696 The Immunoassay Handbook
The clinical condition that results from the excessive secre-
tion of cortisol is termed Cushing’s syndrome. Tradi-
tionally, the causes of Cushing’s syndrome are divided into:
G ACTH dependent—the hypercortisolism is due to
hypersecretion of ACTH, such as pituitary tumors
(also referred to as Cushing’s disease), and ectopic
ACTH syndrome (where ACTH is secreted by
non-endocrine tumors, such as lung cancer).
G ACTH independent—where hypercortisolism is pres-
ent, yet ACTH concentrations are very low or even
undetectable due to an adrenal tumor (adenoma or
carcinoma) or to exogenous administration of
Pseudo-Cushing is a clinical condition that has all the
clinical and most of the biochemical features of hypercor-
tisolism but is obviated after treating the predisposing
cause. The pathophysiology has yet to be fully established
but the two commonest causes occur in patients with
severe depression or with alcohol abuse.
Cushing’s syndrome is more common in women
(4F:1M), and the chronic excess of cortisol that character-
izes the illness leads to a number of symptoms and signs
including obesity (mainly of the face and trunk), easy
bruising, purple abdominal striae, hirsutism, acne and
greasy skin, hypertension, muscular weakness, menstrual
disturbances, depression, and osteoporosis.
The diagnosis of Cushing’s syndrome depends on:
G demonstrating the presence of excessive cortisol
G establishing the cause of the hypercortisolemia.
This diagnostic process often involves difﬁcult and pro-
longed investigations, including the measurement of
serum cortisol and plasma ACTH, ﬁrst under basal condi-
tions (circadian rhythm studies), and also after a number of
provocative dynamic tests of both stimulation and sup-
pression, such as the CRH stimulation test, dexametha-
sone suppression test (DST), and venous catheter studies.
A number of radiological imaging procedures are also
required. This analytical process may appear simple, but
the syndrome has an element of cyclicity making diagnosis
more difﬁcult. It is therefore essential that such patients
are cared for in medical centers with special expertise in
dealing with this condition.
Treatment of Cushing’s syndrome depends on the
cause. Hypercortisolemia is most effectively controlled
with surgery but can also be managed using medical
therapy (metyrapone, ketoconazole) or radiotherapy.
In primary hyperaldosteronism, which is usually due to
an adrenal adenoma and known as Conn’s syndrome,
there are high levels of aldosterone in plasma, often result-
ing in hypokalemic alkalosis with muscle weakness,
polyuria and polydipsia, and high blood pressure.
Hypocortisolemia is caused by adrenocortical insufﬁciency
which is either primary (disease of the adrenal cortex) or
secondary (due to pituitary or hypothalamic lesions).
A life-threatening acute form of primary adrenocortical
insufﬁciency may occur following operations or overwhelm-
ing systemic infections with hemorrhage. However, it is
more commonly chronic and is termed Addison’s disease.
The commonest cause of Addison’s disease in the western
world is autoimmunity. Other causative factors include sar-
coidosis, tuberculosis, amyloidosis, hemochromatosis, and
secondary malignant deposits.
Patients with Addison’s disease suffer from generalized
tiredness, anorexia and nausea, pigmentation or white
patches in the skin, weight loss, dizziness and hypotension,
and slow recovery from illness. Most of these features are
due to the deﬁciency of cortisol. Excessive skin pigmenta-
tion, which may be the earliest sign of the disease and is
characteristic of this condition, is thought to be due to the
high levels of plasma ACTH (or its related peptides). In
secondary hypoadrenalism, there is no skin pigmentation
and little change in blood pressure.
Diagnosis of Addison’s disease relies on the demonstra-
tion of low or undetectable serum cortisol levels that fail to
rise following stimulation with ACTH (synacthen; tetra-
cosactrin). The basal plasma ACTH concentrations in
these patients are typically very elevated. In contrast,
patients with secondary adrenocortical insufﬁciency have
low or undetectable serum cortisol with an inappropriately
low concentration of plasma ACTH.
Treatment of adrenocortical insufﬁciency is with hydro-
cortisone or prednisolone (a synthetic glucocorticoid)
given in dosage forms so as to mimic the normal circadian
rhythm of serum cortisol. A typical dose would be 20mg
hydrocortisone in the morning and 10mg in the evening,
although in some patients, glucocorticoids given three
times a day is a more appropriate treatment.
Another, rare disease of the adrenal cortex resulting in
hypocortisolemia is congenital adrenal hyperplasia. This
is a family of congenital enzymatic defects in the pathway of
cortisol synthesis. The commonest deﬁciency (~92% of all
causes) is that of 21-hydroxylase enzyme leading to hypocor-
tisolemia with elevated ACTH levels; the latter leads to
bilateral adrenal enlargement (hyperplasia). There is a con-
comitant deﬁciency of aldosterone in over 60% of cases
resulting in salt loss. The enzyme block, coupled with excess
ACTH, leads to increased androgen secretion by the adre-
nals resulting in virilization. The diagnosis relies on ﬁnding:
1. Low serum cortisol.
2. Elevated levels of 17α-hydroxyprogesterone (a pre-
cursor of cortisol before the enzyme block) either
basally or following stimulation with ACTH.
Androstenedione and ACTH levels are also raised.
Early diagnosis and treatment with cortisol (and ﬂudro-
cortisone, a synthetic mineralocorticoid) can be life
Cortisol is the most important steroid hormone. It is pro-
duced by the adrenal cortex from low-density lipoprotein
cholesterol by several enzymatic reactions (see Fig. 2).
697CHAPTER 9.3 The Adrenal Cortex
Cortisol is an essential hormone and has a wide variety of
effects on most tissues in the body (see NORMAL ADRENO-
CORTICAL FUNCTION). It is particularly important through
its gluconeogenic action, in coping with situations of men-
tal or physical stress such as infections and operations.
Most of the cortisol circulates bound to protein in
plasma with small amounts circulating in the free, biologi-
cally active form. Predominantly, immunoassays measure
the total cortisol in serum so concentrations can seem high
in situations where concentrations of CBG are raised such
as pregnancy and estrogen therapy. Some commercial ana-
lyzer reagents have been developed to measure the “free”
cortisol fraction only. These assays have been developed
for the measurement of cortisol in saliva samples.
The quoted reference intervals will vary with different
manufacturers’ analyzers and reagents, due to the calibra-
tion of the method. Generally, the 0900h range is
Serum: 160–600nmol/L at 0900h.
: <50nmol/L at 2400h (asleep).
Saliva: 10–27nmol/L at 0900h.
The measurement of cortisol in serum is used in the diag-
nosis of disorders of the HPA axis. In contrast to other
endocrine disorders where assay of single or basal samples
is sufﬁcient for diagnosis, dynamic tests (stimulation and/
or suppression tests) are almost always required in the
diagnosis of disorders of the HPA axis.
As stated above, hypercortisolemia together with the loss
of the normal circadian rhythm of cortisol secretion and
disturbed feedback of the HPA axis are the cardinal bio-
chemical features of Cushing’s syndrome. Tests to con-
ﬁrm the diagnosis are based on these principles. It is
important to realize that the validation of the published
test criteria employed has been for speciﬁc assays, and
Pregnenolone 17OHP5 DHEA
Progesterone 17OHP4 A4 Testosterone
11-DOC 11-deoxycortisol Estrone Estradiol
Major pathway for cortisol synthesis
17OHP5 : 17-Hydroxypregnenolone
17OHP4 : 17-Hydroxyprogesterone
11-DOC : 11-Deoxycorticosterone
A4 : Androstenedione
DHEA : Dehydroepiandrosterone
FIGURE 2 Adrenal steroid biosynthetic pathways.
698 The Immunoassay Handbook
thus, test responses should ideally be validated on the local
assay used before the results can be interpreted in particu-
lar patients. In Cushing’s syndrome, where there is an
excessive secretion of cortisol, the following diagnostic
strategy is recommended:
1. Screening tests (as outpatient) for overproduction
of cortisol by either:
G An overnight (1mg single dose) DST: patients
with Cushing’s syndrome fail to suppress their
serum cortisol to <50nmol/L following the inges-
tion of dexamethasone, a synthetic glucocorticoid.
G Or a 24h urinary free cortisol measurement:
expected to be elevated in patients with Cushing’s
G Night time salivary cortisol can be used as a
screening test for Cushing’s syndrome. Salivary
cortisol concentrations follow the serum circa-
dian rhythm pattern. This simple, noninvasive
test can be performed at home and offers advan-
tages as it is possible to monitor patients over
time—a useful feature for a syndrome that is rec-
ognized to be clinically active and then “cycle”
into a nonactive phase.
In the event of an abnormal screening result or if a nor-
mal result was seen on screening, but the clinical suspi-
cion of Cushing’s syndrome is very high, then the patient
needs to have in-patient investigation tests to conﬁrm
overproduction of cortisol.
2. Conﬁrm overproduction of cortisol and establish
the cause of Cushing’s syndrome by performing in
this order the following tests:
a) Measure plasma ACTH concentration: if ACTH
is undetectable, an adrenal tumor is indicated;
detectable ACTH suggests a pituitary or ectopic
b) Measure serum cortisol to assess for a circadian
rhythm: rhythm of cortisol is lost in Cushing’s
syndrome, i.e., in normal subjects serum cortisol
levels at midnight are <50nmol/L provided the
patient is asleep.
c) CRH test: 100µg human CRH-41 injected
intravenously and serum cortisol (and ACTH)
measured. An exaggerated response (rise in cor-
tisol to above 600nmol/L) indicates pituitary
disease. In contrast, patients with the ectopic
ACTH syndrome do not respond (see Fig. 3).
This is carried out prior to the low-dose DST.
Originally, the CRH test was done after the low-
dose dexamethasone suppression, but on occa-
sion, some patients had a prolonged suppressive
effect of the low-dose DST leading to misinter-
pretation of the CRH test.
d) Perform a low-dose DST: 2mg orally every 6h
for 48h; normal subjects suppress their serum
cortisol to <50nmol/L; patients with Cushing’s
syndrome do not (Fig. 4).
e) Venous catheter studies: see ADRENOCORTICO-
TROPIC HORMONE, CLINICAL APPLICATIONS.
The high-dose DST and the insulin-induced hypoglyce-
mia test are rarely used to investigate patients with sus-
pected Cushing’s syndrome. The high-dose DST consists
of 2mg dexamethasone orally 6 hourly for 48h; 50% or
more suppression of serum cortisol indicates pituitary dis-
ease. This test is now rarely used due to the improvements
in imaging techniques. The insulin-induced hypoglycemia
test was used for excluding depression as the cause of
hypercortisolemia: patients with Cushing’s syndrome
show no cortisol rise during hypoglycemia.
1. Morning (0900h) serum cortisol: low or
2. ACTH (tetracosactrin; synacthen) stimulation test:
short and long synacthen tests. In healthy individu-
als, serum cortisol levels rise to >500nmol/L (or an
increment of >200nmol/L over the basal sample) or
more 30–60min after 0.25mg synacthen is given
intramuscularly. No cortisol rise is seen in Addi-
son’s disease. A delayed rise (6–24h) is seen in sec-
ondary hypoadrenocorticism (due to pituitary or
FIGURE 3 Examples of cortisol response in CRH test. FIGURE 4 Examples of responses in DST.
699CHAPTER 9.3 The Adrenal Cortex
G Serum cortisol levels are affected by changes in the
concentrations of CBG; cortisol concentrations can
seem high in situations where concentrations of CBG
are raised such as pregnancy and estrogen therapy.
G Cortisol measurements can be affected by one or more
of the synthetic or natural steroids and so it is unreliable
in patients taking prednisolone, a commonly prescribed
glucocorticoid. Some immunoassay methods for corti-
sol cross-react signiﬁcantly with 11-deoxycortisol,
the immediate precursor of cortisol. This compound is
excessively elevated in patients with Cushing’s syn-
drome on metyrapone treatment (metyrapone inhibits
the conversion of 11-deoxycortisol to cortisol). This is
a serious drawback because this cross-reactivity results
in apparently normal or even elevated cortisol levels in
patients who have been rendered hypoadrenal. Such
assays must therefore be used with great care.
G Some methods for cortisol measurement that use non-
isotopic immunoassays have a serious problem of non-
speciﬁc interference, often associated with reagents
used to denature cortisol-binding proteins before assay.
UKNEQAS (2010) identiﬁed a possible male and
female difference in results returned by users of this
EQA scheme dependent on which analyzer was used.
When male specimens were dispatched, a Gaussian dis-
tribution was observed. However, for the female speci-
men pools, there is a bimodal result distribution. The
higher peak was with the Roche Elecsys analyzer.
However, when this was followed up with a mass spec-
trometry method, the Roche analyzer demonstrated
good agreement with MS, while the other assays under-
estimated the cortisol concentrations. This gender dif-
ference may be due to the effect of different blocking
agents used or due to a protein problem in the com-
mercial assays. It is important to remember therefore
that the normal cortisol response to a speciﬁc provoca-
tive test should ideally be validated on the local assay
used before the results can be interpreted in particular
patients. This is best illustrated with the short synac-
G A national audit of the short synacthen test in the UK
has shown that although the preanalytical procedures
were similar in most laboratories, there is a require-
ment to recognize the effect that method bias may have
on the reference intervals and consequently on the
diagnosis of adrenal insufﬁciency. There is a need to
develop consensus guidelines, which can aid both clini-
cians and laboratories, and the impact of MS may help
to minimize the method-related bias and generate har-
monization of cortisol reference intervals during a
short synacthen test.
G Many of the automated, non-extraction immunoassays
for UFC are modiﬁcations of the serum cortisol assay.
There are many structurally similar compounds and
metabolites of cortisol in urine which can result in an
overestimate of the true 24h UFC result (look at the
24h UFC reference intervals quoted in the kit inserts
of the major analyzers). Many laboratories (and manu-
facturers) try to minimize this nonspeciﬁcity by incor-
porating an organic solvent extraction step prior to
immunoassay. Increasingly though, LCMS is used to
measure UFC, which overcomes the previous prob-
lem for immunoassays of cross-reactivity of other
structurally similar compounds.
The commonly used methods for cortisol measurement
are all automated robotic instruments and include Seimens
ACS™: 180, Abbott Architect, Siemens IMMULITE™,
DELFIA™, Roche Diagnostics Modular, and Tosoh.
Serum cortisol by isotopic immunoassays has virtually dis-
appeared in routine service laboratories. Very recently,
there has been interest in major service laboratories to
move to tandem mass spectrometry as the preferred ana-
lytical technique for serum cortisol to obviate the well-
recognized speciﬁcity problems of immunoassay. Saliva
cortisol assays in general use modiﬁed serum cortisol
radioimmunoassay, enzyme-linked immunoassay, or tan-
dem mass spectrometry.
Desirable Assay Performance
Knowledge of the degree of cross-reactivity with other ste-
roids especially 11-deoxycortisol, prednisolone, and dexa-
methasone is vital. Current automated assays for serum
cortisol can reproducibly measure down to 10nmol/L. An
analyzer should generate a result within 60min (for urgent
cases, e.g., some patients in intensive care units with occult
adrenocortical insufﬁciency). Saliva cortisol assays have
different reference intervals and diagnostic cutoff values
because of the different assays and the reader should refer
to the laboratory method derived values. The same will
apply to the 24h UFC sample.
Types of Sample
Serum, plasma, or saliva. Ideally, specimen collection
should be done at 0800–1000h since reference intervals
are usually established for this time of day. A 24h urine
collection can be used, but the patient must be given writ-
ten instructions on the sample collection protocol.
Frequency of Use
ACTH (corticotropin) is a 39 amino acid peptide with a
relative molecular mass of 4500 (4.5kDa) that is derived
from a larger (31kDa) precursor, pro-opiomelanocortin,
which is secreted by the corticotroph cells of the anterior
pituitary. ACTH exists in the circulation in different
molecular forms, some of which may be inactive
biologically and undetected by immunoassays.
ACTH is secreted by the anterior pituitary to stimulate the
adrenal cortex directly, to synthesize and secrete glucocor-
ticoids, the most important of which is cortisol. ACTH in
700 The Immunoassay Handbook
turn is secreted in response to CRH and vasopressin from
the hypothalamus. Serum cortisol exerts a negative feed-
back effect on the hypothalamus and pituitary to control
the level of ACTH in the circulation. The bioactivity of
ACTH resides in its ﬁrst 24 amino-terminal amino acids.
10–60ng/L at 0900h (2.2–13.2pmol/L).
The measurement of plasma ACTH simultaneously
with serum cortisol is recommended to help to deter-
mine the etiology of Cushing’s syndrome, primary and
secondary adrenal insufﬁciency, and rarer disorders such
as adrenoleukodystrophy and Nelson’s syndrome.
G Basal ACTH: measurement of ACTH at 0900h helps
to establish the cause of Cushing’s syndrome. Periph-
eral plasma ACTH levels are consistently <10ng/L if
the underlying pathology is an adrenal tumor (both
adenomas and carcinomas) because these tumors
secrete cortisol independently of ACTH and lead to
suppression of plasma ACTH levels through the nega-
tive feedback mechanism. In contrast, if plasma ACTH
concentrations are either detectable (>15ng/L) or high,
then a diagnosis of adrenal tumor is excluded, and the
underlying pathology is most likely to be either a pitu-
itary or non-pituitary (ectopic) tumor such as lung can-
cer. Excessively elevated levels (>300ng/L) are highly
indicative of an ectopic source of ACTH.
G CRH test: used to differentiate the diagnosis of
ACTH-dependent Cushing’s syndrome. Pituitary
ACTH-secreting adenomas retain their response to
CRH, while ectopic ACTH tumors lack CRH recep-
tors and therefore do not respond to CRH. Globally,
there have been many protocols used including the
type of CRH used and sampling time points, conse-
quently, there is no uniﬁed agreement when interpret-
ing the ACTH response to this test. The test involves
the intravenous administration of 100µg (human or
ovine) CRH-41 and the measurement of serum cortisol
and plasma ACTH thereafter. Patients with active
pituitary disease usually respond by elevating their
ACTH levels to above 100ng/L, whereas patients with
the ectopic ACTH syndrome do not show a response.
G Venous catheter studies: ACTH measurements dur-
ing venous catheter studies are considered the “gold
standard” test to differentiate between Cushing’s dis-
ease and an ectopic ACTH-secreting source. Thus, if a
pituitary cause is suspected, the inferior petrosal sinuses
(veins that drain the pituitary) can be cannulated and
blood obtained for ACTH measurements before and
after an injection of CRH. This procedure helps in the
diagnosis of pituitary-dependent Cushing’s disease and
in localizing the tumor within the pituitary itself. A
central (inferior petrosal) to peripheral plasma ACTH
gradient of 2:1 or greater pre-CRH or a gradient 3:1 or
greater post-CRH is consistent with Cushing’s disease.
Plasma ACTH measurement also helps in the follow-
up of patients treated for Cushing’s disease (see Fig. 5).
If the cause of Cushing’s syndrome is suspected to be an
ectopic (non-pituitary) tumor, then a whole-body catheter
Time after CRH
Sampling site Baseline 3–5 min 8–10 min
1. Right low internal jugular X X
2. Left low internal jugular X X
1+2 peripheral X X
3. Right high internal jugular X X
4. Left high internal jugular X X
3+4 Peripheral X X
5. Right inferior petrosal sinus
6. Left inferior petrosal sinus
FIGURE 5 Venous sampling for ACTH with CRH administration in ACTH-dependent Cushing’s syndrome. The peripheral sample is collected
simultaneously to the respective jugular or petrosal sinus sample for ACTH and cortisol. Samples marked “X” are not required.
701CHAPTER 9.3 The Adrenal Cortex
study may need to be done where different veins in the
chest and abdomen are cannulated and blood drawn for
ACTH measurement. High concentrations of ACTH will
be demonstrable in the veins draining the tumor and thus
aid in tumor localization. This procedure is rarely used
these days due to the signiﬁcant improvement in imaging
techniques. See MANAGEMENT OF PATIENTS WITH CUSH-
Nelson’s syndrome: is a rare but serious ACTH-secret-
ing pituitary tumor that arises as a complication of bilateral
adrenalectomy, a surgical procedure employed in difﬁcult
cases of Cushing’s syndrome where the primary source
cannot be identiﬁed, but there is a need to control the
effects of the hypercortisolism. These cases on the grounds
of probability are usually pituitary Cushing’s disease. Post-
adrenalectomy, plasma ACTH measurement pre- and 2h
post-hydrocortisone are extremely valuable to evaluate the
aggressiveness of the Nelson’s tumor; the more aggressive
the tumor the less it responds to hydrocortisone.
G Plasma ACTH levels are very useful in discerning the
etiology of adrenocortical failure. ACTH levels are
elevated (usually >150ng/L but sometimes grossly so
[>300ng/L]) in primary adrenocortical insufﬁciency
(Addison’s disease), whereas in secondary adrenocorti-
cal insufﬁciency (due to pituitary or hypothalamic
causes), ACTH concentrations are inappropriately low
or undetectable, with a concurrent low serum cortisol.
G CRH test: this test occasionally will help to differenti-
ate the pituitary from hypothalamic causes of adreno-
cortical insufﬁciency. Thus, patients with hypothalamic
lesions will have raised ACTH levels after CRH injec-
tion, whereas patients with pituitary disease show no
G ACTH is an unstable peptide in plasma due to degra-
dation by endogenous peptidases. Therefore, process-
ing of the blood specimen must be done quickly so
you will require a blood collection tube with antico-
agulant (e.g., EDTA or lithium–heparin), a refriger-
ated (4°C) centrifuge and solid CO2 to freeze the
plasma immediately, once it has been transferred to a
clean labeled tube. It is recommended that the entire
process of venepuncture to freezing the separated
plasma sample should be done within 20min to avoid
a spuriously low result.
G The assay is useful for the differential diagnosis but not
the diagnosis of Cushing’s syndrome where only
cortisol measurement is required.
G The absolute concentrations of ACTH are not very
useful in distinguishing pituitary from ectopic causes of
Cushing’s syndrome. In the majority of such cases, the
ACTH concentrations lie between 30 and 200ng/L.
Thus, other ways to differentiate these two entities are
G Not all of the ACTH assays available are sensitive
enough to detect levels in all healthy individuals and
are therefore not useful in differentiating low normal
levels from the suppressed levels recorded in patients
with adrenal tumors.
Early assays used for the measurement of ACTH were
competitive radioimmunoassays that used a plasma extrac-
tion procedure, such as the use of Vycor glass to adsorb the
peptide. These extraction assays were time-consuming,
labor intensive and had low sample throughput. They have
been superseded with non-isotopic immunometric assay
methods that have the required sensitivity and are rapid to
perform. Speciﬁcity can be a problem as some immuno-
metric assays are so speciﬁc for ACTH 1–39 that they do
not detect the larger forms of ACTH or its smaller frag-
ments. These forms may be the only ones circulating in
patients with the ectopic ACTH syndrome and would
therefore be reported as undetectable instead of grossly
elevated. This could lead to disastrous errors in diagnosis.
It is therefore mandatory to check for the speciﬁcities of
ACTH assays in relation to their clinical performance
before they can be used for patient samples.
Most smaller laboratories send specimens to specialized
centers for ACTH measurement. The popular methods
used include Siemens IMMULITE® and Roche Modular
analyzer assays. As previously mentioned under serum cor-
tisol, it is important to remember that there may be differ-
ences in plasma ACTH results from different commercial
analyzer manufacturers due to the lack of an international
Desirable Assay Performance
Clinical assay sensitivity of 5–10ng/L is necessary. Detailed
knowledge of assay speciﬁcity is mandatory to avoid errors
Type of Sample
Plasma: blood is collected into plastic tubes containing
heparin or EDTA; glass tubes must not be used for blood
collection as ACTH will be adsorbed to glass and lost.
Owing to the instability of the peptide, blood needs to be
centrifuged at 4°C immediately after venepuncture and
plasma decanted, ﬂash frozen, and stored at −20°C until
assay (stable for 6 months). Ideally, sample collection
should be performed at 0800–1000h since reference ranges
are usually established for this time of day.
Frequency of Use
Management of Patients
with Cushing’s Syndrome
G Diagnosis of Cushing’s syndrome suspected on clinical
702 The Immunoassay Handbook
G Diagnosis of Cushing’s syndrome highly likely bio-
chemically due to failure to suppress serum cortisol by
low-dose DST or by elevated urinary free cortisol
concentration. Ideally, obtain a plasma sample for
ADMIT TO ENDOCRINE WARD
(E.G., ON MONDAY)
Admit patient to ward by noon. Routine chemistry (plasma
potassium, bicarbonate, and glucose in particular), hema-
tology, and chest X-ray. Blood sample for plasma ACTH.
Take blood at midnight (while patient asleep).
Circadian rhythm study: samples for cortisol at 0900,
1800h, and midnight (while asleep). CRH test: give 100µg
hCRH intravenously and measure cortisol and ACTH.
Finish circadian rhythm study: samples for cortisol at
0900h. This sample is also the start of the low-dose DST:
0.5mg dexamethasone orally 6 hourly for 48h.
Take blood at 0900h for serum cortisol. This sample ends
the low-dose DST.
By Following Monday
All results of tests are available and provide provisional
G No suppression on low-dose DST=Cushing’s syn-
drome. If pseudo-Cushing due to depression is sus-
pected, perform an insulin hypoglycemia test.
G Plasma ACTH undetectable: diagnostic of adrenal
tumors. Proceed to radioimaging of abdomen and
G Plasma ACTH detectable but not very grossly elevated:
pituitary or ectopic ACTH-producing tumor.
a. Serum cortisol rises during CRH test and shows
inadequate suppression during low-dose DST=pitu-
itary tumor. Proceed to pituitary radioimaging,
petrosal sinus catheter study, and hypophysectomy.
b. Serum cortisol does not rise during CRH test and
does not suppress on low-dose DST=ectopic
ACTH-producing tumor. Plasma ACTH may be
grossly elevated (greater than 300ng/L) plus hypo-
kalemia, alkalosis, and hyperglycemia. Proceed to
radioimaging studies (and venous catheters) of chest
and abdomen to localize tumor followed by its surgi-
c. If results of CRH and high-dose DST are discrep-
ant, i.e., do not point to a uniform diagnosis as in (a)
or (b), it becomes imperative to proceed to petrosal
catheter study since pituitary disease is much more
common than ectopic ACTH-producing lesions. If
petrosal catheterization is not possible, patient needs
to be referred to a specialist center capable of per-
forming the procedure.
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