J Vet Diagn Invest 22:544–552 (2010)

Evaluation of three automated human immunoturbidimetric assays for

the detection of C-reactive protein in dogs
Stefanie Klenner,1 Natali Bauer, Andreas Moritz

Abstract. C-reactive protein (CRP) is a major, acute-phase protein in dogs; however, there is a need for
automated assays to ensure in-time patient monitoring. Three automated immunoturbidimetric assays
(Randox, Thermo, and Wako) developed for human beings were evaluated for their ability to detect canine
CRP, including method validation, evaluation of diagnostic use, and establishment of exploratory reference
intervals. Sera from 36 healthy dogs and 82 diseased dogs were included for method comparison with the
enzyme-linked immunosorbent assay (ELISA; Tridelta) serving as the reference method. A nonparametric
estimate of the 1-sided 95% reference interval was established (n 5 36). Precision study revealed good intra-
assay coefficients of variation (CVs) of 1–10%, 0–9%, and 2–13% for the Randox, Thermo, and Wako assays,
respectively. Interassay CVs were 18%, 24%, and 19% respectively. Because of a low linear range, the Thermo
test was considered unsuitable for use with canine specimens. No significant differences were present between
the results obtained with the Randox and Wako assays with CRP concentrations less than 15 mg/l; however,
median CRP results differed significantly between the Thermo test and the ELISA (P 5 0.03). Bland–Altman
analysis detected a proportional bias of 0.28, 20.59, and 0.61 mg/l for the Randox, Thermo, and Wako assays,
respectively. For all tests, median CRP values were significantly different between healthy dogs and dogs with
neoplasia. The upper limit of the reference intervals were 8.2 and 9.9 mg/l for the Randox and Wako assays,
respectively. In contrast to the Thermo test, the Randox and Wako assays were suitable for detection of
abnormally high canine CRP concentrations; however, improvement of assay precision and evaluation of
accuracy are warranted before their clinical use with canine specimens.
Key words: C-reactive protein; dogs; human; immunoturbidimetry; validation.

Introduction eases,1,15,23 neoplasia,31,36,38,47 and inflammatory bow-

C-reactive protein (CRP) is well known as a major,
acute-phase reactant in dogs and is part of the
increasing field of research in acute-phase proteins.5
Infection, inflammation, or tissue damage lead to an
increased formation of proinflammatory cytokines,
such as interleukins (IL-6 or IL-1), and subsequent
synthesis of acute-phase proteins, mainly in the
liver.12,46 C-reactive protein is an opsonin and is,
therefore, a member of the classical complement
pathway of the host.33 It induces a protective effect by
several potential mechanisms including down-regula-
tion of the inflammatory response, opsonization of
apoptotic or necrotic cells,32,34 and binding to
bacterial proteins.37 In dogs, increased CRP concen-
trations have been observed in a wide variety of
different diseases and conditions, including infectious
diseases,24,26–28,35,43,44,49,50 immune-mediated dis-
From the Department of Veterinary Clinical Sciences, Clinical
Pathology, and Clinical Pathophysiology, Justus-Liebig-Univer-
sity Giessen, Giessen, Germany.
1 neous and that may impair their detection using human
Corresponding Author: Stefanie Klenner, Department of
Veterinary Clinical Sciences, Clinical Pathology, and Clinical
Pathophysiology Justus-Liebig-University, Frankfurterstrasse 126,
35392 Giessen, Germany. Stefanie.Klenner@vetmed.uni-giessen.de
544
el disease,29 as well as after surgery8,11,21 or after
experimental injury.2
Over the years, many assays have been developed for
the detection of canine CRP. These have included
immunodiffusion assays,52 a time-resolved immuno-
fluorimetric assay,39 capillary tests, and slide reverse-
passive latex agglutination tests.20,30,45 The test de-
scribed by McGrotty is available as a rapid in-house
test for general veterinary practice. Currently, a canine-
specific enzyme-linked immunosorbent assay (ELISA)a
is frequently used as a substantially validated reference
method18; however, ELISAs are labor intensive if
performed without an automated ELISA processing
system. Additionally, high between-run imprecision
restricts the use of this ELISA.18,25 To avoid these
disadvantages and to ensure prompt determination of
acute-phase protein concentrations, rapid, automated
detection methods for human specimens have been
developed for use with biochemical analyzers. In
contrast to human CRP, canine CRP is glycosylated
in 2 of 5 subunits4 that make the molecules heteroge-
CRP tests. Thus, some human tests failed to detect
canine CRP.10 Few studies have reported the use of
automated immunoturbidimetric assays for CRP
 Automated assays for canine C-reactive protein detection
determination in dogs, including 2 human tests
obtained from Bayerb,16 and Olympus,c,3 as well as 2
canine-specific tests.9,10 Use of a species-specific test
would be preferable, but unfortunately, one of the
tests10 is no longer marketed commercially and the
other9 is not commercially available.
The Bayer CRP turbidimetric immunoassayb
(hereafter, Bayer assay) for human beings has been
proven to reliably detect canine CRP,16 but Bayer no
longer produces this assay. The same antibody,
however, is used in the Randox CRP testd (Randox
Laboratories Ltd., personal communication, 2010).
In 2008, the Olympus human immunoturbidimetric
assayc was used to detect CRP in dogs with
hyperadrenocorticism.3 Although excellent correla-
tion to the canine-specific ELISAa and acceptable
intra-assay and interassay coefficients of variation
(CV) of ,10% were reported for the Olympus assay,3
full assay validation was not detailed by the authors
because that was not the primary aim of their
investigation.
In the current study, 3 automated immunoturbidi-
metry CRP assaysd–f originally developed for use with
human specimens were evaluated for use with canine
serum samples. Test evaluation included method
validation, assessment of clinical validity (i.e., the
capacity of the tests to differentiate between dogs
suffering from various diseases and healthy dogs),
and establishment of exploratory reference intervals
for the tests.
Materials and methods
Study design
The study was carried out between August 2007 and
April 2008. Three automated CRP assays developed for
human beings were evaluated in the present study. The first
assay from Randox Laboratoriesd (hereafter, Randox
assay) consisted of a high-linearity test using an unspeci-
fied–anti-human CRP antibody. The second assay from
Thermo Clinical Labsystemse (hereafter, Thermo assay)
used a goat–anti-human CRP antibody. The third test kit
from Wako Chemicalsf (hereafter, Wako assay) used an
ovine–anti-CRP antibody. The manufacturers of the 3 tests
did not clarify whether a monoclonal or a polyclonal
antibody was used. All 3 CRP assays were run on an
automated biochemical analyzerg according to the manu-
facturers’ recommendations and were calibrated with test-
specific calibration material. Because no test-specific
material for internal quality control was available for the
Wako assay, the Randox control was used.
The study was divided into 3 parts, beginning with
method validation. Method validation included evaluation
of linearity and assay precision and comparison of the new
methods to the reference method (canine CRP ELISAa),
which was previously validated.18 In the second part of the
study, clinical validity was tested by comparing CRP
545
concentrations of dogs with different diseases to those
obtained in healthy dogs. In the third part of the study,
exploratory reference intervals were established if all
previous examinations were acceptable.
Method validation
Linearity. Linearity was assessed by serial dilution of
the following human and canine specimens with markedly
increased CRP concentrations: the human calibration
standardh containing a highly abnormal CRP concentra-
tion (153 mg/l) and a canine serum sample with high CRP
concentration (.45 mg/l). Both specimens were serially
diluted with saline (0.9%) to achieve samples with 6
different CRP concentrations (undiluted CRP; 1:1; 1:2;
1:4; 1:16; saline 0.9%). All samples were analyzed in
triplicate using the 3 human CRP assays.
Intra-assay and interassay repeatability. A precision
study evaluated the random error by investigating intra-
assay and interassay repeatability (CV). The repeatability
of a 20-run intra-assay was assessed by repeated measure-
ments of patient serum samples with 3 different CRP
concentrations (high: .45 mg/l; medium: ,15 mg/l; low:
,8 mg/l).
The 15 consecutive observations from serum samples
with similar high, medium, and low CRP levels were
included for assessment of interassay repeatability. Serum
samples with 3 different CRP levels (high: .45 mg/l;
medium: ,15 mg/l; low: ,8 mg/l) were prepared with
specimens obtained from 1 healthy and 4 diseased dogs;
aliquots were frozen at 280uC until analysis. Only vials
needed for each analytical run were thawed to avoid
repetitive freeze–thaw cycles, thus minimizing a source of
potential variation. For each level, standard deviation (SD)
was calculated as the square route of the estimated variance
components. In the next step, CV was calculated as (SD/
global mean) 3 100 to evaluate imprecision of the assays.
Method comparison. Serum samples from 118 dogs (36
healthy, 82 diseased) were available for the method
comparison part of the study. Samples were first measured
with the 3 automated CRP assays and, afterward, were
frozen at 280uC until ELISA analysis. For ELISA analysis,
all samples were thawed, thoroughly mixed, and the ELISA
was manually performed by one of the authors (SK) and by
a trained technician as recommended by the manufacturer.
A standard curve was prepared using serial dilutions of the
calibrator with the standard diluent buffer (both provided
in the test kit) according to the manufacturers’ instruction.
Absorbance was detected at 450 nm with an ELISA reader.i
Clinical validation
Eighty-two dogs presented to the Small Animal Clinic
(Faculty of Veterinary Sciences, Justus-Liebig-University,
Giessen, Germany) with different diseases were included in
the study to evaluate CRP concentrations in various
illnesses. Depending on the clinical signs, various diagnos-
tic tests and procedures were performed to establish the
final diagnosis. These included biochemical profiles,
radiography, ultrasonography, endocrine tests, microbial
tests, bronchoalveolar lavage, analysis of cerebrospinal
546 Klenner, Bauer, Moritz
fluid, cytology, and biopsy. Diseased dogs were assigned to
the following groups based on their final diagnoses:
neoplasia (n 5 16), inflammatory disease (n 5 17), and
miscellaneous diseases (n 5 48), those that did not match
either of the previous diagnoses.
Automated CRP assays were performed on the day of
sampling; subsequently, the serum samples were frozen at
280uC until ELISA analysis. The dogs included for
calculation of reference intervals served as controls.
Establishment of reference intervals
Thirty-six clinically healthy, adult (.1 year old) dogs
were sampled to establish reference intervals. The dogs
comprised the following breeds: 8 Beagles, 3 German
Shepherd Dogs, 2 Rottweilers, 2 Australian Shepherd
Dogs, 2 Great Danes, 2 German Wirehaired Pointers, 2
Golden Retrievers, 1 Bernese Mountain Dog, 1 Saint
Bernard, 1 Austrian black and tan hound (Austrian
brandlbracke), 1 Flat-Coated Retriever, 1 Kuvasz, 1
Labrador Retriever, 1 Newfoundland, 1 German spaniel,
1 Weimaraner, and 5 mixed-breed dogs. Females were
slightly overrepresented with 22 out of 36 dogs (15 intact, 7
spayed), whereas 14 out of 36 were males (9 intact, 5
neutered). The median age of the dogs was 2 years (range:
1–8 years). Dogs were regularly vaccinated and dewormed
blood donors, staff-owned dogs, or healthy dogs presented
for routine radiologic examination to screen for hereditary
hip or elbow dysplasia at the Clinic for Small Animals
(Faculty of Veterinary Medicine, Justus-Liebig University,
Giessen, Germany). Special care was taken in the selection
of the healthy dogs for study. All dogs were clinically
healthy and had no recent history of acute illness. As CRP
is a major acute-phase protein, which increases in a wide
variability of acute inflammatory diseases, traditional
markers of inflammation (i.e., white blood cell count
[WBC], erythrocyte sedimentation rate [ESR], and fibrin-
ogen) were determined to exclude dogs with underlying
inflammation that was not detected by routine clinical
examination. Therefore, inclusion criteria for study were an
unremarkable physical examination; lack of increase in
WBC, ESR, and fibrinogen concentration; and a hemato-
logic profile within reference range. Erythrocyte sedimen-
tation rate was performed via the Westergren methodol-
ogy.j An automated hematology analyzerk was used for
hematological analysis. Fibrinogen concentration was
determined by the Clauss method, using a coagulometer.l
Statistical analysis
All data were evaluated for normal distribution using the
Anderson–Darling test.m
Method validation. Descriptive statistics were per-
formed to calculate medians, means, SDs, and CVs.
Calculation of the Pearson’s product moment correlation
coefficient was used to evaluate the linearity of the tests. In
the method comparison experiment, possible differences
between the means of pairs were detected using the
Wilcoxon signed rank test. Additionally, Bland–Altman
analysis was used to characterize the bias and to visually
evaluate the difference plot.40
Establishment of the reference intervals. Data of the
healthy dogs were described by calculating medians, means,
SDs, and minimums and maximums, as well as by depicting
histograms. If the data were not normally distributed,
logarithmic transformation was done.
The 95% 1-sided reference interval was calculated using
commercial software.m Possible differences between females
and males in the reference range population were investi-
gated using the Mann–Whitney U-test.
Clinical validation. A 1-way analysis of variance (AN-
OVA) and Tukey’s posttest were used for assessment of
potential differences between the different disease groups
when CRP was measured with the 3 assays. For analysis of
the impact of the factors test and disease and the interaction
between test and disease, a 2-way ANOVA was applied. All
tests were performed after logarithmic transformation of
the data.n A P-value of ,0.05 was considered significant.
Results
Method validation
All 3 CRP tests showed excellent linearity (r .
0.99) within a calibration range of ,10–153 mg/ml for
serially diluted human specimens. The Randox and
Wako assays also showed linear calibration graphs
with correlation coefficients of r . 0.98 in serially
diluted canine samples; however, the Thermo assay
had a linear range less than about 8.5 mg/l, with a
markedly lower correlation coefficient than the other
assays (r 5 0.76). As shown in Figure 1, the Randox
and Wako assays mildly overestimated CRP mea-
surement in the lower range of concentrations (10–
30 mg/l). Intra-assay CVs were 1–10%, 0–9%, and 2–
13% for the Randox, Thermo, and Wako assays,
respectively. Interassay precision showed slightly
higher CVs at 8–18%, 9–24%, and 4–19% for the
Randox, Thermo, and Wako assays, respectively
(Tables 1, 2).
Sample volume of the sera available for the method
comparison experiment was too low to perform
duplicate measurements. After the ELISA analysis,
24 sera had to be excluded from the statistical analysis
because results exceeded the upper limit of linearity of
the ELISA (.15 mg/l). Because of the low sample
volume, dilution of the samples and reanalysis with
the ELISA was not possible. Thus, 94 samples were
finally included in the statistical analysis. There was
no significant difference between the median results
obtained with the Randox assay and the ELISA (P 5
0.20) nor between the Wako assay and the ELISA (P
5 0.36). In contrast, a significant difference was
detected (P 5 0.03) between the median results
achieved with the Thermo assay and the ELISA.
The Bland–Altman difference plot revealed a mean
proportional bias of 0.28 mg/l for the Randox assay,
20.59 mg/l for the Thermo assay, and 0.61 mg/l for
the Wako assay. For all tests, the 95% limits of
 Automated assays for canine C-reactive protein detection 547

Figure 1. Linearity range of 3 immunoturbidimetric assays originally developed for human beings determined by serial dilution of
a human calibration standard with a C-reactive protein (CRP) concentration of 153 mg/l (A1, Randox; A2, Thermo; A3, Wako) and
serial dilution of a canine serum sample with a CRP concentration of 50 mg/l (B1, Randox; B2, Thermo; B3, Wako).

agreement were large, with a range of 212.64 to
13.21 mg/l (Randox vs. ELISA), 29.96 to 8.79 mg/l
(Thermo), and 211.67 to 12.90 mg/l (Wako).
Clinical validation
Data from the diseased dogs were not normally
distributed. The 2-way ANOVA revealed a highly
significant impact of the factor disease on results (P
, 0.0001), as well as of the applied test (P 5
0.0136). There was no significant interaction be-
tween the factors (P 5 0.2129). As depicted in
Figure 2, all 3 CRP assays detected significantly
higher CRP values in dogs with neoplastic diseases
compared with the control. Both the Randox and

Table 1. Intra-assay repeatability of 3 automated immuno-
turbidimetric assays (Randox, Thermo, and Wako) originally
developed for human beings for detection of a high (.45 mg/l),
medium (,15 mg/l), and low (,8 mg/l) concentrations of canine
C-reactive protein.*{
Table 2. Interassay repeatability of 3 automated immuno-
turbidimetric assays (Randox, Thermo, and Wako) originally
developed for human beings for detection of a high (.45 mg/l),
medium (,15 mg/l), and low (,8 mg/l) concentrations of canine
C-reactive protein.*{

High Medium Low High Medium Low

Test (.45 mg/l) (,15 mg/l) (,8 mg/l) Test (.45 mg/l) (,15 mg/l) (,8 mg/l)

Randox Randox
Mean 6 SD (mg/l) 47.81 6 0.38 17.73 6 0.31 5.23 6 0.51 Mean 6 SD (mg/l) 44.35 6 2.79 15.75 6 1.27 8.01 6 1.42
CV (%) 1.0 2.0 10 CV (%) 6.0 8.0 18
Thermo Thermo
Mean 6 SD (mg/l) 16.0 6 0.0 12.3 6 0.47 8.65 6 0.81 Mean 6 SD (mg/l) 28.27 6 6.70 10.07 6 0.88 9.60 6 0.74
CV (%) 0.0 4.0 9.0 CV (%) 24 9.0 8.0
Wako Wako
Mean 6 SD (mg/l) 53.91 6 1.32 22.95 6 1.16 8.23 6 1.10 Mean 6 SD (mg/l) 45.80 6 1.81 16.29 6 3.16 9.30 6 1.35
CV (%) 2.0 5.0 13 CV (%) 4.0 19 15
* CV 5 coefficient of variation; SD 5 standard deviation. * CV 5 coefficient of variation; SD 5 standard deviation.
{ Mean, SD, and CV were calculated from 20 replicate { Mean, SD, and CV (coefficient of variation) were calculated
measurements of the different levels. from 15 replicate measurements of the different concentrations.
548 Klenner, Bauer, Moritz

Figure 2. C-reactive protein (CRP) concentration measured with the Randox, Thermo, and Wako assays in healthy dogs (n 5 36),
dogs with neoplasia (n 5 16), dogs with inflammatory diseases (n 5 17), and dogs with miscellaneous diseases (n 5 48). Black horizontal
lines express the median value.

Wako assays were able to detect a significant Establishment of reference intervals

increase in CRP in patients with inflammatory
illnesses. Pronounced overlap between the different
disease groups was present. Table 3 summarizes the
significant differences between the groups and the
tests. The tests evaluated in the current study
generally detected a CRP concentration ,50 mg/l.
The CRP concentrations measured with the Thermo
assay did not exceed 27 mg/l.
The Thermo assay was not included in the
construction of reference intervals because data from
the method validation were not acceptable. Data
from the Randox and Wako assays showed non-
normal distribution even after logarithmic transfor-
mation. Descriptive statistics are shown in Figure 3.
The upper reference limits were 6.8 mg/l CRP for the

Table 3. Median and range of the C-reactive protein (CRP) concentrations in healthy dogs and dogs with neoplastic, inflammatory,
and miscellaneous diseases.*

CRP (mg/l), median (range)

Disease class Randox Thermo Wako

Healthy (n 5 36) 1.0 (0–10.5) 0.0 (0–12.0) 1.1 (0–12.1)

Neoplastic (n 5 16) 17.4 (0–55.1){1 10.5 (0–22){ 24.8 (0–56.2){1
Inflammatory (n 5 17) 6.3 (0–31.3){ 8.0 (0–22) 12.5 (0–40.2){1
Miscellaneous diseases (n 5 48) 3.9 (0–81.0) 6.0 (0–27.0) 4.5 (0–61.7)
* Significant differences between the groups of diseased dogs and the controls evaluated by a 1-way analysis of variance with post hoc
Tukey’s test are depicted as follows:
{ P , 0.01.
{ P , 0.05.
1 Significant differences between Randox and Wako assays, respectively, and the Thermo test are shown using 1 P , 0.05.
 Automated assays for canine C-reactive protein detection
Figure 3. Frequency histograms demonstrating the distribu-
tion of data obtained for calculation of 95% 1-sided reference
intervals (n 5 36 healthy dogs) for the Randox (A) and Wako (B)
assays. Upper 95% reference limits depicted as gray vertical bars.
CRP 5 C-reactive protein.
Randox assay and 9.9 mg/l CRP for the Wako assay.
The Mann–Whitney U-test did not show any
significant difference between females and males
(Randox, P 5 0.11; Wako, P 5 0.18).
Discussion
The Randox assayd (formerly Bayerb; Randox
Laboratories Ltd., personal communication, 2010) as
well as a CRP test from Wako Chemicalsf were able to
detect canine CRP reliably. Linearity of the Randox
and Wako assays was present over a range of 10–
153 mg/l. In the present study, the limit of detection
was not assessed because CRP concentrations below
the reference interval are generally of no clinical
relevance. Intra-assay CVs obtained for the Randox
and Wako assays were comparable to the CVs
reported in the veterinary literature for canine
specimens (Bayer assayb CVs: 5.2–10.8%16; Olympus
assayc CVs: ,10%3; ELISAa CVs: 6.9–10.1%18;
previously developed CRP assay CVs: ,5%9). In the
549
current study, between-run CVs of the Randox and
Wako assays were slightly higher than those reported
previously for dogs (Bayer assay CVs: 3–10.2%16;
Olympus assay CVs: ,10%3; previously developed
CRP assay CVs: 4.8–11%9). However, the between-run
CVs obtained in the current study were still markedly
lower than those reported for the ELISA (7.5–
29.0%).18 It should be pointed out, however, that the
interassay CVs of the Randox assay were nearly twice
as high as those reported in the literature for the
formerly known Bayer assay. Likely causes for the
discrepancy between the previous results and the
current findings include variability between different
batches of the assay, the use of a different analyzer
from the previous study, or changes of components of
the test (e.g., the antibody) during the course of time.
Furthermore, interassay imprecision of the tests
evaluated herein was above the limits of the objective
analytical performance standard for imprecision of
CRP derived from data on biological variation of
canine CRP.19 Therefore, an assessment of reducing
the imprecision of the test is warranted.
The Thermo assay evaluated herein failed to
accurately detect canine CRP as reflected by a low
linear range and a comparatively low correlation
coefficient in the linearity study, as well as a high
imprecision and inability to detect high canine CRP
concentrations. The likely cause for this finding is the
low cross-reactivity between the antibody used in the
Thermo assay and the canine CRP molecule, which
may be explained by the canine CRP is glycosylation
in 2 of 5 subunits in contrast to the human
unglycosylated molecule.4
Human immunoturbidimetric tests evaluated in the
current study were able to detect a significant rise in
median CRP values in dogs with neoplastic diseases.
Given the results of the current study, the Randox
and Wako assays were also able to differentiate
between healthy dogs and dogs with inflammatory
diseases, which is in accordance with results obtained
for other CRP assays.6,16,27,31,38 In the dogs evaluated
in the present study, a marked overlap of CRP results
was present in the different disease groups, which
might be explained by patients being sampled in
different stages of illness rather than in the acute
phase of disease only. In the current study, results of
the patients usually did not exceed a CRP concentra-
tion .50 mg/l. This is in contrast to the established
canine ELISA detecting CRP concentrations of
approximately 100 mg/l.17,10 A higher range of CRP
concentrations (up to ,240 mg/l) has been reported
in septic dogs using the human Olympus assay.3 C-
reactive protein concentrations up to 90 mg/l have
been frequently observed in canine patients with
infectious diseases when using the Bayer assay, which
550 Klenner, Bauer, Moritz
is still higher than the results obtained in the current
study.16 This discrepancy between results is surprising
because the Randox and Bayer assays are identical
(Randox Laboratories Ltd., personal communication,
2010). One explanation might be that because
different patient groups were studied, absolute CRP
concentrations were not directly comparable. Differ-
ences between the reagent batches used in the present
study and the previous investigation might also
contribute. A further possibility might be that
antibodies or other components of the test kit have
been changed by the manufacturer, although this is
not the case according to the information provided by
Randox. The presence of lower CRP results in human
CRP tests than in assays specifically designed for use
in canine specimens indicates a lower antibody-
binding capacity.
The reference intervals established herein have been
derived from a slightly lower number of individuals
(36 instead of 40 dogs) than recommended previ-
ously.14 Additionally, nonparametric statistics had to
be used, which would have required a sample size of n
. 120.41 Unfortunately, it was not possible, econom-
ically, to include such a high number of healthy dogs.
Therefore, the reference intervals have to be seen as
preliminary, and the available data are reported
graphically as suggested previously for small sam-
ples.13 Similar to the slightly lower results obtained
from a similar low numbers of dogs of ,7.1 mg/l (n 5
19),39 ,6.3 mg/l (n 5 8),18 and ,8.4 mg/l (n 5 14)16
have been described in the literature for healthy dogs.
No significant influence by sex on reference intervals
was detected. This is in accordance with other studies
where no sex-related differences in CRP concentra-
tions could be found in healthy Beagles.22,51 Median
age of the healthy control group in the current study
was quite young, but significant age-related differ-
ences in CRP concentrations have not been found
previously.51 To the authors’ knowledge, only 1 study
investigated breed-related differences in acute-phase
proteins,7 and no significant difference in the CRP
concentration between retired racing Greyhounds and
dogs of other breeds was found. Because 16 different
breeds were included in the current study, breed-
specific differences in CRP plasma concentration
were not examined.
The present study had several limitations. It had
been recommended that canine-specific control ma-
terial be used, but the material was not available in
the authors’ laboratory at the time of the experiment.
Additionally, test-specific control material could not
be used for the Wako assay. The Randox control was
used instead. It was considered to be adequate
because it fulfilled the defined criteria of a control,
including an acceptable stated CRP concentration,
and because it demonstrated the stability of the
material over a certain period of time (e.g., 1 year).48
Moreover, test principals of the Wako and Randox
assays were equal, which made the use of the Randox
control possible. A similar principle is also applied in
external quality assessment, where control material
from 1 company or reference laboratory is used for
assays from different manufacturers.47 A further
limitation of the study is that the CRP concentration
of the canine serum used for the linearity study should
have been approximately the same as the CRP
concentration of the human-calibration standard.
However, serum with a similar high canine CRP level
was not observed in the dogs evaluated in the current
study, even in patients with severe underlying
inflammatory diseases. A reason for this might be
that, even in the Wako and Randox assays, the cross-
reactivity between the antibodies and canine CRP was
not as high as for human CRP.
For the ELISA performed in the method compar-
ison experiment, sera had to be frozen and analyzed in
a batch. According to the literature, CRP is stable for
at least 4 months if frozen at 210uC.42 In accordance
with other studies, a negative influence from freezing
on CRP stability was, therefore, considered un-
likely.16,17 Simultaneous measurements of the auto-
mated assays and the ELISA would have been
optimal. However, this procedure was not performed
in the current study because the preliminary results of
the automated CRP assays were directly compared
with the actual disease status of the patients to gain
further experience with the different new tests,
whereas the ELISA analyses had to be performed in
a batch. The ELISA was performed by 2 examiners
(SK and staff member); however, 1 person would have
been optimal to keep interassay variation as low as
possible. Interassay and intra-assay repeatability of
the ELISA could have been further reduced by using
an automated ELISA processing system, but an
automated system was not available in the authors’
laboratory. For interpretation of the ELISA results,
sera with high CRP concentrations that exceeded the
upper limit of the ELISA’s linearity (.15 mg/l) could
not be diluted and reanalyzed because of the low
sample volume. Thus, no information on method
agreement in higher concentrations can be given.
Further studies assessing the linear relationship
between the new automated tests and the ELISA as
the gold standard are recommended, especially for
higher CRP concentrations.
In summary, the current study revealed that
automated CRP tests developed for human beings
from Randox and Wako demonstrated cross-reactiv-
ity with canine CRP and, thus, could be used to detect
an increase in canine CRP concentration from
 Automated assays for canine C-reactive protein detection
neoplasia or inflammation. Further studies are
required to minimize the imprecision of the tests
and to gain deeper insight into the accuracy of the
assays before their routine use in veterinary labora-
tories. The Thermo assay cannot be recommended for
use with canine specimens because of a low cross-
reactivity with canine CRP.
Acknowledgements
The authors wish to thank Dr. Klaus Failing, Institute
for Biomathematics and Data Processing, Justus-Liebig-
University, Giessen, Germany, for his kind advice in
statistical questions. The authors affirm that they do not
have any affiliations or financial involvement in any
organization or entity with a direct financial interest in
the tests discussed in this manuscript.
Sources and manufacturers
a. PhaseTM Range canine CRP, Tridelta Development Ltd.,
Maynooth, County Kildare, Ireland.
b. Bayer CRP TIA, Bayer plc, Newbury, Berkshire, United
Kingdom.
c. CRP OSR 6147, Olympus Life and Material Science Europe
GmbH, Lismeehan, O’Callaghan’s Mills, Ireland.
d. High Linearity CRP (catalog CP7950), Randox Laboratories
Ltd., Crumlin, United Kingdom.
e. KonelabTM CRP (catalog 981699), Thermo Clinical Labsys-
tems Oy, Vantaa, Finland.
f. CRP-HS (catalog 419-22007), Wako Chemicals GmbH,
Neuss, Germany.
g. Horiba ABX Pentra 400, Horiba ABX, Montpellier, France.
h. CRP Plus Calibrator, Thermo Clinical Labsystems Oy,
Vantaa, Finland.
i. Dade Behring, Siemens Healthcare Diagnostics GmbH,
Eschborn, Germany.
j. SediplusH BSG/ESG/VS-Westergren, Sarstedt, Nürnbrecht,
Germany.
k. ADVIA 2120, Siemens Healthcare Diagnostics GmbH,
Eschborn, Germany.
l. Amelung-Coagulometer KC4A, Heinrich Amelung GmbH,
Lemgo, Germany.
m. Analyse-it for Microsoft Excel (version 2.00), Analyse-it
Software Ltd., Leeds, United Kingdom.
n. BMDP Statistical Software 8.1., Statistical Solutions Ltd.,
Cork, Ireland.
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