Microparticle Enzyme Immunoassay MEIA) ) & Thyroid function tests 1

1. Microparticle Enzyme Immunoassay MEIA))&Thyroid function tests 1

2. MEIA • Microparticle Enzyme Immunoassay (MEIA) is an immunoassay method that utilizes the isolation of antibody/antigen complexes on a solid phase surface of small beads called microparticles. • MEIA has been widely adapted to automate the measurement of large molecules such as markers associated with cardiac, fertility, cancer, metabolic, hepatitis, and thyroid testing.

3. Assay components • The components of MEIA include the following, all suspended in a specific buffer optimized for the assay: • Microparticle-Antibody Solid Phase: Latex microparticles that are coated with antibody to bind the specific analyte being measured; • Antibody-Enzyme Conjugate: Alkaline Phosphatase enzyme conjugated to antibody; • Enzyme Substrate: Fluorescent 4-Methyl Umbelliferyl Phosphate (MUP) in solution that is available for a reaction with the enzyme on the antibody.

4. Assay principle • MEIA: technology uses a solution of suspended, submicron sized latex particles to measure analytes. • The particles are coated with capture molecule specific for the analyte being measured. • The effective surface area of micro particles decreases assay incubation time. This permits MEIA assays to be completed in less time than other immunoassay.

5. Reactants and sample for one assay are transferred to a reaction vessel. • Reagents and sample are incubated to allow them to come to reaction temperature. • The reagents and sample are combined and the reaction mixture is transferred to an inert glass fiber matrix. Irreversible binding of the microparticles causes the immune complex to be retained by glass fibers while the reaction mixture flows rapidly through the large pores in the matrix.

6. An alkaline phosphate- labeled conjugate is added to the glass fiber matrix prior to the addition of 4- methylumbelliferyl phosphate MUP. The conjugate catalyzes the hydrolysis of MUP to methylumbelliferone(MU). • Measurement of the fluorescent MU as it is generated on the matrix is proportional to the concentration of the analyte in the test sample.

8. MEIA reaction sequences • There are two types of reaction sequences or formats for MEIA assays: • One step: Sample, microparticles and conjugate are combined in the incubation well of the reaction vessel. • Two step: Sample and microparticles are combined in the incubation well of the reaction vessel and the conjugate reaction takes place on the matrix cell.

9. SummaryMEIA procedure • Analytes bind to micro particles: Samples and micro particles are combined and incubated at reaction temperature. During the incubation period analytes bind to the micro particles creating an immune complex. • Immune complex binds to glass fiber matrix. The processing probe aspirates the reaction vessel and dispenses it onto the matrix cell. The immune complex binds irreversibly to glass fiber matrix. A matrix cell wash removes unbound materials. The immune complex is retained by the glass fibers while the excess reaction mixture flows rapidly through the large ores in the matrix.

10. CMIA • Chemiluminescent Magnetic Immunoassay (CMIA): Chemiluminescent label conjugated to the antibody or antigen, and it produces light when combined with it’s substrate. This method is similar to MEIA, thought the chemiluminescent reaction offers high sensitivity and ease of measurement.

11. CMIA used for quantitative determination of human thyroid stimulating hormone in serum and plasma. • In ARCHITECT instrument, TSH assay is a two-step immunoassay to determine the presence of TSH in serum or plasma by CMIA technology with flexible assay protocols referred to as chemiflex.

12. CMIA principle • In the first step, sample, anti TSH antibody coated paramagnetic micro particles and TSH assay diluent are combined. TSH present in the sample binds to the anti TSH antibody coated micro particles. After washing, anti- alpha TSH acridinium labeled conjugate is added in the second step. • Pre-trigger and trigger solutions are then added to the reaction mixture: the resulting chemiluminescent reaction is measured as relative light units(RLUs). • A direct relationship exists between the amount of TSH in the sample and the RLUs detected by the instrument optical system.

15. Thyroid hormones

16. The thyroid glandThe thyroid gland is a small butterfly-shaped gland at the base of the neck. It weighs only about 20 grams. However, the hormones it secretes are essential to all growth and metabolism. The gland is a regulator of all body functions. Thyroid disorders are found in 0.8-5% of the population and are 4 to 7 times more common in women. • Types of thyroid diseaseThere are many types of thyroid disease. However, the main conditions present in most thyroid illnesses are hypothyroidism (thyroid under activity) and hyperthyroidism (thyroid over activity).

17. Thyroid hormones release

18. TSH • TSH is a member of the glycoprotein hormone family. It is produced by the pituitary thyrotrophs and released to the circulation in a pulsatile manner. • It stimulates thyroid functions using specific membrane TSH receptor (TSHR) that belongs to the superfamily of G protein-coupled receptors (GPCRs). • TSH synthesis in the anterior pituitary is stimulated by thyrotropin-releasing hormone (TRH) and inhibited by thyroid hormone in a classical endocrine negative-feedback loop

19. TSH Function • TSH stimulates the production and secretion of the metabolically active thyroid hormones, thyroxine (T4) and triiodothyronine (T3), by interacting with a specific receptor on the thyroid cell surface. • T3 and T4 are responsible for regulating diverse biochemical processes throughout the body that are essential for normal metabolism and neural activity.

20. Clinical uses of TSH • The principal clinical use for hTSH measurement is for the assessment of thyroid status. In patients with intact hypothalamic-pituitary function, hTSH is measured to: • Exclude hypothyroidism or hyperthyroidism • Monitor T4 replacement treatment in primary hypothyroidism or antithyroid treatment in hyperthyroidism; • Follow T4 suppression of the trophic influence of hTSH in “cold nodules” and non-toxic goiter; • Assess the response to thyrotropin-releasing hormone (TRH) stimulation testing.

21. TSH • Concentrations are usually lowered in thyroxicosis, due feedback inhibition of hypothalamus and pituitary • Conversely, plasma TSH is increased in hypothyroidism, except for the rare cases of hypothyroidism secondary to pituitary disease. • A TSH between 0.4 and 4.0 mIU/L gives 99% exclusion of hypo- or hyperthyroidism, while the TSH is considered more sensitive than FT4 to alterations of thyroid status in patients with primary thyroid disease. • Thyrotropin (TSH)-secreting adenomas These rare tumors make too much thyroid-stimulating hormone (TSH), which then causes the thyroid gland to make too much thyroid hormone. This can cause symptoms of hyperthyroidism (overactive thyroid).

22. The amino acid tyrosine is the starting point in the synthesis ofT3 & T4

23. T3 • Is more biologically active than T4 but both hormones have similar actions in the body. • T3 is not usually used in confirming the diagnosis of suspected hypothyroidism because other tests can demonstrate hypofunction of the thyroid gland. • T3 tests are often useful to diagnosis hyperthyroidism or to determine its severity. • Sometimes, however, a patient may have clinical signs of thyrotoxicosis with a normal T4. Measurement of the T3 is then needed, because T3 may be elevated in thyrotoxicosis while other thyroid tests are still the normal range. • T3 testing rarely is helpful in the hypothyroid patient, since it is the last test to become abnormal.

24. T3 • The main value of FT3 is in the evaluation of the 2 to 5% of patients who are clinically hyperthyroid, but have normal FT4. In this situation, an elevated FT3 would be suggestive of T3 toxicosis, in which the thyroid secretes increased amount of T3 or there is excessive conversion of T4 to T3.

25. T4 • The total T4 test measures the concentration of thyroxine in the serum, including both the protein bound and free hormone. It is dependent on the concentration of thyroid transport proteins, specifically thyroid binding globulin (TBG), albumin, and thyroid binding prealbumin (transthyretin). • The interpretation of results for serum T4 needs to take into account alterations in the thyroid-binding proteins. • The free thyroxine index (FTI) is determined by the following calculation: • FTI = Thyroxine (T4)/Thyroid Binding Capacity • The FTI is a normalized determination that remains relatively constant in healthy individuals and compensates for abnormal levels of binding proteins. • Hyperthyroidism causes increased FTI and hypothyroidism causes decreased value.

26. FT4: This test measures the metabolically active, unbound portion of T4. Measurement of FT4 eliminates the majority of protein binding errors associated with measurement of the outdated total T4. • In developing hypothyroidism, T4 (free T4) is the more sensitive indicator of developing disease than is T3 (Free T3), and is therefore preferred for confirming hypothyroidism that has already been suggested by an elevated TSH result.

30. Commonly used thyroid tests • Radioactive iodine tests, testing uptake by the thyroid gland • Hormones concentration T3,T4 • Thyroid Binding Globulin • TSH • Urinary excretion of thyroid hormones • Tests influenced by the actions of thyroid hormones.(glucose tolerance test, calcium, cholestrol) • Thyroid antibody tests for cases autoimmune.

31. Thyroid hormones • It must be emphasized that a single thyroid function test is NOT absolute in diagnostic accuracy and thus, a careful selection of tests, so that their combination can give comprehensive data, would enhance the diagnostic accuracy.

32. Sample collection • Type of sample: blood, urine or saliva • Most blood tests involving hormones measure the bound protein.  However, saliva-based testing measures the free level of hormone.  • Conditions for test: you should ask the patient if he/she under treatment and receive drug or made surgery thyroidectomy recently. • Preparation the patient: be kind when you deal with patient. • You should read the diagnosis status on request.

33. Reference ranges

34. Case study • A 63-year-old woman has Hashimoto’s disease. Her thyroid laboratory values today include the following: She feels consistently run down and has dry skin that does not respond to the use of hand creams. The hormones levels: • TSH 10.6 mIU/L (normal (0.5–4.5 mIU/L) • A free T4 concentration of 0.5 ng/dL (normal 0.8–1.9 ng/dL).

35. That’s all