practical provocative test

2. For some of the hormones, a substance is given that would
normally affect hormone production and then the level of the
hormone is measured. For example, if insulin is injected, the levels
of ACTH, GH and prolactin should increase. Sometimes, rather
than measuring GH levels directly, another hormone, insulin like
growth factor I (IGF-1) is often measured. GH is produced in
bursts and its levels quickly fall, but IGF-1 levels reflect the
overall daily production of GH.
A stimulation/provocation test is a test that is performed to reveal
a clinical condition; suspected but not fully manifested clinically.
Any procedure in
which
a
suspected
pathophysiological abnormality is deliberately induced by
manipulating conditions known to provoke the abnormality.
The test can be either stimulation for hypoactive medical problem,
or suppression for hyperactive function.

3. In general
GH
TSH
Stairs up
ACTH

4. Indications
Confirming suspected diagnosis.
Monitoring the course of disease.
Following the effect of therapy.
Establishing the significance of borderline low values by
stimulation tests.
Document the presence of hyperfunction by suppression
tests.
Distinguish primary from secondary causes of endocrine
dysfunction.

5. Clinical applications
Almost every system disorder in clinical evaluation of disease
could use a stimulation test.
It can be the only test available in certain situations like in “drug
induced diseases” by challenging the patient with the suspected
drug.
Stimulation testing is commonly used in endocrinology based on
the feedback system.
There are several different types of stimulation testing that can be
ordered to help the doctor diagnose child’s medical condition.
All stimulation testing can take several hours to complete. The
test should take from 1 to 3 hours, including registration, the test,
and a snack afterward.
Most stimulation tests are “fasting,” meaning that child should
not eat or drink before the test. Follow specific instructions given
about eating and drinking before the test.

6. The child will need to have an intravenous (IV) line placed to
start the testing. Blood samples will be removed from the IV
line at different times throughout the test.
Stimulation testing measures the response of certain glands
within the endocrine system to different types of
hormones. Some of the stimulation tests that might be ordered
include:
Growth Hormone Stimulation — used to find out if child’s
pituitary gland is producing enough growth hormone
Lupron Stimulation — used to diagnose precocious (too
early) or delayed (too late) puberty in boys and girls
Glucose Tolerance — used to rule out diabetes, hypoglycemia
and insulin resistance
ACTH Stimulation — used to find out if your child’s adrenal
glands are producing enough cortisol and to rule out congenital
adrenal hyperplasia

8. PURPOSE
Growth hormone tests are ordered for the following reasons:
to identify growth deficiencies, including delayed puberty and
small stature in adolescents that result from pituitary or thyroid
malfunction
to aid in the diagnosis of hyperpituitarism that is evident in
gigantism or acromegaly
to screen for inadequate or reduced pituitary gland function
to assist in the diagnosis of pituitary tumors or tumors related
to the hypothalamus, an area of the brain
to evaluate hGH therapy

9. GROWTH HORMONE
Quantitative GH
Fasting level
Provocative test for suspected deficiency
Hypoglycemia causes GH levels to rise
 Give
insulin, L-dopa or arginine
 take samples at 0, 15, 39, 45, 60, 90, and 120 minutes
 Small rise  partial deficiency
 No rise  inability to secrete GH
Suppression test
Hyperglycemia suppresses GH but not in autonomous or
elevated GH
 Give glucose

10. NORMAL RESULTS
Results are reported in nanograms per milliliter (ng/ml). Normal
results may vary from laboratory to laboratory depending upon the
method used for measurement, but results are usually within the
following ranges.
SOMATOTROPIN (hGH):
men: 5 ng/ml
women: less than 10 ng/ml
children: 0–10 ng/ml
newborn: 10–40 ng/ml
SOMATOMEDIN C:
adult: 42–110 ng/ml
child: 0–8 years; girls 7–110 ng/ml; boys 4–87 ng/ml
9–10 years: girls 39–186 ng/ml; boys 26–98 ng/ml
11–13 years: girls 66–215 ng/ml; boys 44–207 ng/ml
14–16 years: girls 96–256 ng/ml; boys 48–255 ng/ml

11. ABNORMAL RESULTS
Somatotropin hormone: Excess hGH is responsible for the
syndromes of gigantism and acromegaly. Excess secretion is
stimulated by anorexia nervosa, stress, hypoglycemia, and
exercise. Decreased levels are seen in hGH deficiency, dwarfism,
hyperglycemia, failure to thrive, and delayed sexual maturity.
Somatomedin C: Increased levels contribute to the syndromes of
gigantism
and
acromegaly.
Stress,
major
surgery,
hypoglycemia, starvation, and exercise stimulate hGH secretion,
which in turn stimulates somatomedin C.
Growth hormone stimulation: Decreased levels are seen in
pituitary deficiency and hGH deficiency. Diseases of the pituitary
can result in failure of the pituitary to secrete hGH and/or all the
pituitary hormones. As a result, the hGH stimulation test will fail
to stimulate hGH secretion.

12. Pitfalls
False negative or positive results if the test is not
conducted properly.
Failure to provide safety measures during a challenge
test may harm the patients.
Inadequate preparation following the exact procedure
for the test can provide misleading results.
Lab errors may confuse the results (wrong sample
labeling, wrong time, poor calibration).

14. Thyroid hormone production is under the control of pituitary
TSH and in turn hypothalamic TRH. Plasma TSH levels
normally increase rapidly (2 to 5 minutes) after an IV bolus of
TRH, with a subsequent more gradual increase in T3 secretion
from the thyroid. The TRH test thus allows the integrity of the
thyroid axis to be tested.
Abnormalities of the prolactin response to TRH may occur in
pituitary tumors (especially GH or prolactin producing) or in
pituitary stalk pathology. Responses are not diagnostically
pathognomonic, but indicate axis disruption or dysregulation.
In GH excess states (gigantism/acromegaly), TRH may cause
an elevation of GH, but not in normal subjects.

15. Indications secretion and thyroid
1.To assess the response of pituitary TSH
hormone production to stimulation. The main indication is in
suspected secondary (pituitary) or tertiary (hypothalamic)
hypothyroidism. Less frequently may be of assistance in mild
primary hypothyroidism.
2.To assess the response of prolactin to stimulation
3.Less commonly used in investigation of gigantism/acromegaly.
Often performed as part of a combined pituitary function test
(triple test).
Dose:
200 micrograms/m2 BSA by slow intravenous injection over 1 min.
Sample:
0 min, 15min, 30min, 45min, 60min, 90min and 120min
Analysis:
TSH, Free T3, Free T4, Prolactin

16. Interpretation
A normal response is a rapid rise in TSH, peaking between 10-
30 mU/l at 20-30 minutes, then gradually declining to reach
baseline after 2 to 3 hrs. T3 values show a rise, but do not peak
until 3-4 hrs (30-70% rise from baseline).
Prolactin levels are age dependent. Above 1 year age, mean
basal levels are approximately 240 mU/l, rising to
approximately 725 mU/l with TRH stimulation (i.e. a 2-3 fold
rise).
In hyperthyroidism (Grave's disease), T3 and T4 are elevated
and TSH levels are suppressed and unresponsive to TRH
stimulation.
In primary hypothyroidism basal T3 and T4 are low, with
elevated basal TSH and an exaggerated TSH response (usually
to a peak > 30 mU/l at 30-40 min).

17. Conti……
In secondary (pituitary) hypothyroidism T3 and T4 levels are
likely to be low and there is a poor TSH response and poor T3
response.
In tertiary (hypothalamic) hypothyroidism, an exaggerated and
prolonged TSH peak may be seen, and a T3 response occurs.
While prolactin responses are variable, an exaggerated
prolactin response suggests hypothalamic disease or stalk
disruption, owing to loss of inhibitory effects of dopamine.
A poor prolactin response suggests pituitary disease.
In normal subjects TRH induces no rise in GH levels, but it
may do so in pituitary gigantism / acromegaly.

18. Adverse effects
Nausea, flushing, dizziness, urinary urgency, unusual taste
in mouth, occasionally headaches.
Increases in BP and pulse rate frequently observed
Caution in heart failure, myocardial ischemia and asthma.
Caution in severe hypopituitarism - risk of hypoglycemia
Certain drugs may diminish response.

20. Cortisol
Cortisol
Cortisol
ACTH
Fasting
Glucose

21. Rationale
ACTH is the primary regulator of glucocorticoid production,
and also plays some role in adrenal androgen production.
 Synacthen is a synthetic form of ACTH, is used to assess the
stimulated cortisol response of the adrenal cortex and is
valuable in diagnosing suspected primary adrenal
insufficiency.
The test is also useful in suspected secondary or tertiary
adrenal insufficiency since chronic CRH/ACTH deficiency or
dysregulation results in temporary quiescence of the adrenal
cortex and inability to respond acutely.

22. Conti…..
Furthermore, the test is not reliable in assessing secondary or
tertiary insufficiency within 2 weeks of surgery to the
hypothalamic-pituitary region or a major alteration in any
glucocorticoid therapy.
In congenital adrenal hyperplasia, Synacthen test is useful in
diagnosing milder or rare enzyme blocks by examining ratios
of various adrenal steroids to their precursor compounds. The
commonest ratio examined is that of 17-hydroxyprogesterone /
cortisol in suspected non-classical or simple virilizing CAH or
the heterozygote state.

23. Standard doze of synacthen
Over 1 yr: Single IM or IV injection of 250 micrograms.
Under 1 yr: Single IM or IV dose of 125 micrograms.
Alternatively a dose of 250 micrograms/m2 BSA may be
used.

24. Blood sampling
Samples are collected at 0 min, 30 min and 60 min.
Cortisol, 17-OHProgesterone, ACTH and Androgens
(DHEAS, Androstendione & Testosterone).

25. Interpretation
Serum cortisol rise of > 280 nmol/l with maximal level >
600 nmol/l.
Normal ratio of 17-OHP to cortisol at 30 mins < 0.023.
Ratios up to 0.08 suggest heterozygosity for 21-
hydroxylase deficiency.
ratios > 0.1 suggest CAH (21-hydroxylase deficiency).

26. Adverse effects
Hypersensitivity or anaphylactic reaction - rare, but full
resuscitation facilities and drugs must be available.

28.  GnRH (gonadotropin releasing hormone) is a decapeptide
secreted by the hypothalamus which stimulates the
production and secretion of LH and FSH by the anterior
pituitary.
 The GnRH stimulation test evaluates the ability of
gonadotropes to secrete LH and FSH after exposure to the
natural hypothalamic releasing hormone, GnRH, or an
analog.
 The GnRH test is extensively evaluated for the discrimination
of disorders of precocious or delayed puberty and
amenorrhea.
 The GnRH test is useful to monitor adequate treatment of
gonadotropin-dependent precocious puberty.

29. Methods
This test may be done with either recombinant GnRH or a GnRH
agonist, such as leuprolide.
Several different methods have been used:
 Administer 100 micrograms of GnRH (or GnRH analogue)
IV x 1 at time 0. Measure serum LH at time -15 minutes, 0,
+15, +30, +45, and +60 minutes after GnRH administration.
 Administer 25 micrograms/M2 of GnRH IV at 8am after an
overnight fast. Measure serum LH and FSH at time 0, 15 min,
30 min, 60 min, and 90 min after GnRH injection.
 Administer leuprolide 20 mcg/kg x 1, measure LH and FSH
at baseline and after 30 and 60 minutes.

30. Assay requirements
In general, assays for LH and sex hormones (estradiol and
testosterone) must be sensitive enough to detect pre-pubertal
levels. Prepubertal LH is less than 0.1 IU/L and pre-pubertal
E2 is <1 pg/L (undetectable with most available assays).
Immunochemiluminometric assays are much more sensitive
than radioimmunoassay for gonadotropin levels in the prepubertal, thus these should be used, especially when measuring
basal gonadotropin levels.

31. Interpretation
 In general, in gonadotropin dependent precocious puberty
(GDPP), basal LH is elevated (pubertal range) and increases
further with GnRH stimulation (peak LH >5-8 IU/L suggests
GDPP), and in gonadotropin independent precocious puberty
(GIPP), basal LH is low (in the pre-pubertal range) and does
not increase with GnRH stimulation. In addition, stimulated
LH/FSH ratio may be help distinguish GDPP (higher LH:FSH)
from nonprogressive PP, which does not require treatment with
exogenous GnRH. Otherwise, the FSH values (basal or
stimulated) are generally not useful.
 With either GnRH or GnRHa tests, the ratio of peak to basal
LH is also used to define precocious puberty. In general, a
ratio of LH (peak): LH (basal) <3.0 is considered normal and a
ratio of LH (peak): LH (basal) >3.0 is consistent with
precocious puberty.

32. Side effects
 The patient may react to the side effect of GnRH, transient
thirst may occur. Abdominal or stomach discomfort, flushing
(lasting only a short time), headaches, lightheadedness, nausea
may also occur but less common.
 Hypersensitivity reactions to GnRH, such as itching, redness or
swelling of skin at place of injection, skin rash, breathing
difficulty etc may occur but they are rare.
 Rarely, GnRH injection has been associated with pituitary
apoplexy.