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Technical Briefs |
ana Rothkrantz-Kos11 Department of Clinical Chemistry, University Hospital Maastricht, 6202 AZ Maastricht, The Netherlands
aaddress correspondence to this author at: Department Clinical Chemistry, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands; fax 0031-43-3874692, e-mail DIEIJEN{at}Klinchem.azm.nl
Because C-reactive protein (CRP) has prognostic value in patients with acute coronary syndromes and in apparently healthy people (1), various high-sensitivity CRP (hs-CRP) methods have been introduced (2)(3). For the clinical laboratory, it is of course most practical if one CRP method can be used for the complete measuring range (0.2–1000 mg/L) to report reliable CRP results regardless of the clinical context.
We report here the evaluation of a hs-CRP method for the Beckman Coulter IMMAGE®, which is compared with the IMMULITE and BNA hs-CRP methods, as well as with the Beckman Synchron LX®20 CRP method for the higher range.
Venous blood samples were collected from 291 ostensibly healthy blood donors, 177 males and 114 females presenting at the Sanguin Blood Bank in Maastricht. Samples for method comparison were collected from 531 patients for whom a CRP was requested for routine analysis. The Medical Ethical Committee of the Hospital approved the procedure followed.
Serum was separated from the red cells by centrifugation at 2500g for 20 min and stored at -70 °C until analysis (4).
The hs-CRP detection method on the IMMULITE automated analyzer (Diagnostic Product Corporation) is a two-site chemiluminescent enzyme immunometric assay with a detection limit of 0.10 mg/L and a measuring range of 0.10–500 mg/L.
hs-CRP analysis is performed on the BNA nephelometer (Dade Behring) by particle-enhanced immunonephelometry with a detection limit of 0.18 mg/L and a measuring range of 0.18–1150 mg/L.
The IMMAGE hs-CRP (trade name, IMMAGE CRPH; Beckman Coulter) is a turbidimetric method based on the peak rate principle (2) measured by a near-infrared particle immunoassay, with a laser diode at 940 nm, a detection limit of 0.20 mg/L, and a measuring range of 0.20–1440 mg/L. To improve sensitivity, the latex particle size is now increased three- to fourfold.
CRP was measured on a routine clinical chemistry analyzer, Synchron LX 20, by immunoturbidimetry, with a detection limit of 5.0 mg/L and a measuring range of 5.0–488 mg/L (Beckman Coulter, Inc.). The serum index measured on the Synchron LX 20 was used to indicate icterus, hemolysis, or lipemia. Sample turbidity was also assessed by macroscopic examination, which appeared to be more sensitive than the serum index.
Samples for imprecision studies were prepared from five serum pools in the range from 0.2–50 mg/L and from two serum pools in the range from 100–350 mg/L as determined on the IMMAGE. Within-run imprecision was obtained by measuring one sample 20 times within a single run. Between-run imprecision was obtained by measuring each concentration on 20 consecutive days on the basis of a single calibration.
Linearity studies were performed with two samples containing 
50 mg/L and 250 mg/L CRP. Using the manufacturer’s diluent, we prepared 11 dilutions of each sample and measured each in five replicates. To assess the agreement between serum and plasma for the hs-CRP IMMAGE method, serum, EDTA–, and heparin–plasma samples were simultaneously collected from a single stick in 25 patients.
Because hs-CRP distributions were skewed rightward, their values were expressed as medians ± SD. Agreement between methods was assessed visually by the method of Bland and Altman (5) and by means of Deming regression analysis (6). Furthermore, percentile values were estimated and compared. For determination of reference values, the 0.95 nonparametric interpercentile interval was calculated (7). Gender differences in samples from blood donors were assessed by the Mann–Whitney U-test, whereas median serum vs plasma comparisons were assessed by the Wilcoxon signed rank test.
The hs-CRP IMMAGE method appeared to be linear over the whole measured CRP concentration range and comparable with the results of BNA. The mean values for each dilution point were plotted vs expected values, and linear regression was performed. Regression analysis for the low range gave a slope of 1.012, an intercept of 0.201, and a correlation of 0.998, and for the high range a slope of 0.989, an intercept of -1.335, and a correlation of 0.999. For five pools with mean concentrations of 0.23, 1.22, 2.18, 16.94, and 44.46 mg/L, between-run (total) variations were 4.9%, 6.7%, 2.7%, 2.7%, and 2.1% and within-run variations were 5.9%, 4.3%, 3.5%, 2.1%, and 3.0%. For risk stratification for vascular disease, the hs-CRP assay imprecision should be <10% at a concentration of 0.2 mg/L (8), which was confirmed by our study. For two additional samples with concentrations of 168 and 342 mg/L, within-run CVs were <6.2% for all methods.
No significant differences were found when serum, heparin–, and EDTA–plasma samples were compared for the hs-CRP IMMAGE method (serum vs heparin plasma, P = 0.52; serum vs EDTA plasma, P = 0.09). Mean recoveries (± SD) of heparin– and EDTA–plasma samples related to serum concentrations were 99.6% ± 3.6% and 106.4% ± 11.2%, respectively.
Table 1
shows patient characteristics and CRP concentrations measured by the hs-CRP IMMAGE method in 291 blood donors. The median CRP concentration was 1.57 mg/L, and the 0.95 interpercentile interval was 0.20–11.24 mg/L for all samples together (males, 0.20–14.29 mg/L; females, 0.20–9.84 mg/L). When values >10 mg/L were omitted, the whole group median value remained higher than median values reported by others (0.58–1.13 mg/L) (2)(8). This is probably attributable to the weak positive correlation already observed between age and hs-CRP concentration (median age of healthy blood donors in our study was 49 years compared with 32 years reported in previous studies) (2). It might also be attributable to unreported episodes of cold within 90 days before the studies (9). The gender difference in hs-CRP was not significant, neither before (P = 0.15) nor after we omitted (P = 0.06) values >10 mg/L, as is in accordance with previous studies (2)(9).
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Turbidity assessed macroscopically (found more sensitive than lipemic index) is presented in Fig. 1
(indicated by closed circles). The evaluated hs-CRP methods were not influenced by turbidity as can be seen from Fig. 1
.
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In the low CRP range (0–10 mg/L), the hs-CRP IMMAGE method showed good agreement with the hs-CRP methods on BNA and IMMULITE, according to visual inspection of Bland–Altman plots and Deming regression analysis. For the Bland–Altman plots, the mean bias and SD were -0.1 and 0.4 for BNA vs IMMAGE, 0.6 and 0.6 for BNA vs IMMULITE, and -0.7 and 0.4 for IMMULITE vs IMMAGE.
The Deming comparison of hs-CRP (range, 0–10 mg/L) gave the following: a slope of 1.10 ± 0.18, an intercept of 0.01 ± 0.17, and a Sy|x of 0.39 (r = 0.991) for IMMAGE and BNA; a slope of 1.37 ± 0.25, an intercept of 0.01 ± 0.18, and a Sy|x of 0.54 (r = 0.986) for IMMAGE and IMMULITE; and a slope of 0.80 ± 0.11, an intercept of -0.01 ± 0.10, and a Sy|x of 0.22 (r = 0.989) for IMMULITE and BNA. Furthermore, percentile analysis was performed to allow comparison with a recent study by Roberts et al. (2). For the percentile comparison, BNA was taken as a reference method, whereas Roberts et al. used the BN II N hs-CRP assay. In the concentration range of 0.2–10 mg/L, the IMMAGE hs-CRP method showed excellent agreement with BNA in all quartiles (data not shown). In our study, performed on 531 samples, all hs-CRP methods demonstrated good agreement for concentrations <100 mg/L.
In comparison studies among all hs-CRP methods, large discrepancies were seen at concentrations >100 mg/L (Fig. 1
). The scatter observed was >2 SD, which could not be explained by proportional bias or imprecision data in the higher range, but was more likely attributable to assay- or system-related issues. Roberts et al. (2) already reported discrepancies among four hs-CRP methods with concentrations >50 mg/L in a smaller study (50 patients). Our extended study certainly confirms this fact and addresses the problem of reliability of the CRP measurements at concentrations >100 mg/L. Comparison of the hs-CRP methods with our routinely used CRP method (Synchron LX 20) led to a good correlation with only the BNA.
In conclusion, evaluation of the hs-CRP method on the IMMAGE yielded good imprecision results and satisfactory linearity, as is required for hs-CRP methods. The method correlated well with the other two compared hs-CRP methods in the range of 0.2–100 mg/L. At concentrations >100 mg/L, however, poor correlations were found between all compared methods. Because we are convinced that for the clinical laboratory it is most practical to use one CRP method for the complete measuring range, further investigation to improve agreement among the different methods in the higher range is required.
Acknowledgments
We thank Beckman Coulter Inc., for its cooperation, and Kim Herzberg of University Hospital Maastricht, The Netherlands, for perfect technical assistance.
References
The following articles in journals at HighWire Press have cited this article:
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  W. L. Roberts CDC/AHA Workshop on Markers of Inflammation and Cardiovascular Disease: Application to Clinical and Public Health Practice: Laboratory Tests Available to Assess Inflammation--Performance and Standardization: A Background Paper Circulation, December 21, 2004; 110(25): e572 - e576. [Abstract] [Full Text] [PDF]
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J. A. Schaar, E. Regar, F. Mastik, E. P. McFadden, F. Saia, C. Disco, C. L. de Korte, P. J. de Feyter, A. F.W. van der Steen, and P. W. Serruys Incidence of High-Strain Patterns in Human Coronary Arteries: Assessment With Three-Dimensional Intravascular Palpography and Correlation With Clinical Presentation Circulation, June 8, 2004; 109(22): 2716 - 2719. [Abstract] [Full Text] [PDF]
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 N. Khuseyinova, A. Imhof, G. Trischler, D. Rothenbacher, W. L. Hutchinson, M. B. Pepys, and W. Koenig Determination of C-Reactive Protein: Comparison of Three High-Sensitivity Immunoassays Clin. Chem., October 1, 2003; 49(10): 1691 - 1695. [Full Text] [PDF]
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S. Rothkrantz-Kos, M. P. van Dieijen-Visser, P. G.H. Mulder, and M. Drent Potential Usefulness of Inflammatory Markers to Monitor Respiratory Functional Impairment in Sarcoidosis Clin. Chem., September 1, 2003; 49(9): 1510 - 1517. [Abstract] [Full Text] [PDF]
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T. B. Ledue and N. Rifai Preanalytic and Analytic Sources of Variations in C-reactive Protein Measurement: Implications for Cardiovascular Disease Risk Assessment Clin. Chem., August 1, 2003; 49(8): 1258 - 1271. [Abstract] [Full Text] [PDF]
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M. M. Kimberly, H. W. Vesper, S. P. Caudill, G. R. Cooper, N. Rifai, F. Dati, and G. L. Myers Standardization of Immunoassays for Measurement of High-Sensitivity C-reactive Protein. Phase I: Evaluation of Secondary Reference Materials Clin. Chem., April 1, 2003; 49(4): 611 - 616. [Abstract] [Full Text] [PDF]
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T. Vainas, T. Lubbers, F. R.M. Stassen, S. B. Herngreen, M. P. van Dieijen-Visser, C. A. Bruggeman, P. J.E.H.M. Kitslaar, and G. W. H. Schurink Serum C-Reactive Protein Level Is Associated With Abdominal Aortic Aneurysm Size and May Be Produced by Aneurysmal Tissue Circulation, March 4, 2003; 107(8): 1103 - 1105. [Abstract] [Full Text] [PDF]
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indicate completely clear samples.