
authors.lastname: Jensch
authors.affiliation: Center for Innovation Competence - Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald, Germany; Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Krystin
authors.initials: K
authors.lastname: Krauel
authors.affiliation: Center for Innovation Competence - Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald, Germany.
authors.forename: Adnan
authors.initials: A
authors.lastname: Alahmad
authors.affiliation: Center for Innovation Competence - Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald, Germany.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Müller
authors.affiliation: Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Greinacher
authors.affiliation: Center for Innovation Competence - Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald, Germany. Electronic address: matthias.hundt@uni-greifswald.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Hundt
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood*; D000921:Antibody-Producing Cells / Q000378:metabolism*; D005260:Female; D006493:Heparin / Q000032:analysis* / Q000276:immunology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010978:Platelet Factor 4 / Q000032:analysis* / Q000276:immunology; D013921:Thrombocytopenia / Q000139:chemically induced* / Q000276:immunology
chemical_list: D000906:Antibodies; D010978:Platelet Factor 4; D006493:Heparin
keywords: enzyme-linked immunoassay; heparin; heparin-induced thrombocytopenia; humoral immune response; mouse model; platelet factor 4