Description - 500 test kit

The hTSH [¹²⁵I] IRMA system provides a direct quantitative in vitro determination of human thyroid stimulating hormone (hTSH) in human serum. hTSH can be assayed in the range 0-100 µIU/ml using 100 or 200 µl serum sample. Each kit contains materials sufficient for 500 assay tubes.

Introduction

The thyroid stimulating hormone (thyrotropin or TSH) is a glycoprotein with a molecular weight of 28000, secreted by the adenohypophysis. Like other glycoprotein hormones (FSH, LH and HCG), TSH contains two different subunits, an alpha- and a ß-chain, linked by noncovalent bounds. The primary structure of alpha subunits of TSH and of the gonadotrophins is the same, whilst their ß subunits are different. The ß subunits are responsible for the immunological and biological specificity of these hormones.

The synthesis and the release of TSH are controlled by the circulatory level of thyroid hormones; triiodothyronine (T3) and thyroxin (T4) and by the hypothalamic thyrotropin releasing hormone (TRH). Thyroid hormones regulate the secretion of TSH by a negative feed-back mechanism. An elevation of T3 or T4 will suppress, and their fall will, in turn, increase the level of TSH in serum. The increased concentration of TSH in the serum is the earliest and best indicator of primary hypothyroidism.

The determination of TSH by immunoassay methods plays a crucial role in the diagnosis of thyroid disorders and in the evaluation of the functional integrity of the hypothalamic-pituitary axis.

The outstanding sensitivity of the present hTSH IRMA system makes it particularly suitable for the measurement of subnormal hTSH levels, a key to both the diagnosis and treatment follow up of hyperthyroid patients.

Principle of method

The technology uses two high affinity monoclonal antibodies in an immunoradiometric assay (IRMA) system.

The ¹²⁵I labelled signal-antibody binds to an epitope of the TSH molecule spatially different from that recognised by the biotin-capture-antibody. The two antibodies react simultaneously with the antigen present in standards or samples, which leads to the formation of a capture antibody - antigen - signal antibody complex, also referred to as a “sandwich”.

During a 1 hour incubation period immuno-complex is immobilized to the reactive surface of streptavidin-coated test tubes. Reaction mixture is then discarded, test tubes washed exhaustively, and the radioactivity is measured in a gamma counter.

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

Contents of the kit

Materials, tools and equipment required

Test tube rack
Precision pipettes with disposable tips (100, 200 µl and 2 ml)
Distilled water
Vortex mixer
Shaker
Plastic foil
Adsorbent tissue
Gamma counter

Recommended tools and equipment

Repeating pipettes (e.g. Eppendorf)
Dispenser with 1-L reservoir (instead of the 2 ml pipette)

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

Add the wash buffer concentrate (100 ml) to 5 L distilled water to obtain 5.1 L wash solution. Upon dilution store at 2-8 °C until expiration.

Store the rest of 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.

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

The Way of Use

The assay can be used in different procedures. There are three options for running the assay. The procedures are labelled as “A”, “B” and “C”.

Working in accordance to “A” and “B” you need a good laboratory shaker. In case of OPTION C you need a water bath thermostat. The test tubes must be in contact with the water so air conditioned thermostat is not applicable.

Patient sample values and expected values are the same for all procedures.

OPTION “A”: The Basic Procedure

It is very economical on sample consumption. Only 100 µl sample volume is needed. The sensitivity attainable is 0.011 µIU/ml. When the kit has less then 3 weeks to its expiration standard 0.06 µIU/ml can be omitted. However if sample consumption is not critical we recommend you to work according to OPTION B. Shaking is needed.

Standard solution 50 µIU/ml can also be omitted if the curve-fitting algorithm of the gamma counter gives similar results with or without this point.

OPTION “B”: The 3rd Generation Method

It works in the same way as OPTION “A” except the sample volume, which is 200 µl. The sensitivity attainable is 0.005 µIU/ml. Shaking is needed.

OPTION “C”: The Water Bath Procedure

No shaking is applied during incubation. A good laboratory thermostat is important. The sensitivity attainable is 0.020 µIU/ml. When the kit has less then 3 weeks to its expiration standard 0.06 µIU/ml can be omitted. Use this method if you have problems with your shakers but you need the results quickly. Use 200 µl sample volume only!

OPTION A - The Basic Procedure

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

Table 1. Assay Protocol, Pipetting Guide - for OPTION A.
All volumes are in microliters (µl).

Characterization of the assay for OPTION A

Figure 1.
OPTION - A. Typical standard curve corresponding to Table 2.
(Do not use to calculate sample values!)

Table 2. Typical assay data for OPTION A

Sensitivity

The analytical sensitivity or minimum detectable limit is calculated by the interpolation of the mean counts of zero standard plus 2 standard deviation from the standard curve. Determination was carried out using 20 replicates of zero standard response.

The value of analytical sensitivity is 0.011 µIU/ml measured using fresh tracer.

The functional sensitivity is a measure of the hTSH concentration that is significantly different from zero as determined by the inter-assay precision profile (22% CV).

The value of functional sensitivity is: 0.07 µIU/ml.

Hook effect

There is no high dose hook effect up to an hTSH concentration 500 µIU/ml.

Precision

The within-assay (intra-assay) precision was determined with 15 replicates within a single run using pooled human serum samples. CV values are summarized below:

Table 3/1.

The between-assay (inter-assay) precision was determined using pooled human serum samples in independent assay runs. The number of measurements on a sample was a function of sample volume available. Three different operators took part in the investigation process and four different tracer batches were used at different ages of the reagents. Four different lots of coated tubes were used to determine the inter-assay precision profile. Results are presented below.

Table 3/2.

Linearity - dilution test

Individual human serum samples were diluted with the zero standard of the kit. The diluted samples were measured according to kit protocol.

Table 4.

Recovery - addition test

Individual human serum samples were spiked with known amount of an elevated stock sample. Recovery % is to be interpreted as = (observed-base)/added*100. The results are summarized below.

Table 5.

OPTION B - The 3rd Generation Method

(For a quick guide, refer to Table 6.)

Table 6. Assay Protocol, Pipetting Guide for OPTION B
All volumes are in microliters (µl).

Characterization of the assay for OPTION B

Figure 2.
OPTION - B. Typical standard curve corresponding to Table 7.
(Do not use to calculate sample values!)

Table 7. Typical assay data for OPTION B

Sensitivity

The analytical sensitivity or minimum detectable limit is calculated by the interpolation of the mean counts of zero standard plus 2 standard deviation from the standard curve. Determination was carried out using 20 replicates of zero standard response.

The value of analytical sensitivity is 0.005 µIU/ml measured using fresh tracer.

The functional sensitivity is a measure of the hTSH concentration that is significantly different from zero as determined by the inter-assay precision profile (22% CV).

The value of functional sensitivity is: 0.03 µIU/ml.

Hook effect

There is no high dose hook effect up to an hTSH concentration 500 µIU/ml.

Precision

The within-assay (intra-assay) precision was determined with 15 replicates within a single run using pooled human serum samples. CV values are summarized below:

Table 8/1.

The between-assay (inter-assay) precision was determined using pooled human serum samples in independent assay runs. The number of measurements on a sample was a function of sample volume available. Three different operators took part in the investigation process and four different tracer batches were used at different ages of the reagents. Four different lots of coated tubes were used to determine the inter-assay precision profile. Results are presented below.

Table 8/2.

Linearity - dilution test

Individual human serum samples were diluted with the zero standard of the kit. The diluted samples were measured according to kit protocol.

Table 9.

Recovery - addition test

Individual human serum samples were spiked with known amount of an elevated stock sample. Recovery % is to be interpreted as = (observed-base)/added*100. The results are summarised below.

Table 10.

OPTION C - The Water Bath Procedure

(For a quick guide, refer to Table 11.)

Table 11. Assay Protocol, Pipetting Guide for OPTION C
All volumes are in microliters (µl).

Characterization of the assay for OPTION C

Figure 3.
OPTION C Typical standard curve corresponding to Table 12.
(Do not use to calculate sample values!)

Table 12. Typical assay data for OPTION C

Sensitivity

The analytical sensitivity or minimum detectable limit is calculated by the interpolation of the mean counts of zero standard plus 2 standard deviation from the standard curve. Determination was carried out using 20 replicates of zero standard response.

The value of analytical sensitivity is 0.020 µIU/ml measured using fresh tracer.

The functional sensitivity is a measure of the hTSH concentration that is significantly different from zero as determined by the inter-assay precision profile (22% CV).

The value of functional sensitivity is: 0.11 µIU/ml.

Hook effect

There is no high dose hook effect up to an hTSH concentration 500 µIU/ml.

Precision

The within-assay precision was determined with 15 replicates within a single run, the between-assay precision was estimated in 15 independent runs carried out in duplicates. CV values are summarized below:

Table 13/1.

The between-assay (inter-assay) precision was determined using pooled human serum samples in independent assay runs. The number of measurements on a sample was a function of sample volume available. Three different operators took part in the investigation process and four different tracer batches were used at different ages of the reagents. Four different lots of coated tubes were used to determine the inter-assay precision profile. Results are presented below.

Table 13/2.

Linearity - dilution test

Individual human serum samples were diluted with the zero standard of the kit. The diluted samples were measured according to kit protocol.

Table 14.

Option independent data

Specificity

No cross reactivity with hFSH and hTSH can be detected in normal physiological concentrations. 2 000 mIU/ml hCG gives an apparent 3.5 µIU/ml increase in hTSH concentration.

Expected Values

Expected euthyroid range is 0.27 µIU/ml - 3.75 µIU/ml.

It is recommended that each laboratory determine a reference range for euthyroids for its own patient population, since this may vary in different laboratories or regions.

Calculation of results

The calculation is illustrated using representative data. The assay data collected should be similar to those shown in Table 2 / 7 / 12.

Calculate the average count per minute (cpm) for each pair of assay tubes. Calculate the normalized percent binding for each standard, control & sample respectively by using the following equation:

Using logarithmic graph paper plot B/T (%) for each standard versus the corresponding concentration of hTSH.

Determine the hTSH concentration of the controls & unknown samples by interpolation from the standard curve. Automated data processing systems are also applicable.

Additional information

Components from various lots of this product, or those of different manufacturers should not be mixed or interchanged.

Note for the washing step: Decantation is the most critical step of the assay procedure. Pay a special attention not to contaminate the outer surface of tubes, when turning the test tube-rack upside down. Even a small contamination may introduce a high, unidentified background resulting in a substantial overestimation of concentration. The error associated may become particularly high in the low range of concentration, which is of vital importance for the reliable determination of subnormal TSH values. On the same reason, regular checking of the instrument background is inevitable. This is particularly important when multi-channel counters are used. Make sure that background values and variation between individual channels are within the range of acceptance as specified in counter's service book.

Note for the shaking step: The rack must hold the tubes tight during shaking in order to shake all the tubes with the same speed and strength.

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 74 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		Wash buffer