Albertine Donker

Standardized Pediatric Hepcidin Values 255 7 SUPPLEMENTAL FILES Laboratory Methods After arrival in the laboratory, samples were aliquoted. Serum hemoglobin (Hb), red cell indices, CRP, ALT, iron, transferrin and ferritin were measured in freshly obtained sample aliquots. Left- over aliquots were stored at -80 C˚ until use. Hb and red cell indices were measured on a Sysmex XN automated hematology analyzer (Sysmex Netherlands, Etten-Leur, the Netherlands). CRP (lower limit < 0.1 mg/L), ALT, iron and transferrin were all measured on a Cobas 8000 C502 chemistry analyzer with kit reagents form Roche. Ferritin was measured on a Cobas 8000 C602 (e-Module) immunochemistry module (Roche Diagnostics Nederland B.V., Transistorstraat 41, 1322 CK, Almere). TIBC (µmol/L) and TSAT (%) were calculated using transferrin (in g/l, MW 79.550 KDa) and serum iron concentrations (µmol/l). Determination of hepcidin and soluble Transferrin Receptor (sTfR) was performed in batches in freshly thawed samples in April 2018. We measured levels of the bioactive form of hepcidin: (hepcidin-25 1 ) by weak cation exchange chromatography followed by time of flight mass spectrometry (WCX -MS-TOF) as described before. 2 This method was recently standardized using 2nd reference material (RM) that was value assigned by a provisional primary RM. 3 Serum concentration of sTfR was measured by immunonephelometry (Behring Nephelometer II Analyzer; BNII, Dade Behring Marburg GmbH, Marburg, Germany).