Multiplex Assay for Profiling Plasma Protein Biomarkers for Improved Diagnosis and Prognosis of Heparin Induced Thrombocytopenia (HIT).

Background: Laboratory diagnosis of heparin-induced thrombocytopenia (HIT) is currently done by using immunologic, platelet aggregation, and serotonin-release assays; however, the diagnostic efficacy of these tests is variable. Although, various clinical scoring systems are currently used to attempt to differentiate patients with HIT from those with other causes of thrombocytopenia, a definite predictive test for HIT is still not available. The pathogenesis of HIT could involve dysregulation of inflammatory mediators (and other immunomodulators) and growth factors from platelets and endothelial cells. Accordingly, with the goal of identifying potential biomarkers of disease, the present study examined the profiles of cytokines, chemokines, and selected growth factors in HIT patients. We are testing a novel multiplex microbead immunoassay approach, developed by Luminex (Austin, TX), for simultaneous detection of multiple plasma protein analytes.

Methods: Plasma samples were obtained from 20 patients diagnosed with HIT and from 15 healthy controls. Patient plasma samples in this study were positive for heparin-platelet factor 4 (PF4) antibodies by ELISA (GTI, Mulwaukee WI). Using the multiplex microbead immunoassay, measurements were made on plasma levels of thirty four cytokines/chemokines, which included tumor necrosis factor (TNF) alpha, interferon (IFN) gamma, IL-1 beta, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p40), IL12(p70), IL-16, Eotaxin, RANTES, MCP-1, MIP-1alpha, MIP-1beta (CCL4), IP-10, MCP-3 (CCL7), GM-CSF, TNF-beta, IL-1RA, soluble form of interleukin (IL) 2 receptor (sIL-2R)alpha, IFNalpha2a, sFAS ligand, GRO, MDC(CCL22), G-CGF in HIT patients and healthy controls; commercial multiplex detection panels were used (Upstate, Lake Placid, NY). In addition, the levels of six growth factors were measured, including FGF-2, VEGF, EGF, Flt-3 Ligand, PDGF-AA and PDGF-AB/BB by the multiplex method (by detection panels also from Upstate).

Results: Multiplex data were analyzed by methods of computational biology to establish hierarchical clustering. In these surveys, the level of sIL-2R, an in vivo marker of T-cell activation was higher in patient plasma than in healthy control subjects (p≤0.002). Plasma levels of the chemokines, MCP-1, MIP-1alpha and IP-10, were significantly reduced in patients compared to controls. Among the growth factors tested, EGF appeared to be significantly decreased in HIT patients compared to the control group. In addition, patients showed lower PDGF-AA and PDGF-AB/BB levels.

Conclusions: Various immunomodulators and growth factors were either increased or decreased in HIT patient plasma. Accordingly, our studies are providing leads for further characterization of plasma biomarkers of HIT in a larger number of patients, including longitudinal samples. In combination with the detection of anti-PF4-heparin antibodies, and other criteria, multiple microbead immunoassays of plasma protein profiles may enhance the accuracy of HIT diagnosis and prognosis. Additionally, the multiplex method, enabling simultaneous detection of multiple protein factors, is also providing novel leads for further studies on the molecular and cellular mechanisms of HIT.