Albertine Donker

Chapter 8 280 controls were included if they had microcytic anemia, according to WHO guidelines, 25 with TSAT ≤15% and ferritin levels ≤40 µg/L (which is higher than the WHO-defined cut- off value of <15 µg/L). Use of either oral or parenteral iron supplementation during the last three months, CRP >10 mg/L, severe renal impairment (defined as an estimated Glomerular Filtration Rate (eGFR) < 30 ml/min/1.73 m2) 26 and chronic liver disease (defined as ALT > 40 IU/L) were exclusion criteria, as these factors have been described to potentially influence hepcidin production. 8,27-29 Of the 161 eligible patients, we excluded 122 subjects because of (a combination of) the above-mentioned exclusion criteria. This resulted in the inclusion of 39 IDA controls. The study was conducted according to the principles of the Declaration of Helsinki and approved by the local ethics committee of the MMC. For all participants, oral and written informed consent was obtained. The study was registered at theNetherlands Trial Register as ‘SATURNUS Study’, an acronym for the ‘Transferrin Satur ation/Hepcidi n ratio: a st u dy on the diagno s tic utility in the differentiation of Iron Refractory Iron Deficiency Anemia from Iron Deficiency Anemia’. 30 Laboratory measurements For IRIDA and IDA patients Hb, red blood cell indices, serum iron parameters, CRP and if applicable serum creatinine and ALT were measured in accredited Dutch hospital laboratories. Glomerular Filtration Rate was estimated (eGFR) with the CKD-EPI formula in patients in whom a serum creatinine was measured. 26 In both IRIDA patients and IDA controls, hepcidin-25 was measured by weak cation exchange time-of-flight mass spectrometry (WCX-TOFMS) in freshly-thawed samples, as described before. 31 This assay was recently standardized, using a secondary reference material (RM) that was value assigned by a provisional primary RM. 32 Reference values for this standardized assay are available. 33 Measurements of serum hepcidin in the IRIDA group were performed before standardization of our assay, while measurements of serum hepcidin in the IDA control group were performed with the same assay, but after standardization. Standardization slightly altered serum hepcidin values, that is, standardized results were found to be 5.4% higher compared to historic results obtained without standardization. 33 Stability of the assay in time was ensured by the use of serum matrix based quality controls.