This Procedure Is Valid for the Following Chemistry Analyzers s3

Procedure: TOTAL BILIRUBIN OSR6112, OSR6212, and OSR6512 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

© Beckman Coulter, Inc. March 2012 CLSIOSR6x12.02 All printed copies are considered to be copies of the electronic original. Page 1 of 11 Procedure: TOTAL BILIRUBIN OSR6112, OSR6212, and OSR6512 PRINCIPLE:

Measurement of the levels of bilirubin, an organic compound formed during the normal and abnormal destruction of red blood cells, is used in the diagnosis and treatment of liver, hemolytic, hematologic, and metabolic disorders, including hepatitis and gall bladder block.

INTENDED USE:

System reagent for the quantitative determination of Total Bilirubin in human serum or plasma on Beckman Coulter AU Clinical Chemistry analyzers.

Total Bilirubin reagent OSR6512 is for use on the AU680, AU2700 and AU5400 analyzers only.

METHODOLOGY:

Total bilirubin in serum is composed of direct (conjugated) bilirubin and indirect (unconjugated) bilirubin. Since the introduction of the diazo method for bilirubin determination by Ehrlich in 1883,1 several modifications have been proposed to enhance the reaction. The primary reason for these modifications is that while direct bilirubin couples directly with the diazotized compound to yield a colored product, the indirect portion of bilirubin requires a solubilizing agent, such as a surfactant.

This Beckman Coulter AU System Total Bilirubin Reagent is a variation of the classical method. A stabilized diazonium salt, 3,5-dichlorophenyl-diazonium tetrafluoroborate (DPD), reacts with bilirubin to form azobilirubin which absorbs maximally at 570/660 nm. Caffeine and a surfactant are used as reaction accelerators.

The absorbance at 570/660 nm is proportional to the bilirubin concentration in the sample. A separate serum blank is performed to eliminate endogenous serum2 interferences.

caffeine

© Beckman Coulter, Inc. March 2012 CLSIOSR6x12.02 All printed copies are considered to be copies of the electronic original. Page 2 of 11 Procedure: TOTAL BILIRUBIN OSR6112, OSR6212, and OSR6512 Bilirubin + DPD  Azobilirubin surfactant SPECIMEN:

PATIENT PREPARATION:

None required.

Additional instructions for patient preparation as designated by this laboratory:

TYPE:

Serum, heparinized or EDTA plasma samples, free from hemolysis, are the recommended specimens. Samples should be protected from light.

Additional type conditions as designated by this laboratory:

HANDLING CONDITIONS:

It has been reported that exposure to direct sunlight can decrease bilirubin in samples by 50% within one hour. When well protected from light, bilirubin in serum is stable for 3 days when stored at 2 - 8°C, or three months when stored at <-20°C.2

Additional handling conditions as designated by this laboratory:

© Beckman Coulter, Inc. March 2012 CLSIOSR6x12.02 All printed copies are considered to be copies of the electronic original. Page 3 of 11 Procedure: TOTAL BILIRUBIN OSR6112, OSR6212, and OSR6512 EQUIPMENT AND MATERIALS:

EQUIPMENT:

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

MATERIALS:

Beckman Coulter AU System Total Bilirubin Reagent

Final concentration of reactive ingredients: Caffeine 2.1 mmol/L 3,5-Dichlorophenyldiazonium Tetrafluoroborate 0.31 mmol/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 Chemistry Calibrator (Cat. No. DR0070)

Storage location of the calibrator in this laboratory:

Precautions:

© Beckman Coulter, Inc. March 2012 CLSIOSR6x12.02 All printed copies are considered to be copies of the electronic original. Page 4 of 11 Procedure: TOTAL BILIRUBIN OSR6112, OSR6212, and OSR6512 1. For in vitro diagnostic use.

2. Do not ingest. Toxicity has not been established.

3. Do not pipette by mouth. Avoid contact with skin, eyes or clothing. In case of contact, immediately flush affected areas with plenty of water for 15 minutes. Obtain medical attention immediately for eye contact or ingestion.

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 Total Bilirubin Reagent is liquid, ready for use. No preparation is needed.

The Beckman Coulter Chemistry Calibrator reconstitution:

 Remove the vial of calibrator and diluent from storage and let stand at room temperature (15-25C) for 5 minutes.

 Remove the cap and stopper from the vial of the lyophilized serum and reconstituting diluent.

 Using a volumetric pipette or a calibrated air-displacement pipettor, add exactly 5.0 mL of reconstituting diluent to DR0070 lyophilized serum vial. DO NOT pour directly from the reconstituting diluent vial.

 Replace the cap and stopper to the vial of lyophilized serum immediately after adding the diluent

 Allow the calibrator to stand for 5-10 minutes. Gently swirl the contents until completely dissolved.

Storage Requirements:

© Beckman Coulter, Inc. March 2012 CLSIOSR6x12.02 All printed copies are considered to be copies of the electronic original. Page 5 of 11 Procedure: TOTAL BILIRUBIN OSR6112, OSR6212, and OSR6512 1. The unopened reagents are stable until the expiration date printed on the label when stored at 2 - 8°C.

2. Opened reagents are stable for 90 days when stored in the refrigerated compartment of the analyzer.

3. Protect from light.

4. Contamination after opening must be avoided.

5. Un-reconstituted calibrator and diluent are stable until the expiration date stated on the label when stored at 2 - 8°C.

6. For Total Bilirubin, reconstituted calibrator materials are stable for 4 days from the date of reconstitution when stored at 2 - 8°C. The materials should be capped and stored upright 2 - 8°C when not in use.

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

Additional storage requirements as designated by this laboratory:

PERFORMANCE PARAMETERS:

The following data was obtained using this Total Bilirubin Reagent on Beckman Coulter AU analyzers according to established procedures. Results obtained at individual facilities may differ.

© Beckman Coulter, Inc. March 2012 CLSIOSR6x12.02 All printed copies are considered to be copies of the electronic original. Page 6 of 11 Procedure: TOTAL BILIRUBIN OSR6112, OSR6212, and OSR6512 PRECISION:7

Estimates of precision, based on CLSI recommendations6, are consistent with typical performance. The within run precision is less than 3%CV or SD  0.07 and total precision is less than 5%CV or SD  0.10. Assays of control sera were carried out and data reduced following CLSI guidelines.

N=60 Within run Total Mean, mg/dL SD CV% SD CV% 0.85 0.01 1.24 0.02 2.65 2.11 0.01 0.61 0.04 2.01 4.78 0.03 0.53 0.16 3.31 9.82 0.03 0.33 0.21 2.13

METHOD COMPARISON:7

Patient samples were used to compare this Total Bilirubin Reagent. Representative performance data on AU analyzers is shown in the next table. Y Method AU640/AU640e X Method Method 2 Slope 1.046 Intercept -0.065 Correlation Coeff. (r) 1.000 No. of Samples (n) 100 Range (mg/dL) 0.08 – 24.90

Sensitivity:

Typical change in absorbance for 1 mg/dL of Total Bilirubin is 88 mAbsorbance.

© Beckman Coulter, Inc. March 2012 CLSIOSR6x12.02 All printed copies are considered to be copies of the electronic original. Page 7 of 11 Procedure: TOTAL BILIRUBIN OSR6112, OSR6212, and OSR6512 CALIBRATION:

STANDARD PREPARATION:

Perform a one-point calibration (AB) using a water blank (blue rack) and the appropriate calibrator in a yellow calibration rack. The frequency of calibration is every 30 days. Calibration of this Total Bilirubin procedure is accomplished by use of the Beckman Coulter Chemistry Calibrator (Cat No. DR0070), which is traceable to the National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 916a.

CALIBRATION PROCEDURE:

Recalibration of this test is required when any of these conditions exist:

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 quality 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.

© Beckman Coulter, Inc. March 2012 CLSIOSR6x12.02 All printed copies are considered to be copies of the electronic original. Page 8 of 11 Procedure: TOTAL BILIRUBIN OSR6112, OSR6212, and OSR6512 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 (µmol/L), multiply the results by 17.1.

REPORTING RESULTS:

REFERENCE RANGES:

Adults 5: 0.3 - 1.0 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 for each sample in mg/dL at 37°C.

Additional reporting information as designated by this laboratory:

LIMITATIONS:

The Beckman Coulter AU System Total Bilirubin procedure is linear from 0 up to 30 mg/dL. 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.

INTERFERING SUBSTANCES:

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

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

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

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

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 Young4 for a compilation of reported interferences with this test.

Laboratory specific procedure notes:

REFERENCES:

1. Ehrlich, P., Charite-Annalen, 8: 140, 1883.

2. Tietz, N.W. (ed), Fundamentals of Clinical Chemistry, Third Edition W.B., Saunders, 1987.

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

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

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

6. CLSI/NCCLS Evaluation Protocol, EP5-A Vol. 19 No. 2, 1999.

7. Data on file for specific AU analyzers.