ORAL GLUCOSE TOLERANCE TEST,



PROTOCOLS

FOR

DYNAMIC TESTS AND

ENDOCRINE INVESTIGATIONS

INDEX

AMMONIUM CHLORIDE LOAD TEST 1

CLONIDINE TEST 2

HYDROCORTISONE (CORTISOL) Day Profile 4

DEXAMETHASONE SUPPRESSION TEST 6

LOW DOSE DEXAMETHASONE SUPPRESSION TEST 7

NON-ISCHAEMIC FOREARM EXERCISE TEST 8

GHRH and ARGININE STIMULATION TEST 10

i.m. GLUCAGON TEST 13

ORAL GLUCOSE TOLERANCE TEST 15

ORAL GLUCOSE TOLERANCE TEST, (ASSESSMENT OF ACROMEGALY) 17

hCG STIMULATION TEST 19

INSULIN STRESS TEST 20

ORAL LACTOSE TOLERANCE TEST 22

LHRH, or GONADOTROPHIN RELEASING HORMONE (GnRH) TEST 23

LIPOPROTEIN LIPASE (Post Heparin) TEST 25

METHIONINE LOAD TEST 26

PENTAGASTRIN TEST 27

PENTOLINIUM TEST 28

PHENTOLAMINE TEST 29

SEX HORMONE PRIMING PRIOR TO GH ASSESSMENT 30

STEROID WITHDRAWAL PROTOCOL 31

SHORT SYNACTHEN TEST 33

LONG SYNACTHEN TEST 35

Supervised THYROXINE ABSORPTION TEST 36

WATER DEPRIVATION TEST 38

XYLOSE ABSORPTION TEST 40

Document Control 41

AMMONIUM CHLORIDE LOAD TEST

|Purpose of test |To confirm the diagnosis of suspected Renal Tubular Acidosis type 1. |

|Rationale |In type 1 RTA, the distal renal tubule is abnormally permeable to H+ ions and the kidneys are unable to |

| |excrete an acid load. The urine is often spontaneously acid in normal individuals before breakfast, |

| |reflecting overnight fasting. If not, the ability to acidify the urine is tested by challenging with an|

| |oral dose of Ammonium Chloride, which is metabolised in the liver to urea with the production of H+. |

|Patient preparation |Fasting from midnight. Patient should not be taking Acetazolamide. |

|Note |Order Ammonium Chloride from pharmacy in advance. |

| |Phone biochemistry lab. (153403) the day before to make prior arrangements for the urine pH tests. |

|Protocol | |Baseline blood sample for renal profile, including bicarbonate and chloride |

| | |(serum or lithium heparin plasma sample). |

| | |Fresh urine sample for pH (plain, sterile universal). Urinary pH changes fairly |

| | |quickly, therefore transport straight to lab. without delay |

| | |Phone lab. (xn 153403) in advance to make arrangements for this to be done. |

| | |If urine pH ( 5.2, test finished, no need to proceed. |

| | |If urine pH > 5.2, proceed: Breakfast, then |

| | | |

| | |Give Ammonium Chloride, 100mg/Kg p.o. May be taken over one hour to reduce |

| | |gastric irritation. |

| |0 min |Patient allowed to eat and drink normally. |

| | | |

| | |Fresh urine sample for pH. |

| | | |

| |hourly, for 8hr |Fresh urine for pH, and blood for electrolytes, bicarbonate |

| | | |

| |2 hours |Fresh urine for pH, and blood for electrolytes, bicarbonate |

| | | |

| |5 hours |Fresh urine for pH, and blood for electrolytes, bicarbonate |

| | | |

| |8 hours | |

|Cautions |Warn patient the capsules may cause nausea. |

| |Contraindications: NH4Cl not to be given to patients with significant liver impairment, nor if plasma |

| |bicarbonate ( 19mmol/L |

|Interpretation |The plasma bicarbonate should fall if the acid load has been successfully absorbed. |

| |Normal individuals should be able to acidify the urine to ( 5.2, between 2 – 6 hours. |

| |In type I RTA urine pH often remains greater than 6.0. |

| |In type 2 (proximal) and in type 4 RTA, the ability to acidify the urine is unaffected. |

| |Incomplete syndromes of type 1 RTA exist, with intermediate response. |

Refs:

1. O M Wrong, H E F Davies. The excretion of acid in renal disease. 1959. Q. J. Med. 28: 259

CLONIDINE TEST

|Purpose of test |To assess GH reserve when IST is contraindicated. |

|Rationale |Clonidine stimulates GH release in normal subjects. This is not seen in hypopituitarism or other forms |

| |of GH deficiency. Although the response is variable even in normals and interpretation is difficult. |

| |Only use in patients where it is essential to assess GH status but IST is impossible (epilepsy, severe |

| |ischaemic heart disease). |

|Patient preparation |Fasting from midnight. |

| |Insert i.v. cannula and allow patient to rest for 60 min before test. |

|Protocol | |Monitor pulse & BP before, and every 15 min throughout the test |

| | |(systolic blood pressure falls by 20-30 mmHg in almost all subjects). |

| | | |

| |-30 min |blood for GH (serum sample, SST tube) |

| | | |

| |0 min |blood for GH |

| | |give Clonidine 0.15mg/m2 body surface area, or 4(g/Kg, p.o. (max. 0.3mg) |

| | | |

| | |blood for GH |

| |30 min |blood for GH |

| |60 min |blood for GH |

| |90 min |blood for GH |

| |120 min |blood for GH |

| |150 min | |

|Cautions |Side effects of Clonodine include hypotension, and drowsiness. Patient to remain in bed for 2 h after |

| |the test and continue to monitor BP every 30 min for this period. |

| |Test can usually be done as a day case, but be prepared to keep in overnight if drowsiness or |

| |hypotension persist. |

| |Contraindications: Porphyria. |

|Normal response |GH should rise to values > 7 (g/L in normal subjects., usually at 60-90 mins. A peak value ( 5(g/L would|

| |be considered abnormal by most. (1.0(g/L = 2.6mU/L). |

Ref.

Principles and Practice of Endocrinology and Metabolism. Ed K L Becker. Lippincott, 1995

Rochiccioli P, Enjaume C, Tauber MT, et al. Statistical study of 5473 results of nine pharmacological stimulation tests: a proposed weighting index. 1993 Acta Paediatrica 82: 245-8

Styne DM A practical approach to the diagnosis of growth hormone (GH) deficiency in patients transitioning to adulthood using GH stimulation testing. 2003 J Pediat Endocrinol 16 Supp3: 637-43.

CLONIDINE TEST

DATE: ……………. HOSPITAL No. …………………. ……………………

NAME ………………… ……………………

D.O.B. ………………… ……………………

CONS ………………… ……………………

Clonidine given at:

|Actual time |P |BP |Specimen No.s |Elapsed time (min.s)|Glucose |GH ((g/L) |

| | | | | |(mmol/L) | |

|⎫ | | | |-30 | | |

|⎫ | | | |0 | | |

| | | | |15 | | |

| | | | |45 | | |

| | | | |75 | | |

| | | | |105 | | |

| | | | |135 | | |

Please send with last specimen in the series to the laboratory, for results

Hydrocortisone (Cortisol) Day Profile

|Purpose of test |Assessment of the adequacy of Hydrocortisone replacement therapy. |

|Patient preparation |Patients should not take breakfast nor their steroid medication before attending for the test. |

| |Remind patients to bring their own medication in with them. |

|Protocol |Patients will normally be taking their steroid medication either twice daily or thrice daily |

| |time |Elapsed |If taking medication 2 x day |If taking medication 3 x day |

| | |Time | | |

| |0800 |0 |Blood for cortisol (SST serum tube): |Blood for cortisol: |

| | | |Patient then takes Hydrocortisone |dose and has breakfast. |

| |0830 |30 min |Blood for cortisol |Blood for cortisol |

| |0900 |60 min |Blood for cortisol |Blood for cortisol |

| |1000 |120 min |Blood for cortisol |Blood for cortisol |

| |1200 |240 min |Blood for cortisol |Blood for cortisol: Patient then |

| | | | |takes lunchtime dose. |

| |1230 |270 min | |Blood for cortisol |

| |1300 |300 min | |Blood for cortisol |

| |1400 |360 min | |Blood for cortisol |

| |1600 |420 min |Blood for cortisol |Blood for cortisol |

| | | |Patient then takes |teatime dose |

| |1630 |450 min |Blood for cortisol |Blood for cortisol |

| |1700 |480 min |Blood for cortisol |Blood for cortisol |

| |1800 |540 min |Blood for cortisol |Blood for cortisol |

| | | |Patient allowed |home |

| | | | | |

| | | |Please ensure the time is clearly |indicated on every sample! |

|Note |The last sample (or two if running late) may need to be taken by the duty House Officer, since the day unit |

| |finishes at 5.00pm. Please make arrangements in advance if this is required. |

|Interpretation: |There is no evidence-base for the optimal levels of steroid replacement. Clinical assessment and serum |

| |electrolytes are equally important. Replacement therapy usually tries to mimic the normal diurnal variation,|

| |except that the pre-dose levels at 8.00am are likely to be undetectable. Avoid high levels throughout the |

| |day. |

| |Interpretation and dose adjustment should be made by an experienced endocrinologist. |

Photocopy (or print) next page, and complete during the day: to be filed in patient’s notes

Ref: DD

Hydrocortisone (Cortisol) Day Profile

|DATE: |……………. | | |HOSPITAL No. |…………………. |…………………… |

| | | | | |NAME |………………… |…………………… |

| | | | | |D.O.B. |………………… |…………………… |

|RESULTS:- | | | | |CONS |………………… |…………………… |

| | | | |Either: |Or: |

| | | | |Twice daily dose |Three daily doses |

|Specimen No. |Time |Elapsed time |Actual Time |CORTISOL |CORTISOL |

| | | | |nmol/L |nmol/L |

| |08:00 |0 min | |⎫ |⎫ |

|HYDROCORTISONE: DOSE = |GIVEN (time): |

| |08:30 |30 min | |⎫ |⎫ |

| |09:00 |60 min | |⎫ |⎫ |

| |10:00 |120 min | |⎫ |⎫ |

| |12:00 |240 min | |⎫ |⎫ |

|LUNCHTIME HYDROCORTISONE: DOSE = |GIVEN (time): |

| |12:30 |270 min | | |⎫ |

| | |(30 min) | | | |

|EVENING HYDROCORTISONE: DOSE = |GIVEN (time): |

| |16:30 |510 min | |⎫ |⎫ |

| | |(30 min) | | | |

| |17:00 |540 min | |⎫ |⎫ |

| | |(60 min) | | | |

| |18:00 |600 min | |⎫ |⎫ |

| | |(120 min) | | | |

- Record doses and sample times as test progresses

- Then forward this sheet to WPH biochemistry dept. with the last sample, for results.

DEXAMETHASONE SUPPRESSION TEST

|Purpose of test |Aa a preliminary screening test for suspected Cushing’s syndrome |

|Rationale |A small dose of the synthetic glucocorticosteroid dexamethasone in the evening should suppress the HPA axis |

| |in normal individuals, leading to low plasma cortisol levels the next morning, when levels would be expected|

| |to be high. Patients with Cushing’s syndrome of whatever cause have lost this normal feedback control, and |

| |cortisol levels will not be suppressed. |

|Patient preparation |None. The test may be performed as an outpatient procedure. |

|Protocol |2300 – 2400h |Patient takes 1mg dexamethasone p.o. |

| | | |

| |0800 – 0900h |Blood for cortisol (serum, SST tube) |

|Assay arrangements |Cortisols are analyzed mostly on the next working day after receipt in the laboratory |

|Interpretation | A cortisol concentration (50nmol/L virtually excludes the diagnosis of Cushing’s syndrome: the sensitivity|

| |of the test is close to 100%. There is a significant false positive rate (failure to suppress), often seen |

| |in obese or depressed patients. |

| |The test should be combined with estimation of the 24h urinary excretion of free cortisol. |

Refs:

1. P J Wood, et al.. Evidence for the low dose dexamethasone suppression test to screen for Cushing’s syndrome – recommendations for a protocol for biochemistry laboratories. 1997 Ann Clin Biochem 34: 222-229.

LOW-DOSE DEXAMETHASONE SUPPRESSION TEST

|Purpose of test |As a screening test to confirm suspected Cushing’s syndrome, if a preliminary overnight test is |

| |inconclusive. To exclude androgen secreting tumour of the adrenal or ovary. |

|Rationale |2mg/d for 2 days of dexamethasone should completely suppress the HPA axis and blood cortisol levels in |

| |normal individuals. This is checked early the next morning, when cortisol levels should be at their highest.|

| |The cortisol levels do not suppress in patients with Cushing’s syndrome of whatever cause. |

| |The test is sometimes used to check the suppressibility of androgens in women with virilising symptoms. |

|Patient preparation |None. The patient should NOT be taking any steroid medication prior to test. The test is usually performed|

| |as an inpatient. It may be performed on an outpatient basis, but the patient will need clear written |

| |instructions, which emphasise the importance of adhering to strict timing of the dexamethasone doses and the|

| |blood samples |

|Protocol |Day 0 | |

| |08.00 - 09.00 |Blood for cortisol (serum, SST tube) and ACTH (5mL (min 2.5mL) blood into EDTA tube: |

| | |rush latter to lab., to be frozen within 10 mins) |

| | |Serum Testosterone, SHBG, LH/FSH also requested if required. |

| | |Sample for 17OH-Progesterone, DHEAS, Androstenedione if required (Plain serum tube with|

| | |no gel). |

| | |Patient then takes 0.5mg Dexamethasone p.o,. qds |

| | | |

| | |Patient continues to take 0.5mg Dexamethasone strictly 6-hourly, e.g. 09.00am, |

| | |15.00pm, 21.00pm, 03.00am for 48hrs. It may preferable to write these up as individual|

| | |doses on the drug chart |

| | | |

| |Day 1 09.00 |Blood for cortisol |

| | | |

| |Day 2 09.00 |i.e. approx 6hrs after last dose of Dexamethasone: Blood for cortisol |

| | |(and sample for 17OH-Progesterone, DHEAS, Androstenedione if required) |

|Assay arrangements |Cortisols are analyzed mostly on the same working day after receipt in the laboratory |

| |ACTH has a very short half-life, samples must be frozen within 10 minutes: make prior arrangements with the|

| |laboratory to do this. (WPH Xn 153403, 153444, 153447). Results from the Royal London usually available in |

| |2 weeks |

|Cautions |Care is required in patients with: Diabetes Mellitus; active peptic ulcer disease. Schizophrenia; severe |

| |affective disorder. |

|Interpretation | A cortisol concentration (50nmol/L at 48hrs virtually excludes a diagnosis of Cushing’s syndrome; the |

| |false-negative rate is ~2%. Beware patients taking enzyme-inducing drugs (eg anticonvulsants) which may |

| |increase the metabolism of the Dexamethasone, or oestrogens (which increase cortisol binding globulins). |

| |False-positive (failure to suppress) may still be seen in severe depression. |

| |Testosterone and adrenal androgens should suppress to low values in normal subjects and in most women with |

| |P.C.O.S. Failure to suppress suggests a possible androgen-secreting tumour of adrenals or ovary: proceed |

| |to imaging. |

Ref.

1. HE Turner, JAH Wass (eds). Oxford Handbook of Endocrinology and Diabetes. Oxford University Press, 2007

2. P J Trainer, M Besser (eds). The Bart’s endocrine protocols. London: Churchill Livingstone, 1995.

NON-ISCHAEMIC FOREARM EXERCISE TEST

|Purpose of test |Diagnosis of McArdles disease, and some other skeletal muscle glycogen storage diseases. |

|Rationale |The forearm muscles are exercised , which will normally cause an increase in venous outflow lactate, due to |

| |anaerobic glycolysis. In patients with impaired muscle glycogenolysis or glycolysis this increase will be |

| |reduced or absent. Instead, degradation of purines and proteins due to disrupted ATP generation will cause a|

| |large increase in Ammonia. In the absence of a mitochondrial defect or pyruvate carboxylase deficiency, the |

| |pyruvate:lactate ratio will remain normal. |

|Preparation |Warn laboratory the day before: special sample handling will be required. Xn 153444. |

| |Fasting from midnight. |

| |The patient should be resting quietly from 30 minutes prior to the test to establish baseline metabolic |

| |conditions. |

|Protocol | |Determine Maximum Voluntary Contraction (MVC) using the Handgrip Dynamometer, if available. |

| | |Best of three squeezes. Then: |

| | |Insert i.v. cannula in a large antecubital vein, with three-way tap: the subsequent blood |

| | |samples must be free-flowing and easily withdrawn. Wait 10 min then: |

| | |Baseline bloods for Lactate*, Ammonia*, and if required Pyruvate*, CK, U/E’s, Phosphate, |

| | |urate. Then, within 2 minutes start:- |

| | | |

| | |Exercise the same forearm for 1 minute: squeeze handgrip dynamometer (if available) to 100% |

| | |of MVC for 1 sec, relax for 1 sec, repeat…. Counting rhythmically whilst doing this may help.|

| | |Squeezing a rubber ball, or bunch of paper towels to maximum effort in the same way will |

| | |suffice, if no dynamometer available. |

| | |If the patient does have a glycogenolytic defect, this is likely to cause a painful cramp. |

| | |Record any symptoms. |

| | |Timing starts from the end of this 1 min exercise. |

| | | |

| | |Immediately withdraw 2-3ml and discard, then take blood for Lactate and Ammonia, and Pyruvate |

| | |if required. Flush cannula with saline after. |

| |time 0 | |

| | |Blood for Lactate and Ammonia, and Pyruvate if required. |

| | |Blood for Lactate and Ammonia, and Pyruvate if required. |

| |1 minute |Blood for Lactate and Ammonia, and Pyruvate if required. |

| |2 mins |Blood for Lactate and Ammonia, and Pyruvate if required. |

| |4 mins |Blood for Lactate and Ammonia, and Pyruvate if required. |

| |6 mins |Blood for Lactate and Ammonia, and Pyruvate if required. |

| |8 mins |Ensure each sample is clearly identified with the time! |

| |10 mins | |

|*Note! |Lactate: min. 200(l blood into a fluoride/heparin (yellow top) tube. Analyse promptly. |

| |Pyruvate: transfer blood to LiHep tube first; immediately pipette exactly 100(l of this whole blood into a |

| |special perchloric acid tube. Mix, keep on ice, rush to lab. for immediate processing. Ask for help from |

| |lab. with this. |

| |Ammonia: min 200(l blood into EDTA tube, on ice. Rush to lab. for immediate analysis. |

|Warning |This full protocol will require at least 25 ml blood (including discarded). Choose carefully which tests are|

| |required. |

|Normal response |Normally the venous plasma lactate will increase to a peak 3 – 5 x baseline values. |

| |Ammonia increases from normal resting values of around 40(mol/l to a peak of about 100(mol/L. |

| |In Glycogenoses types III (debranching enzyme), V (McArdle’s, muscle phosphorylase deficiency) and VII |

| |(muscle phosphofructokinase) the lactate barely increases. Ammonia increases much more than normal, to about|

| |300 – 400 (mol/l, and this is a good check of adequate exercise. |

| |In myoadenalate kinase deficiency the lactate will increase normally, but ammonia will not increase |

| |significantly. |

| |The Lactate:Pyruvate ratio is normally 10–20, and may increase to about 30-40 on exercise. It is not |

| |affected in glycogenoses. |

Ref.

1. P Kazemi-Esfarjani, E Skomorowska, TD Jensen, RG Haller, J Vissing. A Nonischemic Forearm Exercise test for McArdle disease. 2002. Ann Neurol 52: 153-159

GHRH-ARGININE STIMULATION TEST

|Purpose of test |Used to confirm GH deficiency: |

| |1. In transition, after achievement of final height, to confirm persistence of childhood GH deficiency in |

| |late adolescence/early adulthood if insulin tolerance test is contraindicated or patient is overweight which |

| |may limit interpretation of insulin tolerance test or glucagon stimulation test. |

| | |

| |2. In adults, if glucagon stimulation test or insulin tolerance test shows GH deficiency (peak GH 65 |

| |BMI (kg/m2) |

| |30 8.5 5.5 4.0 |

| | |

| |In transition (adolescence and young adults 95 kg). 15(g/Kg in children. |

| | | |

| | |blood for glucose, GH and Cortisol |

| |90 min |blood for glucose, GH and Cortisol |

| |120 min |blood for glucose, GH and Cortisol |

| |150 min |blood for glucose, GH and Cortisol |

| |180 min |blood for glucose, GH and Cortisol |

| |210 min |blood for glucose, GH and Cortisol |

| |240 min | |

|Cautions |Warn patients they may experience nausea at the end of the test. Rebound hypoglycaemia may occur up to 3|

| |hours after start of test; children should eat a meal before discharge. |

|Normal response |Glucose should peak around 90 mins, and then fall. |

| |Cortisol should reach a maximum of (550 nmol/l.; 500-550nmol/L represents a borderline response. |

| |Cut-offs for the diagnosis of GH deficiency are somewhat arbitrary. Peak GH values are usually seen |

| |90-120mins after injection, and in normal individuals should be ( 10(g/L, often up to 15(g/L or more; a |

| |peak value (5(g/L would be considered abnormal by most. (1.0(g/L = 2.6mU/L). |

Photocopy (or print) next page, and complete during the day.

Please send with last specimen in the series to the laboratory, for results

Ref.

Strich D, Terespolsky N, Gillis D. Glucagon stimulation test for childhood growth hormone deficiency: timing of the peak is important. 2009 J Pediatrics 154: 415-9

Styne DM A practical approach to the diagnosis of growth hormone (GH) deficiency in patients transitioning to adulthood using GH stimulation testing. 2003 J Pediat Endocrinol 16 Supp3: 637-43.

M Vanderscheuren-Lodeweyckx, R Walter, P Malvaux, E Eggermont & R Eeckels. The Glucagon stimulation test: effect on plasma growth hormone and on immunoreactive insulin, cortisol, and glucose in children. 1974 J Pediatrics 85: 182-7.

i.m. GLUCAGON TEST

DATE: ……………. HOSPITAL No. …………………. ……………………

NAME ………………… ……………………

D.O.B. ………………… ……………………

CONS ………………… ……………………

i.m Glucagon: dose:………..., given at:……………

|Time of sample|P |BP |capillary |Specimen No.s |Elapsed time (min.s) |Glucose |Cortisol |GH |

| | | |glucose | | |(mmol/L) |(nmol/L) |((g/L) |

|⎫ | | | | |-30 | | | |

|⎫ | | | | |0 | | | |

| | | | | |30 | | | |

|⎫ | | | | |120 | | | |

|⎫ | | | | |150 | | | |

|⎫ | | | | |180 | | | |

|⎫ | |

|Rationale |oral glucose load raises plasma glucose and stimulates insulin secretion |

|Patient preparation |Give 150-200 gram Carbohydrate diet for 3 days prior to the test - i.e do not restrict CHO in diet prior to the|

| |test. |

| |fast from midnight |

| |refrain from smoking |

|Protocol | |Ensure patient is fasting. Check capillary blood glucose from a finger-prick, with bedside |

| | |glucose meter. |

| | |If capillary blood glucose is 10.0: no need to proceed. Take blood for glucose |

| | |(Fluoride-EDTA sample tube, grey top), send to lab. If capillary blood glucose is 3.0-10.0, |

| | |then: |

| | | |

| | |Patient lying on bed at rest, insert i.v. cannula |

| | | |

| |0 min |blood for glucose |

| | |give 75grams anhydrous glucose orally (( 82.5g of BP Glucose monohydrate) made up to a min |

| | |300mL fluid; or a Pharmaceutical grade glucose polymer equivalent (eg ‘Rapilose OGTT drink’), |

| | |For children, 1.75g Kg-1 anhydrous glucose, or equivalent. |

| | |Patient may drink water freely, BUT MUST NOT EAT UNTIL THE TEST IS COMPLETE! |

| | |blood for glucose |

| | |blood for glucose |

| |60 min |- Please ensure all three samples are clearly identified by time and sequence. |

| |120 min | |

Interpretation: WHO criteria for Diabetes Mellitus:

| |Glucose concentration (mmol/l) |

| |capillary whole blood |Venous plasma |

|Normal |

|Fasting |( 5.6 |( 6.1 |

|and: 2h after oral glucose |( 7.8 |( 7.8 |

|Impaired Glucose Tolerance |

|Fasting |( 6.1 |( 7.0 |

|and: 2h after oral glucose |7.8 - 11.0 |7.8 - 11.0 |

|Diabetes |

|Fasting |( 6.1 |( 7.0 |

|or: 2h after oral glucose |( 11.1 |( 11.1 |

Note: Results from WPH laboratory are usually for venous plasma

In Children the same criteria apply.

- Fasting plasma glucose ( 6.1mmol/l but ( 7.0mmol/l, and 2h post glucose load plasma glucose (7.8mmol/l is described as Impaired Fasting Glycaemia. IFG and IGT are not clinical entities, but indicators of risk of future cardiovascular disease and/or diabetes.

- In Pregnancy, the OGTT is usually performed around 24 – 28 weeks gestation. Gestational Diabetes (GDM) is diagnosed if either fasting venous plasma glucose ≥5.6mmol/L or 2hr post load glucose ≥7.8mmol/L.

- In 2011, the WHO recommended that Hba1c can also be used in the diagnosis of Type 2 diabetes mellitus. An Hba1c level of 48 mmol/mol or above indicates type 2 diabetes. However, Hba1c is not a straightforward alternative to the glucose tolerance test – e.g. Hba1c cannot be used to diagnose Impaired Glucose Tolerance and is not suitable for use in pregnancy or diagnosis of type 1 diabetes.

Refs.

-Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglycaemia, WHO/IDF, 2006.

-Use of Glycated Haemoglobin (HbA1c) in the Diagnosis of Diabetes Mellitus, WHO, 2011.

- NICE NG3 (2015): Diabetes in Pregnancy

ORAL GLUCOSE TOLERANCE TEST,

ASSESSMENT OF ACROMEGALY

|Purpose of test |investigation of acromegaly |

| |diagnosis of diabetes |

|Rationale |elevated blood glucose suppresses GH release in normal subjects, an effect lost in acromegaly |

|Patient preparation |Give 150-200 gram Carbohydrate diet for 3 days prior to the test - i.e do not restrict CHO in diet prior to the|

| |test. |

| |fast from midnight |

| |refrain from smoking |

|Protocol | |patient lying on bed at rest, insert i.v. cannula |

| | | |

| |0 min |blood for glucose (Fluoride-EDTA sample tube, grey top) and GH (Serum sample, red/gold top), and|

| | |for IGF1 if required (serum sample) |

| | |give 75grams anhydrous glucose orally (( 82.5g of BP Glucose monohydrate), or pharmaceutical |

| | |grade equivalent. |

| | |Patient may drink water freely, BUT MUST NOT EAT UNTIL THE TEST IS COMPLETE! |

| | |blood for glucose, and GH |

| |30 min |blood for glucose, and GH |

| |60 min |blood for glucose, and GH |

| |90 min |blood for glucose, and GH |

| |120 min | |

|Interpretation: | |Ensure the plasma glucose concentration shows the expected rise and fall. Patients with excess |

| | |GH will often also have disordered glucose homeostasis, and the plasma glucose concentrations |

| | |may show IGT or diabetes mellitus. |

| | |In normal individuals the increase in plasma glucose will suppress GH secretion: a GH |

| | |concentration of ( 1(g/L at any time excludes the diagnosis. In acromegaly a ’paradoxical rise’|

| | |in GH from baseline levels may be seen. |

| | |Following treatment: There is no universally agreed biochemical definition of successful |

| | |treatment in acromegaly. Ideally basal GH levels should be (2(g/L, and suppress normally during|

| | |the OGTT; many endocrinologists use the OGTT to monitor patients, and expect mean GH levels |

| | |throughout the test to remain below 2.0(g/L |

Refs:

Williams textbook of Endocrinology

Photocopy (or print) next page, and complete during the day.

Please send with last specimen in the series to the laboratory, for results:

ORAL GLUCOSE TOLERANCE TEST

DATE: ……………. HOSPITAL No. …………………. ……………………

NAME ………………… ……………………

D.O.B. ………………… ……………………

CONS ………………… ……………………

75gm anhydrous glucose, given at:

|Time of |Specimen No.s |Elapsed time (min.s) |Glucose (mmol/L)|GH ((g/L) |

|sample | | | | |

| | |0 | | |

| | |30 | | |

| | |60 | | |

| | |90 | | |

| | |120 | | |

Please send with last specimen in the series to the laboratory, for results

hCG STIMULATION TEST

|Purpose of test |To detect presence of functioning testicular tissue in boys with impalpable scrotal testes. |

| |Differential diagnosis of hypogonadal males, and investigation of sexual ambiguity before puberty |

|Rationale |The low levels of testosterone pre-puberty will not differentiate between normal and hypogonadal males. |

| |Gonadotrophin levels should be elevated in primary hypogonadism, but may not reliably differentiate |

| |between male secondary hypogonadism and female. |

| |If functional testicular tissue is present, it will respond to human chorionic gonadotrophin (hCG), |

| |which is a close analogue of LH, by producing testosterone. If there is a testosterone biosynthetic |

| |defect, testosterone precursors will rise instead. |

|Preparation |None required |

|Protocol | | |

| |Day 0 |Baseline bloods for Testosterone and LH/FSH (serum sample, min vol 1.0mL blood)1. |

| | |If required, blood also for Dihydrotestosterone (serum sample, min 1.5mL blood)2 |

| | |and Androstenedione/DHEAS (serum sample, min 1.5mL)3. |

| | | |

| | |Give hCG 1000units i.m.daily, for 3 days. (Adults, 2000units) |

| |Days 0,1,2 | |

| | |Blood for Testosterone, and if required DHT, and AD/DHEAS |

| |Day 3 | |

| | |1,2,3These tests are performed in different laboratories, therefore a separate |

| | |sample is required for each. |

|Normal response |If normal functioning testicular tissue is present, testosterone levels should increase at least |

| |two-fold, and peak > 7nmol/L1, or > 5nmol/L3. |

| |The T:DHT ratio in normal infants is in the range 1.5 – 172. |

Note. There are a large number of different hCG test protocols in use; ref 2 shows that there is no clear relationship between the dose of hCG used and the increment in T or DHT.

Ref.

1. Principles and Practice of Endocrinology and Metabolism. Ed K L Becker. Lippincott, 1995

2. S F Ahmed, A Cheng, I A Hughes. Assessment of the gonadotrophin-gonadal axis in androgen insensitivity syndrome. 1999 Arch Dis Child. 80: 324-329.

3. M Davenport, C Brain, C Vandenberg, S Zappala, P Duffy, P G Ransley, D Grant. The use of the hCG stimulation test in the endocrine evaluation of cryptorchidism. 1995 Br J Urol 76: 790-794

INSULIN STRESS TEST

|Purpose of test |Assess pituitary ACTH and GH responses to stress |

|Rationale |Hypoglycaemic stress induces secretion of ACTH (hence cortisol) and GH |

|Patient preparation |ensure no epilepsy or IHD ( ensure normal TFT’s, or adequate |

| |normal urea and electrolytes thyroid replacement. |

| |normal ECG |

| |patient fasting from midnight |

| |consent form signed |

| |i.v. 50% dextrose & i.v. hydrocortisone available |

|Protocol | |Weigh patient. Lie patient down, BP cuff one arm, IV cannula in the other; check the |

| | |pulse and BP |

| |-30 min |Blood for GH |

| | | |

| |0 min |Blood for glucose (Fluoride-EDTA sample, grey top), cortisol & GH (Serum sample, SST |

| | |tube(red/gold top)) |

| | |Give ACTRAPID 0.1 U/kg i.v.. This is most easily done by drawing up of|

| | |100U/mL actrapid, inject this into 10mL N/Saline: give one mL of this dilution. |

| | |If definite or strong suspicion of hypopituitarism give half dose, 0.05 U/kg. |

| | |Acromegalics and obese patients may need up to 0.3 U/kg |

| | | |

| | |Pulse and BP |

| |every10 min |Capillary blood glucose & laboratory glucose for the first hour |

| | |Expect symptomatic hypoglycaemia between 20-40 mins: document any physical signs |

| | | |

| | |If symptomatic with low glucomter reading, sample straight after for glucose, cortisol & |

| | |GH. Record time. |

| |at any time | |

| | |Blood for glucose, cortisol & GH |

| | | |

| |30 min |Blood for glucose, cortisol & GH |

| | |Monitor glucostix every 20 min for the 2nd hour |

| |60 min | |

| | |Blood for glucose, cortisol & GH |

| | | |

| |90 min |Blood for glucose, cortisol & GH |

| | |Test ends at 120 min |

| |120 min |Keep patients in bed for a further 2h and give them a good meal. monitor pulse & BP |

| | |every 30 min during these 2 hours |

|Interpretation |Ensure adequate hypoglycaemia was achieved, i.e. a plasma glucose ( 2.2mmol/L or classic symptoms. |

| |Cortisol response should reach a peak value of ( 550nmol/L with an increment of (200nmol/L. If pituitary or|

| |hypothalamic pathology is suspected, a response of (600nmol/L is suspicious, and an endocrinological opinion|

| |should be sought. |

| |GH should rise to a peak value of ( 10(g/L after hypoglycaemia, with an increment of ( 5(g/L over baseline, |

| |in subjects with normal pituitary function. |

Notes: A doctor must remain with the patient for all 120 min of the test.

If the patient becomes hypoglycaemic (with impaired conscious level), they should receive oral glucose (or i.v. glucose if necessary). However, the test need not be discontinued as the samples until 120 minutes will be useful.

If patient does not become hypoglycaemic, do not give extra doses of actrapid. Patient will need to be re-booked for repeat test at a higher dose.

Refs

Principles and Practice of Endocrinology and Metabolism. Ed K L Becker. Lippincott, 1995

Photocopy (or print) next page, and complete during the day: to be filed in patient’s notes.

INSULIN STRESS TEST

DATE: ……………. HOSPITAL No. …………………. ……………………

NAME ………………… ……………………

D.O.B. ………………… ……………………

CONS ………………… ……………………

ensure no epilepsy or IHD

normal urea and electrolytes

normal TFT’s

normal ECG

patient fasting from midnight

|WEIGHT = Kg |

consent form signed

i.v. 50% dextrose & i.v. hydrocortisone available

ACTRAPID (see protocol), dose given = at (time)

|elapsed time |actual time |P |BP |

|180 | | | |

|210 | | | |

|240 | | | |

ORAL LACTOSE TOLERANCE TEST

|Purpose of test |Investigation of Lactose intolerance |

|Rationale |An oral dose of Lactose is normally digested into galactose and glucose, which are absorbed leading to a slight |

| |rise in plasma glucose. Patients with a congenital or acquired deficiency of the intestinal enzyme Lactase |

| |cannot absorb the disaccharide, and little increase in plasma glucose is seen. |

|Patient preparation | Patient should fast overnight prior to test. |

|Protocol | |patient lying on bed at rest, insert i.v. cannula |

| | | |

| |0 min |blood for glucose. |

| | |Give lesser of 50g or 1.0g Kg-1 BP Lactose, dissolved in water, orally. The drink must be at |

| | |room temperature! |

| |30 min |blood for glucose |

| |45 min |blood for glucose ( Record any symptoms experienced. |

| |60 min |blood for glucose ( |

| |120 min |blood for glucose |

|Cautions |Note! |Ensure patient knows where the lavatory is. |

| | |Patient should not leave the premises until the test is complete. |

|Interpretation | | A rise of blood glucose of < 1.1mmol/L over the baseline value is suggestive of Lactase |

| | |deficiency, especially if associated with abdominal pain or diarrhoea. |

| | |Abnormal results may also occur with malabsorption: if suspected, a control test should be |

| | |repeated with an equivalent dose of oral Glucose in place of Lactose. |

| | |If blood glucose rises by 1.6mmol/L or more over baseline at any time, the test may be |

| | |discontinued. |

Ref.s:

A D Newcomer, D B McGill, P J Thomas. 1975. New Eng J Med 293: 1232.

[which demonstrates: optimal discrimination @ 45 min.; optimal cut-off ( 20mg/dl; all but one normals > 30mg/dl; the H2 breath test is more sensitive and more specific]

K Peuhkuri, H Vapaatalo, R Nevala, R Korpela. 2000. Scand J Clin Lab Invest 60: 75-80

LHRH TEST

Syn.: Gonadotrophin Releasing Hormone (GnRH) TEST

|Purpose of test |1. To differentiate primary gonadal failure from secondary hypogonadotrophic hypogonadism, when basal |

| |LH/FSH levels are not diagnostic or before puberty. |

| |2. Assessment of the degree of maturity of the hypothalamic-pituitary axis around the onset of puberty |

|Rationale |Synthetic GnRH (LHRH) stimulates the release of LH and FSH from the anterior pituitary. In primary |

| |gonadal failure, if basal LH/FSH levels are not clearly elevated already, this response is exaggerated. |

| |The responsiveness of the pituitary to GnRH is muted (but not absent) before puberty. Assessment of |

| |this responsiveness may be useful in differentiating central (CNS) causes from peripheral causes of |

| |precocious puberty, and delayed puberty. |

|Note! |Since GnRH exerts a trophic affect on pituitary gonadotrophs, hypothalamic pathology will lead to a |

| |secondary blunting of the LH/FSH response. This test therefore does not reliably differentiate between |

| |hypothalamic and pituitary disease. |

|Protocol | |Establish i.v. access, maintain cannula patency throughout. |

| | | |

| |0 min |Baseline bloods for LH, FSH, Testosterone and Oestradiol. (Serum sample. Min vol.|

| | |1.7mL) |

| | |Give 100(g/m2 (max. 100(g) LHRH i.v. bolus. |

| | | |

| |30 min |Blood for LH and FSH |

| | | |

| |60 min |Blood for LH and FSH |

| | | |

| |90 min |Blood for LH and FSH |

| | | |

| |120 min |Blood for LH and FSH, Testosterone and Oestradiol |

|Normal response |LH concentrations normally increase three to six times basal values; FSH increase is less marked, up to|

| |approx. 50% above basal values. |

| |The pituitary response is inducible, and in pre-pubertal subjects or GnRH deficiency, a normal response |

| |may be seen after several repeat GnRH tests. |

Ref.

Principles and Practice of Endocrinology and Metabolism. Ed K L Becker. Lippincott, 1995

Photocopy (or print) next page, and complete during the day.

Please send with last specimen in the series to the laboratory, for results:

LHRH TEST

DATE: ……………. HOSPITAL No. …………………. ……………………

NAME ………………… ……………………

D.O.B. ………………… ……………………

CONS ………………… ……………………

i.v LHRH: dose:………..., given at:……………

|Time of sample |Specimen No.s |Elapsed time (min.s) |LH |FSH (IU/L) |Oestradiol |Testosterone (nmol/L)|

| | | |(IU/L) | |(pmol/L) | |

| | |0 | | | | |

| | |30 | | | | |

Please send with last specimen in the series to the laboratory, for results

LIPOPROTEIN LIPASE TEST

(Post Heparin)

|Check first! |Check with the Laboratory that this test is still available. In 2019, the sole provider in Glasgow had |

| |temporarily withdrawn their assays for LPL and Apo CII |

|Purpose of test |To assess Lipoprotein Lipase activity in cases of Type V hyperlipidaemia |

|Rationale |Hypertriglyceridaemia or excess Chylomicronaemia may be due to inherited deficiency of Lipoprotein |

| |Lipase (LPL), or even less often due to inherited deficiency of Apolipoprotein CII, which is an |

| |essential co-factor for LPL. |

| |LPL is bound to the endothelial surface in fat and muscle, and normally its activity in plasma is very |

| |low. This bond is easily disrupted by Heparin, and following a single i.v. bolus free LPL will be |

| |released into the circulation. |

|Contraindications |The test should not be performed in individuals on aspirin, with a history of bleeding diatheses or |

| |sensitivity to heparin, nor with a history of CVA, rheumatic fever or if patient has peptic ulceration. |

|Preparation |Check contraindications. |

| |Overnight Fast |

| |Refrain from alcohol for 24 hours prior to test |

| |Refrain from heavy exercise for 48 hours prior to test |

| |Ice available; warn laboratory. |

|Protocol | |Weigh patient |

| | |Establish i.v. access, and flush with saline |

| | | |

| | |Baseline bloods for Lipid Profile (SST serum tube, red/gold top), and min 5mL blood|

| | |into EDTA tube (purple top) for ApoCII. |

| | | |

| |0 min |Give Heparin, 70 IU/Kg body weight, as i.v. bolus. Flush through with 10mL saline |

| | | |

| | |10mL Blood for LPL: Two LiHep samples (green/gold top), keep on ice |

| |12 min | |

| | |Test finished |

| | | |

| | |Note! Samples should be transported on ice, and must be centrifuged, and plasma |

| | |frozen within 10 minutes! Make prior arrangements with lab. to do this, on Xn |

| | |153402/153403, or 153444. |

| | | |

|Interpretaion |Normal plasma reference ranges: |

| |LPL 2 – 12 (mol FA/mL/hr |

| |ApoCII 2 – 10 mg/dL |

| |Type I hyperlipidaemia: LPL typically 200ng/l makes the diagnosis almost certain. Peak values between 30-100ng/l are |

| |considered non-diagnostic, and the test should be repeated or genetic analysis pursued. |

N.B. Consider also genetic analysis of RET oncogene for MEN II or familial MTC.

Ref.

1. Principles and Practice of Endocrinology and Metabolism. Ed K L Becker. Lippincott, 1995

2. P J Trainer, M Besser (eds). The Bart’s endocrine protocols. London: Churchill Livingstone, 1995.

3. Hammersmith Endocrine laboratory protocol, 2008.

PENTOLINIUM TEST

|Purpose of test |To confirm or exclude the presence of Phaeochromocytoma, when conventional measurement of catecholamine and |

| |metabolite excretion in urine is not possible and initial measurement of plasma catecholamines is not clearly |

| |diagnostic. |

|Rationale |Pentolinium is a ganglion blocking drug acting at the Nicotinic AchR . Administration will normally cause an |

| |acute decrease in catecholamine secretion, an affect not seen in the presence of autonomic secretion of |

| |noradrenaline or adrenaline from a tumour. |

|Patient preparation |Patient should ideally be fasted overnight. Other anti-hypertensive medication should be discontinued for 24hrs|

| |prior to test. |

| |Ensure ice tray is available for samples |

| |Ensure facilities are available to monitor the patient’s BP |

|Protocol | |Patient lying supine, at rest; insert i.v. cannula into forearm vein: wait 30 mins for return |

| | |to basal conditions. |

| | |Patient should remain supine, and be monitored throughout the test |

| | | |

| |-5 min |Blood sample for plasma catecholamines: Li Hhep plasma (green top) tube, on ice |

| | | |

| | |Blood sample for plasma catecholamines |

| |0 min |Give 2.5mg Pentolinium i.v. |

| | |Monitor patient’s pulse and BP regularly for 1hr |

| | | |

| | |Blood sample for plasma catecholamines |

| |10 min |Blood sample for plasma catecholamines |

| |20 min |Blood sample for plasma catecholamines |

| |60 min | |

| | |Note! Blood samples for catecholamines must be centrifuged, and plasma frozen within 15 |

| | |minutes! Make prior arrangements with lab. to do this, on Xn 153402/153403. |

|Cautions |Transient hypotension, is possible. Not recommended for patients with severe carotid or coronary arterial |

| |disease. |

| |Check lying/standing BP has recovered before allowing patient home. |

|Interpretation |Both Adrenaline and Noradrenaline normally suppress to 410 nmol/L proceed to DAY 4 |

| | | |if all cortisols < 410 nmol/L test has ended: patient must remain on |

| | | |hydrocortisone (but may be possible to reduce dose). |

| | | | |

| |Day 4 | |Insulin stress test with glucose and cortisol measurements. Only measure |

| | | |growth hormone if required |

|Discharge |Discharge the patient on their usual steroid therapy pending the results, with their steroid card. |

Photocopy (or print) next page, to compile all results: to be filed in patient’s notes

STEROID WITHDRAWAL PROTOCOL

DATE: ……………. HOSPITAL No. …………………. ……………………

NAME ………………… ……………………

D.O.B. ………………… ……………………

CONS ………………… ……………………

Pre-admission steroid regime:

|DAY |SAMPLE |

|Rationale |Chronic ACTH deficiency results in quiescent adrenal glands. Both this and primary adrenal failure result |

| |in an inadequate cortisol response to the exogenous ACTH analogue Synacthen (tetracosactrin, N-terminal ACTH|

| |1-24). |

|Patient preparation |None. The test may be performed as an outpatient procedure. The test may be performed at any time or day, |

| |unless ACTH is also required in which case early a.m. (after breakfast) is preferred for the peak ACTH |

| |drive. |

|Protocol | |Insert i.v.cannula; wait approx. 10 min for return to basal conditions. |

| | | |

| |0 min |Blood for basal cortisol (SST serum tube, red/gold top); if required, also 5-10 mL (min |

| | |2.5mL) blood into EDTA tube for ACTH: rush latter to lab., to be frozen within 10 mins. |

| | |If Congenital Adrenal Hyperplasia is suspected, separate serum sample for 17-hydroxy |

| | |progesterone also. |

| | |Give Synacthen 250 (g i.m. or i.v. |

| | |(The endocrinologists will sometimes request a “1.0(g Synacthen” test: transfer all of |

| | |the 250(g vial into a 250mL bag of N/Saline and mix well. Inject 1.0mL of this dilution |

| | |i.v. Otherwise the protocol is the same.). |

| |30 min |Blood for cortisol. If required, separate serum sample for 17OH-prog. |

| |60 min |Blood for cortisol. If required, separate serum sample for 17OH-prog. |

|Sample handling |The samples for serum cortisol and 17OH-Prog. need no special handling. Ensure the 3 tubes are clearly |

| |identifiable with regard to sequence/time. |

| |Samples for ACTH must be collected into plastic EDTA (purple top) tubes, use no glass. ACTH has a very |

| |short half-life, samples should be frozen within 10 minutes: make prior arrangements with the laboratory to|

| |do this. (WPH Xn 153403, 153444, 153447) |

|Assay arrangements |Cortisols are analyzed mostly on the next working day after receipt in the laboratory |

| |The ACTH samples will be held frozen in the laboratory for 1 month: they will only be referred for ACTH |

| |assay if the cortisol response is abnormal. Return of results from referral laboratory may take up to 4 |

| |weeks. |

|Interpretation | A basal cortisol concentration ( 450nmol/L makes the diagnosis of primary or secondary adrenal failure very|

| |unlikely. |

| |The Roche immunoassay for cortisol currently in use in the laboratories in Berkshire has a modest negative |

| |bias compared to other assays. A normal response is defined as a 30 min. peak of ( 410nmol/L. There should|

| |also normally be an increment over basal of ( 200nmol/L. 60min values >445nmol/L are considered normal |

| |(locally derived reference values). |

| |If secondary adrenal failure (pituitary/hypothalamic dysfunction) is suspected, a 30 min peak ( 500nmol/L is|

| |suspicious and warrants further endocrine assessment. |

| |The 60 min values are less reliable in diagnosis: if 30 min values are borderline with an adequate response|

| |at 60 min, patients should be referred to an endocrinologist. |

| |High ACTH values (ref. Range 10 – 80ng/L) at the same time as a low cortisol, and poor response confirms |

| |primary adrenal failure. |

| |17-OH prog. increases modestly in normal individuals (increment (10 nmol/L), to less than twice basal levels|

| |at 60 mins. In patients with CAH there is an exaggerated response ((20nmol/L) to clearly abnormal |

| |concentrations. Heterozygotes for 21-OH’ase deficiency show an intermediate response, with considerable |

| |overlap with the normal range. |

Refs:

1. S J Hure. Short Synacthen test and insulin stress test in the assessment of the hypothalamic pituitary adrenal axis. 1996 Clin Endocrin 44: 141-146.

2. F Amari, et al. A comparison between short synacthen test and insulin stress test for assessing hypothalamic pituitary adrenal function. 1996 Clin Endocrin 44: 473-476.

3. A M Wallace. Analytical support for the detection and treatment of Congenital Adrenal Hyperplasia. 1995 Ann Clin Biochem 32: 9-27.

4. BIO-WPH-VAL-003: Deriving the lower reference interval for 30 minute cortisol in the Short Synacthen test (WPH) (version 1.0). Nov 2015.

LONG SYNACTHEN TEST

|Purpose of test |to assess adrenal reserve in those patients who fail to respond during a short synacthen test, and have |

| |been taking exogenous steroids |

|Rationale |patients with primary adrenal failure do not respond to synacthen |

| |patients with secondary adrenal failure (pituitary pathology or after treatment with exogenous steroids) |

| |may have a delayed response. |

|Patient preparation |None. Patient should not be taking any oral steroids; if this means discontinuing long term steroid |

| |prescription, patient must be monitored in hospital throughout the test. If necessary, steroid cover with|

| |Dexamethasone may be given, which does not interfere with the assay. |

|Protocol | |Insert i.v. cannula and flush regularly with Hepsal |

| | | |

| |0 min |Blood for cortisol (SST tube, serum sample) |

| | |give Synacthen depot 1 mg i.m. |

| | | |

| |1 h |Blood for cortisol |

| | | |

| |4 h |Blood for cortisol |

| | | |

| |8 h |Blood for cortisol |

| | | |

| |24 h* |Blood for cortisol |

| | | |

| |48 h* |Blood for cortisol |

|Note: |*Further injections of depot Synacthen (1 mg) may be given every 24 hours for 3 days, if the cortisol |

| |response is inadequate. |

|Predicted responses |The normal response is a brisk rise in serum cortisol: 95% confidence intrervals:- |

| |at 1hr 605 – 1265 nmol/L |

| |at 4hr 960 – 1650 nmol/L |

| |at 8hr 1025 – 1600 nmol/L |

| |at 24hr 610 – 1500 nmol/L |

| |In primary adrenal failure cortisol remains low < 300 nmol/L throughout the test, with an increment < |

| |10% over baseline |

| |In secondary adrenal failure cortisol levels remain low for the first 4 - 6 hours and then rise to a |

| |maximum level > 550 nmol/L. The difference between primary and secondary adrenal failure increases with |

| |further doses of synacthen. |

| |Note this does not test the “top end”, ACTH reserve. |

Refs:

1. P J Trainer, M Besser (eds). The Bart’s endocrine protocols. London: Churchill Livingstone, 1995.

2. C R W Edwards. Adrenocortical diseases. In D J Weatherall, J G G Ledingham, D A Warrell (eds) Oxford textbook of medicine, 3rd edn. Oxford: OUP, 1996.

Supervised THYROXINE ABSORPTION TEST

|Purpose of test |Used to assess patients with persistently low serum levels of thyroid hormones, despite prescribed high |

| |doses of l-Thyroxine. To differentiate problems with absorbance of thyroxine, from erratic compliance. |

|Rationale |After an adequate oral dose of l-Thyroxine, serum levels of free-T4 rise to normal within 90-120 minutes. |

|Patient preparation |Exclude: |

| |confounding medications or dietary factors (eg oral iron, calcium, espresso, etc..) |

| |Malabsorption states, eg Coeliac, Lactose intolerance, achlorhydria. |

| |Obvious non-compliance (failure to regularly renew prescriptions) |

| |Assay interference |

| |Check no contraindications to high-dose Thyroxine – heart disease, frail elderly. Perform ecg |

| |Patient must be fasting from midnight. |

| |Record patient’s weight |

| |Empty bladder immediately prior to test commencing (to allow uninterrupted observation for 1 hour) |

|Protocol | |Insert i.v. cannula and flush with Hepsal. |

| | | |

| | |Prepare oral dose of l-Thyroxine: 1.6 x weight (Kg) x 7 (g. Round off to nearest |

| | |50(g. The tablets can be dispersed in a small amount of water – e.g. 20-30 mL. |

| | |Under direct supervision, patient drinks this oral dose of l-Thyroxine, followed by|

| | |200mL water. |

| | | |

| | |Continue to observe patient for 60 minutes. Patient must not eat during this first|

| | |hour. |

| | | |

| | |Blood for TSH, FT4 and FT3 (SST serum tube, red/gold top) |

| |0 min |Blood for TSH, FT4 and FT3 |

| |30 min |Blood for TSH, FT4 and FT3 |

| |45 min |Blood for TSH, FT4 and FT3 |

| |60 min |Blood for TSH, FT4 and FT3 |

| |90 min |Blood for TSH, FT4 and FT3 |

| |120 min |Blood for TSH, FT4 and FT3 |

| |240 min |Blood for TSH, FT4 and FT3 |

| |360 min | |

|Interpretation |FT4 and TSH normalize by end of test: suggests previous non-compliance. Explore reasons why, and |

| |consider supervised weekly dosing. |

| |Inadequate rise in FT4: Investigate for causes of malabsorption. |

| |Consistent rise in FT4 post dose, but no change in TSH levels after repeated dosing: consider dose |

| |adjustment, and/or further investigation into disorders of Thyroid hormone metabolism |

Refs:

1. O Koulouri, C Moran, D Halsall, K Chatterjee, M Gurnell. Pitfalls in the measurement and interpretation of thyroid function tests. Best Practice & Research Clinical Endocrinology & Metabolism, 2013. 27 (6): 745.

Supervised THYROXINE ABSORPTION TEST

DATE: ……………. HOSPITAL No. …………………. ……………………

NAME ………………… ……………………

Weight: …..……Kg D.O.B. ………………… ……………………

CONS ………………… ……………………

l-Thyroxine: dose:…………...., given at:……………

|Time of sample |Specimen No.s |Elapsed time (min.s) |TSH |FT4 |FT3 (pmol/L) |

| | | |mU/L |(pmol/L) | |

| | |0 | | | |

| | |30 | | | |

| | |45 | | | |

| | |60 | | | |

| | |90 | | | |

| | |120 | | | |

| | |240 | | | |

| | |360 | | | |

Please send with last specimen in the series to the laboratory, for results

WATER DEPRIVATION TEST

|Purpose of study |Investigation of polyuria (urine output > 3 l per 24h). |

| |To differentiate primary polydipsia, cranial diabetes insipidus (DI) and nephrogenic DI. |

|Rationale |Depriving subject of any fluid intake for up to eight hours should stimulate ADH secretion and thus a small |

| |volume, concentrated urine. Plasma osmolality should be maintained within the normal range. |

|Preparation |Warn the Laboratory several days before, so that staffing can be arranged to support the testing required. |

| |Ensure no adrenocortical or thyroid deficiency. |

| |Stop DDAVP/ lysine vasopressin 8 h before hand in patients already on treatment. |

| |Other anterior pituitary hormone replacement continued throughout. |

| |Encourage fluid intake overnight before the test. Patient should not fast prior to test. |

| |No coffee, tea or cigarettes from midnight. |

|Protocol | |Patient to be carefully supervised throughout. |

| | | |

| |08:00 h |Light breakfast |

| | |Insert IV cannula |

| | | |

| |08:30 h |Weigh patient, patient empties bladder, measure urine volume and send for osmolality (ask for|

| | |result of this to be telephoned back (Xn 153402/153403) as the test can be terminated if |

| | |urine osmolality > 750 before fluid deprivation commences). |

| | |Take blood for U/E’s, creat and osmolality (LiHep plasma sample, green/gold top) |

| | |No fluids for 8 h (watch carefully). Give dry food freely. |

| | | |

| | |Weigh patient hourly - test to be discontinued if > 3% body weight is lost |

| | |Monitor pulse and BP hourly |

| |hourly |Hourly bloods - 10 ml into Lithium heparin for sodium and osmolality |

| | |Hourly urine samples for osmolality and record volume on chart or in the notes |

| | |Send each pair of blood and urine samples to lab immediately after taking – ensure all |

|Note! Day unit finishes at | |samples are identified with their time. |

|1700pm, arrangements for these| | |

|samples must be made with the | |Give desmopressin 40 (g intranasally or 2 (g i.m.. Free fluids now allowed |

|duty H.O. | | |

| |16:30 h |Urine volume and osmolality |

| | | |

| |19:30 h |Urine volume and osmolality |

| | |Blood for plasma sodium and osmolality |

| |21:30 h | |

| | |Stop test if Urinary osmolality ever greater than 800. |

|Interpretation | |Urine osmolality > 750 mmol/kg excludes cranial DI, especially if the plasma osmolality |

| | |remains < 295 mmol/kg. |

| | |Hypotonic urine after dehydration followed by urine osmolality > 650 mmol/kg after exogenous |

| | |DDAVP suggests cranial DI |

| | |Hypotonic urine after dehydration with no response to DDAVP suggests nephrogenic DI. |

| | |Submaximal urine concentration (500 - 700 mmol/kg) with no response to DDAVP suggests |

| | |compulsive drinking or partial DI. |

Note! For children, the paediatricians will often use an alternate protocol, which commences in the evening and the child is deprived of water overnight. Consult with the child’s consultant.

Photocopy (or print) next page, and complete during the day: to be filed in patient’s notes.

|DATE: |……………. | | |HOSPITAL No. |…………………. |…………………… |

| | | | | |NAME |………………… |…………………… |

| | | | | |D.O.B. |………………… |…………………… |

| | | | | |CONS |………………… |…………………… |

| | | | | | | | |

| | | | | | | | |

| | |WATER DEPRIVATION TEST | | | |

| | | | | | | | |

| |Patient preparation as per protocol: | | | |

| | |No coffee, tea, or smoking from midnight | | |

| | |Patient does not need to fast prior to test | | |

| | | | | | | | |

| | | | | | | | |

| | | |= baseline weight less 3% | | |

|TIME |ACTUAL TIME |WEIGHT |HR |BP |URINE VOLUME |URINE OSMOLALITY |PLASMA OSMOLALITY |

|8:30 | |Kg | | |mL | | |

|9:30 | | | | | | | |

|10:30 | | | | | | | |

|11:30 | | | | | | | |

|12:30 | | | | | | | |

|13:30 | | | | | | | |

|14:30 | | | | | | | |

|15:30 | | | | | | | |

|16:30 | | | | | | | |

|17:30 | | | | | | | |

|18:30 | | | | | | | |

|19:30 | | | | | | | |

|20:30 | | | | | | | |

|21:30 | | | | | | | |

| | | | | | | | |

| | | | | | | | |

| | |DDAVP given at |(state time) |- |patient then allowed to drink. |

| | | | | | | | |

XYLOSE ABSORPTION TEST

|Purpose of test |Investigation of suspected malabsorption. |

|Rationale |The absorptive capacity for this pentose in the proximal small intestine is much smaller than for other |

| |sugars, and therefore it is more susceptible to malabsorption in upper G.I. pathology. About half of the |

| |D-xylose absorbed is excreted unchanged in the urine and can be measured, the remainder is metabolised or |

| |excreted in bile. |

|Preparation |Overnight fast. |

| |Order D-Xylose from pharmacy at least one day before the test. |

| |Collect Urine container with benzoic acid preservative from the laboratory. |

|Protocol | | |

| |time 0 |Empty bladder, discard. |

| | |Give 5g D-Xylose in a glass of water (children 10g m-2, max 5g). |

| | |Patient may drink freely, but no food. |

| | | |

| | |Collect all urine over the next 5 hours into the special container. |

| | | |

| |[1 h |Optional blood sample for Xylose (Fluoride oxalate (grey-top) sample)] |

| | | |

| | |Empty bladder, add to collection. Note start and finish times on the container, |

| |5 h |and deliver to laboratory with request form. |

|Interpretation |At least 23 % of the administered dose (1.1g / 7.3mmol) should appear in the urine over 5 hours. |

| |If urine is difficult to collect in children, or there is significant renal impairment, the blood xylose |

| |60 min post oral dose should be at least 1.33mmol/l. |

| |Values below these figures suggest significant carbohydrate malabsorption. |

|Note: |The test has a significant false-positive (20-50%) and false negative rate (20-30%). Results may be |

| |affected by ascites, intestinal hurry or bacterial overgrowth, or renal impairment. |

| |Its use has largely been eclipsed by anti-gliaden antibody tests and direct endoscopic biopsy. |

Ref.

M H Z Labib & B J M Jones. The assessment of gastrointestinal function. In D L Williams & V Marks (eds) Scientific foundations of Biochemistry in Clinical Practice, 2nd edn. Oxford: Butterworth-Heinemann, 1994.

DOCUMENT CONTROL

|TITLE |PROTOCOLS FOR DYNAMIC TESTS AND ENDOCRINE |

| |INVESTIGATIONS |

|FOR |Frimley Health Trust, WPH site |

|DATE |January 2020 |

|(Please destroy previous version, November 2017) |

|NEXT REVISION |January 2021 |

|AUTHORISED |Dr I A L Walker |

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