History of assay achievements

A major research focus within the Department of Laboratory Medicine of the Radboud University Medical Center is on iron metabolism regulators, and the translation of new findings into diagnostic assays that can be clinically implemented.  Our initial work focused on the measurement of iron-regulatory-protein hepcidin in human urine and serum and resulted in two publications in the Blood-journal. In one of these studies, an in vivo human endotoxemia model was used to analyse the effects of endotoxemia as an upstream inflammation activator of hepcidin. These studies underscored the important role of hepcidin as the mediator of anemia of inflammation. This prompted us to develop a novel mass spectrometry (MS)-based assay for quantification of hepcidin in urine and serum. In fact, in 2005 we were the first to publish the potential of this MS approach, which paved the way for hepcidin measurements in clinical studies .
 
Our recent improvements quantitative serum/plasma and urine hepcidin measurements opened novel opportunities for studies on hepcidin in humans resulting in further insights in the clinical implications of hepcidin-mediated regulation of iron metabolism. Hepcidin measurements are now routinely performed by weak cation exchange chromatography in combination with time-of-flight mass spectrometry (WCX-TOF MS) on a Microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics).
 
For high throughput plasma and serum hepcidin measurements, we have developed an in-house competitive ELISA .  We moreover developed a competitive bioanalytical assay for plasma and serum hepcidin using the Anticalin protein Hep-Dx as a next-generation diagnostic antibody mimetic and biotin-labeled hepcidin-25 as a tracer (Enzyme Linked antibody-Mimetic Assay, ELMA). Both these method measures the total of hepcidin isoforms and are especially appropriate for large sample numbers.
 
To date, we have taken two initiatives (round robins) to harmonize hepcidin methods throughout the world.

 

References

Engineered human lipocalin as an antibody mimetic: application to analysis of the small peptide hormone hepcidin Grebenchtchikov N, Geurts-Moespot AJ, Trentmann S, Andersen N, Bel Aiba RS, Allersdorfer A, Laarakkers CM, Sweep FC, Tjalsma H, Hohlbaum AM, Swinkels DW. Clin Chem 2014;60(6):897-9.
Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform Laarakkers CM, Wiegerinck ET, Klaver S, Kolodziejczyk M, Gille H, Hohlbaum AM, Tjalsma H, Swinkels DW. PLoS One 2013;8(10):e75518.
Second round robin for plasma hepcidin methods: first steps toward harmonization Kroot JJ, van Herwaarden AE, Tjalsma H, Jansen RT, Hendriks JC, Swinkels DW. Am J Hematol 2012;87(10):977-83
Immunochemical and mass-spectrometry-based serum hepcidin assays for iron metabolism disorders Kroot JJ, Laarakkers CM, Geurts-Moespot AJ, Grebenchtchikov N, Pickkers P, van Ede AE, Peters HP, van Dongen-Lases E, Wetzels JF, Sweep FC, Tjalsma H, Swinkels DW. Clin Chem 2010;56(10):1570-9.
Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization Kroot JJ, Kemna EH, Bansal SS, Busbridge M, Campostrini N, Girelli D, Hider RC, Koliaraki V, Mamalaki A, Olbina G, Tomosugi N, Tselepis C, Ward DG, Ganz T, Hendriks JC, Swinkels DW. Haematologica 2009;94(12):1748-52.
Advances in quantitative hepcidin measurements by time-of-flight mass spectrometry Swinkels DW, Girelli D, Laarakkers C, Kroot J, Campostrini N, Kemna EH, Tjalsma H. PLoS One 2008;3(7):e2706.
Hepcidin: from discovery to differential diagnosis Kemna EH, Tjalsma H, Willems HL, Swinkels DW. Haematologica 2008;93(1):90-7.
Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications Kemna EH, Tjalsma H, Podust VN, Swinkels DW. Clin Chem 2007;53(4):620-8.
Novel urine hepcidin assay by mass spectrometry Kemna E, Tjalsma H, Laarakkers C, Nemeth E, Willems H, Swinkels D. Blood 2005;106(9):3268-70.
Time-course analysis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS Kemna E, Pickkers P, Nemeth E, van der Hoeven H, Swinkels D. Blood 2005;106(5):1864-6.