The document discusses various dynamic tests used in endocrinology, detailing their purposes, protocols, and interpretations. Key tests include the Insulin Tolerance Test, ACTH Stimulation Test, Growth Hormone Provocative Test, Water Deprivation Test, and others aimed at diagnosing endocrine dysfunction. Emphasis is placed on the importance of methodical execution, accuracy in hormonal assays, and careful interpretation of results.

1. DYNAMIC TESTS IN ENDOCRINOLOGY
Dr. Pranb Kumar Sahana
Associate Professor
Department of Endocrinology
NRS Medical College
Kolkata
Faculty Lecture
Date - 02.08.2021

2. Learning objectives
 What are dynamic endocrines tests ?
 How the tests are done ?
 How the tests are interpreted ?

3. Introduction
 Dynamic testing is integral to the diagnosis of endocrine
dysfunction.
 Dynamic function tests (DFT's) involve either stimulating
or suppressing a particular hormonal axis and observing
the appropriate hormonal response.
 In general, if a deficiency is suspected a stimulation test
should be used whilst if excess is considered likely, a
suppression test is required.

4. Overview of presentation
 Insulin Tolerance Test (ITT)
 (ACTH) stimulation /Synacthen Test
 Growth Hormone Provocative Test
 IGF1 Generation Test
 Overnight Dexamethasone Test (ONDST)
 GNRH stimulation Test
 HCG stimulation Test
 Androgen sensitivity Test
 Water Deprivation Test (WDT)

5. Insulin Tolerance Test (ITT)
 Principle: The stress of insulin induced hypoglycaemia
triggers the release of GH and ACTH from the pituitary
gland in normal subjects.
 GH response is measured directly. ACTH is typically
measured indirectly using cortisol, but may be measured
directly.
 It is considered as the gold standard for assessing GH and
ACTH reserve ( seconday adrenal insufficiency)

6. Insulin Tolerance Test
Indications:
 Assessment of cortisol
reserve
 Assessment of GH reserve
Contraindications:
 Ischaemic heart disease
 Epilepsy
 Untreated hypothyroidism
 Basal Cortisol < 6.5 μg/dl
Prerequsites:
 Euthyroidism
 Cortisol >6.5 μg/dL,
 Normal ECG

7. Protocol of ITT
 IV canulation
 Keep 25% Dextrose 100 ml ready at hand
 Inject IV Insulin @ 0.1 – 0.15 units/kg
 Check CBG every 5 minutes and maintain CBG chart.
 Adequate hypoglycemia blood glucose < 40 mg/dl with signs of
hypoglycemia (sweating, tachycardia etc.) is necessary for adequate
stimulation.
 If hypoglycemia is not achieved at 30 minutes ,increase dose by 50%.
 Draw sample (GH, Cortisol, glucose ) at 0,15,30,45,60,90,120 minutes .
 Reverse hypoglycemia by iv glucose after taking the the first sample.
Patient need not be hypoglycemic throughout the procedure.

8. Interpretation of ITT
 Test can not be interpreted unless adequate
hypoglycemia is achieved.
 Adequate cortisol respose : >18 μg/dL.
 Adequate GH response > 6 ng/ml.
 5-15% of normal individuls show suboptimal response.
 Concerns: Convulsion and increased mortality.

9. Adrenocorticotropic hormone (ACTH) stimulation
test/Synacthen test
 It tests the adequacy of the HPA axis
 Principle: Cortisol response is determined by the endogeneous ACTH drive to
the adrenal cortex.
 Indications:
1. To confirm the diagnosis of adrenal insufficiency (primary and long-standing
secondary insufficiency)
2.To confirm the diagnosis of CAH
 This test is performed using synthetic ACTH 1–24 (Synacthen).
 Test is not necessary if serum cortisol is < 5.0 μg/dL and ACTH is elevated by
more than two fold.
 Basal cortisol > 14.5 μg/dL indicates an intact HPA axis.

10. Protocol of Short Synacthen Test
(standard)
 Fasting is not required and the test can be performed
irrespective of the time of the day.
 Synacthen dose: age: <1 year: 15 μg/kg, 1–2 year: 125 μg,
 >2 year: 250 μg.
 Administration: Intramuscular or intravenous.
 Collect blood at 0, 30 min and 60 min after the injection
for measurement of cortisol (and 17-OH progesterone, if
needed).

11. Interpretation of SST
 A peak serum cortisol ≥ 20 μg/dL at 30 or 60 min
indicates normal HPA axis.
 (30 or 60 min) serum 17-hydroxyprogesterone ≥10 ng/mL
rules out CAH.
 Low dose synacthen test with 1 μg synacthen test is done
to detect subtle adrenal insufficiency.
 Recent onset (3 months, Surgery,apoplexy)adrenal
insufficiency / secondary adrenal insufficiency, LDSST has
better sensitivity (83% vs 64 %).

13. SSST (standard) VS LDSST (low dose)

14. SSST VS LDSST

15. Growth Hormone provocative test
 Assessment of GH secretion is difficult as GH secretion is
pulsatile.
 GH secretion varies with age, gender, pubertal stage,
nutritional status.
 GH secretion is minimum in between pulses (< 0.1 ng/ml)
 Random GH measurement is useless in diagnosis of GHD.
 Stimulation test forms the foundation for the diagnosis of GHD.
 It is indicated in short stature by auxological criteria due to
suspected GHD.

16. Growth Hormone provocative tests
Stimulus Dosage Sampling
(minutes)
Sensitivity,
Specificity
Insulin
(IV)
0.05-0.15 units/kg 0,15,30,60,90,120 80-90%
Levodopa < 15 kg-125 mg
15-30 kg- 250 mg
>30 kg-500 mg
0,30,60,90,120 85%,80%
Clonidine 0.15 mg/m2 0,30,60,90,120 70%,85%
Glucagon(
IM)
0.03 mg/kg(max 1
mg)
0,30,60,90,120,150
,180
86%, 95%

17. Sex steroid priming
 Sex steroid priming: It increases the specificity of the test.
 Indicated in
1. Children with bone age of ≥10 years or with delayed puberty.
2. Prepubertal boys >11 years, Girls>10 years.
How priming is done?
 In boys: 100 mg depot testosterone IM 3 days prior.
 Girls: Ethinyl estradiol 100 μg or 1-2 mg micronized estradiol daily for 3 consecutive
days.

18. Interpretation of GH provocative test
 Lower cut off, higher specificity.
 10 ng/ml is the current accepted cut off in general.
 Higher cut off will diagnose partial GHD also.

19. Limitations
 The tests are nonphysiologic.
 The cutoff level of "normal" is arbitrary and may depend on the specific
provocative agent used.
 Adiposity also influences GH response to the stimulation test, such that obese
children show diminished GH responses to all stimuli
 The tests rely upon GH assays of variable accuracy.
 Test reproducibility has not been adequately documented.
 The tests are expensive, uncomfortable, and carry some risks.
 The ability of the tests to discriminate between normal short children and
children with partial GHD is limited.

20. IGF1 generation test
 Indication: Short stature due to suspected GHI (Low IGF1
but high basal GH).
 Protocaol:
 Day 1 –Collect basal IGF1
 Inj rGH 0.1 unit/kg/day SC for day 1 to day 4
 Day 5 – Collect sample for IGF1
Inerpretation: An increase of IGF1 by < 15 μg/L from the
baseline value indicates GHI.

21. Water deprivation test
 Raising the plasma osmolality (and serum sodium concentration) leads to a
progressive elevation in ADH release and an increase in urine osmolality in
normal individuals.
 Once the plasma osmolality reaches 295 to 300 mosmol/kg (normal 280 to 290
mosmol/kg) or the plasma sodium is 145 mEq/L or higher, the effect of
endogenous ADH on the kidney is maximal.
 Indication:
1. Diagnosis of Diabetes Insipidus .
2. Differential diagnosis of polyuria (> 4ml/kg/hr or 24 hour urine output >3 L or
50 ml/kg in adult or 2 L/m2 in children.

22. What is normal?
 Normal plasma osmolality: 280- 295 msom/kg.
 An individual with a normal diet and normal fluid intake has a urine
osmolality of approximately 500-850 mosm/kg water.
 A healthy kidney can concentrate urine to 800-1400 mosm/kg. of water
 U/P osmolality ratio is > 2.0
 Exclude other causes of polyuria (hypercalcemia ,hypokalemia).

23.  If the basal plasma osmolality >295 msom/kg , Na > 145 mmol/L and urine is
hypotonic (< 300 msom/kg) water deprivation can be skipped.
 Random urine osmolality> 750 msOm/kg rules out DI.
 Prerequisites:
 DDAVP should be stopped 24 hours prior.
 Thyroid and adrenal function should be normal.
 Individuals with severe polyuria and a urine osmolality <100 mosmol/kg (who
likely have complete DI) should not undergo overnight water restriction.

24. Water deprivation test (WDT)
PROTOCOL:
 Test must be undertaken with strict supervision and following protocol.
 Start the test at 8 AM. Weigh the patient, check BP and vitals. Calculate 5% of
weight. Insert IV cannula.
 Collect sample for plasma and urine osmolality and plasma Na.
 Do not allow fluid but dry food .
 Collect and measure urine hourly and weigh hourly.
 Monitor vitals and look for volume status.

Hours of projected water restriction = Weight (kg) x (0.03) x 1000 ÷ Measured
urine volume/hour (mL/hour).

25. Termination of the test -WDT
 Test is continued for 8 hours or
> 5 % weight loss.
Signs of hypovolemia present.
Urine osmolality exceeds 750 msom/kg.
Plasma osmolality > 295 msom/kg.
 Take sample for plasma osmolality, Na, AVP and urine osmolality at the time of
termination.
 Patient now can eat and drink.
 Inj Aqueous AVP 5 U/m2 SC or DDAVP 10 μg intranasally or DDAVP 2 μg im/sc.
 Collect urine sample for osmolality hourly for 4 hours.

26. Interpretation of Water Deprivation Test
Basal Urine
osmolality()
Urine
osmolality
msom/kg.
(post
dehydration)
Urine
osmolality
msom/kg.
(Post DDAVP)
% Change in
urine
osmolality
> 750 >750 Normal
<300 <300 >750 >50 % CDI
<300 300-750 300-750 9-50% Partial DI/PP
<300 <300 300-750 <9% NDI
Normal response: Progresive rise of urine osmolality with water deprivation
and plasma osmolality rises but remains below 295 mosm/kg.
When well performed the test has a sensitivity and specificity of 95% for severe CDI and NDI

27. Partial CDI/Primary polydipsia
 If the water restriction test is nondiagnostic, we perform a therapeutic trial
of desmopressin, as follows.
 For three days, desmopressin (10 mcg intranasally) is given in the evening
and patients are advised to restrict their fluid intake to less than 1.5 to 2
L/day.
 Assessments of symptoms (thirst and polyuria), urine osmolality, and serum
sodium (obtained as "stat") are made twice daily.
 In patients with partial central DI, the polyuria and polydipsia will reverse,
the urine will become concentrated and, if the patient was adherent to fluid
restriction, the serum sodium should remain normal .
 In patients with primary polydipsia, desmopressin will not relieve the thirst,
although the urine will be concentrated and, because of persistent water
intake, the patient can become hyponatremic.

28. CDI/Primary polydipsia
 Copeptin is the C-terminal glycoprotein moiety of the AVP prohormone and
therefore a surrogate marker of ADH.
 To distinguish central DI from primary polydipsia, hypertonic saline is infused
to raise the serum sodium to >145 mEq/L, at which point plasma copeptin is
measured.
 Central DI is diagnosed if the plasma copeptin is ≤4.9 pmol/L.
 Primary polydipsia is diagnosed if the plasma copeptin is >4.9 pmol/L.
 A low plasma sodium concentration with a low urine osmolality (eg, less than
one-half the plasma osmolality) is indicative of water overload due to primary
polydipsia.

29. Overnight Dexamethasone Suppression Test
 Principle:The test assesses the hypothalamic and pituitary corticotroph
cell responses to glucocorticoid negative feedback inhibition of
corticotropin-releasing hormone (CRH) and ACTH secretion.
 Indication: Endogeneous Cushing syndrome
 Outpatient basis
 Precautions: Stop enzyme inducing drugs
 Administer tab dexamethasone 1mg or 30 μg/kg at 2300 hours and
measure cortisol at 8 AM in the next morning.
 Interpretation: < 1.8 μg/dl indicates good suppression.
 Problem: High false positivity. Needs repeat test or 48 h LDDST.
 Sensitivity- 95%,Specificity-80% (97% in 2day LDDST)

30. Spurious results - ONDST
 Pseudocushing’s
 Acute stress
 Altered bioavailability of dexamethasone- malabsorption,
drugs, non compliance
 Altered CBG binding

31. Gonadotropin-releasing hormone
(GnRH) agonist stimulation test
 Indications:
1. To differentiate CPP from precocious pseudo puberty.
2. To differentiate between CDGP and HH.
3. To assess the pubertal status.
 Principle of the test: A primed pituitary will respond to GnRH
 Protocol : Inject 100 µg/m2 triptorelin (maximum 100 µg) IV or subcutaneously.
 Measure serum LH,FSH at baseline , and at 30 min, 60 min (IV)
 Or at 1 h, 2 h and 4 h ( if injected SC) .

32. Interpretation of GnRH stimulation
test
 Suggestive of puberty or GnRH dependent Precocious Puberty or good
pituitary reserve or CDGP
 Peak LH ≥10 IU/L (≥8)
 Peak FSH ≥ 2 IU/L
 or ≥ 2-3 fold increase from baseline.
 In HH – response will be subnormal.

33. HCG stimulation test
 This test is used to evaluate Leydig cell function in prepubertal boys
 Indications:
1. To detect functioning testicular tissue in patients with Cryptorchidism
2. Evaluation of patients with testosterone biosynthetic defects
 Protocol: Three-day stimulation test
 Dose: <1 year: 500 IU intramuscular HCG for three consecutive days
(days 1–3). 1–10 year: 1000 IU, > 10 years: 1500 IU.
 Sampling: Obtain serum testosterone (T) at baseline and 24 h after
the last injection (day 4).
 In addition, while evaluating for testosterone biosynthetic defects,
blood may be sent for dihydrotestosterone (DHT) androstenedione (A)
measurement.

34. Interpretation of HCG stimulation test
 In normal individuals, peak serum testosterone level of 2.3–2.8 ng/mL
 Stimulated testosterone <1.0–1.4 ng/mL considered as an abnormal
response.
 A cutoff value of 1.4 ng/mL may be used during the evaluation of
cryptorchidism, and 1.0 ng/mL for differentiation between CDGP and
HH.
 A stimulated T/DHT ratio >10 indicates 5-alpha-reductase type 2
deficiency.
 T/A ratio<0.8 is suggestive of 17β-HSD3 deficiency. Biochemical
findings can be variable and genetic testing is required for the
confirmation of the diagnosis in both conditions.

35. Stanozolol- Sex Hormone-binding Globulin (SHBG)
test (Androgen sensitivity test)
 Indication:
 To support the diagnosis of androgen insensitivity syndrome.
 Protocol
 Administer stanozolol (e.g., menabol, tanzol, neurobol) orally (0.2 mg/kg/day)
for 3 consecutive days in a single evening dose (days 1–3).
 Obtain serum SHBG at baseline (before the first dose) and on days 5, 6, 7, and
8. All samples should be taken between 2 pm–6 pm. The lowest serum SHBG
measured on days 5–8 represents the nadir and is representative of the largest
response to stanozolol.
 Calculate the ratio of nadir serum SHBG to baseline serum SHBG.

36. Interpretation : Androgen sensitivity test
 A nadir SHBG ≤63.4% of the baseline level is considered
normal.
 The nadir SHBG levels range between 73 to 89% and 93 to
97% of the baseline in subjects with PAIS and CAIS,
respectively.

37. Take home
 Dynamic tests are essential tests in diagnosis of Endocrine diseases
 Dynamic tests should be done methodically
 Hormonal assays must be accurate
 Test results should be interpreted carefully