Description

The ¹²⁵I- L-3,5,3'-triiodothyronine (T3) assay system provides the quantitative in vitro determination of T3 in human serum in the range of 0-12 nmol/l (0-780 ng/dl).

Introduction

Among the thyroid hormones produced in the thyroid gland triiodothyronine (T3) is regarded as the most biologically active molecule, produced up to 80% by the deiodination of tetraiodothyronine (T4) in peripheral tissues.

T3 is found in the bloodstream in a major (99.7%) protein-bound, and a minor (0.3%) unbound, fraction. Variations in total thyroid hormone in blood may result from either changes of binding proteins concentrations, or thyroid hormone production.

T3 contributes significantly to the maintenance of the euthyroid state, and the total T3 level has a role in screening for thyroid disease in conjuction with other tests. T3 alone cannot diagnose hypothyroidism, but it may be more sensitive than T4 for hyperthyroidism.

Principle of method

This assay is based on the competition between unlabelled T3 and fixed quantity of ¹²⁵I-labelled T3 for limited number of binding sites on T3 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 T3, a calibration curve is constructed, from which the unknown concentration of T3 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 tissue
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 lyophilized 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 T3. Figure 1 shows a typical standard curve.
Determine the T3 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

T3 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 values are shown below.

Sensitivity

Better than 0.3 nmol/l, corresponding to the 0-2xSD value.

Precision

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

Reproducibility

To determine interassay precision 5 control samples were measured in duplicates in 7 independent assays. Values obtained are shown below.

Recovery

Recovery was defined as the measured increase expressed as percent of expected increase upon spiking serum samples with known amount of T3. The mean (± SD) recovery % for added T3 (5 samples, 2.5 nM added T3) was 102.1 ± 3.9.

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 T3 were 2.18 ± 0.5 (as mean ± SD). Sample values were found scattered in a range of (1.25 - 3.91). As a guide, 1.4 - 3.3 nmol/l (91 – 215 ng/dl) can be interpreted as reference range for normal patients.

In a population (n = 118) of adult male blood donors serum concentrations of T3 were 1.80 ± 0.38 (as mean ± SD). Sample values were found scattered in a range of (0.55 – 2.75). As a guide, 1.0 – 2.6 nmol/l (65 – 169 ng/dl) can be interpreted as reference range for normal patients.

For female and male (n = 238) the mean (± SD) serum concentration of T3 was 1.99 ± 0.49 in a range (0.55 - 3.91). As a guide, 1.0 - 3.3 nmol/l (65 – 214.5 ng/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 = 0.65 ng/ml
1 ng/ml = 1.54 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 14.5 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 instructions for use		Catalogue number		Antiserum