A Novel PF4-Heparin Microbead Assay and Plasma Protein Biomarker Profiling for Improved Diagnosis of Heparin Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening, immune-mediated complication of heparin administration resulting in a hypercoagulable state. The diagnosis is made using clinical scoring systems with confirmatory laboratory testing. However, current diagnostic criteria may lack predictive value for HIT-associated thrombosis and need for alternative anticoagulation. Therefore, there is a need to develop diagnostic methods that will have a better predictive value for HIT diagnosis. The pathogenesis of HIT involves platelet-activating antibodies that recognize platelet factor 4 (PF4) -heparin complex, and may also involve dysregulation of inflammatory mediators and growth factors from platelets and endothelial cells. Luminex multiplex microbead assay allows for the simultaneous detection of multiple serum analytes. The aim of this study is to develop a multiplex method to detect anti-PF4-heparin antibodies and to generate profiles of cytokines, chemokines, growth factors and adhesion molecules with better predictive value for the diagnosis of HIT than current diagnostic methods. In combination with measurement of antibodies to (PF4)-heparin complex, such a panel is expected to enhance the accuracy of HIT diagnosis. To develop the anti-PF4-heparin IgG microbead assay, carboxylated Luminex microbeads were coated at various concentrations with the optimal molar ratio of the PF4/polyvinyl sulfonate (PF4/PVS) complex, the antigenic determinant from the GTI ELISA kit, (provided by GTI, Waukesha WI). The anti-PF4-heparin microbead assay was standardized using plasma samples from patients and healthy controls. Furthermore, we compared the anti-PF4-heparin microbead assay to traditional commercial HIT antibody ELISA kits from GTI, Diagnostica Stago and Aniara. To identify potential plasma biomarkers for HIT, measurements were made on plasma levels of 63 cytokines, chemokines, growth factors and adhesion molecules in patients with and without HIT (as defined by the Chong’s score) and healthy controls using commercial multiplex detection panels (Bio-Rad, Upstate, Linco). Multiplex data were analyzed using multivariate statistical methods and compared with their respective clinical scores. Preliminary anti-PF4-heparin microbead assay results show an overall concordance rate of 80% with GTI (Polyclonal) ELISA; 91% with Diagnostica Stago (Polyclonal); 78% with Aniara Polyclonal and 76% with Aniara IgG ELISA. The anti-PF4-heparin microbead assay results also show good correlation with clinical determination of HIT disease status as determined by Chong’s scoring (72% sensitivity, and 58% specificity). Preliminary biomarker profiling suggest that Il-2Ra, b-NGF, GRO-a, TNF-b, PDGF AB/BB, PDGF AA, and s-ICAM1 are potentially useful markers in further analysis for distinguishing HIT status. We have identified these candidate biomarkers to evaluate in a larger number of patient samples with suspected HIT. Our data show that our novel microbead assay for anti-PF4-heparin antibodies correlate well with the existing conventional ELISA results, and also correlate with HIT diagnosis as determined by the Chong’s score. Furthermore, we have identified a panel of biomarkers that could be useful for improved diagnosis and prognosis of HIT, and will establish a basis for further understanding the pathophysiology of this coagulation disorder.