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. 2020 Sep 1;5(5):943-953.
doi: 10.1093/jalm/jfaa038.

Clinical Immunoassay for Human Hepcidin Predicts Iron Deficiency in First-Time Blood Donors

Patrick Gutschow  1 , Huiling Han  1 , Gordana Olbina  1 , Keith Westerman  1 , Elizabeta Nemeth  2 , Tomas Ganz  2 , Karen Copeland  3 , Mark Westerman  1 , Vaughn Ostland  1
Affiliations

Clinical Immunoassay for Human Hepcidin Predicts Iron Deficiency in First-Time Blood Donors

Patrick Gutschow et al. J Appl Lab Med. .

Abstract

Background: Serum markers currently used as indicators of iron status have clinical limitations. Hepcidin, a key regulator of iron homeostasis, is reduced in iron deficiency (ID) and increased in iron overload. We describe the first CLIA-validated immunoassay with excellent accuracy and precision to quantify human serum hepcidin. Its diagnostic utility for detecting ID in first-time blood donors was demonstrated.

Methods: A monoclonal competitive ELISA (C-ELISA) was developed for the quantitation of human hepcidin and validated according to CLIA guidelines. Sera from nonanemic first-time blood donors (n = 292) were analyzed for hepcidin, ferritin, transferrin, and serum iron. Logistic regression served to determine the utility of hepcidin as a predictor of ID.

Results: The C-ELISA was specific for human hepcidin and had a low limit of quantitation (4.0 ng/mL). The hepcidin concentration measured with the monoclonal C-ELISA was strongly correlated with a previously established, extensively tested polyclonal C-ELISA (Blood 2008;112:4292-7) (r = 0.95, P < 0.001). The area under the receiver operating characteristic curve for hepcidin as a predictor of ID, defined by 3 ferritin concentration thresholds, was >0.9. For predicting ID defined by ferritin <15 ng/mL, hepcidin <10 ng/mL yielded sensitivity of 93.1% and specificity of 85.5%, whereas the same hepcidin cutoff for ferritin <30 ng/mL yielded sensitivity of 67.6% and specificity of 91.7%.

Conclusion: The clinical measurement of serum hepcidin concentrations was shown to be a potentially useful tool for diagnosing ID.

Keywords: Anemia; ELISA; Hepcidin; Iron Deficiency.

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Figures

Fig. 1
Fig. 1
Development and characterization of hepcidin C-ELISA. (A) Dose response curve starting at 400 ng/mL with serial dilutions. Structurally similar peptides were compared to assess selectivity. Hepcidin-10 to -25 is an N-terminally truncated hepcidin peptide. (B) Comparison of monoclonal hepcidin C-ELISA (n = 292) with a previously described polyclonal C-ELISA (1).
Fig. 2
Fig. 2
Evaluation of first-time blood donors. (A) Donor comparison of matched serum hepcidin and ferritin concentrations (n = 292) (Demming regression). (B) Hepcidin by sex in donors with normal iron status (female, n = 104; male, n = 113; P < 0.0002). (C) Hepcidin in donors with low, normal, or high iron status classified using serum ferritin and TSAT (P < 0.0001, Steel-Dwass method).
Fig. 3
Fig. 3
Age- and sex-dependent variation of hepcidin. Healthy donors were distributed into 4 bracketed age groups. Serum hepcidin (A) and ferritin (B) concentrations demonstrate similar distributions across age groups for both sexes.
Fig. 4
Fig. 4
ROC curve for hepcidin as a predictor of ID. The AUCROC for hepcidin compared with ferritin cutoffs of <15, <20, or <30 ng/mL was 0.94, 0.93, and 0.90, respectively.
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