CPSL National Guidelines / Endocrinology 1 2 CPSL National Guidelines / Endocrinology

INDEX
1. Dynamic Function Tests in
Endocrinology CONTENT PAGE NUMBER

1.1 Introduction 03
Compilation and editing of this volume:
1.2 Investigation of Growth Hormone deficiency 03
Dr. Eresha Jasinge (Consultant Chemical Pathologist)
1.3 Investigation of Growth Hormone excess 16

List of contributors 1.4 Investigation of Thyroid disorders 19

Consultant Chemical Pathologists 1.5 Investigation of disorders of Cortisol

Dr. Saroja C.Siriwardene metabolism 52
Dr. Meliyanthi Gunatillaka
Dr. Deepani Siriwardhana 1.6 Investigation of disorders of Aldosterone
Dr. Chandrika Meegama metabolism 68

Consultant Physicians 1.7 Water Deprivation Test 74

Dr. Noel Somasundaram
Dr. Bandula Wijesiriwardane
Dr. Rushika Lanerolle

Consultant Paediatrician
Dr. Shyama De Silva

Coordinators
Consultant Histopathologists
Dr. Siromi Perera
Dr. Kamani Samarasinghe
Dr. Modini Jayawickrama
 CPSL National Guidelines / Endocrinology 3 4 CPSL National Guidelines / Endocrinology

1.1 Introduction Available tests

Children (<18 yrs) (short stature)
Endocrinology is the study of intra- and
extracelluar communication by messenger molecules as • Glucagon stimulation test
hormones (from the Greek hormon, meaning to excite, to
arouse to activity). Second line test Clonidine stimulation test or
Hormones vary widely with respect to their Exercise stimulation test
composition, transport, metabolism and mechanism of
action. Investigations for anterior pituitary reserve
Tests of endocrine functions are of two types.
For adults only
• The basal secretion of the hormone is measured
using a single blood or urine sample. Check both GH and ACTH reserve

• Dynamic tests, on the other hand, require two or • Insulin hypoglycaemic test or Glucagon
more samples and are used to test the integrity of stimulation test
the control mechanism of the hypothalamic- • Second line test Clonidine stimulation test
pituitary-end organ axis.
or children and adults with contraindications for
ITT
1.2 Tests for Growth Hormone • Glucagon stimulation test
Reserve (Deficiency)
These tests should be performed under the Choice of appropriate specimens for analysis will
supervision of a paediatician or a physician. be decided by the time and degree of achievement of
Blood samples should be ( serum separated) sent hypoglycaemia.
to the relevant laboratory.

Basal growth hormone (GH) is usually < 1mU/l

in normal individuals except during pulses of secretion.

Therefore measurement of random growth

hormone levels is unhelpful.
 CPSL National Guidelines / Endocrinology 5 6 CPSL National Guidelines / Endocrinology

Insulin Hypoglycemic Test (IHT)/ Insulin Draw the basal blood sample (0 min) for GH and glucose
Insulin (soluble) i.v bolus at 09.00
Tolerance Test (ITT) Usual dose: 0.15U/Kg
Cushing’s and acromegaly: usually 0.3 U/Kg
Indication
Assessment of ACTH/cortisol and GH reserve
Draw further blood samples at 30. 60. 90 and 120
min for venous plasma glucose (analyzed in the lab)and
Rationale
serum GH.
The stress of insulin induced hypoglycaemia triggers the
(If insulin dose is repeated 30, 60, 90, 120 and
release of GH and ACTH from the pituitary gland in
150 min)
normal subjects.
GH response is measured directly; cortisol is measured
If cortisol deficiency is suspected draw samples
as the indicator of ACTH secretion.
for cortisol as well. However the analysis of cortisol in
the lab will depend on achievement of hypoglycaemia
Contraindications
and the response of growth hormone to it.
Epilepsy or unexplained blackouts
During the test patient should be observed for
Ischemic heart disease or cardiovascular insufficiency
signs of hypoglycemia (tremors, sweating, tachycardia)
Severe long standing hypoadrenalism (liver glycogen
to ensure that adequate stress has occurred.
stores are depleted → severe hypoglycaemia during ITT) If not clinically hypoglycaemic at 45 min then
Glycogen storage disease consider repeating insulin dose in full. The patient must
be awake throughout and be able to answer simple
Precautions questions.
ECG must be normal With severe and prolonged hypoglycaemia
Serum cortisol(09.00) must be > 100 nmol/L (>20 min), or impending or actual loss of consciousness,
Normal serum T4 (replace first if low) or fits, it may rarely be necessary to terminate the test.
If above tests are abnormal, or in doubt, perform
glucagon test Give 25 ml of 25% dextrose i.v followed by an
5% & 25% dextrose and i.v hydrocortisone 100 mg infusion of 5% dextrose but continue sampling if
ampoules should be available possible (the hypoglycaemic stimulus has been adequate).
Consider hydrocortisone 100 mg i.v at the
Procedure end of test.
asting from midnight (review medication) Give lunch and sweet drink at the end of the test.
Weigh patient Observe the patient for 2h after the test.
Insert the IV cannula at 08.30
 CPSL National Guidelines / Endocrinology 7 8 CPSL National Guidelines / Endocrinology

Time(min) GH Plasma Recommendations

glucose A. Should be carried out under the supervision and
0 √ √ recommendation of a consultant. Samples should
30 √ √ be sent to the relevant laboratory for analysis.
60 √ √ Please discuss with the relevant laboratory before
90 √ √ sending the specimens.
120 √ √
References
150 √ √
(only if insulin is
1. Tietz Text book of clinical Chemistry 3rd edition
repeated)
2. Clinical chemistry in Diagnosis and Treatment,
Normal response Philp D. Mayne, 6th edition
Venous plasma glucose concentration has fallen to
3. The Bart’s Endocrine protocols. Peter J. trainer,
< 2.2 mmol/l, this is a satisfactory evidence of sufficient
Michael Besser
stress.
Serum cortisol rises by > 200 nmol/L to at least
550 nmol/l Glucagon Stimulation Test
Serum GH rises to > 20 mU/L
Indication
Assessment of ACTH/cortisol and GH reserve when ITT
Interpretation
If adequate hypoglycaemia wasn’t achieved cortisol or is contraindicated.
GH deficiency cannot be diagnosed.
Rationale
Untreated hypothyroidism can also give Glucagon stimulates GH release.
subnormal results-treatment with thyroxine may be A safer test than insulin hypoglycemic test in young
necessary for 3 months before ITT becomes normal. children and infants as it doesn’t usually cause the same
degree of hypoglycaemia as is induced during an ITT.
Peak GH response < 10 mU/L suggests GH .
deficiency. Contraindications
Responses 10-20 mU/L suggests partial • Recent or intercurrent illness
deficiency. • Severe cortisol deficiency
¾ > 20 mU/L is regarded as normal • Glycogen storage disease
• Patients who haven’t eaten for 48 h
 CPSL National Guidelines / Endocrinology 9 10 CPSL National Guidelines / Endocrinology

In all these situations glycogen stores are low or If the result shows GH deficiency same sample
cannot be mobilized and hypoglycemia may occur, can be utilized to measure cortisol, if the clinician has
especially in children requested.
Administer sc glucagon 0.01mg/Kg, up to a
Side effects maximum of 1mg at 0900 (1.5 mg if > 90 Kg)
Nausea, vomiting, abdominal cramps and a Draw blood samples at 60, 120, 150 and180min
feeling of apprehension which may occur in the first 1-2 Child fed and must have normal blood sugar prior
hours after injection. to discharge.
Glucagon induces a rise in blood glucose ( 2-3 Observe minimum of 2 hrs after test.
fold), maximal in the first hour, but this may be followed Leave i.v cannula in situ until after child has eaten and
by symptomatic hypoglycaemia. . blood sugar level returns to normal limits.
If bed side blood sugar < 4 mmol/L, give 0.5g/Kg
Precautions dextrose as 25% dextrose i.v
Serum cortisol > 100 nmol/L
Serum T4 must be normal (replace first if low for several Time(min) glucose GH
weeks) 0 √ √
Patient must be supervised for at all times 60 √ √
90 √ √
Preparation 120 √ √
asting from midnight 150 √ √
(fasting should not be longer than 4-6 hrs in infants and 180 √ √
young children)
Review medication as these may need to be withheld Normal response
until after the test is completed. plasma glucose: usually rises to peak around 90 min and
Accurate weight then falls
The patient must be on a bed for the duration of the test. Cortisol: rises by > 200 nmol/L to above 550 nmol/L
Water only is allowed until completion of the test. GH: rises to> 20 mU/L
Blood glucose should be monitored at the bedside Interpretation
throughout the test. As for the ITT
Insert i.v canula at 0830 h Peak GH is usually at around 120 min.
Baseline blood (0 min) is collected for serum growth Peak GH response < 10 mU/l suggests GH deficiency.
hormone and venous plasma glucose. Responses of 10-20 mU/l suggests partial deficiency.
Response > 20 is regarded as normal.
 CPSL National Guidelines / Endocrinology 11 12 CPSL National Guidelines / Endocrinology

Precautions
References Systolic blood pressure (BP) falls by 20-25 mmHg in all
subjects
1. Endocrine test manual, department of endocrinology, In the event of more significant BP fall, elevation
Westmead Hospital for Children, Sydney, Australia
of the legs is recommended and record the BP every 15
2002
min.
Patients should lie down during and 2h after test
2. The Bart’s Endocrine Protocols: Peter J. Trainer &
or until BP is satisfactory.
Michael Besser

Recommendations Adverse reactions

A. Should be carried out under the supervision and Drowsiness; all in blood pressure is expected,
recommendation of a consultant. Samples should may last several hours. Effect will be prolonged in renal
be sent to the relevant laboratory for analysis. failure.
Please discuss with the relevant laboratory before
sending the specimens. Preparation
asting from mid night
However a minimum fasting time of only 2 hours
Clonidine Stimulation Test is required and short fasting times(< 4-6 hrs) should be
applied in infants and young children.
Indication Accurate height and weight and calculate body
Assessment of GH reserve surface area(BSA).
Insert i.v cannula at 0830 h.
Rationale Draw the baseline blood (0min) for glucose and
Clonidine is a potent stimulus to Growth growth hormone at 0900 h
hormone release via Growth hormone releasing hormone Give 0.15 mg/m2 oral dose of Clonidine at 0900h
secretion. Draw samples of blood for growth hormone and
In this test clonidine is administered orally and glucose at 60, 90 120and 150 min.
the growth hormone response is in peripheral blood is Water is allowed during the test.
measured.

Contraindications
• Sick sinus syndrome
• Compromised intravascular volume
 CPSL National Guidelines / Endocrinology 13 14 CPSL National Guidelines / Endocrinology

Time(min) GH Plasma glucose Exercise Stimulation test

0 √ √ Indication
60 A screening test of GH secretion
√ √
90 √ √
Rationale
120 √ √ Exercise is a physiological stimulant of GH
150 √ √ secretion.
Exercise to approximately 50% of maximal
Interpretation capacity is required and this is usually achieved on riding
Since the mechanism and locus of action is an exercise bike or by repeated stair climbing for about
unclear, interpretation is of uncertain significance. 15 min.
This test has a relatively high incidence of false
• Peak GH response < 10 mU/l suggests GH positives for GH deficiency often due to inadequate
deficiency; exercise, yet is a safe and inexpensive test.
• Responses of 10-20 mU/l suggest partial
deficiency. Contraindications
• Response > 20 is regarded as normal. Limitation of exercise capacity by cardiovascular,
References respiratory or other systemic disease. Children under 8
often do not tolerate the enforced exercise well.
1. Endocrine test manual, Department of endocrinology,
Westmead hospital for children, Sydney, Australia Preparation
2001 fasting for at least 2 hours; in the morning.
2. The Bart’s Endocrine Protocols: Peter J. trainer&
IV sampling cannula
Michael Besser Patients with exercise induced asthma who
normally take prophylactic medication before exercise
should do so.
Recommendations
A. Should be carried out under the supervision and
Method
recommendation of a consultant. Samples should Draw the pre exercise blood sample for growth hormone
be sent to the relevant laboratory for analysis. and glucose
Please discuss with the relevant laboratory before
Record baseline heart rate
sending the specimens. Child is exercised vigorously for 20 min
 CPSL National Guidelines / Endocrinology 15 16 CPSL National Guidelines / Endocrinology

Measure heart rate at approximately 5 min 1.3 Investigation of Growth Hormone

interval.
A heart rate of 140-160 is usually achieved. Excess
The test should be stopped if the heart rate
exceeds 180 or the child is markedly distressed or Biochemical diagnosis of acromegaly/gigantism
exhausted. is made by assessing autonomous secretion of growth
Offer cool water during test, but continue hormone. (GH)
exercising.
After 20 min of exercise, child rests, collect the This is done by measuring growth hormone levels
second sample at 40 min. (20 min post exercise) for during a 2-hour period after a standard 75-g oral glucose
growth hormone and glucose. load. (glucose-tolerance test).

Interpretation Random or basal GH level is not a specific or

Peak GH response < 10 mU/l suggests GH deficiency. sensitive test for acromegaly given the pulsatile nature of
Responses 10-20 mU/l suggest partial deficiency. GH secretion.
>20 mU/l is regarded as normal.
Elevation of random GH levels also occurs in
References stress, chronic renal failure, liver failure, diabetes
mellitus and malnutrition.
1. Duty Biochemist manual, Laboratory Medicine, Royal
Perth Hospital, Australia,2001 Growth Hormone Assay
2. Test Manual,Department of Endocrinology, An optimal GH assay should have the following:
Westmead Hospital for Children, Sydney,
Australia,2002
1. The sensitivity limit of the assay should be < 0.26
mU/L with interassay coefficient of variation of <
Recommendations 15%.
A. Should be carried out under the supervision and 2. Assay should be validated with a normal range
recommendation of a consultant. Samples should for suppressed GH after an oral glucose load
be sent to the relevant laboratory for analysis. (OGTT).
Please discuss with the relevant laboratory before 3. Have information on the antibody specificity to
sending the specimens. the GH isoform (the most abundant in circulation
being the monomeric 22KDa isoform).
 CPSL National Guidelines / Endocrinology 17 18 CPSL National Guidelines / Endocrinology

As there is lack of standardization of GH assays, Draw samples of blood for growth hormone and
this limits the comparisons of results between plasma glucose at 30, 60, 90 and 120 min.
laboratories.
Interpretation
Specimen collection for Growth Hormone Serum GH suppresses to < 0.8mU/L (as measured
Serum is the preferred specimen. by current two-site immunometric assays) after glucose
Serum specimens should be stored at 2 to 800 C if in normal subjects.
they are not to be tested within 8h ailure of GH to suppress after glucose is highly
If they must be stored for long periods, samples suggestive of GH excess.
are frozen at – 2000C. ailure to suppress also occurs in
Patient must be fasting and at complete rest for • chronic liver disease
30 min before collection. • renal disease
• uncontrolled diabetes
Glucose Tolerance Test • malnutrition
• anorexia nervosa
Indication • pregnancy
Diagnosis of acromegaly • estrogen therapy
Contraindications • puberty
None
Precautions References
Cases with known diabetes 1. Tietz text book of clinical chemistry 3rd edition
A basal blood sugar must be checked Carl A Burtis and E. R. Ashwood
Procedure
The test should be performed after an overnight 2. The Bart’sEndrocrine protocols: P.J Trainer and
fast with the patient maintained at bed rest. Michael Besser
Insert an iv cannula
20-30 min after inserting the cannula draw 3. Lim E.M, Pullan P. Cosensus statement review.
baseline blood samples for glucose and growth hormone. Biochemical assessment and long term monitoring in
Note the time. (Time 0) patients with acromegaly: Statement from a Joint
Consensus conference of the growth hormone
75g of oral glucose load at time 0
research society and the pituitary society. The clinical
(Dissolve 75 g in 300 ml of water)This should be Biochemist Reviews 2005; 26: 41-43
taken within 3-4 min
Paediatric dose 1.75g/Kg bodyweight
 CPSL National Guidelines / Endocrinology 19 20 CPSL National Guidelines / Endocrinology

Recommendations Hypothyroidism
A. Should be carried out under the supervision and
recommendation of a consultant. Samples should Primary Hypothyroidism
be sent to the relevant laboratory for analysis.
Please discuss with the relevant laboratory before Screening
sending the specimens. • Serum TSH (2nd or 3rd generation) as first line.

1.3 Thyroid Disorders This strategy may be cost-effective for a wide range
Thyroid function tests can be carried out in of clinical purposes but is not the best as it may be
any hospital.Seperated serum should be sent to insufficient in situations where the pituitary-thyroid axis
relevant laboratories for the analysis. is disturbed such as, secondary hypothyroidism
(hypopituitarism), during optimization of therapy in
Thyroid disorders are amongst the most prevalent newly diagnosed primary hypothyroidism or
of medical conditions, especially in women. Disorders of hyperthyroidism.
the thyroid include both overt and mild/subclinical hypo The most appropriate initial screening would be a
and hyperthyroidism, goiter and thyroid cancer. combination of serum (2nd or 3rd generation) TSH and
T4.
Thyroid function tests (TFT)
Diagnosis
Measurement of serum hormone levels are
utilized to establish the diagnosis of diseases of the • The diagnosis of primary hypothyroidism requires
thyroid gland, or to monitor the response to therapy. The the measurement of both TSH and T4.
best tests are,
3rd generation Serum Thyroid stimulating • Subjects with a TSH of > 10 mU/L and T4 below
hormone (TSH) and Serum ree Thyroxine ( T4) the reference range have overt primary
Alternately in the absence of the above, 2nd hypothyroidism and should be treated with thyroid
generation Serum Thyroid stimulating hormone (TSH) hormone replacement.
and Serum Total Thyroxine (TT4) are acceptable.
Serum Total T3 or, Serum ree T3 measurements • Subjects with subclinical hypothyroidism should
are only useful in specific situations and therefore are have the pattern confirmed within 3-6 months to
not recommended for routine testing. exclude transient causes of elevated TSH.
 CPSL National Guidelines / Endocrinology 21 22 CPSL National Guidelines / Endocrinology

• The measurement of thyroid antibodies in subjects • The measurement of T3 is of no value in patients on

with subclinical hypothyroidism helps to define the tri-iodothyronine replacement because of the
risk of developing overt hypothyroidism. variability after taking the replacement dose.

Guiding treatment with thyroxine Assessing response to thyroxine therapy

• The measurement of both TSH and T4 is required to • In determining the optimal dose of thyroxine the
optimize thyroxine replacement therapy. biochemical target is a TSH result that is detectable,
not elevated and preferably within the reference
• The primary target of thyroxine replacement therapy range.
is to make the patient well and to achieve a serum
TSH within the reference range. Long-term follow-up of patients on thyroxine

• The corresponding T4 will be within or slightly • Patients stabilized on long-term therapy should have
above its reference range. serum TSH checked annually as a change in
requirement for thyroid hormone can occur with
• The minimum period to achieve stable concentrations ageing.
of TSH after a change in dose of thyroxine is two
months and thyroid function tests should not Subclinical (Mild) Hypothyroidism
normally be requested before this period has
elapsed. Diagnosis

Guiding treatment with tri-iodothyronine • Subclinical hypothyroidism is characterized by a

TSH above the reference range with a T4
• If tri-iodothyronine is used as a replacement hormone measurement within the reference range.
increasing doses should be used until serum TSH is
within the reference range. • Should be confirmed by repeat thyroid function
testing 3-6 months after the original test.
• The measurement of TSH is required to optimize tri-
iodothyronine replacement therapy.
 CPSL National Guidelines / Endocrinology 23 24 CPSL National Guidelines / Endocrinology

Guiding Treatmant • Subjects with subclinical hypothyroidism who are

thyroid peroxidase antibody negative should have
• If the serum T4 concentration is normal but the repeat thyroid function testing approximately every 3
serum TSH is > 10 mU/L, then treatment with yrs.
thyroxine is recommended.
Secondary hypothyroidism
• If the serum T4 concentration is normal and the
TSH is elevated but, <10 mU/L then thyroxine Diagnosis
therapy is not recommended as a routine therapy.
• irst line TSH and T4 are required to identify
• However, thyroxine may be indicated in non- secondary hypothyroidism.
pregnant patients having a goiter or in those who are • Secondary hypothyroidism should be distinguished
seeking pregnancy. from
o non-thyroidal illness on the basis of
Assessing response to therapy clinical history, and ree T3 measurement,
o from hyperthyroid patients on antithyroid
• The aim should be to restore and maintain TSH therapy from the drug history.
within the reference range.
Measurement of other anterior pituitary hormones are
• TSH should be measured in 2-3 months, following a useful in the diagnosis of hypopituitarism as a cause of
change in thyroxine dose. secondary hypothyroidism. Referral to an
endocrinologist and selection of suitable dynamic
Long- term Follow-up function tests are recommended.

In patients who receive thyroxine therapy Guiding treatment

• TSH should be measured annually and the thyroxine
dose altered to maintain TSH within the reference • Establish degree of hypopituitarism before
range. commencing thyroxine replacement.

In patients who do not receive thyroxine therapy • Thyroid hormone replacement shouldn’t be
• Subjects with subclinical hypothyroidism who are commenced in patients with cortisol deficiency as
thyroid peroxidase antibody positive should have an this could provoke an Addisonian crisis.
annual thyroid function test.
 CPSL National Guidelines / Endocrinology 25 26 CPSL National Guidelines / Endocrinology

Assessing response to therapy • Confirmation of the diagnosis of congenital

hypothyroidism involves measurement of serum TSH
• Essential to monitor treatment by estimating T4 and and T4 in both mother and neonate and TSH
maintain the thyroid hormone concentration within receptor antibody in the mother.
the reference range.
• All hypothyroid neonates should be treated as early a
• Measurement of TSH cannot be used to assess the possible. Treatment must be started within the first
response in patients with hypopituitarism. 18 days of life.

Long term follow –Up Guiding treatment /follow-Up

• An annual check of serum thyroxine concentration • The measurement of both TSH and T4 are needed to
should be performed in all patients with secondary optimize thyroxine replacement in infants.
hypothyroidism, stabilized on thyroxine replacement
therapy. • Use age related reference ranges.

Congenital hypothyroidism Hyperthyroidism

Diagnosis Primary Hyperthyroidism

• All newborn babies should be screened for Diagnosis

congenital hypothyroidism by measurement of filter
paper blood spot TSH using a sample collected • The single most important biochemical test is serum
within 2-8 days of birth, as part of a national TSH.( by a reputable second or third generation TSH
screening programme. In the absence of such a assay with a functional sensitivity of < 0.02 mU/L)
national screening programme, clinically suspected
babies should have a sample of venous blood • If the serum TSH concentration is within the
collected for serum TSH assay. The result should be reference range then a diagnosis of hyperthyroidism
interpreted with the reference range for an age- is effectively ruled out.
matched (in days) healthy group. o Exceptions are rare TSH dependant causes of
hyperthyroidism such as,
• The measurement of TSH should be restricted to ƒ TSH producing tumours of the pituitary
specialist laboratories and should have a turnaround ƒ Thyroid hormone resistance
time of < 5 days.
 CPSL National Guidelines / Endocrinology 27 28 CPSL National Guidelines / Endocrinology

• In patients suspected of having hyperthyroidism all Guiding Treatment

subnormal TSH results should trigger the
measurement of T4. • The degree of elevation of serum T4 or T3 provides
an indication of the severity of hyperthyroidism and
• If T4 is not elevated in patients with subnormal TSH, should be interpreted in the context of clinical
T3 should be measured to identify cases of T3- symptoms and signs to direct first-line therapy.
thyrotoxicosis.
Assessing response to therapy
• The finding of a serum TSH concentration below the
reference is not, however, specific for the diagnosis Thionamide therapy
of hyperthyroidism.
o Low serum TSH, especially if low but > 0.10 • T4 (or T3 in cases of T3-thyrotoxicosis) will be the
mU/L, often reflects “non-thyroidal illness” or marker of choice to guide thionamide therapy.
therapy with a variety of commonly prescribed
drugs. • Serum TSH alone is not adequate since TSH may
remain suppressed for weeks-months after initiation
o The co-existence of hyperthyroidism and non- of thionamides.
thyroidal illness may result in the finding of a
normal T3. • Thyroid function tests should be performed every 4-6
weeks after commencing thionamides. The frequency
Once a biochemical diagnosis of hyperthyroidism of testing should be reduced to every 3 months once a
has been made, following tests may be needed to indicate maintenance dose is achieved.
the cause and specialist referral should be sought.
Radioiodine therapy
Thyroid peroxides antibodies (TPOAb)
TSH -receptor antibodies (TSH-RAb) • Measure serum T4 and TSH in all patients. In most
cases the T4 will be the marker of choice to guide
The measurement of above antibodies is not therapy.
routinely required to determine the cause of • Thyroid function tests should be performed every 4-6
hyperthyroidism if the treatment plan is not altered. weeks for at least 6 months. Reduce the frequency of
testing when the T4 remains within the reference
range, although an annual T T is still needed.
 CPSL National Guidelines / Endocrinology 29 30 CPSL National Guidelines / Endocrinology

Long –term Follow-Up • Untreated subclinical hyperthyroidism should be

followed into the long term by testing thyroid
• Life –long thyroid function testing is needed for all function every 6-12 months.
patients who have received radioiodine therapy or
surgery for hypethyroidism. Inappropriate TSH

• Regular thyroid function testing is required for all • Elevation of T4 and /or T3 is associated with an
patients being treated with long-term thionamides. “inappropriately” detectable or elevated serum TSH
concentration.
Subclinical (Mild) Hyperthyroidism • This should stimulate the laboratory to consider
errors or assay artifacts. Confirmation by repeat,
Diagnosis including another assay is good practice.
• Once the laboratory has excluded such explanations
• Low serum TSH in the presence of normal then the cause of “true” inappropriate TSH should be
concentrations of T4 and T3 considered.
• Exclude non thyroidal illness and drug therapies • The measurement of serum SHBG and circulating
free alpha subunit and other anterior pituitary
Guiding treatment/follow- up hormones help to distinguish TSH-oma from thyroid
hormone resistance.
Patients withsubclinical hypothyroidism
that cannot be explained by non-thyroidal illness or Thyroid Function Tests in Pregnancy
drug therapy should have repeat serum TSH with T4
and T3 Introduction
• The timing of repeat assessment should be based on • During pregnancy TT4 and TT3 are elevated. TSH is
the clinical picture elevated in the first trimester.
• Both TSH and FT4 (and T3 also when TSH below
• More frequent testing may be appropriate if the the detection limit of a reputable assay) should be
subject is elderly or has underlying vascular disease, used to assess thyroid status and monitor thyroxine
otherwise repeat biochemical testing after 3-6 months therapy in pregnancy.
may be appropriate. • Trimester- and method-specific reference
intervals should be used when reporting thyroid test
• Persistent subclinical hyperthyroidism should prompt values for pregnant patients.
specialist referral.
 CPSL National Guidelines / Endocrinology 31 32 CPSL National Guidelines / Endocrinology

Hypothyroidism * before conception if possible

• The thyroid status of hypothyroid patients should be * at time of diagnosis of pregnancy

checked with TSH and T4 during each trimester.
Measurement of T3 is not appropriate. * at antenatal booking

• Normal TSH and T4 concentrations for the * at least once in second and third trimesters
gestational age should be maintained. and again after delivery

• In hypothyroid patients the TSH should be checked • the newly diagnosed hypothyroid patient will need to
and the thyroxine dose should be adjusted as soon as be tested frequently( every 4-6 weeks) until stabilized
pregnancy is diagnosed.
Hyperthyroidism
• The dose of thyroxine will usually require a small
increase, to ensure that the T4 level is in the upper • If possible, thyroid function tests should be
reference range and the TSH in the low / normal performed, prior to conception, in hyperthyroid
range. women taking antithyroid drugs, and therapy
modified if appropriate.
• An increase in the dose of T4 is especially important
for women who have been treated for thyroid cancer, • Hyperthyroid women taking antithyroid drugs should
to ensure that the TSH remains fully suppressed. have thyroid function tests checked at the time of
diagnosis of pregnancy or at antenatal booking, when
• After delivery the TSH should be checked, at 2-4 the therapy may need to be modified and the dose
weeks post-partum. ( at which time the dose of reduced.
thyroxine can usually be reduced back to the pre-
pregnancy level) • The newly diagnosed hyperthyroid patients should be
monitored by measuring T4 (rather than TSH)
• Ideally, the following sequence of T T should be monthly until stabilized.
performed in the hypothyroid woman during
pregnancy • Monthly measurement of serum T4 is required in
pregnant women receiving antithyroid drugs.
 CPSL National Guidelines / Endocrinology 33 34 CPSL National Guidelines / Endocrinology

• Women who have been successfully treated Screening for thyroid disease during pregnancy
previously for hyperthyroidism and are euthyroid at
antenatal booking may be checked again in the • Pregnant women in the following categories should
second and third trimesters. have thyroid function assessed either at diagnosis or
at antenatal booking or even before conception, if
• All previously hyperthyroid females should be feasible.
retested after delivery, as there is a higher chance of
relapse at this time. * type-1 diabetes
* previous history of thyroid disease
• Do not measure TSH-RAb again if it is low or * current thyroid disease
negative at antenatal booking. A very high titer can * family history of thyroid disease
predict the chance of intrauterine or neonatal * goiter
thyrotoxicosis developing. * symptoms of hypothyroidism

Post –Partum Thyroiditis • Thyroid function tests during pregnancy should

comprise both TSH and T4
• Post-partum patients should have TSH and T4
measured at 6-8 weeks post-partum (or post-abortum) Neonatal thyroid assessment
if they have any of the following.
• Goiter Neonatal screening for congenital hypothyroidism
• Non-specific symptoms that may suggest thyroiditis
• Previous history of post partum thyroiditis • All newborn babies should be screened for congenital
• Previous history of autoimmune thyroid disease hypothyroidism by measurement of blood spot TSH
• Positive TPO-Ab using a sample collected within 2-7 days after birth
as part of a national screening programme.
• If the initial T T show a thyrotoxic pattern, do
further tests to differentiate post-partum thyroiditis • The peak TSH surge occurs within 30 min of birth
from Grave’s disease. and start to fall by 1-2 hours so that the
concentrations reach adult levels by 2-3 days.
• Start thyroxine therapy in a symptomatic patients if
thyroid tests show hypothyroidism. • The laboratories should be able to issue a result
within 5 days.
• Confirmation of the diagnosis of congenital
hypothyroidism involves measurement of serum TSH
•
 CPSL National Guidelines / Endocrinology 35 36 CPSL National Guidelines / Endocrinology

and T4 in both mother and neonate and TSH Monitoring treatment and long term follow-up
receptor antibody in the mother.
• All hypothyroid patients should be treated as early as • After thyroidectomy for thyroid cancer the TSH
possible. Treatment must be started within the first should be suppressed to and maintained at a level of
18 days of life. <0.1 mU/L , in a reputable second or third generation
TSH assay.
Neonatal hypothyroidism
Thyroglobulin(Tg) and thyroglobulin
• The measurement of both TSH and T4 are required antibodies(TgAb)
to optimize thyroxine replacement in infants. Age
related reference ranges should be used. Diagnosis

Neonatal hyperthyroidism • The measurement of serum thyroglobulin has no role

in the diagnosis of thyroid cancer.
• The diagnosis of neonatal hyperthyroidism requires
measurement of both TSH and T4. Both should be Monitoring therapy and long term follow-up
measured at regular intervals to guide treatment.
• Samples for thyroglobulin assay should not be
collected for at least 4-6 weeks after thyroidectomy
Thyroid Function Tests in Thyroid Cancer or iodine therapy.

Differentiated thyroid cancer • In a sensitive assay (RIA or IRMA) detectable

thyroglobulin usually indicates the need for further
The role of TSH and thyroid hormones investigation to identify the source. The laboratory
should advise of the cut-off level foe a particular
Diagnosis assay method.
• Perform T T whenever thyroglobulin and
• Thyroid function tests do not directly aid the thyroglobulin antibodies are measured. .
diagnosis of thyroid cancer, as patients are generally
euthyroid. • The requesting clinician should indicate on the
request form whether the patient is on thyroxine
therapy.
 CPSL National Guidelines / Endocrinology 37 38 CPSL National Guidelines / Endocrinology

• The sensitivity of serum Tg measurement for Medullary thyroid cancer

detecting recurrence is enhanced by an elevated TSH
level. Hence, Tg should be preferably measured Calcitonin
when the serum TSH is > 30 mU/L( after thyroxine
withdrawl or the use of recombinant human TSH). Diagnosis
There is no need for TSH stimulation if the serum Tg
is already detectable. • A pre-operative value for serum calcitonin
(preferably fasting) should be measured in patients
• If TgAb are detectable, measurement should be with medullary thyroid cancer (MTC) to establish a
repeated at regular intervals.(6monthly) If baseline for the long- term follow up.
undetectable they should be measured at follow-up
when thyroglobulin is measured. The development of Monitoring therapy
increasing TgAb may indicate recurrence of tumor.
• The response to primary surgery can be assessed both
• or routine follow-up of patients in remission, serum clinically and by the measurement of serum
Tg can be measured while the patient is taking TSH calcitonin.
suppressive treatment.
• Post-operative samples should be collected at least 10
• The frequency of Tg measurement during follow-up days after thyroidectomy and should be fasting if
of thyroid cancer depends on the clinical condition of possible.
the patient.
• Life long calcitonin measurement is recommended
• Tg results are method dependent. Clinicians should for patients with medullary cancer. The frequency of
use the same laboratory and Tg assay on a long-term measurement will depend on both clinical status and
basis, to ensure the continuity in monitoring. the previous calcitonin result.

• Laboratories should not change methods without

prior consultations with clinical users of the service. Thyroid function tests in medullary cancer

• T T have no place in the diagnosis of MTC.

• During follow-up T T should be monitored.
 CPSL National Guidelines / Endocrinology 39 40 CPSL National Guidelines / Endocrinology

• TSH suppression is not appropriate for the treatment Tests to establish if there is thyroid dysfunction
of MTC. So thyroid hormone replacement should
follow the guidelines for treating hypothyroidism. Thyrotrophin(TSH)

Laboratory Investigations for Thyroid Dysfunction • The measurement of TSH by a sensitive

immunometric assay provides the single most
Provision of Laboratory Tests sensitive, specific and reliable test of thyroid status in
both overt and subclinical primary thyroid disorders.
• The laboratory should be able to issue T T results • TSH alone is not a reliable test for detecting thyroid
within 48 hours from receipt of the specimen though dysfunction arising from hypothalamic-pituitary
in the majority of cases there is no urgency for dysfunction.
receipt of routine T T. • It is essential that laboratories use a reliable and
• More rapid response is desirable in thyrotoxic crisis sensitive method for TSH that meets the following
and myxodema coma as they are medical conditions.
emergencies.
1. The functional sensitivity should be used to
Grouping of Thyroid Function Tests define the lowest concentration of TSH that can
be determined in routine use.
• Blood tests which establish if there is thyroid
dysfunction(TSH, T4, T3 , TT4, TT3) 2. unctional sensitivity is defined from the 20%
• Tests to elucidate the cause of thyroid between- run coefficient of variation (CV)
dysfunction(thyroid auto antibodies) determine by a recommended protocol.

Measurement of TSH and thyroid hormones 3. Laboratories should use a TSH method with a
should be performed to determine the patient’s functional sensitivity of < 0.02 mU/L
thyroid status before ordering the more demanding 4. Prior to the introduction of a TSH method, the
tests that seek to determine the cause of the thyroid laboratory should validate the functional
dysfunction. sensitivity quoted by the manufacturer. Quality
assurance procedures should be in place to ensure
that the functional sensitivity of the assay is
regularly monitored.
 CPSL National Guidelines / Endocrinology 41 42 CPSL National Guidelines / Endocrinology

Free T4 (FT4) and Free T3 (FT3) Total T4 (TT4) and total T3 (TT3)
• Laboratories should be aware of how their assay • It is common to find abnormal TT4 and TT3 in some
performs in a variety of clinical situations including euthyroid patients.
thyroid disorders, pregnancy, non-thyroidal illness, Eg Changes in serum thyroid binding proteins
certain mediactions (heparin, phenytoin, frusemide, (pregnancy)
carbamazepin, salicylate) and familial binding Changes in affinity for hormones (non-thyroidal
protein abnormalities. illness drugs such as salicylates)
• Clinicians should be made aware, by the laboratory,
of the expected assay performance in the clinical Reference ranges
settings listed above.
• Laboratories should obtain from kit manufactures Adults
details of how their assay compares with equilibrium • TSH 0.4- 4.5 mU/L
dialysis in the clinical situations listed above.
• Free hormones can increase in samples on storage • T4 9.0-25 ρmol/L
but because of assay design (eg inclusion of albumin
in reagents) not all free hormone methods detect such • T3 3.5-7.8 ρmol/L
changes. Laboratories should be aware of how
storage affects free hormone concentrations when • TT4 60-160 nmol/L
measured by their own method. Appropriate action
should be taken to minimize such sample • TT3 1.2-2.6 nmol/L
deterioration.
• reeze samples that cannot be assayed within 48 TSH (3rd generation immunochemiluminometric assay)
hours of collection.
* Premature (28-36 wk) 0.7-27.0(miu/L)
• Interference from anti-thyroid antibodies is method
dependent. Laboratories should know how the * Cord blood (>37 wk) 2.3-13.2
presence of such antibodies would affect their assay.
* Children birth to 4 days 1.0-39.0

2-20 wk 1.7-9.1

21wk-20y 0.7-6.4
 CPSL National Guidelines / Endocrinology 43 44 CPSL National Guidelines / Endocrinology

* Adults 21-54 y 0.4-4.2 1-2 wk 126-214

55-87 y 0.5-8.9 1-4 mo 93-186

* Pregnancy irst trimester 0.3-4.5 4-12 mo 101-213

Second trimester 0.5-4.6 1-5 yr 94-194

Third trimester 0.8-5.2 5-10 yr 83-172

TSH surges immediately after birth, peaking at 30 10-15 yr 72-151

min at 25-160 mIU/L and values decline back to adult
values in the first week of life. Adults (15-60 yr)

FT4 (by direct equilibrium assay) Males 59-135

ρmol/L
emales 65-138
* Newborn ( 1-4 days) 28.4-68.4
Adults> 60 yr 65-138
Children (2wk-20yr) 10.3-25.8
New born screen (whole blood)
Adults ( 21-87y) 10.3-34.7
1-5 days >97
Pregnancy ( irst trimester) 9.0-25.7
6 days > 84
nd rd
(2 & 3 trimester) 6.4-20.6

Total T4 nmol/L • Where possible manufactures reference ranges

should be confirmed locally using an adequate
Cord 95- 168 population size of at least 120 ambulatory subjects.
• Since TSH, free and total thyroid hormones change
Children during pregnancy. trimester related reference ranges
should be available.
1-3 days 152-292
 CPSL National Guidelines / Endocrinology 45 46 CPSL National Guidelines / Endocrinology

Such protocols may include:

Quality control and quality assurance
dilution tests
Internal quality control (IQC)
Removal of heterophilic antibody and HAMA using
• All laboratories must run IQC that comprise human commercial tubes(for hormone assay)
serum pools
• The analyte concentrations for the pools should be Remove of antihtyroid antibodies using polyethylene
chosen to ensure that assay performance is monitored glycol precipitation
within the euthyroid, hyperthyroid and hypothyroid
ranges. Laboratories should define allowable limits Confirmation by an alternative assay which if
of error for each of these results. possible should have been validated against a
reference method

External quality control (EQC) Laboratory tests used to determine the cause of
thyroid dysfunction
• All laboratories must participate in an accredited
EQA scheme. 1. Thyroid peroxidase antibodies (TPOAb)
• Laboratories should ensure that assay performance
meets the minimum performance specified by the • The measurement of TPOAb is of clinical usein
EQA scheme. diagnosis of autoimmune thyroid disorders
As a risk factor for autoimmune thyroid disorders
As a risk factor for hypothyroidism during treatment
Follow-up of unusual test results with interferon alpha, interleukin-2 or lithium
As a risk factor for thyroid dysfunction during
Unusual combinations of TSH and thyroid lithium or amiodarone therapy.
hormone results may have a pathological source but • TPO results are method dependent and this should
more commonly result from poor compliance or assay be recognized.
interference in one or more assays.
• unctional sensitivity should be determined for the
TPOAb method using the same protocol as for TSH
• Laboratories should have protocols available to
• A sensitive and specific immunoassay should be
determine if results are analytically valid or due to
used to measure TPOAb , not an agglutination test.
assay interference.
 CPSL National Guidelines / Endocrinology 47 48 CPSL National Guidelines / Endocrinology

2. Thyroglobulin antibodies(TgAb) To identify neonates with transient

hypothyroidism due to TSH blocking antibodies
• In iodine sufficient areas it is of no value to measure
both TgAb and TPOAb in non-neoplastic conditions. 4. Thyroglobulin (Tg)

• The only reasons to measure Tg antibodies are • In patients with differentiated thyroid cancer
a. in differentiated thyroid cancer to determine measurement of serum Tg is used in monitoring, but
possible interference from these antibodies in is not of value in the initial diagnosis.
immunoassays for thyroglobulin.
b. Serial measurements may prove to be useful as a • Samples for Tg measurement should be preferably be
prognostic indicator taken when the TSH is elevated either after
withdrawal of T4 therapy or following administration
• Assays should be performed using a sensitive of recombinant human TSH. or patients in
immunoassay not an agglutination method remission and under routine follow-up it is
acceptable in the first instance to sample during T4
• Determine functional sensitivity as for TSH administration.

• Thyroglobulin and TgAb be measured in the same • Laboratories and manufactures should inform
specimen. clinicians of the possibility of interference due to
endogenous TgAb and indicate the most likely nature
• If Tg Ab is being used for monitoring purposes the of the interference( false elevation/false reduction in
method should not be changed without consultation measured Tg)
with the users.
5. Calcitonin
3. TSH receptor antibodies(TSH-RAb)
Specimen collection
• The measurement of TSHR-Ab is particularly helpful
in pregnancy • Ideally a fasting morning specimen should be
to investigate hyperthyroidism of uncertain origin obtained to enable optimal comparison with reference
to investigate patients with suspected “ euthyroid values.( If this is not possible specimens can be
Graves” ophtalmopahty. collected at any time of day)
or pregnant women with a past or present Calcitonin in serum is unstable. Specimens should
history of Graves disease be kept on ice. Red cells then should be separated
 CPSL National Guidelines / Endocrinology 49 50 CPSL National Guidelines / Endocrinology

within 30 min of collection and serum frozen Triiodothyronine

immediately. Serum is the preferred specimen.
• Post-operative samples should be collected at last 10 Serum is best stored at 2 to 80C if they will not
days after thyroidectomy and should also be fasting be tested within 24 hours.
samples if possible. If longer periods of storage are necessary, freeze
• or provocative testing samples are usually collected the specimen.
5 min prior to administration of calcium/pentagastrin rozen specimens are stable for at least 30 days.
and then at intervals of 2.5 and 7 min after. Avoid repeat freezing and thawing.

Methodology Thyroid binding globulin

• Laboratories must decide whether to use a method Serum is the preferred specimen.
that recognizes primarily monomeric calcitonin Serum is best stored at 2 to 80C if they will not
(IMA) or a method with broader specificity ( RIA). be tested within 24 hours.
If longer periods of storage are necessary, freeze
Specimen collection and storage the specimen.
rozen specimens are stable for at least 30 days.
TSH Avoid repeat freezing and thawing.
Serum that is free of haemolysis and signs of Patients should discontinue thyroid hormone
lipaemia is preferred. replacement therapy long enough (6 weeks) to have an
Specimens are stable for 5 days at 2 to 80 C and elevated TSH level prior to specimen collection.
for at least 1 month when stored frozen.
or newborn screening, whole blood can be Antiyhyroid antibodies
collected by heel prick 48 to 72 hours after birth. Serum is the preferred specimen.
Serum is best stored at 2 to 80C if they will not
Thyroxine be tested within 24 hours.
Serum is the preferred specimen If longer periods of storage are necessary, freeze
Serum is best stored at 2 to 80C if they will not the specimen.
be tested within 24 hours.
If longer periods of storage are necessary, freeze Calcitonin
the specimen. Serum is the preferred specimen.
rozen specimens are stable for at least 30 days. Ideally a fasting morning specimen should be
Avoid repeat freezing and thawing. obtained.
Do not analyse grossly haemolysed specimens.
 CPSL National Guidelines / Endocrinology 51 52 CPSL National Guidelines / Endocrinology

Calcitonin results may be affected by visible • The measurement of TSH with T4 should allow the
haemolysis or lipaemia and assay of such specimens detection of almost all cases of thyroid dysfunction as
should be avoided if possible. long as the results of both tests are correctly
Calcitonin in serum is unstable. Specimens interpreted.
should be kept on ice. • Additional tests such as T3 may be needed in some
Red cells then should be separated within 30 circumstances.
mns of collection and serum frozen immediately.
Post operative samples should be collected at References
least 10 days after thyroidectomy. 1. Tietz text book of Clinical chemistry. Carl A. Burtis,
Edward R. Ashwood, 3rd edition
Selective use of thyroid function tests
• The use of first-line TSH will fail to identify some Recommendations:
patients with thyroid disorders. B. Thyroid function tests can be carried out in any
• Situations in which TSH usually provides the correct hospital lab. Samples should be sent to the
estimate of thyroid status relevant laboratory for analysis. Please discuss
with the relevant laboratory before sending the
I. In overt primary hyperthyroidism TSH is specimens.
nearly always below 0.10 mU/L

II. In overt primary hypothyroidism serum TSH 1.5 Investigation of Disorders of Cortisol
is always increased.
Metabolism
III. In subclinical disorders, TSH will be the most
sensitive indicator of failing thyroid function, • Cortisol excess
and serum T4 and T3 are often normal.
Before the diagnosis of subclinical thyroid • Cortisol deficiency
disorders can be made, causes of abnormal A random cortisol estimation is difficult to
TSH other than thyroid disorders such as interpret due to the variability of cortisol excretion
pregnancy, non-thyroidal illnesses, drug during the day and should be avoided if possible.
treatment and assay interference must be As with cortsol ,17 hydroxyprogesterone
excluded. (17OHP) has a marked circadian rhythm.
Diurnal rhythm and adult values are reached by 3
months.
 CPSL National Guidelines / Endocrinology 53 54 CPSL National Guidelines / Endocrinology

Cortisol Excess Principle

Dexamethasone is a potent glucocorticoid which,
Patient assessment when given as a 1 mg oral dose at night, will normally
suppress ACTH secretion and hence suppress the cortisol
Laboratory tests shouldn’t be used in isolation, level the following day morning.
results should be interpreted together with a full clinical It is a useful initial screening test for Cushing’s
assessment of the patient. syndrome.

Choice of test Patient preparation Not required

Sample Serum
irst-line screening tests must have high Procedure
sensitivity to minimize the incidence of false negative
results and reference (cut-off) values chosen to achieve The patient is given 1 mg Dexamethasone( small
these aims. children may require a relatively smaller dose) to be
taken at at 11.00 pm( ± 1 hour)
Blood is taken at 9.00 am ± 1 hour) the following
Screening tests morning for cortisol measurement.
Overnight Dexamethasone Suppression
Test is particularly recommended Interpretation
Or Normal subjects will have a 9 am cortisol of < 50
24-hour urinary free cortisol nmol/L after 1 mg of Dexamethasone.

Definitive Tests ollowing are the causes for serum cortisol level
Are indicated if a positive result is found on to be > 50 nmol/L (failure to suppress) following
screening. overnight dexamethasone test.

Overnight Dexamethasone Suppression Test 1. Cushing’s syndrome

2. Stress
This test can be performed in any hospital. 3. Obesity
Separated serum should be sent to relevant 4. Oral contraceptive use
laboratory for analysis. 5. Pregnancy
6. Estrogen therapy
7. Alcoholism
8. Acute or chronic illness
 CPSL National Guidelines / Endocrinology 55 56 CPSL National Guidelines / Endocrinology

9. ailure to take dexamethasone This test is useful in

10. Treatment with phenobarbitone, phenytoin,
carbamazepin • pregnancy
11. Glucocorticoid therapy with prednosolone or • Patients on oral contraceptive treatment or
hydrocortisone estrogens
12. Endogenous depression
Recommendations
Recommendations A. Should be carried out under the supervision and
A. Should be carried out under the supervision and recommendation of a consultant. Samples should
recommendation of a consultant. Samples should be sent to the relevant laboratory for analysis.
be sent to the relevant laboratory for analysis. Please discuss with the relevant laboratory before
Please discuss with the relevant laboratory before sending the specimens.
sending the specimens.

24-hour Urine Free Cortisol (UFC) Low Dose Dexamethasone Suppression Test
This should be performed under the
1. This should be determined using an assay which supervision of a Consultant. Separated serum should
incorporates an extraction step. be sent to relevant laboratory for the analysis.

2. Creatinine should be measured in the 24-hour Indications

sample to verify the adequacy of collection. Establishment of diagnosis of Cushing’s
syndrome.
3. Results should be interpreted with caution in
young children and in patients with significant Contraindications None
renal dysfunction
Precautions
4. The reference range should be quoted on reports; Care in diabetes mellitus and active peptic
the upper limit should not exceed 300 nmol/24 ulceration.
hours. or inpatients each dose of dexamethasone
should be written up as an individual dose.
* UFC may not identify patients with mild
hypercortisolism and therefore UFC cannot be Procedure
considered a universal single screening test for Draw a sample of blood at 9.00 am on day 0 for
the detection of Cushing’s syndrome. serum cortisol.
 CPSL National Guidelines / Endocrinology 57 58 CPSL National Guidelines / Endocrinology

(Basal sample)Give dexamethasone 0.5 mg orally Severe stressful illness /infection Do not
strictly 6-hourly at 9.00, 15.00, 21.00 and 3.00 (9 am, 3 perform the test
pm, 9 pm and 3 am) for 48 hours, commencing Failure to take dexamethasone correctly Check
immediately after the basal sample. with the patient
Oestrogen therapy Pregnancy, Oral
(Children < 10 years 5µg/Kg) contraceptives due to high globulin binding
proteins
Blood sample for cortisol at 9.00 hours (9.00 am) Corticosteroid therapy Prednisolone &
after 48 hours on day 2 (6 hrs after last dose) hydrocortisone cross react in the cortisol assay.

Normal response Recommendations

Basal serum cortisol is in the normal resting am A. Should be carried out under the supervision and
range at 9.00(170-700 nmol/L) recommendation of a consultant. Samples should
After 48 hours serum cortisol is suppressed to < be sent to the relevant laboratory for analysis.
50 nmol/L Please discuss with the relevant laboratory before
sending the specimens.
Interpretation
A serum cortisol < 50 nmol/L after 48 hours
excludes Cushing’s syndrome unless clinical suspicion is Dexamethasone suppression of cortisol
very high (extended)
Very rarely patient’s with Cushing’s syndrome Test for differential diagnosis of Cushing’s
may show normal suppression.
syndrome.
• A number of conditions may cause non-
This should be performed under the
suppression with low –dose dexamethasone in the
supervision of a Consultant. Separated serum should
absence of Cushing’s syndrome.
be sent to relevant laboratory for the analysis.
Severe endogenous depression Patients
Principle
may have an abnormal circadian rhythm but show
The multiple Dose Dexamethasone suppression
normal cortisol response to an ITT.
text (DST) consists of administration of Dexamethasone
Alcoholism(Alcoholic psudo- Cushing’s )Rapidly
reverses on stopping drinking
 CPSL National Guidelines / Endocrinology 59 60 CPSL National Guidelines / Endocrinology

0.5 mg every 6hrs for 48 hours (low does Day Time Dosage Cortisol
Dexamethasone suppression test) followed by 2mg Day 1 0900 0.5mg 3
Dexamethasone every 6hrs for 48 hrs 1500 0.5mg
2100 0.5mg
Clinical use Day 2 0300 0.5mg
The test aids in the differential diagnosis of 0900 0.5mg 3
hypercortisolism in patients who do not suppress after an 1500 0.5mg
overnight Dexamethasone suppression text. 2100 0.5mg
Day 3 0300 0.5mg
Sample Serum for cortical 0900 2.0 mg 3
1500 2.0 mg
Side effects some subject report sleep disturbances 2100 2.0 mg
Day 4 0300 2.0 mg
Procedure
0900 2.0 mg
The test is performed as an in-patient procedure.
1500 2.0 mg
No preparation is required but the ward staff must
be aware of the importance of correctly timed collection 2100 2.0 mg
of specimens. Day 5 0300 2.0 mg
Insert an iv cannula 0900 2.0 mg 3
Blood is collected daily at 9.00a.m. for cortisol
measurements.
At 9.00a.m on day 1, commence Dexamethasone The test is carried out according to the table
0.5 mg orally 6hrs for 48hours after drawing the basal above. It is important to collect the 0900 hour blood
blood sample at 9.00.m. sample before giving oral Dexamethasone.
At 9.00 a.m. on day3, increase the dose to 2mg
every 6hrs for 48 hours.
Guide to interpretation

Patients with pituitary dependent Cushing’s

syndrome( Cushing’s disease) usually do not suppress
(<50% of basal) with low dose but the majority of
patients will suppress with high dose.
(A significant minority of patient’s (15%) with
Cushing’s disease do not suppress)
 CPSL National Guidelines / Endocrinology 61 62 CPSL National Guidelines / Endocrinology

Suppression is defined as 50% reduction in Clinical stigmata of Cushing’s

basal cortisol.
Patients with false positive results in the
overnight Dexamethasone suppression test should 1. Overnight dexamethasone suppression test
or
suppress in the low dose period. 2. 24 h urinary excretion of cortisol

Patients with adrenal tumors or ectopic ACTH 1. 9am plasma cortisol 1. 9am plasma cortisol
production tumors fail to suppress with rare exceptions. <50nmol/l >50nmol/l
2. urinary free cortisol < 2. urinary free cortisol
300nmol/d<100µg/d <300nmol/d>100µg/d

Patients with adrenal adenomas have ACTH Low dose dexamethasone suppression test
Confirmation
levels below the reference range, while patients with
ectopic ATCH producing tumors have ATCH levels
within or above the reference range. No suppression
Suppression

Cushing syndrome &

Cushing syndrome excluded other causes
Recommendations
A. Should be carried out under the supervision and
Localization
recommendation of a consultant. Samples should High dose dexamethasone suppression test

be sent to the relevant laboratory for analysis.

Please discuss with the relevant laboratory before
sending the specimens. Suppression
No suppression

Inconclusive

Bilateral inferior
MRI or CT petrosal sinus sampling

No central to central to peripheral

peripheral gradient gradient

Ectopic ACTH Primary cushing

Adrenal tumor secreting tumor disease
 CPSL National Guidelines / Endocrinology 63 64 CPSL National Guidelines / Endocrinology

Sample collection and storage which the specimen is taken and stress from a poorly
performed venipuncture, must be taken into account.
Serum cortisol
Blood specimen should be collected into
Serum is the preferred specimen. Collect prechilled polystyrene(plastic) tubes containing EDTA.
blood(2ml) into a bottle without a preservative. No Immediately placed on ice, and centrifuged at 40C .
special handling procedures are necessary. Specimens Supernatant is then transferred to another plastic
must be stored refrigerated overnight at 20C to 80 C. tube and stored at -200C or colder.
reezing is preferred for long-term stability. Immediately prior to setting up the ACTH
assay ,frozen specimen should be thawed and centrifuged
24-hour free cortisol to remove any fibrin clots, which may interfere with the
assay.
A complete 24-hour urine specimen is collected
with or without 10g boric acid to maintain pH < 7.5. Precision and Bias
If collected without preservative uine should be
refrigerated during the collection period. Each laboratory should ensure that
After measuring the total volume, a thoroughly appropriate internal quality control and external quality
mixed aliquot (∼ 10 ml) is stored frozen at -200C . control assessment procedures are in place.
Care should be taken to ensure an appropriately Any laboratory consistently unable to
timed, complete 24-hour collection because an meet the following criteria and which cannot change to a
incorrectly timed sample is the largest source of error superior assay should refer samples elsewhere.
with this method.
ree cortisol determination on randomly collected a) or serum cortisol, the precision should be les than
urine are discouraged because of the variation and 15% and bias < 15%.
pulsatile characteristic of cortisol secretion. b) or urine free cortisol, the precision should be <
25%.
Adrenocorticotropic Hormone(ACTH)
References
ACTH is easily oxidized, strongly adsorbs to
glass surfaces, and can be rapidly degraded by plasma 1. Queensland health pathology services: Royal Brisbane
Hospital , Tests list, collection details 2001
proteases into immunoreactive fragments during freezing
and thawing of the specimen.
actors that influence plasma ACTH, such as 2. All Wales Clinical biochemistry audit group standards
for screening for Cushing’s syndrome, 2001
prior administration of corticosteroids, time of day at
 CPSL National Guidelines / Endocrinology 65 66 CPSL National Guidelines / Endocrinology

3. C.Bhagat. Consensus Statement Review: Diagnosis and Contraindications

Complication of Cushing’s Syndrme: a consensus • Known sensitivity to ACTH
statement. The Clinical Biochemist Reviews 2004: 25;
• Pregnancy
149-151

4. Tietz Text Book of Clinical Chemistry. Carl A. Burtis, Patient preparation

Edward R. Ashwood. 3rd edition The following drugs should be withheld 24 hours
prior to the test:
5. Bonstein S.R, Stratakis C.A., . Chrousos G.P. • Glucocorticoids eg prednisolone or cortisone
Adrenocortical tumours: Recent Advances in Basic • Metapyrone
Concepts and Clinical Management. Annals of If steroids are required within this 24 hour period,
Internal Medicine 1999; 130:759-771 preferred drug is Dexamethasone.
Patient doesn’t require to fast, nor be at rest.
Cortisol Deficiency
Procedure
Synacthen Stimulation of Cortisol-(short) Cannulate the patient and take baseline sample (0
Test for Adrenal Insufficiency min) for cortisol.
Administer the synacthen dose
Rationale 250 µg Synacthen im or as a slow iv bolus
A low plasma/serum cortisol that does not rise 0-6 months 36 µg/Kg
after ACTH administration (Short Synacthen test) 6 months-2 yrs 125 µg
confirms impaired adreno-cortical reserve. ≥2 years 250 µg
If the impaired response persists after prolonged
ACTH administartion (Long SynacthenTest), primary Draw further two blood samples at 30 and 60 min
adreno-cortical insufficiency is indicated rather than for cortisol.
secondary adrenal atrophy due to impaired ACTH 17OHP may also be requested (indications,
secretion; the latter results either from prolonged congenital adrenal hyperplasia CAH, hirsuitism,
corticosteroid therapy or hypothalamic-pituitary disease. infertility)

Tetracosactrin (Synacthen or Cortrosyn) is the Interpretation

synthetic analogue of ACTH that is now most commonly A normal response is an increase in the serum
used for the ACTH stimulation test. cortisol level of . 200 nmol/L over the baseline and or the
final value > 550 nmol/l.
 CPSL National Guidelines / Endocrinology 67 68 CPSL National Guidelines / Endocrinology

A failure to respond suggests adrenal failure 1.6 Investigation of Disorders of

primary or secondary. A long tetracosactrin test is
required to confirm primary adrenal failure Aldosterone Metabolism

A peak 17OHP response of > 30 nmol/l is Aldosterone-producing tumours are usually

indicative of CAH.In addition, a 30 min 17OHP; cortisol associated with hypertension with or without
ratio >0.1 suggests CAH, < 0.023 is normal. hypokalaemia.
Screening tests for an aldosterone producing
Values between 0.023 and 1.0 suggest tumour include evaluation of hypertencsion and
heterozygosity for 21 hydroxylase deficiency. measurement of serum potassium.

References Screenig tests

1. Tietz text book of Clinical biochemistry 3rd • Serum potassium

editionDuty Biochemists Manual, Department of
clinical biochemistry, Royal Perth hospital, Sydney, • Urinary potassium
Australia, 2001 To demonstrate renal losses of potassium in the
presence of hypokalaemia the patient should be on at
Recommendations least 120 mmol/l of sodium for 3 days before
A. Should be carried out under the supervision and investigation, since a low sodium intake may normalize
recommendation of a consultant. Samples should mild hypokalaemia.
be sent to the relevant laboratory for analysis.
Please discuss with the relevant laboratory before Samples Spot Urine
sending the specimens. (needs simultaneous blood and
urine specimens)
Long Synacthen Test 24-hour urine
• Plasma aldosterone : rennin ratio in an upright
There are two protocols. Please contact the following position
laboratories.(NHSL, MRI, Faculty of Medicine Galle, • >20 suggests primary hyperaldosteronism
Faculty of Medicine Peradeniya)
Confirmatory tests
These should be performed under the
supervision of a Consultant. Separated serum should
be sent to relevant laboratory for the analysis.
 CPSL National Guidelines / Endocrinology 69 70 CPSL National Guidelines / Endocrinology

• Saline suppression test Diltiazem can be used to control blood pressure

during testing for aldosterone excess.
• Oral salt loading test Serum potassium should be maintained within the
reference range (>3.5 mmol/L) prior to commencement
Recommendations and during the test.
A. Should be carried out under the supervision and The subject is awakened at 0600 h and kept in an
recommendation of a consultant. Samples should upright posture for 2h.
be sent to the relevant laboratory for analysis. Record Blood Pressure.
Please discuss with the relevant laboratory before BP must be < 190/110 mm/Hg before proceeding
sending the specimens. Insert IV line and send blood for urgent
Saline Suppression Test potassium
Blood is drawn for determination of plasma
Rationale aldosterone and electrolytes at 0800h. The subject then
Aldosterone is the major mineralocorticoid assumes a supine position, and 2L of isotonic saline, 0.9
synthesized in the adrenal cortex. g%, is infused over a 4-h period.
Rapid volume expansion with intravenous saline Record BP, pulse every 15 min during the
should suppress plasma aldosterone in normal subjects infusion for first hour, every half an hour thereafter.
but not in patients with primary aldosetronism. Blood is drawn from the non-IV infusion arm
for plasma aldosterone and electrolytes determination at
Procedure 1200h
The test is carried out in the ward under Potassium should be ≥ 3.5 mmol/L before patient
supervision. leaves.
Patients may have fluids but otherwise should be Observe patient one hour prior to discharge.
fasted on the day of the test. Patient may have coffee/tea and food at this time.
Withhold the following drugs Cancel the test if
• K+ < 3.5 mmol/L
• β-adrenoceptor blocking drugs - 2 weeks before • BP>190/110 mm/Hg
testing
• dihydropyridine calcium channel blocker - 2 weeks Contraindications
before testing • Heart failure
• Spironolactone - 6 weeks before testing • Uncontrolled hypertension
• Loop diuretics - 6 weeks before testing
 CPSL National Guidelines / Endocrinology 71 72 CPSL National Guidelines / Endocrinology

Interpretation Diagnostic algorithm for primary

Normal individuals show a plasma aldosterone hyperaldosteronism
level of ≤ 140 ρmol/L after saline infusion. Hypertention / hypokalaemia
Levels ≥ 277.4 ρmol/L are usually seen in
patients with autonomously functioning aldosterone – Check aldosterone:renine ratio in upright position
secreting tumours.
>20 < 20
Recommendations Suggests primary hyperaldosteronism Normal
A. Should be carried out under the supervision and Confirmation
recommendation of a consultant. Samples should
Saline infusion test (2 L isotonic saline over 4 hours) and measure plasma aldosterone
be sent to the relevant laboratory for analysis. Or
Please discuss with the relevant laboratory before Oral salt loading test and measure 24 hour urinary aldosterone (12g NaCl tablets for 3 days)

sending the specimens.

Plasma aldosterone >10ng/dl Plasma aldosterone <10ng/dl
Or Plasma aldosterone level or
24 h urinary aldosterone >10-14 µg/d 5-10ng/dl 24 hour urinary aldosterone
with urine sodium >250 nmol/d <8µg/d
References
1. Tietz textbook of Clinical chemistry 3rdedition. Diagnostic Borderline Exclude primary
Duty Biochemist Manual, Royal Perth Hospital, hyperaldosteronism

Australia, 2001
Localization

2. Bonstein S.R, Stratakis C.A., . Chrousos G.P. 1.

2.
CT or MRI adrenals
Postural stimulation test
Adrenocortical tumours: Recent Advances in 3. 18-hydroxycorticosterone

Basic Concepts and Clinical Management.

Annals of Internal Medicine 1999; 130:759-771 1. Unilateral 1. Normal or bilateral nodules
abnormality 2. Increase in aldosterone
2. Decrease in 3. <100ng/dl
aldosterone
3. > 100ng/dl

Inconclusive

Adrenal venous
sampling

Autonomous primary Lataralization No lateralization Idiopathic

hyperaldosteronism hyperaldosteronism
 CPSL National Guidelines / Endocrinology 73 74 CPSL National Guidelines / Endocrinology

1.7 Water Deprivation Test

Specimen collection and storage

Aldosterone These should be performed under the

supervision of a Paediatrician or a Physician.
Plasma (preserved with heparin, EDTA) or serum Separated serum should be sent to relevant
can be used. If an upright blood specimen is to be laboratory for the analysis.
collected, the subject should be in an upright position
(standing or seated) for at least 2 h before collection. Osmolality should be analyzed only by an osmometre.
Specimen should be stored frozen in an airtight container
and are stable for up to 2y at -200C. Transport the samples to the laboratory as
or urine assays, a complete 24-hour urine soon as they are collected.
specimen is collected with boric acid as a preservative. Laboratory should analyze them promptly.
50% acetic acid should also be added to the urine
collection to achieve a pH between 2.0 and 4.0. Rationale
Specimens should not be acidified with strong mineral This test is to establish the diagnosis of diabetes
acids, such as hydrochloric acid. insipidus in a patient with polyuria, polydipsia, and
Aliquots are stored frozen at -200C. It is possibly hypernatremia.
recommended that a urine sodium also be performed to With dehydration and increased plasma
facilitate the interpretation of the result. osmolality, arginine vasopressin (AVP) is released from
the hupothalamus/posterior pituitary gland. Under the
influence of AVP, water is reabsorbed from the
Renin
collecting duct lumen into the hyper osmotic renal
Blood should be collected in to an EDTA medulla along the osmotic gradient. This results in the
containing bottle. production of concentrated urine.
After centrifugation at room temperature, the
plasma is removed and frozen at -200C. or lower. Contraindications
reeze thaw cycles should be avoided because of the If the urine osmolality is > 800 mOsmol/Kg water
possible activation of prorenin. without exogenous 1-desamino-8-D arginine vasopressin
Plasma should be transported frozen to the laboratory. (dDAVP), the test is unnecessary and the patient is
considered normal.
At the time of collection, blood should not be
chilled or placed on ice because irreversible Before commencing the test hypothyroidism,
cryoactivation of prornin can occur, leading to falsely cortisol deficiency and osmotic diuresis must be
high estimates of plasma rennin activity. excluded.
 CPSL National Guidelines / Endocrinology 75 76 CPSL National Guidelines / Endocrinology

DDAVP should not be used in patients with Interpretation

vascular disease especially coronary atherosclerosis and If
renal failure. Serum osmolality is 275-295 mosmol/Kg
Urine osmolality > 600 mosmol/Kg
It is important to monitor vital signs during the Urine/serum osmolality ratio >2
dehydration procedure. The patient will be considered as having normal
If weight loss exceeds 2Kg (or 5% in children) or posterior pituitary reserve and further testing is not
the clinical condition deteriorates, the test must be indicated.
stopped. If the test is performed as above, adverse effects
are rare. Precautions
Before commencing the test hypothyroidism,
To ensure adequacy of dehydration, plasma cortisol deficiency and osmotic diuresis must be
osmolality before administration of dDAVP should be > excluded.
288 mOsmol/Kg water.
dDAVP (1-desamino=8-D-arginine vasopressin)
Side effects should not be used in patients with vascular disease
dDAVP may produce water intoxication only if especially coronary atherosclerosis and renal failure.
there has been an increase in urine osmolality. Because
of this, patients must not be allowed to drink freely after It is important to monitor vital signs during the
completion of the test if they have responded to the drug. dehydration procedure.
The early sign of water intoxication are drowsiness,
listlessness and headache. If weight loss exceeds 2Kg (or 5% in children) or
the clinical condition deteriorates, the test must be
Economization of resources stopped. If the given protocol is followed, adverse effects
could be minimised.
The following patients can be exempted from
following the complete test protocol after assessment of Water deprivation test is not indicated if serum
Posterior Pituitary Reserve in the following manner. sodium is > 145 mmol/L. Check the serum osmolality.

Basal investigations If the serum osmolality is > 300 mosm/Kg water

Serum osmolality in a patient suspected of having diabetes insipidus, or in
Urine osmolality very small children, testing can be done without
(Obtained simultaneously on rising or as soon as subjecting the patient to a period of dehydration.
possible thereafter in the OPD)
 CPSL National Guidelines / Endocrinology 77 78 CPSL National Guidelines / Endocrinology

Check basal serum and urine osmolality Continue until urine osmolality shows < 30
Give dDAVP (see below for dose) mOsmol/Kg water difference between three successive
Test serum and urine osmolality 2 hours later. collections.
.
Procedure Draw a sample of blood (1ml) for measured
The laboratory must be given notice in advance osmolality and sodium when the plateau is reached. The
of commencement of test/specimen collection laboratory will inform this to the ward.
.
Ensure availability of a ward environment in After blood has been collected, inject 5 units of
which the patient can be observed and in which the aqueous vasopressin subcutaneously or administer
patient does not have access to any water, including hand intranasal desmopressin acetate in the following doses.
washing basins, toilets etc.
Adults 0.4 ml (40 micrograms)
If mild polyuria exists, the subject should be Children 0.2 ml (20 micrograms)
fasted from 2200 hours (10.00 pm) the night before the Infants 0.1 ml (10 micrograms)
test, if moderate to severe polyuria exists, the subject
should be fasted from 0700 hours on the day of the test. One hour later collect urine for measured
In children the test should be carried out in the day time osmolality and blood sample for measured osmolality
under supervision. and sodium.
If there is no definite change in urine osmolality
Prior to fasting (before 0700 hours- 7.00 am) , repeat measurement of osmolality in urine one hour later.
weigh patient, take random urine( 1-2 ml) for osmolality
(do not use any preservative) and collect blood (2 ml) for
osmolality and serum sodium.

Collect urine hourly from 0800 hours (8.00 am)

on day of the test into a clean dry container and send
immediately to the laboratory for measured osmolality.

Weigh patient hourly from 0800 hours until

completion of the test.
 CPSL National Guidelines / Endocrinology 79 80 CPSL National Guidelines / Endocrinology

In complete nephrogenic diabetes insipidus, AVP

Guide to Interpretation levels exceed 3 ng/L when plateau osmolality is reached,
Urine osmolality Increase in Serum whereas in complete central diabetes insipidus, AVP
(mosmol/Kg urine osmolali levels do not exceed 1.5 ng/l.
water) before osmolality ty(mosm
dDAVP after dDAVP ol/ Kg Psychogenic polydipsia can be excluded when
water)
before 1. Serum Na > 145 mmol/L or serum osmolslity >
dDAVP 300 mosmol/Kg water
Normal ≥ 800 ( > 400 < 9%( No < 300 2. Urine/ plasma osmolality < 1.5
usually) rise in urine Serum
osmolaity) Na is Osmolality should be measured by an osmometer.
normal
Diabetes <800( < than that May be Recommendations
Insipidus of serum) > 50% to > 300 A. Should be carried out under the supervision and
recommendation of a consultant. Samples should
-Central reach ≥ 800
hypothala be sent to the relevant laboratory for analysis.
mic Please discuss with the relevant laboratory before
-Nephro usually < 300 sending the specimens.
< 10%
genic usually < 300
Sample Collection
Partial Urine osmol> Increase to <
central plasma 800 ( peak of
Osmolality
300-600)
Collect fresh urine (1 ml) and serum (1 ml)
Partial 300< urine < 800 No response
samples into a plain bottle without a preservative.
nephroge
Transport to the laboratory as soon as they are
nic
collected.
Laboratory should analyze them promptly.
 CPSL National Guidelines / Endocrinology 81

Antidiuretic hormone (ADH) or (AVP)

Blood specimens for ADH are collected into

chilled tubes containing EDTA as an anticoagulant.
Specimens should be transported to the laboratory
on ice and centrifuged at 40C within 30 min of collection.
Plasma is then removed and stored at frozen at
-200C until analysis is performed.
Significant deterioration occurs with prolonged
storage.

Reference ranges

Serum

neonate may be as low as 266 mOsmol/kg H2O

child and adult 275-295 mOsmol/kg H2O
> 60 yr 280-301 mOsmol/kg H2O

Urine random 50-1200 depending on the fluid intake

References
1. Tietz text book of Clinical chemistry Burtis CA,
AshwoodER (Eds) 3rd edition

2. Biochemistry manual, Department of Clinical

Biochemistry, Westmead Hospital for children,
Australia 2002