Iron regulatory hormone Hepcidin

Dorine W. Swinkels  from the Department of Laboratory Medicine of the Radboud university medical center is presenting a poster about harmonization and standardization of Hepcidin methods at the International Congress on Quality in Laboratory Medicine 2020 in Helsinki. This poster is based on their work started in 2005  when they were the first to employ time-of-flight mass spectrometry to measure hepcidin in urine. After that they have continued to improve this assay towards a current quantitative assay to measure hepcidin in human serum and urine samples using an stable hepcidin-25 (+40) isotope as internal standard for quantification. In 2010, they have also developed and validated a competitive ELISA, that facilitates measurements for studies with large sample sizes. Using both assays, they have gained thorough expertise on biomedical and pre-analytical factors that influence hepcidin levels in clinical and biological samples and their assay has been used in several biomedical studies. Now they coordinate the harmonization of hepcidin methods that are currently in use throughout the world. Read more from 

Abstract: Clinical translation through harmonization of a novel biomarker: the iron regulatory hormone Hepcidin. A clinical chemist perspective

Authors: Dorine W. Swinkels, Laura Diepeveen and Ellis Aune
Presenter: Dorine W. Swinkels
Organization: Laboratory Medicine, Radboudumc, Nijmegen, The Netherlands

The liver-derived peptide hormone hepcidin is responsible for maintaining iron homeostasis and can be an important diagnostic biomarker and therapeutic target in a range of pathologies, including iron disorders such as hereditary hemochromatosis and anemias and in cases of inflammation and infection. Fundamental studies on structure and function of hepcidin revealed a 25-amino-acid peptide that modulates ferroportin, a cellular iron exporter present in macrophages and enterocytes. In cases of high body iron  levels that can be damaging to various tissues, hepcidin is elevated to reduce total ferroportin activity; conversely, when total body iron is low, hepcidin concentrations decrease to promote iron availability. Hepcidin also exists in vivo in isoforms (hepcidin-24, 22, and 20) which are especially elevated in diseased states such as chronic kidney disease and sepsis, but the clinical significance of these isoforms remains unclear.

Development of a validated hepcidin assay in body fluids proved challenging. Hepcidin adheres to laboratory plastics and is only stable at room temperature for six to eight hours, requiring creativity in developing measurement protocols. Furthermore, hepcidin is influenced by various factors, including diurnal rhythm, prolonged fasting, gender, age, inflammation, iron stores, iron supplementation, renal function, erythropoiesis, chronic liver diseases, red blood cell transfusions, various hormones, and genetic factors. Despite these difficulties, we and others have developed and validated both mass spectrometry (MS) assays and  enzymelinked immunosorbent assays (ELISAs) that allow hepcidin quantification in serum and plasma. We established reference values for hepcidin in adults and children and – since hepcidin as a hormone is subject to complex regulatory feedbacks- also for hepcidin levels relative to circulating (transferrin saturation) and stored iron (ferritin).

However, as the prevalence of hepcidin assays has increased, highly variable results among international laboratories have made apparent the urgent need for harmonization and standardization in order to facilitate the use of hepcidin as a clinically relevant diagnostic parameter. We developed a commutable secondary reference material (sRM) derived from human plasma and our international study showed mathematical harmonization was possible with both MS and ELISA techniques in 10 measurement procedures. Subsequently, we optimized and produced the sRM in human serum for low and middle physiologic concentrations and a primary reference material (synthetic human hepcidin-25) was created to assign values to the sRM, completing a traceability chain to standardize the measurement techniques. Additional international studies confirmed the validity of the sRM as a calibrator, allowing measurementsin 11 procedures to be brought within an optimal bias range. This reference material is now available from our laboratory.

The future of clinical hepcidin assay implementation is twofold. First, addition of a high hepcidin standard that represents the high-normal physiological range of hepcidin would further improve standardization across laboratories. Some obstacles must be overcome, including ethically obtaining highhepcidin serum and confirming whether it proves commutable across assays. Second, our pilot international proficiency test will provide further insight into the current practices of hepcidin measurement worldwide and provide a framework to establish laboratory accreditation and quality control procedures to further validate hepcidin in clinical diagnostics.

Labquality Days – International Congress on Quality in Laboratory Medicine

Welcome to the International Congress on Quality in Laboratory Medicine on 6-7 February 2020. The scientific program of the next International Congress on Quality in Laboratory Medicine will be built around the theme of optimizing quality. The program covers the topics on harmonization of medical practices, how to communicate the results to clinicians and patients and the quality assurance of new measurement technologies. Welcome to Helsinki!