Results of the First International Round Robin for the Quantification of Urinary and Plasma Hepcidin: Need for Standardization

The hepatic peptide hormone hepcidin plays a central role in body iron metabolism. Quantification of hepcidin concentrations in urine and plasma holds promise as a biomarker for diagnosis and monitoring of disorders of iron metabolism. To date, various mass spectrometry (MS, time of flight and liquid chromatography-MS/MS) as well as immunochemical (IC, competitive radio-immunoassay and ELISA) and functional methods exploiting an internal or external standard, have been developed to measure hepcidin levels in both plasma and urine. Only a few of them have been published. The hepcidin levels quantified by these methods are likely to differ substantially in analytical characteristics. Therefore, comparative studies are imperative for progress in the field by allowing meaningful exchange of hepcidin results derived from various laboratories. In this study we compared the analytical characteristics of several available urinary and plasma hepcidin assays as a first step towards standardization of hepcidin quantification methods. Six and 8 laboratories participated in an international inter-laboratory evaluation of hepcidin levels from a common human urinary (n=8) and plasma (n=7) sample set, respectively, exploiting MS (n=5) and IC (n=3) based methods. Participating laboratories were requested to perform 3 hepcidin measurements for each sample on 4 different days, making a total of 96 urine and 84 plasma measurements per laboratory. The methods differed widely in mean urine and plasma hepcidin level, i.e. range 2.9–427.1 nmol/mmol creatinine and 11.4–124.6 nM, respectively. Coefficient of variation (CV)-ranges for the between-sample and withinsample variation were 126.1–191.5 % and 10.8–33.8%, respectively, for urine and 40.4–96.6 % and 5.5–31.0%, respectively, for plasma. The ratio between-sample CV/within-sample CV varies between 15.3 and 4.1 for urine and 15.1 and 2.4 for plasma. Hepcidin results of most methods mutually correlated (plasma: R range 0.82–1.00; urine: R range 0.73–0.99). We conclude that for the various methods

• absolute hepcidin values differ widely, but generally correlate and

• the ratio between-sample and within sample CV vary greatly, suggesting relevant differences in ability to detect small differences in hepcidin concentrations.

To allow future comparison, exchangeability and transferability of hepcidin data, we recommend

• publication of all hepcidin results according to STARD (www.stard-statement.org) to ensure complete reporting of analytical characteristics and

• to develop initiatives for standardization of assays.