This Procedure Is Valid for the Following Chemistry Analyzers s7

Procedure: C-REACTIVE PROTEIN OSR6x47 This procedure is valid for the following chemistry analyzers:

 AU400/AU400e  AU640/AU640e  AU480  AU680  AU600  AU2700/AU5400

Prepared By Date Adopted Supersedes Procedure #

Review Date Revision Date Signature

# of # of Distributed to Copies Distributed to Copies

C-reactive protein (CRP) has long been recognized as one of the most, if not the most, sensitive of the acute-phase reactants. C-reactive protein levels in plasma can rise dramatically after myocardial infarction, stress, trauma, infection, inflammation, surgery, or neoplastic proliferation. The increase occurs within 24 to 48 hours and the level may be 2000 times normal. Because the increase is nonspecific; however, it cannot be interpreted without a complete clinical history and even then only by comparison with previous values. Cord blood normally has low CRP concentrations (0.1 – 0.35 mg/L), but in intra-uterine infection, levels may be as high as 260 mg/L.

For unknown reasons, the degree of C-reactive protein response varies in some diseases that are otherwise apparently similar. For example, the C-reactive protein response in systemic lupus and ulcerative colitis, even when there are obvious signs and symptoms of inflammation, is slight in contrast to its very large response in rheumatoid arthritis and Crohn’s disease.

INTENDED USE:

System reagent for the quantitative determination of C-Reactive Protein in human serum on Beckman Coulter AU Clinical Chemistry analyzers.

METHODOLOGY:

Immune complexes formed in solution scatter light in proportion to their size, shape, and concentration. Turbidimeters measure the reduction of incidence light due to reflection, absorption, or scatter.

In this Beckman Coulter AU procedure, the measurement of the rate of decrease in light intensity transmitted (increase in absorbance) through particles suspended in solution is the result of complexes formed during the antigen-antibody reaction.

PATIENT PREPARATION:

An 8 to 12-hour fast is recommended but not required. A fasting sample should minimize any possible lipemia.

Additional instructions for patient preparation as designated by this laboratory:

TYPE:

Serum, free from hemolysis, is the recommended specimen. Avoid highly lipemic samples, which may produce excessively high scatter signals.

Additional type conditions as designated by this laboratory:

HANDLING CONDITIONS:

Serum specimens are stable for two weeks when stored at 2 - 8C. Freeze serum at <- 20C for longer storage,. However, please note that it has been reported that frozen specimens may give false-positive results.2

Additional handling conditions as designated by this laboratory:

EQUIPMENT:

Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e, AU680, AU2700, and AU5400 analyzers.

MATERIALS:

Beckman Coulter AU System C-Reactive Protein Reagent.

Final concentration of reactive ingredients: Tris buffer (pH 7.5) 80 mmol/L Sodium Chloride 125 mmol/L Polyethylene glycol 6000 1.5 % Goat anti-CRP antibodies  0.6 g/L Also contains preservatives.

Reagent storage location in this laboratory:

Test tubes 12 -16 mm in diameter or sample cups (Cat No. AU1063).

Storage location of test tubes or sample cups in this laboratory:

Beckman Coulter Serum Protein Multi-Calibrator (Cat No. ODR3021).

Storage location of the calibrator in this laboratory:

© Beckman Coulter, Inc. 2010 Page 4 of 11 All printed copies are considered to be copies of the electronic original. Rev #1, Dec 01, 10 Procedure: C-REACTIVE PROTEIN OSR6x47 Precautions: 1. For in vitro diagnostic use.

2. Do not ingest reagent or calibrator. Harmful if swallowed.

3. Contains sodium azide as a preservative that may react with lead joints in copper plumbing to form explosive compounds. Even though the reagent contains minute quantities of sodium azide, drains should be well flushed with water when discarding the reagent or calibrators.

4. WARNING: POTENTIAL BIOHAZARDOUS MATERIAL. The calibrator is manufactured from human serum. No test method can offer complete assurance that HIV- 1/2, HCV, Hepatitis B, or other infectious agents are absent from biological materials, all calibrator material should be handled at the Biosafety Level 2 as recommended for any infectious human serum or blood specimen in the CDC/National Institutes of Health manual, Biosafety in Microbiological and Biomedical Laboratories, 1993.

PREPARATION:

The Beckman Coulter AU System CRP Reagent is ready for use. No preparation is needed.

The Beckman Coulter Serum Protein Multi-Calibrators are liquid, ready for use as supplied. Mix by gentle inversion to achieve a homogenous mixture prior to use.

Storage Requirements: 1. The unopened reagents and calibrators are stable until the expiration date printed on the label when stored at 2 - 8C.

2. Opened bottles of reagent are stable for 90 days when stored in the refrigerated compartment of the Beckman Coulter AU analyzers.

3. Opened bottles of calibrator are stable for 30 days provided stoppers and caps are replaced immediately after each use. The Multi-calibrators should be stored at 2 - 8C when not in use.

Additional storage requirements as designated by this laboratory:

Indications of Deterioration: Discoloration of the reagent or calibrators, visible signs of microbial growth, turbidity or precipitation in reagent or calibrators may indicate degradation and warrant discontinuance of use.

PERFORMANCE PARAMETERS:

The following data was obtained using the CRP Reagent on Beckman Coulter AU analyzers according to established procedures. Results obtained in individual laboratories may differ.

PRECISION7:

Estimates of precision, based on CLSI recommendations6, are consistent with typical performance. The within run precision is less than or equal to 5% CV or SD < 1 and the total precision is less than 5% CV or SD < 1 Assays of serum pools and control sera were performed and the data reduced following CLSI guidelines.

N=100 Within run Total Mean, mg/L SD CV% SD CV% 12.8 0.4 3.4 0.5 3.7 20.1 0.5 2.4 0.5 2.7

COMPARISON7:

Patient samples were used to compare the C-Reactive Protein Reagent. Representative performance data on AU analyzers is shown in the next table. Y Method AU640/AU640e X Method AU600 Slope 1.017 Intercept -0.4 Correlation Coeff. (r) 0.9968 No. of Samples (n) 101 Range (mg/L) 5 - 103

CALIBRATION:

STANDARD PREPARATION:

Perform a multi-point calibration (6AB) using a water blank (blue rack) and the appropriate calibrators in a yellow calibration rack. The frequency of calibration is every 90 days. Calibration of this C-Reactive Protein procedure is accomplished by use of the Beckman Coulter Serum Protein Multi-Calibrator (Cat No. ODR3021), which are traceable to IFCC (International Federation of Clinical Chemistry) standard CRM 470 (RPPHS). The Beckman Coulter AU Serum Protein Multi-Calibrator is a 6-level calibrator for serum proteins for CRP.

This calibrator has not been tested for use with any other Chemistry Systems other than those listed above.

The results obtained using this calibrator are dependent upon several factors, including proper storage of the calibrator and proper technique in use of the Beckman Coulter AU Clinical Chemistry analyzers and their respective reagents.

CALIBRATION PROCEDURE:

Recalibration is required when any of the following conditions occur:

© Beckman Coulter, Inc. 2010 Page 7 of 11 All printed copies are considered to be copies of the electronic original. Rev #1, Dec 01, 10 Procedure: C-REACTIVE PROTEIN OSR6x47 1. A reagent lot number has changed or there is an observed shift in control values.

2. Major preventative maintenance was performed on the analyzer.

3. A critical part was replaced.

QUALITY CONTROL:

During operation of the Beckman Coulter AU analyzer at least two levels of an appropriate control material should be tested a minimum of once a day. In addition, controls should be performed after calibration, with each new lot of reagents, and after specific maintenance or troubleshooting steps described in the appropriate AU User’s Guide. Quality control testing should be performed in accordance with regulatory requirements and each laboratory’s standard procedure.

Location of controls used at this laboratory.

ANALYZER PARAMETERS:

A complete list of test parameters and operating procedures can be found in the appropriate User’s Guide and at www.beckmancoulter.com.

CALCULATIONS:

To work in mg/dL, divide the results by 10.

REPORTING RESULTS:

REFERENCE RANGES:

Adults4: < 5 mg/L

© Beckman Coulter, Inc. 2010 Page 8 of 11 All printed copies are considered to be copies of the electronic original. Rev #1, Dec 01, 10 Procedure: C-REACTIVE PROTEIN OSR6x47 Expected values may vary with age, sex, diet and geographical location. Each laboratory should determine its own expected values as dictated by good laboratory practice.

Expected reference ranges in this laboratory:

PROCEDURES FOR ABNORMAL RESULTS:

Abnormal results are flagged by the listed analyzers according to the normal values entered by the user into the instrument parameters.

REPORTING FORMAT:

Results are automatically printed out for each sample in mg/L at 37o C.

Additional reporting information as designated by this laboratory:

LIMITATIONS:

The Beckman Coulter AU C-Reactive Protein assay is linear from 5 to 300 mg/L using Beckman Coulter Serum Protein MultiCalibrator (Cat No. ODR3021) 6pt calibration. Samples exceeding the upper limit of linearity should be diluted and repeated. The sample may be diluted, repeated and multiplied by the dilution factor automatically utilizing the AUTO REPEAT RUN.

© Beckman Coulter, Inc. 2010 Page 9 of 11 All printed copies are considered to be copies of the electronic original. Rev #1, Dec 01, 10 Procedure: C-REACTIVE PROTEIN OSR6x47 This kit is not recommended for cardiovascular risk assessment or diagnostic evaluation of neonates. Results of this test should be interpreted with other clinical and laboratory findings.

Samples with very high CRP concentrations (> 750 mg/dL) can generate false low results without appropriate “Z” flagging due to excess antigen in the sample.

In very rare cases Gammopathy, especially monoclonal IgM (Waldenstrom’s macroglobulinemia), may cause unreliable results.

Note: Samples from patients with abnormal lipoprotein metabolism such as those seen in cholecystitis or obstructive liver disease may give artificially elevated CRP results. These samples are characterizes by having extremely elevated Cholesterol values (>387 mg/dL) and elevated Bilirubin. Such samples should be diluted 1 part sample to 4 parts deionized water prior to analysis and the result multiplied by 5.

INTERFERING SUBSTANCES:

Results of studies3 show that the following substances interfere with this CRP procedure.

The criteria for no significant interference is recovery within 10% of the initial value. Bilirubin: No significant interference up to 40 mg/dL Bilirubin Hemolysis: No significant interference up to 500 mg/dL Hemolysate Lipemia: No significant interference up to 1000 mg/dL Intralipid*

* Intralipid, manufactured by KabiVitrium Inc., is a 20% IV fat emulsion used to emulate extremely turbid samples.

Please Note: Oral contraceptives have been reported to affect results.1

The information presented is based on results from Beckman Coulter AU studies and is current at the date of publication. Beckman Coulter Inc., makes no representation about the completeness or accuracy of results generated by future studies. For further information on interfering substances, refer to Young5 for a compilation of reported interferences with this test.

Laboratory specific procedure notes:

REFERENCES:

1. Ashwood, E.R., Burtis, C. A., Tietz Textbook of Clinical Chemistry, Second Edition, W. B. Saunders, 1994.

2. Rose, N.R. et al., (ed), Manual of Clinical Laboratory Immunology, American Society of Microbiology, Washington, D.C., 1986

3. CLSI/NCCLS, Interference Testing in Clinical Chemistry, EP7-P, 1986.

4. Beckman Coulter, Inc. data on samples collected from 200 blood donors in North Texas.

5. Young, D.S., Effects of Drugs on Clinical Laboratory Tests, Fifth Edition, AACC Press, 2000.

6. CLSI/NCCLS Evaluation Protocol EP5-T2, 1992.

7. Data is on file for specific AU analyzers.