Erythroferrone as a sensitive biomarker to detect stimulation
Kelvin Ramirez Cuevas
| Céline Schobinger
| Emeric Gottardo
Sven Christian Voss
| Tiia Kuuranne
| Jean-Daniel Tissot
| Bernard Favrat
| Nicolas Leuenberger
Swiss Laboratory for Doping Analyses,
University Center of Legal Medicine, Lausanne
and Geneva, Centre Hospitalier Universitaire
Vaudois and University of Lausanne,
Anti-Doping Lab Qatar, Sports City Road,
Sports City, P.O. Box 27775, Doha, Qatar
Transfusion interrégionale CRS, site
Department of Ambulatory Care and
Community Medicine, University of Lausanne,
Athlete Health and Performance Centre,
Aspetar Orthopaedic and Sports Medicine
Hospital Doha, Qatar
Nicolas Leuenberger, Swiss Laboratory for
Doping Analyses, Ch. Des Croisettes 22, 1066
Fondation Dr Henri Dubois-Ferrière Dinu
Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in
response to erythropoietin stimulation. ERFE suppresses the hepatic synthesis of
the master iron-regulatory hormone, hepcidin. The impact of erythropoiesis stimu-
lation on ERFE secretion in humans is poorly understood. This paucity of infor-
mation is due in part to the lack of available means for ERFE quantification in
serum samples. The present study tested a new sensitive sandwich immunoassay
for human ERFE. This assay was used to demonstrate that injection of various
erythropoiesis stimulating agents (ESAs) increased the blood ERFE levels in
healthy volunteers. After exogenous stimulation of erythropoiesis, ERFE increased
up to 8-fold with a detection window of 13 days. The impact of one unit of
blood withdrawal on erythropoiesis stimulation of ERFE was also tested. ERFE
significantly increased after blood withdrawal in subjects injected with both iron
and saline solution, suggesting that iron supplementation did not mask the ERFE
increase after blood withdrawal. The effects of exercise-induced muscle damage
on ERFE was assessed by comparing ERFE levels with creatine kinase levels in
samples from subjects with heavy exercise loads, and determined that this was
not a confounder. The ERFE assay is a sensitive means to investigate the connec-
tion between iron metabolism and erythropoiesis in humans, and to detect ESA
abuse in the antidoping field.
blood doping, erythroferrone, immunoassay
The greatest deterrent to blood doping with erythropoiesis stimu-
lating agents (ESAs) is the hematological module of the Athlete
Biological Passport (ABP).
The hematological module focuses on
long-term monitoring of specific blood parameters, such as hemo-
globin concentration (Hb) and reticulocyte percentage (Ret%), to
detect abnormal absolute and/or relative changes in individual pro-
files that may indicate doping with ESAs. Stimulation of
erythropoiesis can be also observed after the blood withdrawal
step necessary for autologous blood transfusion, which is an alter-
native means for blood doping.
However, the shift towards blood
transfusions and micro-dose injections of ESAs necessitates the
development of new markers that can be integrated into the ABP
hematological module to increase its sensitivity.
in erythropoiesis and iron metabolism have been suggested as
potential biomarkers for ESA abuse.
An example for this is
hepcidin which is downregulated by recombinant human
Received: 5 July 2019 Revised: 9 October 2019 Accepted: 13 October 2019
Drug Test Anal. 2019;1–7. wileyonlinelibrary.com/journal/dta © 2019 John Wiley & Sons, Ltd. 1
erythropoietin (rhEPO) administration.
In 2014, erythroferrone
(ERFE) was discovered as a novel erythroid regulator of iron
metabolism in a mouse model.
However, ERFE has been described
as having the same or a similar structure as myonectin.
In its role
as an iron metabolism regulator, ERFE mediates hepcidin suppres-
sion during increased erythropoietic activity stimulated by endoge-
nous and exogenous EPO, and facilitates compensatory iron
acquisition during recovery from hemorrhage-induced anemia.
These observations suggested that ERFE could be a viable bio-
marker for the detection of blood doping. However, because these
experiments were almost exclusively performed in mice, compara-
tive studies in humans are necessary, yet the quantification of
ERFE with a validated immunoassay remains challenging.
Previously, different ERFE immunoassays were tested to inves-
tigate the link between iron metabolism and erythropoiesis in
However, the data presented suggested that these
assays were not valuable for the detection of ESA abuse in the
antidoping context, mainly due to sensitivity. Furthermore, the
influence of various confounding factors was not explored in these
In the present study, testing and validation of a new commercial
available sensitive sandwich immunoassay for human ERFE were per-
formed. The impact of ESA administration and blood withdrawal on
ERFE levels in human subjects, as well as potential confounding fac-
tors such as iron supplementation and exercise-induced muscle dam-
age, were investigated.
2|MATERIAL AND METHODS
2.1 |Human ERFE ELISA
The commercial ERFE (human) Matched Pair Detection Set (AG-
46B-0012-KI01; AdipoGen Life Sciences, Epalinges, Switzerland)
was used per the manufacturer's instructions (https://adipogen.
set.html). Briefly, 100 μL of standard and serum samples was
added to the ELISA plate and detected by absorbance. The limit of
detection was determined by adding three standard deviations to
the mean value of 50 background level (zero concentration of
ERFE) standards. The standard of the ELISA kit is the full-length
erythroferrone recombinant protein with a His-tag expressed in
mammalian cells. This protein has been validated by AdipoGen
Life Sciences and is characterized to be stable for at least 1 year
when stored at 4C. To assess the intra-assay precision, four sam-
ples of known human recombinant ERFE concentration were
assayed in replicate six times. To test the inter-assay precision,
three samples of known human ERFE concentration were assayed
in four separate assays. The limit of detection of the immunoassay
was calculated as an apparent concentration of 40 pg/mL. The
intra- and inter-day variation ranges were 2–6% and 5–9%,
2.2 |Clinical study samples
2.2.1 |Clinical studies: administration of
Details regarding the participants and the time of serum sample col-
lection of the different clinical studies are described in previous stud-
ies which are summarized below.
For the first generation rhEPO clinical study, six healthy
Caucasian males (mean age, 27.0 years (SD, 4.1); mean body mass
index (BMI), 23.9 kg/m
(SD, 2.66)) received a single intravenous
injection of rhEPO delta (5000 IU; Dynepo™, Dynepo Shire Pharma-
ceuticals, Basingstoke, UK) on days 1, 3 and 5 of the study as
For the third generation rhEPO clinical study, six healthy
Caucasian men with a mean age of 23.0 years (SD, 2.97) and a mean
BMI of 23.3 kg/m
(SD, 1.48) received a single subcutaneous or intra-
venous injection of 200 mg of CERA (MIRCERA; Roche Pharma AG,
Reinach, Switzerland) as described previously.
In another study, six healthy Caucasian males received a single
intravenous injection of the EPO analog hematide/peginesatide
(Affymax, Inc. Palo Alto, CA) at 50 μg/kg as described previously.
2.2.2 |Clinical study: blood withdrawal and iron
A randomized, single-blind, placebo-controlled trial was approved by
the Human Research Ethics Committee of the Canton of Vaud in
Switzerland (Protocol: 2016–00324). Participants were randomized to
receive an i.v. injection of either iron or placebo at a ratio of 1:1 using
R studio software (Version 1.0.44). Volunteers were blinded to the
study treatment by covering blood bags with opaque bags. The
ClinicalTrials.gov identifier is NCT03014921, and the study is entitled
“Impact of Iron Injection on Blood Donation: a Randomized and
Controlled Clinical Trial.”The study design is shown in Figure S1.
Written informed consent was obtained from each subject prior to
enrolment in the study.
Male Caucasian volunteers aged 20–35 years with a ferritin con-
centration ≤50 μg/L ± 10% and a BMI of 18–30 who were eligible for
blood donation according to national regulations were invited to
The iron supplement used was a 250 mL perfusion of 0.9% NaCl
(B. Braun Medical AG, Crissier, Switzerland) combined with 10 mL of
a 500 mg ferric carboxymaltose complex (Ferinject
, Vifor Pharma,
Villars-sur-Glâne, Switzerland). The placebo used in the control group
was a 250 mL perfusion of 0.9% NaCl (Figure S1).
2.2.3 |Clinical study: impact of exercise-induced
muscle damage on serum ERFE levels
Ten healthy, Caucasian middle- and long-distance runners took part in
this study. The subjects provided written informed consent, and the
2RAMIREZ CUEVAS ET AL.
study was approved by the institutional review board (E2015000073)
of ADL Qatar. Serum samples were collected at sea level at various
time points over a period of 2 weeks. To assess the level of muscle
damage, serum samples were analysed for creatine kinase (CK), a cir-
culating protein that is considered an indirect marker for muscle dam-
age. To reflect individual responses to exercise-induced muscle
damage, three samples per subject were included in this study: the
sample with the lowest and the two samples with the highest CK
values. The highest concentration of CK obtained in this study was
2.3 |Clinical chemistry, immunology, and
All venous blood samples were obtained from an antecubital vein
according to standard procedures. Blood was drawn into 8.5 mL
serum tubes (SST II Advance, BD Vacutainer, ref. 366644). Serum
tubes were centrifuged for 15 min at 1500 rcf after collection and
stored at −20Cor−80C until further analysis. Transferrin satura-
tion (TSAT) was measured using an automated Dimension EXL 2000
technology system (Siemens Healthcare Diagnostic SA Zurich, Swit-
zerland) according to the manufacturer's instructions.
The ferritin concentration was measured with a Centaur instru-
ment. Erythropoietin (EPO) was measured in serum using an Immulite
system (Siemens Healthcare Diagnostic SA, Zurich, Switzerland). Crea-
tine kinase was measured on a fully automated Cobas Integra 400 plus
analyser (Roche Diagnostics, Rotkreuz, Switzerland). The reticulocyte
percentage (Ret%) was measured using a fully automated hematology
analyser (Sysmex XN 2000, Sysmex AG, Yverdon-les-Bains, Switzer-
land). The serum hepcidin concentration was measured by liquid chro-
matography coupled with high-resolution mass spectrometry (LC-
HRMS) according to a method described by Leuenberger et al.
2.4 |Statistical analyses
A Shapiro test was used to determine the normality of the data. For
normally distributed data, comparisons between and within groups
were performed using an ANOVA followed by post-hoc pairwise com-
parisons (t-test adjusted by the Bonferroni correction and Tukey
HSD). Non-parametric comparisons were performed using the
Kruskal-Wallis and Wilcoxon tests. Correlations were assessed using a
Spearman's correlation test. A value of P< 0.05 was considered signif-
icant. All tests were conducted using R studio software (Version
1.0.44). Data are presented as mean ± SEM, unless otherwise noted.
After injection of recombinant EPOs and the analog peginesatide,
ERFE substantially increased in a minimum of 50% in all subjects
(Figure 1A–C). In the Dynepo study, ERFE reached the highest
concentrations on day 3 after the second injection. Interestingly, the
responses were dependent on the individual, with two of the six sub-
jects showing only small changes. Additional injections did not further
increase ERFE levels. For peginesatide, ERFE was increased by an
average 8-fold on day 6 from 0.9 to 6.3 ng/mL, with concentrations
decreasing back to the baseline on day 13. In the CERA group, sub-
jects had an average 6-fold increase from 0.64 to 3.9 ng/mL, which
returned to baseline on day 10. A positive significant Spearman's cor-
relation (rho = 0.73; P= 1.34e-08) was observed between the ERFE
concentration and Ret% after stimulation by C.E.R.A. injection
Table 1 summarizes the iron (ferritin, TSAT, and hepcidin) and
erythropoiesis variables at baseline and after blood withdrawal pre-
ceded by saline (saline group) or iron (iron group) injection. The clinical
study design is shown in the supporting information (Figure S1). The
baseline concentration of iron markers was significantly higher in the
iron group than in the saline group, presumably due to the 14 day pre-
ceding iron injection. In both groups, ferritin and TSAT were
decreased after blood withdrawal. As expected, EPO was significantly
induced from day +1 up to day +15 to compensate for blood loss due
to withdrawal. By contrast, hepcidin was significantly decreased in the
saline group, but no change was observed in the iron-treated group,
after blood withdrawal.
In the saline group, the ERFE concentration rose on day +1
(Figure 2A). This elevation lasted until day +9, after which the ERFE
concentration returned close to the initial value of the baseline. For
the iron group, a significant ERFE increase was observed from day +3
until day +6 (Figure 2B).
Healthy blood donors (30 females and 40 males) were subjected
to ERFE measurements (Figure S3). The median ERFE concentration
in males and females was 0.57 ng/mL (95% CI, 0.53–0.65 ng/mL) and
0.48 ng/mL (95% CI, 0.45–0.55 ng/mL), respectively. The maximum
and minimum concentration in males and females were 0.17 and 1.11
and 0.16 and 1.1 ng/mL, respectively. No significant sex difference in
ERFE concentrations was observed (Figure S3).
To assess the impact of exercise-induced muscle damage, CK
values from human subjects with a high exercise load were compared
with ERFE concentrations (Figure 3A). No significant correlation
(Spearman's rho = 0.30, P= 0.1) was observed between ERFE and CK
concentrations. Also, ERFE concentration was unchanged in iron-
injected subjects (Figure 3B). By contrast, other hematological and
iron biomarkers, such as Ret%, TSAT, ferritin, and hepcidin, were
impacted by iron injection (Table S1).
4.1 |Impact of ESAs injection and blood
withdrawal on ERFE level
This study investigated whether serum ERFE levels could be used to
detect small changes in erythropoiesis. The impacts of different ESAs
and the withdrawal of one bag of blood (450 mL) on ERFE
RAMIREZ CUEVAS ET AL.3
concentration were investigated. The goal was to test and validate a
new specific and sensitive sandwich immunoassay. Previously, the
association between ERFE and biomarkers of erythropoiesis and iron
metabolism has been evaluated using a commercially available sand-
wich ELISA kit constituting mouse monoclonal antibodies.
ously evaluated this commercially available kit, and found that ERFE
quantification by this method was not reproducible.
following rhEPO administration were highly variable, and not coher-
Other commercial ELISAs produced with mouse antibodies
were also tested unsuccessfully.
Previously, Ganz et al. developed a rabbit monoclonal antibody-
based sandwich immunoassay for the detection of human ERFE.
14 ng/mL limit of quantification was reported for this assay. Contrast-
ingly, the limit of quantitation for our validated ELISA was
0.04 ng/mL. However, both assays and standards should be directly
compared in order to draw conclusions.
The response to blood withdrawal was examined in subjects
injected previously with iron or saline solution (placebo). ERFE was
increased in both contexts, reaching a maximum concentration after
6 days. In placebo patients, this ERFE increase was coincident with a
decline in serum hepcidin concentrations, but this correlation was not
present when iron was supplemented before blood withdrawal. Previ-
ously, Mirciov et al. demonstrated that high circulating iron levels
overcome ERFE inhibition of hepcidin in mice.
This effect did not
alter the expression of ERFE itself, and seemed linked to the interfer-
ence of di-ferric transferrin. This explanation could be also extrapo-
lated to the present study, due to the high TSAT in our iron injection
group. Our data suggest that, in humans, the inhibitory effect on
hepcidin is not needed when enough circulating iron is available, simi-
lar to mice.
The personalized follow-up of Ret% is the most common indirect
biomarker for the detection of erythropoiesis stimulation in the
FIGURE 1 ERFE levels after injection
of different ESAs. (A) Effect of
recombinant erythropoietin (rEPO) delta
administration on individual (left panel)
and mean (right panel) ERFE
concentrations. (B) Effect of peginesatide
administration on individual (left panel)
and mean (right panel) ERFE
concentrations. (C) Effect of CERA
administration on individual (left panel)
and mean (right panel) ERFE
concentrations. Vertical black lines
represent ESA administration times. D,
days. *P< 0.05, **P< 0.01. Variance was
calculated within groups compared
4RAMIREZ CUEVAS ET AL.
context of the ABP. Due to the cellular feature of Ret%, analyses
should be performed within approximately 48 hours after collection.
By contrast, many serum biomarkers can be analysed up to 10 years
after storage at −20C. In fact, some samples used in the present
study were collected 10 years ago in previous clinical studies
Although the ERFE concentration significantly corre-
lates with Ret%, the relative increase observed in the ERFE concentra-
tion was higher than that of Ret%. Indeed, after ESA injection
(peginesatide) an increase in ERFE up to 8-fold was observed.
4.2 |Assessment of different confounding factors
on ERFE concentration
ERFE was identified as an erythroid regulator of iron metabolism.
However, ERFE had earlier been described as myonectin, or C1q
tumor necrosis factor α-related protein isoform 1500 (CTRP15), and in
mice, was highly expressed in muscle tissue.
In this context, muscle
ERFE levels were increased after physical activity in rodents. Because
myonectin is produced also in the muscle of humans, serum samples
from a previously conducted exercise study were used to determine if
muscle damage could cause myonectin to potentially leak into the
blood, affecting circulating ERFE. In the present study, no increase of
circulating ERFE was detectable after muscle damage, as indicated by
the CK levels (Figure 3A). These data suggest that this approach is a
specific measurement of circulating ERFE secreted from erythroid ori-
gin after erythropoietic stimulation, rather than regulation of ERFE by
myocyte metabolism. The apparent specificity of our sandwich immu-
noassay is currently under investigation in our laboratory. However, a
difference of ERFE isoforms caused by specific glycosylation in ery-
throid cells or a different secretion of the quaternary structure (mono-
meric vs multimeric proteins) in muscle versus erythroid cells is a
theoretically possible explanation.
A single infusion of iron was reported to improve fatigue, mental
health, cognitive function, and erythropoiesis in iron-deficient women
with normal or borderline Hb concentrations.
Some endurance ath-
letes inject iron to maintain a high serum ferritin concentration.
Intravenous iron supplementation improves fatigue and overall mood
in runners without a clinical iron deficiency.
Iron injection is not
considered to be a doping method, but could potentially interfere with
antidoping biomarkers involved in iron metabolism. In the present
study, we identified that iron injection did not affect the ERFE con-
centration (Figure 3B). As expected, iron injection dramatically
affected ferritin, TSAT and hepcidin (Table S1). Interestingly, increased
Ret%, an actual hematological marker of the ABP, was observed after
iron injection, as previously demonstrated in females.
these data reinforce the specificity of the presented ERFE immunoas-
say for antidoping purposes.
Regarding the assessment of other confounding factors, no
influence of sex on ERFE concentrations was observed (Figure S3).
To ensure the specificity of the ERFE response to ESA administra-
tion, the impact of altitude exposure as a confounding factor on
ERFE should be characterized. Because ERFE and reticulocytes
TABLE 1 Iron and erythropoiesis biomarker analyses after blood withdrawal
Saline (n = 8) Baseline Day 0 Day +1 Day +2 Day +3 Day +6 Day +9 Day +15 Day +30
Ferritin (μg/mL) 50.39 ± 9.42 48.29 ± 8.48 44.51 ± 8.57 41.46 ± 7.86 40.28 ± 7.99* 31.13 ± 5.30* 26.79 ± 4.71** 19.93 ± 3.05** 22.76 ± 4.12**
TSAT (%) 26.98 ± 3.97 27.01 ± 3.02 27.28 ± 3.58 28.71 ± 4.26 25.11 ± 3.07 21.04 ± 3.69 15.70 ± 1.95* 15.31 ± 1.65* 19.08 ± 4.22
Hepcidin (nM) 1.50 ± 0.30 1.45 ± 0.40 1.12 ± 0.39* 0.70 ± 0.20** 0.56 ± 0.14** 0.55± 0.11** 0.43 ± 0.10** 0.39 ± 0.15** 0.43 ± 0.10**
EPO (mUI/mL) 10.01 ± 0.93 11.68 ± 0.97 16.16 ± 2.08** 16.38 ± 2.08** 15.69 ± 1.86** 16.15 ± 1.76** 15.75 ± 1.80** 13.71 ± 1.07** 11.16 ± 0.92
Iron (n = 8) Baseline Day 0 Day + 1 Day + 2 Day + 3 Day + 6 Day + 9 Day + 15 Day + 30
Ferritin (μg/mL) 224.51 ±22.27
220.29 ± 20.41 176.56 ± 18.26* 158.99 ± 17.69* 154.29 ± 18.99* 121.50 ± 13.79* 102.01 ± 13.29** 80.69 ± 12.97** 64.69 ± 12.66**
TSAT (%) 34.84 ± 5.64
34.81 ± 5.19 38.81 ± 5.43 34.53 ± 4.15 30.12 ± 3.64 34.93 ± 3.32 32.35 ± 5.42 27.66 ± 5.11 23.30 ± 4.06*
Hepcidin (nM) 3.69 ± 1.90
3.49 ± 1.62 3.61 ± 1.27 3.40 ± 2.14 1.95 ± 0.99 2.59 ± 1.09 1.99 ± 1.05 3.07 ± 1.49 3.36 ± 2.01
EPO (mUI/mL) 11.81 ± 1.36 10.01 ± 1.81 19.46 ± 1.74** 16.36 ± 1.60** 18.24 ± 1.10** 14.70 ± 1.93* 14.14 ± 1.43* 16.60 ± 2.62* 10.75 ± 1.67
Data are expressed as mean ± SEM. Significant changes relative to baseline (the mean of day −4 and day −1) are indicated by bold text with grey highlighting. Day 0 corresponds to the mean of hours +3, +6
and + 12 after saline or iron injection. Saline, saline-supplemented group; iron, iron-supplemented group. TSAT, transferrin saturation; EPO, erythropoietin. *p< 0.05, **p< 0.01. Variance was calculated within
groups compared with baseline.
P< 0.01. Variance was calculated between groups compared with baseline.
RAMIREZ CUEVAS ET AL.5
correlate closely (Figure S2), the influence of altitude exposure
on Ret% measurement should also be valuable for ERFE as
In conclusion, we tested a new immunological assay for human
ERFE that can sensitively detect the stimulation of erythropoiesis,
including ESA abuse and blood withdrawal. In an antidoping context,
this assay could be a useful tool for retrospective investigations in
which analysis of Ret% is no longer possible due to sample degrada-
tion. Moreover, this assay could be useful to investigate the interac-
tions between erythropoiesis and iron metabolism in human clinical
The authors thank the staff of Transfusion Interrégionale CRS for their
assistance. The authors thank Prof. Thomas Ganz for providing the
hERFE plasmid. We are grateful to Fondation Dr Henri Dubois-Fer-
rière Dinu Lipatti for financial support.
Nicolas Leuenberger https://orcid.org/0000-0001-7106-6304
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FIGURE 3 Muscle damage and
iron injection did not affect ERFE
levels. (A) Effect of muscle damage
(creatine kinase: CK) on ERFE
concentration. (B) ERFE levels in eight
individuals after iron injection
(500 mg of Ferinject
FIGURE 2 ERFE levels after blood withdrawal. Effect of blood withdrawal on ERFE concentration in (A) saline (B) and iron groups. Vertical
red lines represent blood withdrawal times. D, days; H, hours. *P< 0.05, **P< 0.01. Variance was calculated within groups compared with
baseline (D-1 and D-4)
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Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Ramirez Cuevas K, Schobinger C,
Gottardo E, et al. Erythroferrone as a sensitive biomarker to
detect stimulation of erythropoiesis. Drug Test Anal. 2019;1–7.
RAMIREZ CUEVAS ET AL.7