Investigations
1st investigations to order
urine pregnancy test
Test
Women of childbearing potential should always have pregnancy excluded in the evaluation of hypercortisolism.
Result
negative
serum glucose
Test
Cushing syndrome commonly leads to diabetes and glucose intolerance.
Result
elevated
late-night salivary cortisol
Test
One of the first-line tests to consider in any patient with suspected Cushing syndrome.[21]
Samples are collected by saturating a collection swab with saliva or by passively drooling into a collection tube between 11 p.m. and midnight.[60][61]
Obtaining multiple (at least two) samples may increase sensitivity, and initial testing should be performed with sampling on two separate nights.[19][24][25][26]
Value greater than the upper limit of normal is considered positive. Normal values vary greatly depending on the assay and clinical laboratory used.
Positive results should be confirmed with dexamethasone suppression testing or 24-hour urinary free cortisol.
Testing of late-night salivary cortisol (LNSC) is more accurate at initial diagnosis, but LNSC can be frequently normal in patients with recurrent/persistent disease after pituitary surgery, thus sometimes more samples are needed.[29]
Result
elevated
1 mg overnight dexamethasone suppression test
Test
One of the first-line tests to consider in any patient with suspected Cushing syndrome.[21]
A positive test is defined as morning cortisol >50 nanomol/L (>1.8 micrograms/dL).
Should not be used in patients taking drugs affecting dexamethasone metabolism (e.g., phenytoin, carbamazepine, rifampin [rifampicin], cimetidine).
Patient is given 1 mg of dexamethasone at 11 p.m., and a plasma cortisol level is obtained the following morning at 8 a.m. Dexamethasone levels are measured simultaneously with cortisol to ensure that appropriate levels are achieved. This may help in severely obese patients whose dexamethasone levels may be sub-optimal.[61]
Positive results should be confirmed with late-night salivary cortisol or 24-hour urinary free cortisol.
In patients with incidentally discovered adrenal nodules without clinical features of Cushing syndrome, the 1 mg dexamethasone suppression test should be the initial diagnostic test because urinary free cortisol and late-night salivary cortisol have a lower sensitivity in these patients.[47][49][50]
Result
morning cortisol >50 nanomol/L (>1.8 micrograms/dL)
24-hour urinary free cortisol
Test
Normal ranges vary by assay method.
Should be considered as a possible first-line test in any patient with suspected Cushing syndrome, except those with renal failure.
Sensitivity may be lower than late-night salivary cortisol or 1 mg overnight dexamethasone suppression testing.
Patients need to be instructed on appropriate 24-hour urine collection, and should avoid excessive fluid intake.[19]
Likelihood ratio positive 10.6; likelihood ratio negative 0.16; diagnostic odds ratio 95.4.[61]
At least two 24-hour urinary free cortisol samples should be collected to increase diagnostic accuracy.[27]
Positive results should be confirmed with late-night salivary cortisol or 1 mg overnight dexamethasone suppression testing.
Result
>50 micrograms/24 hour
48-hour 2 mg (low-dose) dexamethasone suppression test
Test
Rarely used in isolation; used in combination with other tests.
A positive test is defined as morning cortisol >50 nanomol/L (>1.8 micrograms/dL).
May be considered as a first-line test in a patient with suspected Cushing syndrome, except those taking drugs known to affect metabolism of dexamethasone.[19] Common examples of such drugs include phenytoin, carbamazepine, rifampin (rifampicin), and cimetidine.
Patients should be given 0.5 mg of dexamethasone at 9 a.m. and at 6-hour intervals for 48 hours, with a plasma cortisol obtained at 9 a.m., 6 hours after the last dose.[36][61]
Positive results should be confirmed with late-night salivary cortisol or 24-hour urinary free cortisol.
Result
morning cortisol >50 nanomol/L (>1.8 micrograms/dL)
Investigations to consider
morning plasma adrenocorticotrophic hormone (ACTH)
Test
Morning plasma ACTH is the test of choice for differentiating ACTH-dependent from ACTH-independent Cushing syndrome (when interpreting values, note that assays differ between different laboratories).[1][2][33] Should be obtained only after biochemical diagnosis of hypercortisolism (Cushing syndrome) has been established.
Unsuppressed ACTH levels (>4 picomol/L [>20 picograms/mL]) suggest ACTH-dependent Cushing syndrome (pituitary adenoma [Cushing's disease] or ectopic ACTH-secreting tumours).
Suppressed/low ACTH levels (<2 picomol/L [<10 picograms/mL]) suggest ACTH-independent Cushing syndrome (adrenal aetiology).
Exercise caution in interpretation of values as assays differ between different laboratories.
ACTH is unstable in blood samples at room temperature, and care must be taken to ensure appropriate handling of samples.
Result
>4 picomol/L (>20 picograms/mL) suggests pituitary or ectopic aetiology; <2 picomol/L (<10 picograms/mL) suggests adrenal aetiology
plasma dehydroepiandrosterone sulphate (DHEAS) level
Test
In patients with adrenocorticotrophic hormone (ACTH) levels in the intermediate range, measurement of DHEAS may be considered. DHEAS is ACTH-stimulated, therefore low-normal or suppressed DHEAS concentrations indicate an adrenal cause (ACTH-independent Cushing syndrome).[1] Elevated plasma DHEAS level is not specific. However, in patients with accelerated virilisation and Cushingoid features, it may point to an adrenal carcinoma.
Result
low-normal or suppressed concentrations suggests an adrenal cause; greatly elevated levels suggest adrenocortical carcinoma
pituitary MRI
Test
Should be ordered as the initial imaging test in patients with confirmed adrenocorticotrophic hormone (ACTH)-dependent Cushing syndrome.
The majority of Cushing's disease adenomas measure <1 cm, and up to 40% of patients with Cushing's disease do not have an adenoma visible on MRI.[62]
Patients with ACTH-dependent Cushing syndrome and an adenoma ≥10 mm on MRI may proceed to treatment. Some clinicians prefer additional biochemical confirmation with high-dose dexamethasone suppression testing prior to initiating surgical therapy.[36] The use of high-dose dexamethasone suppression testing is an area of debate.[37]
Patients with adenomas 6-9 mm on MRI should undergo inferior petrosal sinus sampling (IPSS) to confirm the diagnosis.[21] Patients without definitive lesions on MRI should also undergo IPSS.[1][2][38] Approximately 40% to 50% of patients with Cushing's disease will not have visible lesions on pituitary/sellar MRI.[1]
Result
may show pituitary adenoma
adrenal imaging
Test
In patients with established adrenocorticotrophic hormone (ACTH)-independent Cushing syndrome, imaging of the adrenal glands is performed to identify adrenal pathology causing hypercortisolism, such as an adenoma.[1][33] Several adrenal abnormalities can produce excess cortisol; however, unilateral adrenal adenoma is the most common cause of ACTH-independent Cushing syndrome.[1]
Adrenal protocol CT is recommended to differentiate adenomas from adrenal carcinoma (unenhanced CT followed by contrast-enhanced CT as indicated).[33] Adrenal adenomas typically have attenuation less than 10 Hounsfield units (HU) on unenhanced CT and rapid contrast washout on contrast-enhanced CT scans. Adrenocortical carcinoma has attenuation more than 10 HU on unenhanced CT scans and delayed contrast washout.[1][2]
Additional evaluation recommended for suspected adrenal carcinoma includes fluorodeoxyglucose positron emission tomography/CT, chest CT with or without contrast, abdomen/pelvis CT or MRI scans with contrast.[33]
Primary bilateral macronodular adrenal hyperplasia is characterised by nodules with an appearance like bunches of grapes in both adrenal glands on adrenal CT.[2] Adrenal glands may appear normal on adrenal CT in patients with micronodular adrenal hyperplasia, or may appear slightly hyperplastic with micronodules, usually smaller than 6 mm in diameter.[2]
Result
may show adrenal adenoma, hyperplasia, or tumour
high-dose dexamethasone suppression test
Test
Can be considered in patients together with inferior petrosal sinus sampling (IPSS) for differentiating pituitary versus ectopic source of adrenocorticotrophic hormone (ACTH)-dependent Cushing syndrome. Rarely used in countries where IPSS is not readily available.
Patients should be given 2 mg of dexamethasone at 6-hour intervals for 48 hours, or as an overnight test using a single dose of 8 mg of dexamethasone at 11 p.m. Plasma cortisol should be obtained at the start of the test and on the following morning. A positive test suggests a pituitary source of ACTH oversecretion. However, the use of high-dose dexamethasone suppression testing is an area of debate because of its variable sensitivity and specificity.[37]
Result
positive test is defined as suppression of cortisol <50% of the baseline value indicative of ACTH-dependent Cushing syndrome
inferior petrosal sinus sampling (IPSS)
Test
IPSS is the only test with sufficient diagnostic accuracy to differentiate Cushing disease from ectopic adrenocorticotrophic hormone (ACTH) production.[39] Should be performed in patients with confirmed ACTH-dependent Cushing syndrome without an obvious pituitary lesion on MRI or those with adenomas 6-9 mm in size on MRI.[1][2][21][38] Should be carried out in a specialised centre because of potential patient risk.[21]
Central/peripheral ACTH ratio >2:1 at baseline or >3:1 after corticotrophin-releasing hormone (CRH) stimulation.
Blood is sampled peripherally and in the inferior petrosal sinuses simultaneously. If the pituitary effluent (blood in the petrosal sinuses) has a concentration of ACTH greater than 2-fold that of peripheral blood at baseline or greater than 3-fold after stimulation with CRH, the source of the hypercortisolism is pituitary ACTH secretion.[38]
If the ACTH ratio does not reach this threshold, ectopic ACTH secretion is likely.
This is a technically demanding study that very few centres can perform well.
Result
elevated central/peripheral ACTH ratio indicates pituitary source
CT of chest, abdomen, and pelvis
Test
Used to determine the source of ectopic adrenocorticotrophic hormone syndrome.
May also be considered in additional evaluation of patients with suspected adrenal carcinoma.[33]
Result
may localise tumour
MRI chest
Test
May be helpful in selected cases of ectopic adrenocorticotrophic hormone syndrome to localise tumour.
Result
may localise tumour
octreotide scanning
Test
May be helpful in selected cases of ectopic adrenocorticotrophic hormone syndrome to localise tumour.
Result
may localise tumour
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