Subject Free Thyroxine (FT4-II) cobas e411 Index Number Lab-1594 Section Regional/Affiliate Subsection Laboratory Category Departmental Contact Goplin, Darcy Last Revised 2/2/2017 References Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers for Medicare and Medicaid (CMS) and/or COLA. Applicable To Employees of the Gundersen Tri-County Hospital laboratories, Gundersen St. Joseph s Hospital laboratories, Gundersen Boscobel Area Hospital laboratories and Gundersen Palmer Lutheran Hospital and Clinics laboratories. Detail PRINCIPLE: The cobas e411 assay uses a competition principle. Total duration of assay: 18 minutes. 1. 1st incubation: Sample (15uL) and a specific anti-t4 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 antibodyhapten 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. 4. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. 5. Results are determined via a calibration curve which is instrument-specific generated by 2-point calibration and a master curve provided via the reagent bar code. CLINICAL SIGNIFICANCE: The thyroid hormone thyroxine (T4) is physiologically part of the regulating system of the thyroid gland and has an effect on general metabolism. The major fraction of the total thyroxine is bound to transport proteins (TBG, prealbumin, and albumin). The free thyroxine (FT4) is physiologically active thyroxine component. The determination of free thyroxine is an important element in clinical routine diagnostics. Free T4 is measured together with TSH when thyroid function disorders are suspected. The determination of FT4 is also suitable for monitoring thyrosuppressive therapy. Page 1 of 5
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 (Tuptake, TBG) is therefore unnecessary. 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. In the cobas e411 FT4 test the determination of free thyroxine is made with the aid of a specific anti-t4 antibody labeled with a sulfonyl ruthenium complex. The quantity of antibody used is so small (equivalent to approx. 1-2% of the total T4 content of a normal serum sample) that the equilibrium between bound and unbound T4 remains virtually unaffected. SPECIMEN: 1. Lithium-Heparinized plasma or serum collected using standard sampling tubes or tubes containing separating gel. 2. Stability: Stable for 5 days at 15-25 o C, 7 days at 2-8 C, 30 days at -20 C. Freeze only once. 3. Samples containing precipitate must be centrifuged before performing the assay. 4. Samples and controls stabilized with azide cannot be used. 5. Ensure that patient samples, calibrators, and controls are at ambient temperature (20-25 C) before measuring. REAGENTS / MATERIALS: Cobas e411 FT4 II reagent kit, 200 tests Cobas e411 FT4 II CalSet. 1. M Streptavidin-coated microparticles (transparent cap), 1 bottle, 12 ml: Streptavidin-coated microparticles, 0.72 mg/ml, preservative. 2. R1 Anti-T4-Ab~Ru (bpy) 3 2+ (gray cap), 1 bottle, 18 ml: Polyclonal anti-t4-antibody (sheep) labeled with ruthenium complex 15 ng/ml; phosphate buffer 100 mmol/l, ph 7.0; preservative. 3. R2 T4~biotin (black cap), 1 bottle, 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. Reagent Handling: The reagents in the kit have been assembled into a ready-for-use unit that cannot be separated. All information required for correct operation is read in automatically via the reagent bar codes. Storage and Stability: Store at 2-8 C. Store the cobas e411 FT4 reagent kit upright in order to ensure complete availability of the microparticles during the automatic mixing prior to use. DO NOT FREEZE. Page 2 of 5
Stability: Unopened at 2-8 C: up to the stated expiration date After opening: twelve weeks at 2-8 C On cobas e411: 4 weeks. EQUIPMENT / INSTRUMENTATION: Roche cobas e411. Refer to the manual for operating instructions, maintenance and trouble shooting. Calibration: Cobas e411 FT4 has been calibrated against Enzymun-Test FT4. This in turn was calibrated using equilibrium dialysis. Every FT4 reagent set has a bar-coded label containing the specific information for calibration of the particular reagent lot. The predefined master curve is adapted to the analyzer by the use of cobas e411 FT4 CalSet. Calibration frequency: Calibration must be carried out 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. at initial installation 2. if necessary after instrument service or repair 3. if dictated by quality control results 4. if instructed to do so by Roche support personnel 5. at a minimum of every 28 days when using the same reagent lot 6. every 7 days when using same reagent kit on the analyzer Calibrators: cobas e411 FT4 II CalSet. Store at 2-8 C. Supplied ready for use. Stable for 5 calibrations or 12 weeks, whichever comes first. QUALITY CONTROL: Two levels 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 Refer to Lab 4405 Gundersen Lutheran Quality Control Criteria for Chemistry for interpretation of QC. Implementation For optimal performance of the assay it is important to follow the directions given for the analyzer used and to check that the system's inventory of assay materials and other consumables is adequate. Resuspension of the microparticles before use and the reading of the test-specific parameters via the reagent bar code take place automatically. No manual input is necessary. If in exceptional cases the bar code cannot be read, enter the 15-digit sequence of numbers. Page 3 of 5
Cobas e411: Bring the cooled reagents to approximately 20 C and 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. Refer to cobas e411 Operators Manual for routine instrument operation. PROCEDURE NOTES: Results are reported to the nearest hundredth in ng/dl. AMR (Analytical Measurement Range): 0.1 7.77 ng/dl (defined by the lower detection limit and the maximum of the master curve). Values below the detection limit are reported as < 0.1 ng/dl. Values above the measuring range 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 proteinbound forms which are in equilibrium. A change in the concentration of the binding proteins alters this equilibrium. CALCULATIONS: The cobas e411 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 ng/dl 1-12 months 1.00-2.17 ng/dl 1.09-2.02 ng/dl LIMITATIONS: The assay is unaffected by icterus (bilirubin <41 mg/dl), hemolysis (Hb < 1.0 g/dl), lipemia (Intralipid <2000 mg/dl) and biotin < 20 ng/ml, (criterion: recovery within +/- 10% of initial value). IgG <7 g/dl and Igm < 1 g/dl. IgA <1/6 g/dl, albumin<6.3 g/dl. In patients receiving therapy with high biotin doses (i.e. >5 mg/day) no sample should be taken until at least 8 hours after the last biotin administration. No interference was observed from rheumatoid factor (up to 1200 IU/mL) and samples from dialysis patients. Of 17 commonly used pharmaceuticals tested in vitro, only furosemide and Levothyroxine caused elevated FT4 findings at the daily therapeutic dosage level. Page 4 of 5
The test cannot be used to determine FT4 in patients receiving treatment with lipid-lowering agents containing D-T4. If the thyroid function is to be checked in such patients, the therapy should be interrupted for 4-6 weeks to allow the physiological state to become re-established. Samples from neonates have not been tested with the cobas e411 FT4 assay. Auto-antibodies to thyroid hormones can interfere with the assay. The risk of interference from potential immunological interactions between these components and rare sera has been minimized by the inclusion of additives. In rare cases, interference due to extremely high titers of antibodies to streptavidin or ruthenium can occur. Cobas e411 FT4 contains additives which minimize these effects. 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, Supervisor, Manager, Medical Director, or designated person. Changes require retyping document or form and review by the Medical Director. REFERENCES: 1. Roche FT4 package insert 2. Roche cobas e411 Reference Manual 3. Pediatric Reference Intervals 5th ed. AACC Press, 2005. Page 5 of 5