Tuesday, December 11, 2012

Thyroid profile testing or Thyroid function test (TFT)

Thyroid blood tests:

The blood tests that may be done as part of a thyroid diagnosis include the following:
  1. Thyroid Stimulating Hormone (TSH) Test
  2. Total T4/ Total Thyroxine
  3. Free T4 / Free Thyroxine
  4. Total T3 / Total Triiodothyronine
  5. Free T3 / Free Triiodothyronine
  6. Thyroglobulin/Thyroid Binding Globulin/TBG
  7. T3 Resin Uptake (T3RU)
  8. Reverse T3
  9. Thyroid Peroxidase Antibodies (TPOAb) / Antithyroid Peroxidase Antibodies
  10. Antithyroid Microsomal Antibodies / Antimicrosomal Antibodies
  11. Thyroglobulin Antibodies / Antithyroglobulin Antibodies
  12. Thyroid Receptor Antibodies (TRAb)
  13. Thyroid-Stimulating Immunoglobulins (TSI)

Test for thyroid dysfunction

a. Measurement of TSH
TSH is measured by sandwich ELISA method by using anti TSH antibodies (Ab against β subunit of TSH) as primary and HRP enzyme conjugated secondary antibody, here TSH is sandwiched between these two antibodies and color is produced when chromogenic substrate like TMB (Tetramethyl Benzidine) is added. Substrate A containing TMB and substrate B containing H2O2 is mixed and added. HRP will release [O] by hydrolysis of H2O2 which will oxidize TMB producing color.

Reference range for TSH is 0.39 – 6.16 µIU/L.

b. Measurement of free T4 and T3
The plasma concentration of free thyroid hormones are extremely small and as most of them are protein bound and especially in NTI or under medication, there is alteration in protein level or hormone itself making their measurement less informative. So, free hormone estimate is used for quantification. The binding of T4 to TBP is overcome by using barbital buffer which will selectively inhibit the binding. Similarly Anilino naphthalene sulfonic acid (ANS) is also used for this purpose. These agents displace T4 from TBG. This is in case of measurement of total T3 and T4.

Equilibrium dialysis and ultrafiltration methods are reference methods for measuring free thyroid hormones. But immunometric methods are used for routine purpose.

Fig. ELISA plate (Color development)
Initially total T4 and T3 (free and protein bound) were measured, but there are various condition where results may be misleading. In pregnancy and oestrogen medication there is increased TBG concentration and thus increases in total T3 and T4. Use of contraceptives also increases TBG. Whereas Androgens, glucocorticoid, malnutrition, malabsorption, illness, liver disease, Cushing’s disease, genetic variant of TBG, transthyretin, albumin with low affinity for T3, T4, etc. there is low TBG which will give false low results. NTI will produce modified TBG with diminished affinity for thyroid hormone.

Nowadays free T3 and T4 are measured by competitive ELISA method. There exists a competitive reaction between native Ag and enzyme-Ag conjugate for a limited number of immobilized binding sites on Ab coated on microwell. After the Ag-Ab reaction has taken place, the fraction of Ag in the conjugate or native Ag from sample which does not bind to Ab coated in well is washed away (during this washing the unbound free and protein bound will be decanted off). The enzymatic activity in the Ab bound fraction, which is inversely proportional to the native Ag concentration, is measured by addition of the substrate. By utilizing calibrators of known concentration of conjugated Ag a dose response curve is generated from which the Ag concentration in a sample can be found out.

Here HRP enzyme is conjugated and TMB and H2O2 are used as substrates. Detergent like tween 20 is used in wash buffer.

Reference range: 
fT3 = 1.4-4.2 pg/mL
fT4 = 0.8-2.7 ng/dL

c. Interpreting results of thyroid function test
Since the level of T3 and T4 vary during different physiological and pathological conditions (thyroidal and non thyroidal) they do no provide the exact picture. TSH measurement is the initial test of thyroid function since it is regulated by thyroid hormones and also regulates their production. A normal TSH effectively excludes primary thyroid dysfunction. If abnormal results are obtained thyroid hormones are measured to confirm thyroid dysfunction. So measurement of both TSH and T4 together can provide a more satisfactory method of assessing thyroid status. Sensitive TSH assay should be used for initial screening of thyroid dysfunction.

TSH low
TSH normal
TSH high
Free T4 low
·  Severe NTI
· Secondary hypothyroidism
·   NTI
· Secondary hypothyroidism
·   Primary Hypothyroidism

Free T4 normal
· Subclinical    hyperthyroidism
·   NTI – drugs

·      Euthyroid
·   Subclinical hypothyroidism
·   Recovery from NTI.
Free T4 high
·   Hyperthyroidism:
·      Artifact
·      NTI- drugs
·      Autoantibodies to T4 and (T3)
·      Resistant to thyroid hormone
·      Testing after 6 hr of thyroxine dose
·   TSH secreting pituitary adenomas
·   Resistant to thyroid hormones:

In hyperthyroidism T3 is elevated greater than T4 due to 2 sources one is increased thyroidal synthesis and another is increased peripheral conversion of T4.

Fig. TSH Assay (Source: Tietz Textbook of clinical chemistry, 4th Edition)
Primary hyperthyroidism:
Here TSH is decreased due to feedback inhibition by increased T3 and T4. Sometimes T4 and fT4 are both normal, whereas both total T3 and fT3 are increased: this condition called T3 hyperthyroidism or T3 thyrotoxicosis.

Primary hypothyroidism:
TSH is invariably increased, as feedback inhibition of pituitary is diminished and fT4 and total T4 are low. fT3 and total T3 measurement are of no value here, since normal concentrations are often observed because of increased peripheral deiodination of T4 to T3.  

Subclinical thyroid disease:
Here TSH is low but normal total and free thyroid hormones and this is called subclinical hyperthyroidism. Sometimes TSH is high with normal thyroid hormones called subclinical hypothyroidism.

d. Common situations in which TSH results may be misleading:
  • Interference from endogenous heterophilic antibodies like in autoimmune disorders
  • Pregnancy: TSH is reliable indicator of thyroid status in 2nd and 3rd trimester of pregnancy but in first trimester a low TSH may be found.
  • Secondary thyroid disorders: TSH is normal in many patients with central (pituitary) hypothyroidism and T4 and T3 are also low. In pituitary adenomas there is persistent TSH secretion without regulation and this will increase T4 and T3 leading to 
  • secondary hyperthyroidism. Abnormal T3 receptor function is another rare cause of hyperthyroidism. 

e. Thyroglobulin measurement:

Its measurement is useful in the follow up of patients with thyroid cancer, in whom elevation of previously suppressed concentration may indicate tumor recurrence. This can be used as tumor marker for thyroid cancer. 

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