Transcript How to measure Total and Free T3?

CASE E
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Hyperthyroidism
Poonam Shrestha
 Veronica Nou
 Mary Tormey
Ainsley Macdonald
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Mr TF, aged 50 years, has a history of asthma
and CAL. He has recently been hospitalised
for a severe chest infection. He has also lost
5kg in weight in the last 3 months, has
tachycardia of 120/min and fine
tremor(hands). He is currently receiving
prednisolone 25mg daily & ceftriaxone 1g
daily
Thyrotoxicosis is suspected
Effect of prednisolone on thyroid
function
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Glucocorticoid group
Decrease TSH secretion
Large dose decrease serum T3 concentration
Long term glucocorticoid therapydecrease in
serum thyroid binding globulin(TBG)slight
decrease of T4 concentration
Thyrotoxicosis vs asthma and
CAL
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Thyrotoxicosis can worsen asthma and CAL
Increase frequency and severity of asthma attack
Increase requirement for medication
Asthma attacks improve in an euthyroid state
Possible cause
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Reactive oxygen species due to hyperthyroidism
may be the contributing factor in exacerbating
asthma
Reactive oxygen species
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Induce an autonomic imbalance between
muscarinic receptor-mediated contraction and
the beta-adrenergic-mediated relaxation of the
pulmonary smooth muscle
Induce bronchoconstriction
Elevate mucus secretion
Microvascular leakage
These conditions worsens the respiratory
conditions.
WHAT CLINICAL
CHEMISTRY TESTS
SHOULD BE
PERFORMED TO
RULE OUT
HYPERTHYROIDISM
& WHY?
HYPERTHYROIDISM?
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An elevation of free T4
An elevation of free T3
A very low TSH
fine tremor (hands)
tachycardia
weight loss
These are diagnostic signs & classic symptoms
of hyperthyroidism
Hyperthyroidism?
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Measurement of serum Thyrotropin Thyroid Stimulating Hormone is considered the
initial screening test in distinguishing
hyperthyroid and primary hypothyroid states
from euthyroid states
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A suppressed TSH is the cornerstone of
diagnosis of hyperthyroidism however, its
secretion is influenced by many factors other
than the negative feed back inhibition by t3 or
t4 bmj 2000;320:1332-1334
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For example TSH can be reduced by fasting,
glucocorticoids -exogenous & endogenous,
stress, nonthyroidal illness, & false negative
results
In the presence of the above the specificity of
se TSH as a screening test is greatly reduced.
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Hyperthyroidism?
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In at least 90% of patients with hyperthyroidism
T4 & T3 are elevated
In 5% of hyperthyroid patients T3 is exclusively
elevated.
In developing hyperthyroidism fT4 & fT3 are
elevated before tT4 & tT3
Increased fT4 & decreased TSH is seen in
conditions other than hyperthyroidism therefore
need to measure fT3 as well
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changes in TSH, T3 & T4 during systemic
illness are poorly understood bmj 2000;320:1332-1334
In very ill patients both T3 &T4 (free & total)
are suppressed
Free hormone assays are preferable to total
levels as there is decreased protein binding of
thyroid hormone - relevant to this patient :
acute severe illness,
protein malnutrition
with the ingestion of steroids
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Free T3 index levels can be measured to
compensate for altered binding levels
These levels are derived from the total hormone
levels & measurement of the distribution of
radiolabelled t3 between unoccupied protein
binding sites in the sample and an absorbent
resin
Expect Free Thyroxine Index decreased in NTI
& steroid administration.
Expect Reverse T3 normal / increased in NTI
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Very systemically ill Patients with low T4 levels
have a poor prognosis/high morbidity
TFTs cannot be interpreted in patients with
systemic illness bmj 2000;320:1332-1334
Doing more indiscriminate biochemical tests will
lead to confusion not clarity bmj 2000;320:1332-1334
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Significant false positive & negative TFT results
seen in the presence of NTI as well as during
the administration of glucorticoids
Repeat TSH, fT3 & fT4 after recovery from
systemic illness.
TSH can remain suppressed for months after
starting treatment for hyperthyroidism even
when T4 & T3 are normal
Prolonged thyrotoxicosis can
cause a number of non-specific
biochemical abnormalities
oxford textbook
of medicine 2003
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Abnormal LFTs
Hypercalcuria
Elevated levels of serum ferritin
less common se calcium & phosphate raised
glucose intolerance
microcytic aneamia or thrombocytopenia
How to measure Total
and Free T3?
Overview
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Over 99% of Triiodothyronine (T3) circulates in blood
bound to carrier proteins: thyroxine- binding globulins
(TGB)
Only the free (unbound) portion of T3 is responsible
for its biological action.
The concentration of the carrier proteins may be
altered but the total concentration of T3 will change so
that the concentration of free T3 wil stay relatively
constant.
Overview (2)
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Thus, the concentration of free T3 correlates
more reliabily than total T3 levels.
Serum T3 measurement has little specificity or sensitivity
for diagnosing hypothyroidism, since enhanced T4 to T3
conversion maintains normal T3 concentrations until
hypothyroidism becomes severe.
Approaches to the measurement
of total and free T3.
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Physical techniques: Equilibrium Dialysis,
Ultrafiltration and Gel Filtration
OR
Assay or Index Approaches: Estimate the free
hormone concentration in the presence of
protein-bound hormone.
Equilibrium Dialysis
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Separates bound from free hormone.
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Time-consuming, expensive, technically demanding and
unavailable in most commercial laboratories.
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Measuring serum free T3 using overnight equilibrium
dialysis of serum containing 125I-T3
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The percentage of free T3 is calculated by determining the
total counts in the dialysate divided by the total 125I-T3
added to the serum multiplied by the total T3 concentration.
Index / Immunoassays
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The free hormone methods used by most clinical
laboratories (indexes and immunoassays) do not employ
physical separation of bound from free hormone and
do not measure free hormone concentrations directly!
These tests are typically binding protein dependent to
some extent and should more appropriately be called
"Free Hormone Estimate" tests, abbreviated FT4E
and FT3E.
Index Methods: FT4I and FT3I
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Require two separate measurements:
1.
One test is a total hormone measurement (TT4
or TT3) the other
2. Is an
assessment of the thyroid hormone binding
protein concentration using either an
immunoassay for TBG or a T4 or T3 "uptake"
test called a Thyroid Hormone Binding Ratio
(THBR).
Indexes Using a Thyroid
Hormone Binding Ratio (THBR)
or "Uptake" Test
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"Classical" uptake tests add a trace amount of
radiolabeled T3 or T4 to the specimen and
allow the labeled hormone to distribute
across the thyroid hormone binding proteins
in exactly the same way as endogenous
hormone.
The distribution of the tracer is dependent
upon the saturation of the binding proteins.
Indexes ctd.
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The free T3 index is then calculated using the
total T3 and the TBG level.
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The index is directly proportional to the free T3
level.
Immunoassays
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The two most commonly used methods are a
two-step and a one-step immunoassay method.
These assays are not completely free of the
influence of binding proteins or substances in
serum that may result in false increases or
decreases in the free T4 / 3 levels
Two Step Immunoassay
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Two-step assays use a physical separation of free
from protein-bound hormone before free
hormone is measured by a sensitive
immunoassay, or alternatively, an antibody is
used to immunoextract a proportion of ligand
out of the specimen before quantitation.
One Step Immunoassay
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One-step ligand assays attempt to quantify free
hormone in the presence of binding proteins.
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Important to maintain free to protein- bound
equilibrium.
Solid Phase competitive ELISA.
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The samples, assay buffer and T3 enzyme conjugate are added to
the wells coated with anti- T3 monoclonal antibody.
FT3 in the patients serum competes with a T3 enzyme conjugate
for the binding sites.
Unbound enzyme conjugate is washed off by washing with
buffer.
Upon the addition of the substrate, the intensity of the colour is
inversely proportional to the concentration to the FT3
The Application Of Total
And Free T3 Levels In
Evaluation Of Patient
Status
Total And Free T3
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Only 0.3% of the total T3 is freely available.
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Most of the T3 in circulation is bound to and
transported by TBG (thyroxine binding globulin).
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Therefore abnormalities in levels of TBG can cause
erroneous results in total and free T3.
Excess or lower TBG levels can be passed down as a
hereditary trait. It causes no problems aside from causing
false test increases or decreases in thyroid hormones.
People with this trait are often diagnosed as having a
thyroid dysfunction in the absence of any real problem or
need for treatment.
Serum free T3 levels are generally considered
more reliable as they are less affected by
carrier proteins (as the free T3 concentration is
preserved by equilibrium.)
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Smaller amounts of T3 are bound to albumin and
prealbumin.
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Normal Levels
 Total T3: 80-180 ng/dl (nanograms/decilitre)
 Free T3: 230-619 pg/dl (picograms/decilitre)
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Measurement of Serum Thyroid Hormones
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an indicator of thyroid function.
The most frequently used test is T4 by radioimmunoassay.
This is referred to as a T7
meaning that a resin T3 uptake or RT3u has been done to
correct for certain medications such as birth control pills,
other hormones, seizure medication, cardiac drugs, or even
aspirin that may alter the routine T4 test.
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The T4 reflects the amount of thyroxine in the blood.
If the patient does not take any type of thyroid
medication, this test is usually a good measure of
thyroid function.
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However thyroxine (T4) only represents 80% of
thyroid hormone produced.
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The other 20% is tri-iodothyronine or T3. T3 levels are
elevated in most patients with thyroid dysfunction and are
therefore unreliable as an exclusive screen.
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T3 may be measured in cases where there is some doubt
about whether the patient has hyperthyroidism or
hypothyroidism after measuring T4 and RT3u.
 where symptoms of hyperthyroidism are apparent but the
thyroid gland is still producing normal levels of T4.
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They are also used to monitor response to therapy.
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Therefore T3 is measured as part of a thyroid function
evaluation to provide a more accurate picture.
T3 Elevation
Greater-than-normal levels may indicate:
 Hyperthyroidism
(e.g., Graves’ disease)
 Serum
T3 elevation parallels serum T4 in 90-95%
of cases
 Isolated serum T3 elevation in ~ 5% of cases
 T3
thyrotoxicosis (rare)
 Isolated
 Thyroid
T3 elevation
Cancer (rare)
 Thyroiditis
T3 Reduction
Lower-than-normal levels may indicate:
- Chronic illness
- Hypothyroidism (e.g., Hashimoto’s disease)
- Starvation
Other Factors That May Affect T3 Measurement
Drugs that can increase T3 measurements include:
- clofibrate, estrogens, methadone, and oral
contraceptives.
Drugs that can decrease T3 measurements include:
- anabolic steroids, androgens, antithyroid drugs (for
example, propylthiouracil), lithium, phenytoin, and
propranolol.