Albertine Donker

Chapter 8 290 Another factor that makes comparison of results between the two studies challenging, is that we reported the TSAT/hepcidin ratio rather than the log-transformed TSAT/ log 10 (hepcidin) ratio, and that both studies used differently calibrated assays. Previous round robin studies show that thepcidin levels of the group of Heeney et al are measured up to approximately a factor 7 higher due to the use of a calibrator in their assay that is not traceable to primary reference material. 39 Indeed, after multiplying our hepcidin values 7 times, we observed a comparable ability of the TSAT/log 10 (hepcidin) ratio in distinguishing IRIDA from IDA as in our untransformed TSAT/hepcidin data. The strengths of our study are: i) we validated the ability of the TSAT/hepcidin ratio to detect both mono-allelic affected IRIDA patients as bi-allelic affected IRIDA patients from IDA controls in a broad iron-deficient population, ii) we established a cut-off value for use of the TSAT/hepcidin ratio as diagnostic test in the work up of iron deficient microcytic anemic patients suspected for the presence of IRIDA, iii) we used a recently standardized assay for hepcidin measurements of our IDA controls enabling future comparison of results of hepcidin assays used elsewhere, provided these are standardized by using the same reference material. This will ultimately allow global uniform decision-making based on TSAT/hepcidin ratios. iv) We described baseline characteristics and comorbid conditions that could have influenced hepcidin production in IDA controls in detail. Our study has several limitations. As we used a case-control design in which IDA controls had an alternative diagnosis explaining their microcytic anemia and the prevalence of TMPRSS6 -related IRIDA is unknown, we could not calculate the positive and negative predictive value. However, since a low TSAT/hepcidin ratio was highly specific for TMPRSS6- related IRIDA, we argue that a ratio of 5.9%/nM or lower strongly indicates the presence of IRIDA. Nevertheless, there was one IRIDA patient who was diagnosed with IRIDA based on microcytic anemia unresponsive to oral iron treatment in combination with a mono-allelic pathogenic TMPRSS6 variant, despite the serum hepcidin level being not discrepantly high (i.e. an hepcidin value of 1.3 nM). This resulted in a TSAT/hepcidin ratio of 9.1%/nM, which would be considered as not suspect for having TMPRSS6 -related IRIDA according to our established cut-off value of 5.9%/nM. One could argue this patient has been diagnosed with IRIDA incorrectly, as inappropriately increased serum hepcidin levels in relation to body iron stores are the cornerstone of diagnosing IRIDA, even after iron supplementation has been