Abstract
Hepcidin measurement advances insights in pathophysiology, diagnosis, and treatment of iron disorders, but requires analytically sound and standardized measurement procedures (MPs). Recent development of a two-level secondary reference material (sRM) for hepcidin assays allows worldwide standardization. However, no proficiency testing (PT) schemes to ensure external quality assurance (EQA) exist and the absence of a high calibrator in the sRM set precludes optimal standardization. We developed a pilot PT together with the DutchEQA organization Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek (SKML) that included 16 international hepcidin MPs. The design included 12 human serum samples that allowed us to evaluate accuracy, linearity, precision and standardization potential. We manufactured, value-assigned, and validated a high-level calibrator in a similar manner to the existing low- and middle-level sRM. The pilot PT confirmed logistical feasibility of an annual scheme. Most MPs demonstrated linearity (R2>0.99) and precision (duplicate CV>12.2%), although the need for EQA was shown by large variability in accuracy. The high-level calibrator proved effective, reducing the inter-assay CV from 42.0% (unstandardized) to 14.0%, compared to 17.6% with the two-leveled set. The calibrator passed international homogeneity criteria and was assigned a value of 9.07±0.24nmol/L. We established a framework for future PT toenable laboratory accreditation, which is essential to ensure quality of hepcidin measurement and its use in patient care. Additionally, we showed optimized standardization is possible by extending the current sRM with a third high calibrator, although international implementation of the sRM is a prerequisite for its success.
Highlights
The liver-derived hormone hepcidin is the key regulator of iron homeostasis by inhibiting the only known cellular iron exporter ferroportin [1, 2]
We showed that calibration using this secondary reference material (sRM) reduced the inter-method coefficient of variation (CV) from 42.1 to 11.0% when standardization was simulated and from 52.8 to 19.1% when standardization was performed in practice
Anonymous sample collection from diagnostic leftovers and therapeutic phlebotomies was efficient, and the process of developing proficiency testing (PT) samples of particular concentrations based on initial hepcidin measurements was successful
Summary
The liver-derived hormone hepcidin is the key regulator of iron homeostasis by inhibiting the only known cellular iron exporter ferroportin [1, 2]. Hepcidin is a therapeutic target for both iron-overload disorders, such as β-thalassemia and hereditary hemochromatosis, and ironrestrictive anemias as observed with iron refractory iron deficiency anemia (IRIDA), inflammatory diseases, certain tumors and chronic kidney disease [5,6,7]. Both mass spectrometry (MS) and immunochemistry (IC) based measurement procedures (MPs) have been developed to quantify hepcidin concentrations. Effective use of hepcidin measurement in patient care and clinical research require both comparability and analytical reliability to establish uniform clinical decision limits and reference ranges [13] This is essential to compare results across studies or monitor a patient’s treatment at different facilities to prevent inconsistent or incorrect conclusions
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