Standard Operating Procedure

Subject Free Thyroxine (FT4) – Cobas e601

Index Number Lab-4045
Section Laboratory
Subsection Chemistry
Category Departmental
Contact Benjamin Michel
Last Revised 10/12/2016

References
Required document for Laboratory Accreditation by the College of American Pathologists.

Applicable To
Employees of the Gundersen Health System clinical laboratory.

Detail
INTENDED USE:
Immunoassay for the in vitro quantitative determination of free thyroxine in human serum and plasma.
Measurements obtained by this assay are used in the diagnosis and treatment of thyroid disease.

PRINCIPLE:
The Elecsys FT4 II assay is based on a competitive test principle using a specific anti-T4 antibody labeled
with a sulfonyl-ruthenium complex to determine the free thyroxine. Total duration of assay: 18
minutes.
1. 1st incubation: 15μL of sample and a T4-specific antibody labeled with a sulfonyl-ruthenium
complex.
2. 2nd incubation: After addition of biotinylated T4 and streptavidin-coated microparticles, the still-
free binding sites of the labeled antibody become occupied, with formation of an antibody-
hapten complex. The entire complex is bound to the solid phase via interaction of biotin and
streptavidin.
3. The reaction mixture is aspirated into the measuring cell where the microparticles are
magnetically captured onto the surface of the electrode. Unbound substances are then removed
with ProCell M. Application of a voltage to the electrode then induces chemiluminescent
emission which is measured by a photomultiplier.
4. Results are determined via a calibration curve which is instrument-specifically generated by 2-
point calibration and a master curve provided via the reagent barcode.

CLINICAL SIGNIFICANCE:
Thyroxine (T4) is the main thyroid hormone secreted into the bloodstream by the thyroid gland.
Together with triiodothyronine (T3) it plays a vital role in regulating the body’s metabolic rate,
influences the cardiovascular system, growth and bone metabolism, and is important for normal
development of gonadal functions and nervous system.

T4 circulates in the bloodstream as an equilibrium mixture of free and serum-bound hormone. Free T4
(FT4) is the unbound and biologically active form, which represents only 0.03% of the total T4. The

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 Standard Operating Procedure

remaining T4 is inactive and bound to serum proteins such as thyroxine binding globulin (75%),
prealbumin (15%), and albumin (10%).

The determination of free T4 has the advantage of being independent of changes in the concentrations
and binding properties of the binding proteins; additional determination of a binding parameter (T-
uptake, TBG) is therefore unnecessary. Therefore free T4 is a useful tool in clinical routine diagnostics
for the assessment of the thyroid status. It should be measured together with TSH if thyroid disorders
are suspected and is also suitable for monitoring thyrosuppressive therapy.

A variety of methods are available for estimating the free thyroid hormone levels. The direct
measurement of FT4 and FT3 via equilibrium dialysis or ultrafiltration is mainly used as a reference
method for standardizing the indirect procedures generally used for routine diagnostic purposes.

SPECIMEN:
Serum or lithium heparin plasma collected using standard sampling tubes or tubes containing separating
gel.

Stability: 5 days at 15-25°C, 7 days at 2-8°C, 30 days at -20°C. Only one freeze/thaw cycle is allowed.
Samples containing precipitate must be centrifuged before performing the assay. Samples and controls
stabilized with azide cannot be used. Ensure that patient samples, calibrators, and controls are at
ambient temperature (15-25°C) before measuring.

REAGENTS / MATERIALS:
Free thyroxine reagent pack, 200 tests – the reagent rackpack is labeled as FT4 II.
M - Streptavidin-coated microparticles (transparent cap), 12 mL: Streptavidin-coated
microparticles, 0.72 mg/mL, preservative.
R1 - Anti-T4-Ab~Ru(bpy)23+ (gray cap), 18 mL: Polyclonal anti-T4-antibody (sheep) labeled with
ruthenium complex 75 ng/mL; phosphate buffer 100 mmol/L, pH 7.0; preservative.
R2 - T4~biotin (black cap), 18 mL: Biotinylated T4 2.5 ng/mL; phosphate buffer 100 mmol/L, pH 7.0;
preservative.

Precautions and warnings: For in vitro diagnostic use. Exercise the normal precautions required for
handling all laboratory reagents. Disposal of all waste material should be in accordance with local
guidelines.

Storage: Store at 2-8°C. Do not freeze. Store the reagent kit upright in order to ensure complete
availability of the microparticles during the automatic mixing prior to use.

Stability: Unopened at 2-8°C: up to the stated expiration date. Opened at 2-8°C: 12 weeks. On-board the
e601: 4 weeks.

EQUIPMENT / INSTRUMENTATION:
Roche Cobas e601 analyzer. Refer to the operator's manual for operating instructions, maintenance, and
troubleshooting.

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 Standard Operating Procedure

Calibration: This method has been standardized against the Elecsys FT4 method. The Elecsys FT4 assay is
traceable to the Enzymun-Test which was standardized using equilibrium dialysis. Every reagent set has
a barcoded label containing the specific information for calibration of the particular reagent lot. The
predefined master curve is adapted to the analyzer using the relevant CalSet.

Calibrators: FT4 II CalSet. Store unopened at 2-8°C.

Preparation: Ready for use. Mix carefully, avoiding foam formation. Transfer reconstituted
calibrators into two sets of labeled snap-cap bottles. One set is used for immediate calibration
and the second set is stored refrigerated.
Stability: 12 weeks at 2-8°C.

Calibration Frequency: Calibration must be performed once per reagent lot using fresh reagent (i.e. not
more than 24 hours since the reagent pack was registered on the analyzer). Renewed calibration is
recommended as follows:
1. After 28 days when using the same reagent lot.
2. After 7 days when using the same reagent kit on the analyzer.
3. If dictated by quality control results.
4. If necessary after instrument service or repair.

QUALITY CONTROL:
BioRad Immunoassay Plus controls, levels 1 and 2

Refer to Lab-4405 Quality Control Criteria for Chemistry for interpretation of QC. Each level of Quality
Control should be performed at a minimum:
1. once every twenty-four hours
2. if a new pack of reagent is put in use
3. if a calibration is performed

Implementation
Resuspension of the microparticles takes place automatically prior to use. Read in the test-specific
parameters via the reagent barcode. If in exceptional cases the barcode cannot be read, enter the 15-
digit sequence of numbers.

Bring the cooled reagents to approximately 20°C by allowing them to sit at room temperature for at
least 45 minutes. Place on the reagent disk of the analyzer. Avoid the formation of foam. The system
automatically regulates the temperature of the reagents and the opening/closing of the bottles.

PROCEDURE NOTES:
Results are reported to the nearest hundredth in ng/dL.
AMR (Analytical Measurement Range): 0.10-7.77 ng/dL.
Values below 0.10 are reported as <0.10 ng/dL.
Values above 7.77 are reported as >7.77 ng/dL.

Dilution: Samples for FT4 determination cannot be diluted, as T4 in the blood is present in free and
protein-bound forms which are in equilibrium. A change in the concentration of the binding proteins
alters this equilibrium.

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 Standard Operating Procedure

CALCULATIONS:
The Cobas 6000 system automatically calculates the FT4 concentration of each sample.

INTERPRETATION:
Expected values: 0.90-1.7 ng/dL

Values for children less than 1 year are higher. See table below:
Age Male Female
1 – 3 days 1.16 – 2.95 ng/dL 1.09 – 2.09 ng/dL
4 – 30 days 0.78 – 2.25 ng/dL 0.85 – 2.09 g/dL
1 – 12 months 1.00 – 2.17 ng/dL 1.09 – 2.02 ng/dL

LIMITATIONS:
Hemolysis: No significant interference up to an H index of 1000 (approximate hemoglobin
concentration: 1.0 g/dL).
Icterus: No significant interference up to an I index of 41 (approximate total bilirubin concentration: 41
mg/dL).
Lipemia: No significant interference up to a triglyceride concentration of 2000 mg/dL. There is poor
correlation between the L index (corresponds to turbidity) and triglycerides concentration.
However, Gundersen Health System has established a L index of 137 as a lipemia screen for this test.
If the L index is above this number, test triglycerides to determine specimen suitability and then
ultracentrifuge specimen if necessary. The triglycerides value of the cleared specimen must be
below the limit in order to report results. It may be necessary to ultracentrifuge multiple times.
See package insert for additional interference and cross-reactivity studies. The results should always be
assessed in conjunction with the patient’s medical history, clinical examination and other findings.

REVIEW AND CHANGES:

This document and all attached forms should be reviewed on an annual basis, with 2 years as the
maximum review date. Review will be done by the Technical Leader, Medical Director, or designated
person. Changes require retyping document or form and review by the Medical Director.

REFERENCES:
1. Roche Free thyroxine package insert
2. Roche FT4 II CalSet package insert
3. Roche Cobas 6000 Operator’s Manual
4. Pediatric Reference Intervals 5th ed. AACC Press, 2005.

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