The document provides information on various diagnostic tests for adrenal disorders in children, including the ACTH test, serum cortisol test, urinary free cortisol test, salivary cortisol test, and different dexamethasone suppression tests. The ACTH test can help differentiate primary and secondary adrenal insufficiency and ACTH-dependent and independent causes. The cortisol tests evaluate cortisol levels at different times to screen for adrenal insufficiency or excess. The dexamethasone suppression tests use dexamethasone to inhibit ACTH and cortisol production, with inability to suppress indicating hypercortisolism.

1. azad82d@gmail.com
azad.haleem@uod.ac
Dr.Azad A Haleem AL.Mezori
MRCPCH,DCH, FIBMS
Assistant Professor
University Of Duhok
College of Medicine
Pediatrics Department
Diagnostic test for Adrenal
disorders in children
Scan
For
Contact

2. Key points
• ACTH Test
• Serum Cortisol
• Urinary free cortisol 24 hours
• Salivary cortisol
• Dexamethasone Suppression test
• Low-Dose Dexamethasone Suppression Test
• High- Dexamethasone Suppression Test

3. Introduction
• Adrenal glands are small, triangular glands
that are located on the top of each kidney.
• This gland is made up of the adrenal cortex,
which is the outer part, and the adrenal
medulla, which is the inside of the gland.
• The glands work with the pituitary gland and
the hypothalamus, while also secreting
hormones affecting the metabolism, body
characteristics, and blood chemicals.

4. ACTH Test
• Role
• To differentiate primary from secondary adrenal insufficiency.
• To differentiate ACTH dependent from an independent cause.
• Pre-analytical issues
• Timing- Draw the sample at 7-9 am. Collect sample simultaneously with
cortisol
• Container- Take in a plastic syringe and transfer to the EDTA tube.
• Transport- Transport immediately on ice.
• Reference range
• Prepubertal- 7-28 pg/ml (1.5-6.2 pmol/L)
• Pubertal- 2-49 pg/ml (0.4-10.8 pmol/L)

5. ACTH Test
• Interpretation
• Adrenal insufficiency
• Levels above 300 pg/ml (66 pmol/L) conclusively
prove primary adrenal insufficiency while low or
normal level indicates secondary adrenal insufficiency.
• Cushing syndrome
• ACTH level below 5 pg/ml (1.1 pmol/L) suggests
ACTH independent cause while those above 20 pg/ml
(4.4 pmol/L) point to ACTH dependent cause.
• Very high levels indicate ectopic ACTH secreting
tumor.

6. ACTH stimulation test
• Role: To determine the secretory capacity of the adrenal cortex.
• Rationale
• Cortisol secretion exhibit diurnal variation and pulsatility limit the value of
a single baseline level. ACTH is an endogenous stimulus for the production
of cortisol and androgens from the adrenal cortex. Exogenous
administration of ACTH stimulates cortisol synthesis and secretion acutely
and chronically. Response to ACTH is compromised in both primary
(adrenal) and secondary adrenal insufficiency (hypothalamic-pituitary
insufficiency).
• Indication
• Suspected adrenal insufficiency with borderline/low cortisol levels.
• Diagnosis and classification of 21 hydroxylase deficiency.
• Protocol
• ACTH stimulation test can be performed at any time of day.
• No fasting is required.

7. Standard-Dose ACTH Stimulation Test
(Short Synacthen Test)
• Dose: Injection synacthen 15 µg/kg for children less than 2 years
and 250 µg for children above 2years intravenous or intramuscular.
• Sampling
• Sample for serum cortisol (adrenal insufficiency) or 17
hydroxyprogesterone (CAH) taken 60 minutes after injection.
• Interpretation
• Cortisol above 550 nmol/L 18 µg/dl) or the rise of cortisol above
190 nmol/L (7 µg/dl) from baseline excludes adrenal insufficiency.
• 17 hydroxyprogesterone above 1000 ng/dl (>30 nmol/L) confirms
21 hydroxylase deficiency.
• Limitation
• Not sensitive for early and subtle cases of secondary (hypothalamic-
pituitary) adrenal insufficiency where adrenal produces some
response to ACTH stimulation.

8. Low-Dose ACTH Stimulation Test
(Low-Dose Synacthen Test)
• Indication
• Suspected subtle cases of secondary adrenal insufficiency.
• Dose
• Injection synacthen 0.5 µg/m2 (maximum 1 mcg)
intravenous or intramuscular.
• Sampling
• Sample for serum cortisol taken 60 minute later.
• Interpretation
• Cortisol above 550 nmol/L 18 µg/dl) or the rise of cortisol
above 190 nmol/L (7 µg/dl) from baseline excludes adrenal
insufficiency.

9. Long ACTH Stimulation Test
• Dose
• Injection Acton prolongatum (1 ml/250 IU,5 ml) 25 IU
intramuscular.
• Sampling
• Sample for serum cortisol at 4, 24 and 48 hours after
injection.
• Interpretation
• Cortisol level at 4 hours above 1000 nmol/L considered
a normal response.
• Cortisol at 4 hours below 1000 nmol/L with no rise at
24 and 48 hours suggest primary adrenal insufficiency.
• Cortisol value at 4 hours below 1000 nmol/L with a
gradual rise at 24 and 48 hours suggests secondary
adrenal insufficiency.

10. Serum Cortisol
• Role
• Screening for adrenal insufficiency or excess.
• Single abnormal basal cortisol level is not diagnostic of a condition and should be
followed up with dynamic tests.
• Sample type: Serum
• Timing
• Morning fasting sample for diagnosis of adrenal insufficiency.
• Midnight sample for diagnosis of cushing syndrome.
• The sample can be obtained at any time of the day in infants due to lack of diurnal
rhythm.
• Container: Plain vacutainer for serum.
• Transport: Transport refrigerated.
• Reference range
• Serum 8 AM
• Preterm newborn- 6.5 µg/dL (179.4 nmol/L)
• Term newborn- 1.7–14 µg/dL (46.9–386.4 nmol/L)
• 1 – 11 months- 2.8–23 µg/dL (77.2–634.8 nmol/L)
• 12 months – 15 years- 3–21 µg/dL (82.8–579.6 nmol/L)

11. Serum Cortisol
• Interpretation
• 8 am Cortisol level less than 140 nmol/L (5 mcg/dL) suggest the possibility
of adrenal insufficiency. Lower cutoffs are used in the first week (100
nmol/L, 3.6 mcg/dL) and from day 7-30 of life (70 nmol/L, 2.5 mcg/dL).
• Low cortisol with ACTH level above 300 pg/ml (66 pmol/L) indicated
primary adrenal insufficiency while low or normal levels of ACTH indicate
secondary adrenal insufficiency.
• During stress/hypoglycemia/hyponatremia, cortisol level less than 500
nmol/L (18 µg/dL) suggest an adrenal insufficient response.
• A low level of cortisol with the clinical picture of Cushing syndrome
suggests suppression of the Hypothalamo-pituitary axis by exogenous
steroids.
• Midnight cortisol levels above 50 nmol/L (1.8 mcg/dL) in sleeping
and 207 nmol/L (7.4 mcg/dL) in an awake sample suggests the diagnosis
of cushing syndrome.

12. Urinary free cortisol 24 hours
• Role
• Screening test for Cushing syndrome.
• Test of choice in patients with altered cortisol binding globulin (pregnancy, oral
contraceptive pills), those on drugs altering dexamethasone metabolism
(antiepileptic drugs) and cyclic Cushing syndrome.
• Not useful for patients with renal failure and those with difficulty in complete urine
collection.
• Procedure
• Complete 24 hour collection is essential.
• Discard the first morning void with collection from the empty bladder.
• All subsequent voids up to the next morning first void should be collected.
• Keep refrigerated (not frozen).
• Avoid excessive water drinking and avoid steroid containing preparations.
• Obtain the sample twice.
• Measure the total urine volume and creatinine for the completeness of sample
collection.
• Container: Plastic container
• Transport: Transport refrigerated.

13. Urinary free cortisol 24 hours
• Reference range
• Adults- 4–50 μg/24 h (11–138 nmol/24 h)
• Children
• 1–4 years- 0.9–8.2 μg/24 h (2.5–22.6 nmol/24 h)
• 5–9 years- 1.0–30.0 μg/24 h (2.8–82.8 nmol/24 h)
• 10–13 years- 1.0–45 μg/24 h (2.8–124.2 nmol/24 h)
• 14–17 years- 3.0–55 μg/24 h (8.3–151.8 nmol/24 h)
• Interpretation:
• A level above 3 times the upper limit suggests Cushing
syndrome.

14. Salivary cortisol
• Role
• Screening test for Cushing syndrome and adrenal
insufficiency.
• Procedure
• Saliva is collected by passive drooling or by chewing cotton
pledget for 1-2 min.
• Collect multiple samples and refrigerate.
• Container
• Plastic container
• Transport
• Transport refrigerated or at room temperature.

15. Salivary cortisol
• Reference range
• Adults
• 8–10 a.m.- 0.04–0.56 mcg/dL (1.1–15.4 nmol/L)
• 4–6 p.m.- Less than 0.15 mcg/dL (<4.1 nmol/L)
• 10–11 p.m.- Less than0.09 mcg/dL (2.5 nmol/L)
• Interpretation:
• Elevated midnight level above 5 nmol/L (0.18 mcg/dL)
suggests Cushing syndrome.

16. Dexamethasone Suppression test
• Role
• Diagnosis and classification of Cushing syndrome.
• Rationale
• Cortisol secretion exhibit diurnal variation and pulsatility limit the
value of a single baseline level.
• Administration of glucocorticoid in supra-physiologic dose inhibits
ACTH secretion reducing cortisol levels.
• Dexamethasone is a potent and synthetic glucocorticoid and it is not
detected by commonly available assay, so it is used for suppression
tests.
• Inability to suppress cortisol production by dexamethasone suggests
hypercortisolism.
• Indication
• Suspected cushing syndrome

17. Dexamethasone Suppression test
• Conditions affecting dexamethasone suppression test
• Dexamethasone metabolized by Cyp3A4 enzyme in liver.
• Drugs inducing Cyp3A4 enzyme (phenytoin, phenobarbitone,
carbamazepine, rifampicin) accelerate dexamethasone metabolism
resulting in false-negative results.
• Drugs inhibiting Cyp3A4 enzyme (itraconazole, cimetidine,
fluoxetine, ritonavir) reduce dexamethasone metabolism giving
false-positive tests.
• Serum cortisol measures both bound and free cortisol.
• Hyperestrogenic states (oral contraceptive pills and pregnancy)
increase cortisol binding globulin levels resulting in a false-positive
diagnosis of Cushing syndrome. Estrogen preparation should be
stopped 6 weeks prior to the test.
• Conditions like nephrotic syndrome, liver disease lower cortisol
binding globulin cause false-negative tests.

18. Dexamethasone Suppression test

19. Overnight Dexamethasone
Suppression Test
• Role
• Screening test for Cushing syndrome.
• Dose
• Tab dexamethasone 0.3 mg/m2 (maximum 1 mg) orally at 11 -12
PM.
• Sampling
• Sample for serum cortisol next day morning 8-9 am.
• Interpretation
• Cortisol above 50 nmol/L (1.8 µg/dl) suggest hypercortisolism.
• Advantage
• Good sensitivity so used for screening purposes.
• Limitation
• Poor specificity.

20. Low-Dose Dexamethasone
Suppression Test
• Role
• Screening test for Cushing syndrome.
• Procedure
• Tab dexamethasone 1.25 mg/m2 (2 mg/day) orally 6 hourly for 2
days.
• Sample for serum cortisol on the third day morning at 8-9 am.
• Interpretation
• Cortisol above 50 nmol/L (1.8 µg/dl) suggest hypercortisolism.
• Advantage
• Good specificity so used for confirmation of Cushing syndrome.
This test cannot differentiate the pituitary, adrenal and ectopic
sources of cortisol excess.

21. High-Dose Dexamethasone
Suppression Test
• Role
• Differentiation of pituitary from the ectopic cause of ACTH
dependent Cushing syndrome.
• Procedure
• Tab dexamethasone 8 mg/m2 (maximum 8 mg) orally at
night 11-12 pm
• Sample for serum cortisol on the next day morning 8-9 am.
• Interpretation
• Cortisol above 50 nmol/L (1.8 µg/dl) after high dose
dexamethasone suppression in the setting of ACTH
dependent Cushing syndrome suggests ectopic ACTH
production.

22. THANKS FOR YOUR
Attention