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.

[heparin-induced thrombocytopenia]

The key feature of heparin-induced thrombocytopenia (HIT ) is the production of antibodies (Ab ) against the platelet factor 4 (PF 4 )/heparin complex . These Ab are directed against neoepitopes of the PF 4 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 PF 4 /heparin complexes , but not PF 4 alone are required .We established fluid-phase enzyme-immunoassays in which the soluble biotinylated antigen , PF 4 /heparin , is firstly captured by specific Ab , and secondly directly detected with enzyme-conjugated streptavidin .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 PF 4 /heparin compared to murine PF 4 alone . This fluid-phase approach applied to the detection of specific murine PF 4 /heparin Ab -secreting cells (ASC ) identified the spleen as the main lymphatic organ that contributes to the PF 4 /heparin Ab response in mice . IgG ASC specific for PF 4 /heparin are very transiently detectable in mice , which might explain why anti-PF 4 /heparin IgG Ab typically disappear within 100 days in humans . Furthermore , this fluid-phase approach was successfully transferred to detect human PF 4 /heparin-specific Ab .T he fluid-phase principle for the specific detection of anti-PF 4 /heparin IgG and IgM Ab enables new and improved assays for HIT research in men and mice . At least in mice PF 4 /heparin antibodies are produced by transient B cells .