Description

The T4 [¹²⁵I] RIA system provides a quantitative in vitro determination of thyroxine (T4) in human serum in the range 0-320 nmol/l (0-24.9 µg/dl).

Introduction

T4 (3,5,3’,5’-tetraiodothyronine, MW 777) is the primary active hormone synthesized within the follicular cells of thyroid gland.

In plasma, ~70% of T4 is bound to thyroxine-binding globulin (TBG), 15-25% to transthyretin, 5-15% to albumin and a small percentage is bound to erythrocytes. Less than 0.1% of total T4 circulates in a free or unbound form.

T4 bounds to specific cell receptors and has diverse cellular and somatic effects. T4 is catabolized by several processes, including deiodination, transamination followed by oxidative decarboxylation and conjugation.

In most patiens the total T4 level is a good indicator of thyroid status, but T4 levels may be altered in conditions affecting the capacity of the thyroid hormone binding proteins, e. g. pregnancy.

Principle of method

This assay is based on the competition between unlabelled T4 and fixed quantity of ¹²⁵I-labelled T4 for limited number of binding sites on T4 specific antibody. Allowing to react a fixed amount of tracer and antibody with different amounts of unlabelled ligand the amount of tracer bound by the antibody will be inversely proportional to the concentration of unlabelled ligand.

During a 2 hour incubation period with continuous agitation immuno-complex is immobilized on the reactive surface of test tubes. After incubation the reaction mixture is discarded, and the radioactivity is measured in a gamma counter.

The concentration of antigen is inversely proportional to the radioactivity measured in test tubes. By plotting binding values against a series of calibrators containing known amount of T4, a calibration curve is constructed, from which the unknown concentration of T4 in patient samples can determined.

Contents of the kit

Materials, tools and equipment required

Test tube rack
Precision pipettes with disposable tip (100 and 1000 µl)
Shaker
Plastic foil
Absorbent issue
Gamma counter

Recommended tools and equipment

Repeating pipettes (e.g. Eppendorf)

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.

Preparation of reagents, storage

Store the reagents between 2-8 °C after opening. At this temperature each reagent is stable until expiry date. The actual expiry date is given on the package label and in the quality certificate.

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. Store at 2-8 °C until expiry date.

CAUTION! Equilibrate all reagents and serum samples to room temperature. Mix all reagents and samples thoroughly before use. Avoid excessive foaming.

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

B₀/T% is an optional quality control parameter unnecessary for determination of sample concentrations.

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 3% of total count.

Using semi-logarithmic graph paper plot B/B₀ (%) for each standard versus the corresponding concentration of T4. Figure 1 shows a typical standard curve. Determine the 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 computerized data processing logit-log, or spline fittings can be used.

Table 2.    Typical Assay Data

T4 concentration nmol/l

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

Characterization of the assay

Assay parameters

Specificity

Cross reactivity walues are shown below.

Sensitivity

Better than 7 nmol/l, corresponding to the 0-2xSD value.

Precision

5 control samples were assayed in 10 replicates to determine intra-assay precision. Values obtained are shown below.

Reproducibility

To determine inter-assay precision 5 control samples were measured in duplicates in 6 independent assays. Values obtained are shown below.

Recovery

Recovery was defined as the measured increase expressed as per cent of expected increase upon spiking serum samples with known amount of T4. The mean (± SD) recovery % for added T4 (5 samples, 100 nM added T4) was 98.2 ± 2.9.

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 = 120) of adult female blood donors serum concentrations of T4 were 99.2 ± 26.3 (as mean ± SD). Sample values were found scattered in a range of (51.8 - 178.4). As a guide, 60 - 170 nmol/l (4.66 - 13.2 µg/dl) can be interpreted as reference range for normal patients.

In a population (n = 118) of adult male blood donors serum concentrations of T4 were 94.9 ± 19.4 (as mean ± SD). Sample values were found scattered in a range of (23.7 - 153). As a guide, 55 - 130 nmol/l (4.27 - 10.1 µg/dl) can be interpreted as reference range for normal patients.

For female and male (n = 238) the mean (± SD) serum concentration of T4 was 97.1 ± 23.1 range (23.7-178.4) As a guide, 55 - 170 nmol/l (4.27 - 13.2 µg/dl) reference range was obtained from normal patients.

The results obtained should only be interpreted in the context of the overall clinical picture. None of the in vitro diagnostic kits can be used as the one and only proof of any disease or disorder.

Conversion of SI units can be performed according to the following formula:

1 nmol/l = 0.078 µg/dl
1 µg/dl = 12.82 nmol/l

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. To minimize this risk, never take more tubes than needed out of plastic box, and put those left after work back to the box. It is recommended to label assay tubes by a marker pen.

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

Components from various lots or from kits of different manufacturers should not be mixed or interchanged.

Precaution

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.

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.

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 72.5 mg.

	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		Antiserum