Medição do telopeptídeo C-terminal do colágeno tipo I (CTX) no soro

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Clinical Biochemistry 45 (2012) 928–935
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Clinical Biochemistry
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Review
Measurement of C-terminal telopeptide of type I collagen (CTX) in serum
S.A. Paul Chubb ⁎
School of Pathology and Laboratory Medicine and School of Medicine and Pharmacology, University of Western Australia, Australia
Department of Biochemistry, PathWest Royal Perth and Fremantle Hospitals, Perth, Western Australia, Australia
Abbreviations: BTM, bone turnovermarker; IOF, Intern
cross-linked telopeptide of type I collagen; sCTX, serumCT
ELISA, enzyme-linked immunosorbent assay; RIA, radioim
variation; GLP-2, glucagon-like peptide-2.
⁎ Department of Biochemistry, PathWest, Fremantle H
E-mail address: paul.chubb@health.wa.gov.au.
0009-9120/$ – see front matter © 2012 The Canadian S
doi:10.1016/j.clinbiochem.2012.03.035
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 23 December 2011
received in revised form 26 March 2012
accepted 28 March 2012
Available online 6 April 2012
Keywords:
CTX
CrossLaps™
Collagen peptides
Collagen C-telopeptide
Serum
Bone resorption
Osteoporosis
Serum CTX assays measure a fragment of the C-terminal telopeptide of type 1 collagen released during
resorption of mature bone. Assay reagents are available in manual and automated formats and give good
analytical performance. However their standardisation is not transparent and significant differences in
results between methods have been demonstrated. CTX is most stable in EDTA plasma, although serum
samples processed promptly would be satisfactory. sCTX shows a profound circadian rhythm, especially in
non-fasting subjects; specimens should be collected from fasting patients at a well-defined time of day to
minimise biological variation. Reference intervals in pre-menopausal women have been well studied but in
other adult groups there is less information. Healthy children show the expected age-related variation
corresponding to growth rate. Serum CTX fulfils or partially fulfils all the criteria of a reference bone turnover
marker. Further studies aimed at reducing inter-method differences in results and establishing the
relationships of sCTX with fracture risk and with fracture risk improvement with treatment are required.
© 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929
Assay development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929
Urine CTX assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929
Serum CTX assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929
Measurement issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930
Specimen type and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930
Biological variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930
Circadian variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930
Effect of fasting status on biological variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931
Longer term biological variation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931
Therapeutic applications of circadian variation of bone turnover. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931
Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931
Optimal sampling protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931
Serum CTX values in reference individuals and other healthy people . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931
Other pre-analytical issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 932
Evidence for bone collagen specificity of sCTX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 932
Serum CTX as a reference bone resorption marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 934
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 934
ational Osteoporosis Federation; IFCC, International Federation of Clinical Chemistry and LaboratoryMedicine; CTX, C-terminal
X; P1NP, N-terminal pro-peptide of type I collagen; ICTP, C-terminal cross-linked telopeptide of type I collagen,MMP generated;
munoassay; ECLIA, electrochemiluminometric assay; EDTA, ethylene diamine tetra-acetate; CVi, within-individual coefficient of
ospital, PO Box 480, Fremantle, WA 6959, Australia. Fax: +61 8 9431 2520.
ociety of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.clinbiochem.2012.03.035
mailto:paul.chubb@health.wa.gov.au
http://dx.doi.org/10.1016/j.clinbiochem.2012.03.035
http://www.sciencedirect.com/science/journal/00099120
929S.A.P. Chubb / Clinical Biochemistry 45 (2012) 928–935
Introduction
Osteoporosis is a major cause of morbidity and mortality among
older people in the developed world. To improve the identification
and treatment of individuals at risk of osteoporotic fracture, a number
of biochemical bone turnover markers (BTMs) have been developed
with the aim of assessing the rates of bone formation and resorption.
However a recent systematic review carried out by the International
Osteoporosis Federation (IOF)/International Federation of Clinical
Chemistry and Laboratory Medicine (IFCC) Bone Marker Standards
Working Group [1] found that because of methodological differences
between studies it was difficult to carry out meta-analyses to
determine whether BTMs make a valid contribution to fracture risk
assessment and treatment monitoring. One recommendation was
that serum CTX (sCTX) and N-terminal pro-peptide of type I collagen
(P1NP) should be included in all future studies of osteoporotic
fracture risk assessment or treatment as reference markers of bone
resorption and formation, respectively. These markers came closest to
meeting ideal criteria.
This review covers the development of the sCTX assay, its
analytical performance, and a number of pre-analytical factors
relevant to interpreting results. It then compares sCTX against the
criteria for an ideal BTM set out in the systematic review mentioned
above [1].
Assay development
The sCTX assay measures cross-linked C-terminal telopeptides of
type I collagen. The telopeptides are non-helical sequences of
collagen at each end of the molecule. For type I collagen α1 C-
terminal telopeptide, two types of assay are available. The ICTP assay
(Orion Diagnostica, Espoo, Finland) measures a sequence that
includes both helical and telopeptide components. In contrast, the
antigen used for raising antibodies for the CTX assay is shorter and is
contained within the telopeptide domain (Fig. 1). The ICTP assay has
not found wide use as a bone resorption marker in osteoporosis
studies, and is not considered further here. Serum CTX is often
referred to as CTX-I to distinguish it from an equivalent peptide in
typeII collagen, CTX-II.
Urine CTX assays
The first published CTX assay was developed for urine samples
and was a competitive enzyme-linked immunoassay (ELISA) [2]. The
antibody was raised in rabbits against an 8 amino acid peptide, glu-
Fig. 1. Schematic presentation of type I collagen showing the amino acid sequence of
the human C-terminal telopeptide α1 chain. Type I collagen consists of 2 α1 chains and
1 α2 chain. The intact C-terminal telopeptide α1 chain consists of a 26 amino acid
sequence; the 8 amino acid CTX peptide starts at position 15. Residue 16 can be
involved in cross-linking; the DG sequence (positions 19–20) is prone to undergo β
isomerisation.
Reproduced from J Biol Chem 272(15), Fledelius C et al. Characterization of Urinary
Degradation Products Derived from Type I Collagen, pages 9755–9763, © 1997 by The
American Society for Biochemistry and Molecular Biology, Inc.
lys-ala-his-asp-gly-gly-arg (EKAHDGGR), which is within the C-
terminal telopeptide of type 1 collagen; the lysine residue partici-
pates in an inter-molecular pyridinoline cross-link. This was devel-
oped by Osteometer BioTech A/S, Herlev, Denmark and marketed
under the trade name CrossLaps™ [2].
In 1996, Bonde et al. [3] described a coated-tube radioimmuno-
assay (RIA) for urine CTX using a monoclonal antibody raised against
the EKAHDGGR peptide, with the code MAbA7. The 8 amino acid
peptide was attached to the tube surface and the same peptide was
used as calibrator. The following year, Fledelius et al. [4] published an
ELISA using the same monoclonal antibody; in this assay the
competitor antigen was collagenase-treated collagen coated onto
the surface of the wells and the calibrator was a child's urine with a
known concentration determined by the original CTX assay [2].
Unexpectedly, results from this ELISA were approximately 40% of
those from the original assay, whereas the RIA gave very similar
results. Urine from patients with Paget's disease gave results that
were proportionally higher in the new ELISA than in the RIA. These
findings suggested that the monoclonal and polyclonal antibodies
were detecting different sets of collagen fragments.
This discrepancy was elucidated in a study that characterised the
forms of CTX present in urine from an 11 year old boy [5]. By using
synthetic octapeptides, it was shown that the original CrossLaps
ELISA measured an isomer of the original 8 amino-acid peptide
bearing a β-peptide bond, EKAHD-βGGR, the aspartyl residue (D)
being linked through its β carboxyl group to the glycine residue
whereas the MAbA7-based assays only reacted to the all-α isomer,
EKAHDGGR. The urine was shown to contain a mixture of CTX
isoforms, including α and β homo- and hetero-dimers, with both
pyridinoline and deoxypyridinoline cross-links. Urine from children
had a higher proportion of αCTX than urine from adults, and bovine
foetal and trabecular bone had relatively more αCTX than adult and
cortical bone, respectively. Based on these data, and by analogy with
other systems, it was suggested that the ratio of αCTX to βCTX
reflects the age of the bone, with older bone having a greater
proportion of βCTX. Subsequent studies have found that L–D
racemisation of the aspartyl α-carbon atom also occurs, leading to 4
possible isomers of the CTX peptide, β-L, α-L, β-L, and α-D [6]. When
measured in urine, these isomers may have relevance for fracture risk
prediction [7].
Serum CTX assays
Blood-based assays for bone resorption markers are attractive
because the specimen is more reproducible and a second assay
(creatinine) to correct for variation in urine concentration, and which
adds imprecision to the result, is not needed. The first serum CTX
assay was a competitive polyclonal antibody ELISA [8]. This employed
the 8 amino acid βCTX peptide (in the β-L conformation) immobi-
lised in the assay wells and a rabbit antibody raised against
collagenase treated type I collagen. It was calibrated using the βCTX
peptide, in mass units. It was recognised that a variety of collagen-
derived peptides containing the CTX peptide sequence would be
measured. Post-menopausal females, hyperparathryoid patients and
patients with Paget's disease had higher results than pre-menopausal
females. Treatment of post-menopausal women with bisphospho-
nates caused a reduction in sCTX at 6 months, and importantly,
Paget's disease patients treated with daily intra-venous pamidronate
showed a marked reduction after 3 days, before bone formation
would be affected. These results indicated that the new assay
reflected bone resorption, not bone formation.
In the following year the same group described an ELISA for sCTX
based on 2 new monoclonal antibodies, specific for the β conforma-
tion of CTX [9]. The calibrator used was desalted urine antigens
referenced to the polyclonal serum βCTX assay, but with results given
in molar units. Subsequently, the assay was re-calibrated using a
Fig. 2. Circadian variation in sCTX concentration in 15 post-menopausal women in
non-fasting and fasting states.
Reprinted from Bone, 26/5, Christgau S et al. Serum CrossLaps for Monitoring the
Response in Individuals Undergoing Antiresorptive Therapy, pages 505–511, © 2000,
with permission from Elsevier.
930 S.A.P. Chubb / Clinical Biochemistry 45 (2012) 928–935
synthetic dimer of the βCTX sequence with results given in mass units
but although a defined relationship to the original assay calibration
has been published [10], it is not clear that the results are
interchangeable. The assay detected only cross-linked CTX dimers.
Alpha homo- and hetero-dimers were not detected. Size-exclusion
chromatography of serum showed that the assay detected antigens
with a range of molecular weights from approximately 1000 Da to
10,000 Da, with a peak at 2900 Da. These are likely to comprise a
mixture of collagen degradation peptides of varying size containing
the dimeric βCTX motif.
Technically this assay performed well, with between-run impre-
cision better than 8%, recovery of calibrator added to serum samples
of 101±4% and linear response on dilution in sera from healthy
adults, children and renal failure patients. In clinical studies, pre- and
post-menopausal women had mean (SD) sCTX values of 1748 (740)
and 2952 (1325) pmol/L. Early post-menopausal women in a small
trial of hormone replacement therapy showed a highly significant fall
in sCTX of 75% after 12 months, compared to no change in a placebo
group. The assay therefore performed as expected for a bone
resorption marker, based on experience with the urine CrossLaps™
assay.
The next important step in the development of the sCTX assay was
automation. The first automated version of the assay was for the
Elecsys 2010 and E170 immunoassay analysers (Roche Diagnostics,
Penzberg, Germany). This system uses an organic ruthenium complex
to generate luminescence in an electrochemiluminescent immuno-
assay (ECLIA). The antibodies used appear to be those described by
Rosenquist et al. [9] and the calibrator was synthetic βCTX dimer.
Calibration was in mass units. In evaluation studies [11,12], between-
run imprecision was better than 5.7%. Dilution of the specimen up to
16 fold yielded results 86–119% of expected values and recovery of
added calibrator was 91–101% [11]. There was a high correlation with
results obtained by the sCTX ELISA (r=0.82) but it was not possible
to assess numerical agreement of the two assays because of the
different calibrations employed [11]. Healthy post-menopausal
women gave sCTX results that were 85% higher than pre-
menopausal women in samples collected at 0730–0930 h in the
fasting state. These studies indicate that the sCTX ECLIA has
satisfactory analytical properties.
The sCTX assay is also available on the automated IDS-iSYS
analyser (Immunodiagnostic Systems, Boldon, UK). However there is
little published data obtained with this method.
In a recent application of the sCTX assay, the reagents were
deployed in a miniaturised ‘chip’ format, along with those for
parathyroid hormone,osteocalcin and P1NP [13]. The assay signal
reagent was a sensitive fluorescent latex conjugate. This allowed for
simultaneous measurement of all four analytes in a 20 μL sample.
Imprecision was comparable to that of the sCTX assay by the ECLIA,
but sensitivity was significantly higher. This assay has not been made
commercially available as yet.
Although the available sCTX assays now use the same units the
exact nature of the calibrators used has not been disclosed. There
have been few direct comparison studies of the manual and
automated sCTX assays but recently, Eastell et al. demonstrated that
the ELISA assay gave significantly higher results for sCTX than the
automated Elecsys 2010 method on samples from pre-menopausal
women, necessitating method-specific reference intervals [14].
Studies are required to clarify the causes of this difference with a
view to result harmonisation.
Measurement issues
Specimen type and stability
Optimal sample handling conditions need to be known when
recommending reference procedures for an analyte. Rosenquist et al.
[9] found that CTX in separated serum was stable for 24 h at room
temperature, but deteriorated thereafter. At 4 °C, it was essentially
stable for 1 week. Okabe et al. [12] found that CTX in uncentrifuged
clotted blood was stable for only 4 h at room temperature, but stable
for 24 h at 4 °C. In EDTA whole blood, CTX was stable for at least 24 h
at room temperature. In a more comprehensive study [15], CTX in
EDTA plasma was shown to be stable for 48 h at room temperature
and for 7 days at 4 °C. In contrast, CTX in uncentrifuged clotted blood
and separated serum was stable for 4 and 8 h, respectively, at room
temperature; in separated serum at 4 °C it was stable for 48 h. The
authors recommended that EDTA plasma be used with rapid
centrifugation and either immediate analysis, or freezing of the
plasma. Most recent studies have used serum samples, but the data
above suggest that if the samples had been processed promptly, this
is unlikely to have caused confounding. Unfortunately only a minority
of studies using serum samples appear to have explicitly stated the
processing time. In the remainder of this review, sCTX refers to CTX
measured in serum or EDTA plasma.
Both Rosenquist et al. [9] and Okabe et al. [12] found that sCTX is
stable after multiple freeze-thaw cycles, an important observation for
retrospective analyses of clinical trials.
Biological variation
Circadian variation
Circadian variation in the urinary output of bone resorption
markers, including urine CTX, was well understood when the sCTX
assay was developed [16,17]. Studies to investigate circadian
variation in sCTX were therefore carried out early in the assay's
development. Non-fasting young adult males [18] and post-
menopausal women [19] showed peak sCTX in the early hours of
the morning and nadir concentrations in the middle of the day (see
Fig. 2). The range of the circadian variation was marked, being
between±30% and±35%. Both studies highlighted the dramatic rate
of change in sCTX during the morning.
The circadian variation in urine BTMs was also known to be larger
in non-fasting patients than fasting patients [16]. Christgau et al. [19]
tested the effect of fasting on serum CTX in post-menopausal women
and showed significantly lower variation in patients who were kept
fasting for the 24 h of the study (fasting, ±8.8%; non-fasting ±35%)
(Fig. 2). A review [20] of several factors that might influence the
circadian variation of sCTX found that the fasting status was the only
factor that had an effect; menopausal status, blindness, taking
glucocorticoids or calcitonin and bed-rest had no effect.
image of Fig.�2
931S.A.P. Chubb / Clinical Biochemistry 45 (2012) 928–935
Effect of fasting status on biological variation
Two studies [19,21] examined the effect of fasting status on short-
term (2 weeks) biological variation (expressed as within-individual
coefficient of variation, CVi) of sCTX. The results are shown in Table 1.
In both studies, women gave lower CVi when they were fasting than
non-fasting, although the estimates of CVi were quite different
(Table 1). The cause of the differences in CVi between these studies
is not clear but may relate to the difference in menopausal status of
the study subjects. Clowes et al. [21] studied pre-menopausal women,
in whom there is evidence for variation in sCTX of approximately 9.5%
between follicular and luteal phases of the menstrual cycle [22]. This
cause of variation would be absent in the post-menopausal women
studied by Christgau et al. [19]. These studies clearly showed that
fasting reduced the within-individual biological variability.
Longer term biological variation
Longer term variation has been assessed in a number of studies as
shown in Table 1 [19,23–25]. Estimates of CVi ranged from 9.3 to
15.1%. Estimations of least significant change varied from 27.67% to
36.2%, however these are not strictly comparable because of
differences in the way they were calculated.
Four studies compared the biological variation of sCTX to that of
other BTMs [21,23–25]. Data for uNTX are included in Table 1. When
the responses of sCTX and uNTX in patients given anti-resorptive
therapy were compared in terms of least significant change, more
patients showed significant changes in sCTX than uNTX [23,24],
suggesting that sCTX was more sensitive for monitoring response to
treatment.
Several studies have found evidence of a significant circannual
variation in sCTX in fasting samples from older women [26–28]. In the
Geelong study [26], the peak sCTX was in women sampled in early
Spring, 2 months later than the seasonal nadir of 25-hydroxyvitamin
D concentrations and approximately coincident with a peak in
fracture incidence. This was a cross-sectional study, so it was not
demonstrated that an individual would show this rhythmicity in
sCTX. Nevertheless the mean amplitude of the sCTX variation was
10.6% of the annual mean, which may have a significant impact on an
individual's biological variation. Similar circannual variation in other
BTMs and rate of BMD loss has been reported in women in New
England and Germany [27,28]. In contrast to these observations, no
circannual variation was seen in a sample of volunteers followed
longitudinally for 1 year [29] nor amongst the men enrolled in the
Geelong Osteoporosis Study [30]. The cause of these discrepancies
between studies is not clear.
Therapeutic applications of circadian variation of bone turnover
Understanding the circadian influences on sCTX has led to some
interesting therapeutic observations. The effect of food consumption
on sCTX can be reproduced by the oral glucose tolerance test [31] and
by eating separately all main food types (fat, sugars, protein) [32].
Studies of some gastro-intestinal peptides showed that the post-
Table 1
Biological variation data for serum CTX.
Study population (n) Number Period Fasting status s
Women, 65–76 y 10 2 weeks Fasting
Non-fasting 1
Women, pre-menopausal 20 2 weeks Fasting 1
Non-Fasting 2
Post-menopausal women 51 1 week Fasting
Men,
women
5,
14
3 months Fasting 1
Post-menopausal women 44 1 y Not stated 1
Pre-menopausal women 12 1 y Fasting
prandial fall in sCTX was associated with a rise in glucagon-like
peptide-2 (GLP-2), and that injection of GLP-2 into healthy volunteers
caused prompt falls in both sCTX and urine deoxypyridinoline
without changing serum osteocalcin [32]. These observations led to
trials of night-time GLP-2 as a means of reducing the nocturnal
increase in bone resorption [33,34]; improvements in BMD were
found after 4 months [34]. Intriguingly, another approach has shown
that the parathyroid hormone analogue, teriparatide, given daily in
the morning, substantially disrupts the circadian variation in sCTX,
whereas it is much less disrupted when given in the evening [35].
Exercise
Elite athletes and endurance-trained military personnel are prone
to stress fractures and spinal osteopenia and there have been reports
of short-termchanges in BTMs in such people. In a study of healthy
young adult men [36], exercise to exhaustion caused a 40% increase in
sCTX on the following 3 days that was not accompanied by changes in
bone formation markers. In a similar study, lesser degrees of exercise
did not result in a change in sCTX [37]. Other studies of the effect of
exercise on sCTX carried out at close to maximal exercise tolerance
produced variable results [38–40] and a study of the impact of
community-based exercise showed no significant effect on sCTX [41].
It seems that only the most extreme exercise regimes will produce
changes in sCTX, so recreational exercise is unlikely to be a significant
confounding factor in most clinical situations in which sCTX will be
measured.
Optimal sampling protocol
As mentioned in Specimen type and stability, CTX is most stable
in EDTA plasma. If serum is used, prompt separation and freezing
are vital, probably within 2–4 h of collection. Collection of samples
from fasting patients at a well-defined time reduces the within-
individual variation to manageable levels, even when collected
during the morning. Collection during the middle of the day might
appear to be optimal to minimise the rate of change, however this
is a much less convenient time to collect fasting specimens.
Furthermore, nearly all clinical studies have used morning collec-
tions for sCTX.
Serum CTX values in reference individuals and other healthy people
Reference intervals in women for sCTX measured by the Roche
ECLIA have been well-studied, particularly prompted by the sugges-
tion that the lower half of the pre-menopausal female reference
interval could be used as a therapeutic target for anti-resorptive
therapies [42]. In their evaluation of the ECLIA, Garnero et al. [11]
established reference values in early morning fasting samples from
women well-characterised as to their menopausal and bone health
status. More recent studies [14,43–46] carried out on carefully
CTX CVi (%) uNTX CVi (%) sCTX least significant
change (as published) (%)
Reference
7.9 – – [19]
4.3 – –
9.1 43.7 45 [21]
6.3 29.9 61
9.99 13.28 27.67 [24]
5.1 27.0 30.2 [23]
3.4 – 31.2 [19]
9.3 17.4 36.2 [25]
Table 4
Serum CTX: manufacturers' suggested reference intervals.
Manufacturer Method Sex Age (y)/menopausal
status
Reference interval
(ng/L)
IDS Ltd ELISA F Pre-menopausal 112–738
IDS Ltd ELISA F Post-menopausal 142–1651
IDS Ltd ELISA M – 115–748
Roche Diagnostics ECLIA F 31–58, pre-menopausal b573
Roche Diagnostics ECLIA F Post-menopausal b1008
Roche Diagnostics ECLIA M 30–50 b584
Roche Diagnostics ECLIA M 51–70 b704
Roche Diagnostics ECLIA M >70 b854
Table 2
High quality reference interval data for sCTX in pre-menopausal women. Reference
intervals were determined by the automated ECLIA method except where indicated.
Age (y) Number Location Reference interval
(ng/L)
Reference
31–58 254 France b573 [11]
28–45 178 USA 94–659 [43]
30–39 637 UK, France, Belgium, USA 114–628 [44]
35–45 153 UK 100–620 [45]
46–50 82 Italy 70–610 [46]
35–45 765 Saudi Arabia 163.6–274.1 [47]
35–45 145 UK 200–900a [24]
35–39 194 France, Denmark 111–791 [14]
35–39 194 France, Denmark 177–862a [14]
a Reference interval determined using the ELISA method.
932 S.A.P. Chubb / Clinical Biochemistry 45 (2012) 928–935
documented samples of healthy, predominantly Caucasian pre-
menopausal women showed reasonable agreement (see Table 2).
Another study, however, carried out in Saudi Arabian women, found a
markedly lower pre-menopausal mean and upper reference limit
[47]. It is not clear whether this difference is due to the stringent BMD
selection criterion used in this study, or whether a true population
difference exists. The above studies all used the ECLIA sCTX method.
The samples in two of these studies were also analysed using the
ELISA method [14,24]; the reference intervals generated were
somewhat higher than other studies.
There is less information about reference intervals for other
patient groups. For older men, reference data are available from a
study carried out in Spain using the ECLIA method [48] (see Table 3).
Descriptive data from control groups in clinical studies, or from
method evaluations, may provide approximate reference information
but cannot be used to generate reference intervals without formal
statistical treatment; available data are shown in Table 3. Manufac-
turers supply suggested reference intervals in product information
sheets and these are shown in Table 4.
For children, at least 4 studies of reference data are available.
Crofton et al. [10] studied plasma specimens from infants and
children of all ages that had been collected for investigation of
minor conditions not likely to affect growth and from a population-
based epidemiological study. More recent studies [49–51] examined
samples collected during the early morning, with or without
fasting. Serum CTX concentrations mirrored the expected growth
pattern across the years of childhood, with high concentrations in
the first year, and another peak corresponding to the pubertal
growth spurt (Fig. 3). There were 2–3 fold differences between the
upper reference limits provided by Alberti et al. [49] and Crofton et
al. [10] that are in line with our understanding of the effects of
collection time and fasting status: the non-fasting samples collected
between 0900 h and 1500 h assayed by Crofton et al. [10] would be
expected to have had lower sCTX results than the fasting samples
collected between 0800 h and 1000 h used by Alberti et al. [49], as
both used the ELISA method. The upper reference limits provided
Table 3
Serum CTX concentrations in groups of healthy men and women.
Sex Menopausal status Age Number Location Mean
F Pre – 65 ns 1748
F Post – 169 ns 2952
M – 31–40 88 France 3.0
M – 41–50 105 France 2.6
M – 51–70 531 France 2.4
M – >49 660 Spain 300
F Pre – 157 France 269
M – 40–59 33 France 234
M – >60 101 Australia 290
M – >65 933 USA 400
ns: not stated.
by Huang et al. [50], obtained by the ECLIA, were between those of
Crofton et al. and Alberti et al. [10,49].
Other pre-analytical issues
Among pre-menopausal women, oral contraceptive use is associ-
ated with reduced sCTX concentrations [43,45,52]. In the reference
interval study of de Papp et al. [43], mean sCTX was 251 ng/L in oral
contraceptive users compared to 304 ng/L in non-users. Phase of the
menstrual cycle was found to have a mild influence on sCTX
concentrations [22,44], with higher concentrations in the follicular
phase and a difference of approximately 9.5% between peak and
trough [22]. Fractures are known to cause increased BTM concentra-
tions; in a study of patients undergoing repair of long bone fracture,
sCTX increased promptly, reaching a maximum increase of approx-
imately 55% at 4 weeks post operation, declining gradually over the
course of a year [53]. Samples for osteoporosis management should
not be collected for 3–6 months following a fracture.
Evidence for bone collagen specificity of sCTX
Type I collagen is found in sites other than bone, such as skin,
dentine and tendon and peptide fragments derived from other types
of collagen are likely to be present in samples for sCTX analysis. Thus
it is important to establish the specificity of sCTX for bone collagen
resorption.
The urine CTX assay was designed to be specific for type I collagen,
in that the 8 amino acid sequence chosen as the assay epitope is only
found in the C-telopeptide sequence of the α1I collagen molecule [2].
The sCTX assay requires two 8 amino acid peptides to be cross-linked
to give an assay signal and both peptides are required to be in the β
conformation; these features exclude fragments from newly synthe-
sised and young collagen from reacting in the assay. The sCTX assay
was therefore designed to have high specificity for type I collagen
derived from mature bone.
The original urine CTX assay was shown to be highly correlated
with urine deoxypyridinoline and hydroxyproline measurements [2],
which were established markers of boneresorption [54]. The
SD Reference interval Units Analyser Reference
740 – pmol/L ELISA [9]
1325 – pmol/L ELISA [9]
1.3 – nmol/L ELISA [68]
1.3 – nmol/L ELISA [68]
1.1 – nmol/L ELISA [68]
171 69–760 ng/L ECLIA [48]
114 112–659 ng/L Multiplex [13]
72 – ng/L Multiplex [13]
120 – ng/L ELISA [62]
210 – ng/L ELISA [57]
Fig. 3. SerumCTX values in relation to age for healthymale (left) and female (right) children and adolescents, andfitted reference curves. Curves represent the 2.5th percentile (−−−−),
50th percentile (……) and 97.5th percentile (− · − · −).
Reproduced with permission of American Association for Clinical Chemistry, Inc., from Clinical Chemistry 57(10), pages 1425–1435, Alberti C et al. Serum concentrations of insulin-like
growth factor (IGF)-1 and IGF binding protein-3 (IGFBP-3), IGF-1/IGFBP-3 ratio, and markers of bone turnover: reference values for French children and adolescents and z-score
comparability with other references. © 2011 Highwire Press.
933S.A.P. Chubb / Clinical Biochemistry 45 (2012) 928–935
polyclonal sCTX assay was shown to behave as other markers of bone
resorption [8]; in particular, the fall within 3 days in sCTX on
pamidronate treatment of Paget's disease patients, before any
increase in bone formation occurs, indicated that the assay does
indeed reflect bone resorption [8]. Furthermore, potent anti-
resorptive agents such as zoledronic acid and the antibody to receptor
activator of nuclear factor κ-B ligand (RANKL), denosumab, (which
specifically targets the inter-cellular control of osteoclast function)
reduced sCTX concentrations by approximately 90% [55,56], indicat-
ing that the amount of non-bone collagen contributing to sCTX
measurement, if any, is small. Taken together, these observations
provide good circumstantial evidence that sCTX is a specific marker of
bone collagen resorption. Studies searching for sCTX production from
non-bone sources do not appear to have been done, however.
Serum CTX as a reference bone resorption marker
In the recent review of BTMs by Vasikaran et al. [1] criteria for the
selection of reference BTMs were proposed. It seems appropriate to
review briefly how well sCTX meets these criteria.
1. The reference BTMs should be adequately characterised and
clearly defined.
The calibrator for sCTX is the 8 amino acid peptide cross-linked
into a dimer. In principle, this should be amenable to establishing
as a primary reference material, although its exact nature has not
been disclosed. Furthermore, the collagen fragments that the assay
measures are probably a mixture of peptides [9]. Thus it may be
difficult to establish a reference material that is fully commutable
with sCTX in native samples. The recently demonstrated difference
in results given by automated and ELISA sCTX assays [14]
highlights the importance of this topic if studies using different
methods are to be comparable with each other.
2. The reference BTMs should be bone specific and should ideally
perform well both in fracture risk prediction as well as in
monitoring treatments used or trialled for osteoporosis treatment
among women and men.
There is good circumstantial evidence that sCTX is specific for
resorption of bone collagen, and no evidence against it.
Studies have been unable to demonstrate that sCTX is a risk
marker for incident osteoporotic fracture independent of BMD
[57–66], although several found it associated in unadjusted
analyses [58–61]. These studies may have been confounded by
the effects of non-optimal sample collection conditions and further
work, using samples collected from fasting patients at a well-
defined time, may show that sCTX is an independent risk marker.
Serum CTX shows a large response to anti-resorptive and
parathyroid hormone-based therapies, as discussed elsewhere in
this special issue [67] and this allows its use in determining
compliance with therapy and prediction of BMD response to
treatment. There is limited evidence that the response of CTX to
bisphosphonate therapy is significantly associated with reduction
in fracture risk, but further studies are needed.
3. The reference analyte assay should be widely available and the
intellectual property covering its use should preferably not be the
monopoly of a single owner.
The sCTX assay is available on widely used automated analyser
systems (Elecsys, Roche Diagnostics; IDS-iSYS, IDS), and in ELISA
format. However the intellectual property appears to belong to one
company.
4. The reference BTMs should have biological and physicochemical
characteristics that make them suitable candidates for practical
laboratory use in terms of biological and analytical variability,
sample handling, stability, ease of analysis etc.
Analytical variability of sCTX assays is acceptable; between-run
imprecision was generally well below 10%. CTX, particularly in
EDTA plasma, shows good stability in storage and following
repeated freeze-thaw cycles. No special handling of blood samples
is required. Analysis on the automated systems is straightforward.
The biological variability of sCTX has been well characterised;
circadian variation is marked, especially in non-fasting subjects
and probably contributes to longer-term variation. Use of fasting,
carefully timed morning samples or, possibly, early afternoon
samples, may reduce this impact in studies of fracture risk. It may
be useful to have a standard sampling protocol for sCTX defined.
5. The reference BTMs should be measurable by methodology
(ideally automated) that is widely available in routine clinical
laboratories.
Serum CTX can be measured on 2 automated systems widely
available in clinical laboratories, and by a manual assay. These
systems can be deployed in most laboratories of reasonable size.
However studies are required to establish that the different
methods yield equivalent results.
6. Whilst the medium of measurement could be either blood or urine,
the ideal medium is blood.
Serum CTX is measured in a blood sample, and serum or plasma
may be used.
Serum CTX clearly or partially fulfils all 6 of these criteria.
Questions remain over the identity of the measured analyte
compared to the calibrator, the ability to predict fracture risk
image of Fig.�3
934 S.A.P. Chubb / Clinical Biochemistry 45 (2012) 928–935
independently of BMD and the optimal protocol for collection of
specimens.
Conclusions
This review has examined the analytical and pre-analytical
properties of sCTX. It is certainly a sensitive marker of bone
resorption that responds promptly to changes in bone metabolism.
However, the marked effects of circadian variation and food
ingestion, a reflection of its acute sensitivity to short-term changes
in bone metabolism, mean that strict attention to detail is required
when collecting samples to maximise the ‘signal-to-noise’ ratio.
Studies directed towards harmonising the results produced by the
different assays, as well as more well-performed studies of its
association with fracture risk and with fracture risk improvement
with treatment are needed. By nominating sCTX as one of two
reference bone turnover markers, the members of the IOF-IFCC
Working Group [1] have given researchers the opportunity to focus
on this marker. This will lead to improved understanding of the
response of bone tissue to therapeutic interventions and the optimal
use of sCTX in clinical practice.
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	Measurement of C-terminal telopeptide of type I collagen (CTX) in serum
	Introduction
	Assay development
	Urine CTX assays
	Serum CTX assays
	Measurement issues
	Specimen type and stability
	Biological variation
	Circadian variation
	Effect of fasting status on biological variation
	Longer term biological variation
	Therapeutic applications of circadian variation of bone turnover
	Exercise
	Optimal sampling protocol
	Serum CTX values in reference individuals and other healthy people
	Other pre-analytical issues
	Evidence for bone collagen specificity of sCTX
	Serum CTX as a reference bone resorption marker
	Conclusions
	References