g

Extended Range C-Reactive Protein (erCRP) Reagent

0.15mg/L >200mg/L 5 - 20mg/L1mg/L 5.78mg/L

Thermo Extended Range CRP

Detection Limit

Median Normals

Median TIMI 11A

Acute Transmural Myocardial Infarction

Bacterial Sepsis Acute Infection

Clinical Diagnostics 189-199 Browns Road PO Box 700 +61 3 9790 4100 phone www.thermo.com Clinical Chemistry Noble Park VIC 3174 Noble Park Vic 3174 +61 3 9790 4155 fax

Australia Australia July 2005

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Table Of Contents

Extended Range CRP Features and Benefits .................................... 3 Extended Range CRP Test ................................................................ 4 Performance Data .............................................................................. 5

Method Comparison ......................................................................... 5 Precision Studies.............................................................................. 6 Assay Analytical Sensitivity .............................................................. 8 Assay Analytical Specificity .............................................................. 8 High Dose Hook Effect ................................................................... 12 Exogenous Interference ................................................................. 12 Sample Type Studies ..................................................................... 13 Reagent Stability ............................................................................ 15 Calibrator Stability .......................................................................... 16 Calibration ...................................................................................... 16 Reference Interval .......................................................................... 17

Background Information ................................................................... 18 C-Reactive Protein ......................................................................... 18 Clinical Significance ....................................................................... 18 CRP Methodology .......................................................................... 19 References ..................................................................................... 20

Literature .......................................................................................... 22

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Extended Range CRP Features and Benefits Features Benefits High Sensitivity To measure very low

concentrations

Precision satisfies AHA and CDC recommendations for high sensitive method (CVs of <10% at levels of 0.3 mg/L to 10 mg/L CRP)

A highly reproducible assay adds to the reliability of measurement

Method Standardized vs. Certified Reference Method (CRM470 BCR)

Accurate results

Wide measurable range To cover the clinically useful range with a single reagent

No need for sample dilution up to 320 mg/L Saves repeating assays and disrupting laboratory workflow

No high dose hook effect up to 1000 mg/L CRP

Avoids erroneous results with extremely high samples

Performance Characteristics Method Comparison Slope: 1.03

Intercept: -0.25 mg/L Correlation Coefficient: 0.995

Precision Within-Run <2% Between- Run <2%

Open reagent stability 90 days

No significant interferences up to levels indicated: Bilirubin Conjugated Bilirubin Unconjugated Hemoglobin Ascorbic acid

60 mg/dL 60 mg/dL 1000 mg/dL 500 mg/dL

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Extended Range CRP Test Assay Principle The Extended Range CRP Reagent is an enhanced latex-agglutination turbidimetric immunoassay. Increased sensitivity at low concentration together with a wide linear range is gained by having latex particles of different sizes.22 Sample is added to a buffer solution and mixed with a suspension of mouse anti-human CRP monoclonal antibody that is bound to latex. CRP binds to the latex-bound antibody and agglutinates. The light scattering caused by the increase in particle size is used as a measure of CRP concentration. The amount of light scattering is proportional to the concentration of CRP in the sample.

Diagnostics Assay ProcedureThe Extended Range CRP method is a liquid, two reagent method for the direct measurement of CRP. The method can be completely automated and applicable to most clinical chemistry analyzers. Below is an example of the Extended Range CRP assay procedure for the Hitachi 912 clinical chemistry analyzer.

CRP Result

Sample: 2.4 µL Reagent 1: 120 µL

5 min. Incubation

Absorbance 1 (570 nm – 800 nm)

4 min. 25 sec Incubation

Reagent 2: 120 µL

Absorbance 2 (570 nm – 800 nm)

CRP molecule

A combination of large and small Anti-human CRP mouse monoclonal antibody-coated latex particles.

Measurement of absorbance change resulting from agglutination

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Performance Data The following section contains performance data for Thermo’s Extended Range CRP reagent. The data presented in this section were generated, using the Hitachi 912 and 911 clinical chemistry analyzers. Method Comparison Performance studies were conducted using the Extended Range CRP Reagent on the Hitachi 912 clinical analyzer and compared to the Dade Behring N High Sensitive CRP method*. CRP was measured over six days in which 229 serum samples, with concentrations between approximately 0.2 and 200 mg/L CRP were used (Figure 1). Samples with results greater than the initial assay range of the Dade Behring N High Sensitive CRP method (>11 mg/L) were diluted on board the Dade analyzer, as per the manufacturer’s instructions. The protocol followed the recommendations of NCCLS EP9, Method Comparison and Bias Estimation Using Patient Samples.23

Figure 1

Comparison: N-geneousTM Wide Range CRP and Dade Behring N High Sensitive CRP

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* at Boston’s Children’s Hospital Table 1 Extended Range CRP Dade Behring N High

Sensitive CRP n 229 229 Mean (mg/L) 9.0 9.0 Range (mg/L) 0.2 - 224 0.2 -198 Regression Analysis y = 1.03x - 0.25 mg/L Correlation Coefficient 0.995

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Precision Studies Precision studies were conducted using the Extended Range CRP Reagent on the Hitachi 912 clinical chemistry analyzer. The protocol followed the recommendations of NCCLS EP5, Evaluation of Precision Performance of Clinical Chemistry Devices.24 Within-run and total precision were calculated from an ANOVA (Analysis of Variance), as recommended by NCCLS, in which each control was run in duplicate, twice a day for 20 days. The studies were performed using serum test sets that were prepared by supplementing CRP-free human serum with a purified preparation of human CRP to the CRP concentrations shown in the table below. These materials were stored frozen (-20ºC) as aliquots and allowed to thaw prior to use. Within-Run Precision Within run precision experiments produced the following results on the Hitachi 912 clinical chemistry analyzer: Table 2

Serum Pool 1 2 3 4 5 n 80 80 80 80 80 Mean CRP level (mg/L) 0.30 1.00 2.97 51.3 202 Standard Deviation (mg/L) 0.02 0.02 0.04 0.61 3.0 Coefficient of Variation (%) 5.5 1.8 1.3 1.2 1.5

Total Precision Total precision experiments produced the following results on the Hitachi 912 clinical chemistry analyzer: Table 3

Serum Pool 1 2 3 4 5 n 80 80 80 80 80 Mean CRP level (mg/L) 0.30 1.00 2.97 51.3 202 Standard Deviation (mg/L) 0.02 0.02 0.05 0.96 3.1 Coefficient of Variation (%) 6.7 2.3 1.7 1.9 1.5

Linearity (Dilution and Recovery) Linearity studies were conducted in two parts: one to verify the larger, full range of concentration prediction and a second to evaluate the CRP concentration at the lower concentration range. Two sets (Full and Low Concentration Range) of admixtures of CRP-stripped serum (base matrix) and the same matrix supplemented with purified human CRP were prepared. The Full Concentration Range set consisted of samples with nominal concentrations ranging from 0 to 350 mg/L CRP. Low Concentration Range set, with nominal concentrations from 0 to 55 mg/L CRP, was used to evaluate the lower range. The protocol followed the recommendations of NCCLS EP6, Evaluation of the Linearity of Quantitative Measurement Procedures, a Statistical Approach.25 Figures 2 & 3 illustrate both linearity studies across the measurable range. Specimens above 320mg/L may be diluted with physiological saline before assaying. Multiply the result obtained from the manual dilution by the appropriate dilution factor.

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Linearity: Full Concentration Range Figure 2

Full Concentration Range Linearity

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Linearity: Low Concentration Range

Figure 3

Low Concentration Range Linearity

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Assay Analytical Sensitivity It has been recommended for clinical utility in predicting cardiovascular risk, CRP assays should be able to reliably measure down to 1 mg/L. For population based studies and clinical research the assays should be able to measure lower concentrations of 0.15 mg/L (the 2.5th percentile of the reference population).22

The sensitivity for the Extended Range CRP was evaluated on the Hitachi 911 clinical chemistry analyzer following the recommendations of NCCLS EP17-P, Protocols for Determination of Limits of Detection and Limits of Quantitation; Proposed Guidelines.26 Samples with decreasing concentrations of CRP were assayed 15 times, along with a saline blank, which was assayed 35 times, each on three lots of reagent. Functional sensitivity (or the Limit of Quantification, LOQ) is the concentration of CRP at which the CV is 20%. The functional sensitivity of the Extended Range CRP Reagent is 0.12 mg/L. Assay Analytical Specificity Endogenous Interference The potential effect of conjugated and unconjugated bilirubin, hemoglobin, Intralipid® and ascorbic acid on the Extended Range CRP assay were evaluated according to the recommendations of NCCLS EP7, Interference Testing in Clinical Chemistry,27 using the dose response method over the varying concentrations, as illustrated in Figures 4 to 8. A serum pool was split with part used as control and part used to prepare a high concentration of each potential interferent. Admixtures were used for testing. The serum pool had a nominal CRP concentration of 3 mg/L. The CRP concentration of the control portion and test portions were measured using the Extended Range CRP Reagent on the Hitachi 912 clinical chemistry analyzer.

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Figure 4

Ascorbic Acid

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No significant ascorbic acid interference as observed at levels up to 500 mg/dL.

Figure 5

Haemoglobin

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No significant hemoglobin interference was observed at levels up to 1000 mg/dL.

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Figure 6

Conjugated Bilirubin

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No significant conjugated bilirubin (ditaurobilirubin) interference was observed at levels up to 60 mg/dL.

Figure 7

Unconjugated Bilirubin

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No significant unconjugated bilirubin interference was observed at levels up to 60 mg/dL.

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Figure 8

Intralipid®

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A CRP bias of > 0.3 mg/L was observed at Intralipid® concentration > 0.8% (~2400 mg/dL triglyceride equivalent). Genzyme Diagnostics The effects of rheumatoid factor (RF) and human anti-mouse antibody (HAMA) on the Extended Range CRP Reagent were evaluated by testing eight patient samples (four sera samples for RF and four plasma samples for HAMA) that were known to have these antibodies present. In addition, a control sample that contained 1080 IU/mL RF was added to the evaluation. For each sample tested, a “spike and recover” experiment was conducted. The same small volume of very concentrated CRP was added to control-pool serum or plasma (for RF and HAMA, respectively) and to the samples to be evaluated. All samples were measured using the Extended Range CRP Reagent on the Hitachi 912 clinical analyzer. The additional CRP nominal concentration of 50 mg/L in the control samples was compared to the results in each of the test samples. The concentration ranges of substances tested for interferences as described above are listed in Table 4. Table 4

Substance Tested Concentration Rheumatoid factor (RF) 153 to 1711 IU/mL Human anti-mouse antibody (HAMA)

100 to 1494 ng/mL

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High Dose Hook Effect High dose hook was evaluated as part of the linearity testing as illustrated in Figure 9. No high dose hook effect was observed at CRP concentration up to 1000 mg/L.

Figure 9

High Dose Hook Effect

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Exogenous Interference The effect of 27 drugs, listed in Table 5, were evaluated according to the recommendations of NCCLS EP7, Interference Testing in Clinical Chemistry,27 using the paired difference method at the concentrations listed. Potential interferents were added to a serum pool with a nominal CRP concentration of 3 mg/L. The CRP concentration of the control portion and test portions of the specimen pool were measured using the Extended Range CRP Reagent on the Hitachi 912 clinical chemistry analyzer. Genzyme Diagnostics Table 5

Substance Tested Concentration (mg/dL) Acetaminophen (paracetamol) 20 Acetylsalicylic acid 50 Ampicillin 5 Caffeine 10 Captopril 6 Chlorpheniramine maleate 0.8 Cimetidine 10 Cyclosporin U 0.8 Doxycycline hyclate 6 Furosemide 2

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Substance Tested Concentration (mg/dL) Ibuprofen 40 Indomethacin 1 Levodopa 160 Lovastatin 1.6 Methotrexate 450 Methyldopa 2.5 Metoprolol tartrate 0.3 Metronidazole 1 Nicotinic acid 2 Omeprazole 7.2 Prednisone 1.2 Promethazine hydrochloride 1 Propranolol hydrochloride 0.5 Quinidine sulphate 5 Simvastatin 0.8 Theophylline 25 Tolbutamide 100

No interference was observed with the substances at the concentrations indicated. Sample Type Studies In conducting the sample type studies for Extended Range CRP, 18 volunteers each had four tubes drawn by standard venipuncture. A serum separator tube, a lithium heparin tube, a sodium heparin tube, and an EDTA anticoagulant tube were collected on each volunteer. Extended Range CRP reagent was used to measure CRP in duplicate with three lots of reagent for all the serum and plasma samples. The percent recoveries for the comparisons of heparin (sodium and lithium), and EDTA are summarized in Table 6. Table 6

Serum vs.

EDTA Plasma Serum vs. Sodium

Heparin Plasma Serum vs. Lithium

Heparin Plasma n 18 18 18 Percent Recovery 100.6% 99.4% 100.9%

Genzyme Diagnostics Sample Stability Testing The effects of sample aging, as well as long-term freezing, were evaluated using the Extended Range CRP Reagent on the Hitachi 912 clinical chemistry analyzer. CRP concentrations of fresh serum and plasma specimens were determined in quadruplicate. Specimens were split into aliquots and subsequently stored at 25°C, 2-8°C and at -20°C and retested in duplicate within approximately 5, 15 and 36 days, respectively (Figures 10 and 11). No significant bias was observed under the storage conditions and time intervals indicated. The effect of freeze-thaw cycles on serum and plasma on the recovery of CRP concentration measurements was determined by testing specimens that had been

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frozen at -20°C. The mean percent recovery of the Extended Range CRP results on fresh specimens was compared to the thawed specimen. No significant bias was observed through two freeze thaw cycles for either serum or plasma using the Extended Range CRP Reagent.

Figure 10

Sample Stability (25oc)

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Reagent Stability Extended Range CRP Reagents were opened and stored at either 2-8ºC or at 25ºC. Combinations of stored and newly opened R1 and R2 were tested using a set of five serum controls, covering a nominal CRP concentration range of 0.3 to 200 mg/L. Once opened, Extended Range CRP Reagents can be stored on board at 2-8ºC for 90 days, see Figure 12. Also, when opened, the reagents can be stored for seven days at 25ºC, see Figure 13.

Figure 12

Open On-Board Stability 2-8oC

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Figure 13

Reagent Stability at 25oC

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Calibrator Stability Extended Range CRP Calibrator Sets were opened and stored at 2-8ºC and tested weekly, see Figure 14. Once opened, the capped Extended Range CRP Calibrator Set was found to be stable for four weeks when stored at 2-8ºC.

Figure 14

Reconstituted Calibrator Stability at 2-8oC

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Calibration The Extended Range CRP Calibrator Set is a stabilized human serum used to calibrate the Extended Range CRP Reagent for the quantitative measurement of CRP in serum or plasma. The set contains 5 levels of CRP calibrators in the nominal range of 3 to 360 mg/L CRP. The Extended Range CRP Calibrator Set is traceable to CRM470 available from Institute for Reference Materials and Measurements (IRMM), and certified by the European Commission, Community Bureau of Reference (BCR).28,29 A master calibrator is prepared, of which aliquots are stored frozen for future use for assignment of Extended Range CRP Calibrator values. The master lot has CRP concentration assigned from multiple measurements with multiple lots of Extended Range CRP Reagent used on a Hitachi 917 clinical analyzer. The master lot, in turn, is used to calibrate Extended Range CRP Reagent on a Hitachi 917 clinical analyzer, from which Extended Range CRP Calibrators are assigned appropriate values for the quantitative measurement of CRP on clinical analyzers.

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Reference Interval The CRP reference interval for Extended Range CRP Reagent was confirmed to be consistent with the large study conducted by Rifai and Ridker,29 in which 22,403 apparently healthy individuals were included. Extended Range CRP Reagent is traceable to the same international reference material (CRM470). Confirmation was done by following recommendations of NCCLS protocol C-28, How to Define and Determine Reference Intervals in the Clinical Laboratory,30 in which samples from 50 apparently healthy subjects (25 male and 25 female) were assayed for CRP concentration. The 5th and 95th percentiles for the Extended Range CRP Reagent are 0.19 mg/L and 9.14 mg/L for females, and 0.28 mg/L and 8.55 mg/L for males, respectively. Genzyme Diagnostics

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Background Information C-Reactive Protein This protein was first identified in 1930 in the sera of acutely ill patients. It is able to bind the C-polysaccharide on the cell wall of Streptococcus pneumoniae. Eleven years later it was shown to be a protein, and was given the name C-reactive protein (CRP).1 It is produced in the liver and consists of five identical, non-glycosylated polypeptide subunits non-covalently linked to form a disc shaped structure with a molecular weight of 115,000 – 140,000. Clinical Significance The Acute Phase Response CRP is one of the most sensitive markers of the acute phase response in which various serum proteins change in concentration following an inflammatory stimulus such as severe bacterial infections.2 After myocardial infarction, stress, trauma, infection, inflammation, surgery or neoplastic proliferation it can rise within 24 to 48 hours to up to 2000 times the normal concentration. Measurement of this acute phase response is clinically useful for monitoring the activity of inflammatory conditions such as rheumatoid arthritis; for detecting infection in cases of leukemia or following surgery; for detecting transplant rejection; screening for organic disease and managing neonatal septicemia and meningitis.1 Although CRP concentrations increase many fold in response to major inflammatory conditions, it is also present at low concentrations in asymptomatic individuals and may reflect the baseline activity of circulating cytokines such as interleukin 6.3 The measurement of these lower concentrations has required the development of a new generation of high sensitivity CRP (hs-CRP) methods. Cardiovascular Risk Coronary Heart Disease is the major cause of death in the developed world. The main underlying cause is atherosclerosis, now recognized as an inflammatory disease4, which develops slowly over decades. Lipid screening is helpful in identifying individuals who are at increased risk of developing future coronary events; it fails to detect almost half of those who develop myocardial infarction. It has now been shown that in adults the measurement of hs-CRP concentration may be especially valuable for identifying individuals at higher risk for cardiovascular disease who have lipid concentrations within reference values.5 Laboratory and clinical evidence has shown that atherosclerosis is not simply a disease of lipids infiltrating the arterial wall. Systemic inflammation also plays a key role in the initiation and development of atheromatous plaques.4,6 Macrophages, monocytes and other inflammatory cells are prominent both in these plaques in the arterial wall7 and may also release lytic enzymes that degrade the fibrous cap causing plaque rupture.8 There have been several studies investigating hs-CRP as a risk factor in those who already have cardiovascular disease. The European Concerted Action on Thrombosis and Disability Angina Pectoris Study Group showed that in 2121 subjects with stable and

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unstable angina, there was a 45% increase in the relative risk of non-fatal MI or sudden cardiac death for every standard deviation increase in hs-CRP.9 Similarly, in the Cholesterol and Recurrent Events trial, studying those who had already suffered a MI, those with hs-CRP in the highest quintile had an 75% higher risk of developing another coronary event within five years.10

As well as being a powerful predictor of adverse events in those with established disease, the value of the test in primary prevention has also been established. Ten prospective studies, six in the US and four in Europe, have consistently shown that hs-CRP is a powerful predictor of future first coronary event in apparently healthy men and women.5 For example, the Physician’s Health Study showed that those in the highest quartile for hs-CRP had a twofold higher risk of stroke, a threefold higher risk of myocardial infarction and a fourfold higher risk of peripheral vascular disease.11,12 Also, the Helsinki Heart Study showed that subjects with the highest quartile of hs-CRP values had more than a threefold increased risk of future myocardial infarction or coronary death.13

Separate studies have shown that hs-CRP is a good prognostic marker of future coronary events in both men14 and women15. CRP is also significantly related to various cardiovascular disease risk factors in children.16

Sampling and interpretation There is no diurnal variation in CRP concentrations, so samples may be taken at any time.17 However, CRP concentrations do show substantial intra-individual variability.18

Consequently, more than one measurement may be needed to better assess a person’s true CRP status. The American Heart association and CDC have recommended that two CRP determinations should be made two weeks apart in people who are metabolically stable and free from obvious inflammatory of infectious conditions to provide a better estimate of CRP concentration.19

Initially it was felt that higher levels of CRP (>10 mg/L) might represent non-specific inflammation and therefore not a useful predictor of cardiovascular risk, and that very low levels might give a false sense of security. However, it has now been shown that hs-CRP gives clinically useful risk predictions across all values from <0.5mg/L to >10mg/L.20

CRP Methodology Original assays for high sensitive CRP were based on ELISA or labeled immunoassay. More recently, nephelometry using polystyrene particles coated with monoclonal antibodies to CRP has been used.21 However, this requires a nephelometer to measure the scattered light which increases as CRP causes the agglutination of the particles. Now, turbidimetric assays such as the Extended Range CRP test have been developed that can be run on conventional clinical chemistry analyzers.

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References 1. Johnson AM, Rohlfs EM, Silverman LM. Proteins. In: Tietz Textbook of Clinical Chemistry Third Edition Eds Burtis CA, Ashwood ER. WB Saunders 1999:493. 2. Morley JJ, Kushner I. Serum C-reactive protein levels in disease. Ann N Y Acad Sci 1982;389:406-18. 3. Castell JV, Gomez-Lechion MJ, David M, Fabra R, Trullenque R, Heinrich PC. Acute-phase response of human hepatocytes: regulation of acute-phase protein symthesis by interleukin-6. Hepatology 1990;12:1179-86. 4. Ross R. Atherosclerosis - an inflammatory disease. N Engl J Med 1999;340:115-26. 5. Rifai N, Ridker PM. Proposed cardiovascular risk assessment algorithm using high-sensitivity C-reactive protein and lipid screening. Clin Chem 2001;47:28-30. 6. Libby P, Ridker PM. Novel inflammatory markers of coronary risk: theory versus practice [Editorial; comment]. Circulation 1999;100:1148-50. 7. Cybulsky MI, Gimbrone MA Jr. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 1991;251:788-91. 8. Moreno PR, Falk E, Palacios IF, Newell JB, Fuster V, Fallon JT. Macrophage infiltration in acute coronary symptoms. Implications for plaque rupture. Circulation 1994;90:775-8. 9. Haverkate F, Thompson SG, Pyke SD, Gallimore JR, Pepys MB. Production of C-reactive protein and risk of coronary events in stable and unstable angina. European Concerted Action on Thrombosis and Disability Angina Pectoris Study Group. Lancet 1997;349:462-6. 10. Ridker PM, Rifai N, Pfeffer MA, Sacks FM, Moye LA, Goldman S, et al. Inflammation, pravastatin, and the riska of coronary events after myocardial events in patients with average cholesterol levels. Cholesterol and Recurrent Events (CARE) Investigators. Circulation 1998;98:839-44. 11. Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med 1997;336:973-9. 12. Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Plasma concentrations of C-reactive protein and risk of developing peripheral vascular disease. Circulation 1998;97:425-8. 13. Roivainen M, Viik-Kajander M, Palosuo T, Toivanen P, Leinonen M, Saikku P, et al. Infections, inflammation, and the risk of coronary heart disease. Circulation 2000;101:252-7. 14. Rohde LEP, Hennekens CH, Ridker PM. Survey of C-reactive protein and cardiovascular risk factors in apparently healthy men. Am J Cardiol 1999;84:1018-22. 15. Ridker PM, Hennekens CH, Buring JE, Rifai N. C-Reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 2000;342:836-43. 16. Cook DG, Mendall MA, Whincup PH, Carey IM, Ballam L, Morris JE, et al. C-reactive protein concentration in children: relation to adiposity and other cardiovascular risk factors. Atherosclerosis 2000;149:139-50. 17. Meier-Ewart HK, Ridker PM, Rifai N, Price N, Dinges DF, Mullington JM. Absence of diurnal variation of C-reactice protein concentrations in healthy human subjects. Clin Chem 2001;47:426-30. 18. Kluft C de Maat MP. Determination of the habitual low blood level of C-reactive protein in individuals. Ital Heart J 2001;2:172-80.

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19. Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon RO 3rd, Criqui M, et al. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Center for Disease Control and Prevention and the American Heart Association. Circulation 2003;107:499-511. 20. Ledue TB, Weiner DL, Sipe JD, Poulin SE, Collins MF, Rifai N. Analytical evaluation of particle-enhanced immunonephelometric assays for C-reactive protein, serum amyloid and mannose-binding protein in human serum. Ann Clin Biochem 1998;35:745-753. 21. Ridker PM, Cook N. Clinical usefulness of very high and very low levels of C-reactive protein across the full range of Framingham risk scores. Circulation 2004;109:1955-9. 22. Ledue TB, Rifai N. Preanalytic and analytic sources of variations in C-reactive protein measurement: implications for cardiovascular disease risk assessment. Clin Chem 2003;49:1258-71. 23. National Committee for Clinical Laboratory Standards. Method Comparison and Bias Estimation Using Patient Samples Approved Guideline. NCCLS document EP9-A. Villanova, PA:2002. 24. National Committee for Clinical Laboratory Standards. Evaluation of Precision Performance of Clinical Chemistry Devices; Approved Guideline. NCCLS document EP5-A. Villanova, PA:1999. 25. National Committee for Clinical Laboratory Standards. Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline. NCCLS document EP6-A. Villanova, PA:2003. 26. National Committee for Clinical Laboratory Standards. Protocols for Determination of Limits of Detection and Limits of Quantitation; Proposed Guideline. NCCLS document EP17-P. Villanova, PA:2004. 27. National Committee for Clinical Laboratory Standards. Interference Testing in Clinical Chemistry: Approved Guideline. NCCLS document EP7-A. Villanova, PA:2002. 28. Kimberly MM et al. Standardization of Immunoassays for Measurement of High-Sensitivity C-Reactive Protein. Phase I: Evaluation of Secondary Reference Materials. Clin Chem 2003;49:611-616. 29. Rifai N and Ridker PM. Population Distributions of C-reactive Protein in Apparently Healthy Men and Women in the United States: Implication for Clinical Interpretation. Clin Chem 2003;49:666-669. 30. National Committee for Clinical Laboratory Standards. How to Define and Determine Reference Intervals in the Clinical Laboratory: Approved Guideline. NCCLS document C28-A2. Villanova, PA:2002.

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Literature Genzyme Diagnostics

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