Laboratory evaluation
of Thyroid function
1. Test
of thyroid hormone in blood
a. Free
T4 level
b. Free
thyroxine index (FT4I)
c. Thyroid
hormone–binding ratio (THBR)
d. Total
T3
e. Free
T3
f.
Reverse T3
g. Thyroglobulin
h. Total
T4
i.
Free T3 index
j.
Thyroid hormone–binding
proteins
k. Thyroid
antibodies
l.
T3 suppression
m. TRH
stimulation
2. Evaluation
of the hypothalamus–pituitary–thyroid axis
a. Thyroid
stimulating hormone
3. Assessment
of iodine metabolism
a. Radioactive
Iodine Uptake (RAIU)
4. Estimation
of gland size
a. Iodine–123
b. Technetium
99m pertechnetate
c. Americium–241
Fluorescent scanning
d. Magnetic
Resonance Imaging (MRI)
5. Thyroid
biopsy
a. Fine
needle aspiration biopsy (FNAB)
6. Observation
of the effects of thyroid hormones on peripheral tissues
a. Basal
Metabolic Rate (BMR)
b. Cardiac
Muscle Contractility
c. Photomotogram
7. Measurement
of Thyroid antibodies
a. Thyroglobulin
antibody (TgAb)
b. Thyroid
peroxidase antibody (TPO Ab)
c. TSH
receptor antibody
****** PROTEIN–BOUND IODINE (PBI) *******
This is the organic function of blood
iodine that precipitates with the serum proteins and consists of thyroxine and
triiodothyronine. The PBI level in serum correlates well with the status of
thyroid activity. The PBI determination is considered the most valuable method
for evaluation of thyroid function.
Steps required among the common
method of PBI determination:
1. Precipitation
of proteins
2. Washing
of the precipitated proteins to separate and remove the inorganic iodine
3. Destruction
of organic matter and conversion of thyroxine to inorganic iodine by:
a. Wet
ashing method using any of the following oxidizing agent
(1) Chromic
acid
(2) Permanganate
(3) Chloric
acid
b. Dry
ashing method
4. Quantitation
of the inorganic iodide formed by its catalytic effect on the ceric arsenious
acid reaction.
Chloric method – Zak et.al and
Bodansky et.al
The PBI is precipitated and washed
with trichloroacetic acid and digested with chloric acid and chromate to
convert iodine to iodate (sodium chromate or chromic acid serves as indicator
and also prevents volatilization of iodic acid). The addition of arsenious acid
reduces the excess chromate to give a colorless reaction and at the same time
the iodate reduced to iodide. The arsenious acid reduces ceric ammonium sulfate
from a yellow to a colorless solution with the iodide acting as a catalyst.
O’neal and Sim modified method
of Zak et.al. and Bodansky et.al
The above procedure was modified by
using perchloric acid as a protein precipitant instead of trichloroacetic acid.
Dry ashing method:
(Foss et.al. modification of
the alkaline incineration method of Barker)
The protein bound iodine is
precipitated and washed by the zinc hydroxide technique; sodium carbonate is
added to make the washed precipitate alkaline and the precipitate is completely
dried in an oven. The protein is then incinerated in a muffled furnace to
convert iodine to iodide. The ash is dissolved and the iodide present is
measured using the ceric arsenious acid reaction.
****** BUTANOL EXTRACTABLE IODINE (BEI) ******
This is more specific method for
measuring thyroxine because it is less subject to interference by drugs
containing organic iodine.
In this method, thyroxine and
triiodothyronine are extracted from acidified serum by butanol. The extract is
washed with alkali to remove inorganic iodine and iodotyrosine and then
evaporated.
The subsequent steps in the oxidation
and spectrophotometric determination may be carried out by method for PBI.
******* FREE THYROXINE (FT4) ******
The equilibrium dialysis was the first
method developed to measure free T4. In this method, a serum sample
was incubated in a dialysis membrane against a standard buffer until
equilibrium of added T4 tracer reached the dialysate. The ratio of
the counts in the dialysate to the dialysant give the dialyzable fraction or %
of free T4. Recent advances in technology have allowed development
of equilibrium dialysis assays that allow direct measurement of the free T4
by RIA after separating the free T4 from the protein–bound T4
with the use of dialysis cells.
Assay methodology:
1. Chemiluminiscence
2. RIA
Normal values: 0.8 – 2.0 ng/dl
****** TOTAL THYROXINE (T4) ******
Elevated T4 values are
characteristically seen in patients with hyperthyroidism, low levels are
associated with hypothyroidism.
T4 values that do not
reflect thyroid status are seen in patients with altered TBG concentration or
in patients receiving medications that affect TBG binding to T4.
Total T4 is elevated in
women who are pregnant, taking oral contraceptives, or are on estrogen therapy
because estrogen increases hepatic production of TBG.
Total T4 is decreased in
cirrhosis and nephrotic syndrome. Glucocorticoids decrease total T4
by inhibiting TSH secretion and by decreasing the concentration of TBG in
serum. Dopamine therapy reduces TSH levels and therefore reduces thyroid
hormone secretion.
Factors influencing decreased T4
values
1. Androgens
and other anabolic steroids
2. Salicylates
(competes in the binding sites)
3. Anticonvulsants
such as phenytoin and carbamazepine
4. Slow
induction of hepatic binding and metabolism of T4
5. Inhibition
of TSH secretion
Assay methodology:
1. RIA
2. Fluorometric
enzyme immunoassay
3. Fluorescence
polarization immunoassay
Normal values: 4.5 – 11.0 ug/dl
****** THYROID HORMONE–BINDING RATIO (THBR) ******
The first of the THBR test was
formerly known as the resin T3 uptake (RT3U), however, in
May 1987, the Nomenclature of the American Thyroid Association recommended RT3U
test as well as other assays designed to estimate the distribution of thyroid
hormone bound to thyroid binding proteins be termed THBR assays.
Increased thyroid–binding
proteins, resulting in decreased RT3U is seen in:
1. Pregnancy
2. Estrogen
therapy
3. Hyperproteinemia
4. Acute
intermittent porphyria
5. Hereditary
TBG increase
6. Acute
hepatitis
Decreased thyroid–stimulating
proteins, resulting in increased RT3U is seen in
1. Nephrotic
renal disease
2. Chronic
debilitating diseases
3. Liver
diseases
4. Treatment
with glucocorticoid
5. Phenytoin
and salicylates
Assay methodology: Fluorescent polarization immunoassay
Normal values: 0.72 – 1.24
****** FREE T4 INDEX (FT4I) ******
The FT4I is a
calculation used for the purpose of normalizing the total T4 and the
THBR values
in the presence of protein alterations. The calculations vary depending on the
design of the THBR assay. Multiplication of the total T4 by the RT3U
provides an index of effective free T4 concentration somewhat
independent of changes in protein binding. It is important to note that it is
not a true free T4 value but only an index of this measurement.
FT4I
= T4 x THBR (THBR = T3U / normal T3U)
Normal values: 1.13 – 3.85
****** TOTAL TRIIODOTHYRONINE ******
T3 is useful in diagnosing
mild hyperthyroidism because T3 rises earlier and more markedly than
does T4 in all common forms of hyperthyroidism. Clinicians typically
use T3 to monitor patients receiving sodium liothyronine. This assay
is of little clinical usefulness in evaluating hypothyroidism.
Assay methodology:
1. Solid–phase
RIA
2. Microparticle
enzyme immunoassay
3. Flourometric
enzyme immunoassay
Normal values: 80 – 180 ng/dl
****** FREE T3 INDEX (FT3I) ******
The Free T3 index is used
to normalize the T3 and THBR values because T3 levels are also affected by
variations in TBG concentrations.
FT3I
= T3 x THBR (THBR = T3U / normal T3U)
Normal values: 20 – 63
****** FREE T3
(FT3) ******
Free T3 is primarily used
as a confirmation test in hyperthyroidism, especially in T3
thyrotoxicosis. Free T3 correlating well with total T3,
may offer the advantage of not being affected by changes in TBG, but it depends
on the methodology of the assay.
Assay methodology: Solid phase RIA
Normal values: 1.4 – 4.4 pg/ml
****** REVERSE T3 (rT3) ******
Reverse T3 (rT3)
is a metabolically inactive isomer of T3 and it is present in serum
almost entirely as a result of generation from T4 in peripheral
tissues. Removal of iodide from an inner phenyl ring of thyroxine produces rT3,
whereas iodide removal from an outer ring produces the metabolically active T3.
Elevated rT3 levels are seen in patients with severe systemic
illness as a result of decreased conversion of T4 to T3
as well as inhibition of T3 degradation, rT3 is elevated
in hyperthyroidism and low in hypothyroidism.
Normal values: 25 – 75 ng/dl
****** THYROID HORMONE–BINDING PROTEINS ******
Alteration in thyroid hormone–binding
proteins affects total but free concentrations of T4 and T3.
Use of the estimation method, FT4I, together with a free T4
determination, demonstrates this effect. However, in patients with large
changes in TBG due to such conditions as acute liver disease or congenital
abnormalities, it may be necessary to directly measure binding proteins.
This is particularly useful in
diagnosing familial dysalbuminemic hyperthyroxinemia. This syndrome is
characterized by elevation in serum thyroxine and the free T4 index.
These elevations are due to an albumin that has an abnormal binding site with a
much greater affinity for T4 as compared with T3. It is
important to differentiate this syndrome, a euthyroid state, from
hyperthyroidism, with which it is confused.
****** THYROGLOBULIN (Tg) ******
Thyroglobulin is the storage form of
thyroid hormone precursors and is measured primarily in the monitoring of
patients with follicular or papillary thyroid cancer. Injury to the thyroid
gland, as occurs in these diseases, results in leakage of thyroglobulin from
the follicle into the patient’s serum. It is a useful tumor marker, if present,
and reflects recurrence or remission of the primary disease.
Thyroglobulin levels are increased
with stimulation by TSH, thyroid stimulating immunoglobulin (TSI), TRH and HCG.
Tg is the last parameter to normalize after thyrotoxicosis and is therefore
useful in resolving thyroid history in the presence of normal T4 and
T3 with clinical symptoms consistent with recent thyrotoxicosis. Tg
may also be useful in differentiating subacute thyroiditis, in which elevated
levels of Tg are present, from thyrotoxicosis factitia, which is characterized
by low Tg.
Normal values: 10 – 40 ng/ml
****** THYROID ANTIBODIES ******
Detecting the presence of thyroid
antibodies in sera is important both for confirming or ruling out
autoimmune disease as well as evaluating their possible interference in
assays of their respective antigens. T4 and T3
autoantibodies are particularly useful in explaining free T4, T4
or T3 results, which are conflicting with clinical evaluation.
1.
Thyroid
microsomal antibodies (TMAb) – are present in about 95% of
patients with Hashimoto’s thyroiditis and in 85% of those with Grave’s disease.
2.
Thyroglobulin
antibodies (TgAb)
– are found in 60% of patients with Hashimoto’s thyroiditis and in 30% of
patients with Grave’s disease.
3.
Antithyroid
antibodies
– titers of these antibodies are low in subacute thyroiditis although it
doesn’t give exact information about thyroid function.
4.
TSH receptor
antibodies (TRAb)
– are of limited clinical use, although they have clarified the pathophysiology
of Grave’s disease substantially.
Assay methodology:
1. Hemagglutination
assay
2. RIA
3. IRMA
4. ELISA
****** T3 SUPPRESSION TEST ******
The principle of the T3
suppression test is that when normal individuals are given thyroid hormone (T3)
in quantities adequate to meet peripheral requirements, suppression of
endogenous thyroid function should occur. This suppression of function,
indicated by iodine uptake, is a result of decreased TS secretion.
After a baseline RAIU is determined, T3
is administered every 8 hours for 7 days. Another RAIU test is then performed.
Absence of normal suppression indicates thyroid autonomy present in conditions
such as Grave’s disease. Because of the limitations of this test, the T3
suppression test has been supplanted by the TRH stimulation test.
There is significant risk to the patient because of the administration of
potent hormones, especially to patients with thyrotoxicosis or cardiovascular
disease, and because of exposing tissue to irradiation.
T3 suppression should reduce RAIU in
normal subjects by at least 50%.
****** TRH STIMULATION TEST ******
The TRH stimulation test take
advantage of the extreme sensitivity of TSH secretion to the feedback mechanism
of the hypothalamus–pituitary–thyroid axis. This test can detect abnormalities
before thyroid hormone concentrations are outside their reference range.
It is useful in distinguishing between
pituitary and hypothalamic hypothyroidism and in confirming mild hypothyroidism
in patients whose free T4 and T3 results are equivocal
and yet whose other clinical findings are suggestive of thyrotoxicosis.
Procedure of the test
1. Injection
of 200–500 ug of synthetic TRH after determining a baseline TSH level.
2. Extraction
of blood for TSH determination after 15, 30 and 60 minutes.
3. A
flat response indicates hyperthyroidism because elevated levels of T4
and T3 override additional stimulation of the pituitary by TRH.
4. A
flat response differentiates hypothyroidism secondary to hypopituitarism from
hypothalamic hypothyroidism in which a delayed peak is seen.
5. The
augmented response seen in primary hypothyroidism provides little additional
information from that derived from the combination of an elevated sTSH and
decreased free T4.
6. A
flat response is also seen in hyperthyroidism since elevated endogenous levels
of T4 and T3 already suppress TSH production to
undetectable levels.
7. A
normal response is a two to fivefold increase over basal TSH at 15–30 minutes.
****** RADIOACTIVE IODINE UPTAKE (RAIU) ******
In this method, the uptake by the
thyroid gland 24 hours after the administration of a tracer dose of radioactive
iodine is measured.
Many factors interfere with the
iodine trapping mechanism of the thyroid gland:
1. Using
organic iodides used for diagnostic X–ray procedures:
a. Bronchograms
b. Intravenous
pyelography (IVP)
c. Lipoidal
substances used in spinal conditions
2. Used
of dessicated thyroid
3. Cortisone
4. ACTH
5. Antithyroid
drugs
6. Inorganic
iodides
Radioactive iodine uptake
measures:
1. Ability
of the gland to trap iodine.
2. Ability
of the gland to concentrate iodine
3. Ability
of the gland to convert iodine to the hormonal form.
4. Ability
of the gland to finally release it.
Increased RAIU is seen in:
1. Hyperthyroidism
2. Endemic
goiter
Decreased RAIU is seen in
1. Thyrotoxicosis
associated with thyroiditis, thyrotoxicosis factitia, and metastases from
thyroid carcinoma.
Erroneous results are recorded
in:
1. Iodine
administration
2. Thiouracil
3. Renal
disease
4. Cardiac
disease
****** RADIONUCLIDE IMAGING ******
Radionuclide imaging is accomplished
using Iodine–123 or Technetium–99m pertechnetate and is useful in determining
the functional activity of the thyroid gland.
a. Iodine–123
is administered orally in a dose of 200–300 uCi and a scan of the thyroid is
obtained at 8 – 24 hours.
b. Technetium
–99 m pertechnetate is administered intravenously in a dose of 1 – 10 mCi and
the scan is obtained at 30–60 minutes.
Images can be obtained with either a
rectilinear scanner or a gamma camera. Radionuclide scans provide information
about both the size and shape of the thyroid gland and the geographic
distribution of functional activity of the gland. Functioning thyroid nodules
are called “hot” nodules while the non–functioning ones are called “cold”
nodules.
****** FLUORESCENT SCANNING ******
The iodine content and an image of the
thyroid gland can be obtained by fluorescent scanning without administration of
a radioisotope. An external source of Americium –241 is beamed at the thyroid
gland and the resulting emission of 28.5 keV x–ray from iodide ion is recorded,
producing an image of the thyroid gland similar to that obtained with Iodine–123.
The advantage of this procedure is that the patient receives no radioisotope
and the gland can be imaged even when it is loaded with iodine. The
disadvantage of this study is that it requires specialized equipment that may
not be generally available.
****** THYROID
ULTRASONOGRAPHY OR MAGNETIC RESONANCE IMAGING (MRI) ******
Thyroid ultrasonography is
particularly useful for measuring the size of the gland or individual nodule
and for evaluating the results of therapy. It is useful also for
differentiating solid from cystic lesions and to guide the operator to a deep
nodule during fine needle aspiration biopsy. Thyroid ultrasonography is limited
to thyroid tissue in the neck, thus, it cannot be used for susbsternal lesions.
MRI provides an excellent image of the
thyroid gland, including posterior or substernal extension of a goiter or
malignancy. Both transverse and coronal images of the gland can be obtained and
lymph nodes as small as 1 cm can be visualized. MRI is invaluable for the
demonstration of tracheal compression for a large goiter, tracheal invasion or
local extension of a thyroid malignancy or metastases to local or mediastinal
lymph nodes.
****** FINE NEEDLE ASPIRATION BIOPSY ******
This is the best method for
differentiation of benign from malignant thyroid disease. It is performed as an
outpatient procedure and requires no preparation
Procedure:
1. The
skin over the nodule is cleansed with alcohol, and, if desired, a small amount
of lidocaine can be injected intracutaneously for local anesthesia.
2. A
number 25 1½ inch needle is then inserted into the nodule and moved in and out
until a small amount of bloody material is seen in the hub of the needle; the
needle is then removed and with a syringe, the contents of the needle are
expressed onto a clean slide.
3. A
second clean slide is placed on top of the first slide and a thin smear is
obtained by drawing the slides apart quickly. It is dried and fixed then
stained.
****** BASAL METABOLIC RATE (BMR) ******
Basal metabolism refers to the heat
production during complete mental and physical rest in the post–absorptive
state and is a measure of the vital processes. It measures the ratio of oxygen
consumption in the resting fasting state. Basal metabolism is proportional to
the surface area of the body which in turn is determined by the height and weight
and it varies somewhat with age and sex. Somnolent metabolic rate (SMR) –
determination of metabolic rate in a sleepy state; patient is in a state of
sleepiness.
****** CARDIAC MUSCLE CONTRACTILITY ******
With echocardiography, it is
relatively easy to measure such indices as the preejection period (PEP), the
time from onset of the QRS complex to the opening of the aortic valve or left
ventricular ejection time (LVET). These are prolonged in hypothyroidism and
shortened in hyperthyroidism. Although these measurements are modified by
coexistent cardiac disease, they may be the best objective tests for measuring
the peripheral effects of the thyroid hormone action.
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