SYNCHRON® System(s) T4

Chemistry Information Thyroxine

Sheet REF 445995

For In Vitro Diagnostic Use

ANNUAL REVIEW

Reviewed by: Date Reviewed by: Date

       

       

       

       

PRINCIPLE

INTENDED USE

T4 reagent, when used in conjunction with SYNCHRON LX® System(s), UniCel® DxC 600/800 System(s)
and T4 Calibrator, is intended for quantitative determination of total thyroxine concentration in human
serum or plasma.

CLINICAL SIGNIFICANCE

Thyroxine (T4) and Triiodothyronine (T3) are thyroid hormones which are essential in the regulation of
various metabolic functions.1,2
Increases in total thyroxine are seen in hyperthyroidism, a condition caused by an excess of circulating
hormone. Decreased levels are seen in hypothyroidism, a condition caused by insufficient amounts of
circulating hormone. Quantization of total thyroxine, along with clinical history and other thyroid tests
such as T-Uptake, is a valuable tool in the evaluation of thyroid function.3

METHODOLOGY

T4 reagent has been designed using recombinant DNA techniques. The enzyme β-galactosidase (β-gal)
is split into two inactive components, an enzyme-donor (ED) and an enzyme-acceptor (EA). When these
two inactive components are mixed together, they spontaneously form active enzyme (β-gal).4 Thyroxine
has been attached to the enzyme donor so that the binding of thyroxine specific antibody (Thyroxine-Ab)
will inhibit the reassociation of the two enzyme components. Thyroxine in the sample competes with
thyroxine-enzyme-donor for thyroxine-specific antibody and regulates the formation of active enzyme. The
amount of active enzyme is measured by the rate of hydrolysis of the substrate O-nitrophenyl-β-D-
galactopyranoside (ONPG) to O-nitrophenol.
The SYNCHRON® System(s) automatically proportions the appropriate sample and reagent volumes into
a cuvette. The ratio used is one part sample to 29 parts reagent. The System monitors the ONPG
hydrolysis by measuring the change in absorbance at 410 nanometers. This change in absorbance is
proportional to the concentration of thyroxine in the sample and is used by the System to calculate the
thyroxine concentration. This calculation is based on a two-point calibration curve.

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CHEMICAL REACTION SCHEME

SPECIMEN

TYPE OF SPECIMEN

Biological fluid samples should be collected in the same manner routinely used for any laboratory test. 5
Freshly drawn serum or plasma are the preferred specimens. Acceptable anticoagulants are listed in the
PROCEDURAL NOTES section of this chemistry information sheet. Whole blood 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 or plasma be physically separated from contact with cells within two hours from the time of
collection.6
2.  Separated serum or plasma should not remain at room temperature longer than 8 hours. If assays are
not completed within 8 hours, serum or plasma 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.6

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.

CRITERIA FOR UNACCEPTABLE SPECIMENS

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

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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 (200 tests when filled as directed)
One Calibrator Diskette
One ED Reconstitution Buffer
One EA Reconstitution Buffer
One ED Reagent (lyophilized)
One EA Reagent (lyophilized)
One 4 mL Low Calibrator/Cal 1 (lyophilized phosphate buffer with bovine serum albumin and
preservative)
One 4 mL High Calibrator/Cal 2 (lyophilized phosphate buffer with bovine serum albumin, L-thyroxine and
preservative)

VOLUMES PER TEST

 Sample Volume  9 µL

 Total Reagent Volume  260 µL

 Cartridge Volumes  

   A  200 µL

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    B  60 µL

   C  – –

REACTIVE INGREDIENTS
REAGENT CONSTITUENTS
 THYROXINE ENZYME-DONOR REAGENT:  
 Enzyme-Donor conjugated to thyroxine o-nitrophenyl-β-  19 mL
D-galactopyranoside (after reconstitution with ED buffer)
 THYROXINE ENZYME-ACCEPTOR REAGENT:  
 Monoclonal anti-Thyroxine Antibodies (mouse) (after  50 mL
reconstitution with EA buffer)
 Also non-reactive chemicals necessary for optimal system performance.

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).

EUROPEAN HAZARD CLASSIFICATION

T4 EA Buffer  Xn;R22  Harmful if swallowed.
 S28  After contact with skin, wash immediately with plenty
of water.
T4 EA Reagent  Xn;R22  Harmful if swallowed.
 S28  After contact with skin, wash immediately with plenty
of water.
T4 ED Reagent  Xn;R22  Harmful if swallowed.
 S28  After contact with skin, wash immediately with plenty
of water.
T4 ED Buffer  Xn;R22  Harmful if swallowed.
 S28  After contact with skin, wash immediately with plenty
of water.
T4 High Calibrator (CAL 2)  Xn;R22  Harmful if swallowed.
 S28  After contact with skin, wash immediately with plenty
of water.
T4 Low Calibrator (CAL 1)  Xn;R22  Harmful if swallowed.
 S28  After contact with skin, wash immediately with plenty
of water.

MATERIALS NEEDED BUT NOT SUPPLIED WITH REAGENT KIT

At least two levels of control material

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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.  Transfer the contents of cold Enzyme-Donor Reconstitution Buffer to the ED Reagent bottle.

B.  Mix by gentle inversion (avoid foaming). Allow to stand at room temperature for 5 minutes. Mix
again.

C.  Transfer the contents of the bottles to the B (middle) compartment of the reagent cartridge.

D.  Record the reconstitution date on the cartridge label.

2.  Enzyme-Acceptor Reagent (EA)

A.  Transfer the contents of cold Enzyme-Acceptor Reconstitution Buffer to the EA Reagent bottle.

B.  Mix by gentle inversion (avoid foaming). Allow to stand at room temperature for 5 minutes. Mix
again.

C.  Transfer the contents of the bottles to the A (large) compartment 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, as defined in the CONTROL PROCEDURES section
of this manual.

REAGENT STORAGE AND STABILITY

T4 reagent when stored lyophilized and unopened at +2°C to +8°C will remain stable until the expiration
date printed on the reagent bottle label. Once reconstituted, the reagent is stable for 30 days at +2°C to
+8°C unless the expiration date is exceeded. DO NOT FREEZE.
Do not expose Thyroxine Reagent to room temperature for greater than 24 hours, either in a single or
cumulative exposure.

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REAGENT STORAGE LOCATION:

CALIBRATION

CALIBRATOR REQUIRED

T4 Calibrator Levels 1 and 2 (included in T4 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).

CALIBRATOR STORAGE AND STABILITY

If unopened, the T4 Calibrator may be stored at +2°C to +8°C until the expiration date printed on the
calibrator bottle. Reconstituted calibrators that are resealed and stored at +2°C to +8°C are stable for 30
days unless the expiration date is exceeded.

CALIBRATOR STORAGE LOCATION:

CALIBRATION INFORMATION

1.  The system must have a valid calibration curve in memory before control or patient samples can be
run.
2.  Under typical operating conditions the T4 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.

TRACEABILITY

For Traceability information refer to the Calibrator instructions for use.

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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, 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.

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.

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REFERENCE INTERVALS

Each laboratory should establish its own reference intervals based upon its patient population. The
following reference intervals were taken from literature and a study performed on SYNCHRON Systems.7
Table 2 Reference intervalsa
INTERVALS SAMPLE TYPE CONVENTIONAL UNITS S.I. UNITS
 Literature  Serum or Plasma (Male)  4.6 – 10.5 µg/dL  59.3 – 135.5 nmol/L
 Serum or Plasma  5.5 – 11.0 µg/dL  71.0 – 141.9 nmol/L
(Female)
 SYNCHRON  Serum or Plasma (Male)  4.6 – 11.1 µg/dL  59.3 – 143.2 nmol/L
 Serum or Plasma  4.7 – 16.1 µg/dL  60.6 – 207.7 nmol/L
(Female)

INTERVALS SAMPLE TYPE CONVENTIONAL UNITS S.I. UNITS

 Laboratory      
     
Refer to References (8, 9, 10) for guidelines on establishing laboratory-specific reference intervals.
Procedures for reporting results to the appropriate personnel can be found in the HOW TO REPORT
RESULTS section of this manual.

ADDITIONAL REPORTING INFORMATION AS DESIGNATED BY THIS LABORATORY:

PROCEDURAL NOTES

ANTICOAGULANT TEST RESULTS

If plasma is the sample of choice, the following anticoagulants were found to be compatible with this
method based on a study of 20 healthy volunteers:
Table 3 Acceptable Anticoagulantsb
ANTICOAGULANT LEVEL TESTED FOR IN VITRO AVERAGE PLASMA-SERUM BIAS
INTERFERENCE (µg/dL)
 Sodium Heparin  14 Units/mL  NSIc
 Lithium Heparin  14 Units/mL  NSI

LIMITATIONS

1.  Specimens containing particulate matter should be clarified by centrifugation.

2.  Ethylene glycol-based controls are not recommended for use with this product.
3.  The incidence of patients having antibodies to E. coli β-galactosidase is extremely low. However,
some samples containing such antibodies can result in artificially high results that do not fit the clinical
profile.

INTERFERENCES

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1.  There are a number of compounds which can interfere directly or indirectly with the synthesis of
thyroid hormones, thereby affecting thyroid levels. These major inhibitors can be classified as follows:

A.  Antithyroid drugs which interfere with hormone synthesis.

B.  Ionic inhibitors which block the iodide transport mechanism.

C.  Iodide, which in high concentration can suppress the thyroid.

D.  Radioactive iodine which damages the thyroid gland with ionizing radiation.

Therefore, the full clinical history should be considered when evaluating thyroxine results.
2.  The following substances were tested for interference with this methodology:
Table 4 Interferencesd
SUBSTANCE SOURCE LEVEL OBSERVED EFFECT
 Hemoglobin  RBC hemolysate  500 mg/dL  NSIe
 Bilirubin  Bovine  24 mg/dL  NSI

 Lipemia  Human  4+  NSI

3.  Refer to References (11,12,13) for other interferences caused by drugs, disease and preanalytical
variables.
4.  For assays employing mouse antibodies, the possibility exists for interference by human anti-mouse
antibodies (HAMA) in the sample. Human anti-mouse antibodies may be present in samples from
patients who have received immunotherapy or diagnostic procedures utilizing monoclonal antibodies
or in individuals who have been regularly exposed to animals.14,15 Additionally, other heterophile
antibodies, such as human anti-goat antibodies may be present in patient samples. Interpretation of
results should be done in the context of the overall clinical presentation of the patient, including
symptoms, clinical history, data from additional tests and other appropriate information.

SPECIFICITY

1.  Specificity of the Thyroxine assay: Cross-reactivity is expressed as a ratio of the thyroxine

concentration giving 50% of the high calibrator response, to the concentration of the cross-reactant
giving the same response.

2.  Cross reactivity is clinically insignificant (<0.8%) with the following compounds:

Table 5 Cross Reactivityf
CROSS-REACTANT CONCENTRATION (µg/dL) % CROSS-REACTIVITY
 Acetylsalicylic Acid  15,000  <0.1%

 Methimazole  15,000  <0.1%

 Phenylbutazone  15,000  <0.1%

 Salicylic acid  15,000  <0.1%

 5, 5'-Diphenylhydantoin  15,000  <0.1%

 DL-Tyrosine  15,000  <0.1%

 6-n-Propyl-2-thiouracil  15,000  <0.1%

 3, 5-diiodo-L-thyronine  15,000  <0.1%

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 CROSS-REACTANT CONCENTRATION (µg/dL) % CROSS-REACTIVITY
 3-iodo-L-thyrosine  15,000  <0.1%

 3, 3', 5-Triiodothyroacetic acid  2,000  <0.77%

 3, 3', 5-Triiodo-L-thyronine (T3)  3,750  <0.02%

 3, 3', 5-Triiodo-D-thyronine (D-T3)  7,500  <0.22%

3.  Cross reactivity was 90% for D-Thyroxine.

PERFORMANCE CHARACTERISTICS

Analytic Range

The SYNCHRON LX System method for the determination of thyroxine provides the analytical range from
2.0 µg/dL to the concentration of the high calibrator (the high concentration value is printed on the high
calibrator bottle for each lot - approximately 20 µg/dL).
Samples exceeding the high end of the analytical range should be diluted one part sample with one part
Low Thyroxine Calibrator and reassayed. The value obtained on reassay should be multiplied by the
dilution factor using the following equation:
Actual Thyroxine value = (Diluted value X 2) - Assigned value of low calibrator

REPORTABLE RANGE (as determined on site):

TABLE 6 REPORTABLE RANGE

SAMPLE TYPE CONVENTIONAL UNITS S.I. UNITS
     

     

     

SENSITIVITY

Sensitivity is defined as the lowest measurable concentration which can be distinguished from zero with
95% confidence. Sensitivity for T4 determination is 2.0 µg/dL (25.8 nmol/L).

EQUIVALENCY

Equivalency was assessed by Deming regression analysis of patient samples to accepted clinical
methods.
Serum or Plasma (in the range of 2.5 to 14.6 µg/dL):
   Y (SYNCHRON LX Systems)  = 0.980X + 0.03
   N  = 78
   MEAN (SYNCHRON LX Systems)  = 8.2
   MEAN (SYNCHRON CX7 DELTA)  = 8.4
   CORRELATION COEFFICIENT (r)  = 0.9928

Refer to References (16) for guidelines on performing equivalency testing.

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PRECISION

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

TABLE 7 PRECISION VALUES

TYPE OF SAMPLE TYPE 1 SD CHANGEOVER VALUEg % CV
PRECISION
µg/dL nmol/L µg/dL nmol/L
 Within-run  Serum  0.4  5.2  8.0  104.0  5.0

 Total  Serum  0.6  7.8  8.0  104.0  7.5

Comparative performance data for a SYNCHRON LX ® System evaluated using the NCCLS Proposed
Guideline EP5-T2 appears in the table below.17 Each laboratory should characterize their own instrument
performance for comparison purposes.

TABLE 8 NCCLS EP5-T2 PRECISION ESTIMATE METHOD

TYPE OF SAMPLE TYPE No. No. Data Test Mean EP5-T2 Calculated
IMPRECISION Systems Pointsh Value Point Estimates
(µg/dL) SD %CV
 Within-run  Serum  Control 1  1  80  4.2  0.2  4.3
 Serum  Control 2  1  80  8.6  0.2  2.4
 Serum  Control 3  1  80  17.1  0.3  1.7
 Total  Serum  Control 1  1  80  4.2  0.4  8.8
 Serum  Control 2  1  80  8.6  0.4  4.3
 Serum  Control 3  1  80  17.1  0.4  2.4

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.

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REFERENCES

1. Femander-Ulloa, M., Murad, F., "Thyroid", Clinical Chemistry, C. V. Mosby Company, St. Louis,
MO (1984).

2. Gornall, A. G., Luxton, A. W., Bhavani, B. R., "Endocrine Disorders", Applied Biochemistry of
Clinical Disorders , J. B. Lippincott Company, Philadelphia, PA (1981).

3. Walmsley, R. N., Penhaligon, J., "Screening Thyrometabolic Disorders Using Total Serum
Thyroxine Level", J. Med. J., 8:821 823 (1976).

4. 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).

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

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

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

8. 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).

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

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

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

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

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

14. Bjerner, J., et al., "Immunometric Assay Interference: Incidence and Prevention", Clin. Chem.
48:613 621 (2002).

15. Kricka, L. J., "Interferences in Immunoassays-Still a Threat", Clin. Chem., 46:1037 1038 (2000).

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

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 17. 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

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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 NSI = No Significant Interference (within ± 0.8 µg/dL or 10%).
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 ± 0.8 µg/dL or 10%).
f Data shown was collected using SYNCHRON CX Systems. Equivalency between SYNCHRON LX
Systems has been established by Deming regression analysis to SYNCHRON CX Systems.
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.

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