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Evaluation of a Turbidimetric Denka Seiken C-Reactive Protein Assay for

Cardiovascular Risk Estimation and Conventional Inflammation Diagnosis

Article  in  Clinical Chemistry · April 2003

DOI: 10.1373/49.3.511 · Source: PubMed

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 Clinical Chemistry 49, No. 3, 2003 511

Evaluation of a Turbidimetric Denka Seiken C-Reactive

Table 1. PPT1 and TPP1 activities in dried blood samples
Protein Assay for Cardiovascular Risk Estimation and
from patients with different forms of neuronal ceroid
Conventional Inflammation Diagnosis, Thomas Christian
lipofuscinosis and from healthy controls. Vukovich,* Stefan Mustafa, Helmut Rumpold, and Oswald
PPT1 activity, TPP1 activity,
Patient Diagnosis nmol/spot nmol/spot
Wagner (Institute of Medical and Chemical Laboratory
Diagnostics, University Hospital of Vienna, AKH Leit-
1 CLN1 0.02 0.22
stelle 5H, Waehringerguertel 18, A-1090 Vienna, Austria;
2 CLN1 0 0.34
* author for correspondence: fax 43-1-40400-5389, e-mail
3 CLN1 0.02 0.39
thomas.vukovich@kimcl.akh.magwien.gv.at)
4 CLN1 0.02 0.27
5 CLN1 0.03 0.31
Measurement of C-reactive protein (CRP) is used for
6 CLN1 0.01 0.3
conventional inflammation diagnosis (1 ) and diagnosis of
7 CLN2 1.13 0
low-grade inflammation for risk estimation of cardiovas-
8 CLN2 0.8 0
cular events (2, 3 ). Because diagnostic measurement
9 CLN2 0.49 0
ranges for those two indications differ by two orders of
10 CLN2 0.41 0
magnitude, different methods or different applications of
11 CLN2 0.88 0
one method must be used at present to cover both
12 CLN2 carrier 0.42 0.05
diagnostic measurement ranges (4, 5 ). The aim of this
13 CLN2 carrier 0.46 0.04
study was to evaluate the analytical performance of the
14 CLN3 0.54 0.21
Denka Seiken turbidimetric CRP assay compared with the
15 CLN3 0.49 0.11
Dade Behring nephelometric assay across a concentration
Controls 0.4–1.52 0.1–0.67 range of 0.2–300 mg/L. For this evaluation, leftover
(n ⫽ 70) material was used, which is in concordance with the
European Law for Medical and Diagnostic Products.
For precision and linearity studies, we prepared serum
Additionally, because the enzyme activities remain stable pools from blood samples with previously measured CRP
over several days, mailing to specialized centers is easier (BN II nephelometer; Dade Behring). The low and high
and less expensive. Furthermore, the assay requires only pools were prepared by combining samples with CRP ⬍1
a few drops of blood in contrast to the 2–5 mL of EDTA and 200 –300 mg/L, respectively. The high pool was
blood needed for leukocyte assays. We consider the dried diluted with the low pool to the following final percent-
blood tests for PPT1 and TPP1 a very useful approach to ages of high pool: 100%, 33%, 11%, 3.7%, 1.2%, 0.41%,
the diagnosis of CLN1 and CLN2. However, the diagnosis 0.14%, and 0%. The dilutions were aliquoted and stored at
should be confirmed by DNA tests, electron microscopy, ⫺20 °C for a maximum period of 4 weeks until use. Both
and enzyme measurements in skin fibroblasts if only very CRP methods were used according the manufacturers’
low or no enzyme activities are detectable. instructions. The turbidimetric wide-range CRP assay
provided by Denka Seiken [CRP-latex (II)X2 assay, cali-
brated against reference preparation CRM 470] was per-
formed on a Hitachi 911 (Roche), and the nephelometric
References
1. Goebel HH, Mole SE, Lake BD, eds. The neuronal ceroid lipofuscinoses assay provided by Dade Behring was performed on a BN
(Batten disease). Amsterdam: IOS Press, 1999:197 pp. II nephelometer (Dade Behring).
2. Van Diggelen OP, Keulemans JL, Winchester B, Hofman IL, Vanhanen SL, To examine the precision of the Denka Seiken method
Santavuori P, et al. A rapid fluorogenic palmitoyl-protein thioesterase assay: compared with the established method, aliquots of serum
pre- and postnatal diagnosis of INCL. Mol Genet Metab 1999;66:240 – 4.
3. Ezaki J, Takeda-Ezaki M, Oda K, Kominami E. Characterization of endopepti-
pool dilutions were measured in duplicate on 10 different
dase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in days (Table 1). CVs were ⱕ6.8% for the Denka Seiken
classical late infantile neuronal ceroid lipofuscinosis. Biochem Biophys Res method and ⱕ4.4% for the Dade Behring method. For all
Commun 2000;268:904 – 8.
4. Chamoles NA, Blanco MB, Gaggioli D, Casentini C. Hurler-like phenotype:
enzymatic diagnosis in dried blood spots on filter paper. Clin Chem 2001; Table 1. Summary of precision and linearity data.
47:2098 –102.
Denka Seiken (Hitachi 911) Dade Behring (BN II)
5. Vanhanen SL, Raininko R, Autti T, Santavuori P. MRI evaluation of the brain in
infantile neuronal ceroid-lipofuscinosis. Part 2. MRI findings in 21 patients. Mean, CV, Target, Deviation, Mean, CV, Target, Deviation,
J Child Neurol 1995;10:444 –50. Pool mg/L % mg/L % mg/L % mg/L %
6. Salonen T, Jarvela I, Peltonen L, Jalanko A. Detection of eight novel palmitoyl 1 254 1.3 243 2.1
protein thioesterase (PPT) mutations underlying infantile neuronal ceroid
lipofuscinosis (INCL; CLN1). Hum Mutat 2000;15:273–9. 2 84.7 1.1 84.2 0.6 82.9 2.5 80.2 3
7. Steinfeld R, Heim P, von Gregory H, Meyer K, Ullrich K, Goebel HH, et al. Late 3 29.7 1.6 28.3 5 28.9 2.3 27 7
infantile neuronal ceroid lipofuscinosis: quantitative description of the clinical 4 10.1 2.1 9.78 3 8.61 2.9 9.28 ⫺7
course in patients with CLN2 mutations. Am J Med Genet 2002;112:347–54.
5 3.62 2.8 3.5 3 3.15 2.5 3.3 ⫺5
8. Kohlschütter A, Laabs R, Albani M. Juvenile neuronal ceroid lipofuscinosis:
quantitative description of its clinical variability. Acta Paediatr Scand 1988; 6 1.57 2.0 1.42 11 1.27 3.1 1.31 ⫺3
77:867–72. 7 0.81 3.7 0.74 9 0.68 2.6 0.66 3
8 0.38 6.8 0.32 4.4
512 Technical Briefs
Fig. 1. Method comparison of selected serum
or plasma samples with results ⬎5 mg/L (A;
n ⫽ 102) and ⬍5 mg/L (B; n ⫽ 108).
pools, values measured by the Denka Seiken method were
somewhat higher than those measured by the Dade
Behring method.
To study the linearity of each method, we calculated the
target concentrations of pools 2–7 from the mean concen-
trations of pool 1 (100% high pool) and pool 8 (100% low
pool) as measured by the respective methods. Table 1
shows the calculated target concentrations of the pool
dilutions as well as the percentages of deviation of the
measured concentrations from the respective targets. The
Denka Seiken method revealed positive deviations,
whereas the comparison method revealed positive as well
as negative deviations. Deming regression of measured-
vs-target pool values revealed that the slopes of the Denka
Seiken method were closer to 1 when calculated for the
whole range (pools 2–7; slope, 1.01; intercept, 0.09 mg/L;
Sy兩x ⫽ 0.43 mg/L; r ⫽ 1) as well as for the low range (pools
4 –7; slope, 1.03; intercept, 0.06 mg/L; Sy兩x ⫽ 0.09 mg/L;
r ⫽ 0.999) than were the values obtained with the Dade
Behring comparison method (pools 2–7; slope, 1.04; inter-
cept, 0.16 mg/L; Sy兩x ⫽ 0.72 mg/L; r ⫽ 0.999; and pools
4 –7; slope, 0.92; intercept, 0.09 mg/L; Sy兩x ⫽ 0.10; r ⫽
0.999).
To study the concordance of results obtained from
plasma and serum samples, we analyzed 30 pairs of
heparin and serum samples from the same blood dona-
tion with the Denka Seiken method. Deming regression
analysis of these measurements revealed optimum corre-
lation: y ⫽ 1.00x ⫺ 0.006 mg/L [r ⫽ 1.000; range, 0.3– 83
mg/L; mean (SD) 24.9 ⫾ 26.6 mg/L in serum vs 24.8 ⫾
26.5 mg/L in plasma].
To study the correlation of the Denka Seiken method
with the comparison method, we collected 191 serum and
19 heparin-plasma samples from routine requests for
traditional CRP analysis or high-sensitivity CRP. Of these,
108 samples had concentrations ⬍5 mg/L and 102 had
concentrations ⬎5 mg/L. The samples were then ana-
lyzed with the Denka Seiken and the comparison method
(Fig. 1). Fig. 1 shows that at concentrations ⬍5 mg/L, the
slope was higher for the Denka Seiken method compared
with the Dade Behring method (slope, 1.15; intercept, 0.09
mg/L; Sy兩x ⫽ 0.14 mg/L; r ⫽ 0.986). At concentrations ⬎5
mg/L, however, the slopes were close to the lines of unity
for both methods (slope, 1.03; intercept, 0.11 mg/L; Sy兩x ⫽
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3.76 mg/L; r ⫽ 0.998). This bend in the slopes might be
attributed to the opposite nonlinearity between the Denka
Seiken method and the Dade Behring method, as shown
in Table 1.
To evaluate the concordance of both method in cardio-
vascular risk assessment, the recently proposed cutoff
values for the Dade Behring method (6 ) were adjusted for
the Denka Seiken method by the regression equation
calculated from patient samples ⬍5 mg/L (Fig. 1B). At
these adjusted cutoff values, 96% of patients were allo-
cated to identical risk groups. No patient was mismatched
more than one adjacent risk group.
In conclusion, the linearity of the Denka Seiken CRP
assay is slightly better than that of the comparison
method. Because of the bend in the linearity curve for the
Dade Behring method, correlation of patient values re-
vealed different slopes at high and low CRP concentra-
tions. Therefore, sufficient concordance between methods
for cardiovascular risk estimation might be obtained only
after adjustment of cutoff values by the regression equa-
tions. The Denka Seiken CRP assay covers in a single
determination the ranges for diagnosis of both conven-
tional and low-grade inflammation. This method there-
fore improves laboratory throughput by reducing the
number of retests with different sample dilutions or a
different method.
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protein, a sensitive marker of inflammation, predicts future risk of coronary
heart disease in initially healthy middle-aged men. Results from MONICA
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