FOR IN VITRO DIAGNOSTIC USE ONLY

1. CLINICAL APPLICATIONS

Disfunction of the thyroid gland may lead to either an increase (hyperthyroiditis) or a decrease (hypothyroiditis) of T4 or T3 release. Thyroid hormones are transported from the thyroid gland to the target organs closely bound to plasma proteins including thyroxine-binding globulin (TBG), thyroxine binding prealbumin (TBPA) and albumin. The free fraction of FT4 (0.03 %) is considered as metabolicaly active and its concentration is maintained independent of binding protein concentration. Furthermore, free T4 is a major precursor to T3. Free T4 level is independent of binding protein concentration and therefore correlates well with the functional thyroid state. The measurement of free T4 can distinguish the euthyroid hyperthyroxinemias from hyper-thyroidism and the euthyroid hypothyroxinemias from hypothyroidism. In borderline cases of suspected thyroid malfunction, it is recommended to perform additional tests such as Free T3 or high sensitive TSH.

2. PRINCIPLE OF THE ASSAY

In this test kit, free T4 of the sample is allowed to compete with a ligand immobilised on the inner wall of tubes for a limited number of binding sites of an anti-T4 murine monoclonal antibody labelled with ¹²⁵I. This antibody can bind both T4 and the immobilised ligand. Quantities of immobilised ligand and labelled antibody have been carefully adjusted so that the antibody can bind less than 1 % of total T4, resulting in a negligible disturbance of the equilibrium. At the end of the incubation, the radioactivity bound to the immobilised ligand is inversely related to the concentration of free T4 in the sample.

3. REAGENTS PROVIDED WITH THE KIT

- The reagents are sufficient for 100 (500) determinations.

- Store the kit and reagents at 2-8°C.

- The expiration date of each reagent is shown on the label.

1.        Radioactive Tracer : 1 (5) vial(s) (52 ml) of ¹²⁵l labelled anti-T4 mouse monoclonal antibody in HEPES buffer containing gelatin and bovine IgG. Radioactivity contents : ±140 kBq. Preservative : NaN₃ (< 0.1%).

2.        Calibrators: 6 (2 x 6) vials of FT4 in human serum. Lyophilized. Reconstitute with 0.7 ml of distilled water. After reconstitution, the calibrators can be stored at 2-8° C for one month. For the exact value, refer to the value indicated on the Quality Control data sheet. Preservative : NaN₃ (< 0.1%).

3.        Control: 1 (2) vial(s) of FT4 in human serum. Lyophilized. Reconstitute with 0.7 ml of distilled water. After reconstitution, the control serum can be stored at 2-8°C for one month. For the exact value, refer to the value indicated on the Quality Control data sheet. Preservative : NaN₃ (< 0.1%).

4.     Coated Tubes : 100 (500) tubes coated with a Triiodothyronine derivate. Unused tubes must be stored at 2-8°C , protected from moisture.

5.     Washing Solution (50 x concentrated) : 1 (5) vial(s) of 20 ml Tris-HCl buffer with detergent and preservative NaN₃ (< 0.1%).

Bring to 1,000 ml with distilled water. The diluted washing solution is stable for 2 months at 2-8°C.

4. MATERIAL REQUIRED BUT NOT SUPPLIED

- Plastic test tubes

- Test tube racks.

- Adjustable, automatic micropipettes with disposable tips.

- Vortex mixer.

- Graduated cylinder.

- Aspiration pump or automated washing device.

- Scintillation gamma counter.

- Distilled water.

- Orbital shaker adjustable at 150 rpm.

5. WARNINGS AND PRECAUTIONS

In order to obtain reproducible results, the following rules must be observed :

- Do not mix reagents of different lots.

- Do not use reagents beyond their expiry date.

- Use thoroughly clean glassware.

- Use distilled water, stored in clean containers.

- Avoid any contamination among samples; to this purpose disposable tips should be used for each sample and reagent.

In order to avoid personal and environmental contamination, the following precautions must be observed :

- Use disposable gloves while handling potentially infectious material and performing the assay.

- Do not pipette reagents by mouth.

- Do not smoke, eat, drink or apply cosmetics during the assay.

- All material of human origin used for the preparation of this kit has been tested and found negative for HBsAg, anti-HIV and anti-HCV. Since no test at present can guarantee complete absence of these viruses, all samples and reagents used for the assay must be considered potentially infectious; therefore, the assay waste must be decontaminated and disposed of, in accordance with established safety procedures. Disposable ignitable material must be incinerated; disposable non-ignitable material must be sterilized in autoclave for at least 1 hour at 121°C. Liquid wastes must be added with sodium hypochlorite at a final concentration of 3%. Let the hypochlorite act for at least 30 minutes. Liquid wastes containing acid must be neutralized with appropriate amounts of base, before treating with sodium hypochlorite.

- Avoid splashing and aerosol formation; in case of spilling, wash carefully with a 3% sodium hypochlorite solution and dispose of this cleaning liquid as potentially infectious waste.

- Some reagents contain sodium azide as preservative; to prevent build-up of explosive metal azides in lead and copper plumbing, reagents should be discarded by flushing the drain with large amounts of water.

- Acquisition, storage, use and disposal of radioactive material (liquid and solid) are subject to regulation and ordination of local authorities.

6. SPECIMEN COLLECTION

It is recommended to use serum. Highly lipemic or hemolyzed samples must be discarded. Keep samples at 2-8°C for 1 day; for longer periods it is advisable to freeze samples in aliquots at -20°C. Repeated freezing and thawing of samples should be avoided.

7. ASSAY PROCEDURE

- Bring all reagents and samples to warm up at room temperature.

- Mix  samples by gentle agitation before use.

- For all calibrators, a duplicated measure is recommended.

1.     Prepare plain tubes for Total Counts, and coated tubes for Calibrators, Samples and Control.

2.     Pipette 25 ml of each Calibrator, Control and Sample into the appropriated tubes. Pipette directly to the bottom of the tubes.

3.     Add 500 ml of Radioactive Tracer to every tube. Mix gently and cover the tubes with parafilm or an aluminium foil.

4.     Incubate for two hours at room temperature on an orbital shaker set at 150 rpm.

5.     Aspirate the contents of each tube, except the tubes for Total Counts.

6.     Add 2 ml of diluted washing solution to every tube, except tubes for Total Counts and aspirate thoroughly or decant the contents of all tubes on absorbent paper.

7.     Count the radioactivity bound to the tubes for 1 minute in a gamma counter. We suggest to control the background of the instrument before counting the assay. In order to avoid variations in the sensitivity of the system, the background must be reduced to a minimum or adjusted properly.

ASSAY SCHEME

8. CALCULATION OF RESULTS

Calculate the binding capacity percentage (Bo/T%), and the binding percentage for Calibrators, Controls and Samples (B/Bo%).

Draw the calibration curve on a logit-log or semilog paper, using the spline fitting method, with the calibration doses on the abcissa and the respective B/Bo % on the ordinate. Calculate the B/Bo % for each sample and read the concentration by interpolation on the calibration curve to obtain FT4 concentration present in the assayed samples, expressed in pg/ml.

EXAMPLE OF CALCULATION

The values reported below must be considered as an example and may not be used in place of experimental data.

9. REFERENCE VALUES

It is recommended that each laboratory determines its own reference interval. Values reported below are only indicative.

Patients treated with L-T4 (levothyroxine) may exhibit very high concentrations of FT4, especially in high dosage.

During pregnancy, there is a slight decrease of the values FT4 as the pregnancy progresses although the values remain generally in the normal range.

Results may be expressed either in pg/ml or in pmol/l. The conversion is calculated as follow :

pmol/l = pg/ml  x 1.287

10. PERFORMANCES OF THE ASSAY

SPECIFICITY

The cross-reactions of the anti-T4 monoclonal antibody used as tracer in this assay have been measured in a total T4 assay on a molar basis, with displacement agent for the binding proteins, obtaining the follows results : 100.0 % with L-T4 and less than 0.50 % with L-T3.

SENSITIVITY

Analytical sensitivity

The sensitivity was calculated based upon the calibration curve and expressed as the minimual dose showing a significant difference from the Zero Calibrator (mean -2 S.D.). This dose is 1.3 pg/ml.

PRECISION

Precision was evaluated upon intra- and inter-assay variability, at different analyte concentrations.

Intra-assay

Inter-assay

11. BIBLIOGRAPHIE – BIBLIOGRAPHY – ΒΙΒΛΙΟΓΡΑΦΙΑ – BIBLIOGRAFIE – BIBLIOGRAFIA - BIBLIOGRAFÍA

1.     Ekins, R. : Measurements of Free Hormones in Blood. Endocrine Reviews, 1990, 11 (1), 5-46.

2.     Maberly G., Waite K., MA G., Soni N., and Eastman C.  Binding Characteristics of Thyroxin Binding Globulin in Serum of Normal, Pregnant, and Severely III Euthyroid Patients. Clin. Chem. 1986, 32 (4), 616-20.