Procedure:
CERULOPLASMIN
OSR6164
This procedure is valid for the following chemistry analyzers:
· AU400/AU400e / · AU640/AU640e· AU480 / · AU680
· AU600 / · AU2700
· AU5400 / · AU5800
Prepared By / Date Adopted / Supersedes Procedure #
Review Date / Revision Date / Signature
Distributed to / # of
Copies / Distributed to / # of
Copies
PRINCIPLE:
Ceruloplasmin is the primary copper-containing protein in plasma. It is a late acute phase reactant synthesized by the liver. Acute phase reactants refers to proteins whose serum concentrations rise significantly during acute inflammation due to causes including surgery, myocardial infarction, infections and tumors. Ceruloplasmin’s main clinical importance is in the diagnosis of Wilson’s disease. Here plasma Ceruloplasmin concentration is reduced while dialyzable copper concentration is increased. Increased ceruloplasmin levels are particularly notable in diseases of the reticuloendothelial system such as Hodgkin’s disease as well as during pregnancy or the use of contraceptive pills. Low plasma levels of ceruloplasmin are found in malnutrition, malabsorption, nephrosis and severe liver disease, particularly biliary cirrhosis.1,2
INTENDED USE:
System reagent for the quantitative determination of Ceruloplasmin (CER) in human serum and plasma 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 incident light due to reflection, absorption, or scatter.
In the Beckman Coulter AU system procedure, the measurement of the decrease in light transmitted (increase in absorbance) through particles suspended in solution as a result of complexes formed during the antigen-antibody reaction, is the basis of this assay.
SPECIMEN:
Patient Preparation:
An 8 to 12-hour fast is recommended, but not required, in order to minimize any possible lipemia.
Additional instructions for patient preparation as designated by this laboratory:Type:
Fasting serum, free from hemolysis, is the recommended samples. Avoid highly lipemic samples, which may produce excessively high scatter signals. Heparinized plasma can also be used for a specimen.
Additional type conditions as designated by this laboratory:Handling Conditions:
Serum samples are stable for 3 days when refrigerated at 2 - 8°C. Samples can be stored frozen ( -20°C) for 1 month.3
Additional handling conditions as designated by this laboratory:EQUIPMENT AND MATERIALS:
Equipment:
Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e, AU680, AU2700, AU5400 and AU5800 analyzers.
Materials:
Beckman Coulter AU System Ceruloplasmin Reagents.
Final concentration of reactive ingredients:
Solution of Polymers in Phosphate Buffered Saline (pH 7.4 – 7.6)Rabbit anti-human Ceruloplasmin antiserum
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 2 (Cat No. ODR3023)
Storage location of the calibrator in this laboratory: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 Ceruloplasmin reagent is liquid, 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 Ceruloplasmin reagent on Beckman Coulter AU analyzers according to established procedures. Results obtained in individual laboratories may differ.
Precision8:
Estimates of precision, based on CLSI recommendations7, are consistent with typical performance. The within run precision is less than 5% CV and the total precision is less than 10% CV. Assays of serum pools and control sera were performed and the data reduced following CLSI guidelines.
N=100 / Within run / TotalMean, mg/L / SD / CV% / SD / CV%
120.3 / 1.10 / 0.94 / 2.90 / 2.44
934.1 / 11.80 / 1.27 / 22.10 / 2.36
1810.5 / 17.10 / 0.94 / 84.80 / 4.68
Comparison8:
Patient samples were used to compare the Ceruloplasmin. Representative performance data on AU analyzers is shown in the next table.
Y Method / AU640/AU640eX Method / Method 2
Slope / 1.020
Intercept / -3.5
Correlation Coeff. (r) / 0.990
No. of Samples (n) / 50
Range (mg/L) / 90.0 – 510.0
CALIBRATION:
Standard Preparation:
Perform a multi-point calibration (5AB) using a water blank (blue rack) and the appropriate calibrators in a yellow calibration rack. The frequency of calibration is every 14 days. Calibration of this Ceruloplasmin procedure is accomplished by use of the Beckman Coulter Serum Protein Multi-Calibrator 2 (Cat No. ODR3023), which is traceable to IFCC International Reference Preparation CRM470 (RPPHS).
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:
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 immunology control material should be tested a minimum of once a day. In addition, these 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:
Values set for working in SI units (mg/L). To work in mg/dL divide by 10.
REPORTING RESULTS:
Reference Ranges:
Adults6: 180 - 580 mg/L
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 System Ceruloplasmin assay is linear from 60-2000 mg/L. 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.
Note: Samples from patients with abnormal lipoprotein metabolism such as those seen in cholecystitis or obstructive liver disease may give artificially negative Ceruloplasmin results. These samples are characterized 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 studies4 show that the following substances interfere with this Ceruloplasmin procedure:
The criteria for no significant interference is recovery within 10% of the initial value.
Bilirubin: / No significant interference up to 40 mg/dL BilirubinHemolysis: / 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.
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. Johnson AM, Rohlfs EM, Silverman LM. Proteins. In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry. Philadelphia:WB Saunders Company, 1999; 490-4922.
2. Kazmierczak SC. Ceruloplasmin. In: Kaplan LA, Pesce AJ eds. Clinical chemistry theory, analysis, correlation. St. Louis: Mosby, 1996:966pp.
3. Tietz NW, ed. Clinical guide to laboratory tests, 3rd ed. Philadelphia: WB Saunders Company, 1995: 122pp.
4. CLSI/NCCLS, Interference Testing in Clinical Chemistry, EP7-P, 1986.
5. Young, D.S., Effects of Drugs on Clinical Laboratory Tests, Fifth Edition, AACC Press, 2000.
6. Beckman Coulter, Inc. data on samples collected from 80 blood donors in North America.
7. CLSI/NCCLS Evaluation Protocol EP5-T2, 1992.
8. Data is on file for specific AU analyzers.
© Beckman Coulter, Inc. March 2012 CLSIOSR6x64.02
All printed copies are considered to be copies of the electronic original. Page 1 of 11