Thyroid function tests

this is a series of notes on clinical pathology, useful for undergraduate and post graduate pathology students. Notes have been prepared from standard textbooks and are in a format easy to reproduce in exams.

1. THYROID FUNCTION
TESTS

2. OVERVIEW

1. Biosynthesis of thyroid hormones
2. Regulation thyroid hormone production
3. What happens to thyroid hormones after release
4. Concept of FT3 and FT4
5. Hypothyroidism
a. Causes
b. Clinical features
c. Laboratory features
6. Hyperthyroidism
a. causes
b. Clinical features
c. Laboratory features
7. Thyroid function tests in detail
a. TSH
b. Total T4 and Free T4
c. Total T3 and Free T3
d. TRH Stimulation test
e. Anti thyroid antibodies
f. RAIU test
g. Thyroid scintigraphy
8. Summary and result interpretation
9. Neonatal hypothyroidism screening

3. *Biosynthesis of thyroid hormones:-










Steps:

1. Iodide (I
-
) enters the thryroid cell via sodium iodide symporter
2. It enters the colloid through pendrin receptor
3. It is oxidized into Iodine (I
0
) by peroxidase enzyme
4. Then it is organified into MIT and DIT (mono and di iodo thyronine)
5. Then after coupling it forms T3 (Tri iodo thyronine) and T4 (Thyroxine)
6. T3 and T4 conjugate with TBG (thyroid binding globulin)
7. conjugated TBG is stored in colloid till required
8. While releasing into blood stream, it is first endocytosed into thyroid cell and then de -
coupled to form, T3 and T4 with MIT and DIT
9. MIT and DIT can be reutilized for coupling
10. T3 and T4 are released into the blood stream

4. *Regulation of thyroid hormone production

5. *What happens to thyroid hormones after release




























Action of thyroid hormone on the body:

6. *Concept of FT3 and FT4

1. Out of the total T3 and T4 in circulation, most of it remains bound to thyroid binding
globulin *, thyroid binding prealbumina nd Thyroid binding albumin. (*note – this is not
thyroglobulin)
2. Only about 0.05% of each T3 and T4 remains free in circulation. This is FT3 and FT4.
3. These are better indicators for thyroid function than total T3 and Total T4.
(total=bound+free)
4. For example in pregnancy, level of thyroid binding globulin rises; hence though total T3
and total T4 remains same, level of FT3 and FT4 decreases.

7. *Hypothyroidism

Causes:

Primary Hypothyroidism
High TSH
In response to low T3 and T4
thyroid problem
Secondary hypothyroidism
Low TSH with normal TSH-RH
i.e. pituitary problem
Tertiary hypothyroidism
LOW TSH, Low TSH-RH
i.e. hypothalamic problem
1. Iodine deficiency
2. Goitrogens
3. Hashimoto’s
(antimicrosomal
antibodies)
4. Iatrogenic – surgery,
antithyroid drugs,
radiation
1. diseases of pituitary

1. diseases of the
hypothalamus

Exaggerated response to TSH-
RH stimulation
No response to TSH-RH
stimulation
Rise and Delayed response to
TSH-RH stimulation



Clinical Features:
1. Lethargy
2. Weight gain
3. Cold intolerance
4. Menstrual disturbances
5. Dry skin
6. myopathy
7. myxedema coma

8. Laboratory features:

Clinical Features of hypothyroidism



Measure TSH and FT4




High TSH High TSH Low TSH
Low FT4 Normal FT4



Primary hypothyroidism Subclinical hypothyroidism Sec or Tertiary
Hypothroidism


Check for antimicrosomal a/w
Antibody 1. Bad obstetric outcome
2. hypercholesterolemia risk TRH Stimulatn
3. Poor cognitive development test
4. Risk of progression to overt Check TSH
Hypothroidism FT4



Increased Normal Little or no response Delayed but
Present TSH
response


Hashimoto’s iodine def Secondary Tertiary
Congenital T4 synth def hypothyroidism hypothyroidism

9. *Hyperthyroidism

Causes:
Primary hyperthyroidism
Low TSH, High T4
Secondary Hyperthyroidism
High TSH, High T4
Pituitary/Paraneoplastic
syndrome
Factitious Hyperthyroidism

1. Grave’s disease
2. Toxicity in
multinodular goiter
3. toxicity in adenoma
4. subacute thyroiditis
1. TSH secreting
pituitary adenoma
2. Trophoblastic tumors
that secrete TSH
(choriocarcinoma, H.
mole)



Clinical Features:
1. anxiety
2. insomnia
3. fine tremors
4. weight loss
5. heat intolerance
6. amenorrhoea and infertility
7. palpitations and tachycardia
8. cardiac arrythmias
9. muscle weakness
10. proximal myopathy
11. osteoporosis

Triad of Grave’s Ophthalmopathy
1. Hyperthyroidism
2. Ophthalmopathy
a. exophthalmos
b. lid retraction
c. lid lag
d. corneal ulceration
e. impaired eye muscle function
3. Pretibial myxedema (dermopathy)

10. Laboratory Features:
Clinical features of hyperthyroidism


Measure TSH and FT4




High FT4, Low TSH Normal FT4, Low TSH High FT4, High TSH
Primary Hyperthyroidism Secondary Hyperthyr



Isotope thyroid scan Measure FT3 TRH test





Diffuse Nodular Irregular Normal High
Uptake Uptake uptake



Grave’s Toxic Toxic Subclinical T3 Thyrotoxicosis
Adenoma multinodular Hyperthyroid
Goiter


a/w
1. atrail fibrillation
2. osteoporosis
3. progression to overt
hyperthyroidism





Increased response No response i.e
i.e. TRH – inc TSH – inc FT4 self controlled


Resistance to thyroid hormone Pituitary
Adenoma/
Paraneoplastic

11. *Thyroid function tests in detail

Rider:
Thyroid levels can be affected by various non thyroidal diseases mentioned below. Hence
thyroid function tests should not be carried out during active diseases.
1. infections
2. liver diseases
3. malignancies
4. trauma
5. surgery
6. renal failure
7. cardiac failure

12. (a) Thyroid stimulating hormone (TSH)

Method: (Radioimmunoassay)(RIA)
The technique was introduced in 1960 by Berson and Yalow as an assay for the
concentration of insulin in plasma. It represented the first time that hormone levels in the blood
could be detected by an in vitro assay.

(known concentration of
I
125
/I
131
labelled TSH)



(anti TSH antibody)


(Known concentration of
unlabelled TSH)



















 A mixture is prepared of
o radioactive antigen
 Because of the ease with which iodine atoms can be introduced into
tyrosine residues in a protein (TSH here), the radioactive isotopes
125
I or
131
I are often used.
o antibodies ("First" antibody) against that antigen.
 Known amounts of unlabeled ("cold") antigen (known unlabelled TSH) are added to
samples of the mixture. These compete for the binding sites of the antibodies.

13.  At increasing concentrations of unlabeled antigen, an increasing amount of radioactive
antigen is displaced from the antibody molecules.
 The antibody-bound antigen (assay sample TSH) is separated from the free antigen
(radioactive TSH) in the supernatant fluid, and
 the radioactivity of each is measured.
 From these data, a standard binding curve, like this one shown in red, can be drawn.






 The samples to be assayed (the unknowns) are run in parallel.
 After determining the ratio of bound to free antigen ("cpm Bound/cpm Free") in each
unknown, the antigen concentrations can be read directly from the standard curve (as
shown above).

This method is used for assaying all thyroid function tests.

Normal levels:
Adults Normal 0.5 to 5 mU/L
Borderline 5 to 10 mU/L
High >10 mU/L
Low <0.1 mU/L



Abnormal values:
Low TSH High TSH
1. primary hyperthyroidism
2. T3 thyrotoxicosis
3. Secondary and tertiary hypothyroidism
1. Primary hypothyroidism
2. Secondary hyperthyroidism (pituitary
adenoma/paraneoplastic syndromes)

14. (b) Total T4 and Free T4
TSH combined with FT4 gives best assessment of thyroid function

Method: Competititve immunoassay
Principle same as for TSH

Free T4:
1. Free T4 constitutes around 0.05% of total T4
2. Levels co relate better with metabolic state than total, because free levels are not affected
by TBG levels –
TBG levels are affected in
a. pregnancy
b. OCP use
c. Nephrotic syndrome

Normals:
Total T4 5-12 μg/dL
Free T4 0.7-1.9 ng/dL

Abnormals:
Causes of increased T4 (Total) Causes of decreased T4 (total)
1. Primary hyperthyroidism
2. Increased thyroid binding globulin

Decreased FT4

Increased TSH

Increased T4


Normal FT4, Elevated total T4

3. Factitious hyperthyroidism
4. Secondary hyperthyroidism (pituitary
adenoma/paraneoplastic syndromes)
1. Primary hypothyroidism
2. Secondary and tertiary hypothyroidism
3. Anti thyroid Drugs, estrogen, danazol
4. Decreased thyroid binding globulin


Increased FT4


Decreased TSH

Decreased T4

Normal FT4, decreased total T4

15. (c) Total T3 and Free T3

For routine assessment, TSH and T4 are enough, T3 levels are very low compared to T4 hence may
not be used.

Method:
Same as for TSH

Free T3:
1. Free T3 constitutes around 0.5% of total T3
2. Levels co relate better with metabolic state than total, because free levels are not affected
by TBG levels –
TBG levels are affected in
a. pregnancy
b. OCP use
c. Nephrotic syndrome

Normals:
T3 80-180 ng/dL
Free T3 0.5% of T3 ie 2.3 to 4.2 pg/ml

Uses:
1. For early diagnosis of hyperthyroidism – in early stages T4 is normal but T3 is elevated
2. For measurement of T3 thyrotoxicosis

16. (d) TRH Stimulation test

Method:
Baseline sample collected for estimation of basal serum TSH levels



Inject TRH (200 to 500 mU/L)



Measure TSH at 20 & 60 mins

Uses:
1. Confirmation of secondary (pituitary/hypothalamic) hypothyroidism
2. suspected hyperthyroidism

Interpretation:

Baseline TSH 20 min TSH 60 min TSH interpretation
Hypothyroidism Normal Rise of
>2mU/L
Small decline normal
Elevated Further rise Small decline Primary hypothyroidism
Low No rise - Secondary
hypothyroidism
(pituitary)
Low rise Further rise
(delayed)
Hypothalamic
hypothyroidism
Hyperthyroidism elevated rise - Thyroid hormone
resistance
elevated No rise - Pituitary
adenoma/paraneoplastic

17. (v) Antithyroid antibodies

Antibodies used:

Anti microsomal antibody
Anti thyroid peroxidase antibody (anti TPO)
Hashimoto
Anti TSH receptor antibody Grave’s

Uses:
For diagnosis and monitoring of autoimmune thyroid disorders

18. (vi) Radioactive Iodine Uptake (RAIU)

Principle:
Radioactive iodine uptake co relates with functional activity of thyroid gland

Method:
Patient is administered tracer dose of I
123
or I
131
orally


The I
123
or I
131
is taken up through Iodine symporters in follicular cells


Radioactivity over thyroid gland is measured at 2 to 6 hours and again at 24 hours


Normals:

RAIU @ 24 hrs 10-30%


Causes:
RAIU separates causes of hyperthyroidism into-

Increased uptake Decreased uptake
1. Grave’s disease
2. Toxic multinodular goiter
3. Toxic adenoma
4. TSH secreting tumor
1. Cryptogenic hyperthyroidism
(exogenous hormone administration)
2. Subacute thyroiditis

19. (vii) Thyroid Scintigraphy

Method:
99m
Tc pertechnate is administered


A gamma counter is used to assess its distribution within the thyroid gland

Interpretation and uses:

1. EVALUATION OF CAUSES OF THYROTOXICOSIS WITH INCREASED RAIU



Uniform/diffuse uptake multiple discrete areas uptake single area of uptake


Grave’s Toxic multinodular goiter Adenoma


2. EVALUATION OF A SOLITARY THYROID NODULE



Hot nodule Cold nodule



Hyperfunctioning Non functioning
(20% malignant)

20. *summary and result interpretation

Sr. No. TSH FT4 RESULT
1 Normal Normal Euthyroid
2. Low Low Secondary hypothyroidism
3 High Normal Subclinical hypothyroidism
4 High Low Primary hypothyroidism
5 Low Normal with
normal ft3
Subclinical hyperthyroidism
6 Low Normal with
raised ft3
T3 thyrotoxicosis
7 Low High Primary hyperthyroidism

21. *Neonatal Hypothyroidism Screening

Rationale:
1. Thyroid hormone deficiency can cause cretinism that can be prevented by early detection
and treatment

Method:
Take dry blood spots on filter paper (3
rd
to 5
th
day of life) OR Cord serum


Test for TSH


If elevated, diagnostic of hypothyroidism


To confirm do I
123
RAIU



Increased uptake No uptake


Dyshormonogenesis thyroid agenesis

Normals:
Neonatal TSH <20 mU/L