Description

The ¹²⁵I-FT4 assay system provides the quantitative determination of free thyroxine (FT4) in human serum. Free T4 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 assay of 42 unknowns and 1 control 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.

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.

The present, simple, and rapid labelled antibody free T4 radioimmunoassay enables the reliable, clinically validated determination of FT4 in the concentration range 0-100 pmol/l.

Principle of method

This assay is based on the competition between free T4 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 bound to the conjugate will be inversely proportional to the concentration of ligand. Upon addition of magnetizable immunosorbent the conjugate - ¹²⁵I-anti T4 complex is bound on solid particles which are then separated by either magnetic sedimentation or centrifugation. Counting the radioactivity of solid phase enables a standard curve to be constructed and samples to be quantitated.

Contents of the kit

Materials and equipment required

Round bottom polystyrene or polypropylene assay tubes, about 12 x 75 mm
Plastic film to cover tubes
Precision pipettes (50 and 500 µl)
Vortex mixer
Magnetic separator (or alternatively centrifuge)
Decanting racks
Gamma counter

Recommended tools and equipment

Orbital shaker
Repeating pipettes

Preparation of reagents

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. Sera can be stored at 2-8 °C for two days after collection. For later analysis they should be stored deep-frozen (-20 °C). 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

Calculate the average counts per minute (CPM) for each pair of assay tubes (Table 2.)
Calculate the percent B₀ / T for zero standard (S₁) by using the following equation:

Calculate the normalised 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 magnetic separator).

Using semi-logarithmic graph paper plot B / B₀ % for each standard versus the corresponding concentration of free T4. (Figure 1)

Determine the free T4 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 computerised data processing logit-log, or spline fittings can be used.

Table 2.    Typical Assay Data

Figure 1.
A 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

Assay parameters

Specificity

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

DL – detection limit

Sensitivity

1.7 pmol/l, defined as the concentration 2 standard deviations from the zero standard.

Precision

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

Expected reference 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.

In a population (n = 140) of adult female blood donors (ages: mean 38.0 ± 11.6, range 19 - 69) serum concentrations of free T4 were 14.77 ± 1.77 pmol/l (mean ± SD). Sample values were found scattered in a range of 8.70 - 19.8 pmol/l.

In a population (n = 140) of adult male blood donors (ages: mean 29.1 ± 10.7 range 19-61) serum concentrations of free T4 were 15.25 ± 1.79 pmol/l (mean ± SD). Sample values were found scattered in a range of 11.3 – 19.9 pmol/l.

For female and male (n = 280, ages: mean 33.6 ± 12.0, range 19 - 69) the serum concentration of free T4 was 15.01 ± 1.79 pmol/l (mean ± SD), range 8.70 - 19.9 pmol/l.

As a guide (mean ± 2*SD), 11.4-18.6 pmol/l reference range was obtained from normal patients.

Additional information

Storage

Store the reagents between 2 and 8 °C. At this temperature each reagent is stable until expiry date. Pay special attention to preventing magnetic immunosorbent suspension from freezing.

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 and warnings

Radioactivity

This kit contains radioactive material. Receipt, acquisition, possession, or use of radioactive materials are subject to regulations, and a licence of (inter)national authorizing bodies. It is the responsibility of the user to ensure that local regulations or codes of practice are satisfied.

Potentially infectious materials

Human blood products provided as components of this product have been obtained from donors tested individually and found negative for Human Immunodeficiency Virus antibody (HIV-Ab) as well as for Hepatitis B surface Antigen (HBsAg) using approved EIA methods.

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), or other infectious agents are absent, and all human blood samples should be considered potentially infectious.

Chemical and other hazard

Some components contain sodium azide (0.1% w/v) as an antimicrobial agent. Dispose the waste by flushing it with copious amounts of water to avoid build up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 124.5 mg.

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

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