Date. Measurement of C-Reactive protein aids in evaluation of stress, trauma, infection, inflammation, surgery, and associated diseases.

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1 SYNCHRON System(s) Chemistry Information Sheet Copyright 2010 Beckman Coulter, Inc. High Sensitivity C-Reactive Protein REF For In Vitro Diagnostic Use ANNUAL REVIEW Reviewed by: Date Reviewed by: Date PRINCIPLE INTENDED USE High Sensitivity reagent, when used in conjunction with SYNCHRON LX PRO System, UniCel DxC 600/800 System(s) and SYNCHRON Systems CAL 5 Plus, is intended for the quantitative determination of C-Reactive Protein in human serum or plasma by rate turbidimetry. CLINICAL SIGNIFICANCE Measurement of C-Reactive protein aids in evaluation of stress, trauma, infection, inflammation, surgery, and associated diseases. SUMMARY AND EXPLANATION Blood levels of C-Reactive Protein (CRP) are known to rise rapidly from normal baseline levels of < 0.3 mg/dl to as high as 50 mg/dl as part of the body s non-specific inflammatory response to infection or injury. 1,2,3,4,5,6 In more recent years, the utility of measuring CRP has expanded from its historical use as a sensitive marker of acute inflammation to include assessment of cardiac events and risk. A prognostic value for measuring CRP has been suggested from studies with cardiac patients where elevated levels of CRP were associated with a higher risk of having a future cardiac event. 7,8,9,10 Elevated levels of CRP have been associated with poor prognosis in cases of stable angina, unstable angina and myocardial infarction. 7,8,9,10 Cardiac disease is believed to be the end result of an interplay between minor changes in the cardiovascular endothelium and the corresponding inflammatory response to these changes. 11 The ability to measure CRP at extremely low concentrations has raised the possibility of using CRP to detect early inflammatory responses and potentially detect cardiac disease in the preclinical stages. Recent evidence supporting this potential application has shown that high baseline values of CRP in individuals without a history of cardiac disease were associated with an increased incidence of subsequent cardiac events. 12,13 It is important to note that baseline CRP values are known to be influenced by various non-pathological factors (age, gender, obesity, hormone replacement therapy, smoking) and a single measurement may lead to an erroneous assessment of early cardiac inflammation. 14,15,16,17,18 Increases in CRP levels are non-specific and should not be interpreted without a complete clinical history. It is recommended, therefore, that any estimations of inflammation be based on changes in CRP values from multiple measurements and be used in conjunction with the values of other cardiac risk indicators (i.e., HDL, cholesterol, etc.). SEPTEMBER 2010 Page 1 of 12

2 METHODOLOGY SYNCHRON System(s) reagent is based on the highly sensitive Near Infrared Particle Immunoassay rate methodology. An anti-crp antibody-coated particle binds to CRP in the patient sample resulting in the formation of insoluble aggregates causing turbidity. The SYNCHRON System(s) automatically proportions the appropriate sample and reagent volumes into a cuvette. The ratio used is one part sample to 26 parts reagent. The system monitors the change in absorbance at 940 nanometers. This change in absorbance is proportional to the concentration of C-reactive protein in the sample and is used by the System to calculate and express C-reactive protein concentration based upon a single-point adjusted, pre-determined calibration curve. CHEMICAL REACTION SCHEME SPECIMEN TYPE OF SPECIMEN Serum samples are recommended. Plasma samples (EDTA, Lithium Heparin, and Sodium Heparin) can be used. Serum or plasma samples should be collected in the manner routinely used for any clinical laboratory test. 6 Freshly drawn serum or plasma from a fasting individual is preferred. Anticoagulants tested are listed in the PROCEDURAL NOTES section of this chemistry information sheet. SPECIMEN STORAGE AND STABILITY 1. Tubes of blood are to be kept closed at all times and in a vertical position. It is recommended that the serum or plasma be physically separated from contact with cells within two hours from the time of collection If serum samples are not assayed within 8 hours, samples should be stored at +2 C to +8 C. If samples are not assayed within 72 hours, samples should be stored frozen at -15 C to -20 C. Frozen samples should be thawed only once. Analyte deterioration may occur in samples that are repeatedly frozen and thawed Plasma samples can be stored at +2 C to +8 C for up to 72 hours. Plasma samples should not be frozen. Additional specimen storage and stability conditions as designated by this laboratory: SAMPLE VOLUME The optimum volume, when using a 0.5 ml sample cup, is 0.3 ml of sample. For optimum primary sample tube volumes and minimum volumes, refer to the Primary Tube Sample Template for your system. CRITERIA FOR UNACCEPTABLE SPECIMENS Refer to the PROCEDURAL NOTES section of this chemistry information sheet for information on unacceptable specimens. Page 2 of 12 SEPTEMBER 2010

3 Criteria for sample rejection as designated by this laboratory: PATIENT PREPARATION Special instructions for patient preparation as designated by this laboratory: SPECIMEN HANDLING Special instructions for specimen handling as designated by this laboratory: REAGENTS CONTENTS Each kit contains the following items: Two Reagent Cartridges (2 x 200 tests) One lot-specific Parameter Card VOLUMES PER TEST Sample Volume 20 µl ORDAC Sample Volume 12 µl Total Reagent Volume 255 µl Cartridge Volumes A 185 µl B 70 µl C SEPTEMBER 2010 Page 3 of 12

4 REACTIVE INGREDIENTS REAGENT CONSTITUENTS CRP Antibody (particle bound goat and mouse anti-crp antibody) Reagent Buffer Sodium Azide (used as a preservative) Bovine Serum Albumin 17.3 ml 47.8 ml <0.1% (w/w) 0.125% (w/v) Also non-reactive chemicals necessary for optimal system performance. CAUTION Sodium azide preservative may form explosive compounds in metal drain lines. See National Institute for Occupational Safety and Health Bulletin: Explosive Azide Hazards (8/16/76). CAUTION Although not composed of substances of human origin, this product may come in contact with human serum during processing. This material and all patient samples should be handled as though capable of transmitting infectious disease. The United States Food and Drug Administration recommends such samples be handled as specified in the Centers for Disease Control s Biosafety Level 2 guidelines. 20 EUROPEAN HAZARD CLASSIFICATION High Sensitivity C-Reactive Protein Reagent (Compartment A) N;R51/53 S60 S61 Toxic to aquatic organisms, may cause long-term adverse effects in aquatic environment. This material and/or its container must be disposed of as hazardous waste. Avoid release to the environment. Refer to special instructions/safety data sheets. MATERIALS NEEDED BUT NOT SUPPLIED WITH REAGENT KIT SYNCHRON Systems CAL 5 Plus Saline At least two levels of control material REAGENT PREPARATION 1. Gently invert the cartridge several times prior to loading onto the system. 2. Check for bubbles or foam in compartments; break any bubbles. ACCEPTABLE REAGENT PERFORMANCE The acceptability of a reagent is determined by successful calibration and by ensuring that quality control results are within your facility s acceptance criteria. Page 4 of 12 SEPTEMBER 2010

5 REAGENT STORAGE AND STABILITY reagent, when stored unopened at +2 C to +8 C, will obtain the shelf-life indicated on the cartridge label. Once opened, the reagent is stable for 60 days at +2 C to +8 C unless the expiration date is exceeded. DO NOT FREEZE. Reagent storage location: CALIBRATION CALIBRATOR REQUIRED SYNCHRON Systems CAL 5 Plus CALIBRATOR PREPARATION No preparation is required. CALIBRATOR STORAGE AND STABILITY SYNCHRON Systems CAL 5 Plus is stable until the expiration date printed on the calibrator bottle if stored capped in the original container at +2 C to +8 C. Calibrator storage location: CAUTION Because this product is of human origin, it should be handled as though capable of transmitting infectious diseases. Each serum or plasma donor unit used in the preparation of this material was tested by United States Food and Drug Administration (FDA) approved methods and found to be negative for antibodies to HIV and HCV and nonreactive for HbsAg. Because no test method can offer complete assurance that HIV, hepatitis B virus, and hepatitis C virus or other infectious agents are absent, this material should be handled as though capable of transmitting infectious diseases. This product may also contain other human source material for which there is no approved test. The FDA recommends such samples to be handled as specified in Centers for Disease Control s Biosafety Level 2 guidelines. 20 CALIBRATION INFORMATION 1. The system must have a lot-specific parameter card and a valid calibration adjustment in memory before controls or patient samples can be run. SEPTEMBER 2010 Page 5 of 12

6 2. Under typical operating conditions the reagent cartridge must be calibrated every 30 days and also with certain parts replacement or maintenance procedures, as defined in the SYNCHRON LX Maintenance Manual and Instrument Log, or the UniCel DxC 600/800 System Instructions for Use (IFU) manual. 3. This assay has within-lot calibration available. For detailed calibration instructions, refer to the SYNCHRON LX Operations Manual, or the UniCel DxC 600/800 System Instructions for Use (IFU) manual. 4. The system will automatically perform checks on the calibration and produce data at the end of calibration. In the event of a failed calibration, the data will be printed with error codes and the system will alert the operator of the failure. For information on error codes, refer to the SYNCHRON LX Diagnostics and Troubleshooting Manual, or the UniCel DxC 600/800 System Instructions For Use (IFU) manual. TRACEABILITY For Traceability information refer to the Calibrator instructions for use. QUALITY CONTROL At least two levels of control material should be analyzed daily. In addition, these controls should be run with each new calibration, with each new reagent cartridge, and after specific maintenance or troubleshooting procedures as detailed in the appropriate system manual. More frequent use of controls or the use of additional controls is left to the discretion of the user based on good laboratory practices or laboratory accreditation requirements and applicable laws. The following controls should be prepared and used in accordance with the package inserts. Discrepant quality control results should be evaluated by your facility. Table 1.0 Quality Control Material CONTROL NAME STORAGE TESTING PROCEDURE(S) 1. If necessary, load the reagent onto the system. A lot-specific parameter card must be loaded one time for each lot. 2. After reagent load is completed, calibration may be required. 3. Program samples and controls for analysis. 4. After loading samples and controls onto the system, follow the protocols for system operations. For detailed testing procedures, refer to the SYNCHRON LX Operations Manual, or the UniCel DxC 600/800 System Instructions For Use (IFU) manual. CALCULATIONS The SYNCHRON System(s) performs all calculations internally to produce the final reported result. The system will calculate the final result for sample dilutions made by the operator when the dilution factor is entered into the system during sample programming. Page 6 of 12 SEPTEMBER 2010

7 REPORTING RESULTS Equivalency between the SYNCHRON LX PRO and UniCel DxC 600/800 Systems has been established. Chemistry results between these systems are in agreement and data from representative systems may be shown. REFERENCE INTERVALS The reference interval values were established using the SYNCHRON System(s) test, for a population of 551 apparently healthy, non-smoking, 18 years of age, male and female adults from a Southern California blood bank. Table 2.0 Reference intervals a a INTERVALS SYNCHRON LX PRO Serum or Plasma CONVENTIONAL UNITS S.I. UNITS < mg/dl < 7.48 mg/l (In 95% of the population tested) Each laboratory should establish its own reference interval(s) based on its patient population. (In 95% of the population tested) INTERVALS CONVENTIONAL UNITS S.I. UNITS Laboratory Refer to References (21,22,23,24) for guidelines on establishing laboratory-specific reference intervals. Additional information as designated by this laboratory: PROCEDURAL NOTES ANTICOAGULANT TEST RESULTS The following anticoagulants were assessed by Deming regression analysis with a minimum of 50 paired serum and plasma samples. Values of serum (X) ranging from mg/dl to mg/dl were compared with the values for plasma (Y) yielding the following results. Table 3.0 Anticoagulant Test Results ANTICOAGULANT LEVEL OF ANTICOAGULANT TESTED DEMING REGRESSION ANALYSIS (mg/dl) Lithium Heparin 14 Units/mL Y = 0.999X ; r = Sodium Heparin 14 Units/mL Y = 0.998X ; r = LIMITATIONS Increases in CRP levels are non-specific and should not be interpreted without a complete clinical history. It is recommended that a baseline be determined on individual patients for comparison. Estimations of inflammation should be based on the changes in CRP values from multiple measurements and used in conjunction with the values of other cardiac risk indicators (i.e., HDL, cholesterol, etc.). SEPTEMBER 2010 Page 7 of 12

8 INTERFERENCES 1. The following substances were tested in serum for interference with this methodology: Table 4.0 Interferences SUBSTANCE SOURCE LEVEL TESTED OBSERVED EFFECT Bilirubin Porcine mg/dl NSI a Lipid Human Triglyceride mg/dl NSI b Hemoglobin Human mg/dl NSI Rheumatoid Factor Human 300 IU/mL NSI a NSI= No Significant Interference (within ±0.02 mg/dl or ±10%). b Quantitation of CRP by turbidimetry may not be possible in lipemic specimens or may produce inaccurate results, due to the extreme light scattering properties of the sample. Lipemic specimens should be delipidated by ultra centrifugation (90,000 x g for 10 minutes) prior to determination of CRP concentration. 2. Dust particles or other particulate matter (i.e. debris and bacteria) in the reaction solution may result in extraneous light-scattering signals, resulting in variable sample analysis. PERFORMANCE CHARACTERISTICS ANALYTIC RANGE The SYNCHRON System(s) method for the determination of this analyte provides the following analytical ranges: Table 5.0 Analytical Range CONVENTIONAL UNITS S.I. UNITS Serum or Plasma mg/dl mg/l Serum or Plasma (ORDAC) mg/dl mg/l Samples with concentrations outside of the analytical range will be reported as "<0.020 mg/dl" ("<0.2 mg/l") or ">8.0 mg/dl" (">80.0 mg/l"). (ORDAC "<6.0 mg/dl" ["<60 mg/l"] or ">38.0 mg/dl" [">380 mg/l"]) Samples reported out as greater than the analytical range should be confirmed by enabling ORDAC, or diluting with saline, and reanalyzing. If manual dilution is used the appropriate dilution factor should be applied to the reported result. Samples reported out as "SUPPRESSED" due to RXN ERROR should be reanalyzed. REPORTABLE RANGE (AS DETERMINED ON SITE): Table 6.0 Reportable Range CONVENTIONAL UNITS S.I. UNITS ANALYTICAL SENSITIVITY Analytical sensitivity is defined as the lowest measurable concentration which can be distinguished from zero with 95% confidence. Analytical sensitivity for determination is 0.02 mg/dl (0.2 mg/l). Page 8 of 12 SEPTEMBER 2010

9 FUNCTIONAL SENSITIVITY Functional sensitivity is defined as the lowest concentration that can be measured with an interassay CV of 20%. Functional sensitivity is estimated to be mg/dl ( 0.18 mg/l). EQUIVALENCY Equivalency was assessed by Deming regression analysis of patient samples to accepted clinical methods. Serum or plasma (in the range of to 8.0 mg/dl): a Y (SYNCHRON LX PRO Systems) = 1.011X N = 141 MEAN (SYNCHRON LX PRO Systems) = MEAN (Nephelometric Immunoassay) a = CORRELATION COEFFICIENT (r) = A product of Dade Behring. Refer to References (25,26) for guidelines on performing equivalency testing. PRECISION A properly operating SYNCHRON System(s) should exhibit precision values less than or equal to the following: Table 7.0 Precision Values a b TYPE OF PRECISION Within-run Total mg/dl 1 SD mg/l CHANGEOVER VALUE a mg/dl mg/l % CV Serum/Plasma Serum/Plasma (ORDAC) NA b NA NA NA 7.5 Serum/Plasma Serum/Plasma (ORDAC) NA NA NA NA When the mean of the test precision data is less than or equal to the changeover value, compare the test SD to the SD guideline given above to determine the acceptability of the precision testing. When the mean of the test precision data is greater than the changeover value, compare the test % CV to the guideline given above to determine acceptability. Changeover value = (SD guideline/cv guideline) x 100. NA = Not applicable. Comparative performance data for the SYNCHRON LX System evaluated using the NCCLS Approved Guideline EP5-A appears in the table below. 27 Each laboratory should characterize their own instrument performance for comparison purposes. Table 8.0 NCCLS EP5-A Precision Estimate Method TYPE OF IMPRECISION Within-run No. Systems No. Data Points a Test Mean Value (mg/dl) EP5-A Calculated Point Estimates SD %CV Serum Level Serum Level Serum Level Serum Level SEPTEMBER 2010 Page 9 of 12

10 Table 8.0 NCCLS EP5-A Precision Estimate Method, Continued a TYPE OF IMPRECISION Total No. Systems No. Data Points a Test Mean Value (mg/dl) EP5-A Calculated Point Estimates SD %CV Serum Level Serum Level Serum Level Serum Level The point estimate is based on the pooled data from one system, run for twenty days, two runs per day, two observations per run on an instrument operated and maintained according to the manufacturer s instructions. Refer to References (25,27) for guidelines on performing precision testing. NOTICE These degrees of precision and equivalency were obtained in typical testing procedures on a SYNCHRON LX PRO System(s) and are not intended to represent the performance specifications for this reagent. ADDITIONAL INFORMATION For more detailed information on SYNCHRON LX Systems or UniCel DxC Systems, refer to the appropriate system manual. This product is licensed per U.S. Patent number 6,040,147 (Ridker et al.). SHIPPING DAMAGE If damaged product is received, notify your Beckman Coulter Clinical Support Center. Page 10 of 12 SEPTEMBER 2010

11 REFERENCES 1. Pepys, M. B., et al., C-Reactive Protein Fifty Years On, Lancet, 21:653 (1981). 2. Pepys, M. B., Baltz, M. L., "Acute Phase Proteins With Special Reference to C-Reactive Protein and Related Proteins (Pentraxins) and Serum Amyloid A Protein", Adv. Immunol., 34:141 (1983). 3. Dati, F., et al., "Consensus of a Group of Professional Societies and Diagnostic Companies on Guidelines for Interim Reference Ranges for 14 Proteins in Serum, based on the Standardization Against the IFCC/BCR/CAP Reference Material", Eur. J. Clin. Chem. Clin. Biochem. 4. Kushner, D. L., Rzewricki, D. L., "The Acute Phase Response: General Aspects", Ballicre s Clinical Rheumatology., 8: (1994). 5. Hind, C.R.H., Pepys, P.M., "The Role of Serum C-Reactive Protein (CRP) Measurement in Clinical Practice", Int. Med., 5: (1984). 6. Burtis, C. A., Ashwood, E. R., eds., "Specimen Collection and Processing: Sources of Biological Variation", Tietz Textbook of Clinical Chemistry, 3rd Edition, W. B. Saunders, Philadelphia PA (1999). 7. Thompson, S. B., et al., "Hemostatic Factors and the Risk of Myocardial Infarction or Sudden Death in Patients with Angina Pectoris", European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group, N. Eng. J. Med., 332: (1995). 8. Liuzzo, G., et al., "The Prognostic Value of CRP and Serum Amyloid A Protein in Severe Unstable Angina", N. Eng. J. Med, 331: (1994). 9. Pietila, K. O., et al., "Serum CRP Concentration in Acute Myocardial Infarction and its Relationship to Mortality During 24 Months of Follow-up in Patients Under Thrombolytic Treatment", Eur. Heart J., 17: (1996). 10. Pietila, K. O., et al., "Acute Phase Reaction Infarct Size", and "In-Hospital Morbidity in Myocardial Infarction Patients Treated With Streptokinase or Recombinant Tissue-type Plasminogen Activator", Ann. Med., 23: (1991). 11. George, D. A., "C-Reactive Protein, Inflammation and Cardiovascular Disease, Part 2: The Predictive Value of C-Reactive Protein in Acute Coronary Syndromes", Scripps News, Volume 14, Number Kuller, L., H., "Relationship of CRP and Coronary Heart Disease in the MRFIT Nested Case-Control Study", Am. J. Epidemiol., 144: (1996). 13. Koenig, W., et al., "CRP, A Sensitive Marker for Inflammation, Predicts Future Risk of Coronary Heart Disease in Initially Healthy Middle-aged Men", MONICA-Augsburg Cohort Study, Circulation, 99: (1999). 14. Hutchinson, W.L., et al., "Immunoradiometric Assay of Circulating C-Reactive Protein: Age Related Values in the Adult General Population", Clin. Chem., 46: (2000). 15. Visser, M., et al., "Elevated C-Reactive Protein Levels in Overweight and Obese Adults", JAMA, 282: (1999). 16. Cook, D.G., et al., "C-Reactive Protein Concentration in Children: Relationship to Adiposity and other Cardiovascular Risk Factors", Atherosclerosis, 149: (2000). 17. Ridker, P. M., et al., "Hormone Replacement Therapy and Increased Plasma Concentration of C-Reactive Protein", Circulation, 100: (1999). 18. Das, I., "Raised C-Reactive Protein Levels in Serum From Smokers", Clin. Chim. Acta, 153:9 13 (1985). SEPTEMBER 2010 Page 11 of 12

12 19. National Committee for Clinical Laboratory Standards, Procedures for the Handling and Processing of Blood Specimens, Approved Guideline, NCCLS publication H18-A, Villanova, PA (1990). 20. CDC-NIH manual, Biosafety in Microbiological and Biomedical Laboratories, U.S. Government Printing Office, Washington, D.C. (1984). 21. National Committee for Clinical Laboratory Standards, How to Define, Determine, and Utilize Reference Intervals in the Clinical Laboratory, Approved Guideline, NCCLS publication C28-A, Villanova, PA (1992). 22. Tietz, N. W., Clinical Guide to Laboratory Tests, 2nd Edition, W. B. Saunders, Philadelphia, PA (1990). 23. Henry, J. B., ed., Clinical Diagnosis and Management by Laboratory Methods, 17th Edition (1984). 24. Statland, Bernard E., "Clinical Decision Levels for Lab Tests", Medical Economic Book, Oradel, New Jersey (1983). 25. Tietz, N. W., ed., Fundamentals of Clinical Chemistry, 3rd Edition, W. B. Saunders, Philadelphia, PA (1987). 26. National Committee for Clinical Laboratory Standards, Method Comparison and Bias Estimation Using Patient Samples, Tentative Guideline, NCCLS publication EP9-T, Villanova, PA (1993). 27. National Committee for Clinical Laboratory Standards, Evaluation of Precision Performance of Clinical Chemistry Devices, Approved Guideline, Vol. 19, No. 2, NCCLS publication EP5-A, Villanova, PA (1999). Beckman Coulter Ireland Inc., Mervue Business Park, Mervue, Galway, Ireland ( ) Beckman Coulter, Inc., 250 South Kraemer Blvd., Brea, CA Page 12 of 12 SEPTEMBER 2010