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Original Report: Laboratory Investigation
Am J Nephrol 2017;45:49–59
DOI: 10.1159/000451060
Distinct in vitro Complement Activation
by Various Intravenous Iron Preparations
JuliaCordeliaHempel a FelixPoppelaars a MarianaGayadaCosta a
CasperF.M.Franssen a ThomasP.G.deVlaam a MohamedR.Daha a,b
StefanP.Berger a MarcA.J.Seelen a CarloA.J.M.Gaillard a
a Department of Internal Medicine, Division of Nephrology, University of Groningen, University Medical Center
Groningen, Groningen , and
b Department of Nephrology, University of Leiden, Leiden University Medical Center,
Leiden , The Netherlands
tran significantly induced complement activation in the
blood of healthy volunteers and HD patients. Furthermore,
in the ex-vivo assay, ferric carboxymaltose and iron sucrose
only caused significant complement activation in the blood
of HD patients. No in-vitro or ex-vivo complement activation
was found for ferumoxytol and iron isomaltoside. IV iron
therapy with ferric carboxymaltose in HD patients did
not lead to significant in-vivo complement activation.
Conclusion: This study provides evidence that iron dextran
and ferric carboxymaltose have complement-activating ca-
pacities in-vitro, and hypersensitivity reactions to these
drugs could be CARPA-mediated. © 2016 The Author(s)
Published by S. Karger AG, Basel
Introduction
A majority of patients with chronic kidney disease
(CKD) receive intravenous (IV) iron for the treatment of
anemia
[1] . However, controversy exists regarding the
safety of IV iron preparations since hypersensitivity reac-
tions have been reported for all iron drugs
[2] . Although
these reactions appear sporadic, they can be acute and life
Key Words
Intravenous iron · Complement activation related pseudo
allergy · Hypersensitivity reaction · Complement activation ·
Iron sucrose · Iron dextran
Abstract
Background: Intravenous (IV) iron preparations are widely
used in the treatment of anemia in patients undergoing he-
modialysis (HD). All IV iron preparations carry a risk of caus-
ing hypersensitivity reactions. However, the pathophysio-
logical mechanism is poorly understood. We hypothesize
that a relevant number of these reactions are mediated by
complement activation, resulting in a pseudo-anaphylactic
clinical picture known as complement activation-related
pseudo allergy (CARPA). Methods: First, the in-vitro comple-
ment-activating capacity was determined for 5 commonly
used IV iron preparations using functional complement as-
says for the 3 pathways. Additionally, the preparations were
tested in an ex-vivo model using the whole blood of healthy
volunteers and HD patients. Lastly, in-vivo complement ac-
tivation was tested for one preparation in HD patients.
Results: In the in-vitro assays, iron dextran, and ferric car-
boxymaltose caused complement activation, which was
only possible under alternative pathway conditions. Iron su-
crose may interact with complement proteins, but did not
activate complement in-vitro. In the ex-vivo assay, iron dex-
Received: April 18, 2016
Accepted: July 17, 2016
Published online: November 26, 2016
Nephrolo
gy
American Journal of
Marc A.J. Seelen, MD, PhD
University Medical Center Groningen
Department of Internal Medicine, Division of Nephrology, AA53
Postbus 196, NL–9700 AD Groningen (The Netherlands)
E-Mail m.seelen @ umcg.nl
© 2016 The Author(s)
Published by S. Karger AG, Basel
www.karger.com/ajn
C.J.H. and F.P. contributed equally.
is article is licensed under the Creative Commons Attribution-
NonCommercial-NoDerivatives 4.0 International License (CC BY-
NC-ND) (http://www.karger.com/Services/OpenAccessLicense).
Usage and distribution for commercial purposes as well as any dis-
tribution of modi ed material requires written permission.
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DOI: 10.1159/000451060
50
threatening. The exact frequency of the hypersensitivity
reactions is unknown. This is attributed to a lack of data,
due to underreporting and differential reporting
[3] .
The underlying mechanism of reactions due to the hy-
persensitivity of IV iron remains unclear. However, elu-
cidating the pathophysiology is critical to improve pre-
diction, prevention and management of these adverse
events. In contrast to the immunoglobulin E (IgE)-medi-
ated anaphylaxis observed in older compounds of IV
iron, hypersensitivity reaction due to new IV iron prepa-
rations are thought to result from complement activa-
tion-related pseudo allergy (CARPA)
[4, 5] . Nonetheless,
this has not been tested systematically. CARPA is an ad-
verse event seen after the administration of monoclonal
antibodies, intravenously administered drugs and
nanoparticle-containing drugs
[4, 5] . CARPA was postu-
lated since all available preparations consist of iron-car-
bohydrate nanoparticles
[6] .
Activation of the complement system occurs via
3pathways: the classical pathway (CP), the lectin pathway
(LP) and the alternative pathway (AP). The CP is acti-
vated by antibody–antigen complexes, the LP by carbo-
hydrates and the AP by microbial surfaces. This results in
the formation of the C3- and C5-convertases and the gen-
eration of anaphylatoxins. Subsequently, activation of the
terminal pathway leads to the formation of the membrane
attack complex (C5b-9)
[7] . In CARPA, such a cascade is
initiated predominantly by the generation of comple-
ment activation products, leading to the stimulation of
mast cells and basophil granulocytes resulting in secre-
tion products, which cause various responses in effector
cells such as platelets, endothelial cells and smooth mus-
cle cells. Clinically, these processes may give rise to bron-
chospasm, laryngeal edema, tachycardia, hypo- or hyper-
tension and hypoxia
[5] .
The aim of this study was to determine the effect of
5currently available IV iron preparations on the comple-
ment system. By evaluating different IV iron drugs in an
in-vitro and ex-vivo model for complement activation, we
intended to test for the probability of CARPA by IV iron
drugs. Lastly, in-vivo complement activation was tested for
one IV iron preparation in hemodialysis (HD) patients.
Materials and Methods
2.1 Subjects
We recruited 2 groups:
• Control subjects (5–10 per experiment, as indicated below).
• Patients on maintenance HD (n = 8). During one dialysis
session, blood samples were taken at 0, 120 and 240 min dur-
ing dialysis. Patients received 100 mg/2 ml ferric carboxy-
maltose (Ferinject
© ) IV over 1 h at 120 min into the dialysis
session.
2.2 Reagents
Iron sucrose (Venofer
©
) and ferric carboxymaltose (Ferinject
©
)
were purchased from Vifor Nederland, Breda, The Netherlands;
ferumoxytol (Rienso
©
) from Takeda Nederland, Hoofddorp, The
Netherlands; low molecular weight iron dextran (CosmoFer
©
) and
iron isomaltoside 1000 (Monofer
©
) from Cablon Medical, Leusden,
The Netherlands.
For the whole blood experiments, lepirudin (Refludan
©
,
Hoechst, Frankfurt am Main, Germany) was used as anticoagu-
lant.
2.3 Normal Human Serum
Blood was taken from 10 healthy volunteers and directly stored
on ice. Samples were centrifuged, then pooled and stored at –80
° C
until further analysis.
2.4 Complement Pathway Activity in Human Serum
Functional assays were used to allow quantification of com-
plement activation via the CP, the LP and AP in human serum.
These assays were previously described
[8] . In brief, 96-well
plates were coated overnight with human immunoglobulin M for
the CP, mannan for the LP or lipopolysaccharide (LPS) for the
AP. Plates were washed 3 times after each step with PBS contain-
ing 0.05% Tween-20. Plates were blocked with 1% bovine serum
albumin (BSA) in PBS for 1 h at 37
° C. Serum was diluted in
gelatin veronal buffer (GVB) buffer adapted specifically for each
pathway. For the CP and LP, serum was diluted in GVB with
Ca
2+ –Mg 2+ . For the AP, serum was diluted in GVB with magne-
sium only. After 1 h at 37
° C, deposition the of properdin, C4, C3
or C5b-9 was detected using rabbit anti-human properdin
(obtained from the laboratory of Nephrology, Leiden, The
Netherlands), mouse anti-human C4 (obtained from the labora-
tory of Nephrology, Leiden, The Netherlands), RFK22 (anti-hu-
man C3, obtained from the laboratory of Nephrology, Leiden,
The Netherlands) and AE11 (anti-human C5b-9, DAKO,
Glostrup, Denmark), respectively. Binding of antibodies was de-
tected using the appropriate primary and secondary antibody.
For visualization, TMB and H
2 SO 4 were added before the absorp-
tion was measured at 450 nm.
Prior to incubation on the enzyme linked immunosorbent as-
say (ELISA) plate, all serum samples were pre-incubated at 37
° C
for 30 min with iron in a dose ranging from 0.0625 to 0.5 mg/ml.
Next, samples were further diluted to the final concentration with
the appropriate buffer.
2.5 Complement Activation Assays by IV Iron
For the complement activation assay, iron preparations or BSA
were coated overnight on a 96-well plate followed by blocking with
1% BSA/PBS at 37
° C for 1 h. The wells were exposed to pooled
human serum diluted in adapted GVB (see 2.3) or with ethylene-
diaminetetraacetic acid (EDTA; 20 m
M ) for 1 h at 37 ° C. The plate
was then incubated with antibodies against properdin, C3 or
C5b-9 (see 2.3). Detection was completed using appropriate pri-
mary and secondary antibody. The plate was washed with PBS
Tween-20 (0.05%) after each step. Visualization was similar as de-
scribed in section 2.3.
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2.6 Complement Pathway Activity in Human Whole Blood
The experimental set-up has previously been described
[9] . In
short, blood was drawn in LPS-free tube with 50 µg/ml lepirudin.
Whole blood was then incubated for 0 or 90 min at 37
° C with IV
iron (0.5 mg/ml ferrous iron) while continuously rotated. PBS was
added to the negative controls. The reaction was stopped with
EDTA (final concentration of 20 m
M ). Samples were then centri-
fuged and plasma was stored at –80
° C until further analysis.
2.7 Quantification of the Antigenic Levels of C1q, C3d, C3
Mannose-Binding Lectin, Properdin and C5b-9
The ELISA for C1q, C3d, C3 mannose-binding lectin (MBL),
properdin, and C5b-9 were performed as described previously
[10–12] .
2.8 Statistics
Statistical analyzes were performed using BM SPSS Statistics
Version 22 and p values <0.05 were considered statistically sig-
nificant. The Kruskal–Wallis test and Mann–Whitney U test were
used to assess differences between groups of non-parametric data
and one-way analysis of variance and t test for normally distrib-
uted data. If needed, data was in-transformed for normality.
2.9 Ethics
All participants gave informed consent. The Medical Ethical
Committee of the University Medical Center Groningen has re-
viewed the study design and it was confirmed that an official ap-
proval of this study by the committee is not required since the
Medical Research Involving Human Subjects Act (WMO) does
not apply.
Results
3.1 In-vitro effect of IV iron preparations on
complement activity
The interaction of the different IV iron drugs with
complement was determined using functional comple-
ment assays for each pathway. Normal human serum
(NHS) was pre-incubated with different IV iron drugs
prior to the assay; subsequently, residual complement ac-
tivity was measured. In this assay, decreased residual ac-
tivity reflects either activation or inhibition of comple-
ment by the IV iron compound during the pre-incuba-
tion period.
3.1.1 Decreased Residual Activity of the CP by Iron
Sucrose
First, residual complement activity was tested for
theCP after incubation with the IV iron drugs ( table1 ).
Iron sucrose was the only preparation that significantly
reduced residual complement activity. Furthermore,
the effect of iron sucrose on CP activity (p = 0.016)
wasdose-dependent ( fig.1 ). At a concentration of 0.5
mg/ml, iron sucrose reduced C4, C3 and C5b-9 deposi-
tion by 92, 88 and 96%, respectively (p < 0.001). For
iron dextran, ferric carboxymaltose, iron isomaltoside
Table 1. Activation of complement components of the CP, LP and AP by IV iron drugs
Residual complement
activity1IV iron preparations, %
control iron dextran ferric carboxy-maltose iron isomaltoside
1000
ferumoxytol iron sucrose
CP 100±5.5 126.8±23.4 94.3±13.8 108.2±3.9 110.8±4.2 10.4±4.5***
LP 100±5.2 88±4.4 88.7±3.5 84.3±2.4 91.4±3.3 4.7±0.4***
AP 100±4.6 6.3±0.9*** 62.3±11.8*75±14.5 89.2±16.5 80±16
Complement
activation2 IV iron preparations, %
positive
contr ol
iron dextran ferric carboxy-maltose iron isomaltoside
1000
ferumoxytol iron sucrose BSA
CP 100±0.5 2.9±0.1 2.5±0.1 2.5±0.1 3.0±0.1 3.0±0.0 2.9±0.1
LP 100±3.4 3.1±0.1 3.0±0.1 4.1±0.1 3.8±0.3 4.0±0.1 4.0±0.1
AP 100±8.1 138.5±5.5*** 122.2±10.9** 9.1±0.4 8.6±0.3 8.1±0.4 9.7±2.1
1Pooled serum was pre-incubated with 0.5 mg/ml ferrous iron for 30 min at 37°C. PBS was used for the controls. The serum was
then used in the functional assay for the CP, LP or AP to measure residual activity. Deposition of C5b-9 was used as readout and the
amount obtained in the control was set at 100%
2
Iron preparations were coated overnight on a 96-well plate. The wells were exposed to pooled human serum diluted in a buffer
adapted specifically for each pathway. Deposition of C5b-9 was used as readout and the amount obtained in the positive control was
set at 100%. Data are shown as mean ± SEM of 3 experiments (*p < 0.05, **p < 0.01, *** p < 0.001).
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and ferumoxytol, there was no change in residual
complement activity, indicating low to no effect on
theCP.
3.1.2 Decreased residual activity of the LP by iron
sucrose
Next, residual complement activity for the LP was as-
sessed ( table1 ). Once again, iron sucrose significantly re-
duced residual complement activity in a dose-dependent
manner (p < 0.001) indicating prominent activation of
the LP during the pre-incubation period ( fig.1 ). Deposi-
tion of C4, C3 and C5b-9 were lowered by 88, 95 and 95%
at 0.5 mg/ml for iron sucrose (p < 0.001). For iron dex-
tran, ferric carboxymaltose, iron isomaltoside and feru-
moxytol, there was no change in residual complement ac-
tivity for the LP.
0
50
100
150
Control 62.5 125
Iron sucrose – CP Iron sucrose – LP
250 500
Residual complement
activity (% C4 deposition)
***
**
0
50
100
150
Control 62.5 125 250 500
Residual complement
activity (% C4 deposition)
***
***
***
**
0
50
100
150
Control 62.5 125 250 500
Residual complement
activity (% C3 deposition)
***
***
***
***
0
50
100
150
Control 62.5 125 250 500
Residual complement
activity (% C5b-9 deposition)
Concentration (njg/ml)
***
***
***
**
0
50
100
150
Control 62.5 125 250 500
Residual complement
activity (% C5b-9 deposition)
Concentration (njg/ml)
***
***
0
50
100
150
Control 62.5 125 250 500
Residual complement
activity (% C3 deposition)
**
***
Fig. 1. The dose-dependent decrease of residual activity of the CP,
LP and AP by iron sucrose, iron dextran and ferric carboxymalt-
ose. Pooled serum was pre-incubated with increasing concentra-
tions of intravenous iron (x-axis, log2 scale) for 30 min at 37°C.
PBS was used for the controls. The serum was then used in the
functional assay for the CP, LP and AP to measure residual activ-
ity. Deposition of C4, properdin, C3 and C5b-9 were used as read-
out and the amount obtained in the control was set at 100% (y-
axis). Data are shown as mean ± SEM of 3 experiments (* p < 0.05,
**p < 0.01, ***p < 0.001).
(Figure continued on next page.)
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3.1.3 Decreased Residual Activity of the AP by Iron
Dextran and Ferric Carboxymaltose
Lastly, residual activity of the AP was analyzed
( table1 ). The addition of iron dextran and ferric carboxy-
maltose caused a significant reduction in residual com-
plement activity at the level of C5b-9 generation ( fig.1 ).
In accordance, pre-incubation with iron dextran and fer-
ric carboxymaltose resulted in a significant dose-depen-
dent reduction of residual complement activity at the lev-
el of properdin and C3 deposition (p < 0.01). For iron
dextran, deposition of properdin, C3 and C5b-9 were
lowered by 71, 85 and 94% at 0.5 mg/ml (p < 0.01) and
lowered by 34, 24 and 30% at 0.5 mg/ml (p < 0.01) for
ferric carboxymaltose, respectively. Ferumoxytol, iron
sucrose and iron isomaltoside did not affect the comple-
ment activity of AP.
3.2 In-vitro Testing of Complement Activation by IV
Iron Drugs
Next, we investigated whether IV iron preparations
can directly activate the complement system. In an
ELISA-based set-up, we immobilized the IV iron drugs
on the plate and added NHS diluted in buffers that al-
low the specific activation of CP, LP or AP. Under these
0
50
100
150
Ctrl 62.5 125
Iron dextran – AP Ferric carboxymaltose – AP
250 500
Residual complement activity
(% properdin deposition)
******
***
***
0
50
100
150
Ctrl 62.5 125 250 500
Residual complement activity
(% properdin deposition)
***
***
**
**
0
50
100
150
Ctrl 62.5 125 250 500
Residual complement
activity (% C3 deposition)
****
0
50
100
150
Ctrl 62.5 125 250 500
Residual complement
activity (% C5b-9 deposition)
Concentration (njg/ml)
*
0
50
100
150
Ctrl 62.5 125 250 500
Residual complement
activity (% C3 deposition)
*
**
** **
0
50
100
150
Ctrl 62.5 125 250 500
Residual complement
activity (% C5b-9 deposition)
Concentration (njg/ml)
***
***
***
**
1
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0
1
2
3
4
C3 deposition (OD450 nm)
Serum (%)
0 5 10 15 20
Ferric carboxymaltose
BSA
Iron dextran
Iron
dextran
Ferric
carboxymaltose
0
1
2
3
C5b-9 deposition (OD450 nm)
BSA 1 5 10 50 1 5 10 50 (μg)
b
0
1
2
3
4
C5b-9 deposition (OD450 nm)
Serum (%)
0 5 10 15 20
cd
0
1
2
3
4
Properdin deposition (OD450 nm)
Serum (%)
0 5 10 15 20
Ferric carboxymaltose
BSA
Iron dextran
Ferric carboxymaltose
BSA
Iron dextran
e
0
1
2
3
4
Iron
dextran
C5b-9 deposition (OD450 nm)
Ferric
carboxymaltose
BSA
15% NHS
MgEGTA
EDTA
a
Fig. 2. a–e AP-mediated complement activation on iron dextran
and ferric carboxymaltose. a ELISA wells were coated with iron
dextran, ferric carboxymaltose at 50 g and 1% BSA as negative
control. Wells were blocked by 1% BSA/PBS for 60 min at 37°C. A
fixed concentration of 15% pooled human serum diluted in GVB++
MgEGTA or EDTA was added to the wells with detection by mouse
anti-human C5b-9 antibody. Data are shown as mean ± SEM of 3
experiments. b Iron dextran and ferric carboxymaltose at various
concentrations or 1% BSA were coated to the wells. All coated wells
had 1% BSA/PBS added for 60 min at 37°C as a blocking agent.
Fifteen percent pooled human serum diluted in GVB++ MgEGTA
was added followed by detection using mouse anti-human C5b-9
antibody. c–e Iron dextran and ferric carboxymaltose were coated
at 50 g and 1% BSA as negative control to the wells. The plate was
blocked using 1% BSA/PBS at 37°C for 60 min. Increasing concen-
trations of pooled human serum diluted in GVB++ MgEGTA were
added to the wells followed by measuring deposition for C5b-9, C3
or properdin.
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conditions, iron dextran and ferric carboxymaltose had
the capacity to activate the AP. Ferumoxytol and iron
isomaltoside showed no complement activation for all
pathways ( table1 ). In this set-up, iron sucrose failed to
show complement activation for the LP or the CP.
3.2.1 AP Activation by Iron Dextran and Ferric
Carboxymaltose
We further determined conditions required for iron
dextran and ferric carboxymaltose-mediated comple-
ment activation. An ELISA plate was coated with iron
dextran, ferric carboxymaltose or BSA and then exposed
to 15% pooled human serum diluted in either magnesium
ethylene glycol tetraacetic acid (MgEGTA) or EDTA.
Subsequently, C5b-9 deposition was assessed. Iron
dextran and ferric carboxymaltose coating caused strong
C5b-9 depositions compared to BSA controls ( fig.2 a).
The addition of EDTA completely inhibited complement
deposition. Hence, complement deposition was the result
of calcium- and magnesium-dependent complement ac-
tivation. The degree of complement activation was de-
pendent on the concentration of iron dextran and ferric
carboxymaltose immobilized on the plate ( fig.2 b). Fur-
thermore, we titrated NHS in MgEGTA and showed that
C5b-9 depositions were dose-dependent when compared
to the negative control, BSA ( fig.2 c). Lastly, we tested
whether AP activation also involves deposition of other
complement components of the AP. We found that simi-
lar to C5b-9 deposition, C3 ( fig.2 d) and properdin depo-
sition ( fig. 2 e) occurred in a dose-dependent manner,
while no C4 deposition was observed (data not shown).
Altogether, these results show that dextran and ferric car-
boxymaltose-mediated complement activation is only
possible under AP conditions.
3.3 Ex-vivo Analysis of the Effect of IV Iron Drugs on
Complement Activation in Healthy Volunteers
The effect of IV iron drugs on fluid phase complement
activation was determined by incubating IV iron prepara-
tion (0.5 mg/ml ferrous iron) for 90 min in human whole
blood. Subsequently, complement activation in the sam-
ples was determined by measuring soluble C5b-9 (sC5b-9)
levels. Increased sC5b-9 levels demonstrate complement
activation. Additionally, properdin, MBL and C1q levels
were measured to determine which pathway was involved.
3.3.1 Ex-vivo Terminal Pathway Complement
Activation by Iron Dextran
The addition of iron dextran to whole blood samples
of healthy volunteers led to the activation of vast terminal
pathways ( fig.3 a). Levels of sC5-b9 were 13-fold higher
than in the controls (p < 0.001). Incubation with iron su-
crose, ferric carboxymaltose, iron isomaltoside or feru-
moxytol did not lead to significant complement activa-
tion.
3.3.2 Ex-vivo Complement Activation by Iron
Dextran Is Mediated via the AP
In order to determine which complement pathway was
activated, C1q, MBL and properdin were measured at
0and 90 min (online suppl. data, see www.karger.com/
doi/10.1159/000451060). For iron dextran, a significant
decrease in properdin concentration of 42% was found
compared to control (p = 0.032). The concentration of
C1q and MBL remained largely unchanged ( fig.3 b). No
significant alterations of in C1q, MBL and properdin con-
centration were found for iron sucrose, ferric carboxy-
maltose, iron isomaltoside and ferumoxytol.
3.4 Effect of IV Iron Drugs on Complement in Whole
Blood from HD Patients
Next, we analyzed whether the observed effects of iron
dextran can be extrapolated from control subjects with-
out CKD onto HD patients with severe CKD, and wheth-
er other iron preparations induce complement activation
similar to iron dextran.
3.4.1 Ex-vivo Terminal Pathway Complement
Activation by Iron Dextran
Similar to healthy controls, iron dextran led to signifi-
cant complement activation in whole blood from HD pa-
tients (p < 0.001), indicated by the marked sC5b-9 gen-
eration ( fig.3 c). Furthermore, ferric carboxymaltose and
iron sucrose led to significant complement activation in
HD whole blood but not in healthy controls. However,
the complement activation by ferric carboxymaltose and
iron sucrose was 2- to 3-fold lower than it was in iron
dextran. Iron isomaltoside or ferumoxytol did not lead to
significant complement activation.
3.5 No in-vivo Complement Activation by Current IV
Iron Treatment in HD Patients
Lastly, we checked if the current IV iron therapy, used
at our dialysis unit, leads to in-vivo complement activa-
tion in HD patients ( fig.3 d, e). Prior to iron therapy, all
patients displayed strong complement activation within
the first 120 min. The sC5b-9 levels ( fig.3 d) increased
from 109 ng/ml (interquartile range (IQR) 85–122) to 247
ng/ml (IQR 211–274), while the C3d/C3-ratio ( fig.3 e) al-
most doubled from 7.68 (IQR 5.52–9.92) to 13.04 (IQR
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0
5,000
10,000
15,000
20,000
25,000
Control
Iron dextran
Ferric carboxymaltose
Iron isomaltoside
Ferumoxytol
Iron sucrose
sC5b-9 (ng/ml)
***
a
–50
–25
0
25
50
Control
Iron dextran
Control
Iron dextran
Control
Iron dextran
© Concentration (%)
C1q MBL Properdin
*
n.s.n.s.
b
0
2,000
4,000
6,000
8,000
10,000
Control
Iron dextran
Ferric carboxymaltose
Iron isomaltoside
Ferumoxytol
Iron sucrose
sC5b-9 (ng/ml)
***
***
**
c
sC5b-9 (ng/ml)
0
100
200
300
400
0120 240
Time after start HD (min)
***
*
d
0
5
10
15
20
25
0120 240
C3d/C3-ratio
Time after start HD (min)
*
e
Fig. 3.
a–e
The ex-vivo effect of iron preparations and in-vivo effect
of ferric carboxymaltose on complement activation. Whole blood
was incubated with 0.5 mg/ml of iron dextran, Iron sucrose, ferric
carboxymaltose, iron isomaltoside and ferumoxytol (x-axis) for 90
min at 37°C. PBS was used for the controls.
a
Concentration of
sC5b-9 was determined in plasma from healthy controls and used as
read-out for complement activation (y-axis). Data are mean and
SEM of 5 experiments using different donors each time.
b
The con-
centration of C1q, MBL and properdin was determined in samples
from healthy controls with 0.5 mg/ml of iron dextran at 0 and 90
min. The difference in concentration was calculated by dividing the
concentration at 90 min, by the concentration at 0 min and then
minus 100% (y-axis).
c
Concentration of sC5b-9 was determined in
plasma from HD patients (y-axis). Data are mean and SEM of 8 ex-
periments using different donors each time.
d
sC5-9 levels and
e
C3d/C3-ratio were determined in HD patients during one dialysis
session, in which they received 100 mg of ferric carboxymaltose at
120 min into the dialysis session. Data are mean and SEM of 8 sub-
jects (*p < 0.05, **p < 0.01, ***p < 0.001).
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6.55–16.32). Patients then received 100 mg of ferric
carboxymaltose intravenously throughout the following
1 h at 120 min into the dialysis session. At the end of
the dialysis, complement levels remained higher than
baseline but did not increase significantly compared to
levels at 120 min. Median sC5b-9 levels at 240 min were
252 ng/ml (IQR 188–264), while C3d/C3-ratio were 15.22
(IQR 11.40–16.29).
Discussion
Current EMA-approved IV iron drugs have markedly
better safety profiles than the traditional IV iron com-
pounds. However, hypersensitivity reactions still occur
and have led to controversy regarding the safety and the
risk–benefit ratio of these preparations
[2] . Unlike the
IgE-mediated reactions by older IV iron compounds, the
majority of hypersensitivity reactions by the new IV iron
preparations are thought to be caused by CARPA
[4–6] .
The results of our study are the first, to our knowledge, to
support this hypothesis by demonstrating the capacity of
several IV iron preparations to activate complement in
in-vitro and ex-vivo models using blood samples of
healthy volunteers and HD patients.
Initially, an in-vitro assay was used to investigate a
possible interaction between IV iron and complement in
serum. In this set-up, interaction (binding) and comple-
ment activation cannot be distinguished. During pre-in-
cubation, the IV iron drug reacts with the complement
system. If the IV iron preparation activates complement,
this consequently leads to decreased residual comple-
ment activity and therefore the deposition on the ELISA
plate will be reduced. However, if IV iron binds the com-
plement proteins, then this effect will also reduce comple-
ment deposition as the drug is only diluted but not re-
moved after the pre-incubation step.
In order to distinguish between true IV iron-mediated
activation and other forms of interaction, ELISA plates
were coated with different concentrations of IV iron prep-
arations and fixed concentrations of NHS were added.
Complement activation was increased in a dose-depen-
dent manner by iron dextran and ferric carboxymaltose
under AP-specific conditions. Combining these results,
we can conclude that the reduced complement deposition
after incubation with iron dextran and ferric carboxymalt-
ose in NHS in the functional assays was indeed due to
complement activation. However, for iron sucrose, we
have to consider an alternative explanation such as a direct
effect of iron sucrose on C2, C4 or the serine proteases.
We also tested the capacity of each drug to activate
complement in an ex-vivo model. By incubating whole
blood with iron, the preparations were not only exposed
to serum components but also to blood cells and mem-
brane-bound complement regulatory factors. In line with
the previous in-vitro experiments, iron dextran induced
significant complement activation, while, surprisingly,
ferric carboxymaltose did not. This might be because the
functional assays measure complement deposition on a
plate and thereby test solid phase activation while the
whole blood model tests fluid phase activation by mea-
suring soluble complement activation products. A similar
discrepancy has been found for LPS and IgA
[8, 13] . Fur-
thermore, the whole blood model and the functional as-
says differ in sensitivity. While coating with the iron prep-
aration and exposing it to NHS serum is a very sensitive
test, the whole blood model does not involve dilution of
the blood sample and is, therefore, a more physiological
approach.
Subsequently, we analyzed the effect of IV iron in a
group of HD patients who are regularly receiving IV iron.
In the ex-vivo experiments, whole blood from HD pa-
tients showed similar activation trends as whole blood
from healthy volunteers. In both groups, iron dextran
caused a significant increase in sC5b-9 generation. How-
ever, the overall complement activation was lower com-
pared to healthy volunteers. This can be considered a sign
of pre-existing chronic complement activation, which is
well described in HD patients
[11, 12, 14] . Concordantly,
in our in-vivo experiments, elevated C3d/C3 ratio and
C5b-9 serum levels were measured in blood samples tak-
en from these patients prior to dialysis.
The IV infusion of ferric carboxymaltose did not lead
to significant additional complement activation in HD
patients. Both, sC5b-9 levels as well as the C3d/C3 ratio
rose during the first half of the dialysis session and then
remained consistently elevated from the start of the IV
iron administration till the end of the HD session. While
these measurements were performed in a small patient
group, the results are in line with the ex-vivo findings,
which did not indicate strong complement activation ca-
pacity for ferric carboxymaltose. Moreover, the slow ad-
ministration as a continuous infusion over 1 h reduces
the risk of massive complement activation
[5, 15] . Lastly,
vast complement activation and subsequently relative de-
pletion of complement factors has taken place during the
first half of the HD session. We would therefore expect to
see more complement activation in non-dialysis CKD
patients after IV iron. In addition, we would hypothesize
that bolus injection would lead to more complement ac-
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Hempel etal.
Am J Nephrol 2017;45:49–59
DOI: 10.1159/000451060
58
tivation than slow administration. This is supported by
previous studies, showing that the rate of infusion is cru-
cial for both the risk of hypersensitivity reactions and
complement activation [5, 15, 16] . As none of our pa-
tients are currently treated with iron dextran, we were
unfortunately not able to test the complement activating
properties of this iron preparation in-vivo. To further un-
ravel the effects of different iron preparations in-vivo, a
trial comparing the ex-vivo and in-vivo effects of differ-
ent IV iron drugs in various patient populations would be
needed. Nonetheless, since these trials will not be able to
observe and compare the very rare clinical severe adverse
events, data of observational cohorts including adequate
sampling need to be gathered. In addition, further in-vi-
tro studies may help to better understand the mechanism
behind hypersensitivity reactions by IV iron prepara-
tions.
Clear guidelines exist regarding the maximum dose
and minimal duration of administration per IV iron drug.
For iron dextran and iron sucrose, the recommended
dose is 100–200 mg, to be administered intravenously
over 2–5 min for 5–10 consecutive HD sessions. Consid-
ering an average post-dialysis blood volume of 3,755 ±
941 ml , final blood concentrations would vary between 42
and 71 µg/ml. Other IV iron drugs are given in higher
doses or administrated more rapidly, resulting inmuch
higher local concentrations at the site of injection than
concentrations measured in the peripheral blood
[16, 17] .
In addition to that, Geisser and Burckhardt
[18] found
higher IV iron blood concentrations after repetitive dos-
ing. Thus, concentrations chosen for the experiments are
considered physiologically reasonable.
A limitation of our study is the extrapolating of our
findings into the clinical setting. Hypersensitivity reac-
tions to IV iron are rare and not in line with the comple-
ment activation seen in the in-vitro and ex-vivo results.
Thus, an extremely important question that remains to be
answered is concerning the difference in frequency of
clinically observed adverse events and the frequency and
magnitude of complement activation in our in-vitro ex-
periments. Factors such as route and rate of administra-
tion and patient characteristic (conditions of pre-existent
complement activation) determine the magnitude of
complement activation. However, mere activation of the
complement system is not sufficient to cause CARPA, but
it is a crucial first step in this reaction. In addition, beyond
the acute effects, it has been hypothesized that repetitive
complement activation, inflammation and oxidative
stress may cause endothelial dysfunction and vascular re-
modeling. Indeed, in an observational study, Michael et
al.
[19] report an 18% increase in mortality in HD patients
receiving high doses of IV iron. However, due to the ob-
servational study design, no conclusion could be drawn
regarding the causal relation between IV iron and mortal-
ity.
Previous studies defined a 5- to 10-fold increase of
complement activation as a realistic predictor for clinical
reactions
[20] . Given this information, it can be assumed
that iron dextran carries a risk of causing CARPA-medi-
ated hypersensitivity reactions. In accordance with our
findings, it has been shown that dextran-coated mag-
neticiron nanoparticles activate the complement system
via the AP. These agents are used as an MRI contrast
agent and are able to cause severe hypersensitivity reac-
tions in patients. The chemical structure of the iron dex-
tran preparation is similar to this contrast agent
[21] . We
hypothesize that the iron–carbohydrate nanoparti-
cleshave complement-activating properties and not the
iron itself, since ferric chloride did not cause significant
complement activation (data not shown). In addition,
there are several clinical studies stating the higher risk of
serious adverse events after the administration of iron
dextran formulations [19, 22] . Recently, Wang et al. in-
vestigated the risk of adverse events among the different
IV iron drugs. A 3 times higher rate of adverse events
was found for iron dextran compared to other IV iron.
Also, more anaphylactic reactions were seen after the
first administration of IV iron compared to repeated ad-
ministration
[23] . This phenomenon is in line with our
results and the description of CARPA
[24] . Ferric
carboxymaltose also showed complement activating ca-
pacity and could shift the regulatory balance in predis-
posed individuals toward unregulated complement acti-
vation.
In conclusion, the present study shows that different
IV iron formulations have the in-vitro capacity to acti-
vate complement in healthy individuals as well as in
HD patients undergoing long-term IV iron treatment.
The major finding of this study is that iron dextran sig-
nificantly activates complement via the AP in-vitro and
ex-vivo. In addition, ferric carboxymaltose also activat-
ed complement in-vitro via the AP. Furthermore, iron
sucrose may interact with complement proteins of the
LP and CP, but did not activate complement. Notably,
slow infusion of ferric carboxymaltose during HD did
not lead to additional complement activation. Our re-
sults indicate that current guidelines are efficient at
avoiding CARPA by IV iron and explain why these
routinely administered drugs show a limited number of
adverse events. Our results are the first to our knowl-
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Complement Activation by IV Iron Am J Nephrol 2017;45:49–59
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59
edge, to provide proof of concept of complement acti-
vation by IV iron and therefore provide new insights
into the pathophysiological mechanism for a well-de-
scribed adverse reaction to IV iron. Mere activation of
the complement system is not sufficient to cause CAR-
PA, but it is a crucial first step in this reaction. Further-
more, long-term complement activation is known to
cause free radical generation and accelerate arterioscle-
rosis. These findings warrant further translational stud-
ies in HD and iron naïve patients in order to gain new
insights into the pathophysiological mechanism of
these clinical adverse events and to develop a safer
treatment.
Acknowledgments
We thank Anita Meter-Arkema for her excellent technical
assistance.
Disclosure Statement
None of the authors have competing interests to declare.
Financial Support
This work was financially supported by the Graduate School of
Medical Sciences of the University of Groningen.
References
1 Saran R, Li Y, Robinson B, Ayanian J,
Balkrishnan R, Bragg-Gresham J, Chen JT,
Cope E, Gipson D, He K, Herman W, Heung
M, Hirth RA, Jacobsen SS, Kalantar-Zadeh K,
Kovesdy CP, Leichtman AB, Lu Y, Molnar
MZ, Morgenstern H, Nallamothu B, O’Hare
AM, Pisoni R, Plattner B, Port FK, Rao P,
Rhee CM, Schaubel DE, Selewski DT, Shahin-
ian V, Sim JJ, Song P, Streja E, Kurella Tamu-
ra M, Tentori F, Eggers PW, Agodoa LY,
Abbott KC: US renal data system 2014 annual
data report: epidemiology of kidney disease in
the United States. Am J Kidney Dis 2015;
65(6suppl 1):A7.
2 European Medicines Agency: New Recom-
mendations to Manage Risk of Allergic Reac-
tions with Intravenous Iron-Containing
Medicines, 2013.
3 Wysowski DK, Swartz L, Borders-Hemphill
BV, Goulding MR, Dormitzer C: Use of par-
enteral iron products and serious anaphylac-
tic-type reactions. Am J Hematol 2010;
85:
650–654.
4 Szebeni J, Fishbane S, Hedenus M, Howaldt S,
Locatelli F, Patni S, Rampton D, Weiss G,
Folkersen J: Hypersensitivity to intravenous
iron: classification, terminology, mechanisms
and management. Br J Pharmacol 2015;
172:
5025–5036.
5 Szebeni J: Complement activation-related
pseudoallergy caused by liposomes, micellar
carriers of intravenous drugs, and radiocon-
trast agents. Crit Rev Ther Drug Carrier Syst
2001;
18: 567–606.
6 Danielson BG: Structure, chemistry, and
pharmacokinetics of intravenous iron
agents. J Am Soc Nephrol 2004;
15(suppl
2):S93–S98.
7 Walport MJ: Complement. First of two parts.
N Engl J Med 2001;
344: 1058–1066.
8 Roos A, Bouwman LH, Munoz J, Zuiverloon
T, Faber-Krol MC, Fallaux-van den Houten
FC, Klar-Mohamad N, Hack CE, Tilanus MG,
Daha MR: Functional characterization of the
lectin pathway of complement in human se-
rum. Mol Immunol 2003;
39: 655–668.
9 Mollnes TE, Brekke OL, Fung M, Fure H,
Christiansen D, Bergseth G, Videm V,
Lappegård KT, Köhl J, Lambris JD: Essential
role of the C5a receptor in E coli-induced oxida-
tive burst and phagocytosis revealed by a novel
lepirudin-based human whole blood model of
inflammation. Blood 2002;
100: 1869–1877.
10 Trouw LA, Seelen MA, Duijs JM, Benedikts-
son H, Van Kooten C, Daha MR: Glomerular
deposition of C1q and anti-C1q antibodies in
mice following injection of antimouse C1q
antibodies. Clin Exp Immunol 2003;132:32–
39.
11 Poppelaars F, Gaya da Costa M, Berger SP,
Assa S, Meter-Arkema AH, Daha MR, et al:
Strong predictive value of mannose-binding
lectin levels for cardiovascular risk of hemodi-
alysis patients. J Transl Med 2016;14:236.
12 Poppelaars F, Gaya da Costa M, Berger SP,
Assa S, Meter-Arkema AH, Daha MR, et al:
Erratum to: Strong predictive value of man-
nose-binding lectin levels for cardiovascular
risk of hemodialysis patients. J Transl Med
2016;14:245
13 Roos A, Bouwman LH, van Gijlswijk-Janssen
DJ, Faber-Krol MC, Stahl GL, Daha MR: Hu-
man IgA activates the complement system via
the mannan-binding lectin pathway. J Immu-
nol 2001;167:2861–2868.
14 DeAngelis RA, Reis ES, Ricklin D, Lambris
JD: Targeted complement inhibition as a
promising strategy for preventing inflamma-
tory complications in hemodialysis. Immu-
nobiology 2012;
217: 1097–1105.
15 Auerbach M, Ballard H: Clinical use of intra-
venous iron: administration, efficacy, and
safety. Hematology Am Soc Hematol Educ
Program 2010;
2010: 338–347.
16 Pai AB, Garba AO: Ferumoxytol: a silver lin-
ing in the treatment of anemia of chronic kid-
ney disease or another dark cloud? J Blood
Med 2012;
3: 77–85.
17 Chaignon M, Chen WT, Tarazi RC, Bravo EL,
Nakamoto S: Effect of hemodialysis on blood
volume distribution and cardiac output.
Hypertension 1981; 3: 327–332.
18 Geisser P, Burckhardt S: The pharmacokinet-
ics and pharmacodynamics of iron prepara-
tions. Pharmaceutics 2011;
3: 12–33.
19 Michael B, Coyne DW, Fishbane S, Folkert V,
Lynn R, Nissenson AR, Agarwal R, Eschbach
JW, Fadem SZ, Trout JR, Strobos J, Warnock
DG; Ferrlecit Publication Committee: Sodi-
um ferric gluconate complex in hemodialysis
patients: adverse reactions compared to pla-
cebo and iron dextran. Kidney Int 2002;
61:
1830–1839.
20 Chanan-Khan A, Szebeni J, Savay S, Liebes L,
Rafique NM, Alving CR, Muggia FM: Com-
plement activation following first exposure to
pegylated liposomal doxorubicin (Doxil):
possible role in hypersensitivity reactions.
Ann Oncol 2003;
14: 1430–1437.
21 Banda NK, Mehta G, Chao Y, Wang G, Inturi
S, Fossati-Jimack L, et al: Mechanisms of
complement activation by dextran-coated su-
perparamagnetic iron oxide (SPIO) nano-
worms in mouse versus human serum. Part
Fibre Toxicol 2014;11:64.
22 Coyne DW, Adkinson NF, Nissenson AR,
Fishbane S, Agarwal R, Eschbach JW, Michael
B, Folkert V, Batlle D, Trout JR, Dahl N, My-
irski P, Strobos J, Warnock DG; Ferlecit In-
vestigators: Sodium ferric gluconate complex
in hemodialysis patients. II. Adverse reactions
in iron dextran-sensitive and dextran-toler-
ant patients. Kidney Int 2003;
63: 217–224.
23 Wang C, Graham DJ, Kane RC, Xie D, Wer-
necke M, Levenson M, et al: Comparative risk
of anaphylactic reactions associated with intra-
venous iron products. JAMA 2015;314:2062–
2068 .
24 Szebeni J: Complement activation-related
pseudoallergy: a stress reaction in blood trig-
gered by nanomedicines and biologicals. Mol
Immunol 2014;61:163–173.
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University of Groningen
129.125.19.61 - 11/28/2016 12:10:28 PM
