Description

The FT4 [¹²⁵I] RIA system provides a quantitative determination of free thyroxine (FT4) in human serum. Using 50 µl serum sample FT4 can be assayed in the range 0-100 pmol/l (0-7.77 ng/dl). Each kit contains materials sufficient for 100 assay tubes permitting the construction of one standard curve and the assay of 42 unknowns in duplicate.

Introduction

Circulating thyroid hormones (thyroxine, T4 and triiodothyronine, T3) are distributed into two, a major, protein-bound, and a minor, (0.03% of T4 and 0.3% of T3) free, compartments. Variations in total thyroid hormone in blood may result from either changes of binding proteins concentrations, or thyroid hormone production. Thyroid disorders are existing only if a net change of free unbound fractions occur persistently. Therefore the clinical utility of total T4 and T3 is dependant on the knowledge of functional levels of binding proteins.

Serum level of free T4 (FT4) correlates very well with secretion and metabolism rate of T4, and has been recommended as the most reliable and meaningful diagnostic indicator of thyroid diseases, mostly in conflicting or borderline instances. Apparent FT4 levels, however, are very sensitive to the analytical method due to the sophisticated multiple equilibrium between various protein compartments of T4.

Principle of method

This assay is based on the competition between FT4 and conjugate (T4 analog bound to biotinylated carrier protein) for a limited number of binding sites on ¹²⁵I-labelled monoclonal anti-thyroxine antibodies (tracer). Allowing to react a fixed amount of conjugate and antibody with different amounts of ligand the radioactivity measured on the solid phase will be inversely proportional to the concentration of ligand.

During a 2 hour incubation period with continuous agitation immuno-complex is immobilized on the reactive surface of test tubes. Decanting the supernatant from all tubes the radioactivity in tubes can be measured in a gamma counter.

By plotting binding values against a series of calibrators containing known amount of FT4, a calibration curve is constructed, from which the unknown concentration of FT4 in patient samples can be determined.

Contents of the kit

Materials, tools and equipment required

Test tube rack
Precision pipettes with disposable tips (50 and 500 µl)
Vortex mixer
Shaker
Plastic foil
Absorbent tissue
Gamma counter

Recommended tools and equipment

Repeating pipettes

Preparation of reagents

Tracer, standard and conjugate solutions are ready to use.
Add 500 µl distilled water to the lyophilised control serum. Mix gently with shaking or vortexing (foaming should be avoided).
Ensure that complete dissolution is achieved, and allow the solution to equilibrate at room temperature for at least 20 minutes.

Specimen collection and storage

Serum samples can be prepared according to common procedures used routinely in clinical laboratory practice. Samples can be stored at 2-8 °C if the assay is carried out within 24 hours, otherwise aliquots should be prepared and stored deep frozen (-20 °C). Frozen samples should be thawed and thoroughly mixed before assaying. Repeated freezing and thawing should be avoided. Do not use lipemic, hemolyzed or turbid specimens.

Assay procedure

(For a quick guide, refer to Table 1.)

Table 1 Assay Protocol, Pipetting Guide (all volumes in microliters)

Calculation of results

The calculation is illustrated using representative data. The assay data collected should be similar to those shown in Table 2.
Calculate the average count per minute (CPM) for each pair of assay tubes.
Calculate the percent B₀ / T % for zero standard (S₁) by using the following equation:

Calculate the normalized percent binding for each standard, control and sample respectively by using the following equation:

For simplicity, these values are uncorrected for non-specific binding (NSB). This is enabled by low NSB being less than 1.5% of total count.

Using semi-logarithmic graph paper plot B / B₀ (%) for each standard versus the corresponding concentration of FT4. Figure 1 shows a typical standard curve.

Determine the FT4 concentration of the unknown samples by interpolation from the standard curve. Do not extrapolate values beyond the standard curve range.

Out of fitting programs applied for computerized data processing logit-log, or spline fittings can be used.

Table 2. Typical assay data

Figure 1.
Typical standard curve
(Do not use to calculate sample values)

Conversion of SI units can be performed according to the following formula:
1 pmol/l = 0.0777 ng/dl

Characterization of the assay

Typical assay parameters

Specificity

T3 and r-T3 were added in 4 different concentrations to T4 free standard (S₁ = B₀) and the concentration of FT4 was measured. The cross reactivities are shown below.

DL – detection limit

Sensitivity

Better than 0.7 pmol/l, corresponding to the 0-2xSD value.

Precision

The within-assay precision was determined with 10 replicates within a single run, the between-assay precision was estimated in 8 independent runs carried out in duplicates, both with 7 samples. CV values are summarized below.

Expected Values

It is recommended that each laboratory establish its own reference intervals. The expected values presented here are based on testing of apparently healthy blood donors. Samples were measured in duplicates unseeing different kit lots.

In a population (n = 243) of adult female blood donors (ages: mean 37.8 ± 11.3, range 19 - 69) serum concentrations of FT4 were 14.26 ± 1.91 pmol/l (mean ± SD). Sample values were found scattered in a range of 10.1 – 22 pmol/l.

In a population (n = 243) of adult male blood donors (ages: mean 29.0 ± 10.5 range 19 - 61) serum concentrations of FT4 were 15.4 ± 2.32 pmol/l (mean ± SD). Sample values were found scattered in a range of 10.1 – 22.5 pmol/l.

For female and male (n = 486, ages: mean 33.4 ± 11.7, range 19 - 69) the serum concentration of FT4 was 14.83 ± 2.2 pmol/l (mean ± SD), range 10.1 – 22.5 pmol/l.

As a guide (mean ± 2*SD), 10.4 – 19.2 pmol/l reference range was obtained from normal patients based on statistical consideration only. Taking into consideration not only statistical results but clinical practice as well more realistic reference range of 10-22 pmol/l can be recommended.

Procedural notes

1) Source of error! Reactive test tubes packed in plastic boxes are not marked individually. Care should be taken of not mixing them with common test tubes.

2) Source of error! To ensure the efficient rotation, tubes should be firmed tightly inside the test tube rack. Never use a rack type with open hole. An uneven or incomplete shaking may result in a poor assay performance.

Additional information

Storage

Store the reagents between 2-8 °C. At this temperature each reagent is stable until expiry date. Control serum should be aliquotted and stored deep frozen (-20 °C) for a repeated use.

Availability

From stock.

Shelf life

The minimum shelf life of kit reagents is usually 8 weeks from the date of manufacturing. The actual expiry date is given on the package label and in the quality certificate. To make the maximum benefit of long-term stability it is recommended to adjust the date of ordering to new-batch manufacturing calendar issued each year. Components from various lots or from kits of different manufacturers should not be mixed or interchanged.

Precautions

Radioactivity

This product contains radioactive material. It is the responsibility of the user to ensure that local regulations or code of practice related to the handling of radioactive materials are satisfied.

Chemical hazard

Components contain sodium azide as an antimicrobial agent. Dispose of waste by flushing with copious amount of water to avoid build-up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 113.5 mg.

Biohazard

Human blood products used in the kit have been obtained from healthy human donors. They were tested individually by using approved methods (EIA, enzyme immunoassay), and were found to be negative, for the presence of both Human Immunodeficiency Virus antibody (Anti-HIV-1) and Hepatitis B surface Antigen (HBsAg).

Care should always be taken when handling human specimens to be tested with diagnostic kits. Even if the subject has been tested, no method can offer complete assurance that Hepatitis B Virus, Human Immunodeficiency Virus (HIV-1), or other infectious agents are absent. Human blood samples should therefore be handled as potentially infectious materials.

	Use by		In vitro diagnostic medical device		Control
	Batch code		Manufacturer		Standard
	Caution, consult accompanying documents		Radioactive material		Coated tube
	Biological risk		Temperature limitation Store between 2-8 °C		Tracer
	Consult operating instructions		Catalogue number		Conjugate

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