3.      PRINCIPLES  OF  THE METHOD

A fixed amount of ¹²⁵I labelled T4 competes with the T4 to be measured present in the sample or in the calibrator for a fixed amount of anti-T4 antibody sites, which are bound to the goat anti mouse (GAM) antibodies immobilized to the wall of a polystyrene tube.  After 1 hour incubation at room temperature, an aspiration step terminates the competition reaction. The tubes are then washed with 2 ml of working wash solution and aspirated again.  A calibration curve is plotted and the T4 concentrations of the samples are determined by dose interpolation from the calibration curve.

4.       REAGENTS PROVIDED

5.      SUPPLIES NOT PROVIDED

The following material is required but not provided in the kit:

1.        Distilled water

2.        Pipettes for delivery of: 20 μl, 100 µl, 200 µl and 500 μl (the use of accurate pipettes with disposable plastic tips is recommended)

3.        Vortex mixer

4.        Magnetic stirrer

5.        Tube shaker

6.        5 ml automatic syringe (Cornwall type) for washing

7.        Aspiration system (optional)

8.        Any gamma counter capable of measuring ¹²⁵I may be used (minimal yield 70%).

6.     REAGENT PREPARATION

a.       Calibrators:  Reconstitute the calibrators with 0.5 ml distilled water.

b.       Controls: Reconstitute the controls with 0.5 ml distilled water.

c.      Anti-T4: Reconstitute the anti-T4 with 11 ml distilled water.

d.       Working Wash solution: Prepare an adequate volume of Working Wash solution by adding 69 volumes of distilled water to 1 volume of Wash Solution (70x). Use a magnetic stirrer to homogenize. Discard unused Working Wash solution at the end of the day.

7.      STORAGE  AND  EXPIRATION  DATING  OF  REAGENTS

-         Before opening or reconstitution, all kits components are stable until the expiry date, indicated on the label, if kept at 2 to 8°C.

-                 After reconstitution, calibrators and controls are stable for 7 days at 2-8°C.  

          For longer storage periods, aliquots should be made and kept at –20°C for maximum 3 months.  Avoid subsequent freeze-thaw cycles.

-         After reconstitution, the anti-T4 antibodies are stable for 6 weeks at 2-8°C.  DO NOT             FREEZE.

-         Freshly prepared Working Wash solution should be used on the same day.

-         After its first use, tracer is stable until expiry date, if kept in the original well-closed vial at 2 to 8°C.

-         Alterations in physical appearance of kit reagents may indicate instability or deterioration.

8.      SPECIMEN  COLLECTION  AND  PREPARATION

-       Serum or plasma samples must be kept at 2‑8°C.

-       If the test is not run within 24 hrs, storage at ‑20°C is recommended.

-       Avoid subsequent freeze-thaw cycles.

-       Serum or Plasma (EDTA or heparin) provides similar results.

9.       PROCEDURE

A.    Handling notes

        Do not use the kit or components beyond expiry date.

        Do not mix materials from different kit lots.

        Bring all the reagents to room temperature prior to use.

        Thoroughly mix all reagents and samples by gentle agitation or swirling.

        Use a clean disposable pipette tip for addition of each different reagent and sample in order to avoid cross-contamination.  High precision pipettes or automated pipetting equipment will improve the precision.

        Respect the incubation times.

Prepare a calibration curve for each run, do not use data from previous runs.

B.     Procedure

1.        Label coated tubes in duplicate for each calibrator, control and sample. For the                  determination of total counts, label 2 normal tubes

2.        Briefly vortex calibrators, controls and samples and dispense 20μl of each into the respective tubes.

3.        Dispense 200 µl of ¹²⁵Iodine labelled T4 into each tube, including the uncoated tubes for total counts.

4.        Dispense 100 µl of anti-T4 into each tube, except tubes for total counts.

5.        Shake the tube rack gently by hand to liberate any trapped air bubbles.

6.        Incubate for 1 hour at room temperature with continous shaking.

7.        Aspirate (or decant) the content of each tube (except total counts).  Be sure that the plastic tip of the aspirator reaches the bottom of the coated tube in order to remove all the liquid.

8.        Wash tubes with 2 ml Working Wash solution (except total counts) and aspirate (or decant). Avoid foaming during the addition of the Working Wash solution.

9.        Let the tubes stand upright for two minutes and aspirate the remaining drop of liquid.

10.     Count tubes in a gamma counter for 60 seconds.

10.      CALCULATION  OF  RESULTS

1.        Calculate the mean of duplicate determinations.

2.        Calculate the bound radioactivity as a percentage of the binding determined at the zero calibrator point (0) according to the following formula :

4.        Computer assisted methods can also be used to construct the calibration curve. If automatic result processing is used, a 4-parameter logistic function curve fitting is recommended.

5.        By interpolation of the sample (B/B0 (%)) values, determine the T4 concentrations of the samples from the calibration curve.

6.        For each assay, the percentage of total tracer bound in the absence of unlabelled T4 (B0/T) must be checked.

11.     TYPICAL DATA

The following data are for illustration only and should never be used instead of the real time calibration curve.

12.    PERFORMANCE  AND  LIMITATIONS

A.       Detection limit

          Twenty zero calibrators were assayed along with a set of other calibrators.

          The detection limit, defined as the apparent concentration two standard deviations below the average counts at zero binding, was < 5 nmol/l.

B.       Specificity

The percentage of cross-reaction estimated by comparison of the concentration yielding a 50% inhibition are respectively:

Note: this table shows the cross-reactivity for the anti T4

C.      Precision

SD: Standard Deviation; CV: Coefficient of variation

D.       Accuracy    

                                             DILUTION  TEST

Samples were diluted with zero calibrator.

RECOVERY  TEST

To the best of our knowledge, no international reference material exists for this parameter.

E.       Time delay between last calibrator and sample dispensing

          As shown hereafter, assay results remain accurate even when a sample is dispensed 60 minutes after the calibrator has been added to coated tubes.

                                                TIME DELAY

13.   INTERNAL  QUALITY  CONTROL

-         If the results obtained for Control 1 and/or Control 2 are not within the range specified on the vial label, the results cannot be used unless a satisfactory explanation for the discrepancy has been given.

-         If desirable, each laboratory can make its own pools of control samples, which should be kept frozen in aliquots.

-         Acceptance criteria for the difference between the duplicate results of the samples should rely on Good Laboratory Practises.

14.       REFERENCE  INTERVALS

These values are given only for guidance; each laboratory should establish its own normal range of values.

T4 concentrations for untreated euthyroid subjects ranged from 64 to 158 nmol/l (n=80).

15.   PRECAUTIONS  AND  WARNINGS

Safety

For in vitro diagnostic use only.

This radioactive product can be transferred to and used only by authorized persons; purchase, storage, use and exchange of radioactive products are subject to the legislation of the end user's country.  In no case the product must be administered to humans or animals.

All radioactive handling should be executed in a designated area. away from regular passage.  A logbook for receipt and storage of radioactive materials must be kept in the lab.  Laboratory equipment and glassware, which could be contaminated with radioactive substances, should be segregated to prevent cross contamination of different radioisotopes.

Any radioactive spills must be cleaned immediately in accordance with the radiation safety procedures.  The radioactive waste must be disposed of following the local regulations and guidelines of the authorities holding jurisdiction over the laboratory.  Adherence to the basic rules of radiation safety provides adequate protection.

The human blood components included in this kit have been tested by European approved and/or FDA approved methods and found negative for HbsAg, anti-HCV, anti-HIV-1 and 2.  No known method can offer complete assurance that human blood derivatives will not transmit hepatitis, AIDS or other infections.  Therefore, handling of reagents, serum or plasma specimens should be in accordance with local safety procedures.

All animal products and derivatives have been collected from healthy animals. Bovine components originate from countries where BSE has not been reported. Nevertheless, components containing animal substances should be treated as potentially infectious.

Avoid any skin contact with reagents (sodium azide as preservative).  Azide in this kit may react with lead and copper in the plumbing and in this way form highly explosive metal azides.  During the washing step, flush the drain with a large amount of water to prevent azide build-up.

Do not smoke, drink, eat or apply cosmetics in the working area.  Do not pipette by mouth.  Use protective clothing and disposable gloves. 

16.  BIBLIOGRAPHY

1.       Davis J.P. (1991)

          Cellullar actions of thyroid hormones.

          The Thyroid.  190-230. 6^th ed. Braverman L.E., Ultiger R.D.

2.       Stockigt J.R. (1996)

Guidelines for diagnosis and monitoring of thyroid disease: nonthyroidal illness.

          Clin. Chem. 42 (1); 188-192.

3.       Fisher D.A. (1996)

Physiological variations in thyroid hormones: physiological and pathophysiological considerations.

          Clin. Chem. 42 (1); 135-139.

4.       Nelson J.C. and Wilcox R.B. (1996)

Analytical performance of free and total thyroxine assays.

          Clin. Chem. 42 (1); 146-154.

5.             Robbins, J. (1973)

        Radioassay and Thyroid Gland.

Metabolism, 22 (8) : 1021.