ROYAL ALEXANDRA HOSPITAL FOR

CHILDREN

POLICY & PROCEDURES MANUAL

(Based on Australian Council of Healthcare Standards Guidelines - 13th Edition)

DEPARTMENT / SECTION Ray Williams Institute of Paediatric Endocrinology, Diabetes

and Metabolism Endocrinology,

Endocrinology and Metabolism

Testing Protocols
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Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page 1
 Endocrinology and Metabolism Testing Protocols
1996

The Ray Williams Institute of Paediatric Endocrinology,

Diabetes and Metabolism

The New Children's Hospital

Royal Alexandra Hospital for Children

Postal address: PO Box 3515, Parramatta NSW 2124, Australia

Visitors address: Cnr Hawkesbury Rd and Hainsworth St, Westmead,
Sydney, Australia.

Phone 61 2 9845 3907 Laboratory 61 2 9845 3190

Fax 61 2 9845 3170

Editors:

Dr Geoffrey Ambler, Staff Specialist in Endocrinology and Diabetes

Mary McQuade, Clinical Nurse Consultant, Endocrinology
Contributors:

Dr Geoffrey Ambler, Staff Specialist in Endocrinology and Diabetes, Deputy Director, Diabetes Centre
Dr Barbara Blades, Manager, Endocrine Laboratory
Dr Christopher Cowell, Staff Specialist in Endocrinology and Diabetes, Deputy Director, Institute of
Endocrinology
Dr Kim Donaghue, Staff Specialist in Endocrinology and Diabetes
Dr Neville Howard, Staff Specialist in Endocrinology and Diabetes
Director, Diabetes Centre
Elizabeth Lawrie Clinical Nurse Specialist, Endocrinology
Mary McQuade, Clinical Nurse Consultant, Endocrinology
Kristine Savage, Clinical Nurse Specialist, Endocrinology
Professor Martin Silink, Staff Specialist in Endocrinology and Metabolism, Director, Institute of Endocrinology

A number of previous staff of the Institute also made valuable contributions to the evolution of these
protocols, including the late Dr Robert Vines.

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1. Foreword
This manual includes Endocrinology and Metabolism testing protocols in common usage in the Ray
Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism at the New Children's Hospital,
Westmead and also includes information on sample collection and handling and local laboratory
reference ranges. As such it is designed mainly as a practical manual for day to day use in our
Endocrine testing ward, but it may be found useful by clinicians external to the Institute. It should be
borne in mind that local laboratories may have different assay procedures and reference ranges. Also,
while great care has been taken in the preparation of these protocols, no responsibility can be taken for
their suitability or application in other centres. The suitability, safety and performance of any test in an
individual patient must remain the responsibility of the treating physician.

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 TABLE OF CONTENTS: Page

1. Foreword.................................................................................................................................. 3

2. Introduction .............................................................................................................................. 6

2.1 Principles of Dynamic Testing ................................................................................................. 6

2.2 Safety considerations ............................................................................................................. 6
2.3 Blood sampling...................................................................................................................... 7
2.3.1 IV cannula insertion............................................................................................................. 7
2.3.2 Sampling from IV cannula .................................................................................................... 7
2.4 Blood volume considerations ................................................................................................... 7
2.5 Specimen collection requirements ........................................................................................... 8

3. Specimen Collection Requirements and Reference Ranges.................................................... 9

3.1 RWI Endocrinology Laboratory Specimen Collection ................................................................. 9

3.2 RWI Endocrinology Laboratory Reference Ranges................................................................... 14
3.2.1 Aldosterone ...................................................................................................................... 14
3.2.2 Androstenedione ............................................................................................................... 14
3.2.3 Cortisol, plasma................................................................................................................ 14
3.2.4 Cortisol, urinary free .......................................................................................................... 14
3.2.5 Dehydroepiandrosterone Sulphate (DHAS)........................................................................... 15
3.2.6 11-Deoxycortisol (DCOR)................................................................................................... 15
3.3 Other RAHC laboratories and external laboratories Specimen Collection ................................... 20

4. Test Protocols......................................................................................................................... 24

4.1 Arginine Stimulation Test...................................................................................................... 24

4.2 Clonidine Stimulation Test .................................................................................................... 25
4.3 Combined Pituitary Function Test (formerly, Triple Test) .......................................................... 26
4.4 Desmopressin Test of Renal Concentrating Ability .................................................................... 1
4.5 Dexamethasone Suppression Test .......................................................................................... 3
4.6 Overnight Low-Dose Dexamethasone Suppression Test............................................................. 4
4.7 Overnight High-Dose Dexamethasone Suppression Test............................................................ 5
4.8 Standard (Long) Dexamethasone Suppression Test .................................................................. 6
4.9 Exercise Stimulation Test....................................................................................................... 8
4.10 Exercise Stimulation Test, with Propranolol (Propranolol Exercise Test).................................... 9
4.11 Fasting Study .................................................................................................................... 10
4.12 Glucagon Stimulation Test (in suspected hypoglycemic disorders) ......................................... 13
4.13 Glucagon Stimulation Test (for pituitary function)................................................................... 15
4.14 Gonadotrophin Releasing Hormone (GnRH) Test (LHRH test)................................................. 18
4.15 hCG Stimulation Test ......................................................................................................... 20
4.16 IGF-I Generation Test ......................................................................................................... 22
4.17 Insulin Stimulation Test (Insulin Tolerance Test).................................................................... 23
4.18 Intravenous Glucose Tolerance Test..................................................................................... 25
4.19 Oral Glucose Tolerance Test ............................................................................................... 27
4.20 Parathyroid Hormone Infusion Test (Ellsworth-Howard test).................................................... 29
4.21 Pentagastrin Stimulation Test.............................................................................................. 31
4.22 Sex Steroid Priming In Growth Hormone Stimulation Tests .................................................... 33
4.23 Short ACTH (Synacthen) Stimulation Test ............................................................................ 34
4.24 Spontaneous Growth Hormone Secretion (Overnight or 24 hr GH sampling) ............................. 36
4.25 Thyrotropin Releasing Hormone (TRH) Test........................................................................... 37
4.26 Water Deprivation Test........................................................................................................ 39

5. Appendices............................................................................................................................. 41

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Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page 5
2. Introduction

2.1 Principles of Dynamic Testing

Basal or unstimulated hormone levels frequently do not provide sufficient diagnostic information in the
investigation of endocrine and metabolic disorders. A range of dynamic or provocative tests are available
to assess the dynamic responses of hormonal and metabolic axes. These tests may involve :

1. Stimulation of a hormonal axis by releasing hormones or other agents

eg. clonidine stimulation of growth hormone
gonadotropin releasing hormone stimulation of LH and FSH

2. Attempted suppression of a hormonal system

eg. cortisol suppression in Dexamethasone suppression test

3. Physiological stimulation or challenge of a metabolic or hormonal system

eg. exercise stimulation of growth hormone
fasting study to assess glucose homeostasis
water deprivation to assess water regulation

This document describes protocols for these tests in common usage in the Institute of Endocrinology.

2.2 Safety considerations

Any dynamic or provocative test has potential for side effects or adverse reactions, although these are
uncommon in experienced hands and if appropriate precautions are taken. Precautions,
contraindications and adverse reactions are indicated in the protocols for each test and should be
reviewed before each test is undertaken. Important adverse reactions in various tests include:

Hypoglycemia
Dehydration
Minor reactions to provocative agents eg. nausea, vomiting
Allergic or anaphylactic reaction to provocative agent
Cannula related complications - blood loss, infection
Hypotension

To minimize potential adverse events the following should be considered:

1. Tests should only be performed and supervised by personnel and centres experienced in their
use in children, and this should be in specialized paediatric endocrine centres.

2. Staff must have detailed knowledge of the particular test protocol and provocative agents.
Specialized nursing staff familiar with these tests are essential if they are to be performed safely
and give accurate results.

3. Tests must be performed in an environment where full paediatric emergency resuscitation

facilities and experience are available. Deaths and serious morbidity have been reported from
such testing in inexperienced hands, particularly with insulin stimulation tests (Shah, Stanhope,
Matthew. Hazards of pharmacological tests of growth hormone secretion in childhood. BMJ 304:
173-4, 1992.) and fasting studies.

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4. It may be necessary to adjust protocols for particular individuals or circumstances, and the
same protocol cannot automatically be safely applied to all patients. Prior to the test,
consideration should be given to any particular customization or precautions required for the
individual patient (see guidelines under individual tests). This should be discussed with the
consultant concerned.

5. Appropriate laboratory back-up is essential, particularly for tests involving fasting, hypoglycemia
or water deprivation. Facilities are required for immediate formal glucose monitoring in the
testing ward ; monitoring by diabetes-type blood glucose monitors is considered unsafe and
sending samples to central hospital laboratories results in unacceptable delay.

6. A medical officer must always be readily available, and in certain tests (eg. insulin stimulation
test) must be immediately available in the ward.

7. Experienced personnel are required to place intravenous cannulas

2.3 Blood sampling

Most tests require the insertion of one IV cannula through which provocative agents are administered
and/or periodic blood samples drawn. A large vein in the cubital fossa is the preferred insertion site.
Occasionally separate infusion and sampling cannulas are required or desirable. Butterfly needles are
useful for single samples, but are not recommended where multiple samples are to be taken. Arterial
sampling via cannulas or needle/syringe should only be used if there is no alternative, and with approval
from the consultant.

2.3.1 IV cannula insertion

• Local anaesthetic cream (EMLA cream or patch) is applied for a minimum of one hour before
in selected cases
• Site disinfected with iodine solution or alcohol-based preparation
• In infants and young children a 22g cannula is desirable if veins of sufficient calibre, otherwise
24g. In older children a 20g cannula is desirable.
• Cannula inserted and taped in cross-over fashion with 1 cm Elastoplast
• Extension piece attached and cannula flushed with 2 mls normal saline or heparinized saline.

2.3.2 Sampling from IV cannula

All samples are drawn using aseptic technique. Gloves should be worn for protection.

When sampling from cannulas it is imperative that sufficient void volume be removed before the blood
sample for analysis is collected, otherwise the sample will be diluted and spurious results obtained. 0.5
to 1 ml should be withdrawn prior to drawing of the blood sample if a standard T-piece is being used - in
infants and young children this can be replaced if volume considerations are critical (see below).
Cannulas should initially be flushed with heparinized saline, then subsequently with normal saline unless
patency difficulties are experienced.

2.4 Blood volume considerations

The circulating blood volume of infants and children is approximately 80 mls/kg and this should be borne
in mind for all studies, especially in the very young. Total blood volumes withdrawn should not exceed

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2.5% of blood volume in any one day study, or 5% over several day studies. As a guide in small
children:

Child's weight Total blood volume Maximum one day test sampling
volume - includes blood removed in
clearing lines prior to sampling

2.5 kg 200 mls 5 mls

5 kg 400 mls 10 mls

10 kg 800 mls 20 mls

15 kg 1200 mls 30 mls

20 kg 1600 mls 40 mls

2.5 Specimen collection requirements

The tables in section 2 indicate sample volumes and collection requirements for various analyses. These
are divided into:

1. Analytes assayed at RWI Endocrine Laboratory

2. Analytes assayed in other laboratories at RAHC, or external laboratories

Where possible, the preferred volume rather than the minimum volume should be collected. This allows
for repeat assays to be performed, or for additional tests to be performed if needed. For most tests in
older children there is no problem in collecting ample volumes, but special consideration must be given
to small children or when collection is technically difficult.

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3. Specimen Collection Requirements and Reference Ranges

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3.1 RWI Endocrinology Laboratory Specimen Collection

Analyte List and Specimen Requirements

Ray Williams Institute of Endocrinology, Endocrine Laboratory
Level 1, Diagnostic Services Building (Bldg 5), Royal Alexandra Hospital for Children (The New Children's Hospital)
Cnr Hawkesbury Rd and Hainsworth St, Westmead NSW 2145
General Instructions
All blood samples for the Endocrine Laboratory are to be centrifuged as soon as possible, the plasma or serum pipetted off immediately into an appropriately labelled tube
and immediately frozen. Delays in centrifuging the sample and freezing the plasma can cause erroneous results. If serum is to be collected, allow the blood to clot
completely before centrifugation. If there is any doubt as to the collection procedure or volume required please contact the Endocrine Laboratory on (02) 845 3190 (RAHC:
ext 53190). All samples from outside the New Children's Hospital are to be transported frozen to the above address, accompanied by a request form.

Note: * "for research purposes only" - contact Endocrine Lab if assay required Note: Serum = collect blood into plain (clotted blood) tubes.

Analyte Abbreviations Sample type required Acceptable Plasma Minimum Preferred

alternative sample volume blood volume blood volume
types required in
assay

Aldosterone Aldo Li-Hep plasma EDTA plasma, 0.4 ml 0.9 ml 2 ml

serum

Androstenedione ∆4A, A'dione Li-Hep plasma EDTA plasma, 0.2 ml 0.5 ml 1 ml

serum

Cortisol - plasma Cort Li-Hep plasma EDTA plasma, 0.05 ml 0.2 ml 0.5 ml
serum

Cortisol - Urinary free UFC 24 hr urine collection with no (1.0 ml urine) Send all urine Send all urine
preservative, total volume collected collected
measured

Dehydroepiandrosterone DHAS, DHEAS Li-Hep plasma EDTA plasma, 0.02 ml 0.2 ml 0.5 ml
sulphate serum

* 11-Deoxycortisol DCOR Li-Hep plasma EDTA plasma, 0.1 ml 0.3 ml 0.5 ml

serum

* 11-Deoxycorticosterone DOC Li-Hep plasma EDTA plasma, 2.0 ml 4.2 ml 8 ml

serum

Dihydrotestosterone DHT Li-Hep plasma EDTA plasma, 0.4 ml 0.9 ml 2 ml

serum

Estradiol (oestradiol) E2 Li-hep plasma Serum 0.2 ml 0.5 ml 1 ml

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 Free fatty acids FFA Li-hep plasma EDTA plasma 0.2 ml 0.5 ml 1 ml

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 Analyte Abbreviations Sample type required Acceptable Plasma Minimum Preferred
alternative sample volume blood volume blood volume
type required in
assay

Follicle stimulating FSH Li-hep plasma Serum 0.1 ml 0.3 ml 0.5 ml

hormone

Growth Hormone GH, hGH Li-hep plasma EDTA plasma, 0.1 ml 0.3 ml 0.5 ml
serum

Haemoglobin A1c - Diamat Diamat HbA1c 5 µl capillary blood Venous whole blood 5 µl whole 5 µl using 5 µl using
method (fingerprick) collected in collected in EDTA, blood collection kit collection kit
special capillary tube and this potassium oxalate or or
("send-in" method - carried placed in special buffer or sodium fluoride 0.5 ml of 1.0 ml of
out in the laboratory) containing tube - collection tubes whole blood whole blood
kits available from the (may be frozen)
Endocrine Lab

Haemoglobin A1c DCA 2000 Fresh capillary blood EDTA, heparin or 1 µl whole 1 µl fresh 1 µl fresh
- DCA 2000 method HbA1c - contact Endocrine citrate preserved blood capillary blood capillary
Laboratory whole blood (contact lab) blood
("on-the-spot" method for (may be frozen) or (contact lab)
use in Diabetes Clinics) 0.5 ml of or
whole blood 1.0 ml of
whole blood

ß-Hydroxybutyrate ßOHB, 1 volume blood + 2 volumes 0.05 ml 0.1 ml blood + 0.1 ml blood +
(Ketones) Ketones 0.6M perchloric acid (volumes 0.2 ml 0.6M 0.2ml 0.6M
accurately measured) perchloric acid perchloric
acid

* 17-Hydroxypregnenolone 17OHPG,17HP Li-hep plasma EDTA plasma, 0.4 ml 0.9 ml 2 ml

G serum

17-Hydroxyprogesterone 17OHP, 17HP Li-hep plasma EDTA plasma, 0.1 ml 0.3 ml 0.5 ml
serum

Insulin INS Li-hep plasma EDTA plasma, 0.2 ml 0.5 ml 1 ml

serum

* Insulin Autoantibodies IAA Serum 0.3 ml 1.0 ml 5 ml

Insulin-like growth factor-1 IGF-1 Li-hep plasma EDTA plasma, 0.2 ml 0.5 ml 1 ml
(Somatomedin-C) serum

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 Analyte Abbreviations Sample type required Acceptable Plasma Minimum Preferred
alternative sample volume blood volume blood volume
type required in
assay

* Islet Cell Antibodies ICA Serum 0.05 ml 0.2 ml 5 ml

(Pancreatic Islet cell (2.0 ml minimum preferred)
autoantibodies)

Luteinising hormone LH Li-hep plasma Serum 0.16 ml 0.45 ml 1 ml

Microalbumin, Urinary Malb Overnight urine collection - (2.1 ml urine) (50 ml sample
special instructions, additive of total
and urine pots obtained from overnight
Endocrine Lab urine
collection -
contact lab
for special
instructions)

Oestradiol (see "Estradiol") E2 Li-hep plasma Serum 0.2 ml 0.5 ml 1 ml

Plasma Renin Activity PRA EDTA plasma Li-hep plasma 0.5 ml 1.0 ml 2 ml
(morning sample preferred)

Prolactin Prol, PRL Li-hep plasma Serum 0.05 ml 0.2 ml 0.5 ml

Testosterone Testo Li-hep plasma EDTA plasma, 0.2 ml 0.5 ml 1 ml

serum

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3.2 RWI Endocrinology Laboratory Reference Ranges

Endocrine Laboratory
Ray Williams Institute of Paediatric Endocrinology
The New Children's Hospital, Westmead NSW
(Royal Alexandra Hospital for Children)

Routine Assay Reference Ranges

3.2.1 Aldosterone

Males and females, all ages : 280 - 2800 nmol/L

3.2.2 Androstenedione

Males and females: Age Androstenedione (nmol/L)

Mean Range
3 months - 8 years 1.1 0.7 - 1.7
8 years - 10 years 2.0 1.0 - 2.9
10 years - 12 years 3.0 1.9 - 4.2
over 12 years 4.9 2.7 -10.2

3.2.3 Cortisol, plasma

Males and females:

(a) Diurnal variation:
Morning: 200 - 600 nmol/L
Afternoon: approximately one third of morning value
(b) Response to Synacthen or hypoglycaemia:
An increase over basal level of greater than 280 nmol/L, with a final level exceeding 600 nmol/L

3.2.4 Cortisol, urinary free

Males and females: Age Urinary free cortisol

nmol/24 hr
2 weeks - 10 years 29 - 78
10 years - 15 years 57 - 145
over 15 years 120 - 432

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3.2.5 Dehydroepiandrosterone Sulphate (DHAS)

Males and females: Age DHAS µmol/L

0 - 3 months 0.5 - 7.0
3 months - 5 years < 0.5
5 years - 9 years 0 - 1.5
9 years - 14 years 0.5 - 6.0
over 14 years 1.8 - 10.0

3.2.6 11-Deoxycortisol (DCOR)

Males and females, all ages: < 30 nmol/L

11-Deoxycorticosterone (DOC)
Males and females, all ages: < 0.6 nmol/L

Dihydrotestosterone (DHT)
Males:
Testosterone/Dihydrotestosterone Ratio Post HCG: mean: 10 (range: 2-20)

Estradiol (oestradiol) (E2)

Age Estradiol (pmol/L)
Mean Range
Females: 1 year - 12 years 56 32 - 80
12 years - 14 years 133 97 - 169
> 14 years 260 100 - 410

Males: 1 year - 12 years 30 17 - 41

12 years - 14 years 50 27 - 73
> 14 years 110 90 -130

Free Fatty Acids (FFA)

Males and females, all ages: 0.8 - 1.0 mmol/L (after overnight fast)

Follicle stimulating hormone (FSH)

The standards have been calibrated against the 2nd International Reference Preparation of Pituitary FSH/LH
(ICSH) human for bioassay - 78/549.
Age FSH (IU/L)
Mean Range
Females: 0 - 1 week 0.39 0.08 - 8.7
1 week - 1 month 3.1 0.80 - 13.2
1 month - 3 years 2.4 0.12 - 8.7
3 years - 9 years 1.2 0.18 - 7.5
9 years - 12 years 2.3 0.38 - 6.4
12 years - 14 years 3.5 1.4 - 5.6
14 years - 18 years 3.1 1.4 - 6.8

Males: 0 - 1 week 0.25 0.11 - 2.0

1 week - 2 months 1.2 0.58 - 5.5
2 months - 9 months 0.52 0.10 - 1.5
9 months - 3 years 0.37 0.10 - 1.2
3 years - 9 years 0.46 0.12 - 1.4
9 years - 12 years 0.93 0.18 - 2.8
12 years - 14 years 1.4 0.20 - 5.4
14 years -18 years 2.1 0.50 - 6.3

Growth Hormone (GH)

The hGH standard is calibrated against 1st International Standard 80/505 from WHO.
Males and females:
Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page 15
Normal Growth Hormone > 20mIU/L following any appropriate stimulus (e.g., sleep, exercise, hypoglycaemia,
arginine). Random Values are usually low.

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Glycosylated haemoglobin (GHb) (This assay is no longer available)
Males and females: GHb (pmol/mg) Glucose Control Level
600 - 1040 Normal range
1040 - 1200 Near Normal range
1200 - 1400 Good
1400 - 1600 Fair
1600 - 2000 Poor
> 2000 Very Poor

Haemoglobin A1c - Diamat and DCA 2000 methods

Males and females: HbA1c Glucose Control Index
4-6% Normal Range
6-7% Near normal glycaemia
7-8% Excellent
8-9% Good
9 - 10 % Unsatisfactory
> 10 % Poor

ß-Hydroxybutyrate (Ketones)
Males and females, all ages: < 0.5 mmol/L

17-Hydroxypregnenolone (17OHPG, 17HPG)

Males and females, all ages: < 45 nmol/L

17-Hydroxyprogesterone (17OHP)
Males and females:
(a) Age 17OHP (nmol/L)
< 3 months < 24
3 months - 18 years <6
(b) Response to Synacthen at 30 minutes
Ratio of 17OHP/CORTISOL < 0.023

Insulin
The Insulin standard (human insulin) is calibrated against "research Standard A for Insulin, human, for
immunoassay, 66/304" from the WHO International Laboratory for Biological Standards.
Males and females, all ages: Insulin values vary with diet and ambient glucose levels.
Please consult endocrinologist for interpretation.
[1 mU/L = 7.5 pmol/L]

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Insulin-like growth factor-1 (IGF-1)
(Somatomedin-C)
Age IGF-1 (nmol/L) Age IGF-1 (nmol/L)
Females: < 6 years 7 - 21 Males: < 6 years 7 - 21
6 - 7 years 7 - 31 6 - 7 years 7 - 28
8 - 9 years 13 - 46 8 - 10 years 11 - 34
10 -11 years 22 - 64 11 - 12 years 19 - 52
12 - 13 years 34 - 106 12 - 13 years 34 - 97
14 - 15 years 34 - 82 14 - 15 years 34 - 79
16 - 18 years 28 - 64 16 - 18 years 27 - 64

Luteinizing hormone
The standards have been calibrated against the WHO 2nd International standard for pituitary LH for
immunoassay (coded 80/552).
Age LH (IU/L)
Mean Range
Females: 0 - 1 week 2.1 0.46 - 17.4
1 week - 1 month 0.57 0.03 - 4.1
1 month - 3 years 0.16 0.01 - 3.5
3 years - 9 years 0.27 0.01 - 4.4
9 years - 12 years 0.95 0.03 - 5.4
12 years - 14 years 3.1 0.45 - 9.9
14 years - 18 years 3.8 0.6 - 9.8

Males: 0 - 1 week 1.2 0.4 - 4.3

1 week - 2 months 3.2 0.93 - 12.1
2 months - 9 months 0.55 0.02 - 3.2
9 months - 3 years 0.07 0.01 - 0.61
3 years - 9 years 0.22 0.01 - 4.7
9 years - 12 years 0.54 0.07 - 5.4
12 years - 14 years 1.7 0.25 - 7.6
14 years - 18 years 1.5 0.21 - 6.1

Oestradiol (see "Estradiol)

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Plasma Renin Activity
Males and females: Age PRA (fmol/L/sec)
Mean Range
1 year 2000 1070 - 2930
1 year - 8 years 1200 600 - 1800
8 years - 18 years 600 300 - 900

Prolactin
The standards have been calibrated against the WHO 3rd International Standard for Prolactin (coded 84/500).
Males and females: Age PRL (mU/L 75/504)
< 2 weeks 608 - 6080
2 weeks - 2 months 304 - 6080
2 months - 6 months 304 - 3040
6 months - 1 year 152 - 1520
> 1 year < 760

Testosterone
Age Testo (nmol/L)
Females: 0 - 2 months <2
2 months - 10 years <1
10 years - 12 years < 1.5
12 years - 14 years 1-2
> 14 years 1 - 2.5
Adult 1-4

Males: at birth 5 - 12
5 - 6 days 1-2
30 - 60 days 7 - 12
7 months - 10 years < 1.0
10 years - 12 years < 1.5
12 years - 14 years 1 - 3.5
> 14 years 3 - 13
Adult 11 - 30

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3.3 Other RAHC laboratories and external laboratories Specimen
Collection

Analyte list and specimen collection

Analyte / sample Abbreviations Sample type (tube) Preferred Minimum Laboratory
and collection blood volume blood
requirements volume

1,25 dihydroxyvitamin D 1,25 OHD Serum (plain) 6 ml 3 ml RNSH

25 hydroxy vitamin D 25 OHD Serum (plain) 6 ml 3 ml RNSH

Adrenocortiocotrophic ACTH EDTA - on ice, 2 ml 1 ml RPAH Endo

hormone separate within 1 hour

Albumin Li hep (plasma) 1 ml 0.5 ml Biochem

Aldosterone - urinary 24 Aldo 24 hour specimen in RNSH

hour 1g thymol as Hypertension
preservative. pH Unit
should be 5-7,
otherwise adjust with
a few drops of acetic
acid. Ring lab prior to
collection.

alpha 1 antitrypsin Serum (plain) 4 ml 2 ml Biochem

phenotype

alpha 1 antitrypsin Serum (plain) 2 ml 1 ml Biochem

alpha-subunit Serum (plain) 4 ml RNSH

RPAH endo

Amino acids AA Li hep (plasma), on 2 ml 1 ml Biochem

ice

Ammonia NH4 Li hep (plasma), on 1 ml 0.5 ml Biochem

ice

Amylase Li hep (plasma), on 1 ml 0.5 ml Biochem

ice

Angiotensin II Na EDTA, collect on 10 ml RNSH

ice, spin at 4 C and Hypertension
separate. Ring RNSH Unit
lab prior to collection

Antibodies - parietal cell Serum (plain) 2 ml 1 ml Immuno

Antibodies - peroxisomal Serum (plain) 2 ml 1 ml Immuno

Antibodies - ovarian Serum (plain) 2 ml 1 ml Immuno

Antibodies - nuclear Serum (plain) 2 ml 1 ml Immuno

Antibodies - TSH receptor Serum (plain) 2 ml 1 ml RNSH Endo

Antibodies - smooth Serum (plain) 2 ml 1 ml Immuno

muscle

Antibodies - thyroglobulin Serum (plain) 2 ml 1 ml Immuno

Antibodies - gliadin ICA Serum (plain) 2 ml 1 ml Biochem

Antibodies - microsomal Serum (plain) 2 ml 1 ml Immuno

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 Antibodies - glutamic anti-GAD Serum (plain) 2 ml 1 ml Endo
acid decarboxylase

Antibodies - adrenal Serum (plain) 2 ml 1 ml Immuno

Antibodies - islet cell ICA Serum (plain) 5 ml 5 ml Endo

Antibodies - insulin IAA Serum (plain) 2 ml 1 ml Endo

Antidiuretic hormone ADH

Atrial natriuretic hormone ANP K EDTA (+2000 KIU 10 ml

trasylol - 1 ml/10ml
blood). Collect on ice,
spin at 4 C and
separate. Ring lab
prior to collection.

C-peptide Serum (plain)

Calcitonin Serum (plain) 4 ml 2 ml RPAH Endo

RNSH Endo

Calcium, phosphate, Ca, PO4, Mg, Plasma (Li hep) 2 ml 1 ml Biochem

magnesium, SAP SAP

Calcium - 24 hour urine Ca No preservative All urine in 24 Biochem

hours

Calcium/creatinine ratio - Ca / Cr ratio Sterile urine jar 10 ml Biochem

urine

Carbamazepine Plasma (Li hep) 2 ml 1 ml Biochem

(Tegretol)

Carnitine Plasma (Li hep) 2 ml 1 ml Biochem

Ring biochem prior

Carotene Serum (plain) 4 ml Biochem

Catecholamines - urine

Catecholamines - serum

Cholesterol Serum (plain) or 1 ml 0.5 ml Biochem

plasma (Li hep)

Chromosomes
(see karyotype)

Clonazepam Plasma (Li hep) 2 ml Biochem

Copper Cu Serum (plain) 2 ml 1 ml Biochem

Creatine phophokinase CPK Plasma (Li hep) 1 ml 0.5 ml Biochem

Digoxin Plasma (Li hep) 1 ml 0.5 ml Biochem

collect 6-12 hrs after
dose

Electrolytes - urine Sterile urine jar > 10 ml Biochem

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page 21
 Electrolytes, urea, EUC Plasma (Li hep) 1 ml 0.5 ml Biochem
Creatinine

Erythrocyte ESR EDTA 0.5 ml Haematology

sedimentation rate

Ferritin Plasma (Li hep) or 1 ml EDTA Haematology

serum (plain) or EDTA 1.5 ml Li hep

Folate - red cell EDTA 1 ml 0.5 ml Haematology

Folate Plasma (Li hep) 4 ml Haematology

Full blood count FBC EDTA 1 ml Heamatology

Gastrin Serum (plain) 2 ml RPAH

Glucose - 24 hour urine No preservayive 24 hour urine Biochem

Glucose Glc Fl oxalate 0.5 ml 0.2 ml Biochem

Hemoblobin A1C HbA1c Capillary tube and 5 µl Endo

(Diamet - HPLC) solution as supplied

Hepatitis serology Serum (plain) 5 ml Haematology

High density lipoproteins HDL

Hydroxyproline - urine RNSH

Immunoglobulins Ig Serum (plain) 2 ml 1 ml Immunology

Immunoreactive trypsin IRT Guthrie paper Fill 3 circles Biochem

Iron (total) binding TIBC Plasma (Li hep) 1 ml Haematology

capacity

Iron Fe Plasma (Li hep) 1 ml Haematology

Karyotype Li hep (sterile tube) 5 ml 2.5 ml Genetics

Send without delay

Lactate and pyruvate Perchloric acid Biochem

Lead

Lipid EPG (LDL, HDL)

Liver function tests LFTs Plasma (Li hep) 1 ml 0.5 ml Biochem

Low density lipoproteins LDL

Metabolic screen - urine Sterile urine jar > 20 ml Oliver

Freeze Latham

Methylmalonic acid MML Plasma (Li hep) 1 ml Oliver

Latham

Monospot Serum (plain) 1 ml 0.5 ml Immunology

Organic acids Plasma (Li hep) 2 ml 1 ml Oliver

Latham

Osmolality - urine Urine osmo Sterile urine jar > 10 ml Biochem

Osmolality - plasma Plasma Plasma (li hep) 1 ml 0.5 ml Biochem

osmo

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page 22
 Osteocalcin Plasma (Li hep) 1 ml 0.5 ml Endo

Pancreatic isoamylase Serum (plain) 2 ml 1 ml Biochem

Parathyroid hormone PTH Serum (plain) 4 ml RNSH

Phenylalanine Plasma (Li hep) 1 ml 0.5 ml Biochem

Phenytoin Plasma (Li hep) 2 ml Biochem

Phosphate - 24 hour PO4 No preservative 24 hr urine Biochem

urine

Primidone Plasma (Li hep) 1 ml 0.5 ml Biochem

Progesterone Prog Plasma (Li hep) 1 ml 0.5 ml RPAH

Protein electrophoresis Serum (plain) 1 ml 0.5 ml Biochem

Protein (total) Plasma (Li hep) 1 ml 0.5 ml Biochem

Pyruvate and lactate

Rheumatoid factor Serum (plain) 2 ml Immuno

Sodium valproate Plasma (Li hep) 2 ml 1 ml Biochem

(Epilim)

Thyroid function tests TFTs Serum (plain) 2 ml Biochem

(Free T4 and TSH)

TORCH titres Serum (plain) 3 ml Immuno

Transferrin Serum (plain) 1 ml 0.5 ml Biochem

Triglycerides Serum (plain) or 1 ml 0.5 ml Biochem

plasma (Li hep)

Tyrosine Plasma (Li hep) at 4 C 1 ml Biochem

(on ice)

Uric acid Plasma (Li hep) 1 ml 0.5 ml Biochem

Vitamin D - 1,25(OH)2 Serum (plain) 6 ml 3 ml RNSH

Vitamin D - 25 OH Serum (plain) 6 ml 3 ml RNSH

VDRL Serum (plain) 1 ml 0.5 ml Biochem

Vitamin B12 Serum (plain) 5 ml Biochem

Zinc Zn Plasma (Li hep) 2 ml 1 ml Biochem

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page 23
4. Test Protocols

4.1 Arginine Stimulation Test

Indications: A test of growth hormone secretion. Often used as part of a combined pituitary function test.

Rationale: Arginine (and some other amino acids) stimulate GH secretion via effects on α-receptors
which influence GHRH and somatostatin secretion from the hypothalamus. In this test,
arginine is infused intravenously and the response of GH is measured in peripheral blood. It
is often a useful test in the neonate or young infant when GH testing is required, since it is
relatively free of adverse side effects compared to other tests at this age.

Contraindications: Severe renal disease. Electrolyte disturbances (especially hyperchloraemia).

Formulation: L-arginine hydrochloride (Ophthalmic Labs) 600 mg/ml, single dose 25 ml vial

Dose: 0.5 g/kg (500 mg/kg), to a maximum dose of 30g. Diluted in normal saline to a 10% solution
(ie. 10g arginine per 100 mls normal saline). Infused intravenously over 30 minutes.

Adverse Rapid IV infusion may cause flushing, nausea, vomiting, numbness, headache and local
reactions: venous irritation.
Allergic reaction - macular rash, anaphylactic reaction (extremely rare).
Elevated potassium in uraemic patients.

Preparation: Patient fasted for 8 hours (2-4 hours only in neonates or young infants). May drink water.

Equipment: Worksheet
IV cannula
Syringes 2 ml and 5 ml
Normal saline for cannula flushes
Normal saline for arginine dilution (500 ml bag)
Tubes - Li heparin and fluoride oxalate (collection) and plain (plasma storage) - labelled with
name, date, time and "Arg stim".

Method: 1. Patient weighed and dose calculated

2. IV cannula inserted and baseline samples collected.
3. Arginine diluted in normal saline as above and infused intravenously over 30 minutes.
4. Blood sampling as below. If performed as part of a combined pituitary test, see
combined protocol.

Sample TubeBlood volume -15 0 30 45mi 60mi 75mi

min min min n n n
Plasma glucose Fl oxalate0.5 ml S S S S S S
GH Li hep0.5 ml S S S S S S
IGF-1 Li hep1 ml S - - - - -
S = Sample at this time point

Interpretation: General principles are: Peak GH response < 10 mU/l suggests GH deficiency; responses of
10-20 mU/l suggest partial GH deficiency; response ≥ 20 mU/l is regarded as normal.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page 24
4.2 Clonidine Stimulation Test
Indication: A screening test of growth hormone secretion
Rationale: Clonidine is a selective α-agonist with central and peripheral actions. Its usual uses are in
hypertension and migraine prophylaxis. Central actions are predominantly via α2-
adrenergic stimulation and it is a potent stimulus to hGH release via GHRH secretion. In
this test clonidine is administered orally and the GH response in peripheral blood is
measured.
Contraindications: Sick sinus syndrome. Compromised intravascular volume.
Formulation: Clonidine tablets 25 micrograms; blue coated (Dixarit, Boehringer Ingelheim Pty Ltd)
Dose: 125 micrograms per m2 BSA orally (calculate amount to the nearest half tablet)
Adverse reactions: Drowsiness; Fall in blood pressure is expected, may last several hours. Effect will be
prolonged in renal failure. Troublesome adverse reactions are rare.
Preparation: Preferably morning test, with nil by mouth (excepting water) from midnight (food intake
suppresses GH secretion). However a minimum fasting time of only 2 hours is required
and short fasting times should be applied in infants and young children.
Accurate height and weight, allowing surface area calculation
IV sampling cannula
Equipment: Worksheet
Sphygmomanometer
IV sampling cannula
Syringes 2 ml and 5 ml, Normal saline for IV flushes
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with Name, date, time,
"Clonidine stim"
Method: 1. Surface area calculated from nomogram or formula
2. RMO to calculate and order dose of clonidine (125 micrograms /m2 BSA to nearest
half tablet)
3. IV cannula inserted; check baseline blood glucose level.
4. Baseline BP at time 0, then at 30 minute intervals
5. Child recumbent and resting during the test; may drink water.
6. Dose given with water after 0 blood sample collected
7. Blood sampling as below

Sample Tube Blood 0 min 30 60 90 120 150

volume min min min min min
GH Li hep0.5 ml S S S S S S
IGF-1 Li hep1 ml S - - - - -
Glucose F1 oxalate S - - - - -
0.5ml
S = Sample at this time point
8. Child fed after test, and only allowed home after BP stabilized to normal levels (not
sooner than 30 min after completion of test).
9. A mild to moderate drop in blood pressure is expected. In the event of more significant
BP fall, elevation of the legs is recommended and 15 minutely recording of BP. The
registrar should be notified. Volume expansion with normal saline or colloid may rarely
be required.

Interpretation: Peak GH response < 10 mU/l suggests GH deficiency; responses of 10-20 mU/l suggest
partial GH deficiency; response ³ 20 mU/l is regarded as normal.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page 25
4.3 Combined Pituitary Function Test (formerly, Triple Test)

Indications: In suspected multiple pituitary deficiencies, this test is provides detailed information on the
integrity of the clinically important anterior pituitary hormone axes - growth hormone, ACTH,
TSH and gonadotropins. It is frequently employed following neurosurgery or other insult to
the hypothalamic-pituitary region, or when other baseline or dynamic tests suggest one or
more pituitary hormone deficiencies and more information is required. In this test, a number
of stimulating agents are administered in a timed protocol, and the responses of hormones
in peripheral blood measured.

Rationale: See information under individual component tests. This test involves the combination of
insulin stimulation test, arginine stimulation test, TRH stimulation test and GnRH
stimulation test. This test is potentially dangerous because of the insulin component and
must only be performed by experienced personnel and closely supervised.

Contraindications: Specific contraindications or relative contraindications are listed in the separate protocols for
the various components of the test. In general this test should not be performed on patients
with unstable medical conditions or significant acute intercurrent illness

Insulin: History of convulsions. Hypoglycemic disorder. Caution in untreated adrenal

insufficiency. Infants and young children - in general is not performed under 5 yrs
except in particular circumstances.
Arginine: Severe renal disease. Electrolyte disturbances (especially hyperchloraemia).
TRH: Uncontrolled heart failure, severe myocardial ischaemia or asthma. Caution in
lesser degrees of these conditions.

Formulations: See individual tests

Doses: L-arginine hydrochloride (Ophthalmic Labs 600 mg/ml, single dose 25 ml vial) 0.5 g/kg (500
mg/kg), to a maximum dose of 30g. Diluted in normal saline to a 10% solution (ie. 10g
arginine per 100 mls normal saline). Infused intravenously over 30 minutes.

Insulin (soluble, regular human; Actrapid or Humulin R) The dose is chosen according
to the suspected diagnosis, as some patients will show greater sensitivity:
Hypopituitarism strongly suspected 0.025 to 0.075 unit/kg
Steroid treated patients 0.025 to 0.075 unit/kg
Standard dose ("Normal" patients) 0.1 unit/kg
Acromegaly / gigantism 0.15 unit/kg
Dose diluted in 5 mls normal saline and given by slow intravenous injection over
1 min.
TRH (Roche, 200 micrograms in 2 ml)
200 micrograms/m2 BSA by slow intravenous injection over 1 min

GnRH (Gonadorelin) ( HRF, Ayerst)

100 micrograms by slow intravenous injection over 1 min. ie. same dose all
ages, all sizes

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page 26
Adverse reactions :Insulin Symptoms of hypoglycemia are expected - pallor, sweating, hunger, headache,
tiredness.Hypoglycemic seizures.
Deaths have occurred, some associated with inappropriate (excessive) glucose
resuscitation.

Arginine: Rapid IV infusion may cause flushing, nausea, vomiting, numbness, headache
and local venous irritation.
Allergic reaction - macular rash, anaphylactic reaction (extremely rare).
Elevated potassium in uraemic patients.

TRH: Nausea, flushing, dizziness, urinary urgency, unusual taste in mouth,

occasionally headaches.
Increases in BP and pulse rate frequently observed
Caution in heart failure, myocardial ischaemia and asthma.
Caution in severe hypopituitarism - risk of hypoglycemia
Certain drugs may diminish response.

GnRH: Significant adverse reactions have not been encountered. Occasionally nausea
and abdominal pain.
Preparation: Patient fasted for at least 6 hours. Remain nil by mouth until after hypoglycemia.

Equipment: Worksheet
IV cannula
Syringes 2 ml and 5 ml
Normal saline for cannula flushes
Normal saline for arginine dilution (500 ml bag) and insulin dilution
Tubes - Li heparin and fluoride oxalate (collection) and plain (plasma storage) - labelled with
name, date, time and "Triple test".
Dextrose for IV administration must be available drawn up for immediate use - 10% dextrose,
2 ml/kg.

Method: 1. Patient weighed and measured. BSA calculated. Doses calculated

2. IV cannula inserted and baseline samples collected. Must be a reliable IV line.
3. Stimulating agents given as indicated below (see shaded boxes)
4. Blood sampling as in table, plus ward glucose testing as in 5.

5. After insulin administered medical officer must not leave the ward until patient
recovered from hypoglycemia. Patient closely observed for symptoms of hypoglycemia
which usually occur after 15 to 30 minutes. The aim is to achieve a plasma glucose
fall to 2.6 mmol/l or less, or symptomatic hypoglycemia with a fall of blood glucose to
< 50% of baseline. Blood glucose measured in ward at 10, 15, 20, 25, 30, 45, 60, 75,
90 and 120 minutes after insulin administration, or at any other time if in doubt.

6. When plasma glucose level < 2.6 mmol/l is recorded or symptomatic hypoglycemia
occurs, the test proceeds in one of two ways, but sampling continues:

a. Mild to moderate symptoms - give sweet drink, followed by food.

b. Severe hypoglycemia - intravenous dextrose - 2 ml/kg 10% dextrose, followed

by continuing infusion of 10% dextrose if slow recovery, or sweet drinks and food.
If poor response consider hydrocortisone 50-100 mg IVI.

7. The child is not allowed home until a glucose containing drink and a meal have been
tolerated, and all observations are satisfactory.

Interpretation: See interpretation of individual tests.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page 27
 Combined -15 0 30 45 60 75 85 90 95 100 105 120 135 150 165 195
Test time
Pituitary
mins
Function Test

(Triple test)

Arginine time -15 0 30 45 60 75

TRH time 0 15 30 45 55 90 120

GnRH time 0 15 30 45 55 90 120

Insulin time 0 10 20 30 45 60 75 90 120

Tube
Sample
Blood volume

Glucose Fl ox 0.5 ml S S S S S S S * S * S S S S S S

GH Li hep 0.5 ml S S S S S S S S S S S S S S

Cortisol Li hep 0.5 ml S S S S S S S

TSH Li hep 0.5 ml S S S S S

Free T4 Li hep 0.5 ml S

Free T3 Li hep 0.5 ml S S

Prolactin Li hep 0.5 ml S S S S S S

LH Li hep 1 ml S S S S S S

FSH Li hep 0.5 ml S S S S S S

IGF-1 Li hep 1 ml S

Testosterone Li hep 1 ml
(males)

S
Oestradiol
Li hep 1 ml S
(females)
* if clinically indicated
S = Sample at this time point
4.4 Desmopressin Test of Renal Concentrating Ability

Indications: A test of renal concentrating ability in congenital or acquired renal disease.

Rationale: This test examines renal concentrating ability by the administration of a synthetic
ADH analogue (desmopressin) and subsequent measurement of urine osmolality.
After passing through the distal tubules, approximately 90 % of filtered water has
been resorbed from the glomerular filtrate and urine is iso-osmolar or hypo-
osmolar. Further water uptake with subsequent concentration of urine occurs when
it passes through the collecting ducts in the renal pyramids. Antidiuretic hormone
controls the permeability to water of the collecting ducts via its action at specific
receptors. Impaired renal concentrating ability may arise because of reduced ADH
effect (nephrogenic DI, toxic or inflammatory processes) or because of adverse
effects on osmotic gradient in pyramidal tissue (eg. circulatory disorders,
hyponatremia, reduced GFR).

Contraindications: Overhydration, cerebral oedema, intercurrent illness. Caution in cardiac failure.

Nasal congestion (may result in poor absorption).

Formulation: Desmopressin nasal solution (Minirin Intranasal, Fisons) 100 µg/ml , delivered with
rhinyle supplied

Dose: Infants < 1 year age: 10 µg (0.1 ml) intranasal

Children and adolescents: 20 µg (0.2 ml) intranasal
Adults: Up to 40 µg (0.4 ml) intranasal

Adverse reactions: Overhydration; limit fluid intake for 12 hours after administration (see below)
Preparation: Before the test normal food and fluid intake is allowed, but this should not be more
than usual. The test is commenced in the morning before 10 am. If there is any
reason to suspect abnormal serum electrolytes or hydration, serum electrolytes
should be known before commencement. In infants, a urine bag is applied, and
consideration may need to be given to bladder catheterization.
Equipment: Worksheet
Urine bag (infants)
Refractometer
Containers for urine samples

Method: 1. Patient weighed

2. The bladder is emptied just before the administration of the desmopressin and
a sample collected for baseline specific gravity (SG) and osmolality. If the
patient empties the bladder within 1 hour of the administration of
desmopressin this can be used as a baseline sample, but in general the test
should not commence until baseline voiding has occurred.
3. Desmopressin administered intranasally in dose as above
4. During the test no fluids or liquid foods should be given to avoid the risk of
overhydration, but dry foods are allowed.
5. Urine samples are collected for specific gravity (ward test) and osmolality
(laboratory) from 1 to 8 hours after commencement. If patients can void on
command, samples should be collected at 4 and 6 hours.
6. The test can be terminated after 6 hours if two urine samples have been
obtained, or in any case at 8 hours after encouragement to void.
7. Before going home patients are advised to restrict fluid and liquid food intake
to approximately half of usual until the next morning, with no restriction on
other food intake.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 Tube
Sample Before test Any samples between 1 and 8 hours
(baseline) (preferably at 4 and 6 hours)

Urine SG - S S
(refractometer)

Urine osmolality Plain screw- S S

(laboratory) top container

S = Sample at this time point

Interpretation: The highest urine osmolality achieved during the test is noted. Maximum urine concentrating
ability increases with age, peaking at adult levels at around 3 years of age. From around 20
years of age gradual decline occurs. There are no sex differences. The mean values and
range (-2SD to +2SD) are:

1 year 840 mosmol/l (525 - 1170)

2 years 1000 mosmol/l (700 - 1300)
> 3 years 1050 mosmol/l (825 - 1400)

References: Marild et al. Pediatr Nephrol 6:254-7, 1992

Feber et al. Am J Nephrol 13:129-131, 1993

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.5 Dexamethasone Suppression Test

Indications: Used in the evaluation of suspected Cushing's syndrome, or in androgen excess states
where adrenal tumour is suspected. Three variations of this test are listed:

Overnight standard (low-dose) dexamethasone suppression test: Used as a

simple screening test for Cushing's syndrome.

Overnight high-dose dexamethasone suppression test: Used in distinguishing the

cause of Cushing's syndrome ie. in differentiating Cushing's disease (pituitary ACTH
hypersecretion) from other causes of Cushing's syndrome - ectopic ACTH or adrenal
tumours. Now an often used alternative to the traditional standard (long)
dexamethasone suppression test - easier and more reliable.

Standard (long) dexamethasone suppression test: Less commonly used now, but
may still have some role, especially in evaluating suppressibility in androgen excess
states.

Rationale: Dexamethasone is a synthetic glucocorticoid which is not detected in other steroid

assay systems. Through negative feedback mechanisms, the administration of
dexamethasone normally causes reduced ACTH secretion via effects on the
hypothalamic-pituitary axis, and hence decreased cortisol secretion. Since adrenal
androgen production is also partially under the control of ACTH, these also are normally
suppressed by dexamethasone. In pathological states of autonomous hormone
production, these feedback responses are lost or impaired.

In general low dose tests are used to establish the diagnosis of Cushing's syndrome
regardless of its cause. High dose tests are used to distinguish Cushing's disease
(pituitary ACTH hypersecretion) from ectopic ACTH and adrenal tumours.

Contraindications: Intercurrent acute illness, systemic infection

Adverse reactions: Adverse reactions are unlikely. Hypersensitivity reaction to IV injection is extremely
rare.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.6 Overnight Low-Dose Dexamethasone Suppression
Test

Formulation: Dexamethasone tabs 0.5 mg, 4 mg (scored) (Dexmethsone, Fisons)

Dose: 1 mg per 1.73m2 body surface area; minimum dose 1mg.

Preparation: Must have had 0800 blood sampling (see below) on that morning
Often performed on an outpatient basis

Equipment: Worksheet
Syringes/needles for blood sampling; sometimes via IV cannula if inpatient
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with name, date, time
and "Overnight dex suppression".

Method: 1. 0800 blood sample collected (see below)

2. Oral dexamethasone 1 mg given at 2300 - 2400 hrs that night
3. 0800 blood sampling next morning (see below)

Sample Tube 0800 0800

Blood volume Day 0 Day 1
(after dex)

Cortisol Li hep S S
0.5 ml

ACTH or other
analytes only if
specified
S = Sample at this time point

Interpretation: General principles are:

In normal subjects plasma concentrations fall to less than 140 nmol/l, while in Cushing's
syndrome they remain above 280 nmol/l. This is a screening test, and is only of value if
suppression occurs. Failure to suppress may occur in normal subjects due to stress,
intercurrent illness, obesity, psychiatric disorder, estrogen treatment or pulsatile release of
cortisol. Failure to suppress should prompt further evaluation as clinically indicated.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.7 Overnight High-Dose Dexamethasone Suppression
Test

Formulation: Dexamethasone tabs 4 mg (scored) (Dexmethsone, Fisons)

Dose: 8 mg/m2 BSA orally at 2300 - 2400 hrs

Preparation: Must have had 0800 blood sampling (see below) on that morning
Often performed on an outpatient basis

Equipment: Worksheet
Syringes/needles for blood sampling; sometimes via IV cannula if inpatient
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with name, date, time
and "Overnight dex suppression".

Method: 1. 0800 blood sample collected (see below)

2. Oral dexamethasone 8 mg given at 2300 - 2400 hrs that night
3. 0800 blood sampling next morning (see below)

Sample Tube 0800 0800

Blood volume Day 0 Day 1
(after dex)

Cortisol Li hep S S
0.5 ml

ACTH or other
analytes only if
specified
S = Sample at this time point

Interpretation: General principles are:

In normal subjects plasma concentrations fall to less than 140 nmol/l. In
Cushing's disease plasma cortisol levels are reduced to less than 50% of
baseline in 95% of patients. In ectopic ACTH syndrome or adrenal tumours,
suppression is less marked or absent.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 4.8 Standard (Long) Dexamethasone Suppression
Test
Formulation: Dexamethasone tabs 0.5 mg, 4 mg (scored) (Dexmethsone, Fisons)
Dose: Dexamethasone is administered successively in a low dosage, then high dosage as
follows (see schedule below):
0.5 mg orally q6hrly on days 3 and 4 (all ages and sizes)
2 mg orally q6hrly on days 5 and 6
(Unless specified use the above doses. Occasionally endocrinologist may specify dose/kg;
Low dose = 20 µg/kg/dose, high dose = 80 µg/kg/dose)
Preparation: Inpatient
IV sampling cannula

Equipment: Worksheet
Urine collection bottles (no additive)
2 ml and 5 ml syringes for blood sampling
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with name, date, time
and "Long dex suppression".

Method: 1. Days 1 and 2 collect *:

24 hour urines for urinary free cortisol (separate collections days 1 and 2)
Blood sampling as below 0800 and 2400 hrs days 1 and 2
2. Commence dexamethasone dosage 0800 day 3
3. Days 3 and 4, collect blood and urine as for days 1 and 2
4. Day 5, increase to high dose dexamethasone dose at 0800
5. Days 5 and 6, collect blood and urine as other days

* Abbreviated 5 day test: If specified by endocrinologist

1 day of pre-dexamethasone collections
Low dose dexamethasone days 2 and 3
High dose dexamethasone days 4 and 5
24 hour urine collections only on days 1, 3 and 5.

Sample Tube Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Volume
0800 2400 0800 2400 0800 0800 0800 0800
Plasma cortisol Li S S S S S S S S
hep0.5
ml
Plasma ACTH S S S S S S S S
Plasma Li S - S - S S S S
androgens hep2.5
(DHAS, ml
androstenedione,
testosterone)
Other steroids if Li hep S - S - S S S S
specified
24 hour UFC Plain S S S S S S
bottles
S = Sample at this time point

Interpretation: General principles are: Normal subjects will have suppressed cortisol production on low
dose, whereas patients with Cushing's syndrome do not. At the high dose, 90 % of patients

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 with Cushing's disease (ACTH dependent Cushing's syndrome) will suppress, whereas
patients with adrenal adenoma, carcinoma or ectopic ACTH syndrome will not. Androgen
levels are similarly interpreted.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.9 Exercise Stimulation Test
Indication: A screening test of growth hormone secretion

Rationale: Exercise is a physiological stimulant of GH secretion, presumed to be mediated via the

adrenergic nervous system. Exercise to approximately 50 % of maximal working
capacity is required and this is usually achieved on a cycle ergometer or by repeated
stair climbing. The test has a relatively high incidence of false positives for GH
deficiency often due to inadequate exercise, yet is a safe and inexpensive screening
test. An inadequate GH response should generally lead to a further (usually
pharmacological) test of GH secretion being performed.

Contraindications: Limitation of exercise capacity by cardiovascular, respiratory or other systemic

disease. Less robust children or children under 8 often do not tolerate the enforced
exercise well.

Adverse reactions: Exhaustion. Asthma

Preparation: Fasted for at least 2 hours; any time of day. IV sampling cannula.
Patients with exercise-induced asthma who normally take prophylactic medication
before exercise should do so.

Equipment: Worksheet
IV sampling cannula
Exercise bicycle of appropriate size for age or motorized treadmill.
Syringes 2 ml and 5 ml
Normal saline for IV flushes
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with Name, date,
time, "Exercise stim"

Method: 1. A pre-exercise 0 blood sample is collected

2. Record baseline heart rate
3. Child is exercised vigorously for 20 mins (approximately 2 watts/kg body weight if
ergometer available). Frequent encouragement is usually required. Measure heart
rate at approximately 5 minutely intervals. A heart rate of 140-160 is usually
achieved. The test should be stopped if the heart rate exceeds 180 or the child is
markedly distressed or exhausted.
4. Offer cool water and flannel during test, but continue exercising.
5. After 20 minutes of exercise, collect second sample, child rests and final sample
collected at 40 mins (20 mins post-exercise).
6. As an alternative, 20 minutes of supervised stair climbing or running may be
performed, but is generally not recommended.

Sample Tube Blood Pre-exercise Immediately 20 minutes post-

volume 0 mins post-exercise exercise
20 mins 40 mins
GH Li hep0.5 ml S S S
IGF-1 Li hep1 ml S - -
S = Sample at this time point

Interpretation: Peak GH response < 10 mU/l suggests GH deficiency; responses of 10-20 mU/l suggest
partial GH deficiency; response ≥ 20 mU/l is regarded as normal. An exercise GH test
suggesting GH deficiency should usually be followed up by further pharmacological testing.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.10 Exercise Stimulation Test, with Propranolol
(Propranolol Exercise Test)
See Exercise stimulation test protocol

Rationale: This test is as for the Exercise stimulation test, but in addition propranolol is
administered 2 hours prior to the exercise. Propranolol administration has been reported
to decrease the incidence of false negative results.
Contraindications: See exercise test. Contraindications to propranolol administration are asthma, and
significant cardiac disease.
Formulation: Propranolol - tablets 10 mg, 40 mg (Inderal, ICI ; Deralin, Alphapharm)
Dose: 0.5 mg/kg to a maximum of 40 mg
Adverse reactions: Bronchospasm, hypotension, fatigue
Preparation: Fasted for at least 2 hours; any time of day. IV sampling cannula. Patients with exercise-
induced asthma who normally take prophylactic medication before exercise should do
so.
Equipment: Worksheet
IV sampling cannula
Exercise bicycle of appropriate size for age or motorized treadmill
Syringes 2 ml and 5 ml
Normal saline for IV flushes
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with Name, date,
time, "Exercise stim"
Method: 1. A 0 blood sample is collected
2. Record baseline heart rate and BP
3 Propranolol given orally; child rests quietly for 2 hours
4. 2 hour blood sample collected; recheck BP
5. Child is exercised vigorously for 20 mins (approximately 2 watts/kg body weight if
ergometer available). Frequent encouragement is usually required. Measure heart
rate at approximately 5 minutely intervals. A heart rate of 140-160 is usually
achieved, but this may be less when propranolol has been administered. The test
should be stopped if the heart rate exceeds 180 or the child is markedly distressed
or exhausted.
6. Offer cool water and flannel during test, but continue exercising.
7. After 20 minutes of exercise, recheck BP, collect further blood sample, child rests
and final sample collected at 20 mins post-exercise.

Sample Tube 0 mins Immediately Immediately 20 mins

Blood vol pre-exercise post-exercise post-exercise
120 mins 140 mins 160 mins
GH Li hep S S S S
0.5 ml
IGF-1 Li hep S - - -
1 ml
S = Sample at this time point

Interpretation Peak GH response < 10 mU/l suggests GH deficiency; responses of 10-20 mU/l suggest partial
: GH deficiency; response ≥ 20 mU/l is regarded as normal. An exercise GH test suggesting GH
deficiency should usually be followed up by further pharmacological testing.

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4.11 Fasting Study

Indication: Suspected hypoglycemic disorders, or monitoring progress in a known hypoglycemic

disorder

Rationale: The diagnosis of hypoglycemia and the elucidation of its cause requires a monitored
fasting study when clinical information and baseline studies are inconclusive. Fasting is
performed under carefully controlled conditions to determine whether or not hypoglycemia
occurs during the fasting period, and if so, to elucidate the cause by analysis of the
relevant metabolites. Studies need to be individually planned according to the age of the
patient and the suspected disorder. In patients with a known hypoglycemic disorder on
therapy, periodic fasting studies are performed to guide further management decisions.

Contraindications: Recent or intercurrent illness

Adverse reactions: Potentially a very hazardous test. Requires very close supervision.
Severe or refractory hypoglycemia. Hypoglycemic seizures
Cardiac arrhythmias (fatty acid oxidation disorders)

Preparation: Admit patient, non-fasted.

Remain on current medications unless otherwise specified by consultant
IV sampling cannula

Equipment: Worksheet
IV sampling cannula
Syringes 2 ml and 5 ml
Normal saline for IV flushes
IV glucose 10% available for immediate use
Tubes - Li heparin, fluoride oxalate, perchloric acid (collection) and plain (plasma storage)
- labelled with Name, date, time, "fasting study"

Method: 1. IV cannula inserted; must be a reliable IV line.

2. Medical officer determines the maximum fasting time and time of commencement of
study. This is an individual judgement based on the clinical history of relationship of
episodes to meals and fasting and the age of the patient. Fast should commence at
a time such that if hypoglycemia occurs, it is anticipated between 9 am and 5 pm
when full staff are available. As a guide to appropriate maximum fasting times:

Neonates and infants < 3 months 4 to 8 hours (usually miss 1 feed only)
Infants 3 to 6 months 8 to 12 hours
6 months to 2 yrs 12 to 16 hours
2 to 10 years 16 to 20 hours
> 10 years 16 to 24 hours

The fast is judged to commence immediately after the last caloric intake.

3. Sample collection (see table below): Medical staff will advise which metabolites are
to be monitored during the fast. Blood and urine are collected at baseline. The
frequency of subsequent measurements is dependent on age and the likely duration
of fast. In general, infants under 6 months should have hourly blood glucoses and
young children 2 hourly. In older children where early hypoglycemia is not
anticipated, blood glucose is measured 4 hourly for 8-12 hours, then 1-2 hourly
depending on progress. Blood glucose measurements must be rapidly available.
Other metabolites are usually measured 2 hourly, except where a short fast is
anticipated where they may be measured hourly.
4. Hydration must be maintained during the study and subjects are given free access
to water.

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 5. All urine passed is tested by Ketodiastix for ketones. Urine samples are kept and
frozen for metabolic screen - pre-fast, all urines during the fast and first urine post-
fast. Not all urine samples will be sent for metabolic analysis and this is decided at
the end of the test.

6. Termination of fast: The fast is terminated when hypoglycemia occurs (plasma

glucose ≤ 2.6 mmol/l) or the previously determined maximum fast time is
completed. A blood sample for all metabolites is collected at this time. The study is
terminated in one of three ways:

a. Child able to eat / drink - child fed

b. Emergency treatment of hypoglycemia

Child unable to eat / drink - severe hypoglycemia with impaired
consciousness or seizures - IV 10 % dextrose 2 mls / kg, followed by
continuing IV infusion of electrolyte solution containing 5% or 10% dextrose
at maintenance volumes. Feed when sufficiently recovered.

c. Glucagon stimulation test: In some circumstances (especially suspected

disorders of hepatic gluconeogenesis) it is useful to determine the glucose
response to administered glucagon - this is performed (see separate protocol)
and the child then fed. This should not be performed if a severe hypoglycemic
episode has occurred for which emergency treatment is warranted.
7. After the study, blood glucose levels should be monitored until stable. The child is
not allowed home until they have eaten/drunk and blood sugars are normal and
stable.

Interpretation: Each study needs to be interpreted in its clinical context. Some general principles are as
follows:

A physiological response to fasting is that as blood glucose falls, plasma FFAs and
ketones rise and there is progressive ketonuria. Serum insulin becomes suppressed. In
the presence of hypoglycemia, cortisol and GH should normally be elevated.

Hypoglycemia with a ketotic response is also seen in hypopituitarism or glucocorticoid

deficiency, and in the exaggerated physiological state termed "ketotic hypoglycemia".

n hyperinsulinemic states, serum insulin does not suppress appropriately and

hypoglycemia occurs in the absence of a significant rise in FFAs, beta-hydroxybutyrate
or urinary ketones.

In disorders of hepatic β-oxidation of fatty acids, hypoglycemia occurs with suppressed

insulin, elevated FFAs and minimal or absent ketone response.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 Sample collection summary:

Note that in young children, infants and babies, the fasting study will be of shorter duration,
and metabolites should be collected 2 hourly from commencement, or 1 hourly if only a short
fast is anticipated. Specific additional measures may be requested by medical staff in certain
clinical circumstances.

Sample Tube Baseline 4 8 10 12 14 16 18 20 22 24

Blood vol 0 mins hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
Glucose Fluoride S Frequency varies according to age and condition. Older children
oxalate usually 4 hourly to 8 hours, then 1-2 hourly depending on
0.5 ml progress. Younger children 2 hourly initially. Babies and young
infants usually hourly and sometimes ½ hourly.
Insulin Li hep S S S S S S S S S S S
0.5 ml
β-hydroxy Perchloric S S S S S S S S S S S
butyrate acid 0.1 ml
Free fatty Li hep S S S S S S S S S S S
acids 1 ml
Lactate and Perchloric S S S S S S S S S S S
acid 0.5 ml
pyruvate *
Cortisol Li hep S Collect again at termination
0.5 ml
Growth Li hep S Collect again at termination
hormone 0.5 ml
Urine for Plain sterile S Unless otherwise specified, collected at 0 (pre-fast), then all
metabolic jar urines during fast, and first post-fast urine.
Freeze
screen *
Urine Ward test S All urines to be tested
ketodiastix
for ketones
S = Sample at this time point

* Suspected inborn errors of metabolism only

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.12 Glucagon Stimulation Test (in suspected
hypoglycemic disorders)
Indications: A test of the ability of hepatic glycogen to be mobilized. Used in suspected disorders of
hepatic glycogen metabolism.
Rationale: Glucagon stimulates hepatic glycogenolysis and hence a rise in blood glucose levels. A
normal response is dependent on glycogen stores being present, and able to be
mobilized by the appropriate enzymatic pathway.
Contraindications: Recent or intercurrent illness. Hyperglycemia.
Formulation: Glucagon - lyophilized powder for reconstitution, administered by intramuscular or
intravenous injection. Three preparations are currently available:

Glucagon (Eli Lilly) 1 unit (1 mg) plus 1 ml solvent (animal origin)

Glucagon (Novo Nordisk) 1 unit (1 mg) plus 1 ml solvent (animal origin)
Glucagen (Novo Nordisk) 1 unit (1 mg) plus 1 ml solvent (biosynthetic human)

Dose: Intravenous: 30 micrograms/kg (0.03 mg/kg) to a maximum of 1 mg

Intramuscular: 0.5 to 1 mg
Adverse reactions: Nausea, vomiting
Rebound hypoglycemia
Persisting hypoglycemia in glucagon non-responsive conditions, necessitating IV
glucose administration.
Preparation: Medical officer will have specified fasting or non-fasting. Often performed as part of a
fasting study at the time of hypoglycemia.
IV sampling cannula
Equipment: Worksheet
IV sampling cannula
Syringes 2 ml and 5 ml
Normal saline for IV flushes
IV glucose 10% available for immediate use
Tubes - Li heparin, fluoride oxylate (collection) and plain (plasma storage) - labelled with
Name, date, time, "glucagon stim"
Method: 1. Patient weighed and glucagon dose calculated
2. Time 0 samples collected as below
3. Glucagon administered - IV diluted in 5 mls normal saline over 1 min, or IM
4. Blood sampling as below
5. After the study child is fed. Blood glucose levels should be monitored until stable.
The child is not allowed home until has eaten/drunk and blood sugars are normal
and stable.

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 Sample Tube 0 min 5 min 10 min 20 min 30 min 60 min 90 min
Blood vol

Glucose Fl oxalate S S S S S S S
0.5 ml

Insulin Li hep S S S S S S S
.5 ml
S = Sample at this time point

Interpretation General principles are:

: A normal response in the non-fasted state is a significant rise in plasma glucose levels; this
also occurs in the fasted state unless the fast has been prolonged enough to deplete hepatic
glycogen stores (depends on age and body size). An absent glycemic response occurs with
disorders of hepatic glycogen metabolism. An exaggerated insulin response (usually > 600
pmol/l suggests hyperinsulinism.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.13 Glucagon Stimulation Test (for pituitary function)
Indication: As a test of GH and ACTH secretion; may be useful in infants and young children or
other situations in which insulin stimulation is contraindicated. Sensitivity is increased by
prior administration of propranolol.
Background: Glucagon stimulates release of GH and ACTH by its effects on α-receptors and
stimulating insulin release. The glucagon stimulation test has been advocated as a safer
test than insulin stimulation in young children and infants especially in the low dose
version. The sensitivity of the test may be enhanced by addition of β-blockers (false
negatives reduced by 10-15 %). In this test, glucagon is administered sc or im (with or
without pre-administration of a β-blocker), and the response of cortisol and GH in
peripheral blood is measured.
Contraindications: Recent or intercurrent illness.
For propranolol co-administration - asthma, cardiac disease
Formulation: Glucagon - lyophilized powder for reconstitution, administered by intramuscular or
subcutaneous injection. Three preparations are currently available:

Glucagon (Eli Lilly) 1 unit (1 mg) plus 1 ml solvent (animal origin)

Glucagon (Novo Nordisk) 1 unit (1 mg) plus 1 ml solvent (animal origin)
Glucagen (Novo Nordisk) 1 unit (1 mg) plus 1 ml solvent (biosynthetic human)
Glucagon dose: 15 micrograms/kg body weight by IM injection to a
maximum of 1 mg

Propranolol - tablets 10 mg, 40 mg (Inderal, ICI ; Deralin, Alphapharm)

Propranolol dose: 0.5 mg/kg to a maximum of 40 mg

Adverse reactions: Nausea, vomiting, abdominal pain, hypoglycemia

If propranolol used - hypotension, hypoglycemia, bradycardia, bronchospasm
Preparation: Nil by mouth 4-6 hours (fasting should not be longer than this in infants and young
children)
Accurate weight
IV sampling cannula
Equipment: IV sampling cannula
Syringes 2 ml and 5 ml
Normal saline for IV flushes
Tubes - Li heparin, fluoride oxalate (collection) and plain (plasma storage) - labelled
with Name, date, time, "Glucagon stim"

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 Method: Without propranolol:

1. Baseline bloods collected, blood glucose measured in ward

2. Glucagon administered by IM injection
3. Blood sampling as in table 2
4. Child fed and must have normal blood sugar prior to discharge. Observe minimum of 2
hours after test.

Table 1 Without propranolol

Tube 0 mins 60 mins 90 mins 120 mins 150 mins 180 mins
Blood vol
Glucose Fl ox S S S S S S
0.5 ml
GH Li hep S S S S S S
0.5 ml
Cortisol Li hep S S S S S S
0.5 ml
IGF-I Li hep S
1 ml
S = Sample at this time point

Method: With propranolol:

1. Baseline bloods collected, BP and blood glucose measured in ward

2. Propranolol given orally
3. Patient rests for 2 hours
4. Blood samples collected at 2 hours and glucagon administered by IM injection; child
rests during test.
5. BP monitored every 30 mins
6. Blood samples as in table 1
7. Child fed and must have normal BP and blood glucose level prior to discharge.
Observe minimum of 2 hours after test.

Table 2 With propranolol

Tube 0 mins 120 mins 180 mins 210 mins 240 mins 270 mins 300 mins
Blood vol
Glucose Fl ox S S S S S S S
0.5 ml
GH Li hep S S S S S S S
0.5 ml
Cortisol Li hep S S S S S S S
0.5 ml
IGF-I Li hep S
1 ml
S = Sample at this time point

Interpretation: General principles are:

Peak GH is usually at around 120 min. Peak GH response < 10 mU/l suggests GH
deficiency; responses of 10-20 mU/l suggest partial GH deficiency; response ≥ 20 mU/l is
regarded as normal. An exercise GH test suggesting GH deficiency should usually be

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 followed up by further pharmacological testing. Peak cortisol level should be > 600 nmol/l.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 4.14 Gonadotrophin Releasing Hormone (GnRH) Test
(LHRH test)
Indications: To assess the pituitary gonadotrophin response in disorders of puberty or
gonadal function. Often used as part of a combined pituitary function test
(triple test).

Rationale: GnRH (also called LHRH) from the hypothalamus stimulates luteinizing
hormone (LH) and follicle stimulating hormone (FSH) release from the
pituitary gland. Evaluation of this response is important in the evaluation of
disorders of puberty.

Contraindications: Pregnancy (relative contraindication)

Formulation: Gonadorelin (HRF, Ayerst)

100 micrograms (plus 2 ml diluent); 500 micrograms (plus 2 ml diluent)
A synthetic decapeptide identical to the naturally occurring hormone.

Dose: 100 micrograms by slow intravenous injection over 1 min ie. same dose all
ages, all sizes

Adverse Significant adverse reactions have not been encountered. Occasionally

reactions: nausea and abdominal pain.

Preparation: Nil
Any time of day.

Equipment: Worksheet
IV cannula
Syringes 2 ml and 5 mls
Normal saline for cannula flushes
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with
name, date, time and "GnRH stim".

Method: 1. IV cannula inserted and baseline samples collected.

2. GnRH administered by slow intravenous injection over 1 min
3. Blood sampling as below. If performed as part of a combined pituitary
test, see combined protocol

Tube
Sample 0 min 15 30 45 60 90 120
Blood volume
min min min min min min
Li hep
LH S S S S S S S
1 ml
Li hep
FSH S S S S S S S
0.5 ml
Testosterone Li hep
S - - - - - -
(males) 1 ml
Oestradiol Li hep
S - - - - - -
(females) 1 ml
S = Sample at this time point

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
Interpretation: General principles are:
LH peak usually occurs 15-45 min after injection and FSH peak later at 45-90 min.
Prepubertal children show a small increase in LH and FSH usually between 2-4 mU/l. An
absent response however is not diagnostic, and can occur in normal prepuberty and pubertal
delay. As puberty progresses, responses become more pronounced. In precocious puberty
responses are exaggerated for age, often with elevated basal levels. In primary
hypogonadism baseline levels are elevated and responses are exaggerated.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.15 hCG Stimulation Test

Indications: A test to determine the Leydig cell responsiveness of the testes.

Rationale: hCG induces an increase in testosterone biosynthesis and secretion by Leydig cells
which can be measured within several days of administration. It is most commonly used
in suspected primary hypogonadism or identifying the presence or absence of testicular
tissue in cryptorchidism. While hCG stimulates ovarian oestrogen and progesterone
secretion, it is not employed as a diagnostic test in females.

Contraindications: Known or suspected androgen sensitive tumours (usually mammary carcinoma or

prostatic carcinoma in the male)

Formulation: human chorionic gonadotrophin - lyophilized powder for reconstitution, administered by

intramuscular injection. Obtained from the urine of pregnant women. Three preparations
are currently available:
Pregnyl (Organon) - 500 IU, 1500 IU, 5000 IU. Each with 1 ml diluent.
APL injection (Ayerst) - 5000 IU with 10 ml diluent.
Profasi (Serono) - 500 IU, 100 IU, 2000 IU, 5000 IU. Each with 1 ml diluent.

Dose: Over 2 yrs: Single intramuscular injection of 5000 IU

Under 2 yrs: Single intramuscular injection of 1500 IU

Note that many other dosage protocols exist, usually employing multiple hCG injections,
but a single dose test has been found to give good results in our hands.

Adverse reactions: Skin rashes, local reaction (both rare)

Other side effects related to prolonged and high dose administration only

Preparation: Nil

Equipment: Needle or scalp vein and syringe for blood sampling

Lignocaine 1%
Needle and syringe for IM administration of hCG
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with name, date,
time and "pre hCG" or "post hCG" as appropriate.

Method: 1. Collect pre-hCG samples

2. Administer hCG mixed with lignocaine 1%, by intramuscular injection
Lignocaine 1% dose: 0.5 ml if weight ≤ 5 kg
1 ml if weight > 5 kg
3. When performed in association with GnRH stimulation test - collect pre-hCG
samples prior to GnRH test and administer hCG injection after GnRH test
4. Arrangements made for patient to have post-hCG sample collected 72-96 hours
after injection (return to ward or local collection and transfer of specimens to RWI)

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 Sample Tube pre-hCG post-hCG
Blood volume 0 hours 72-96 hrs
Testosterone Li hep 2 ml S S
Dihydrotestosterone
DHAS Li hep 1.5 ml S -
Androstenedione
LH Li hep 1 ml S
S *
FSH Li hep 0.5 ml S -
S = Sample at this time point

* hCG cross-reacts with the LH antibodies in the RIA, and hence an elevated post-hCG LH
levels can be used to confirm administration and absorption of hCG if desired.

Interpretation: General principles are:

Normal testosterone response if stimulated testosterone level more than 3-fold baseline, or if
baseline level < 1 nmol/l then peak > 4 nmol/l. A lesser response suggests Leydig cell
failure or absence.
Normal T/DHT ratio 10 (mean), 2-20 (range). Poor DHT response and elevated post-hCG
T/DHT ratio suggests 5-α reductase deficiency.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 4.16 IGF-I Generation Test
Indications: A test to evaluate the response of growth hormone-dependent growth factors (IGF-I and
IGFBP-3) to growth hormone administration. Mainly used in the evaluation of growth
hormone insensitivity syndromes.
Rationale: Under normal circumstances, GH administration over several days is associated with
significant rises in serum IGF-I and IGFBP-3. In conditions with growth hormone
insensitivity, these responses are absent or attenuated, depending on the severity of the
defect.
Contraindications: Intercurrent illness
Formulation: Recombinant human growth hormone
(Saizen - Serono, Humatrope - Eli Lilly, Genotropin - Pharmacia, Norditropin - Novo
Nordisk)

Dose: Recombinant human growth hormone 0.1 IU/kg body weight daily by subcutaneous
injection for 4 consecutive days

Adverse Rare, usually trivial. Rarely, oedema may occur with initiation of GH therapy due to
reactions: sodium and water retention.
Preparation: Nil by mouth from midnight on Days 1 and 5 until blood samples collected. May be
performed following a GH provocation test, but not if sex steroid primed.
Equipment: Tubes - Li heparin and plain - labelled with name, date, time and "IGF gen".
Method: 1. On day 1, morning baseline blood samples collected after nil by mouth overnight
2. GH administration begins that evening before bed (usually administered at home by
the family), and continues for 4 days.
3. On day 5, final blood samples collected in the morning after nil by mouth from
midnight.

Sample Tube Day 1 Day 5

Blood volume
Insulin-like growth factor-I Li hep S S
(IGF-I) 1 ml
Insulin-like growth factor I Plain S S
binding protein-3 (IGFBP-3) 5 ml
Growth hormone binding Plain S _
protein (GHBP) 5 ml (if assay available)
S = Sample at this time point

Interpretation General principles are as follows:

:
In subjects without resistance to GH action, the following is usually observed:
IGF-I rise of greater than 15 ng/ml (2.0 nmol/l)
IGFBP-3 rise of greater than 0.4 mg/l
Overlap may occur between GH deficient and GH resistant patients
Reference: Blum et al. Improvement of diagnostic criteria in growth hormone insensitivity
syndrome: pitfalls and solutions. Acta Paediatr Suppl 399: 117-24, 1994.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.17 Insulin Stimulation Test (Insulin Tolerance Test)

Indications: A test of growth hormone secretion and of response of the hypothalamic-pituitary-

adrenal axis. Usually used as part of a combined pituitary function test (triple test).

Rationale: Insulin-induced hypoglycemia induces GH secretion through effects on central α-

adrenergic pathways. The test is also the most rigorous available for assessing
adrenal cortisol response, since it tests the integrity of the entire HPA axis. This
test is potentially dangerous and must only be performed by experienced personnel
and closely supervised.

Contraindications: History of convulsions. Hypoglycemic disorder. Infants and young children - in

general is not performed except in particular circumstances.

Formulation: Soluble human insulin (Actrapid, Novo Nordisk or Humulin R , Lilly) 100 Units/ml.

Dose: The dose is chosen according to the suspected diagnosis, as some patients will
show greater sensitivity:
Hypopituitarism strongly suspected 0.025 to 0.075 unit/kg
Steroid treated patients 0.025 to 0.075 unit/kg
Standard dose ("Normal" patients) 0.1 unit/kg
Acromegaly / gigantism 0.15 unit/kg
Dose diluted in 5 mls normal saline and given by slow intravenous injection over 1
min.

Adverse reactions: Symptoms of hypoglycemia are expected - pallor, sweating, hunger, headache,
tiredness.
Hypoglycemic seizures.
Deaths have occurred, some associated with inappropriate (excessive) glucose
resuscitation.

Preparation: Patient fasted at least 6 hours. Remain nil by mouth until after hypoglycemia.
Equipment: Worksheet
IV cannula
Syringes 2 ml and 5 ml
Normal saline for cannula flushes
Tubes - Li heparin and fluoride oxalate (collection) and plain (plasma storage) -
labelled with name, date, time and "Insulin stim".
Dextrose for IV administration must be available drawn up for immediate use - 10%
dextrose, 2 ml/kg.

Method: 1. Patient weighed and dose calculated

2. IV cannula inserted and baseline samples collected. Must be a reliable IV line.
3. Insulin administered by intravenous injection over 1 min
4. Blood sampling as in table, plus ward glucose testing as in 5. If performed as
part of a combined pituitary test, see combined protocol.
5. After insulin administered medical officer must not leave the ward until patient
recovered from hypoglycemia. Patient closely observed for symptoms of
hypoglycemia which usually occur after 15 to 30 minutes. The aim is to achieve
a plasma glucose fall to 2.6 mmol/l or less, or symptomatic hypoglycemia with
a fall of blood glucose to ≤ 50% of baseline. Blood glucose measured in ward at
10, 15, 20, 25, 30, 45, 60, 75, 90 and 120 minutes after insulin administration,
or at any other time if in doubt.
6. When plasma glucose level < 2.6 mmol/l is recorded or symptomatic
hypoglycemia occurs, the test proceeds in one of two ways, but sampling
continues:

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 a. Mild to moderate symptoms - give sweet drink, followed by food.

b. Severe hypoglycemia - intravenous dextrose - 2 ml/kg 10% dextrose,

followed by continuing infusion of 10% dextrose if slow recovery, or sweet
drinks and food. If poor response consider hydrocortisone 50-100 mg IVI.
7. The child is not allowed home until a glucose containing drink and a meal has
been eaten and tolerated, and all observations are satisfactory.

Sample Tube 0 10 20 30 45 60 75 90 120

Blood vol mins mins mins mins mins mins mins mins mins
Plasma Fl oxalate S S S S S S S S S
glucose 0.5 ml
GH Li hep S S S S S S S S S
0.5 ml
Cortisol Li hep S - S S - S - S S
0.5 ml
IGF-1 Li hep S - - - - - - - -
1 ml
S = Sample at this time point

Interpretation: General principles are:

Peak GH response < 10 mU/l suggests GH deficiency; responses of 10-20 mU/l suggest
partial GH deficiency; response ≤ 20 mU/l is regarded as normal.
Cortisol rise in response to hypoglycemia should be > 280 nmol/l, with a peak > 600 nmol/l.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.18 Intravenous Glucose Tolerance Test

Indication: Study of the dynamics of insulin secretion, usually in suspected pre-diabetes or

glucose intolerance. A test of longer duration may be used to study insulin sensitivity
(see below).
Background: This test provides a detailed assessment of the dynamics of insulin secretion and
glucose disappearance. A known bolus of glucose is injected and samples collected at
specified intervals for measurement of plasma glucose and insulin. A number of indices
can be calculated from the data, but most commonly first phase insulin release is
employed. The test described here is the consensus reached by the ICARUS (Islet cell
Antibody Register User's Study) working group (reference: Bingley et al, Diabetes
Care, 1992, 15:1313-16). A more prolonged test with sampling to 40 or 60 minutes can
be used to measure insulin sensitivity, but this is not a routine clinical test.
Contraindications: Hyperglycemia, overt diabetes
Formulation: 50 % Glucose ; ampoules
Dose: 0.5 g/kg body weight to a maximum of 35g. Administered as a 25 % solution by dilution
with equal volume of sterile water
Adverse reactions: A flushing sensation may occur during glucose injection.
Adverse reactions are not expected
Preparation: Unrestricted diet (containing at least 150g carbohydrate per day) for at least 3 days
prior. Normal physical activity, no significant intercurrent illness. Test performed in the
morning after an overnight fast of 10 - 16 hours.
Accurate weight
IV sampling cannula
Equipment: IV sampling cannula; cannulae in both arms preferable if tolerated by subject
Stop watch or other accurate timer
Syringes 2 ml and 5 ml
Normal saline for IV flushes
Tubes - Li heparin, fluoride oxalate (collection) and plain (plasma storage) - labelled with
Name, date, time, "IVGTT"
Method: 1. IV cannula(e) inserted and patient rests 30 mins
2. Dose calculated and glucose diluted to 25 % with sterile water
3. Pre-glucose samples collected as below
4. Glucose injected over 3 minutes ± 15 seconds, using timer (calculate amount to
be injected every 30 seconds as a guide for injection rate). Time 0 is immediately
after the glucose injection. Then flush cannula with 20 ml normal saline over 15-20
seconds (important to flush all glucose through before sampling begins especially
if using one cannula), and prepare for sampling at 1 minute by commencing to
withdraw void volume 10 seconds before the 1 minute mark.
5. IV glucose to be injected by medical officer or accredited nurse.
6. Child recumbent and resting during the test. Water is permitted. No smoking.
7. Collect samples at accurately timed intervals as below. Note that samples are
closely spaced and timing is critical
8. Child fed before discharge.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 Sample Tube -15 mins -5 mins 0 mins = 1 min 3 min 5 min 10
Blood vol Immediately after min
glucose injection.
Mark time and begin
saline flush

Glucose Fluoride S S No sample S S S S

oxalate
0.5 ml
Insulin Li hep S S No sample S S S S
0.5 ml
S = Sample at this time point

Interpretation: General principles are as follows:

The sum of the insulin levels at 1 and 3 minutes is termed the first-phase insulin secretion .
The first percentile for first phase insulin secretion is 48 mU/l (360 pmol/l) (reference Vardi et
al, Diabetologia, 1991,34:93-102) and values below this are very likely to be abnormal, but
must be considered in association with other available clinical and laboratory information (eg.
family history, ICA titre). Since there is a coefficient of variation of 36 % within subjects,
changes within the normal range (> 5th percentile) cannot be interpreted as pathological.

More detail on percentiles in normal subjects (Vardi et al):

1st percentile 48 mU/l (360 pmol/l)
3rd percentile 56 mU/l (420 pmol/l)
5th percentile 64 mu/l (480 pmol/l)
10th percentile 81 mU/l (608 pmol/l)
50th percentile 162 mU/l (1215 pmol/l)

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.19 Oral Glucose Tolerance Test

Indications: Suspected impaired glucose tolerance or diabetes mellitus, not otherwise diagnosable by
clearly elevated fasting or random blood glucose levels. Frequently employed in
suspected gestational diabetes mellitus.
Less frequently employed to assist in the diagnosis of acromegaly.

Background: A known oral glucose load is administered, allowing the ability of the pancreas to
appropriately secrete insulin in response to be determined. A 3-hour test is usually
performed. A 5-hour oral GTT has been used in investigation of suspected post-absorptive
hypoglycemia, but is rarely employed now.
In gigantism or acromegaly, the usual suppression of GH in response to a glucose load
is lost or impaired.

Contraindications: Hyperglycemia, overt diabetes

Formulation: LucozadeT M (= 5.18g glucose per 100 ml), or

Other oral glucose preparations (eg. Glycosol liquid - 25g per 100 ml)

Dose: Glucose 1.75 g/kg BW, to a maximum of 75g, drunk within 5 minutes

Lucozade TM dose in mls = (grams of glucose required / 19.3) x 100

eg. 25 kg child ; glucose dose = 1.75 x 25 = 43.75g
Lucozade dose = (43.75 / 19.3) x 100 = 227 mls

Adverse reactions: Nil

Preparation: Unrestricted diet rich in carbohydrates for at least 3 days before test. Normal physical
activity, no intercurrent illness. test performed in the morning after 10-16 hours fast.
Accurate weight.
Sampling cannula.

Equipment: IV sampling cannula

Syringes 2 ml and 5 ml
Normal saline for IV flushes
Tubes - Li heparin, fluoride oxalate (collection) and plain (plasma storage) - labelled with
Name, date, time, "OGTT"

Method: 1. IV cannula inserted

2. Dose calculated and glucose solution measured out
3. Pre-glucose samples collected as below
4. Glucose drink taken over no more than 5 minutes
5. Child recumbent and resting during the test. Water is permitted. No smoking.
6. Samples collected at timed intervals as below for 3 hours.
7. Child fed before discharge.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 Sample Tube 0 min 30 min 60 min 90 min 120 min 150 min 180 min
Blood vol

Glucose Fluoride S S S S S S S
oxalate
0.5 ml
Insulin Li hep S S S S S S S
0.5 ml
Li hep
GH * 0.5 ml
S S S S S S S

S = Sample at this time point

* Only if specifically requested in investigation of suspected GH hypersecretion

Interpretation: Results are interpreted according to the WHO criteria. The following values are for glucose
levels performed on venous plasma. Values differ if venous whole blood or capillary blood is
collected.

Fasting (time 0) plasma 2 hour plasma glucose level

glucose (mmol/l) (mmol/l)

Normal < 7.2 < 7.8

and no other sample
exceeds 11.1

Impaired glucose tolerance < 7.8 7.8 - 11.1

Diabetes mellitus ≥ 7.8 ≥ 11.1

and one other sample exceeds
11.1

There are no well-defined criteria for interpreting insulin values in response to oral GTT, and there
is great variation between normal subjects. Fasting insulin will be suppressed (usually less than
15 pmol/l), with a rise usually to levels of > 250 pmol/l at the time of peak glucose levels.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.20 Parathyroid Hormone Infusion Test (Ellsworth-
Howard test)

Indications: A test to assist in the evaluation of hypoparathyroidism, especially in suspected

pseudohypoparathyroidism. Improved PTH assays have greatly reduced the need for
this test.

Rationale: Under normal circumstances, PTH infusion elicits an increase in renal phosphate
excretion and increased serum and urine cAMP concentrations reflecting receptor
activation via the G-protein system. These responses are modified in defects of the
receptor or second messenger systems.

Contraindications: Intercurrent illness

Formulation: 1-38 human PTH (synthetic)

(bovine PTH (Eli Lilly) - largely replaced by use of synthetic fragments)

Dose: 1-38 hPTH

0.5 µg/kg BW diluted in 10-20 ml normal saline. Administered by IV injection over 2
minutes.

Adverse reactions: As with administration of any intravenous agent, allergic reaction is possible

Preparation: Patient fasted for 8 hours (4 hours or less in neonates or young infants). May drink
water as desired. Test performed in the morning.

Equipment: Worksheet
IV cannula
Syringes 2 ml and 5 ml
Normal saline for cannula flushes
Tubes - Li heparin and EDTA (collection) and plain (plasma storage) - labelled with
name, date, time and "PTH infusion".

Method: 1. Patient weighed and dose calculated

2. IV cannula inserted
3. Bladder emptied (discarded). Encourage oral intake (100-150 mls hourly if
possible).
4. After 2 hrs, baseline urine sample collected (voluntary voiding) and baseline blood
collected as below.
5. PTH administered - IV over 2 mins in 10-20 ml normal saline
6. Blood and urine samples collected as below

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 Sample Tube 0 5 10 30 60
Blood volume mins mins mins mins mins
Serum calcium, Li hep S - - - S
phosphate, creatinine 1 ml
Plasma PTH Plain S - - S S
3 ml
Serum cAMP EDTA S S S S S
1 ml
Urine - for cAMP, Plain S S S
phosphate and creatinine
S = Sample at this time point

Interpretation: General principles are as follows:

In normal and hypoparathyroid subjects, PTH induces a rise in plasma cAMP from a basal
level of around 15 nmol/l to a peak over 100 nmoll/l, and a rise in urine cAMP from a basal
level of around 2.5 nmol/dl GF to at least 60. Tubular reabsorption of phosphate * (TRP)
decreases.
In pseudohypoparathyroidism, the rise in plasma cAMP is markedly impaired (maximal 40
nmol/l), as is the rise in urine cAMP (maximal 10.7 nmol/dl GF). TRP decrease is less
marked, but overlaps with normal and HP subjects.
* TRP(%) = 100 (1 - urine phosphate x serum creat )
serum phosphate urine creat

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.21 Pentagastrin Stimulation Test

Indications: Evaluation of suspected medullary thyroid carcinoma, or in screening families with

Multiple endocrine neoplasia.
Rationale: Calcitonin is a polypeptide produced by the parafollicular cells of the thyroid. Calcitonin
levels are increased in medullary thyroid carcinoma, however some patients may have
normal basal levels with an abnormality revealed on provocative testing with
pentagastrin. In this test, pentagastrin is administered IV, and the response of
calcitonin in peripheral blood measured.
Contraindications: Previous severe idiosyncratic response to Pentagastrin
Pregnancy
Caution in known peptic ulcer disease
Formulation: Pentagastrin - a synthetic pentapeptide containing the carboxyl terminal tetrapeptide
responsible for the actions of the natural gastrins (Peptavlon, ICI Pharmaceutical 250
micrograms/ml)

Dose: 0.5 micrograms/kg BW diluted in 20 ml normal saline. Administered by IV injection over

30-60 seconds, and flushed through with 10 mls normal saline.

Adverse reactions: As with administration of any intravenous agent, allergic reaction is possible
Adverse reactions rare, but may include nausea, vomiting, hypotension, dizziness,
abdominal discomfort, headaches. palpitations
Preparation: Fasting not necessary, but avoid full stomach because of possible nausea and
vomiting. Hence, 2 hours fasting is recommended.
Equipment: Worksheet
Stopwatch or accurate clock
IV cannula
Syringes 2 ml and 5 ml
Normal saline for cannula flushes
Tubes - Li heparin and EDTA (collection) and plain (plasma storage) - labelled with
name, date, time and "PTH infusion".

Method: 1. Patient weighed and dose calculated

2. IV cannula inserted
3. Pentagastrin administered by IV injection over 30-60 seconds and flushed through.
4. Timed samples collected as below

Tube 0 min 1 min 2 min 3 min 5 min 10 min 15 min 30 min

Blood volume

Calcitonin Plain S S S S S S S S
2 ml, collect
on ice

Calcium Li hep S S
1 ml
S = Sample at this time point

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
Interpretation: General principles are as follows:
An elevated basal or rise of calcitonin to > 200 pg/ml is abnormal (depending on the
particular assay used).

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.22 Sex Steroid Priming In Growth Hormone
Stimulation Tests

Rationale: Sex steroid priming prior to GH stimulation is recommended in some centres,

especially in subjects with delayed puberty, although is uncommonly used at our
Institute. The rationale for priming is that a proportion of prepubertal children with
slow growth (especially those with delayed puberty) may have subnormal GH levels
during stimulation, but when they are re-tested later or after onset of puberty, normal
levels are found. This may avoid some false diagnoses of GH deficiency and
decrease the number of additional tests required, but does not really aid in
determining which subjects are GH-insufficient.

If used, should generally be reserved for patents with delayed puberty.

Contraindications: Age < 10 years (relative contraindication)

Precocious puberty

Formulations: Males:
Testosterone depot preparation (Sustanon, Primoteston depot) 100 mg IMI given 2-8
days prior to test.

Testosterone undecanoate (Andriol) 80 mg daily for 5 days prior to test

Females:
Ethinyloestradiol (Estigyn) 50 - 100 µg daily for 3 days prior to test

Adverse reactions: Significant side-effects are not anticipated, except possible minor manifestations of
temporarily increased sex steroid levels.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.23 Short ACTH (Synacthen) Stimulation Test

Indications: To assess the response of the adrenal cortex to stimulation in suspected

adrenocortical insufficiency (primary, secondary or tertiary) or in the diagnosis of
congenital adrenal hyperplasia.
Rationale: ACTH is the primary regulator of glucocorticoid production, and also plays some role in
adrenal androgen production. Tetracosactrin (Synacthen), a synthetic form of ACTH, is
used to assess the stimulated cortisol response of the adrenal cortex and is valuable in
diagnosing suspected primary adrenal insufficiency. The test is also useful in
suspected secondary or tertiary adrenal insufficiency since chronic CRH/ACTH
deficiency or dysregulation results in temporary quiescence of the adrenal cortex and
inability to respond acutely. The test is not reliable in assessing secondary or tertiary
insufficiency within 2 weeks of surgery to the hypothalamic-pituitary region or a major
alteration in any glucocorticoid therapy. In congenital adrenal hyperplasia, the
synacthen test is useful in diagnosing milder or rare enzyme blocks by examining
ratios of various adrenal steroids to their precursor compounds. The commonest ratio
examined is that of 17-hydroxyprogesterone / cortisol in suspected non-classical or
simple virilizing CAH or the heterozygote state.
Contraindications: Known hypersensitivity to ACTH. Pregnancy. Other listed contraindications apply to
ongoing treatment with Synacthen only.
Formulation: Tetracosactrin (Synacthen, Ciba-Geigy) 250 micrograms in 1 ml
A synthetic polypeptide consisting of the first 24 amino acids of the ACTH molecule

Dose: Standard dose synacthen test:

Over 1 yr: Single IM or IV injection of 250 micrograms
Under 1 yr: Single IM or IV dose of 125 micrograms
Alternatively a dose of 250 micrograms/m2 BSA may be used
These are all supramaximal stimulus doses.

Low dose synacthen test:

Use if specified by consultant. Reportedly more sensitive in detecting adrenal
suppression from exogenous steroids - dose 0.5 µg/1.73 m2 BSA. Take 1 ml of 250
µg/ml tetracosactrin and dilute under sterile conditions with 49 ml of normal saline to
make a concentration of 5 µg/ml. Take 1 ml of 5 µg/ml solution and 19 mls normal
saline to make a 0.25 µg/ml solution. Do not store solution for later use.
To calculate dose in mls of 0.25 µg/ml solution = (Patient BSA/1.73) x 2

Adverse reactions: Hypersensitivity or anaphylactic reaction - rare, but full resuscitation facilities and drugs
must be available.
Preparation: Nil
Any time of day.
Equipment: Worksheet
IV cannula
Syringes 2 ml and 5 ml
Normal saline for cannula flushes
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with name, date,
time and "Syn stim".
Method: 1 Medical officer to order dose and indicate clearly on request form which adrenal
steroids to be measured
2. IV cannula inserted
3. Synacthen administered IM by nursing staff, or IV by medical staff
4. Blood sampling as below

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 Sample Tube 0 min 30 min 60 min
Blood volume

Cortisol Li hep S S S
0.5 ml

17-OHProgesterone Li hep
0.5 ml
S * S * S *
Other adrenal steroids See reference
list
S * S * S *
S = Sample at this time point

* if requested for investigation of virilizing disorders

Interpretation: General principles are:

Standard dose test:
The following are normal responses -
Serum cortisol rise of > 280 nmol/l with maximal level > 600 nmol/l.
Normal ratio of 17-OHP to cortisol at 30 mins < 0.023. Ratios up to 0.08 suggest
heterozygosity for 21-hydroxylase deficiency and ratios > 0.1 suggest CAH (21-
hydroxylase deficiency).
Detailed references are available for other ratios.

Low dose test:

The following are normal responses:

Serum cortisol rise of > 200 nmol/l with maximal level > 500 nmol/l

Reference: Broide et al J Clin Endocrinol Metab 80: 1243-1246, 1995

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.24 Spontaneous Growth Hormone Secretion
(Overnight or 24 hr GH sampling)
Indication: To evaluate spontaneous GH secretory patterns in short stature (suspected
neurosecretory dysfunction or GH insensitivity) or in tall stature (suspected GH excess).
Uncommonly used now.

Rationale: Growth hormone is secreted in a pulsatile pattern, with the majority of pulses at night;
hence sleep is a physiological stimulus to GH secretion. Significant relationships have
been described between height and height velocity and spontaneous GH secretion,
although considerable inter- and intra- individual variation exists. The study of
spontaneous GH secretion has largely been a research tool. The test still has some
useful application in evaluation of suspected GH excess (pituitary gigantism /
acromegaly), in suspected GH neurosecretory dysfunction (a concept described by some
authors in which GH may respond normally to pharmacological stimuli, but is not
secreted normally under physiological conditions) or GH insensitivity syndromes.

In this test, samples for GH are collected every 20 mins for either 12 hours (overnight) or
24 hours, either manually or using timed-withdrawal apparatus. Secretion patterns can
then be analyzed mathematically if desired. Some units also perform EEG tracings to
compare secretion to sleep stages.

Contraindications: Intercurrent illness.

Adverse reactions: Nil. Secretion patterns may be affected if patient sleeps poorly in hospital. In smaller
patients care must be taken to avoid excessive volume depletion from blood sampling
(see introduction).

Preparation: Patient admitted to hospital - late afternoon if 12 hour overnight sampling, or early
morning if 24 hours sampling.
No fasting, usual oral intake
IV sampling cannula

Equipment: Worksheet
IV sampling cannula
Syringes 2 ml and 5 ml
Normal saline for IV flushes
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with Name, date,
time, "12 hr GH" or "24 hr GH"

Method: 1. Overnight sampling every 20 mins 8 pm to 8 am; 24 hour sampling every 20 min
from 8 am to 8 am.
2. Normal oral intake.
3. When each sample is withdrawn the state of sleep or wakefulness is recorded on
worksheet.

Sample Tube 0 mins Samples collected every 20 mins for 12 or 24 hours

Blood vol
GH Li hep S S
0.3 ml
IGF-1 Li hep S -
1 ml

Interpretation: Under normal circumstances, GH levels during waking hours are low. During sleep there are
usually several pulses of GH ≥ 20 mU/l, usually associated with slow wave sleep. Detailed
mathematical analysis is undertaken for research purposes.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.25 Thyrotropin Releasing Hormone (TRH) Test

Indications: 1. To assess the response of pituitary TSH secretion and thyroid hormone production
to stimulation. The main indication is in suspected secondary (pituitary) or tertiary
(hypothalamic) hypothyroidism. Less frequently may be of assistance in mild
primary hypothyroidism.
2. To assess the response of prolactin to stimulation
3. Less commonly used in investigation of gigantism/acromegaly
Often performed as part of a combined pituitary function test (triple test).
Rationale: Thyroid hormone production is under the control of pituitary TSH and in turn
hypothalamic TRH. Plasma TSH levels normally increase rapidly (2 to 5 minutes) after
an IV bolus of TRH, with a subsequent more gradual increase in T3 secretion from the
thyroid. The TRH test thus allows the integrity of the thyroid axis to be tested.
Abnormalities of the prolactin response to TRH may occur in pituitary tumours
(especially GH or prolactin producing) or in pituitary stalk pathology. Responses are not
diagnostically pathognomonic, but indicate axis disruption or dysregulation.
In GH excess states (gigantism/acromegaly), TRH may cause an elevation of GH, but
not in normal subjects.
Contraindications: Uncontrolled heart failure, severe myocardial ischaemia or asthma. Caution in lesser
degrees of these conditions.
Formulation: Thyrotropin-releasing hormone (TRH, Roche) 200 micrograms in 2 ml (ampoules).
A synthetic tripeptide identical to the naturally occurring hormone. An oral preparation
is also available but infrequently used and requires a different sampling protocol.
Dose: 200 micrograms/m2 BSA by slow intravenous injection over 1 min.
Adverse reactions: Nausea, flushing, dizziness, urinary urgency, unusual taste in mouth, occasionally
headaches.
Increases in BP and pulse rate frequently observed
Caution in heart failure, myocardial ischaemia and asthma.
Caution in severe hypopituitarism - risk of hypoglycemia
Certain drugs may diminish response.
Preparation: Nil
Any time of day.
Equipment: Worksheet
IV cannula
Syringes 2 ml and 5 ml
Normal saline for cannula flushes
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with name, date,
time and "TRH stim".

Method: 1. Calculate BSA and dose. Medical officer to indicate if prolactin levels required.
2. IV cannula inserted
3. TRH administered by intravenous injection over 1 min
4. Blood sampling as below. If performed as part of a combined pituitary test, see
combined protocol

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 Sample Tube 0 15 30 45 60 90 120
Blood min min min min min min min
volume

TSH Li hep S S S S S S S
1 ml

Free T3 Li hep S - - - - - S
1 ml

Free T4 Li hep S - - - - - -
1 ml

Prolactin Li hep 1 S S S S S S S
(if ordered) ml
S = Sample at this time point

Interpretation General principles are:

:
A normal response is a rapid rise in TSH, peaking between 10-30 mU/l at 20-30 minutes,
then gradually declining to reach baseline after 2 to 3 hrs. T3 values show a rise, but do not
peak until 3-4 hrs (30-70% rise from baseline). Prolactin levels are age-dependent - above 1
year age, mean basal levels are approximately 240 mU/l, rising to approximately 725 mU/l
with TRH stimulation (ie. a 2-3 fold rise).
In hyperthyroidism (Grave's disease), T3 and T4 are elevated and TSH levels are suppressed
and unresponsive to TRH stimulation. In secondary (pituitary) hypothyroidism T3 and T4
levels are likely to be low and there is a poor TSH response and poor T3 response. In tertiary
(hypothalamic) hypothyroidism, an exaggerated and prolonged TSH peak may be seen, and
a T3 response occurs. In primary hypothyroidism basal T3 and T4 are low, with elevated
basal TSH and an exaggerated TSH response (usually to a peak > 30 mU/l at 30-40 mins).
While prolactin responses are variable, an exaggerated prolactin response suggests
hypothalamic disease or stalk disruption, owing to loss of inhibitory effects of dopamine. A
poor prolactin response suggests pituitary disease.
In normal subjects TRH induces no rise in GH levels, but it may do so in pituitary gigantism /
acromegaly.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
4.26 Water Deprivation Test

Indications: Investigation of suspected diabetes insipidus

Rationale: Under normal circumstances, water deprivation is associated with declining urine
volumes, increasing urine osmolality and maintenance of normal serum osmolality.
Such effects are mediated by increased ADH (vasopressin) secretion by the posterior
pituitary and its action on the collecting ducts of the kidney. Water deprivation is most
commonly used in patients presenting with polyuria and polydipsia to assist in
distinguishing central diabetes insipidus, nephrogenic diabetes insipidus and
psychogenic (habitual) water drinking.
Contraindications: Existing dehydration or electrolyte abnormality; intercurrent illness.
Formulation: Toward the end of the test, desmopressin may be administered intranasally or
subcutaneously (see below)
Desmopressin (Minirin intranasal) 100 micrograms per ml
Desmopressin injection 4 micrograms per ml (1 ml ampoules)
Adverse reactions: Excessive water deprivation may cause significant dehydration and electrolyte
disturbance, especially hypernatremia.
Preparation: Biochemistry laboratory notified several days in advance to ensure adequate staffing for
multiple samples requiring rapid analysis.
Food and fluids as desired up until start of test. Commencement time to be planned
carefully, taking into account age and degree of polyuria (see below)
Accurate weight (note clothing and other items included eg. armboard), and calculation
of 5% dehydrated weight (an indication to cease test)
Equipment: Worksheet
IV cannula
Syringes 2 ml and 5 ml
Normal saline for cannula flushes
Tubes - Li heparin (collection) and plain (plasma storage) - labelled with name, date,
time and "Water dep".
Method: 1. An individual decision is made by the consultant on commencement time, taking
into account age and anticipated rate of dehydration based on the clinical history.
Where rapid dehydration may be anticipated or in young children, the test is
commenced at 8 am. Where less rapid dehydration is expected (eg. child normally
sleeps through the night without drinking) the test may commence in the evening
or sometime during the night.
2. IV cannula inserted
3. Baseline weight, urine sample and blood sample collected (see below).
4. No food or drink is allowed during the test, and the child must be observed to
ensure that surreptitious water intake is prevented.
5. Samples collected and patient weighed at intervals as indicated. Thirst sensation
and behaviour during the test should be recorded.
The
6. duration of water deprivation is seldom longer than 12-16 hours in children and 6-8
hours in young infants. In any case, the test is terminated if there is either:

≥5% dehydration (5% weight loss), or

Serum osmolality > 300 mosmol/l
7. Children with normal responses to water deprivation (see below) are given drink at
the end of the study, and are allowed home after fluid and food has been taken.
8. In those with inadequate urinary concentration, desmopressin is administered:
Children < 1 year: 10 µg desmopressin intranasally
Children > 1 year: 20 µg desmopressin intranasally

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 As an alternative if specified by the consultant, desmopressin may be
administered by injection: dose 0.5 µg/m2 BSA
The response to DDAVP is monitored over the next 1-2 hours, with collection of
urine samples when passed and simultaneous blood samples. The consultant will
indicate when to cease the test and monitoring.

Sample Tube Baseline Every 2 hours Every 1 hour as After

Blood 0 hours from time weight loss desmopressin
volume specified by approaches 5% administration
medical
officer

Urine specific gravity S S S S

(refractometer)

Urine osmolality Plain 1 ml S S S S

Plasma Na, K, Cl, Li hep 1 ml S S S S

HCO3, urea,
creatinine

Plasma osmolality Li hep 1 ml S S S S

Plasma antidiuretic EDTA 2ml S In addition to baseline, collect ADH at end of test
hormone (ADH) (on ice) and (before DDAVP) and one other time-point beyond
plain 1ml (on the estimated halfway point of test
ice), spin
and freeze
immediately
S = Sample at this time point

Interpretation General principles are:

:
In subjects without DI, urine volume drops and urine osmolality increases usually to at least
twice to three times plasma osmolality (age-dependent). Serum osmolality does not rise
significantly. Plasma ADH levels rise.
In subjects with ADH deficiency (central DI), urine losses continue and dehydration ensues,
plasma osmolality increases, with no or little rise in urine osmolality. In partial DI, urine
osmolality may rise to a peak of 300 - 600 mosm/l. ADH rise may be poor, but this is not
diagnostic. Administration of DDAVP leads to urinary concentration.

In nephrogenic DI, findings are similar to ADH deficiency, except there is no or poor response
to DDAVP administration, and ADH levels are usually clearly elevated.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
5. Appendices

TESTING WORKSHEET
Name MRN

DOB
DATE OF TEST:

TEST: _____________________________________________________

HEIGHT: ___________________ WEIGHT: __________________ SURFACE AREA: ______________

TEST ACTUAL LABORATORY BLOOD BP TEST URINE GENERAL

TIME TI M E LABEL GLUCOSE FOR OBSERVATIONS
KETONES

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Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
ARGININE / INSULIN / LHRH / TRH STIMULATION
TEST WORKSHEET Name MRN
DOB
DATE OF TEST:

TEST: _____________________________________________________

HEIGHT: ___________________ WEIGHT: __________________ SURFACE AREA: ______________

Test
Arginine/insulin
Time/ Clock time LHRH/ Blood Notes General
BGL Lab
Insulin GnRH (mls) Observations
Labels
time
-15 0 5.0 Baseline
samples
0 2.0 Give
Arginine
30 2.0 Give TRH
& GnRH

45 15 5.0

60 30 5.0

75/0 45 5.0 Give

insulin

85/10 55 5.0

90/15 BGL if
indicated

95/20 2.0

100/25 BGL if
indicated

105/30 2.0

120/45 90 5.0

135/60 2.0

150/75 120 5.0

165/90 2.0

195/120 2.0

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
BODY SURFACE AREA CALCULATION

Body surface area can be calculated by the formula below, or by reference to the attached
nomograms.

2
BSA (m ) = sqrt(( Height (cm) x Weight (kg)) / 3600)

Ref: Mosteller RD. Simplified calculation of body surface area. N Engl J Med 1987; 317:1098.

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page
 Intravenous Glucose Tolerance........................ 27
A
L
Arginine Stimulation...........................................25
LHRH .................................................................... 19
B
O
Blood sampling......................................................8
Blood volume considerations..............................8 Oral Glucose Tolerance...................................... 29
Overnight High-Dose Dexamethasone
C
Suppression....................................................... 5
Clonidine Stimulation..........................................26 Overnight Low-Dose Dexamethasone
Combined Pituitary Function (formerly, Triple Suppression....................................................... 4
Test)..................................................................27
P
D
Parathyroid Hormone Infusion (Ellsworth-
Desmopressin, of Renal Concentrating Ability 1 Howard test).................................................... 31
Dexamethasone Suppression ..............................3 Pentagastrin Stimulation.................................... 33
Dynamic Testing , Principles ...............................7
R
E
Reference Ranges , RWI Endocrinology
Ellsworth-Howard................................................31 Laboratory ....................................................... 14
Exercise Stimulation ..............................................7
S
Exercise Stimulation, with Propranolol
(Propranolol Exercise Test)..............................9 Safety considerations........................................... 7
Sex Steroid Priming In Growth Hormone
F
Stimulation....................................................... 35
Fasting Study.......................................................10 Short ACTH (Synacthen) Stimulation ............. 36
Specimen collection
G
Other RAHC laboratories and external
Glucagon Stimulation (for pituitary function).16 laboratories ................................................. 20
Glucagon Stimulation (in suspected Requirements..................................................... 9
hypoglycemic disorders)...............................14 RWI Endocrinology Laboratory................... 11
Gonadotrophin Releasing Hormone (GnRH) Spontaneous Growth Hormone Secretion
(LHRH test)......................................................19 (Overnight or 24 hr GH sampling)................ 38
Standard (Long) Dexamethasone Suppression 6
H
T
hCG Stimulation...................................................21
Thyrotropin Releasing Hormone (TRH).......... 39
I
W
IGF-I Generation ..................................................23
Insulin Stimulation (Insulin Tolerance Test)...24 Water Deprivation .............................................. 41
Insulin Tolerance.................................................24

Ray Williams Institute of Paediatric Endocrinology, Diabetes and Metabolism Testing protocols Page