The ability to measure low levels of C-reactive protein may prove to be an effective tool in predicting cardiovascular and peripheral vascular disease. An assay that can detect these low levels has recently been developed.
C-reactive protein (CRP) is an "acute-phase" protein, the serum or plasma levels of which rise during the body's general, nonspecific response to infectious and noninfectious inflammatory processes, such as rheumatoid arthritis.1
Within four to eight hours after an acute event that results in tissue injury, infection, or inflammation, CRP values may rise above the normal level of less than 5 mg/L to 20 to 500 mg/L.2 Recent research has suggested that even a CRP level as low as 3 mg/L is a possible indication of inflammation that should be investigated through additional testing.
Many physicians now believe that CRP can be used as an aid in assessing the risk of cardiovascular and peripheral vascular disease. This conclusion is a result of recent medical research, combined with technology that can quantitatively measure extremely low levels of CRP in the blood.3–7 The same research has also confirmed the predictive value of measuring low-level CRP to assess atherosclerosis and other cardiovascular diseases.
Primary Risk in Asymptomatic Individuals
Among the more important medical studies sparking interest in the utility of CRP are results from the 1997 Physicians' Health Study (PHS).3 In the PHS, 22,000 male U.S. physicians aged 40–84 years without prior cardiovascular disease history were enrolled and randomized (double blind) into groups that were given either a placebo or a regimen of aspirin and ß-carotene. As part of a nested, case-control study, 543 randomly selected samples from men in both groups were assayed for CRP.
Because the CRP assays then available could not detect levels below 11 mg/L, a special home-brew test was developed for this study by Russell Tracy, director of the laboratory for clinical biochemistry research at the University of Vermont. This manual ELISA test was capable of measuring CRP at levels from 0.1 to 4.0 mg/L.
In the PHS, CRP levels in samples obtained during a baseline draw between 1982 and 1984 were compared with the incidence of recorded cardiovascular events. Men who later suffered an acute myocardial infarction (AMI) had a median baseline CRP level of 1.51 mg/L. Those who didn't experience cardiac problems had a median baseline level of 1.13 mg/L (see Figure 1).
Figure 1. The relative risk of future myocardial infarction, per quartile of CRP baseline plasma concentration.3
The study results suggested that a patient's risk of having an AMI increased with increasing levels of CRP. Compared with subjects in the lowest quartile, that risk was nearly threefold greater for subjects in the upper quartile. Such risks did not diminish over long periods, were not affected by whether the subject smoked cigarettes, and were independent of other lipid-related and non-lipid-related risk factors.
The PHS findings for men were confirmed for women in another paper from the Women's Health Study.4 The study was conducted with postmenopausal subjects with no history of prior cardiovascular disease. In comparing this study with the PHS, the risk associated with increasing levels of CRP was even more pronounced in women than in men, with a fivefold increase in risk for any vascular event and a sevenfold increase in risk for AMI or stroke in the highest quartile (see Figure 2). However, CRP levels in general were higher in this female population, with an upper quartile of 7.3 mg/L, compared with 2.1 mg/L in the PHS. The study indicated that risk-prediction models which included measurement of CRP levels could better predict the occurrence of a cardiovascular or peripheral vascular event than models that did not measure CRP.
Figure 2. Future risk of CVD in apparently healthy women is predicted by CRP concentration, by quartile, when adjusted for BMI, diabetes, hypertension, hypercholesterolemia, exercise, family history, and treatment assignment.4
A German study on a random sample of 936 initially healthy men aged 45–64 years revealed similar results to the PHS.5 Both studies seem to support the assertion that small elevations in CRP levels occur long before traditional symptoms of heart disease are noticed. Meanwhile, other studies have evaluated and shown the therapeutic effect of taking both aspirin and pravastatin, a cholesterol-lowering drug, to modify CRP levels.6,7
These research efforts, among others, effectively established the market need for an automated version of the manual ELISA CRP assay. Such a test could be used not only for research, but to support what could eventually be an assay routinely performed in conjunction with more-traditional tests (e.g., LDL and HDL cholesterol) to identify individuals at increased risk of cardiovascular and peripheral vascular disease.
Behring Diagnostics began its efforts to develop such an assay for commercial use in 1996. The starting point was to redesign the company's standard CRP assay for use on nephelometers—automated, analytical instruments based on the nephelometric analysis of antigen antibodies. The new test used a monoclonal antibody to offer low-end sensitivity for measurements of CRP (0.175 mg/L) not possible with the original assay.
The assay was introduced in late 1996 in Germany and was successful for three reasons. First, nephelometers are more widely used in European clinical laboratories than in the United States because plasma protein analysis is routinely done in Europe. In the United States the analytical focus is more toward clinical chemistry.
Second, the laboratory procedures required to perform the new assay are virtually the same as those for the older, less-sensitive test. Reagent preparation, dilution, and automated performance on the nephelometer are identical. This enabled the new assay to fit in very well with established laboratory practice of drawing plasma or serum from the patient in standard 5-ml or 3-ml tubes, manually removing it from the tube, and centrifugating it prior to running it on the analyzer.
Finally, cardiology conferences in Italy, England, and The Netherlands helped to generate interest in the new assay. Since its introduction in 1996, the test has become widely used in Europe for cardiac review.
Meanwhile, a clinical study conducted in the United States compared results from the automated CRP assay to those from the manual ELISA test.8 Because the manual assay had previously been shown to be a good predictor of future peripheral vascular disease in asymptomatic populations, correlating the results of the automated test would indicate whether it was also efficacious.
The results of this study indicated that the two CRP assays were highly correlated. All but two of 144 apparently healthy men were classified into concordant quartiles or varied by only one quartile. Findings indicated that the automated assay is as effective as the previously validated ELISA for classifying patients according to cutoff points for risk of cardiovascular and peripheral vascular disease.
Principle of the Method
FDA approved Dade Behring's N High Sensitivity CRP assay for marketing in the United States in November 1999. It is the first automated IVD to quantitatively determine CRP in human serum, heparin, and EDTA-plasma using particle enhanced immunonephelometry. The assay is designed specifically for use on Dade Behring nephelometer systems. Nephelometry is the immunochemical technique most commonly used for measuring protein levels in serum, urine, or cerebrospinal fluid.
Blood samples for the assay are obtained using the standard venipuncture technique into standard collection tubes. As with most immunoassays, sample centrifugation is required before processing.
The assay consists of a suspension of polystyrene particles coated with murine monoclonal antibodies to CRP. The concentration of suspended particles is optimal for agglutination measurement by nephelometry. When reagents are mixed with samples containing CRP, the intensity of the scattered light in the nephelometer depends on the CRP content of the sample. Therefore, the CRP concentration can be determined by comparison with dilutions of a standard of known concentration.
The instrument automatically handles the test mixtures and quality controls. Samples are diluted as defined in the test protocol and are automatically calculated using a stored reagent curve. The system contains a computer and proprietary software whereby a stored reference curve is compared to each patient sample run. The assay's dynamic range is 0.175 to 1100 mg/L. First results are available in six minutes on the Dade Behring BN II system, a random-access analyzer with holding capacity for up to 100 samples. The system can provide throughput of up to 225 tests per hour, depending on processing parameters set by the operator.
The implications of this new assay are significant. It is low cost (individual tests cost less than $50) and demonstrates a good coefficient of variance. By combining this assay with standard cholesterol screening, researchers believe that they will be better able to identify people at highest risk for cardiovascular and peripheral vascular disease prior to its occurrence.
In the United States—as elsewhere in the world—the trend in most clinical and commercial reference laboratories is toward workstation consolidation, whereby practically all testing is performed on a single instrument or multiple instruments of the same type.
To take advantage of this trend, Dade Behring is actively researching methods to integrate plasma protein testing (including high-sensitivity CRP) on its existing testing platforms. There are currently more than 2000 Dimension RxL systems installed worldwide, and the ability to perform high-sensitivity CRP testing on this established platform would further increase the test's value to both physicians and patients. A full cardiac panel from a single patient sample could then include high-sensitivity CRP in addition to HDL cholesterol, troponin I, mass CK-MB, myoglobin, and other cardiac-related assays currently on the analyzer.
The current timeline is to integrate the high-sensitivity CRP assay late this year or early in 2001.
1. Dade Behring, Clinical Significance of Tests Available from Dade Behring (March 1995): 132–133.
2. I Kushner, DL Rzewricki, "The Acute Phase Response: General Aspects," Ballicre's Clinical Rheumatology 8 (1994): 513–530.
3. PM Ridker et al, "Inflammation, Aspirin and the Risk of Cardiovascular Disease in Apparently Healthy Men," New England Journal of Medicine 336 (1997): 973–979.
4. PM Ridker et al, "Prospective Study of C-Reactive Protein and the Risk of Future Cardiovascular Events Among Apparently Healthy Women," Circulation 98 (1998): 731–733.
5. W Koenig et al, "C-Reactive Protein, a Sensitive Marker of Inflammation, Predicts Future Risk of Coronary Heart Disease in Initially Healthy Middle-Aged Men," Circulation 99 (1999): 237–242.
6. PM Ridker et al, "Inflammation, Pravastatin, and the Risk of Coronary Events After Myocardial Infarction in Patients with Average Cholesterol Levels," Circulation 98 (1998): 839–844.
7. PM Ridker et al, "Long-Term Effects of Pravastatin on Plasma Concentration of C-Reactive Protein," Circulation 100 (1999): 230–235.
8. N Rifai, RP Tracy, and PM Ridker, "Clinical Efficacy of an Automated High-Sensitivity C-Reactive Protein Assay," Clinical Chemistry 45 (1999): 2136–2141.
Thomas A. Luhr is director of marketing for central laboratory methods, and Jitu Modi is product manager for plasma protein methods at Dade Behring Inc. (Deerfield, IL).