Harmonisation of

Endocrine Dynamic
Testing (HEDT)
This manual is a joint initiative from ESA/AACB/RCPA and is freely available as a resource
for Endocrinologists and Biochemists. Information provided is a guide only and needs to be
verified and modified according to local procedures (e.g. patient consent, sample type, name
of test set). Queries can be directed to the chair of the HEDT working party. A separate
paediatric endocrine dynamic testing protocol is in progress with the HDET-P working party.

CHANGE TO SITE LOGO

LLlOGOLOGO

1
HEDT Version 1.5 2018
Table of Contents
HEDT Working Group Members:.........................................................................................................4
Endocrine Analyte Reporting Unit and Sample Tube............................................................................5
1 Adrenal..........................................................................................................................................8
1.1 SHORT SYNACTHEN TEST.......................................................................................................8
1.2 PRIMARY ALDOSTERONISM INVESTIGATION.......................................................................11
1.3 SALINE SUPPRESSION TEST..................................................................................................13
1.4 FLUDROCORTISONE SUPPRESSION TEST..............................................................................15
1.5 ORAL SODIUM LOADING TEST.............................................................................................17
1.6 ADRENAL VENOUS SAMPLING.............................................................................................18
2 Cushing Overview........................................................................................................................22
2.1 OVERNIGHT DEXAMETHASONE SUPPRESSION TEST (1 mg DST)..........................................23
2.2 LATE NIGHT SALIVARY CORTISOL.........................................................................................24
2.3 24 HOUR URINARY FREE CORTISOL (UFC)............................................................................25
2.4 2-DAY LOW DOSE DEXAMETHASONE SUPPRESSION TEST (LDDST).....................................26
2.5 DEXAMETHASONE-CRH TEST...............................................................................................28
2.6 IV 4 mg DEXAMETHASONE SUPPRESSION TEST...................................................................30
2.7 ACTH Dependent Cushing’s Syndrome................................................................................31
2.7.1 HIGH DOSE DEXAMETHASONE SUPPRESSION TEST (HDDST)...........................................31
2.7.2 PERIPHERAL CRH TEST.....................................................................................................32
2.7.3 BILATERAL INFERIOR PETROSAL SINUS SAMPLING..........................................................33
3 Hypopituitarism...........................................................................................................................36
3.1 INSULIN TOLERANCE TEST...................................................................................................36
3.2 OVERNIGHT METYRAPONE TEST..........................................................................................40
3.3 GLUCAGON STIMULATION TEST..........................................................................................42
3.4 GONADOTROPHIN RELEASING HORMONE STIMULATION TEST..........................................44
4 Acromegaly.................................................................................................................................46
4.1 GROWTH HORMONE SUPPRESSION TEST............................................................................46
5 Hyperglycaemia investigation.....................................................................................................48
5.1 ORAL GLUCOSE TOLERANCE TEST (OGTT)............................................................................48
6 Hypoglycaemia investigation.......................................................................................................51
6.1 MIXED MEAL TEST................................................................................................................51
6.2 PROLONGED OGTT...............................................................................................................55

2
HEDT Version 1.5 2018
 6.3 72 HOUR FAST......................................................................................................................56
6.4 CALCIUM STIMULATION TEST FOR INSULINOMA................................................................59
7 Diabetes Insipidus........................................................................................................................62
7.1 WATER DEPRIVATION TEST..................................................................................................62
8 Thyroid.........................................................................................................................................67
8.1 TRH TEST..............................................................................................................................67
8.2 T3 SUPPRESSION TEST.........................................................................................................69
8.3 CALCIUM STIMULATION TEST FOR MEDULLARY THYROID CANCER.....................................71
9 Phaeochromocytoma...................................................................................................................73
9.1 CLONIDINE SUPPRESSION TEST............................................................................................73
Acknowledgements:............................................................................................................................75
Amendment history:............................................................................................................................76

3
HEDT Version 1.5 2018
 HEDT Working Group Members:

Members Site Affiliations Stream email

Dr Cherie Chiang (chair) VIC ESA, AACB, RCPA Adult [Link]@[Link]
Assoc Prof Warrick Inder QLD ESA Adult [Link]@[Link]
Prof Mathis Grossmann VIC ESA Adult mathisg@[Link]
Assoc Prof Rory Clifton-Bligh NSW ESA Adult jclifton@[Link]
Dr Penny Coates SA ESA, AACB, RCPA Adult [Link]@[Link]
Dr Ee Mun Lim WA ESA, AACB, RCPA Adult [Link]@[Link]
Mr Peter Ward NSW AACB Adult [Link]@[Link]
Dr Phoebe Stanford NSW ESA, AACB, RCPA Adult phoebestanford@[Link]
Assoc Prof Chris Florkowski NZ AACB, RCPA Adult [Link]@[Link]
Dr James Doery VIC AACB, RCPA Paediatric [Link]@[Link]

4
HEDT Version 1.5 2018
 Endocrine Analyte Reporting Unit and Sample Tube
The following table is provided as a guide, check with local laboratory for sample type.

Test Standard Alternate Conversion Collection Collection comment

unit unit to alternate tube
unit
ACTH pmol/L pg/mL 4.54 EDTA Collect on ice
ng/L
Aldosterone pmol/L ng/dL 0.036 EDTA State erect (seated for 10
mins) or supine (for 30
mins) on request slip
Aldosterone- pmol/L ng/dL 0.036 plain Urine 24 h
Urine container Keep the container
refrigerated during the
collection period

AVP / ADH pmol/L pg/mL 0.923 2 x 4 ml Collect on ice

EDTA
Androstenedione nmol/L ng/dL 28.65 Li heparin
βHCG IU/L mIU/mL 1 Li heparin
C-peptide nmol/L ng/mL 0.331 Li heparin Fasting

Calcitonin pmol/L pg/mL 0.292 Li heparin Fasting specimen is

or serum preferred.
tube Collect on ice.
NOT
EDTA.
Chromogranin A ug/L Serum Fasting specimen preferred.
Collect on ice.
Copeptin pmol/L Li heparin
Cortisol nmol/L µg/dL 27.588 Li heparin/ Morning 8 – 9 am sample
serum
Cortisol - Saliva nmol/L µg/dL 27.588 Salivette 11 pm to midnight
collection. Nil by mouth/ no
teeth brushing 30 mins
prior.
DHEAS µmol/L µg/dL 0.027 Serum
Estradiol pmol/L pg/mL 0.272 Serum
Free T3 pmol/L pg/dL 64.9 Li heparin/
serum
Free T4 pmol/L ng/dL 0.0775 Li heparin/

5
HEDT Version 1.5 2018
 serum
FSH IU/L mIU/mL 1 Li heparin
Gastrin pmol/L pg/mL 0.481 Serum Fasting, collect on ice.
Proton pump inhibitors
elevate result.
Glucagon ng/L pg/mL 1 EDTA & Fasting. Collect on ice
trasylol
containing
GLASS
tube (Use 6
mg Na
EDTA +
2,500 KIU
trasylol per
5 ml blood)
Glucose mmol/L mg/dL 18 Fluoride Li heparin/ serum if rapid
oxalate transport to laboratory
GH µg/L mU/L 3 Li heparin
or Serum
tube
Insulin pmol/L mU/L 0.144 Serum Fasting. Collect on ice
Insulin Ab unit Serum
IGF-1 nmol/L ng/mL 0.131 Serum or Li
heparin
IGF BP3 nmol/L Serum Collect on ice
LH IU/L Li heparin/
serum
17-OHP nmol/L Serum
Metanephrines Li heparin Fasting, collect on ice,
(plasma) supine for 30 mins.
3-methoxytyramine might
need to be specified on
request if required
Osteocalcin µg/L Serum, Li-
heparin or
K3 EDTA
Pancreatic pmol/L Serum Fasting, morning sample
polypeptide
PTH pmol/L pg/mL 9.4 Serum or Serum tubes to send
ng/L EDTA immediately
PTH-rp pmol/L EDTA with Collect on ice
Aprotinin
additive
Progesterone nmol/L ng/dL 31.44 Li heparin/
serum

6
HEDT Version 1.5 2018
Prolactin mIU/L µg/L 43.478 Li heparin/ EDTA plasma possible for
serum most assays for IPSS
Renin mass mIU/L EDTA Do NOT collect on ice.
State erect or supine on
request slip as per
aldosterone
SHBG nmol/L µg/mL 8.896 serum or Li
heparin
Steroid profile- plain 24-hr urine
Urine container
Sulphonylurea serum Collect during
Screen hypoglycemia, only detect
ingestion within 24 hrs
VIP pmol/L pg/mL 3.38 EDTA & Fasting, collect on ice
trasylol
containing
GLASS
tube (Use 6
mg Na
EDTA +
2,500 KIU
trasylol per
5 ml blood)
Testosterone nmol/L ng/dL 28.8 Li heparin/ Fasting, morning 8-9 am
serum sample
TSH mIU/L Li heparin/
serum

7
HEDT Version 1.5 2018
1 Adrenal
1.1 SHORT SYNACTHEN TEST

RATIONALE:

The cortisol response to Synacthen stimulation will be low or absent due to primary adrenal pathology
(e.g. Addison’s disease, bilateral adrenal infiltration) or adrenal atrophy secondary to severe ACTH
deficiency of at least 4 weeks' duration. (1) This test does not assess adequacy of ACTH/ CRH
response to stress if pathology was of short duration. This is assessed by the ITT or overnight
metyrapone test.

Also used for diagnosis of non-classical 21-hydroxylase deficiency, if a morning, screening follicular
phase 17 OH progesterone is > 6 nmol/L. For other causes of congenital adrenal hyperplasia, contact
laboratory for required tests.

PREPARATION AND PROCEDURE:

1) Withhold any steroid treatment for 24 hours prior to the test (patients treated with dexamethasone
require at least 48 hours of steroid withdrawal) if appropriate.

2) Baseline blood is collected for cortisol and ACTH. Procedure should be performed between 8 -
9:30am when cortisol peak is present.

3) IM or IV Synacthen 250 µg

4) Blood for cortisol collected at 30 and 60 minutes

Time Procedure/ Test Comment

Baseline ACTH, cortisol Also 17-OH progesterone if CAH queried
0 minute IV or IM Synacthen 250
µg
30 minutes cortisol Also 17-OH progesterone if CAH queried
60 minutes cortisol Also 17-OH progesterone if CAH queried

INTERPRETATION:

 Normal SST requires a cortisol from at least one time point to exceed the minimum
peak cortisol cut-off specified for that assay. The concentration of peak cortisol cut-
off is assay dependent, and for female, OCP raises total cortisol level due to rise in
CBG. (1)
 The use of historical peak cortisol of 550 nmol/L in newer cortisol-specific assays
may result in false positive results. (2) Previous requirement for a minimum cortisol
increment from baseline (e.g. 250 nmol/L) is also redundant as normal individuals
with high baseline cortisol will not achieve this increment.
Laboratories need to determine their own individual cut-off. The table below
describes the minimum cortisol level achieved post synacthen at 30 minutes for

8
HEDT Version 1.5 2018
 different immunoassays. (3) The 60 minutes cortisol level was reported to be around
15% higher than the 30 minutes level. (4)

Minimum peak cortisol cut-off (2.5th centile) for healthy subjects 30 mins post IV
Synacthen (2):

Assay Male Female Female (OCP)

GC-MS 420 420 640
Siemen Centaur 450 450 620
Abbott Architect 430 420 580
Roche E170 * 400 * 400 * 600
Beckman Access 460 460 600
Siemen Immulite 470 480 690
* Roche Cort II assay was not available at time of published study, but correlation study
indicated results similar to MS methods, the above cut-offs were used by most New Zealand
laboratories.

Test outcome Suggested comment:

Normal SST The short Synacthen test was normal, stimulated cortisol level exceeded
(the minimum peak cortisol cut-off specified for that assay). This does
not exclude acute secondary hypocortisolism.
Abnormal SST The short Synacthen test was abnormal, stimulated cortisol levels were
below (the minimum peak cortisol cut-off specified for that assay).

INTERPRETATION for 21 HYDROXYLASE DEFICIENCY CAH: (5)

Test outcome Suggested comment:

Stimulated 17 OH An exaggerated rise of 17 OH progesterone post ACTH stimulation
progesterone > 43 (>43 nmol/L) is seen in 21 hydroxylase deficiency CAH, and some
nmol/L patients with adrenal adenomas.

Stimulated 17 OH A moderate rise of 17 OH progesterone post ACTH stimulation (30-43

progesterone 30 - 43 nmol/L) constitutes a grey zone whereby genetic tests might be required
nmol/L to confirm or exclude 21 hydroxylase deficiency CAH.
Stimulated 17 OH A mild rise of 17 OH progesterone post ACTH stimulation (< 30
progesterone < 30 nmol/L) is a normal physiological response and not consistent with 21
nmol/L hydroxylase deficiency CAH.

The cut-offs for 17 OH progesterone are based on radioimmunoassay, consult laboratory if

LCMS method is used.

NOTES:

 Nausea, palpitation, hot flushes or allergic reaction can rarely occur with synacthen.
 Although IV administration is preferred, IM administration is also valid, however
cortisol at 30 minutes is more variable. (6)
 SST result is difficult to interpret in critically ill patients due to difficulties in
interpreting total cortisol results from immunoassays. (7)

9
HEDT Version 1.5 2018
REFERENCES:

1. Courtney CH, McAllister AS, Bell PM, McCance DR, Leslie H, Sheridan B, Atkinson AB.
Low- and standard-dose corticotropin and insulin hypoglycemia testing in the assessment of
hypothalamic-pituitary-adrenal function after pituitary surgery. The Journal of clinical
endocrinology and metabolism 2004; 89:1712-1717

2. El-Farhan et al. Method-specific serum cortisol responses to the adrenocorticotrophin test:

comparison of gas chromatography mass spectrometry and five automated immunoassays.
Clinical Endocrinology (2013) 78, 673–680

3. Lim EM, Wardrop R, Hadlow N, Fletcher S, Joseph J, Henley D. Cortisol conundrum:

Caution on 550 nmol/L cut-off for Synacthen stimulation tests. Time to adopt method
specific diagnostic cut-off! Clin Biochem Revs. 2016 Dec; 37(4): S1–S56.

4. Chitale A et al. Determining the utility of the 60 min cortisol measurement in the short
synacthen test. Clinical Endocrinology (2013) 79, 14 - 19

5. El-Maouche D, Arlt W, Merke DP. Congenital adrenal hyperplasia. Lancet (London,

England) 2017

6. Longui CA, Vottero A, Harris AG, Chrousos GP. Plasma cortisol responses after
intramuscular corticotropin 1-24 in healthy men. Metabolism: clinical and experimental
1998; 47:1419-1422

7. Cooper MS et al. Corticosteroid insufficiency in acutely ill patients. NEJM. (2003); 348
(8):727.

10
HEDT Version 1.5 2018
 1.2 PRIMARY ALDOSTERONISM INVESTIGATION

PATIENT PREPARATION

Algorithm for the detection, confirmation, subtype testing, and treatment of Primary
hyperaldosteronism (PA). Adapted from Management of Primary Aldosteronism: Case Detection,
Diagnosis, and Treatment: An Endocrine Society Clinical Practice Guideline. (1)

 Interfering drugs which can affect renin, aldosterone or both should be stopped for at
least
o 4 weeks: Spironolactone, eplerenone, amiloride, and triamterene, potassium-
wasting diuretics, licorice.
o 2 weeks: Angiotensin-converting enzyme inhibitors, angiotensin receptor
blockers, renin inhibitors, and dihydropyridine calcium channel antagonists,
clonidine, methydopa, beta-blockers.
 Drugs which do not affect renin, aldosterone for blood pressure control includes:
verapamil slow-release, prazosin, hydralazine, moxonidine.
 Hypokalemia needs to be corrected.
 Aldosterone:renin ratio (ARR) is the preferred screening test. Preferably two elevated
values should be obtained prior to confirmation testing. The ARR test is most

11
HEDT Version 1.5 2018
 sensitive when samples are collected in the morning after patients have been out of
bed for at least 2 hours, usually after they have been seated for 5–15 minutes.
 There is no gold standard for confirmation testing. Of the 4 testing procedures
available, captopril challenge test can have false negative equivocal results and
therefore not mentioned in this document. (1) Oral sodium loading test requires
sensitive and specific urinary aldosterone measurement (LC-MSMS) in patients
without renal impairment.
 In florid Primary aldosteronism (hypokalemia, suppressed renin, elevated aldosterone
> 550 pmol/L), confirmation tests might not be required.

12
HEDT Version 1.5 2018
 1.3 SALINE SUPPRESSION TEST

RATIONALE:

Confirmation test when aldosterone:renin ratio (ARR) elevated. Test should not be performed
in patients with uncontrolled hypertension, hypokalaemia, arrhythmias, severe CCF or renal
failure. Saline infusion acts as a salt and fluid load, suppresses aldosterone production in
normal subjects but not in subjects with primary aldosteronism.

PROCEDURE:

1) Seated procedure

Time Procedure Comment

-30 minutes Patient seated for 30mins prior to
and during infusion
Baseline Renin, aldosterone, U+Es, cortisol Patient remains seated throughout
procedure
0 minute 2L 0.9% Normal Saline IV over
4hours
4 hours Renin, aldosterone, U+Es, cortisol

2) Recumbent procedure

Time Procedure Comment

-60 minutes Patient recumbent for 60mins
prior to and during infusion
Baseline Renin, aldosterone, U+Es, cortisol Patient remains recumbent throughout
procedure
0 minute 2L 0.9% Normal Saline IV over
4hours
4 hours Renin, aldosterone, U+Es, cortisol

INTERPRETATION:

Primary aldosteronism is confirmed if all of the conditions below are met:

1) K+ level normal (at least 4.0 mmol/L)

2) Post infusion cortisol is less than basal cortisol to exclude confounding ACTH effect

3) Post infusion aldosterone cut off:

A) Seated procedure >170 pmol/L

B) Recumbent procedure >140 pmol/L

(140 – 280 pmol/L = borderline, >280 pmol/L = PA very likely)

13
HEDT Version 1.5 2018
NOTES:

 Fluid status check should take place during infusion, particularly for those prone to
fluid overload.
 Seated normal saline suppression test was found to have higher sensitivity compared
to supine normal saline suppression and has good agreement with the more
cumbersome fludrocortisone suppression test. (2)
 The above aldosterone cut offs are for immunoassays, values are lower if measured
using LCMS, please consult laboratory for local cut-off.

14
HEDT Version 1.5 2018
 1.4 FLUDROCORTISONE SUPPRESSION TEST

RATIONALE:

Confirmation test when aldosterone:renin ratio (ARR) elevated. Test should not be performed
in patients with uncontrolled hypertension, hypokalaemia, arrhythmias, severe CCF or renal
failure. Fludrocortisone, a potent mineralocorticoid, suppresses aldosterone production in
normal subjects but not in subjects with primary aldosteronism.

PROCEDURE:

1) Most patients require admission to monitor BP and K+ status

2) Ensure normokalaemia throughout the procedure with oral Slow K QID (target K+ = 4
mmol/L)
3) Salt loading is required, e.g. a liberalized dietary sodium intake, supplemented by Slow-
Na 10 mmol three tablets TDS, target urine Na excretion > 3 mmol/kg/day
4) Collect 24 hr urine for aldosterone, sodium, potassium and creatinine levels 1 day prior to
fludrocortisone administration and again on the last 24h
5) Give Fludrocortisone 0.1 mg every 6 hours for 4 days (0400,1000,1600,2200)
6) Daily blood test for renin, aldosterone, U+Es, cortisol. Extra blood test might be required
for Slow K dosing
7) Blood test at 0700 and 1000 on Day 5 are required for interpretation

Time Procedure Comment

- 1 Day 24 hr urine aldosterone, sodium, Ensure normokalaemia (target K+ = 4
potassium and creatinine mmol/L) and salt loading for the 4 days
of test. Check K+ to adjust slow K dose
Day 1: 0700 Recumbent: Renin, aldosterone,
U+Es, cortisol
Day 1: 1000 Upright: Renin, aldosterone, Fludrocortisone 0.1 mg every 6 hours
U+Es, cortisol (1000,1600,2200)
Day 1: 1600 Check K+ Optional to ensure K remains at target
Day 2: 0700 Recumbent: Renin, aldosterone, Fludrocortisone 0.1 mg every 6 hours
U+Es, cortisol (0400,1000,1600,2200)
Day 2: 1000 Upright: Renin, aldosterone,
U+Es, cortisol
Day 2: 1600 Check K+ Optional to ensure K remains at target
Day 3: 0700 Recumbent: Renin, aldosterone, Fludrocortisone 0.1 mg every 6 hours
U+Es, cortisol (0400,1000,1600,2200)
Day 3: 1000 Upright: Renin, aldosterone,
U+Es, cortisol
Day 3: 1600 Check K+ Optional to ensure K remains at target
Day 4: 0700 Recumbent: Renin, aldosterone, Fludrocortisone 0.1 mg every 6 hours
U+Es, cortisol (0400,1000,1600,2200)
Day 4: 1000 Upright: Renin, aldosterone,
U+Es, cortisol
24 hr urine aldosterone, sodium,
potassium and creatinine
Day 4: 1600 Check K+ Optional to ensure K remains at target

15
HEDT Version 1.5 2018
Day 5: 0700 Recumbent: Renin, aldosterone, Last dose of fludrocortisone Day 5 at
U+Es, cortisol 0400.
Day 5: 1000 Upright: Renin, aldosterone,
U+Es, cortisol

INTERPRETATION:

Primary aldosteronism is confirmed if all of the conditions below are met:

1) upright aldosterone levels on Day 5 (4 days of fludrocortisone) is > 170 pmol/L (1)
2) upright renin on Day 5 is suppressed.
3) K+ level normal (at least 4.0 mmol/L) on Day 5
4) Plasma cortisol on Day 5 does not increase significantly from 0700h to 1000h
(increase may indicate ACTH stimulation of aldosterone production that may have
prevented suppression).

NOTES:

 Blood pressure and fluid status check should take place during fludrocortisone and
salt loading.
 Aldosterone cut off is lower (down to 130 pmol/L) if measured using LCMS rather
than immunoassay, consult laboratory for cut-off.

16
HEDT Version 1.5 2018
 1.5 ORAL SODIUM LOADING TEST

RATIONALE:

Confirmation test when aldosterone:renin ratio (ARR) elevated. Test should not be performed
in patients with uncontrolled hypertension, hypokalaemia, arrhythmias, severe CCF or renal
insufficiency. Oral sodium suppresses aldosterone production in normal subjects but not in
subjects with primary aldosteronism.

PROCEDURE:

1) Ensure adequate potassium to maintain normokalaemia

2) Oral sodium 200 mmol or 6 g daily
3) 24 hr urine collection for aldosterone and Na starting on Day 3
4) Urine aldosterone needs analysis on a specific assay

Time Procedure Comment

Day 1 Oral sodium 200 mmol or 6 g
daily
Day 2 Oral sodium 200 mmol or 6 g
daily
Day 3 Oral sodium 200 mmol or 6 g Start 24 hr urine (aldosterone and Na)
daily collection 8am after discarding first void
urine
Day 4 Complete 24 hr urine (aldosterone and
Na) collection 8am

INTERPRETATION:

Elevated 24 hr urine aldosterone > 33 nmol/day by LCMS method makes primary

hyperaldosteronism likely, providing 24 hr urine Na is elevated (urine Na excretion >3
mmol/kg/day). Consult laboratory for local cut-off.

NOTES:

Non-specific aldosterone methods may blunt diagnostic accuracy due to cross-reactivity with
other metabolites in urine.

17
HEDT Version 1.5 2018
 1.6 ADRENAL VENOUS SAMPLING

RATIONALE:

In patients with confirmed primary aldosteronism (PA) who are surgical candidates, adrenal
venous sampling (AVS) is the gold standard in lateralisation of the source of aldosterone
excess and differentiates between unilateral adrenal adenoma from bilateral adrenal
hyperplasia. All patients should have adrenal CT prior to AVS to exclude large adrenal
masses.
CT and MRI can misdiagnose the cause of PA. Therefore, AVS is still required for
lateralisation with the exception of younger patients < 35 years with spontaneous
hypokalaemia, marked aldosterone excess, and unilateral adrenal cortical adenoma on CT
who might be able to proceed directly to unilateral adrenalectomy. (1)
AVS should be performed by experienced interventional radiologist. The use of ACTH
stimulation is used to improve successful cannulation rate and minimise stress induced
fluctuation in sequential adrenal vein sampling. Point of care cortisol kit during AVS also
increased cannulation rates. (4)

PROCEDURE:

1) Book AVS with experienced interventional radiologist. Notify laboratory of test.

2) AVS can be
a) Unstimulated: only baseline AVS samples collected in early morning after
overnight recumbency.
b) Stimulated with Synacthen: baseline and post ACTH AVS samples collected.
Synacthen protocols include:
I. Bolus 250 µg Synacthen 15 mins before stimulated AVS collection
II. Continuous 50 µg/hr Synacthen (250µg in 500ml N saline, 100ml per
hr) 30 mins before stimulated AVS collection and continued until AVS
completion
III. Bolus 250 µg Synacthen followed by 50µg/hr Synacthen 15- 30 mins
before stimulated AVS collection and continued until AVS completion
3) Tubes should be pre-labelled with site, baseline/ stimulated samples and collected in
duplicate at each site for each timepoint.

Baseline AVS collection

Time Procedure Comment

Document time on Common femoral vein is punctured
tube and a 5 French sheath inserted.
Peripheral cubital fossa blood taken
for aldosterone and cortisol
Document time on Selective catheterization of left
tube adrenal vein and take blood for
aldosterone and cortisol
Document time on Selective catheterization of right Right adrenal vein is more

18
HEDT Version 1.5 2018
tube adrenal vein and take blood for difficult to cannulate.
aldosterone and cortisol
Stimulated AVS collection (post Synacthen stimulation)

Time Procedure Comment

Synacthen stimulation See procedure above
Document time on Peripheral cubital fossa blood Procedure starts 15 to 30 mins post
tube taken for aldosterone and Synacthen administration depending
cortisol on protocol used
Document time on Selective catheterization of Right adrenal vein is more difficult to
tube right adrenal vein and take cannulate
blood for aldosterone and
cortisol
Document time on Selective catheterization of left
tube adrenal vein and take blood for
aldosterone and cortisol

INTERPRETATION:

Lateralisation of aldosterone excess is present if all of the following are present:

1) Both adrenal veins were successfully cannulated (adrenal vein cortisol: peripheral cortisol
≥ 2 at baseline, ≥ 3 post ACTH).

2) Aldosterone:cortisol ratio (ACR) between the two adrenals is > 4 (if gradient < 2: no
evidence of lateralisation, if gradient 2-4: borderline)

3) The unaffected adrenal gland should have an ACR < periphery ACR to indicate
suppression by the contralateral unaffected side.

Lack of lateralisation can occur in a) bilateral aldosterone producing adenoma, b) bilateral

adrenal hyperplasia, c) Glucocorticoid Remediable Aldosteronism.

NOTES:

 Sample worksheet on next page

 Adrenal haemorrhage can occur in up to 2.5% of AVS procedures
 In patients < 20 years old with confirmed PA or in those who have a family history of
PA or strokes < 40 years old, genetic testing for FH-I (Glucocorticoid Remediable
Aldosteronism, hybrid CYP11B1/CYP11B2 mutation) should be considered. (1)
 In patients with confirmed PA presenting in childhood, germline mutations in KCNJ5
causing FH-III should be considered.
 Glucocorticoid Remediable Aldosteronism mutation testing replaces indirect test such
as dexamethasone suppression test.

19
HEDT Version 1.5 2018
AVS Worksheet Template

Pre-Synacthen Post-Synacthen

Right AV Time: Time:

Aldosterone pmol/L
Cortisol nmol/L
Aldo:Cort Ratio ^ #DIV/0! ###### #VALUE! #VALUE!
Left AV Time: Time:
Aldosterone pmol/L
Cortisol nmol/L
Aldo:Cort Ratio ^ #VALUE! ###### #VALUE! #VALUE!
Periphery Time: Time:
Aldosterone pmol/L
Cortisol nmol/L
Aldo:Cort Ratio ^ #VALUE! ###### #VALUE! #VALUE!
# #DIV/0! ###### #VALUE! #VALUE!
R A:C to L A:C
#
L A:C to R A:C #VALUE! ###### #VALUE! #VALUE!

R:P C:C* #VALUE! ###### #VALUE! #VALUE!

L:P C:C * #VALUE! ###### #VALUE! #VALUE!

20
HEDT Version 1.5 2018
REFERENCES:

1. Funder JW, Carey RM, Mantero F, Murad MH, Reincke M, Shibata H, Stowasser M,
Young WF, Jr. The Management of Primary Aldosteronism: Case Detection, Diagnosis, and
Treatment: An Endocrine Society Clinical Practice Guideline. The Journal of clinical
endocrinology and metabolism 2016; 101:1889-1916

2. Ahmed AH, Cowley D, Wolley M, Gordon RD, Xu S, Taylor PJ, Stowasser M. Seated
saline suppression testing for the diagnosis of primary aldosteronism: a preliminary study.
The Journal of clinical endocrinology and metabolism 2014; 99:2745-2753

3. Kempers MJ, Lenders JW, van Outheusden L, van der Wilt GJ, Schultze Kool LJ, Hermus
AR, Deinum J. Systematic review: diagnostic procedures to differentiate unilateral from
bilateral adrenal abnormality in primary aldosteronism. Annals of internal medicine 2009;
151:329-337

4. Page MM, Taranto M, Ramsay D, van Schie G, Glendenning P, Gillett MJ, et al. Improved
technical success and radiation safety of adrenal vein sampling using rapid, semi-quantitative
point-of-care cortisol measurement. Ann Clin Biochem. Jan 1 2018

21
HEDT Version 1.5 2018
2 Cushing Overview

Screening investigations: (exclude exogenous glucocorticoids)

1.1 Overnight 1 mg dexamethasone suppression test
1.2 Late night or 11pm salivary cortisol
1.3 24-hour UFC

Consider additional screening tests (if above results are equivocal or discrepant or to exclude
pseudo-Cushing’s)

1.4 2-day low dose oral dexamethasone suppression

1.5 Dexamethasone-CRH test
1.6 IV 4 mg dexamethasone suppression test

Cushing’s syndrome confirmed – measure plasma ACTH

 Suppressed ACTH (< 10ng/L or 2 pmol/L) – adrenal imaging studies for ACTH
independent Cushing’s
 Normal or elevated ACTH (> 20ng/L or 4 pmol/L) – proceed to further differential
diagnostic tests for ACTH dependent Cushing’s

Differential diagnosis of ACTH-dependent Cushing’s syndrome

Differentiate between pituitary and ectopic source of ACTH

A. High Dose 8 mg Dexamethasone Suppression Test (not required if IV 4mg

dexamethasone test already performed)
B. Peripheral CRH Test
C. Bilateral Inferior Petrosal Sinus Sampling

22
HEDT Version 1.5 2018
 2.1 OVERNIGHT DEXAMETHASONE SUPPRESSION TEST (1 mg
DST)

RATIONALE:

Almost all sources of inappropriate ACTH or cortisol hypersecretion secretion will not
be inhibited by 1 mg dexamethasone, therefore this is an excellent screening test.

PREPARATION & PROCEDURE:

1. Exclude exogenous glucocorticoid use and medications that may induce metabolism
of dexamethasone. Ensure female patients are not on oestrogen therapy.
2. Dexamethasone 1 mg (2x 0.5 mg tablets) to be given to patient (prescription of
dexamethasone may be required)
3. Patient instructed to take dexamethasone at 11 pm.
4. Patient to present to laboratory collection centre for blood test between 8-9am for
serum cortisol and ACTH.

Time Procedure Comment

11pm Take 1 mg dexamethasone tablets Ensure strict compliance.
with water
8-9am the next Cortisol Dexamethasone level available if
morning compliance queried

INTERPRETATION:

Test outcome Suggested comment:

Normal 1mg DST A morning cortisol of < 50 nmol/L after 1 mg dexamethasone
administered overnight indicates normal suppression of the
hypothalamic pituitary adrenal axis.
Abnormal 1mg DST A morning cortisol ≥ 50 nmol/L after 1 mg dexamethasone administered
overnight suggests hypercortisolism.

False positive responses – non-compliance, malabsorption, drugs inducing the hepatic

metabolism of dexamethasone (phenytoin, carbamazepine, phenobarbital, rifampicin), drugs
elevating CBG (oestrogens), chronic kidney disease, acute illness and pseudo-Cushing’s
(depression, alcohol abuse, eating disorders).

False negative responses – nephrotic syndrome (↓CBG), renal dialysis, chronic liver disease
(reduced metabolism and clearance of dexamethasone).

Sensitivity 98-100%, Specificity 85%

NOTES:

23
HEDT Version 1.5 2018
25% of hospitalised inpatients will have an abnormal 1 mg dexamethasone suppression test,
if possible, investigations should be delayed until acute illness has subsided.

2.2 LATE NIGHT SALIVARY CORTISOL

PREPARATION & PROCEDURE:

1. Patient to collect Salivettes from clinic or laboratory collection centre.

2. Patient should chew on Salivette for 2 minutes (or until saturated with saliva) between
11pm to midnight prior to brushing their teeth, and not within 30 mins of eating or
drinking.
3. Minimum of two separate samples should be collected.
4. Salivary cortisol is stable at room temperature. Patient should drop or post Salivettes
back to laboratory collection centre.
5. Smokers should avoid cigarette smoking on the day of the test.

Time Procedure Comment

11pm Chew on salivette provided for 1- Collect prior to brushing teeth and nil by
2 minutes until saturated. mouth for at least 30 mins prior to
collection.

INTERPRETATION:

Normal response – Consult reference intervals provided by the laboratory.

False positive responses – smokers and especially patients who chew tobacco,
contamination of salivettes with corticosteroid, bleeding of the gum, shift workers.

False negative responses - non-compliance with collection procedure (drinking water dilutes
the sample), cyclical Cushing’s.

NOTES:

An excellent test to use in the investigation of cyclical Cushing’s syndrome where initial
screening tests are negative. Repeat frequently over expected cycle e.g. weekly for 1-2
months as required.

24
HEDT Version 1.5 2018
 2.3 24 HOUR URINARY FREE CORTISOL (UFC)

PREPARATION & PROCEDURE:

1. Patient to collect urinary bottle (no preservative) from the collection centre.
2. Ensure complete 24-hour urine collection (laboratory will provide instruction).
3. Avoid excessive water drinking (>3L daily) and avoid glucocorticoid containing
preparations.
4. Suggest 2 x UFC on two separate occasions.

Time Procedure Comment

Day 1 Discard first morning void in
toilet, patient record starting time,
subsequent urine collected into
bottle.
Day 2 Last urine collected into bottle is Send bottle to laboratory for UFC and
starting time of Day 1 creatinine

INTERPRETATION :

Normal response – Consult the reference interval provided by the laboratory. The
laboratory should measure urine creatinine to assess adequacy of collection.

False positive responses – Over 24-hour urine collection. Excessive urine volume.

False negative responses - Inadequate 24-hour urine collection, renal impairment, cyclical
Cushing’s.

25
HEDT Version 1.5 2018
 2.4 2-DAY LOW DOSE DEXAMETHASONE SUPPRESSION TEST
(LDDST)

PREPARATION & PROCEDURE:

1. Exclude exogenous glucocorticoid use and medications that may induce metabolism of
dexamethasone. Ensure female patients are not on oestrogen therapy.
2. Baseline serum cortisol and plasma ACTH to be taken prior to administration of
dexamethasone.
3. Dexamethasone 0.5 mg (require total of eight tablets of 0.5 mg tablets for this test) to be
given to patient (prescription of dexamethasone may be required)
4. Patient instructed to take dexamethasone 0.5 mg at exactly 6-hourly intervals.
Option A: 9am, 3pm, 9pm, 3am (patient to set alarm clock for 3am), 9am, 3pm, 9pm,
3am, last blood test 9am (6 hrs after last dexamethasone dose)
Option B: 8am, 2pm, 8pm, 2am (patient to set alarm clock for 2am), 8am, 2pm, 8pm,
2am, last blood test 8am (6 hrs after last dexamethasone dose)
5. Patient to present to laboratory collection centre for blood test at exactly 9am for serum
cortisol for two consecutive days. Two separate request forms should be given to patient.
1st request form “Baseline serum cortisol and plasma ACTH”.
2nd request form “2-day Dex Suppression – Day 2 serum cortisol”

Time Procedure Comment

Day 0 (Baseline) 8- Blood test for cortisol and ACTH
9 am (baseline)
Bring completed urine collection
to lab.
Day 1: 9 am Patient to take 0.5 mg
dexamethasone
Day 1: 3pm Patient to take 0.5 mg
dexamethasone
Day 1: 9pm Patient to take 0.5 mg
dexamethasone
Day 1: 3 am Patient to take 0.5 mg Patient to set alarm clock for 3am to
dexamethasone take the Dex tablet.
Day 2: 9am Patient to take 0.5 mg
dexamethasone.
Start second urine collection if
requested.
Day 2: 3pm Patient to take 0.5 mg
dexamethasone
Day 2: 9pm Patient to take 0.5 mg
dexamethasone
Day 2: 3 am Patient to take 0.5 mg Patient to set alarm clock for 3am to
dexamethasone take the Dex tablet.
Day 3: 9 am (end of Blood test for cortisol and ACTH
test) (day 2)

26
HEDT Version 1.5 2018
INTERPRETATION:

Normal response = Serum cortisol < 50 nmol/L

False positive responses – non-compliance, malabsorption, drugs inducing the hepatic

metabolism of dexamethasone (phenytoin, carbamazepine, phenobarbital, rifampicin), drugs
elevating CBG (oestrogens), chronic kidney disease.

False negative responses – nephrotic syndrome (↓CBG), renal dialysis, chronic liver disease
(reduced metabolism and clearance of dexamethasone).

Sensitivity 96%, Specificity 70%.

27
HEDT Version 1.5 2018
 2.5 DEXAMETHASONE-CRH TEST

RATIONALE:

Patients with pseudo-Cushing's maintain sensitivity to negative feedback with

glucocorticoid and will be unable to mount a pituitary-adrenal response to CRH when
pre-treated with low dose dexamethasone. Conversely, patients with CS are mostly
insensitive to low-dose dexamethasone suppression and will display unsuppressed
cortisol levels and, in patients with a pituitary ACTH-secreting tumour, the pituitary will
respond to CRH stimulation.

PREPARATION & PROCEDURE:

1. Exclude exogenous glucocorticoid use and medications that may induce metabolism
of dexamethasone. Ensure female patients are not on oestrogen therapy.
2. Baseline serum cortisol and plasma ACTH to be taken prior to administration of
dexamethasone.
3. Dexamethasone 0.5 mg (require total of eight tablets of 0.5 mg tablets for this test) to
be given to patient (prescription of dexamethasone may be required)
4. Patient instructed to take dexamethasone 0.5 mg at exactly 6-hourly intervals.
(1200, 1800, 2400, 0600, 1200, 1800, 2400, 0600)
5. Blood test at 0800 for cortisol and ACTH
6. Inject CRH (1 µg/kg up to 100µg) at 0800 immediately after blood test (see peripheral
CRH test protocol for more details)
7. Blood test for cortisol 15 minutes after CRH

Time Procedure Comment

Day 1 (Baseline) 8- Blood test for cortisol and ACTH
9 am
Day 1: 1200 Patient to take 0.5 mg dexamethasone
Day 1: 1800 Patient to take 0.5 mg dexamethasone
Day 1: 2400 Patient to take 0.5 mg dexamethasone
Day 2: 0600 Patient to take 0.5 mg dexamethasone
Day 2: 1200 Patient to take 0.5 mg dexamethasone
Day 2: 1800 Patient to take 0.5 mg dexamethasone.
Day 2: 2400 Patient to take 0.5 mg dexamethasone
Day 3: 0600 Patient to take 0.5 mg dexamethasone
Day 3: 0800 Blood test for cortisol and ACTH

Day 3: 0800 Inject 1 µg/kg CRH

Day 3: 0815 Blood test for cortisol

INTERPRETATION:

Normal or pseudo-Cushing :
Post dexamethasone : cortisol < 38 nmol/L AND

28
HEDT Version 1.5 2018
Post dex-CRH : cortisol < 38 nmol/L

Cushing’s Disease:
Post dexamethasone : cortisol > 38 nmol/L
Post dex-CRH : cortisol > 38 nmol/L

False positive responses – non-compliance, malabsorption, drugs inducing the hepatic

metabolism of dexamethasone (phenytoin, carbamazepine, phenobarbital, rifampicin), drugs
elevating CBG (oestrogens), chronic kidney disease

False negative responses – nephrotic syndrome (↓CBG), renal dialysis, chronic liver disease
(reduced metabolism and clearance of dexamethasone).

29
HEDT Version 1.5 2018
 2.6 IV 4 mg DEXAMETHASONE SUPPRESSION TEST

PREPARATION & PROCEDURE:

Patient admitted for day procedure

1. Ensure female patients are not on oestrogen therapy

2. Insertion of IV cannula
3. Samples for baseline serum cortisol to be collected at two time-points (-60mins, -5
mins) prior to dexamethasone infusion. Commence -60 mins sampling at 0830h
4. Dexamethasone infusion 1 mg/h for 4 hours (1x4 mg ampoule of dexamethasone in
500 ml Normal Saline running at 125 ml/hr) commencing at 0930h
5. Samples for serum cortisol to be collected 3, 4 and 5 hours post infusion. Ensure
blood collection is taken opposite the infusion site.
6. Patient to present to the lab the following day for serum cortisol at 9:00 am and 9:30
am (unless patient remains an inpatient)

Time Procedure Comment

Day1 Insertion of IV cannula
-60 minutes cortisol
(Baseline, 08:30)
-5 minutes cortisol
0 minutes (09:30) IV Dex infusion commences for 4 4mg dexamethasone in 500 ml Normal
hours duration. Saline at 125 ml/hr
+ 3 hrs cortisol Ensure blood collection is taken opposite
the infusion site
+4 hrs cortisol Completion of the 4 hr Dex infusion
+5 hrs cortisol Patient sent home after blood test
Day 2 cortisol Patient returns for blood test
+23.5h (9:00)
+24h (9:30) cortisol

INTERPRETATION:

Diagnosis of Cushing’s syndrome: (2)

 Day 2 serum cortisol level (mean of +23.5h and +24h cortisol values) >130 nmol/L or
>20% of baseline cortisol (Day 1 at -60 minutes)
 Sensitivity 100%, Specificity 96%
 Cushing’s disease tends to partially suppress on Day 1 with rebound increase on Day 2,
while ectopic ACTH patients rarely suppress during the infusion.

30
HEDT Version 1.5 2018
 2.7 ACTH Dependent Cushing’s Syndrome

2.7.1 HIGH DOSE DEXAMETHASONE SUPPRESSION TEST (HDDST)

Overnight High Dose 8 mg Dexamethasone Suppression Test

PREPARATION & PROCEDURE:

1. Exclude exogenous glucocorticoid use and medications that may induce metabolism of
dexamethasone. Ensure female patients are not on oestrogen therapy.
2. Ensure patient already had baseline morning serum cortisol
3. Dexamethasone 8mg (2x 4 mg tablets) to be given to patient (prescription of
dexamethasone may be required)
4. Patient instructed to take dexamethasone at 11 pm.
5. Patient to present to laboratory collection centre for blood test between 8-9am for serum
cortisol and ACTH.

Time Procedure Comment

8 am cortisol Ensure patient already had baseline
morning cortisol.
11pm Take 8 mg dexamethasone tablets Ensure strict compliance.
8-9am the next cortisol Dexamethasone level available if
morning compliance queried

INTERPRETATION:

Decrease in serum cortisol >50% is suggestive of pituitary Cushing’s disease. Most

Cushing’s disease patients have a cortisol < 140 nmol/L, normal subjects have undetectable
cortisol post 8mg dexamethasone suppression.

HOWEVER, 10% of patients with ectopic ACTH secretion suppress with high dose
dexamethasone and some patients with pituitary tumours fail to suppress. (Sensitivity 81%,
Specificity 79%)

31
HEDT Version 1.5 2018
 2.7.2 PERIPHERAL CRH TEST

RATIONALE:

This test may be combined with bilateral inferior petrosal sinus sampling (BIPSS) or can be
performed prior to BIPSS to provide a diagnosis of Cushing’s Disease along with appropriate
MRI finding and suppressed high dose dexamethasone test to avoid the need for BIPSS.

PREPARATION & PROCEDURE:

1. Order CRH (100 µg)

2. Baseline weight and blood pressure
3. Patient should be supine during the test
4. Insertion of IV cannula
5. Baseline plasma ACTH and serum cortisol collection (-5 and -1 minutes)
6. CRH to be given as an IV bolus over 30-60 seconds (1 µg/kg up to a maximum of 100
g).
7. Warned patient of potential side-effects from CRH: flushing, metallic taste in mouth,
transient hypotension
8. Collect plasma ACTH and cortisol post CRH injection at +15, +30, +45, +60 and +90
minutes.

Time Procedure Comment

.
-5 min ACTH and cortisol
-1 min ACTH and cortisol
0 min IV CRH over 30-60 seconds Warn patient of side-effects
15 min ACTH and cortisol
30 min ACTH and cortisol
45 min ACTH and cortisol
60 min ACTH and cortisol
90 min ACTH and cortisol

INTERPRETATION:

There is no universal agreement on blood test interval and cut off.

Pituitary Cushing’s disease is more likely than ectopic ACTH production if:

 Peak ACTH increment of >50% from mean basal values (Sensitivity 86%,
Specificity 90%). (8,9)
 increase in peak cortisol concentration ≥ 30% above the mean basal values
(Sensitivity 61%, Specificity 70%). (8)
Note :

 CRH used in Australia is recombinant human CRH rather than Ovine CRH used in
Nieman study. (3)
 Peak ACTH has higher diagnostic accuracy than cortisol.

32
HEDT Version 1.5 2018
 2.7.3 BILATERAL INFERIOR PETROSAL SINUS SAMPLING

RATIONALE:

In ACTH dependent Cushing’s syndrome, to differentiate whether the source of excessive

ACTH is central pituitary Cushing’s disease or peripheral ectopic ACTH syndrome. IPSS
might not be required if pituitary adenoma > 6mm AND cortisol suppressed to high dose
dexamethasone AND ACTH/ cortisol rose with peripheral CRH test.

PREPARATION & PROCEDURE:

1. Book experienced interventional radiologist, discuss if patient on blood thinning agents.

2. Confirm active phase of Cushing’s by saliva cortisol at midnight prior to the test, liaise
with laboratory regarding turn-around time and alert them to the BIPSS booking.
Metyrapone and ketoconazole should be stopped 1 week prior to test.
3. Fast the patient from midnight for a morning procedure
4. Under radiological guidance in the Radiology Department, catheters are placed in the left
and right inferior petrosal sinuses.
5. Blood is collected at -5 min and -2 min before CRH administration, from left, right
petrosal catheters and peripheral vein to measure ACTH and prolactin.
6. CRH (1 µg/kg, maximum dose up to 100 µg) is given intravenously at 0 min.
7. Further samples to measure ACTH and Prolactin are collected from left and right petrosal
sinus and peripheral vein at + 2, 5, 10 and 15 minutes post CRH.

Time Procedure Comment

- 5 min ACTH and prolactin from left, 1 x 4 ml EDTA tube from each site
right petrosal sinus and peripheral
vein
- 2 min ACTH and prolactin from left, 1 x 4 ml EDTA tube from each site
right petrosal sinus and peripheral
vein
0 min IV CRH (1 µg/kg, up to 100 µg) Warn patient of side-effects of
flushing and hypotension
+ 2 min ACTH and prolactin from left, 1 x 4 ml EDTA tube from each site
right petrosal sinus and peripheral
vein
+ 5 min ACTH and prolactin from left, 1 x 4 ml EDTA tube from each site
right petrosal sinus and peripheral
vein
+10 min ACTH and prolactin from left, 1 x 4 ml EDTA tube from each site
right petrosal sinus and peripheral
vein
+15 min ACTH and prolactin from left, 1 x 4 ml EDTA tube from each site
right petrosal sinus and peripheral
vein

33
HEDT Version 1.5 2018
INTERPRETATION

 A central to peripheral ACTH ratio of ≥ 2 pre CRH and / or a ratio of ≥ 3 post CRH is
consistent with Cushing’s disease. (5) Sensitivity and specificity 94%
 IPSS has limited utility in localization of ACTH-secreting pituitary adenomas
 Maximal IPS/ peripheral ratio is achieved at 5 minutes in 90% of Cushing’s Disease,
1% achieved the maximum ratio at 15 minutes. The 2 minutes time point was found to
have the best diagnostic accuracy. (5)
 If the above ratios were not achieved on at least one side, then check for adequacy of
petrosal sinus catheterisation. Petrosal sinus prolactin level > 1.8 times peripheral
prolactin level indicates adequate petrosal sinus cannulation. (see diagram below) (7)
In that setting, pituitary ACTH/PRL to peripheral ACTH/PRL gradient of >0.8 is
suggestive of Cushing’s disease. A pituitary ACTH/PRL to peripheral ACTH/PRL
gradient of <0.6 is indicative of ectopic ACTH syndrome.
 False results occur if patient was not in active phase of hypercortisolism at the time of
testing. Late night saliva cortisol before IPSS can assist in determining whether
cyclical Cushing patients are in active phase before proceeding with the test.

Figure adapted from reference 7.

34
HEDT Version 1.5 2018
NOTES:

 ACTH and prolactin can be analysed using a single 4 ml EDTA tube in most centres.
This vastly reduces the number of tubes and volume of blood required for IPSS and
need to be discussed with the laboratory.
 Side effects of CRH includes flushing and hypotension. Rare complications during
IPSS include brain stem injury, (6) deep venous thrombosis, pulmonary embolism and
venous subarachnoid haemorrhage.
 Anticoagulation with heparin can reduce prothrombotic complications.

REFERENCES:

1. Nieman LK, Biller BMK, Findling JW, Newell-Price J, Savage MO, Stewart PM and
Montori VM. J Clin Endocrinol Metab 2008;93(5):1526-1540.
2. Jung C, Alford FP, Topliss DJ, Burgess JR, Gome JJ, Stockigt JR and Inder WJ. The 4-
mg intravenous dexamethasone suppression test in the diagnosis of Cushing’s syndrome.
Clin Endocrinol 2010;73(1):78-84
3. Nieman LK, Oldfield EH, Wesley R, Chrousus GP, Loriaux DL and Cutler GB. A
simplified morning ovine corticotropin-releasing hormone stimulation test for the
differential diagnosis of adrenocorticotropin-dependent Cushing's syndrome. J Clin
Endocrinol Metab 1993;77:1308-12
4. Loriaux DL. Diagnosis and Differential Diagnosis of Cushing’s Syndrome. N Engl J
Med 2017;376:14519.
5. Oldfield EH, Chrousos GP, Schulte HM, Schaaf M et al. Preoperative lateralization of
ACTH- secreting pituitary microadenomas by bilateral and simultaneous inferior
petrosal venous sinus sampling. N Engl J Med 1985; 312: 100–103.
6. Gandhi CD, Meyer SA, Patel AB, Johnson DM, Post KD. Neurologic Complications of
Inferior Petrosal Sinus Sampling. Am J Neuroradiol 2008; 29: 760–5.
7. Sharma ST, Raff H, Nieman LK. Prolactin as a marker of successful catheterization
during IPSS in patients with ACTH-dependent Cushing's syndrome. The Journal of
clinical endocrinology and metabolism 2011; 96:3687-3694
8. Reimondo G, Paccotti P, Minetto M, Termine A, Stura G, Bergui M, et al. The
corticotrophin-releasing hormone test is the most reliable noninvasive method to
differentiate pituitary from ectopic ACTH secretion in Cushing's syndrome. Clinical
endocrinology. 2003;58(6):718-24.
9. Invitti C, Pecori Giraldi F, de Martin M, Cavagnini F. Diagnosis and management of
Cushing's syndrome: results of an Italian multicentre study. Study Group of the Italian
Society of Endocrinology on the Pathophysiology of the Hypothalamic-Pituitary-Adrenal
Axis. The Journal of clinical endocrinology and metabolism. 1999;84(2):440-8

35
HEDT Version 1.5 2018
3 Hypopituitarism
3.1 INSULIN TOLERANCE TEST

RATIONALE

To assess the integrity of the hypothalamic pituitary adrenal axis in patients with suspected
secondary adrenal insufficiency

To assess the integrity of the growth hormone axis in patients with suspected growth
hormone deficiency

PREPARATION AND PROCEDURE:

1. The test should not be undertaken in patients ischaemic heart disease, cerebrovascular
disease, cardiac arrhythmias or epilepsy. The test should only be done with caution in an
experienced unit in patients with morning cortisol < 100 nmol/L, or >70 years of age.

2. Patients should provide written informed consent prior to the procedure.

2. Fast (water only) and no smoking from midnight the night before the test. Omit
glucocorticoids; hydrocortisone after 1600h the day before (at least 16h) and prednis(ol)one
from 0800h the day before (24h).

3. Baseline weight, pulse and BP.

4. ECG.

5. Insert an 18-20g cannula with a three-way tap into an antecubital vein. Secure venous
access is crucial prior to commencing the test. The cannula should both flush freely and draw
easily.

6. The dose of insulin should be determined by the requesting endocrinologist prior

to the procedure. The table below serves as a guide only.

Insulin doses for adults ≥18 years:

Dose Classification Condition Insulin dose

(units/kg)
Low dose High probability of hypopituitarism 0.1
Standard dose BMI <30 kg/m2, non-diabetic 0.15

Insulin resistant Obese (BMI >30 kg/m2) and/or metabolic syndrome 0.2
dose with fasting glucose >5.5 mmol/L
High dose Active acromegaly or Cushing’s syndrome, type 2 0.3
diabetes

36
HEDT Version 1.5 2018
8. The insulin should be diluted to 10 units/mL in 0.9% saline to ensure accurate dosing.

9. Regular human insulin (Novorapid, Actrapid, Humulin R) 100 units/ml. 0.5 mL (50
units) + 4.5 mL 0.9% saline in a 5 ml syringe. The final dose should then be drawn up in a 1
mL or 2 mL syringe according to whether the final dose is <10 units or >10 units.

10. Point of care (measured on the venous sample) and plasma glucose should be
measured throughout the test, but the final determination of adequate hypoglycaemia should
be made on the basis of the plasma glucose result.

11. Take baseline samples at -5 mins (Glucose, cortisol, GH) and 0 min (Glucose,
cortisol, GH, ACTH), then insulin iv over 1 minute immediately following blood sampling

12. Repeat samples at 20, 30 and 40 minutes

13. If glucose has not fallen to ≤2.2 mmol/L, administer second insulin dose 50% higher
than the initial dose

* Repeat insulin dosing in the event of inadequate hypoglycaemia:

If glucose remains >2.2 mmol/L and there are no hypoglycaemic symptoms at 40 min, a
second dose of iv insulin should be given at 50% higher than the initial bolus.

This should represent a new time 0 minutes, with sampling at 20, 30, 40, 60, 90 and 120
minutes after the second injection.

If glucose remains >2.2 mmol/L after the second dose, a third dose of insulin double the
initial dose can be considered. However, by then both patient and investigator may be more
willing to abandon the procedure.

14. An oral carbohydrate solution (e.g. lemonade‡) and 50% glucose for intravenous use
must be available to treat hypoglycaemia if required. Hydrocortisone 100 mg for intravenous
use should also be available if required. (see notes for glucose rescue)

15. A carbohydrate meal should be given at the end of the test. For patients at high risk of
hypopituitarism in whom the serum glucose has been slow to recover, consider
hydrocortisone 50 mg IV at the completion of sampling.

37
HEDT Version 1.5 2018
PROCEDURE:

Time Procedure Comment

-15 minutes Glucose, cortisol, GH
0 minutes Glucose, ACTH, cortisol, GH,
then insulin iv over 1 minute
immediately following blood
sampling
20 minutes Glucose, cortisol, GH
30 minutes Glucose, cortisol, GH
40 minutes Glucose, ACTH, cortisol, GH If glucose has not fallen to ≤2.2
mmol/L, administer second insulin
dose 50% higher than the initial
dose *
60 minutes Glucose, cortisol, GH
90 minutes Glucose, cortisol, GH
120 minutes Glucose, cortisol, GH

INTERPRETATION:

 Adequate hypoglycaemia defined as serum glucose ≤2.2 mmol/L

 Normal response for cortisol: peak cortisol ≥ 500 nmol/L (or cortisol cut-off specified
for that assay) at any time of the test
 Abnormal response for cortisol: peak cortisol < 500 nmol/L
(Cortisol cut-off is assay dependent. Ideally local reference interval should be established
based on cortisol assay used.)

 GH > 5 µg/L excludes GH deficiency, GH <3 µg/L consistent with GH deficiency.

Proposed PBS criteria for treatment of adult GH deficiency in Australia is a peak of
<2.5 µg/L.

NOTES:

Glucose rescue:
Intravenous glucose should be administered in the event of severe hypoglycaemia defined as
any of the following:
• Plasma glucose ≤1.5 mmol/L
• Altered level of consciousness
• Seizure
Initial dose recommended is 25 mL of 50% glucose.
If point of care glucose <3.0 mmol/L after 5 minutes, repeat IV dose (if patient is unable to
ingest oral liquid) OR administer lemonade‡ 200 mL orally.
For patients with mild-moderate hypoglycaemic symptoms, rescue with an oral carbohydrate
solution is unnecessary. Early rescue may blunt the stress response and result in a falsely
abnormal result.
If patient has definite hypoglycaemic symptoms for >10 minutes and point of care glucose
remains ≤2.2 mmol/L, administer lemonade‡ 200 mL orally.
38
HEDT Version 1.5 2018
‡ Concentration of sugars in Sprite is 10.1g/100 mL (sucrose), so approximately 5g glucose
and 5g fructose per 100 ml. Concentration in Schweppes lemonade is similar at 11g/100 ml.

REFERENCES:

1. Sarlos S and Inder WJ. Selective use of the insulin tolerance test to diagnose
hypopituitarism. Int Med J 2013; 43:89-93.

2. Lange M et al. An audit of the insulin-tolerance test in 255 patients with pituitary disease.
Eur J Endocrinol 2002; 147:41-7.

3. Fincuane FM et al. Clinical insights into the safety and utility of the insulin tolerance test
(ITT) in the assessment of the hypothalamo-pituitary-adrenal axis. Clin Endocrinol 2008;
69:603-7.

39
HEDT Version 1.5 2018
 3.2 OVERNIGHT METYRAPONE TEST

RATIONALE:

 Patients with suspected secondary adrenal insufficiency due to pituitary or hypothalamic

dysfunction may have a normal cortisol response to Synacthen and require a test of the
entire HPA axis. The overnight metyrapone test provides a good alternative to the ITT,
particularly if there are contraindications to performing an ITT or assessment of GH
status is not required.
 Metyrapone inhibits the last step (11-hydroxylation) in the synthesis of cortisol. The
negative feedback inhibition of cortisol on ACTH is thereby reduced, leading to elevated
ACTH and an increased 11-deoxycortisol in normal individuals.

PREPARATION & PROCEDURE:

 If patient is taking glucocorticoid replacement, the morning glucocorticoid tablets should

be taken but the evening dose is NOT TO BE TAKEN.
 Metyrapone comes as a 250 mg capsule
 Between 11pm and midnight, patient is to have 30 mg/kg Metyrapone, rounded up to the
nearest 250 mg and maximum dose 3 g.
 e.g. an 80 kg person would take 2.5 g (10 capsules)
 Take with a glass of milk and a snack.
 Remind patient that if they forget to have the metyrapone tablets, then not to present for a
blood test the following morning.
 The patient is not to have their morning glucocorticoid tablets prior to blood test.
 Between 0800h and 0900h, take blood sample for 11-deoxycortisol, cortisol and ACTH.
 The patient should then have their usual morning glucocorticoid medication if prescribed.

PROCEDURE:

Time Procedure Comment

Day 0 Withhold evening dose of
glucocorticoid
Day 0 Metyrapone 30 mg/kg (Max 3 g) Take with a glass of milk and a
23:00-24:00 snack.
Day 1 Withhold morning dose of
glucocorticoid
Day 1 11-deoxycortisol, cortisol and After blood test, can take usual
08:00 – 09:00 ACTH morning glucocorticoid
medication if prescribed

INTERPRETATION:

If cortisol <200 nmol/L, metyrapone inhibition of cortisol and subsequent ACTH stimulation
has been adequate (i.e. test interpretable)
11-deoxycortisol: >200 nmol/L – normal.
<200 nmol/L – secondary adrenal insufficiency

40
HEDT Version 1.5 2018
NOTES

 In a large series from Ireland1, side effects only occurred in 7/398 patients having 576
tests. Side effects include nausea and vomiting, dizziness, nightmares.
 The risk of adrenal crisis from acute cortisol deficiency is very low, but the test should
not be performed in patients with suspected primary adrenal insufficiency.
 Some centres advocate giving the patients oral hydrocortisone or cortisone acetate to take
home in case of severe symptoms of acute cortisol deficiency

REFERENCES:

1. Fiad TM, Kirby JM, Cunningham SK, McKenna TJ. The overnight single-dose
metyrapone test is a simple and reliable index of the hypothalamic-pituitary-adrenal axis.
Clin Endocrinol 1994; 40:603-609

2. Soule S, van Zyl C, Parolis G, Attenborough S, Peter D, Kinvig S, Kinvig T, Coetzer E.

The low dose ACTH stimulation test is less sensitive than the overnight metyrapone test
for the diagnosis of secondary hypoadrenalism. Clin Endocrinol 2000; 53:221-227

3. English K, Inder WJ, Weedon Z, Dimeski G, Sorbello J, Russell AW, Duncan EL, Cuneo
R. Prospective evaluation of a week one overnight metyrapone test with subsequent
dynamic assessments of hypothalamic-pituitary-adrenal axis function after pituitary
surgery. Clin Endocrinol 2017; 87:35-43.

41
HEDT Version 1.5 2018
 3.3 GLUCAGON STIMULATION TEST

RATIONALE:

Glucagon is a hormone that stimulates glycogenolysis in the liver as well as ACTH and growth
hormone release from the pituitary with peak GH response after 90-180 minutes. Glucagon
stimulation test is recommended as the alternative to ITT for diagnosis of adult GH deficiency based
on its reproducibility and safety, Glucagon also stimulated adrenal production of cortisol in subjects
with adequate endogenous ACTH. (6)

Rarely glucagon may be associated with headaches or vomiting and there is a small risk of late
hypoglycaemia. This test should not be performed in malnourished subjects.

PREPARATION:

 Test performed in the morning between 08:00 to 09:00 after fasting from midnight.
 In view of duration and potential late hypoglycaemia best performed as a Day
Admission.
 Collect baseline GH, IGF-1, IGFBP-3, Cortisol, glucose.
 Give Glucagon 1 mg (1.5 mg if weight > 90 kg)
 Take additional blood samples at 90, 120, 150, 180, 210 and 240 minutes.

PROCEDURE:

Time Procedure Comment

Baseline GH, IGF-1, IGFBP-3
Cortisol, glucose
0 minute IM glucagon
90 minutes GH, cortisol, glucose Monitor capillary glucose
120 minutes GH, cortisol, glucose Monitor capillary glucose
150 minutes GH, cortisol, glucose Monitor capillary glucose
180 minutes GH, cortisol, glucose Monitor capillary glucose
210 minutes GH, cortisol, glucose Monitor capillary glucose
240 minutes GH, cortisol, glucose Monitor capillary glucose

NOTE:

In the event of hypoglycaemia (glucose <3) following glucagon obtain an immediate blood sample for
glucose and GH followed by 10% IV dextrose (2 ml/kg) over 3 minutes. After 5 minutes recheck
glucose using a point of care glucometer.

All patients should have a small snack before discharge.

INTERPRETATION:

Normal response:

 Any GH > 3 ug/L. (5, 7)

42
HEDT Version 1.5 2018
  Any Cortisol ≥ 248 nmol/L (cortisol cut-off based on Siemens Centaur assay). (7)
REFERENCES:

1. Corneli G, Gasco V, Prodam F, Grottoli S et al. Growth hormone levels in the diagnosis of
growth hormone deficiency in adulthood. Pituitary 2007; 10:141–149.

2. Alba-Roth J, Muller OA, Schopohl J, Von Werder K. Arginine Stimulates Growth

Hormone Secretion by Suppressing Endogenous Somatostatin Secretion. J Clin
Endocrinol Metab 1988; 67: 1186 - 1189.

3. Richmond EJ, Rogol AD. Growth hormone deficiency in children. Pituitary 2008; 11(2):
115-20.

4. Cohen P, Rogol AD, Deal CL, Saenger P et al. Consensus Statement on the Diagnosis and
Treatment of Children with Idiopathic Short Stature: A Summary of the Growth Hormone
Research Society, the Lawson Wilkins Pediatric Endocrine Society, and the European
Society for Paediatric Endocrinology Workshop. J Clin Endocrinol Metab 2008; 93: 4210–
4217.

5. Gomez JM, Espadero RM, Escobar-Jimenez F, Hawkins F, Pico A, Herrera-Pombo JL, et

al. Growth hormone release after glucagon as a reliable test of growth hormone assessment in
adults. Clinical endocrinology. Mar 2002; 56(3):329-34.

6. Yuen KC, Tritos NA, Samson SL, Hoffman AR, Katznelson L. American Association of
Clinical Endocrinologists and American College of Endocrinology disease state clinical
review: update on growth hormone stimulation testing and proposed revised cut-point for the
glucagon stimulation test in the diagnosis of adult growth hormone deficiency. Endocrine
practice : official journal of the American College of Endocrinology and the American
Association of Clinical Endocrinologists. 2016;22(10):1235-44.

7. Hamrahian AH et al. Revised GH and cortisol cut-points for the glucagon stimulation test
in the evaluation of GH and hypothalamic-pituitary-adrenal axes in adults. Pituitary. 2016;
19:332-341.

43
HEDT Version 1.5 2018
 3.4 GONADOTROPHIN RELEASING HORMONE STIMULATION
TEST

RATIONALE:

This test assists to differentiate severe delayed puberty vs hypogonadotrophic hypogonadism.

Gonadotrophin releasing hormone (GnRH, LHRH) is a hypothalamic decapeptide stimulating
synthesis and rapid release of LH and FSH in the anterior pituitary. Leuprorelin (Lucrin, Abbott) is a
synthetic GnRH analogue with similar action. An absent or sub-optimal response to its administration
indicates hypofunction of the hypothalamus or pituitary.

PREPARATION:

 No specific preparation.
 Generally performed in the morning but may be at any time of the day.
 Leuprorelin (Lucrin, Abbott or alternative equivalent) 5,000 g/ml is available in a
multidose vial. Dose is 0.004 ml/kg given subcutaneously (e.g. 0.2 ml/50kg).
Maximum dose 0.2 ml (1,000 ug).

PROCEDURE:

Time Procedure Comment

Baseline LH, FSH plus
E2(F)/Testosterone (M)
0 minute Leuprorelin administration
30 minutes LH, FSH
60 minutes LH, FSH Typically peak response
120 minutes LH, FSH

INTERPRETATION:

Pre-pubertal peak LH response <5 mIU/L

Normal adult peak response 20-100 mIU/L

NOTES:

Patient should be observed for 5 minutes in case of allergic reaction although no such reaction has
been documented.

Some historic protocols have utilised more frequent samples with no obvious benefit.

REFERENCES:

1. Kletter GB. Commentary: How Should We Diagnose and Monitor Central Precocious
Puberty? Journal of Pediatric Endocrinology & Metabolism 2008; 21: 1105 - 6.

44
HEDT Version 1.5 2018
2. Ibanez L, Potau N, Zampolli M, Virdis R, Gussinye M, Carrascosa A, Saenger P &
Vicens-Calvet E. Use of Leuprolide acetate response patterns in the early diagnosis of
pubertal disorders: Comparison with the gonadotropin - releasing hormone test. JCEM 1994;
78 (1): 30-5.

3. Resende EA, Lara BH, Reis JD, Ferreira BP, Pereira GA, & Borges MF. Assessment
of Basal and Gonadotropin-Releasing Hormone-Stimulated Gonadotropins by
Immunochemiluminometric and Immunofluorometric Assays in Normal Children. JCEM
2007; 92(4): 1424–9.

4. Neely EK, Hintz RL, Wilson DM, Lee PA, Gaultier T, Argente J, Stene M. Normal
ranges for immunochemiluminometric gonadotropin assays. J Pediatr 1995; 127: 40–6.

5. Eckert KL, Wilson DM, Bachrach LK, Anhalt H, Habiby RL, Olney RC, Hintz RL,
Neely EK. A Single-sample, Subcutaneous Gonadotropin-releasing Hormone Test for Central
Precocious Puberty. Pediatrics 1996; (4): 517-519.

6. Brito VN, Batista MC, Borges MF, Latronico AC, Kohek MB, Thirone AC, Jorge B,
Arnhold IV, Mendonc BB. Diagnostic value of fluorometric assays in the evaluation of
precocious puberty. J Clin Endocrinol Metab 1999; 84: 3539–44.

7. Street ME, Bandello MA, Terzi C, Ibanez L, Ghizzoni L, Volta C, Tripodi C, Virdis
R. Leuteinizing hormone responses to leuprolide acetate discriminate between
hypogonadotropic hypogonadism and constitutional delay of puberty. Fertility & Sterility,
2002; 77(3): 555-60.

45
HEDT Version 1.5 2018
4 Acromegaly
4.1 GROWTH HORMONE SUPPRESSION TEST

RATIONALE:

To diagnose acromegaly (growth hormone (GH) excess) when IGF-1 elevated or discordant
with clinical presentation. Normal subjects suppress GH secretion in response to a glucose load,
whereas acromegalic subjects fail to suppress GH secretion or show a paradoxical rise. Random GH
has no role for diagnosis.

In post-op acromegalic patient with elevated IGF-1 or random GH ≥ 1.0 µg/L more than 12
weeks post-surgery, OGTT suppression is used to assess residual disease. This test cannot be
used in patients already on somatostatin analogue.

PREPARATION:

1. The patient should fast overnight prior to the test.

2. Control hyperglycaemia prior to testing.

PROCEDURE:

1. Patient attends for blood test after 10 hr fast

2. Collect baseline glucose, GH and IGF-1
3. Give 75g oral glucose load
4. Collect Glucose and GH every 30 minutes for 2 hours

Time Procedure/ Test Comment

0 minute Glucose, GH, IGF-1

0 minute 75g oral glucose load

30 minutes Glucose, GH
60 minutes Glucose, GH
90 minutes Glucose, GH
120 minutes Glucose, GH

INTERPRETATION:

Normal response: GH < 1.0 µg/L at any time point

A nadir GH post glucose load < 0.4 µg/L has been considered, but due to variable
performance of the commercial GH assays, the 2014 Endocrine Society Clinical Practice
Guideline recommended using a universal cut-off < 1.0 µg/L for acromegalic diagnosis.
Nadir GH post glucose load < 0.4 µg/Lis used as the definition for post-operative remission.

46
HEDT Version 1.5 2018
NOTES:

Poorly controlled diabetes mellitus, nutritional disorders (malnutrition, malabsorption,

anorexia nervosa), renal disease and liver failure are associated with acquired GH
insensitivity to glucose suppression, associated with risk of false-positive OGTT results.

REFERENCES:

1. Carmichael JD, Bonert VS, Mirocha JM, Melmed S. The Utility of Oral Glucose
Tolerance Testing for Diagnosis and Assessment of Treatment Outcomes in 166 Patients
with Acromegaly. J Clin Endocrinol Metab 2009; 94: 523–527.

2. Katznelson L, Laws ER, Jr., Melmed S, Molitch ME, Murad MH, Utz A, Wass JA,
Endocrine S. Acromegaly: an endocrine society clinical practice guideline. The Journal
of clinical endocrinology and metabolism 2014; 99:3933-3951

47
HEDT Version 1.5 2018
5 Hyperglycaemia investigation
5.1 ORAL GLUCOSE TOLERANCE TEST (OGTT)

RATIONALE:

To diagnose diabetes mellitus or gestational diabetes.

PREPARATION:

1. The oral carbohydrate intake should be normal for three days prior to testing.

2. The patient should fast from 10 pm the day prior to the morning OGTT test (water
allowed).

3. For diagnosis of gestational diabetes, the test is conducted between 26-28 weeks.

PROCEDURE:

1. The test is performed in the morning, patient should rest during the test and may not eat or
smoke, drinking water is permitted.

2. At baseline, collect blood for fasting glucose measurement. Concurrently test glucose on a
glucometer if available. If results are >10.0 mmol/L, consider terminating the test after
discussion with requesting doctor as per local procedure.

3. Give glucose solution 75 g orally (check with laboratory for dose in children), consume
within 5 minutes.

4. Collect blood hourly for 2 hours.

5. If the patient has any abnormal symptoms during the test (vomiting, sweating, tremors,
unwell), discuss with medical personnel as per local procedure.

Time Procedure/ Test Comment

Baseline Glucose Consider terminating test if point of care fasting
(glucose testing on glucometer glucose >10 mmol/L as per local procedure
if available)
0 minute 75g oral glucose load within 5
minutes
60 minutes glucose
120 minutes glucose

48
HEDT Version 1.5 2018
INTERPRETATION:

OGTT results in the non-pregnant patient are interpreted as below according to WHO
criteria (4):

Plasma venous glucose (mmol/L)

Test Outcome: Fasting 2 hrs post glucose load
Normal glucose tolerance ≤6.0 AND <7.8
Impaired fasting glycaemia 6.1 – 6.9 AND < 7.8
Impaired glucose tolerance < 7.0 AND 7.8 – 11.0
Diabetes Mellitus ≥7.0 OR ≥11.1

OGTT results in the pregnant patient are interpreted as below according to ADIPS criteria
(1):

Plasma venous glucose (mmol/L)

Test Outcome: Fasting 1 hr 2 hrs post glucose load

Normal ≤ 5.0 AND < 10.0 AND < 8.5

Gestational diabetes 5.1–6.9 OR >10.0 OR 8.5–11.0

mellitus
Diabetes mellitus in ≥ 7.0 OR * OR ≥11.1
pregnancy

NOTES:

*there are no established criteria for the diagnosis of diabetes based on the 1-h post-load
value

The above ADIPS criteria are not used in New Zealand, gestational diabetes mellitus is
diagnosed if fasting ≥ 5.0 mmol/L and 2-h glucose ≥ 9 mmol/L.

49
HEDT Version 1.5 2018
REFERENCES:

1. Nankervis A, McIntyre HD, Moses R, Ross GP, Callaway L, Porter C, Jeffries W,

Boorman C, De Vries B, McElduff A for the Australasian Diabetes in Pregnancy
Society. ADIPS Consensus Guidelines for the Testing and Diagnosis of Gestational
Diabetes Mellitus in Australia.

2. ADPSG Consensus Panel International association of diabetes and pregnancy study

group’s recommendations on the diagnosis and classification of hyperglycemia in
pregnancy. Diabetes Care 2010; 33:676‐682

3. HAPO Collaborative Research Group. Hyperglycemia and adverse pregnancy outcomes.

New Eng J Med 2008; 358:1991-2002

4. World Health Organisation / International Diabetes Federation, Definition and

Diagnosis of Diabetes Mellitus and Intermediate Hyperglycaemia, 2006. (Web
address: [Link]

50
HEDT Version 1.5 2018
6 Hypoglycaemia investigation
6.1 MIXED MEAL TEST

RATIONALE:

To trigger a hypoglycaemic episode which can be captured with plasma glucose and insulin
in patients with post prandial hypoglycaemia. This is preferred to the OGTT for capturing
hypoglycaemia.

PREPARATION:

Patient to fast (may drink water) from 2400 the night before test. Avoid smoking, undue
exercise and alcohol. Withhold all non-essential medications.
Patient brings the Test Meal (Similar to one which provokes hypoglycemic symptoms) or a
commercial meal can be provided (see Notes below).

PROCEDURE:

1. Insert intravenous cannula - usually into cubital fossa.

2. Take baseline blood for glucose, insulin, C-peptide, proinsulin

3. Give Test Meal (provided by patient, if possible). Test meal to be similar to that which the
patient reports has caused symptoms. Record test meal on work sheet.

4. Collect blood specimens for glucose, insulin, C-peptide at times 0, +30, +60, +90, +120,
+180, +210, +240, +270, +300 mins. Extra specimens taken if symptoms or signs of
hypoglycaemia.

5. Record any symptoms experienced by patient. If hypoglycaemic symptom occurs prior to

300 minutes, take samples for Glucose, insulin, C-peptide, proinsulin, confirm glucose < 3.0
mmol/L, then correct hypoglycaemia.

6. If Whipple’s triad is demonstrated a sample should also be collected for sulphonylurea

analysis and insulin antibodies. Add request for proinsulin in baseline sample and hypoglycaemic
sample only

51
HEDT Version 1.5 2018
Time Procedure/ Test Comment
Baseline Glucose, insulin, C-peptide Analyse proinsulin in baseline sample and
hypoglycaemic sample only
0 minute Mixed meal
30 minutes Glucose, insulin, C-peptide If hypoglycaemia occurs prior to 300
minutes, take samples for Glucose, insulin,
c-peptide, proinsulin, confirm glucose < 3.0
mmol/L, then correct hypoglycaemia.
60 minutes Glucose, insulin, C-peptide
90 minutes Glucose, insulin, C-peptide
120 minutes Glucose, insulin, C-peptide
180 minutes Glucose, insulin, C-peptide
210 minutes Glucose, insulin, C-peptide
240 minutes Glucose, insulin, C-peptide
270 minutes Glucose, insulin, C-peptide
300 minutes Glucose, insulin, C-peptide

INTERPRETATION:

Inappropriate endogenous hyperinsulinaemia findings are:

Plasma glucose < 3.0 mmol/L AND both of:
Insulin  20 pmol/L (≥ 3.0 IU/L)
C-peptide  200 pmol/L ( 0.2 nmol/L)

Ratios of insulin and glucose should not be used.

NOTES:

A standardised 470 kcal (1966 kJ) mixed meal (71 g carbohydrate, 8.5 g fat, 20 g protein) from
commercially available solid and liquid supplements was recently validated (2). For comparison, the
75-gram glucose drink in a standard oral glucose tolerance test provides 300 kcal (1255 kJ).

52
HEDT Version 1.5 2018
 Sample work Sheet – MIXED MEAL TEST

NAME: DATE:

UR:
LOCATION:
DOB:

MINS ACTUAL MEDS. BSL BLOOD COMMENTS

TIME GIVEN TESTS
Glucose
-5 Insulin
CPep
Test meal Test Meal Contents:
0 (provided
by patient)
Glucose
Insulin
CPep
Glucose
Insulin
CPep
Glucose
Insulin
CPep
Glucose
Insulin
CPep
Glucose
Insulin
CPep
Glucose
Insulin
CPep
Glucose
Insulin
CPep
Glucose
Insulin
CPep
Glucose
Insulin
CPep
Glucose
Insulin
CPep

53
HEDT Version 1.5 2018
 Glucose
Insulin
CPep
Glucose
Insulin
CPep
Glucose
Insulin
CPep
Glucose
Insulin
CPep

REFERENCES:

1. Cryer PE, Axelrod L, Grossman AB et al. Evaluation and management of adult

hypoglycaemic disorders: an Endocrine Society clinical practice guideline. J Clin
Endocrinol Metab 2009; 94 (3):709-728.

2. Shankar SS, Vella A, Raymond RH et al. Foundation for the National Institutes of Health
beta-Cell Project T: standardized mixed-meal tolerance and arginine stimulation tests
provide reproducible and complementary measures of beta-cell function: results from the
foundation for the national institutes of health biomarkers consortium investigative
series. Diabetes Care. 2016;39:1602–13.

54
HEDT Version 1.5 2018
 6.2 PROLONGED OGTT

RATIONALE:

To capture a hypoglycaemic episode in patients with post-prandial hypoglycaemia, however a

mixed meal test is preferable.

PREPARATION:

1. The oral carbohydrate intake should be normal for three days prior to testing.
2. The patient should fast from 10 pm the day prior to the morning OGTT test (water
allowed).

PROCEDURE:

1. The test is performed in the morning, patient should rest during the test and may not eat or
smoke, drinking water is permitted.
2. At baseline, collect blood for fasting glucose measurement.
3. Give glucose solution 75 g orally.
4. Collect blood glucose, insulin and C-peptide hourly for 3 hours.
5. If the patient has any abnormal symptoms during the test (vomiting, sweating, tremors,
unwell), take blood sample immediately and discuss with medical personnel as per local
procedure.

Time Procedure/ Test Comment

Baseline Glucose, insulin, C-peptide
0 minute 75g oral glucose load Take blood samples immediately if
hypoglycaemic symptoms.
60 minutes Glucose, insulin, C-peptide
120 minutes Glucose, insulin, C-peptide
180 minutes Glucose, insulin, C-peptide

INTERPRETATION:

Inappropriate endogenous hyperinsulinaemia findings are the same for the mixed meal test
and 72 hr fast:

Plasma glucose < 3.0 mmol/L AND both of:

Insulin  20 pmol/L (≥ 3.0 IU/L)
C-peptide  200 pmol/L ( 0.2 nmol/L)

55
HEDT Version 1.5 2018
 6.3 72 HOUR FAST

RATIONALE:

To trigger a fasting hypoglycaemic episode which can be captured with plasma glucose and
insulin in patients in whom samples could not be captured during spontaneous
hypoglycaemia.

PREPARATION:

1. IV access inserted and patient admitted to hospital.

2. The patient is to fast (nothing to eat) and is allowed only water, black tea or black coffee
for the duration of the test. Patient is to remain active during waking hours.
3. Baseline blood tests for glucose, insulin, C-peptide (add proinsulin and beta-
hydroxybutyrate if hypoglycaemic episode occurs during fast)
4. Capillary blood glucose taken 3-hourly and recorded.
5. Throughout the investigation blood samples are taken every 6 hours for:
 2 ml Fluoride EDTA - glucose
 8 ml Serum - ON ICE for insulin and C-peptide.
 If capillary glucose falls below 3.3 mmol/L, increase blood sampling frequency to
hourly.

6. If there are symptoms of hypoglycaemia during the test:

a. Notify the Endocrine Team
b. Record symptoms (eg. drowsiness, mood change, anxiety, hunger, sweating or
symptoms previously experienced by patient)
c. Take blood samples BEFORE treating hypoglycaemia for:
i. 2 ml Fluoride EDTA - glucose
ii. 2x 8 ml Serum - ON ICE for insulin, C-peptide, and beta-hydroxybutyrate.

Do not terminate the test before medical review.

The medical officer may terminate the test if one of these conditions is met.

A) Symptoms and/or signs of hypoglycaemia AND plasma glucose <2.5 mmol/L

measured by either plasma glucose or whole blood on blood gas analyser OR

B) If 72 hours have elapsed without symptoms

Once decision made to terminate fast.

7. Take blood samples for

 Glucose
 2 x 8 ml Serum - ON ICE for insulin and C-peptide and beta-hydroxybutyrate,
proinsulin, sulphonylurea screen and insulin antibody levels.

56
HEDT Version 1.5 2018
8. Administer 1mg glucagon IV before patient is fed and collect blood for glucose, beta-
hydroxybutyrate at 10, 20, 30 minutes after glucagon.
9. Feed patient and ensure normoglycaemia

Time Procedure/ Test Comment

0 minute Baseline glucose, insulin, C- allowed only water, black tea or black
peptide, (add proinsulin only if coffee
positive test)
Commence Fast,
4-hourly capillary blood
glucose.

6 hours Glucose, insulin, C-peptide. If capillary glucose <3.3 mmol/L, check

plasma glucose
If plasma glucose <3.3 mmol/L, blood
sampling hourly
If symptomatic AND plasma glucose <
2.5 mmol/L, terminate fast
12 hours Glucose, insulin, C-peptide.
18 hours Glucose, insulin, C-peptide.
24 hours Glucose, insulin, C-peptide.
30 hours Glucose, insulin, C-peptide.
36 hours Glucose, insulin, C-peptide.
42 hours Glucose, insulin, C-peptide.
48 hours Glucose, insulin, C-peptide.
54 hours Glucose, insulin, C-peptide.
60 hours Glucose, insulin, C-peptide.
66 hours Glucose, insulin, C-peptide.
72 hours Glucose, insulin, C-peptide. If asymptomatic, plasma glucose ≥ 2.5
mmol/L, terminate fast at 72 hours
Termination of fast Glucose, insulin, C-peptide,
beta-hydroxybutyrate,
proinsulin, sulphonylurea
screen, and insulin antibody
levels.
After samples taken, IV 1 mg
glucagon
10 minutes Glucose, beta-hydroxybutyrate
20 minutes Glucose, beta-hydroxybutyrate
30 minutes Glucose, beta-hydroxybutyrate
Feed patient Ensure normoglycaemia before discharge

INTERPRETATION:

Inappropriate endogenous hyperinsulinaemia (insulinoma, nesidioblastosis, post gastric

bypass hypoglycaemia) findings are:

 Plasma glucose < 3.0 mmol/L AND:

 Insulin  20.8 pmol/L (≥ 3.0 IU/L)
 C-peptide  200 pmol/L
 Proinsulin ≥ 5 pmol/L
 beta-hydroxybutyrate < 2.7 mmol/L
57
HEDT Version 1.5 2018
  plasma glucose increased by 1.4 mmol/L post glucagon injection

Patterns of findings during prolonged fasting

Symptoms Glucose Insulin C-peptide Proinsulin β- Glucose Circulating Antibodies Diagnostic

and/or mmol/L mIU/L pmol/L pmol/L Hydroxy- increase oral hypo- to insulin interpretation
signs butyrate after glycaemic
mmol/L glucagon
mmol/L

No < 3.0 <3 <200 <5 > 2.7 < 1.4 No Neg Normal

Yes < 3.0 >> 3 <200 <5 ≤ 2.7 > 1.4 No Neg (Pos) Exogenous
insulin

Yes < 3.0 ≥3 ≥200 ≥5 ≤ 2.7 > 1.4 No Neg Insulinoma,

NIPHS, PGBH

Yes < 3.0 ≥3 ≥200 ≥5 ≤ 2.7 > 1.4 Yes Neg Oral hypo-
glycaemic agent

Yes < 3.0 >> 3 >>200 >> 5 ≤ 2.7 > 1.4 No Pos Insulin
autoimmune

Yes < 3.0 <3 <200 <5 ≤ 2.7 > 1.4 No Neg IGF

Yes < 3.0 <3 <200 <5 > 2.7 < 1.4 No Neg Not insulin (or
IGF)- mediated

Neg, negative; Pos, positive; NIPHS, noninsulinoma pancreatogenous hypoglycaemia syndrome ; PGBH, post gastric bypass hypoglycaemia.

Table copied from Journal of Clinical Endocrinology & Metabolism, March 2009, 94(3): 709-728 – “Evaluation and Management of Adult
Hypoglycemic Disorders: An Endocrine Society Clinical Practice Guideline”

NOTES:

 Insulin, C-peptide and proinsulin clearance is reduced in renal failure, above cut-offs for
these analytes might not apply.
 75% of insulinomas are diagnosed after 24 hours fast, 90% at 48 hours.

REFERENCES:

Cryer PE, Axelrod L, Grossman AB et al. Evaluation and management of adult hypoglycaemic
disorders: An Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2009; 94
(3):709-728.

58
HEDT Version 1.5 2018
 6.4 CALCIUM STIMULATION TEST FOR INSULINOMA

RATIONALE:

Reserved for cases of suspected insulinoma that do not localize by conventional noninvasive
means or if confirmation is required. The calcium stimulation test may also be useful in
differentiating insulinoma from diffuse nesidioblastosis, especially if imaging is negative or
inconclusive1. The decision to proceed with calcium stimulation testing should be made in
conjunction with the operating Endocrine surgeon. The test is based on the observation that
exogenous intra-arterial calcium injection stimulates the release of insulin from tumor beta
cells, but not from normal beta cells2.

PREPARATION AND PROCEDURE:

1. Discontinue calcium channel blockers (may lead to severe hypoglycaemia during test) as
well as non-essential medications and those with the potential to interfere with insulin
secretion by insulinoma cells (such as diazoxide), 5 half-lives prior to the procedure.
2. Overnight fast – run dextrose infusion as required to avoid hypoglycaemia.
3. Visceral arteriography into
a. Superior mesenteric artery
b. Proximal splenic artery
c. Midsplenic artery
d. Gastroduodenal artery
e. Proper hepatic arteries
4. 10% calcium gluconate diluted to volume of 5 mL with normal saline is directly injected
as a bolus into individual artery at a dose of 0.0125 mmol Ca 2+/kg body weight
(maximum total dose for entire procedure is 11.63 mmol or 93 mg Ca2+).
5. 5 mL blood samples are taken at 0, 20, 40 and 60 sec after calcium injection from the
right hepatic vein for insulin levels.
6. Double check samples are labeled correctly with the time and arterial site injected with
calcium.

Time Procedure
Baseline Insulin checked from right hepatic vein
0 Calcium bolus injected into selected artery, repeat
procedure for each artery.
1. Superior mesenteric Proximal artery
2. Splenic artery
3. Midsplenic artery
4. Gastroduodenal artery
5. Proper hepatic artery
20 seconds Insulin checked from right hepatic vein
40 seconds Insulin checked from right hepatic vein
60 seconds Insulin checked from right hepatic vein

59
HEDT Version 1.5 2018
INTERPRETATION:

A greater than 2-fold rise in right hepatic vein insulin levels from 0 at times 20, 40 and /or 60
sec is required to localize an insulin secreting tumor in that portion of the pancreas supplied
by the artery studied.

Artery from which positive response Tumour Localisation Site

achieved from calcium stimulation

Gastroduodenal artery or superior mesenteric Pancreatic head and neck region

artery

Proximal splenic or midsplenic artery Pancreatic body and tail region

Proper hepatic arteries Liver metastases

If a greater than 2-fold rise in insulin levels occurs with calcium injection into more than one
artery, the dominant site is used to predict tumour localization (may represent overlap in
tumour arterial supply). In a large (retrospective) study 3, this correctly predicted tumour
location in 84% (38 of 45 cases). False negative results occurred in 5 of 45 cases (11%)
attributed to arterial anomalies/technical flaws. False positive occurred in 2 of 45 cases (4%)
attributed to tumour necrosis or unexplained reasons. In a retrospective analysis of 240
patients with insulinoma at the Mayo Clinic, calcium stimulation test was performed in 25%
of patients, with a sensitivity of 93% for the regionalization of functioning insulinomas 4.
Nesidioblastosis is suggested by a >2-fold rise in right hepatic vein insulin level following Ca
injection in the gastroduodenal, superior mesenteric and splenic arteries but not the proper
hepatic artery.

NOTES:

Procedure should be performed by experienced interventional radiologists. Hypoglycaemia

can occur during the procedure.

REFERENCES:

1. Thompson SM, Vella A, Thompson GB, Rumilla KM, Service FJ, Grant CS, et al.
Selective Arterial Calcium Stimulation With Hepatic Venous Sampling Differentiates
Insulinoma From Nesidioblastosis. The Journal of clinical endocrinology and
metabolism. 2015;100(11):4189-97.

60
HEDT Version 1.5 2018
 2. Doppman JL, Chang R, Fraker DL, Norton JA, Alexander HR, Miller DL, et al.
Localization of insulinomas to regions of the pancreas by intra-arterial stimulation
with calcium. Annals of internal medicine. 1995;123(4):269-73.
3. Guettier JM, Kam A, Chang R, Skarulis MC, Cochran C, Alexander HR, et al.
Localization of insulinomas to regions of the pancreas by intraarterial calcium
stimulation: the NIH experience. The Journal of clinical endocrinology and
metabolism. 2009;94(4):1074-80
4. Morera J, Guillaume A, Courtheoux P, Palazzo L, Rod A, Joubert M, et al.
Preoperative localization of an insulinoma: selective arterial calcium stimulation test
performance. J Endocrinol Invest. 2016;39(4):455-63.

61
HEDT Version 1.5 2018
7 Diabetes Insipidus

7.1 WATER DEPRIVATION TEST

RATIONALE:

For the diagnosis of polyuria-polydipsia syndrome.

PATIENT PREPARATION:

1. Document polyuria (i.e. > 3 L/24 hours or 40-50 ml/kg/24 hours)

2. Exclude other reasons for polyuria such as hyperglycaemia and hypercalcaemia
3. Check renal function and electrolytes and pituitary function including fT4, TSH and basal
cortisol
4. Check complete electrolytes and serum osmolality, copeptin (if available) and urine
osmolality (i.e. 2nd urine sample after one hour) after overnight water deprivation (> 8 hours).
- If urine osmolality is > 800 mOsm/kg, no further testing is required. In all other cases a
classic water deprivation test is indicated. Note maximal urine osmolality ~1200 mOsm/kg.
* Depending on age and renal impairment, urine osmolality of > 600 mOsm/kg can be
acceptable according to clinical judgment.
If copeptin testing is available:
- If copeptin levels are < 2.6 pmol/L after overnight fluid deprivation diagnosis of central
diabetes insipidus is suggested
- If copeptin levels are ≥ 21.4 pmol/L without prior thirsting the diagnosis of nephrogenic
diabetes insipidus is likely and no further testing is required.
5. Patients should refrain from tobacco, alcohol and caffeine 24 hours prior to test.
6. Stop DDAVP 24 hours prior to test

PROCEDURE:

1. Start water deprivation at 8pm for mild polyuria (i.e. 3-5 L/24h), at 12am for moderate
polyuria (i.e. 5-8 L/24h) and at 8am for severe polyuria (i.e. >8 L/24h)
2. Before starting water deprivation, take baseline weight, blood pressure and pulse, baseline
blood test for serum osmolality, electrolytes, renal function, copeptin (if available) plus
baseline urine osmolality and urine sodium
3. Repeat weight, blood pressure, pulse, urine osmolality and urine sodium hourly from 8am
onwards
4. Repeat blood test every 4 hours (i.e. 8am, 12pm and 4pm) and at termination of test
immediately prior to injection of DDAVP
5. Duration of water deprivation period will vary for different patients. Indication for
termination of water deprivation are any of the following:
a. - Urine osmolality plateaus (i.e. <30 mOsm/kg increase between two consecutive
hourly measurements). This indicates maximal urinary concentrating ability.
b. - Weight loss of more than 3% of baseline weight
c. - Serum sodium levels > 150 mmol/L

62
HEDT Version 1.5 2018
 d. - Urine osmolality > 800 mOsm/kg
6. Intravenous injection of 2 g DDAVP if termination urine osmolality is <800 mOsm/kg
7. 1 hour after DDAVP administration: repeat blood test with serum osmolality and
electrolytes plus repeat urine osmolality and urine sodium

Time Procedure Comment

Baseline (8am) Weight, BP, HR
serum osmolality, U+Es (± copeptin)
urine osmolality, sodium
Hourly weight, BP, HR
urine osmolality and urine sodium
12:00 serum osmolality, U+Es (± copeptin) In the setting of ongoing severe polyuria,
more frequent checks of serum osmolality,
electrolytes and renal function may be
required according to the treating
physician
16:00 serum osmolality, U+Es (± copeptin) In the setting of ongoing severe polyuria,
more frequent checks of serum osmolality,
electrolytes and renal function may be
required according to the treating
physician
Termination serum osmolality, U+Es (± copeptin) - Urine osmolality plateaus (i.e. <30
mOsm/kg increase between two
consecutive hourly measurements). This
indicates maximal urinary concentrating
ability
- Weight loss of more than 3% of
baseline weight
- Serum sodium levels > 150 mmol/L
- Urine osmolality > 800mOsm/kg
IV 2 g DDAVP
1 hour post Weight, BP, HR Patient to limit fluid intake to 500-800ml
DDAVP serum osmolality, U+Es (copeptin) for next 24 hrs if urine concentrated post
urine osmolality, sodium DDAVP

63
HEDT Version 1.5 2018
INTERPRETATION:

Primary Nephrogenic Complete Partial Central

polydipsia Diabetes Central Diabetes Diabetes
Insipidus Insipidus Insipidus
Baseline 5 ≥ 21.4 < 2.6 <5
copeptin
(pmol/L)
Serum osmol < 300 > 300 > 300 > 300
(mOsm/kg)
Urine osmol > 800 < 300 < 300 300-800
(mOsm/kg) (300 – 800 if
chronic)
Post DDAVP < 10% < 50% > 50% 10-50%
urine osmol
Rise (%)

Figure: Diagnostic algorithm for the differential diagnosis of the polyuria-polydipsia syndrome
(3)

NOTES:

 In the setting of severe ongoing polyuria, more frequent checks of serum osmolality,
electrolytes and renal function may be required according to the treating physician.
 Hyponatraemia can occur due to excess water retention after administration of
desmopressin, therefore patients should be instructed to restrict oral intake to 500-800
ml for the next 24 hours if urine is concentrated post DDAVP. (4)

64
HEDT Version 1.5 2018
REFERENCES:

1. Miller M, Dalakos T, Moses AM, Fellerman H, Streeten DH. Recognition of partial defects
in antidiuretic hormone secretion. Annals of internal medicine. 1970;73(5):721-9.

2. Fenske W, Quinkler M, Lorenz D, Zopf K, Haagen U, Papassotiriou J, et al. Copeptin in

the differential diagnosis of the polydipsia-polyuria syndrome--revisiting the direct and
indirect water deprivation tests. The Journal of clinical endocrinology and metabolism.
2011;96(5):1506-15.

3. Nigro, N., M. Grossmann, C. Chiang and W. J. Inder (2018). "Polyuria-polydipsia

syndrome: a diagnostic challenge." Intern Med J 48(3): 244-253.

4. Fenske W, Refardt J, Chifu I, Schnyder I, Winzeler B, Drummond J, et al. A Copeptin-

Based Approach in the Diagnosis of Diabetes Insipidus. N Engl J Med. 2018;379(5):428-39.

65
HEDT Version 1.5 2018
WATER DEPRIVATION TEST – Worksheet

Patient Label:
Date of Test:

Patient thirsting since:

Time BP/HR Weight kg Urine Vol Urine Na Serum Na Serum Osm

mL/hr mmol/L mmol/L mosm/Kg

08:00

09:00

10:00

11:00

12:00

13:00

14:00

15:00

16:00

IV 2 g DDAVP

17:00

66
HEDT Version 1.5 2018
8 Thyroid

8.1 TRH TEST

RATIONALE:

The sole indication for the TRH test is the evaluation of elevated thyroid hormones (free T4 and free
T3) in the setting of a normal or elevated TSH – the differential diagnosis of resistance to thyroid
hormone versus a TSH secreting pituitary adenoma (TSH-oma).

The following should be considered:

1. It is essential to exclude heterophil antibody interference within the discordant thyroid

function panel.
2. Once interference is excluded, and temporal pattern of discordant TFTs persists, other
helpful tests include:
a. SHBG and CTX are elevated in hyperthyroidism (TSH-oma)
b. Alpha subunit elevated in 70% of TSH-oma (not helpful in post-menopausal
women)
c. TFT testing in first degree relatives if available (thyroid hormone resistance)
d. THRB gene test is available for testing of thyroid hormone resistance and
is positive in 90% of RTHβ.

PREPARATION AND PROCEDURE:

1) The patient need not be fasting but should empty their bladder immediately prior to the
test.
2) TSH is collected at baseline, 20 and 60 mins after IV bolus of 200 µg of TRH over 1
minute

Time Procedure Comment

Baseline TSH

0 minute IV bolus of 200 µg of TRH over 1 Side effects: nausea, flushing, headache,
minute * micturition urgency

20 minutes TSH

60 minutes TSH

INTERPRETATION:

In normal individuals TSH increases 4-14 fold with a mean of 8.3 fold. 1
Autonomously secreted TSH from a TSH-oma rarely increases following TRH. 2, 3
In the setting of thyroid hormone resistance there is a normal to exaggerated TSH response. 2

67
HEDT Version 1.5 2018
Proposed diagnostic criteria: †
TSH-oma – < two fold elevation in serum TSH
Thyroid hormone resistance – > four fold increase in serum TSH
NOTES:

Some protocols have used 400-500 µg of TRH in adults 3

† Absolute incremental criteria proposed include “absent response” TSH rise <2 mU/L or
“reduced response” TSH rise <5 mU/L 3. Incremental TSH response to TRH may be normal
in TSH-oma patients with prior thyroid ablation 3

68
HEDT Version 1.5 2018
 8.2 T3 SUPPRESSION TEST

RATIONALE:

For investigation of suspected cases of resistance to thyroid hormone (RTH) linked to thyroid
hormone receptor (THR) beta mutation. As per rationale in TRH stimulation test, analytical
interference needs to be excluded first. T3 suppression test might be useful in cases where
other available results are contradictory, for example a) TRH test is abnormal (TSH rise <2)
and MRI pituitary is normal, b) TRH test is normal and MRI revealed pituitary adenoma
(adenoma occurs in 20% of RTH), c) thyroidectomised or patients with thyroid ablation. 4
However due to cardiac side effects from T3, obtaining thyroid function tests in first degree
relatives and genetic testing for a mutation in the thyroid hormone receptor gene
(identified in around 90% of RTH cases) may be considered preferential to confirm the
diagnosis where possible.

PROCEDURE:

Time Procedure Comment

Baseline TSH, FT4, FT3 (Cholesterol, CK, Ferritin,

SHBG)

Day 1 -3 Patient to take Tertroxin 30µg mane, 20µg

nocte (total 50 µg daily)

Day 4 TSH, FT4, FT3 (Cholesterol, CK, Ferritin, If TSH suppressed, TSH-
SHBG, CTX) oma excluded with high
certainty, no need to
proceed with test

Day 4-6 Patient to take Tertroxin 30µg mane, 20µg

midi, 30µg nocte (total 80 µg daily)

Day 7 TSH, FT4, FT3 (Cholesterol, CK, Ferritin, If TSH suppressed, TSH-
SHBG, CTX) oma excluded with high
certainty, no need to
proceed with test

Day 7-9 Patient to take Tertroxin 40 µg mane, 20 µg

midi, 40 µg nocte (total 100 µg daily)

Day 10 TSH, FT4, FT3 (Cholesterol, CK, Ferritin,

SHBG, CTX)

69
HEDT Version 1.5 2018
INTERPRETATION:

Patient with RTH typically display partial, dose dependant suppression of baseline TSH with
exogenous T3. Failure to suppress TSH to any degree is suggestive of a TSHoma. Partial,
dose dependant suppression of TSH with LT3 is consistent with RTH. With short term 100
ug LT3 daily >80% reduction in TSH from baseline suggests RTH while <40% suggests
TSHoma.2

Additional analytes may be useful to assess the peripheral tissue effects of thyroid hormones.
A normal response to administration of T3 is an increase in SHBG, ferritin and CTX and a
decrease in cholesterol and CK. In patients with RTH these responses are reduced, or
paradoxical.

CONTRAINDICATIONS:

T3 suppression test should not be performed in patients with severe pulmonary/

cardiovascular disease/ psychiatric illness/ potential to decompensate from a short period of
hyperthyroidism.

REFERENCES:

1. Dare G et al. Hypothalamic-pituitary axis and peripheral tissue responses to TRH

stimulation and liothyronine suppression tests in normal subjects evaluated by current
methods. Thyroid 2008; 18:401-9.
2. Macchia E et al. Clinical and genetic characteristics of a large monocentric series of
patients affected by thyroid hormone (Th) resistance and suggestions for differential
diagnosis in patients without mutation of Th receptor. Clin Endocrinol 2014; 81:921-8.
3. Brucker-Davis F et al. Thyrotropin-secreting pituitary tumors: diagnostic criteria, thyroid
hormone sensitivity and treatment outcome in 25 patients followed at the National
Institutes of Health. J Clin Endocrinol Metab 1999; 84:476-86.
4. Beck-Peccoz P, Lania A, Beckers A, Chatterjee K, Wemeau JL. 2013 European thyroid
association guidelines for the diagnosis and treatment of thyrotropin-secreting pituitary
tumors. European thyroid journal 2013; 2:76-82
5. Beck-Peccoz P, Persani L, Lania A. Thyrotropin-Secreting Pituitary Adenomas. In: De
Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C,
Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, eds.
Endotext. South Dartmouth (MA): [Link], Inc.; 2000. Last updated 2015.
6. Sarne D, Sobieszczyk S, Ain K et al. Serum Thyrotropin and Prolactin in the Syndrome
of Generalized Resistance to Thyroid Hormone: Responses to Thyrotropin-Releasing
Hormone Stimulation and Short Term Triiodothyronine Suppression*. J Clin Endocrinol
Metab 1990; 70: 1305-1311

70
HEDT Version 1.5 2018
8.3 CALCIUM STIMULATION TEST FOR MEDULLARY THYROID
CANCER

RATIONALE:

The test is used in patients with mildly raised basal calcitonin levels and remaining diagnostic
uncertainty to distinguish medullary thyroid carcinoma (MTC) from C-cell hyperplasia
(CCH) and from (for example in MEN-1 patients) co-existing calcitonin-producing
neuroendocrine tumours (NET). C-cells (like parathyroid cells) express the Ca-sensing
receptor and acute increases in ionized calcium will lead to a greater increase of calcitonin
from MTC vs. CCH patients (1). In patients with NET or those who have elevated calcitonin
due to interfering antibodies there will be limited calcitonin increase.

PREPARATION:

1. Patient fast overnight.

2. Check electrolytes and serum calcium at baseline.
3. i.v. Cannula, 18-20g.
4. Saline flush.
5. Calcium gluconate 10% (10 - 20 ml required = 93 to 186 mg of elemental calcium).

PROCEDURE:

1. Insert cannula and flush.

2. Take baseline sample for serum calcitonin and calcium.
3. Give elemental calcium 2.3 mg/kg (0.06 mmol/kg) body weight using 10% calcium
gluconate at 10 ml per minute*. (4)
4. Flush cannula.
5. Take samples at 1, 2, 3, 5 and 10 and 15 minutes for calcitonin and calcium.
6. Send immediately on ice to the lab for centrifugation and freezing.

*70 kg patient should be infused with 161 mg or 4.2 mmol of elemental calcium. This
amount corresponds to 18.4 ml of the 10% Ca gluconate solution

INTERPRETATION:

 In a study of healthy volunteers (95 th percentile of basal calcitonin values 5.0 pg/ml in
males and 5.7 pg/ml in females) 95th percentile maximally stimulated calcitonin
values were 131 pg/ml in men and 90 pg/ml in women (2).
 In a study of >100 patients with MTC (n=42), RET gene mutation carriers (n=14),
multinodular goitre (n=69) and healthy volunteers (n=16), basal calcitonin values >68
pg/ml in males and >18.7 pg/ml in females and stimulated calcitonin values >1,620
pg/ml in males and >184 pg/ml in females had the highest accuracy to distinguish
MTC cases from CCH and normal (4).

71
HEDT Version 1.5 2018
NOTES:

Rapid calcium infusion can lead to vasodilatation and arrhythmias - cardiac arrest after iv
calcium stimulation in a healthy young man without known cardiac disease has been reported
(3). While larger case series have not reported serious adverse effects, use with caution and
consider cardiac monitoring, and avoid in patients with significant cardiac disease.

More transient side effects lasting up to 15 minutes include flushing sensation/ feeling of
warmth (98%), facial/ extremity paraesthesia, altered gustatory sensation (20%).

REFERENCES:

1. Fudge NJ et al, Physiological studies in heterozygous calcium sensing receptor

(CaSR) gene-ablated mice confirm that the CaSR regulates calcitonin release in vivo.
BMC physiology, 2004;4:5
2. Doyle P, Duren C, Nerlich K, Verburg FA, Grelle I, Jahn H, et al. Potency and
tolerance of calcitonin stimulation with high-dose calcium versus pentagastrin in
normal adults. The Journal of clinical endocrinology and metabolism.
2009;94(8):2970-4.
3. Russo M, Scollo C, Padova G, Vigneri R, Pellegriti G. Cardiac arrest after
intravenous calcium administration for calcitonin stimulation test. Thyroid : official
journal of the American Thyroid Association. 2014;24(3):606-7
4. Colombo C, Verga U, Mian C, Ferrero S, Perrino M, Vicentini L, et al. Comparison
of calcium and pentagastrin tests for the diagnosis and follow-up of medullary thyroid
cancer. The Journal of clinical endocrinology and metabolism. 2012;97(3):905-13.

72
HEDT Version 1.5 2018
9 Phaeochromocytoma

9.1 CLONIDINE SUPPRESSION TEST

RATIONALE:

To exclude the diagnosis of phaeochromocytoma / paraganglioma (PPGL) in patients with

hypertension and borderline raised catecholamines or catecholamine metabolites. The test intends to
discriminate patients with mildly elevated test results for plasma normetanephrine due to increased
sympathetic activity from patients with elevated test results due to a PPGL. Alternate causes for
elevated plasma metanephrines (e.g. medications, collection procedure) must be excluded prior to
clonidine suppression test. The clonidine suppression test is only recommended in patients with
equivocal biochemistry on initial testing and in situations where the diagnosis remains uncertain.
Clonidine can cause hypotension and is contraindicated in frail patients with a history of hypotensive
episodes/ severe coronary/ carotid disease.

PREPARATION:

1. Obtain the clonidine from pharmacy:

2. Clonidine hydrochloride 100 µg or 150 µg tablets for oral administration
3. Test Dose = 300 µg orally (4.3 µg / kg) (1, 2)
4. Stop sympatholytic medications (e.g. beta blockers) for at least 48 hours before the test.
5. No paracetamol, diuretics or tricyclics anti-depressants for 5 days. Medications including
diuretics, tricyclic antidepressants and β-blockers are known to interfere with
noradrenaline responses to the clonidine suppression test
6. No smoking or caffeine for 24h
7. Fast overnight.
8. Quiet environment.
9. Cancel test if baseline blood pressure is <110/60 mmHg or in volume-depleted patients.
Profound hypotensive responses to clonidine can also occur in patients taking other
antihypertensive medications.

PROCEDURE:

Time Procedure Comment

-30 minutes Insert cannula Patient to lie supine during the test where
possible and must rest in the supine position
rest for 30 minutes prior to baseline measures.
Baseline Collect 2 samples for plasma Two baseline blood samples taken on ice.
metanephrines at 5 minutes apart.

Record blood pressure and pulse.

0 minute Give 300µg clonidine orally.
+60 minutes Record blood pressure and pulse.
+120 Record blood pressure and pulse.
minutes

73
HEDT Version 1.5 2018
+180 Collect blood for plasma Blood needs to be taken ice.
minutes metanephrines.
Record blood pressure and pulse.
INTERPRETATION:

Normally, by activating α2-adrenoceptors in the brain and on sympathetic nerve endings,

clonidine (an α2-adrenoceptors agonist) suppresses catecholamine release by sympathetic
nerves in patients without PPGL. However, due to autonomous tumoral secretion of
catecholamines, PPGL are not influenced by clonidine-induced suppression of the
sympathetic nervous system (1).
An abnormal test result indicating a PPGL includes an elevation of plasma normetanephrine
at 3 h after clonidine administration and a less than 40% decrease in levels compared with
baseline (2). In this retrospective analysis (2) of 48 patients with and 49 patients without
PPGL who underwent clonidine suppression testing, a positive result with normetanephrine
(both elevated plasma concentration after clonidine and lack of suppression) had a sensitivity
of 96% and a specificity of 100%. If there was either only an elevated plasma concentration
after clonidine or a lack of suppression of normetanephrine, sensitivity remained 96% but
specificity dropped to 67% to 96%. Noradrenaline response to clonidine suppression was less
sensitive and specific (2).

NOTES:

Hypotension and sedation can occur with clonidine.

REFERENCES:

1. Lenders et al., Pheochromocytoma and Paraganglioma: An Endocrine Society Clinical

Practice Guideline. Journal of Clinical Endocrinology & Metabolism 2014; 99:1915–1942.
2. Eisenhofer G, Goldstein DS, Walther MM, Friberg P et al. Biochemical diagnosis of
pheochromocytoma: how to distinguish true- from false-positive test results. Journal of
Clinical Endocrinology & Metabolism 2003; 88: 2656-66.

74
HEDT Version 1.5 2018
Acknowledgements:

The working party acknowledge the following institutions for their generosity in sharing their
departmental protocols to improve the harmonisation process.

Austin Hospital

Mater Children’s Hospital

Monash Medical Centre

Pathology Queensland

PathWest

Prince of Wales Hospital

Royal Children Hospital

Royal Melbourne Hospital

Royal North Shore Hospital

St Vincent’s Hospital (Sydney)

St Vincent’s Hospital (Melbourne)

Westmead Hospital

75
HEDT Version 1.5 2018
Amendment history:

Version Date Author/s Comments

1.0 2017 HEDT Working group

1.5 9/2018 HEDT Working group Open for comments on ESA and AACB
website

76
HEDT Version 1.5 2018