For in Vitro Diagnostic Use s7

SYNCHRON® System(s) TU Chemistry Information T-Uptake Sheet REF 445999

For In Vitro Diagnostic Use

ANNUAL REVIEW

Reviewed by: Date Reviewed by: Date

PRINCIPLE

INTENDED USE

TU reagent, when used in conjunction with SYNCHRON LX® System(s), UniCel® DxC 600/800 System(s) and T-Uptake Calibrator, is intended for the quantitative determination of thyroxine binding capacity in human serum.

CLINICAL SIGNIFICANCE

Changes in the concentration of thyroid-binding proteins affect free hormone levels, triggering the depression or stimulation of hormone production until a new equilibrium is reached. The clinical usefulness of total thyroid hormone measurement is compromised if there are marked changes in the binding capacity or concentration of thyroxine-binding proteins.1

METHODOLOGY

TU reagent is used to measure unoccupied thyroxine-binding sites. This reagent used recombinant DNA technology. The enzyme β-galactosidase (β-gal) is split into two inactive components, an enzyme-donor (ED) and an enzyme-acceptor (EA), which, when mixed together, spontaneously form active enzyme. Enzyme-donor-thyroxine conjugate binds directly to the unoccupied thyroxine-binding protein (TBP) sites in serum, preventing spontaneous reassociation of the two enzyme fragments to form active enzyme. 2 Thyroid-binding proteins regulate the amount of active β-galactosidase formed. The unoccupied thyroxine-binding sites in the sample are inversely proportional to the amount of β-galactosidase formed from the reassembly of the remaining ED and EA as monitored by the hydrolysis of the substrate o- nitrophenyl-β-d-galactopyranoside (ONPG). The SYNCHRON® System(s) automatically proportions the appropriate sample and reagent volumes into a cuvette. The ratio used is one part sample to 17 parts reagent. The System monitors the ONPG hydrolysis by measuring the change in absorbance at 410 nanometers. This change in absorbance is inversely proportional to the unoccupied thyroxine-binding sites in the sample and is used by the System to calculate and express T-Uptake as a percentage based upon a two-point calibration curve. The free thyroxine index (FTI) is a means of normalizing the effect of thyroxine-binding protein concentrations on total T4 levels. The FTI yields an index which is related to the biologically active free T4 concentration.

TU A18563AF EN 08/26/2010 1 / 12 The FTI calculation, as recommended by the American Thyroid Association is:

The mean TU% referred to in the above calculation refers to the mean % T-Uptake of the normal range distribution which should be established by each laboratory.

CHEMICAL REACTION SCHEME

SPECIMEN

TYPE OF SPECIMEN

Biological fluid samples should be collected in the same manner routinely used for any laboratory test. 3 Freshly drawn serum is the preferred specimen. Whole blood, plasma or urine are not recommended for use as a sample.

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 be physically separated from contact with cells within two hours from the time of collection.4 2. Separated serum should not remain at room temperature longer than 8 hours. If assays are not completed within 8 hours, serum should be stored at +2°C to +8°C. If assays are not completed within 48 hours, or the separated sample is to be stored beyond 48 hours, samples should be 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.4

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.

TU A18563AF EN 08/26/2010 2 / 12 CRITERIA FOR UNACCEPTABLE SPECIMENS

Refer to the PROCEDURAL NOTES section of this chemistry information sheet for information on unacceptable specimens.

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: One Empty Reagent Cartridge (250 tests when filled as described) One Calibrator Diskette One EA Reagent (lyophilized) One ED Reagent (lyophilized) One EA Reconstitution Buffer One ED Reconstitution Buffer One 4 mL Low Calibrator/Cal 1 (lyophilized) One 4 mL High Calibrator/Cal 2 (lyophilized)

VOLUMES PER TEST 15 µL 252 µL

TU A18563AF EN 08/26/2010 3 / 12 A 209 µL B 43 µL C – –

REACTIVE INGREDIENTS

REAGENT CONSTITUENTS T-Uptake Enzyme-Donor (ED) Reagent: Enzyme-Donor conjugated to thyroxine o-nitrophenyl-β- 14 mL (after reconstitution with ED buffer) D-galactopyranoside T-Uptake Enzyme-Acceptor (EA) Reagent: Enzyme-Acceptor 62 mL (after reconstitution with EA buffer)

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).

MATERIALS NEEDED BUT NOT SUPPLIED WITH REAGENT KIT

At least two levels of control material

REAGENT PREPARATION

NOTICE

To ensure solubility of combined reagents, reconstitute immediately upon removal from refrigerator.

Reconstitute in the following order: 1. Enzyme-Donor Reagent (ED)

A. Open cold ED Reconstitution Buffer bottle and snap adapter on tightly.

B. Connect the bottle of ED Reagent to the bottle of ED Reconstitution Buffer.

C. Mix by gentle inversion to ensure all the ED Reagent is transferred into the ED Buffer bottle. Avoid foaming. Allow to stand at room temperature for 5 minutes. Mix again.

D. Detach the ED Reagent bottle and adapter from the ED Buffer bottle and discard.

E. Transfer the entire contents of the bottle to compartment B (middle) of the reagent cartridge.

F. Record the reconstitution date on the cartridge label.

TU A18563AF EN 08/26/2010 4 / 12 2. Enzyme-Acceptor Reagent (EA)

A. Open cold EA Reconstitution Buffer bottle and snap adapter on tightly.

B. Connect the bottle of EA Reagent to the bottle of EA Reconstitution Buffer.

C. Mix by gentle inversion to ensure all the EA Reagent is transferred into the EA Buffer bottle. Avoid foaming. Allow to stand at room temperature for 5 minutes. Mix again.

D. Detach the EA Reagent bottle and adapter from the EA Buffer bottle and discard.

E. Transfer the entire contents of the bottle to compartment A (large) of the reagent cartridge.

Cross contamination of reagents must be avoided. The enzyme-donor solution should appear colorless to very pale yellow. A deep yellow or yellow-orange color indicates that the reagent has been contaminated and must be discarded. The enzyme-donor and enzyme-acceptor reagents are matched sets. Do not mix bottles from different kit lot numbers in the same cartridge. Always add freshly reconstituted reagents to an unused cartridge.

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.

REAGENT STORAGE AND STABILITY

T-Uptake reagent when stored lyophilized and unopened at +2°C to +8°C will remain stable until the expiration date printed on the kit label. Once reconstituted, the reagent is stable for 21 days at +2°C to +8°C unless the expiration date is exceeded. DO NOT FREEZE. Do not expose T-Uptake Reagent to room temperature for more than 24 hours, either in single or cumulative exposure.

REAGENT STORAGE LOCATION:

CALIBRATION

CALIBRATOR REQUIRED

T-Uptake Calibrator Levels 1 and 2 (included in the T-Uptake Reagent Kit)

CALIBRATOR PREPARATION

1. Reconstitute each bottle with 4.0 mL of deionized water using a volumetric pipette. 2. Let stand 45 minutes to dissolve and equilibrate. Mix by gentle inversion (avoid foaming).

TU A18563AF EN 08/26/2010 5 / 12 CALIBRATOR STORAGE AND STABILITY

If unopened, T-Uptake Calibrators should be stored at +2°C to +8°C until the expiration date printed on the calibrator bottle. Prepared calibrators that are resealed and stored at +2°C to +8°C are stable for 21 days unless the expiration date is exceeded.

NOTICE

Prior to the first calibration of a specific lot number of T-Uptake Reagent, the lot specific calibrator diskette that accompanies the kit must have been loaded onto the system.

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.5

CALIBRATOR STORAGE LOCATION:

CALIBRATION INFORMATION

1. The system must have valid calibration factors in memory before controls or patient samples can be run. 2. Under typical operating conditions the TU reagent cartridge must be calibrated every 24 hours 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. 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.

TU A18563AF EN 08/26/2010 6 / 12 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.

NOTICE

Quality control material containing ethylene glycol is not recommended for use with this assay.

TABLE 1 QUALITY CONTROL MATERIAL CONTROL NAME SAMPLE TYPE STORAGE

TESTING PROCEDURE(S)

1. If necessary prepare reagent as defined in the Reagent Preparation section of this chemistry information sheet and load the reagent onto the system. 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.

TU A18563AF EN 08/26/2010 7 / 12 REPORTING RESULTS

Equivalency between the SYNCHRON LX 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

Each laboratory should establish its own reference intervals based upon its patient population. The reference intervals listed below were taken from literature and a study performed on SYNCHRON Systems.6 Table 2 Reference intervalsa INTERVALS SAMPLE TYPE CONVENTIONAL UNITS Literature Serum (Male) 30.6 to 40.4% uptake Serum (Female) 25.3 to 40.4% uptake SYNCHRON Serum (Male) 27.9 to 39.5% uptake Serum (Female) 21.6 to 34.1% uptake

INTERVALS SAMPLE TYPE CONVENTIONAL UNITS Laboratory

Refer to References (7,8,9) for guidelines on establishing laboratory-specific reference intervals.

ADDITIONAL REPORTING INFORMATION AS DESIGNATED BY THIS LABORATORY:

PROCEDURAL NOTES

ANTICOAGULANT TEST RESULTS

Serum is the specimen of choice. The following anticoagulant data was derived from a study of plasma: Table 3 Incompatible Anticoagulantsb ANTICOAGULANT LEVEL TESTED FOR IN VITRO AVERAGE PLASMA-SERUM BIAS (%)c INTERFERENCE Lithium Heparin 14 Units/mL ≤+2.3 Sodium Heparin 14 Units/mL ≤+2.6

LIMITATIONS

1. Specimens containing particulate matter should be clarified by centrifugation. 2. Specimens quantifying outside of the assay range should be repeated for validity, but not diluted. They should be reported greater than the Hyperthyroid-assigned (Calibrator 2) or less than the Hypothyroid- assigned (Calibrator 1) values. 3. Ethylene glycol-based controls are not recommended for use with this product.

TU A18563AF EN 08/26/2010 8 / 12 INTERFERENCES

1. The following substances were tested for interference with this methodology: Table 4 Interferencesd SUBSTANCE SOURCE LEVEL OBSERVED EFFECT Bilirubin Bovine 24 mg/dL NSIe Hemoglobin RBC hemolysate 500 mg/dL NSI Lipemia Intralipidf 300 mg/dL NSI 2. Refer to References (10,11,12) for other interferences caused by drugs, disease and preanalytical variables.

PERFORMANCE CHARACTERISTICS

Analytic Range

The analytical range is defined by the % uptake values printed on the low and high calibrator bottles. Values outside this range should not be diluted but reported out as less than the low calibrator value or higher than the high calibrator value.

REPORTABLE RANGE (as determined on site):

TABLE 5 REPORTABLE RANGE SAMPLE TYPE CONVENTIONAL UNITS S.I. UNITS

SENSITIVITY

Sensitivity for the thyroxine binding capacity is defined by the % uptake values printed on the low calibrator bottle.

EQUIVALENCY

Equivalency was assessed by Deming regression analysis of patient samples to accepted clinical methods. Y (SYNCHRON Systems) = 0.993X + 0.33 N = 77 MEAN (SYNCHRON Systems) = 34.6 MEAN (SYNCHRON CX7 DELTA) = 34.5 CORRELATION COEFFICIENT (r) = 0.9967 Refer to References (13) for guidelines on performing equivalency testing.

PRECISION

A properly operating SYNCHRON® System(s) should exhibit precision values less than or equal to the following:

TU A18563AF EN 08/26/2010 9 / 12 TABLE 6 PRECISION VALUES

TYPE OF SAMPLE TYPE 1 SD CHANGEOVER VALUEg % CV PRECISION % uptake % uptake Within-run Serum 0.65 26.00 2.50 Total Serum 0.98 26.00 3.75 Comparative performance data for a SYNCHRON LX® System evaluated using the NCCLS Proposed Guideline EP5-T2 appears in the table below.14 Each laboratory should characterize their own instrument performance for comparison purposes.

TABLE 7 NCCLS EP5-T2 PRECISION ESTIMATE METHOD

TYPE OF No. No. Data Test Mean SD %CV IMPRECISION Systems Pointsh Value (% uptake) Within-run Serum Control 1 1 80 31.7 0.30 0.94 Serum Control 2 1 80 41.7 0.49 1.19 Serum Control 3 1 80 48.0 0.44 0.92 Total Serum Control 1 1 80 31.7 0.68 2.15 Serum Control 2 1 80 41.7 0.67 1.60 Serum Control 3 1 80 48.0 0.72 1.51

NOTICE

These degrees of precision and equivalency were obtained in typical testing procedures on a SYNCHRON LX® System 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.

SHIPPING DAMAGE

If damaged product is received, notify your Beckman Coulter Clinical Support Center.

TU A18563AF EN 08/26/2010 10 / 12 REFERENCES

1. Henry, J. B., Kreig, A. F., "Endocrine Measurements", Clinical Diagnosis by Laboratory Methods, W. B. Saunders Company, Philadelphia, PA (1974).

2. Henderson, D. R., Friedman, S. B., Harris, J. D., Manning, W. B., Zoccoli, M. A., "CEDIA, A New Homogeneous Immunoassay System", Clin. Chem., 32(9):1637 1641 (1986).

3. Tietz, N. W., "Specimen Collection and Processing; Sources of Biological Variation", Textbook of Clinical Chemistry, 2nd Edition, W. B. Saunders, Philadelphia, PA (1994).

4. National Committee for Clinical Laboratory Standards, Procedures for the Handling and Processing of Blood Specimens, Approved Guideline, NCCLS publication H18-A, Villanova, PA (1990).

5. CDC-NIH manual, Biosafety in Microbiological and Biomedical Laboratories, U.S. Government Printing Office, Washington, D.C. (1984).

6. Tietz, N. W., Clinical Guide to Laboratory Tests, 3rd Edition, W. B. Saunders, Philadelphia, PA (1995).

7. 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 (1995).

8. Tietz, N. W., ed., Fundamentals of Clinical Chemistry, 3rd Edition, W. B. Saunders, Philadelphia, PA (1987).

9. Henry, J. B., Clinical Diagnosis and Management by Laboratory Methods, 18th Edition, W. B. Saunders Company, Philadelphia, PA (1991).

10. Young, D. S., Effects of Drugs on Clinical Laboratory Tests, 4th Edition, AACC Press, Washington, D. C. (1995).

11. Friedman, R. B., Young, D. S.,Effects of Disease on Clinical Laboratory Tests, 3rd Edition, AACC Press, Washington, D.C. (1997).

12. Young, D. S., Effects of Preanalytical Variables on Clinical Laboratory Tests, 2nd Edition, AACC Press, Washington, D. C. (1997).

13. National Committee for Clinical Laboratory Standards, Method Comparison and Bias Estimation Using Patient Samples, Approved Guideline, NCCLS publication EP9-A, Villanova, PA (1995).

14. National Committee for Clinical Laboratory Standards, Precision Performance of Clinical Chemistry Devices, Tentative Guideline, 2nd Edition, NCCLS publication EP5-T2, Villanova, PA (1992).

Beckman Coulter Ireland Inc., Mervue Business Park, Mervue, Galway, Ireland (353 91 774068)

Beckman Coulter, Inc., 250 South Kraemer Blvd., Brea, CA 92821

TU A18563AF EN 08/26/2010 11 / 12 ENDNOTES a Data shown was collected using SYNCHRON CX Systems. Equivalency between SYNCHRON LX Systems has been established by Deming regression analysis to SYNCHRON CX Systems. b Data shown was collected using SYNCHRON CX Systems. Equivalency between SYNCHRON LX Systems has been established by Deming regression analysis to SYNCHRON CX Systems. c Bias is based on worst case instead of average. Plus (+) or minus (-) signs in this column signify positive or negative bias. d Data shown was collected using SYNCHRON CX Systems. Equivalency between SYNCHRON LX Systems has been established by Deming regression analysis to SYNCHRON CX Systems. e NSI = No Significant Interference (within ± 1.3% uptake or 5%). f Intralipid is a registered trademark of KabiVitrum, Inc., Clayton, NC 27250. g 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. h 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.

TU A18563AF EN 08/26/2010 12 / 12