Validation of the Intrinsic Hepcidin IDxTM Test, an LDT for Clinical Hepcidin Assessment

Abstract Introduction: Hepcidin is the principal regulator of iron absorption and iron recycling, and is required to provide iron for erythropoiesis. Hepcidin is a peptide hormone that binds to the sole iron channel ferroportin (Fpn), and blocks iron efflux by degrading Fpn, trapping iron stored in the cell. Dysregulation of hepcidin by genetic mutations affecting hepcidin expression in hepatocytes is responsible for hereditary hemochromatosis where too little hepcidin leads to iron-overload disease, and iron refractory iron deficiency anemia (IRIDA) where hepcidin expression is inappropriately high leading to anemia. Hepcidin itself is regulated by iron and the inflammatory cytokine IL-6. Thus, hepcidin is dysregulated in most inflammatory diseases and chronic infections. Interest in developing diagnostic applications for hepcidin in iron disorders is high. Objective: We developed a high-affinity monoclonal antibody (mAb) to hepcidin and used it to produce a competitive ELISA (C-ELISA) similar to our first RUO test (Ganz et al. 2008). We determined the performance characteristics using CLIA analytical method validation guidelines. The Intrinsic Hepcidin IDx Test is used for serum and Li-Heparin plasma. We determined the reference range in adult first time blood donors (n=292) and studied an iron deficient (ID) sub-set of the donors. Methods: We developed mouse anti-human hepcidin monoclonal antibodies and coated, blocked, and dried the plates in a temperature and humidity controlled incubator. We used highly purified synthetic hepcidin as reference standard and a biotinylated hepcidin analog as the competitive tracer for the C-ELISA (Peptides International, Louisville, KY). The Intrinsic Hepcidin IDx Test is fully automated on the Biomek FX platform. Serum from first time blood donors (n=292) was obtained, and ferritin, transferrin, plasma iron, CRP, and hepcidin were measured. The reference range for hepcidin was determined and the normal iron status range determined by eliminating iron deficient (ID) donors or those shown to be iron overloaded. Receiver operator characteristic curves (ROC) were plotted to determine the utility of hepcidin and to identify optimal cutoffs of hepcidin as a test of iron deficiency. Results: The mAb used in the Intrinsic Hepcidin IDx Test is specific for human hepcidin. The analytical measurement range (AMR) is 4 - 200 ng/ml. The intra-assay precision CVs range between 7% (at 12 ng/ml) and 4% (at 99 ng/ml). The between-day precision CVs range between 7% (12 ng/ml) and 9% (100 ng/ml). The mean spike recovery across the AMR is 3%. Hepcidin was stable at 4oC for up to 7 days and if specimens are stored at -20oC, for at least 6 months. Hepcidin concentrations determined in blood donors were highly correlated with those measured using our polyclonal RUO C-ELISA (r = 0.95, p<0.001). The reference range is 4.4-54.1 ng/ml for women and 6.1-91.2 ng/ml for men. Clear gender differences were observed: hepcidin was higher in men (median 28.5 ng/ml, n = 143) than in women (median 13.1 ng/ml, n = 149). In addition, the ID group had lower hepcidin (median 6.7 ng/ml) than the normal iron status group (median 21.4 ng/ml, p<0.0001). The area under the ROC curve for hepcidin compared with various ferritin concentrations was greater than 0.90. An optimal hepcidin cutoff less than 10 ng/ml was determined to predict ID with a correct classification rate of greater than 86%. Conclusion: We developed and validated a sensitive, accurate and reproducible C-ELISA, the Intrinsic Hepcidin IDx test, using CLIA validation guidelines. We determined the hepcidin reference ranges by analyzing first-time blood donor serum. We found a significant difference in median hepcidin values between genders. Also, our data show that hepcidin was lower in donors with ID compared to iron replete, healthy donors. Disclosures Gutschow: Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment. Han:Intrinsic Lifesciences.: Employment, Equity Ownership. Ostland:Intrinsic LifeScienc s: Employment, Equity Ownership. Olbina:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership.