Diagnostic procedures and dynamic tests

DIAGNOSTIC PROCEDURES
AND DYNAMIC TESTS
IN
CHEMICAL PATHOLOGY
By
Dr. Basil, B. C – MBBS (Nig),
Department of Chemical Pathology/Metabolic Medicine,
Benue State University Teaching Hospital, Makurdi.
February 2016.
1

INTRODUCTION
• Hormones vary widely with respect to their composition, transport,
metabolism and mechanism of action.
• Endocrine functions are of two types:
• The basal secretion of the hormone is measured using a single blood or urine sample
• Dynamic tests, on the other hand, require two or more samples and are used to test
the integrity of the control mechanism of the hypothalamic-pituitary-end organ axis
• Dynamic tests are carried out under the supervision of a Metabolic
Medicine Physician or Endocrinologist
• We shall look at a few of the numerous Dynamic tests that abound in
clinical chemistry
2

DEFINITIONS:
• Dynamic test:
• Evaluation of biochemical changes or responses that occur within an individual to
various adjustments/alterations (physiological or therapeutic) that are not constant
and change with time
• Stress Test:
• Evaluation of the biochemical response of the body to stress
• Stimulation Test:
• Measures how well a gland respond to its stimulant (a trophic or tropic hormone, or
its analogue).
• Usually employed when assessing for a hypo-functioning state
• Suppression Test:
• Assesses the inhibitory (negative) feedback pathway of a hormone system (e.g HPA)
in an individual
• Usually employed when assessing for a hyper-functioning state.
3

SCOPE
• Investigation of Disorders of Growth Hormone Metabolism
• Investigation of Disorders of Cortisol metabolism
• Investigation of Disorders of Aldosterone metabolism
• Water Deprivation Test
• Oral Glucose Tolerance Test
NB: Investigation of Thyroid disorders have been treated in THYROID
FUNCTIONS AND DISORDERS presentation.
4

INVESTIGATION OF DISORDERS OF GROWTH
HORMONE METABOLISM:
• An optimal GH assay have the following:
• Sensitivity limit of the assay should be < 0.26mU/L with interassay coefficient of
variation of < 15%
• Assay should be validated with a normal range for suppressed GH after an OGTT
• Information on the antibody specificity to the GH isoform – the most abundant in
circulation being the monomeric 22KDa isoform
• Comparison of results btw laboratories is limited by lack of standardization
of GH assays
• Specimen collection:
• Preferred specimen – serum
• Storage – 2 to 8oC if they are not to be tested within 8hrs
• For long storage – frozen at -20oC
• Patient must be fasting and at complete rest for 30mins before sample collection
5

TESTS FOR GH DEFICIENCY:
• Normal basal GH is usually <1mU/L, except during pulses of secretion
• Thus, measurement of random GH levels is unhelpful
• Available tests:
• Short stature (children<18yrs)
• Glucagon stimulation test
• Second line: Clonidine stimulation test OR Exercise stimulation test
• Anterior pituitary reserve (adults only) – check both GH and ACTH:
• Insulin hypoglycemic (Tolerance) test OR Glucagon stimulation test
• Second line: Clonidine stimulation test
• For children and adults with contraindications for IHT, >> Glucagon Stimulation test –
safer as it doesn’t cause hypoglycemia
• Choice of specimens time of achievement and degree of hypoglycemia
6

Insulin Hypoglycemic (Tolerance) Test:
• Indication:
• Assessment of ACTH/Cortisol and GH deficiency
• Principle:
• Stress of insulin-induced hypoglycemia triggers release of GH and ACTH normally
• GH is measured directly, while Cortisol is measured as the indicator of ACTH
secretion
• Contraindications:
• Epilepsy or unexplained blackouts
• IHD or Cardiovascular insufficiency
• Severe longstanding hypoadrenalism
• Glycogen storage disease
7

• Precautions:
• ECG must be normal
• Serum cortisol (9.00) must be >100nmol/L
• Normal serum T4 (replace first if low)
• If above tests is questionable, perform glucagon test
• IV dextrose infusions and IV hydrocortisone 100mg should be available
• Procedure:
• Fast patient from midnight and review medications if any
• Weigh patient
• Insert IV cannula at 8:30
• Draw basal blood sample (0 min) for GH and Glucose; and give IV (soluble) insulin
bolus at 9:00
• Usual dose – 0.15IU/Kg
• Draw 4 more samples at 30min intervals; 5 if insulin was repeated.
8

• Procedure (contd):
• If cortisol deficiency is suspected draw samples for cortisol as well (depending on
achievement of hypoglycemia and GH response)
• Observe for signs of hypoglycemia to ensure that stress is adequate
• If not clinically hypoglycemic at 45min, consider repeating insulin dose in full
• The patient must be awake and able to answer simple questions
• It may rarely be necessary to terminate the test if:
• Severe and prolonged hypoglycemia(>20min)
• Impending or actual loss of consciousness or fits
• Resuscitate with glucose infusion but continue sampling if possible
• Consider IV hydrocortisone 100mg at the end of the test
• Give lunch and soft drink after collecting the last sample and observe patient for 2hrs
• Tabulate your results.
9

• Normal Response:
• Venous plasma glucose of <2.2mmol/L and signs of hypoglycemia is a
satisfactory evidence of sufficient stress
• Serum Cortisol rises by >200nmol/L to at least 55onmol/L
• Serum GH rises to >20mU/L
• Interpretation:
• Cortisol or GH deficiency cannot be diagnosed if hypoglycemia was not
adequate
• Untreated hypothyroidism can also give subnormal results – treatment with
T4 may be necessary for 3months before IHT becomes normal
• Peak GH response < 10mU/L = GH deficiency
• Peak GH response 10 – 20 mU/L = Partial deficiency
10

Glucose Stimulation Test:
• Indication:
• Assesment of ACTH/Cortisol and GH deficiency when IHT is contraindicated
• Principle:
• Glucagon stimulates GH release
• Recent or intercurrent illness
• Severe cortisol deficiency
• Glycogen storage disease
• Patient who haven’t eaten for 48h
• Side effects:
• Nausea, vomiting, abdominal cramps, and a feeling of apprehension which may
occur in the first 1-2hrs after injection
• Glucagon induces a 2-3 fold rise in blood glucose, max in the first hr, but may be
followed by symptomatic hypoglycemia.
11

• Precautions:
• Serum cortisol > 100nmol/L
• Serum T4 must be normal (replace first for several weeks if low)
• Procedure:
• Fasting from midnight (not longer than 4-6hrs in infants and young children) and
review medications if any
• Obtain accurate weight
• Patient must be on bed for the duration of the test
• Water intake only is allowed until completion of the test
• Monitor blood glucose at the bedside throughout the test
• Insert IV cannula at 8:30
• Take basal sample at 9:00 ( 0 min) for serum GH and Venous plasma glucose (same
sample may be utilized for serum cortisol if requested or indicated)
• Administer SC Glucagon 0.01mg/Kg, up to a max of 1mg at 9:00 (1.5mg if >90kg)
12

• Draw blood samples at 60, 120, 150 and 180min
• If bed side blood sugar <4mmol/L, give 0.5g/Kg dextrose via infusion
• Child should be fed and have normal blood sugar prior to discharge (leave IV in-situ
until discharge)
• Observe minimum of 2hrs after test
• Tabulate your results
• Normal Response:
• Plasma glucose – usually rises to peak around 90min and then falls
• Cortisol – rises by >200nmol/L to above 550nmol/L
• GH – rises to >20mU/L
• Interpretation:
• Peak (around 120min) GH < 10mU/L >> GH deficiency
• Peak GH 10 – 20 mU/L suggests partial deficiency
13

Clonidine Stimulation Test:
• Indication:
• Assesment of GH hypofunction
• Principle:
• Clonidine is a potent stimulus for GH release via GHRH secretion
• Contraindication:
• Sick sinus syndrome
• Compromised intravascular volume
• Precautions:
• Systolic BP falls by 20 – 25mmHg in all subjects, if higher, elevate the legs and
monitor BP every 15min
• Patient should lie down during and 2hrs after the test or until BP is satisfactory
• Adverse reactions:
• Drowsiness, reduced BP may last for several hours
• Effect will be prolonged in renal failure.
14

Clonidine Stimulation Test:
• Procedure:
• Fast patient from mid-night; minimum of 2hrs and maximum of <4 – 6 should be
applied to infants and young children
• Obtain weight, height and calculate body surface area
• Insert IV cannula at 8:30hr and draw baseline blood for glucose and GH at 9:00hr
• Give 0.15mg/m2 oral dose of Clonidine at 9:00h
• Draw samples for blood GH and glucose at 60,90,120 and 150mins.
• Interpretation:
• Since the mechanism and locus of action is unclear, interpretation is of uncertain
significance
• Peak GH response < 10mU/L >> GH deficiency
• 10 – 20nU/L >> partial deficiency
• Response >20 is regarded as normal
15

Exercise Stimulation Test:
• Indication:
• A screening test for GH secretion
• Principle:
• Exercise is a physiological stimulant of GH secretion
• Approx 50% of maximum capacity is required and is usually achieved by riding and
exercise bike or thread mill for about 15min
• Has high incidence of false positive due to inadequate exercise
• Safe and inexpensive
• Limitation of exercise capacity by cardiovascular, respiratory or other systemic
disease
• Children under 8 often do not tolerate the enforced exercise well
16

Exercise Stimulation Test:
• Procedure:
• Fast for at least 2hrs; in the morning
• Insert IV cannula
• Draw the pre-exercise blood sample for GH and Glucose
• Record basal heart rate
• Exercise child vigorously for 20min
• Measure heart rate at 5min interval; a heart rate of 140 – 160 is usually achieved
(stop test if it exceeds 180, or child is markedly distressed)
• Offer cool water during test but continue exercising
• After exercise, rest child for 20mins and collect another sample (i.e 40min sample)
• Interpretation:
• Peak GH < 10mU/L >> GH deficiency
• Responses 10 – 20mU/L >> partial deficiency
• >20mU/L is regarded as normal
17

TESTS FOR GH EXCESS:
• Biochemical diagnosis of Acromegaly or Gigantism is made by
assessing autonomous secretion of GH
• This is done by measuring GH levels during a glucose tolerance test
• Random or basal GH level is not specific or sensitive for given the
pulsatile nature of GH secretion
• Elevated random GH levels also occurs in stress, chronic renal failure,
DM and malnutrition.
• The glucose tolerance test is useful in the evaluation of patients with
signs suggestive of GH excess
18

Glucose Tolerance Test:
• Indication:
• Diagnosis of Acromegaly
• Principle:
• Stress of fasting cause elevation of GH which is suppressed by sudden elevation
blood Glucose level
• None
• Precautions:
• Known cases of diabetes mellitus
• Basal blood glucose level must be checked
• Procedure:
• Fast patient overnight
• Maintain bed rest and insert IV cannula
19

Glucose Tolerance Test:
• Draw baseline blood sample for glucose and GH assay 20 – 30mins after insertion of
cannula and note the time (Time – 0)
• Administer 75g of Oral glucose load (dissolved in 300mL of water) at time – 0; which
is to be taken over 3-4min
• Paediatric dose – 1.75g/Kg bodyweight.
• Draw samples for GH and plasma glucose at 30, 60, 90 and 120min
• Interpretation:
• Normal response – Serum GH suppresses to <0.8mU/L (as measured by current two-
site immunometric assays)
• Failure of GH to suppress is highly suggestive of GH excess
• Other causes include:
• CLD, Renal disease, Uncontrolled DM, Malnutrition, Anorexia nervosa, Pregnancy, Estrogen
therapy, Puberty
20

INVESTIGATION OF DISORDERS OF CORTISOL
METABOLISM:
• A random cortisol estimation is difficult to interpret due to variability of
cortisol secretion during the day and should be avoided if possible
• 17-hydroxyprogesterone also have marked circadian rhythm
• Diurnal rhythm and adult values are reached by 3 months
• Sample collection and Storage:
• Store specimens at 2-8oC
• Freezing is preferred for long term stability
• After measuring total volume of 24hr urine collection, a thoroughly mixed aliquot
(abt 10mL) is stored frozen at -20oC
• ACTH is easily oxidized, strongly adsorbs to glass surfaces and can rapidly be
degraded by plasma proteases into immunoreactive fragments during freezing and
thawing of specimen
• Sample for ACTH should be collected into pre-chilled EDTA bottle, immediately
placed on ice, and separated using a cold centrifuge
21

INVESTIGATION OF DISORDERS OF CORTISOL
METABOLISM:
• ACTH sample (Contd):
• The supernantant is then transferred to another plastic tube and stored at -
20oC or colder
• After setting up the ACTH assay, frozen specimen should be thawed and
centrifuged to remove any fibrin clots
• Precision and Bias:
• For serum cortisol, the precision MUST be <15% and bias <15%
• For urine free cortisol, the precision MUST be <25%
22

TEST FOR CORTISOL EXCESS:
• Result should be interpreted with full clinical assessment of the
patient
• Screening tests:
• Overnight Dexamethasone Suppression test (recommended) – useful initial
screening test for Cushing’s syndrome
OR
• 24-hour Urinary free cortisol
• Definitive tests:
• Recommended if a positive result is found on screening
23

Overnight Dexamethasone Suppression Test:
• Principle:
• Dexamethasone (a potent glucocorticoid) given as a 1mg oral dose at night,
will normally suppress ACTH secretion, and hence suppress the cortisol level
the next morning.
• Sample:
• Serum
• Procedure:
• 1mg Dexamethasone (children lower dose) to be taken at 11:00pm (± 1hr)
• Draw blood sample at 9:00am (± 1hr) the following morning for cortisol
measurement
24

Overnight Dexamethasone Suppression Test:
• Interpretation:
• 9am cortisol level of < 50nmol/L is observed in normal subjects after 1mg of
Dexamethasone
• > 50nmol/L indicates Failure to suppress; causes may include:
• Cushing’s syndrome, Stress, Obesity, Oral contraception use, Pregnancy, Estrogen
therapy, Alcoholism, Acute and Chronic illnesses,
• Preanalytical: Failure to take the dexamethasone, Glucocorticoid therapy with
Prednisolone or hydrocortisone,
• Drugs: Phenobarbitone, Phenytoin, Carbamazepine
• Endogenous depression
25

24-hour Urine Free Cortisol:
• Indication:
• Sensitive test for Suspected hypercortisolism
• Use full in pregnancy and in patients on Oral Contraceptive treatment or estrogen
• Principle:
• Increased plasma cortisol >> increased plasma free cortisol which is then filtered
through the glomerulus
• Sample collection:
• 24hr urine collection with 10g boric acid added at the start of collection as
preservative, if collected without preservative, refrigerate during collection peroid
• Specimen collection – universal container
• Urine creatinine should be measured on the 24hr sample to verify adequacy of
collection
• Assay which incorporates an extraction step should be used during
measurement
26

24-hour Urine Free Cortisol:
• Interpretation:
• Result should be interpreted with caution in young children and in patients
with significant renal dysfunction
• Age Reference range should be quoted in reports and should not exceed
300nmol/24hrs
• >300nmol/24hrs is indicative of further definitive testing
• Elevated urine free cortisol is also seen in:
• Stress
• Exogenous glucocorticoid usage
• Pseudohyperaldosteronism
• Licorice consumption
• Chewing tobacco use
27

Low Dose Dexamethasone Suppression Test:
• Indication:
• Establishment of diagnosis of Cushing’s syndrome
• Severe stressful illness/infection
• Precautions:
• DM and active PUD
• For inpatients, each dose should be written up as an individual dose
• Procedure:
• Draw sample at 9:00am on day 0 for serum cortisol (basal sample)
• Give Dexamethasone 0.5mg orally strictly 6-hourly at 9am, 3pm, 9pm, and 3am for
48hrs commencing immediately after the basal sample – in children <10yrs, use
5µg/Kg
• Obtain another blood sample for cortisol at 9am (6hrs after last dose)
28

Low Dose Dexamethasone Suppression Test:
• Interpretation:
• Normal Basal serum cortisol: 170 – 700nmol/L; After 48hrs, serum cortisol is
suppressed to <50nmol/L
• Excludes Cushing’s syndrome unless clinical suspicion is very high
• Other conditions that may cause non-suppression apart from Cushing’s
syndrome:
• Severe endogenous depression – abnormal circadian rhythm with normal cortisol
response to IHT
• Alcoholism (Alcoholic pseudo-cushing’s) – rapidly reverses on cessation of drinking
• Failure to take the dexamethasone correctly
• Oestrogen therapy, pregnancy, OCDd – high globulin binding proteins
• Corticosteroid therapy – may cross react in the cortisol assay
29

Dexamethasone suppression of Cortisol
(Extended):
• Indication:
• Aids in the differential diagnosis of hypercortisolism in patients who do not suppress
after an overnight Dexamethasone suppression test
• Principles:
• Patients are subjected to a low dose followed by a high dose suppression test and
the response is dependent on the condition
• Sample:
• Serum
• Side effect:
• Some patients report sleep disturbances
• Test is performed as an in-patient procedure
• No preparations are required but the ward staff must be aware of the
importance of correctly timed collection of specimens
30

(Extended):
• Procedure:
• Insert IV cannula
• Basal blood sample is collected at 9am for cortisol measurements and low dose
Dexamethasone 0.5mg commenced at same time – to be given orally 6hrly for 48hrs
• At 9am on day 3, increase the dose to 2mg 6hrly for 48hrs
• Ensure 9am sample is collected before giving dexamethasone
• Interpretation:
• Suppression is said to occur when post-dexamethazone cortisol level is <50% of basal
cortisol level
• Patients with Cushing’s disease usually do not suppress with low dose but majority
will suppress with high dose (about 15% do not suppress)
• Patients with false positive results in the overnight Dexamethasone suppression test
should suppress in the low dose period
31

(Extended):
• Interpretation (Contd):
• Patients with adrenal tumors or ectopic ACTH production fail to suppress with
very rare exceptions
• However, patients with adrenal adenomas have ACTH levels below the
reference range, while patients with ectopic ACTH producing tumors have
ACTH levels within or above normal range.
32

Evaluation of Cortisol Excess – Algorithm:
33

TEST FOR CORTISOL DEFICIENCY:
Synacthen Stimulation of Cortisol:
• Principle:
• A low plasma cortisol that does not rise after ACTH stimulation (short synacthen test)
confirms impaired adreno-cortical reserve
• If the impaired response persists after prolonged ACTH administration (Long
synacthen test), primary adreno-cortical insufficiency is indicated.
• Secondary adrenal atrophy due to impaired ACTH secretion may result from
corticosteroid therapy or hypothalamic-pituitary disease.
• Tetracosactrin (Synacthen or Cortrosyn) is the synthetic analogue of ACTH
most commonly used now.
• Known sensitivity to ACTH
• pregnancy
34

Synacthen Stimulation of Cortisol – (short):
• Patient preparation:
• Withhold Glucocorticoids e.g Prednisolone or hydrocortisone 24hrs prior to the test;
if steroids are required within this 24hr period, use dexamethasone
• Patient doesn’t require to fast or be at rest
• Procedure (short):
• Insert IV cannula and take baseline sample for cortisol
• Administer the synacthen dose; 250µg IM or as slow IV bolus
• 0-6months – 36µg/Kg
• 6months-2yrs – 125µg
• ≥ 2yrs - 250µg
• Draw 2 more samples at 30 and 60mins for cortisol
• 17OHP may also be requested:
• Indications: CAH, Hirsutism, Infertility
35

Synacthen Stimulation of Cortisol:
• Interpretation:
• Normal response: increase in serum cortisol level of 200nmol/L over the
baseline, and or the final value > 550nmol/L
• Failure to respond to a short test suggests adrenal failure (primary or
secondary)
• A long tetracosactrin test is required to confirm primary adrenal failure
• A peak 17OHP response of >30nmol/L is indicative of CAH
• In addition, a 30min 17OHP:Cortisol ratio > 0.1 suggests CAH, < 0.023 is
normal
• Values btw 0.023 and 0.1 suggests heterozygosity for 21-Hydroxylase deficiency.
36

Other Tests for accessing Cortisol Deficiency:
• Metyropone Stimulation Test:
• Actually a direct assay for ACTH reserve and indirectly cortisol and it is based
in the fact that decreasing serum cortisol concentration leads to increase in
ACTH secretion
• Metyropone blocks formation of cortisol from 11-deoxycortisol which does
not poses glucocorticoid activity, thus there is loss of negative feedback
• The resultant secretion of ACTH leads to accumulation of cortisol precursors
which is measured
• Insulin Tolerance Test:
• Gold standard for evaluating the integrity of Hypothalamo-pituitary axis
• Lysine Vasopressin Test:
• Stimulates increase on plasma ACTH and Glucocorticoids
37

INVESTIGATION OF DISORDERS OF
ALDOSTERONE METABOLISM:
• Aldosterone-producing tumors are usually associated with hypertension
with or without hypokalemia
• Screening tests include evaluation of HTN and measurement of Serum
Potassium
Specimen collection and Storage:
• Aldosterone –
• Sample – plasma (Heparin, EDTA) or serum can be used
• If an upright blood specimen is to be used, patient should be in an upright position
for at least 2hrs before collection
• Stored in airtight container at -20oC
• For urine assays, 24hr collection with boric acid; 50% acetic acid should be added to
achieve a pH btw 2.0 and 4.0 – DO NOT use strong mineral acids (e.g HCl)
• Aliquots are stored frozen at -20oC
• Its recommended to also perform urine sodium to facilitate interpretation of results.
38

• Renin –
• Sample – EDTA
• Centrifuged at room temp, separated and stored at -20oC or lower
• Freeze and thaw cycles should be avoided because of the possible activation
of prorenin
• Screening Tests:
• Serum potassium
• Urinary potassium
• To determine renal losses of potassium in the presence of hypokalemia, the patient
should be on at least 120mmol/L of sodium for 3 days before the investigation, since a
low sodium intake may normalize mild hypokalemia
39

• Samples
• Spot urine (needs simultaneous blood and urine specimens)
• 24-hour Urine
• Plasma Aldosterone-Renin ratio in an upright position:
• >20 suggests primary hyperaldosteronism
• Confirmatory Test:
• Saline suppression test
• Oral salt loading test
40

Saline Suppression Test:
• Principle:
• Rapid volume expansion with IV saline should suppress plasma aldosterone in
normal subjects but not in patients with Primary aldosteronism
• Heart failure
• Uncontrolled HTN
• Preparation:
• Test is carried out in the ward under supervision
• Patients may have fluids but otherwise should be fasted on the day of the test
• Withhold the following drugs:
• Beta-adrenoceptor blocking drugs – 2wks before testing
• Dihydropyridine calcium channel blocker – 2wks before testing
• Loop Diuretics, Spirinolactone – 6wks before testing
• Diltiazem can be used to control BP during testing
41

• Serum potassium should be maintained within the reference range (>3.5mmol/L)
prior to commencement and during testing
• Procedure:
• The subject is awakened at 6am and kept in an upright posture for 2hrs
• Record BP
• BP must be <190/110mmHg before proceeding
• Insert IV line and send blood for urgent potassium
• Blood is draw for determination of plasma aldosterone and electrolytes at 8am
• The subject then lies supine and 2L of N/S infused over 4hrs
• Record BP, pulse every 15min during the first hr of infusion and every 30min
thereafter
• Blood is drawn from the non-cannulated hand for aldosterone and electrolytes at
12noon
42

• Potassium should be ≥3.5mmol/L before patient leaves
• Cancel test if:
• Potassium < 3.5mmol/L
• BP > 190/110mmHg
• Observe patient one hour prior to discharge.
• Interpretation:
• Normal individuals show a plasma aldosterone level of ≤140pmol/L after
saline infusion.
• Levels ≥ 277.4pmol/L are usually seen in patients with autonomously
functioning aldosterone-secreting tumors.
43

Oral Salt Loading Test:
• Patients increase sodium intake to more than 6g/d for 3 days with diet and
NaCl tabs
• Potassium supplementation and daily monitoring for patients with
hypokalemia
• 24hr urine collection starting from day 3 for Sodium and Aldosterone
• Interpretation:
• 24hr urinary aldosterone secretion >12mcg/d is consistent with Primary
aldosteronism, whereas
• 24hr urinary sodium of 200mmol/24hr indicate adequate intake
• Renal insufficiency may confound the interpretation of the results
• Uncontrolled HTN, CCF, Arrythymias
44

Diagnostic Algorithm for Primary Aldosteronism:
45

WATER DEPRIVATION TEST:
• Aim:
• To establish the diagnosis of DI in a patient with polyuria, polydipsia and possibly
hypernatremia
• Principle:
• Dehydration and increased osmolality causes release of AVP free water reabsorption
from the renal tubules and production of concentrated urine.
• DDAVP should be avoided in patients with vascular disease esp CAD and Renal failure
• Not applicable in hypothyroidism, cortisol deficiency, and osmotic diuresis
• Precautions:
• Monitor vital signs during the dehydration procedure
• Stop the test if weight loss exceeds 2kg (or 5% BW in children) or clinical condition
deteriorates
• To ensure adequacy of dehydration, plasma osmolality should be >288mOsm/kg
before DDAVP administration
46

• Complete test protocol would not be required if:
• Serum osmolality is 275 – 295mOsm/Kg; or Sodium > 145mmol/L
• Urine Osmolality > 600mOsm/Kg
• Urine/Serum Osmolality ratio > 2
• If the serum osmolality is > 300mOsm/Kg water in a patient suspected of
having DI, or in very small children, testing can be done without the
dehydration step:
• Check basal serum and urine osmolality
• Give DDAVP
• Test serum and urine osmolality 2hr later.
• Side effect:
• DDVP may produce water intoxication only if there has been an increase in urine
osmolality, thus, patients who respond must not be allowed to drink freely
• Early signs of water intoxication: Drowsiness, Listlessness and headache.
47

• Procedure:
• Ensure patient is in an environment where they have no access to ANY water
• Weigh patient and take random urine for osmolality (no preservatives) and blood for
osmolality and serum sodium estimation
• If polyuria is mild, fast patient from 10pm the night before, but is moderately or
severely polyuric, fast from 7am on the day of the test
• Collect urine hourly from 8am in a universal bottle and measure osmolality
immediately on each
• Weigh patient hourly from 8am to the completion of the test
• Continue until osmolality shows < 30mOsm/Kg difference btw three successive
collections
• Draw a sample of blood (1mL) for osmolality and sodium when the plateau is
reached
48

• Next, inject 5 units of SC vasopressin or IN desmopressin acetate as follows:
• Adults – 0.4mL
• Children – 0.2mL
• Infants – 0.1mL
• Collect blood for sodium and serum osmolality and urine for osmolality
measurement 1hr later
• If no definite change in urine osmolality is observed, repeat measurement of
osmolality in urine one hour later.
• Osmolality should be measured using an osmometer.
• Interpretation:
• See table
49

50
• Reference ranges:
• Neonate may be as low as 266mOsm/Kg water
• Child and Adult 275 – 295mOsm/Kg water
• > 60yrs = 280 – 301mOsm/Kg water
• Random Urine = 50 – 1200 depending on the fluid intake
INTERPRETATION OF
WATER DEPRIVATION
TEST
Urine osmolality
(mosmol/Kg water)
before DDAVP
Increase in urine
Osmolality after dDAVP
Serum osmolality
(mosmol/ Kg water)
Before DDAVP
Normal ≥800 (>400 usually) <9% (No rise in urine
osmolality)
<300
Serum Na is normal
Diabetes Insipidus:
-Central (hypothalamic)
-Nephrogenic
<800 (<serum osmolality)
Usually < 300
Usually < 300
>50% (to reach ≥ 800)
<10%
May be > 300
Partial Central Urine > Plasma
Osmolality
Increase to < 800 (peak of
300 – 600)
Partial Nephrogenic 300 < urine < 800

• Interpretation (contd):
• In complete nephrogenic DI, AVP levels exceed 3ng/L when plateau osmolality is
reached
• In complete centralDI, AVP levels do not exceed 1.5ng/L
• Psychogenic polydipsia can be excluded when:
• Serum Na > 145mmol/L or Serum osmolality > 300mOsm/Kg
• Urine/Plasma Osmolality < 1.5
Sample Collection:
• Osmolality:
• Collect fresh urine (1mL) and serum (1mL) samples into a plain bottle without
preservatives and analyze promptly
• Antidiuretic Hormone (ADH orAVP):
• Collect blood into chilled EDTA bottle and transport to lab on ice, centrifuged at 4oC
within 30min of collection
• Store at -20oC until analysis is performed; significant deterioration occurs with
prolonged storage.
51

ORAL GLUCOSE TOLERANCE TEST:
• Aim:
• To measure body’s response to a glucose load
• Involves serial measurement of plasma glucose load
• Indications for OGTT:
• Individuals suspected of having diabetes with FPG of 6.1 – 6.9 mmol/L, to
determine glucose tolerance status
• NB: FPG value < 5.5mmol/L is sufficient to rule out DM
• Contraindications to OGTT:
• Patients with Reactive Hypoglycemia
• Acutely ill, hospitalized or inactive patients (bed rest impairs OGTT)
52

• Preparations for OGTT:
• Stop medications known to affect glucose concentration where possible, and
morning medications can be omitted and taken after the test
• Schedule for the test after 3 days of unrestricted diet (containing at least 150g
of carbohydrate per day) and activity
• Patient should observe 10 – 16hrs fast (overnight fast from 10pm) but water is
allowed
• Test should commence between 7 – 9am
• Procedure:
• Patient should be seated for some minutes before the test and remain so
during the test (no smoking)
• Collect sample for FPG by venipuncture and record the time of collection, and
53

• Procedure for OGTT:
• Immediately administer 75g of Anhydrous glucose (children – 1.75g/kg up to 75g
max) dissolved in 300mL of water and ingested over 5min
• 410mls of Lucozade from a standard Lucozade bottle (70kcal/100mmls) is an equivalent of
75g anhydrous glucose.
• Take sample for plasma glucose measurement every 30mins for 2hrs after ingestion
of glucose
• In some cases, samples for plasma insulin, C-peptide and GH are drawn concurrently
• If patient is hypoglycemic or has finger prick glucose of <2.2mmol/L, take blood for
glucose, insulin and C-peptide immediately and terminate the test
• NB: POCT in OGTT is less accurate compared to routine lab assay
• Plot the results of Plasma Glucose measurements on a graph of Blood Glucose Level
vs Time
54

• OGTT Curve:
55

56
• Interpretation:
Diagnosis Fasting Plasma Glucose Glucose Level after 2hr
DM ≥ 7.0 mmol/L ≥ 11.1 mmol/L
IGT 6.1 – 6.9 mmol/L 7.8 – 11.1 mmol/L
DM unlikely < 6.1 mmol/L < 7.8 mmol/L

• Interpretation (contd):
• If the highest level is reached at 30min and then falls below normal levels at
60 and 90min – Lag Curve; Due to rapid glucose absorption followed by a
burst of insulin production which over compensates, resulting in
hypoglycemia; occurs in:
• Post-gastrectomy or gastrojejunostomy
• Hyperthyroidism
• Severe liver disease
• Normal variant
• If the blood glucose rises only a small amount – Flat curve; occurs in:
• Malabsorption
• Addison’s disease
• GH deficiency
• Normal variant
57

REFERENCES:
• Tietz Text Book of Clinical Chemistry and Molecular Diagnostics by Carl A. Burtis,
Edward R. Ashwood and Bruns. 5th edition.
• Clinical Chemistry; Principles, Techniques and Correlations by Bishop et al. 7th
edition.
• Bolarin’s Aids to Chemical Pathology by Debayo M. Bolarin. New Edition, 2012.
• Endocrine test manual, Department of endocrinology, Westmead hospital for
children, Sydney, Australia 2001
• The Bart’s Endocrine Protocols: Peter J. trainer & Michael Besser
• Duty Biochemist manual, Laboratory Medicine, Royal Perth Hospital,
Australia,2001
• Test Manual, Department of Endocrinology, Westmead Hospital for Children,
Sydney, Australia,2002
59

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