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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.

SUBMITTER: Diepeveen L

PROVIDER: S-EPMC8291690 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Eosinophilic esophagitis (EoE) is diagnosed by symptoms, and at least 15 intraepithelial eosinophils per high power field in an esophageal biopsy. Other pathologic features have not been emphasized. We developed a histology scoring system for esophageal biopsies that evaluates eight features: eosinophil density, basal zone hyperplasia, eosinophil abscesses, eosinophil surface layering, dilated intercellular spaces (DIS), surface epithelial alteration, dyskeratotic epithelial cells, and lamina propria fibrosis. Severity (grade) and extent (stage) of abnormalities were scored using a 4-point scale (0 normal; 3 maximum change). Reliability was demonstrated by strong to moderate agreement among three pathologists who scored biopsies independently (P ≤ 0.008). Several features were often abnormal in 201 biopsies (101 distal, 100 proximal) from 104 subjects (34 untreated, 167 treated). Median grade and stage scores were significantly higher in untreated compared with treated subjects (P ≤ 0.0062). Grade scores for features independent of eosinophil counts were significantly higher in biopsies from untreated compared with treated subjects (basal zone hyperplasia P ≤ 0.024 and DIS P ≤ 0.005), and were strongly correlated (R-square >0.67). Principal components analysis identified three principal components that explained 78.2% of the variation in the features. In logistic regression models, two principal components more closely associated with treatment status than log distal peak eosinophil count (PEC) (R-square 17, area under the curve (AUC) 77.8 vs. R-square 9, AUC 69.8). In summary, the EoE histology scoring system provides a method to objectively assess histologic changes in the esophagus beyond eosinophil number. Importantly, it discriminates treated from untreated patients, uses features commonly found in such biopsies, and is utilizable by pathologists after minimal training. These data provide rationales and a method to evaluate esophageal biopsies for features in addition to PEC.

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

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