Biomarkers

ISSN: 1354-750X (Print) 1366-5804 (Online) Journal homepage: https://www.tandfonline.com/loi/ibmk20

Long-term stability of biomarkers of the iron

status in human serum and plasma

Eugène H. J. M. Jansen, Piet K. Beekhof & Erna Schenk

To cite this article: Eugène H. J. M. Jansen, Piet K. Beekhof & Erna Schenk (2013) Long-term
stability of biomarkers of the iron status in human serum and plasma, Biomarkers, 18:4,
365-368, DOI: 10.3109/1354750X.2013.781223

To link to this article: https://doi.org/10.3109/1354750X.2013.781223

Published online: 29 Apr 2013.

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ISSN: 1354-750X (print), 1366-5804 (electronic)

Biomarkers, 2013; 18(4): 365–368

! 2013 Informa UK Ltd. DOI: 10.3109/1354750X.2013.781223

SHORT COMMUNICATION

Long-term stability of biomarkers of the iron status in human serum

and plasma
Eugène H. J. M. Jansen, Piet K. Beekhof, and Erna Schenk

Centre for Health Protection, National Institute of Public Health and the Environment, Bilthoven, The Netherlands

Abstract Keywords
Context: In epidemiological research, it is very important to test the stability of biomarkers as Ceruloplasmin, ferritin, haptoglobin, storage,
function of both storage time and temperature. total iron, transferrin, transferrin receptor,
Objective: In this study, the stability of biomarkers of the iron status was tested up to 1 year of UIBC
storage.
Materials and methods: The biomarkers include total iron, unsaturated iron binding capacity, History
ferritin, transferrin, soluble transferrin receptor, ceruloplasmin and haptoglobin.
Results: The concentrations of all biomarkers tested remain constant upon storage at 20, 70 Received 21 February 2013
and 196  C. Accepted 26 February 2013
Conclusion: All biomarkers of the iron status were stable at the temperatures tested for 1 year. Published online 29 April 2013

Introduction involved in the binding to hemoglobin and prevents the loss of

In epidemiological research, the stability of the serum or
plasma biomarkers during long-term storage is of great
importance. This is part of the whole process of blood
withdrawal, blood processing of serum or plasma and storage
of these end products (Lippi et al., 2011). Biomarkers of iron
metabolism are often determined to assess the nutritional
status in the population (Al-Delaimy et al., 2006; Beard,
2001; Cade et al., 2005; Fleming et al., 2001; Wish, 2006) or
to establish the relation with chronic diseases (Corti et al.,
1997; Hercberg et al., 2005; Joosten et al., 2008; O’Doherty
et al., 2010; Ramakrishnan et al., 2002; Salonen et al., 1992;
Sempos et al., 1994; Weinberg, 1996). For determination of
the iron status, several biomarkers can be used. Often the
binding of iron to the iron-binding protein transferrin is used.
As an alternative, the ratio of serum iron and the unsaturated
iron binding capacity (UIBC) can be used. Another biomarker
for the iron status in the tissues is the serum ferritin
concentration, which is mainly used as a biomarker for the
iron status in large-cohort studies. Transferrin receptor
regulates the uptake of iron by cells. The amount of
transferrin receptor expressed on a cell is proportional to
the cell’s need for iron. A proteolytic product of the
transferrin receptor circulates in plasma as soluble transferrin
receptor and is proportional to the total concentration of the
cellular form. Ceruloplasmin is also an iron binding protein
involved in cupper related diseases, whereas haptoglobin is
Address for correspondence: Eugène H.J.M. Jansen, Centre for Health
Protection, National Institute of Public Health and the Environment,
PO Box 1, 3720 BA Bilthoven, The Netherlands. Tel: +31-302742940.
Fax: +31-302744446. E-mail: eugene.jansen@rivm.nl
heme bound iron.
In the present study the stability of total iron (Fe), UIBC,
ferritin (Fer), transferrin (Trf), soluble transferrin receptor
(TrfR), ceruloplasmin (Cer) and haptoglobin (Hapt), in serum
or plasma of human volunteers have been tested during
storage up to 12 months at different temperatures (20, 70
and 196  C).
Materials and methods
Human volunteers
For the stability of Fe, UIBC, Fer, Cer and Hapt, 16
volunteers gave blood and serum samples were prepared
within 2 h after blood withdrawal, divided in aliquots and
stored immediately at 20, 70 and 196  C. The studies
were conducted with approval of the Medical Ethical 13
20
Committee TNO under no 98/36 and by the Blood
Laboratory of Mid-Netherlands in Utrecht with written
permission of the volunteers coordinated by dr EF van
Leeuwen under no. A99.003X.
For the stability study of Trf and TrfR citrate plasma
samples of 16 human volunteers (blood donors) were used.
Samples were obtained from the Central Blood Laboratory of
the Red Cross in Amsterdam with written permission of the
volunteers. At the day of blood withdrawal, the serum samples
were prepared within two hours, divided in aliquots and
stored immediately at 20, 70 and 196  C.
The initial concentrations for the biomarkers at timepoint
zero (T ¼ 0) have been determined in duplicate within 4 h
after centrifugation. Samples stored at 20  C were kept in a
cold room and samples at 70  C were kept in a refrigerator,
both equipped with temperature recorder and sound alarm.
366 E. H. J. M. Jansen et al.

Samples stored at 196 C were kept in a container with
liquid nitrogen equipped with an automatic refill device and a
sound alarm.
Measurements of parameters
Fe, Fer and Cer were determined in serum samples with
an auto-analyzer (Cobas Fara, Roche Diagnostics, Woerden,
the Netherlands) using dedicated kits from Roche
Diagnostics. Measurements of UIBC and Hapt were per-
formed in serum samples, Trf and TrfR in citrate plasma with
an auto-analyzer (Hitachi 912, Roche Diagnostics) using
dedicated kits from Roche Diagnostics.
Results
Serum iron (Fe)
The long-term stability for Fe was determined for storage
conditions at 20, 70 and 196  C up to 1 year. In Figure 1,
the mean values of Fe from 16 volunteers are shown,
expressed as percentage relative to the mean value of T ¼ 0.
For Fe, the long-term stability is very good. At all time points,
up to 1 year, almost no statistical significant changes were
observed compared with the initial value at all temperatures.
Only at the measurements of 6 months, the samples stored at
70  C showed a statistically significant increase compared
with the other two temperatures, but not at the other time
points.
Figure 1. Long-time stability of Fe upon storage at different tempera-
tures. Data are expressed as percentage (mean  s.d.) relative to the value
at T ¼ 0.
Figure 2. Long-time stability of UIBC upon storage at different
temperatures. Data are expressed as percentage (mean  s.d.) relative
to the value at T ¼ 0.
Biomarkers, 2013; 18(4): 365–368
Unsaturated iron binding capacity
The long-term stability for UIBC was determined for storage
conditions at 20, 70 and 196  C up to 1 year. In Figure 2,
the mean values of UIBC from 16 volunteers are shown,
expressed as percentage relative to the mean value of T ¼ 0.
The long-term stability is excellent because at all time points
no statistical significant changes were observed relative to the
initial value. Only at 12 months, a statistically significant
decrease of about 6% was observed compared to the original
value. The average values at 12 months, however, measured in
samples stored at 20 and 70  C are not different from that
stored at 196  C. The quality control sample showed also a
decrease to 96% at this time point.
Transferrin
The long-term stability for Trf was determined for storage
conditions at 20, 70 and 196  C up to 1 year. In Figure 3,
the mean values from 16 volunteers are shown, expressed as
percentage relative to the mean value of T ¼ 0. For Trf, the
long-term stability is very good. Only at time points at one
week and one month a small decrease was observed to 94%
compared with the initial value, but not for the other time
points. The QC sample, however, also showed a small
decrease at these time points to about 96% relative to the
value at t ¼ 0.
Transferrin receptor
The measurements of the TrfR have started after 3 months
and continued at time points 6 and 12 months. The long-term
stability for the TrfR is good as is shown in Figure 4. At the
time points of 6 and 12 months, a statistically significant
decrease of about 8% was observed compared to the original
value at 3 months. But the average value measured in samples
stored at 20, 70 and 196  C were not statistically
different.
Ferritin
The long-term stability for Fer was determined for storage
conditions at 20, 70 and 196  C up to 1 year. In Figure 5,
the mean values from 16 volunteers are shown, expressed as
percentage relative to the mean value of T ¼ 0. For Fer, the
long-term stability is also very good. At all time points, up to
Figure 3. Long-time stability of Trf upon storage at different tempera-
tures. Data are expressed as percentage (mean  s.d.) relative to the value
at T ¼ 0.
DOI: 10.3109/1354750X.2013.781223 Long-term stability of the iron status in human serum and plasma
Figure 4. Long-time stability of TrfR upon storage at different
temperatures. Data are expressed as percentage (mean  s.d.) relative
to the value at T ¼ 3 months.
Figure 5. Long-time stability of Fer upon storage at different tempera-
tures. Data are expressed as percentage (mean  s.d.) relative to the value
at T ¼ 0.
1 year, no statistically significant change compared with the
initial value was observed between the temperatures. Only
from 6 months onwards, a significant decrease of 8% was
observed for all temperatures. The quality control sample also
decreased with 5% at the time point of 12 months. At 9 and 12
months, the Fer concentration in samples stored at 20  C are
somewhat lower than those which have been stored at 70
and 196  C, but the difference was not statistically
significant.
Ceruloplasmin
The long-term stability for Cer was determined for storage
conditions at 20, 70 and 196  C up to 1 year. In Figure 6,
the mean values from 16 volunteers are shown, expressed
as percentage relative to the mean value of T ¼ 0. For Cer,
the long-term stability is very good. At all time points, up to
1 year, almost no statistical significant change compared with
the initial value was observed at all temperatures. Except at
9 months, a statistically significant increase of about 6% was
observed with samples which have been stored at 20  C.
Also at 12 months, a significant decrease to 95% was
observed for all temperatures. The quality control sample
showed also a decrease to 93% at this time point.
Haptoglobin
The long-term stability for Hapt was determined for storage
conditions at 20, 70 and 196  C up to 1 year. In Figure 7,
the mean values from 16 volunteers are shown, expressed as
percentage relative to the mean value of T ¼ 0. For Hapt, the
367
Figure 6. Long-time stability of Cer upon storage at different tempera-
tures. Data are expressed as percentage (mean  s.d.) relative to the value
at T ¼ 0.
Figure 7. Long-time stability of Hapt upon storage at different
temperatures. Data are expressed as percentage (mean  s.d.) relative
to the value at T ¼ 0.
long-term stability is excellent, because at all time points, up
to 1 year, no statistical significant changes compared with the
initial value were observed at all temperatures.
Discussion
The stability of seven biomarkers that are related to the iron
status was tested, being Fe, UIBC, Trf, TrfR, Fer, Cer and
Hapt. The stability is good for the three temperatures tested
(20, 70 and 196  C) up to 1 year of storage. Only for Fe
and Cer, there are only at a few time points, some small
deviations of the mean values from those measured at T ¼ 0,
but these deviations can be explained by considering the
quality control parameters. The small deviations disappear
when a correction was made based on the value of the control
data. Even storage at 20  C for up to 1 year had no
measurable effect on the concentrations of all seven bio-
markers. At almost all time points the mean values of the
three temperatures are statistically not significant from each
other. This observation makes expensive storage in liquid
nitrogen unnecessary. Although storage of serum or plasma
samples at 20  C is sufficient to remain the original
concentrations of all iron-related parameters tested in his
study, storage at 70  C recommended for longer periods,
because of the possible instability of other biomarkers which
may be of interest, such as folate (Jansen et al., 2012), vitamin
C (Bobrowicz et al., 2001) and maybe others, that are less
stable on storage at 20  C.
In literature, not many studies on the long-term stabi-
lity of biomarkers of the iron status have been reported.
368 E. H. J. M. Jansen et al.
Gislefoss et al. (Gislefoss et al., 2008) and Donnelly et al.
(Donnelly et al., 1995) showed that Fe was stable for 2 and 25
years at 25  C and 4 months at 20  C, respectively.
Transferrin proved to be stable to repeated freezing and
thawing and storage at 20  C for 43 d (Stoddard et al., 1984).
Mathew et al. (Mathew et al., 2009) tested the storage
stability of iron biomarkers, Cer, Fer, Fe Trf and sTfR in
serum with and without gel contact at 80  C for 12 months.
For these parameters there were no statistical differences in
sera with and without gel contact, but they did not check the
concentrations at T ¼ 0.
Other studies showed a good short-term stability of Fer and
TrfR at 11  C (Drammeh et al., 2008), UIBC at þ 4  C for 7 d
and Fe and Fer for 5 d at þ4  C, but after 7 d a statistically
significant increase was observed (Liyanarachcy, 2000).
Tanner et al. (Tanner et al., 2008) showed an increase of Fe
and Fer for 24 h at þ35  C, whereas Trf remained stable under
these conditions.
In conclusion, the concentrations of all iron status
biomarkers tested are constant upon storage at 20, 70
and 196  C. Between the three temperatures at all time
points also no statistically significant differences were
observed. Storage of serum and plasma samples at 20  C
is sufficient to remain a stable outcome of measurements of
iron status biomarkers. Storage at 196  C in liquid nitrogen
and even at 70  C is not necessary if the samples are used
within 1 year.
Declaration of interest
The authors stated that there are no conflicts of interest
regarding the publication of this article. Research support
played no role in the study design; in the collection, analysis
and interpretation of data; in the writing of the report; or in
the decision to submit the report for publication.
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