This Procedure Is Valid for the Following Chemistry Analyzers s1

Procedure: -1-Antitrypsin OSR6163 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

# of # of Distributed to Copies Distributed to Copies

-1-Antitryspin is an acute phase reactant. This term refers to proteins whose serum conc entrations rise significantly during acute inflammation due to causes including surgery, m yocardial infarction, infections and tumors. (-1-Antitryspin has anti-protease activity. An inherited deficiency in this protein is associated with lung and liver disease and is used in the diagnosis of cirrhosis of the liver. Low levels of (-1-Antitryspin are found in neonatal respiratory distress syndrome, in severe protein losing disorders and in congenital deficie ncy.1

INTENDED USE:

System reagent for the quantitative determination of -1-Antitrypsin (-1-AT) 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 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.

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:

Fasting serum specimen, free from hemolysis, is the recommended specimen. Avoid highly lipemic samples, which may produce excessively high scatter signals.2

Additional type conditions as designated by this laboratory:

HANDLING CONDITIONS:

Serum specimens are stable up to one week when stored at 2 - 8C3 and up to 2 months when stored at <-20C.

Additional handling conditions as designated by this laboratory:

EQUIPMENT:

Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e, AU680, AU2700, AU5400 and AU5800 analyzers.

MATERIALS:

Beckman Coulter AU System 1-Antitrypsin Reagent

Final concentration of reactive ingredients: Tris buffer 99 mmol/L Goat anti-human -1-Antitrypsin antiserum Polyethylene Glycol 6000 Tween-20

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)

© Beckman Coulter, Inc. March 2012 CLSIOSR6x63.02 All printed copies are considered to be copies of the electronic original. Page 4 of 11 Procedure: -1-Antitrypsin OSR6163 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 1-Antitrypsin Reagent is ready for use. No preparation is needed.

The Beckman Coulter Serum Protein Multi-Calibrators are liquid, ready for use as supplied. Gently mix by 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.

© Beckman Coulter, Inc. March 2012 CLSIOSR6x63.02 All printed copies are considered to be copies of the electronic original. Page 5 of 11 Procedure: -1-Antitrypsin OSR6163 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 1-Antitrypsin 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 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 Total Mean, mg/dL SD CV% SD CV% 80.7 0.89 1.1 1.36 1.7 153.8 1.41 0.9 1.79 1.2 225.1 2.11 0.9 2.58 1.1

COMPARISON8:

Patient samples were used to compare the 1-Antitrypsin. Representative performance data on AU analyzers is shown in the next table.

Y Method AU640/AU640e X Method AU600 Slope 0.970 Intercept 2.8 Correlation Coeff. (r) 0.993 No. of Samples (n) 151 Range (mg/dL) 54 - 274

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 90 days. Calibration of this -1-Antitrypsin 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).

© Beckman Coulter, Inc. March 2012 CLSIOSR6x63.02 All printed copies are considered to be copies of the electronic original. Page 7 of 11 Procedure: -1-Antitrypsin OSR6163 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:

For SI units (g/L), multiply the results by 0.01.

REFERENCE RANGES:

Adults6: 84 - 218 mg/dL

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/dL at 37o C.

Additional reporting information as designated by this laboratory:

LIMITATIONS:

The Beckman Coulter AU System 1-AT assay is linear from 30 - 500 mg/dL. Samples exceeding the upper limit of linearity should be diluted and repeated. The sample may be

© Beckman Coulter, Inc. March 2012 CLSIOSR6x63.02 All printed copies are considered to be copies of the electronic original. Page 9 of 11 Procedure: -1-Antitrypsin OSR6163 diluted, repeated and multiplied by the dilution factor automatically utilizing the AUTO REPEAT RUN.

INTERFERING SUBSTANCES:

Results of studies4 show that the following substances interfere with this -1- Antitrypsin procedure.

The criteria for no significant interference is recovery within 10% of the initial value. Ascorbate: No significant interference up to 20 mg/dL Ascorbate 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.

The information presented is based on results from Beckman Coulter 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. Tietz N.W., Textbook of Clinical, Second Edition, Burtis, C.A., and Ashwood E.R., editors, W.B. Saunders Company, 1994.

© Beckman Coulter, Inc. March 2012 CLSIOSR6x63.02 All printed copies are considered to be copies of the electronic original. Page 10 of 11 Procedure: -1-Antitrypsin OSR6163 2. Rose, N.R., Friedman, H, and Fahey, John L., Manual of Clinical Laboratory Immunology, Third Edition, American Society for Microbiology, Washington, DC, 1986.

3. Kaplan L.A. and Pesce A.J. ,Clinical Chemistry Theory, Analysis, and Correlation, Third Edition, Mosby, St. Louis, Missouri, 1996

4. CLSI/NCCLS, Interference Test in Clinical Chemistry, EP7-P, 1986.

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

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

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

8. Data on file for specific AU analyzers.