Differentiating iron-loading anemias using a newly developed and analytically validated ELISA for human serum erythroferrone

Abstract

Erythroferrone (ERFE), the erythroid regulator of iron metabolism, inhibits hepcidin to increase iron availability for erythropoiesis. ERFE plays a pathological role during ineffective erythropoiesis as occurs in X-linked sideroblastic anemia (XLSA) and β-thalassemia. Its measurement might serve as an indicator of severity for these diseases. However, for reliable quantification of ERFE analytical characterization is indispensable to determine the assay’s limitations and define proper methodology. We developed a sandwich ELISA for human serum ERFE using polyclonal antibodies and report its extensive analytical validation. This new assay showed, for the first time, the differentiation of XLSA and β-thalassemia major patients from healthy controls (p = 0.03) and from each other (p<0.01), showing the assay provides biological plausible results. Despite poor dilution linearity, parallelism and recovery in patient serum matrix, which indicated presence of a matrix effect and/or different immunoreactivity of the antibodies to the recombinant standard and the endogenous analyte, our assay correlated well with two other existing ERFE ELISAs (both R2 = 0.83). Nevertheless, employment of one optimal dilution of all serum samples is warranted to obtain reliable results. When adequately performed, the assay can be used to further unravel the human erythropoiesis-hepcidin-iron axis in various disorders and assess the added diagnostic value of ERFE.

Circulating erythroferrone has diagnostic utility for acute decompensated heart failure in patients presenting with acute or worsening dyspnea

“…The performance of this assay with respect to variability is not yet fully optimised, but even so, for the purpose of this study, circulating levels of ERFE were well-separated in ADHF compared with other diagnoses (4.9 vs. 1.4 ng/ml) allowing good discrimination of ADHF from non-ADHF phenotypes. This is in agreement with other validation studies of human ERFE assays, whereby although certain assay parameters had limitations, pathologies could be differentiated based on ERFE measurement ( 22 ). Second, whilst our sample size is satisfactory for deriving the diagnostic potential for ERFE, it requires validation in a larger, multicentre recruited set of patients with sufficient comorbid cases (e.g., AF, obesity) as well as accurate current iron and/or ferritin measurements.…”

Partial Parallelism Plots

“…These authors clarify in the abstract poor "dilution linearity" attributed to "presence of a matrix effect and/or different immunoreactivity of the antibodies to the recombinant standard and the endogenous analyte". The partial parallelism plot in Figure 9 is based on the raw data provided in Table 2 in [14]. Consistent with the author's conclusion, this graph convincingly shows absence of parallelism.…”

“…Lack of parallelism has also been reported explicitly [14]. These authors clarify in the abstract poor "dilution linearity" attributed to "presence of a matrix effect and/or different immunoreactivity of the antibodies to the recombinant standard and the endogenous analyte".…”

“…Figure 9. Literature example for reported lack of parallelism for erythroferrone quantified by ELISA from human serum samples [14]. Note that in this example an inverse logarithmic scale was used compared to what was presented in Figure 2B.…”

Erythropoiesis–hepcidin–iron axis in patients with X‐linked sideroblastic anaemia: An explorative biomarker study

“…7 In our previous ERFE-assay validation study, we showed increased ERFE levels in XLSA patients compared to controls. 8 In the current study, we have enriched the ALAS2patient population from our iron expertise centre and included several iron-related parameters to describe the role of the erythropoiesis-hepcidin-iron axis in the pathogenesis of XLSA. Understanding the relationship between this axis and iron loading will help to prevent iron-related toxicity.…”