Author: Dr Mandy Pickersgill/Dr Kathryn Brownbill Date: July 2013 Version: 1 Authorised: A Pickersgill Review date: July 2015

Transcription

1 Author: Dr Mandy Pickersgill/Dr Kathryn Brownbill Date: July 2013 Version: 1 Authorised: A Pickersgill Review date: July 2015 Adult DFT Protocols Authorised: July 2013

2 Table of contents Short Synacthen Test... 3 Cortisol Day Curve... 5 Overnight Dexamethasone Suppression... 7 Low dose 48 hour Dexamethasone Suppression Test... 9 Short Synacthen Test with 17-hydroxyprogesterone Thyrotropin Releasing Hormone (TRH) Test Arginine Stimulation Test for Growth Hormone Glucagon Stimulation Test for Growth hormone Oral Glucose Tolerance test (OGTT) with Growth Hormone GnRH Stimulation Test for LH and FSH Oral Glucose Tolerance Test Prolonged Oral Glucose Tolerance Test hour Prolonged Fast Hypoglycaemia monitoring chart for 72 hour fast Lactose Tolerance Test Water Deprivation Test Water Deprivation Test Template Water Deprivation Test Part 2:

3 Short Synacthen Test Principle Adrenal glucocorticoid secretion is controlled by adrenocorticotrophic hormone (ACTH) released by the anterior pituitary. The Short Synacthen Test evaluates the ability of the adrenal cortex to produce cortisol after stimulation by synthetic ACTH (tetracosactide; Synacthen ). It does not test the whole pituitary-adrenal axis. Indications The investigation of adrenal insufficiency (primary or secondary). This test alone can not distinguish between primary and secondary adrenal insufficiency. The test is not required if a random cortisol (in a non-stressed individual) is > 450nmol/l Contraindications Results are unreliable within 2 weeks of pituitary surgery. Patient Preparation Glucocorticoid replacement on the day of the test invalidates the test. Prednisolone should be stopped 24 hours before the test. The final dose of hydrocortisone prior to the test should be taken at midday on the day before Where possible consider stopping Oestrogen containing medication 6 weeks prior to the test, otherwise document details of this medication with the test request. Side Effects There are rare reports of hypersensitivity reactions to Synacthen particularly in patients with history of allergic disorders. Requirements 250 µg Synacthen (1 vial) 2 brown gel tubes Procedure This test should be performed preferably in the morning between 0800 and 0900 hours as the cortisol responses in the morning and afternoon may differ by as much as 100nmol/l. Minutes Procedure Sample 0 Take 5ml blood for Cortisol, label with patients name and time of collection and then administer 250µg Synacthen i/m immediately after (note time of injection on form) 30 Take 5ml blood for Cortisol, label with patients name and time of collection Interpretation of results Adrenal insufficiency is excluded by a 30 min value > 550 nmol/l. 3 1 x brown gel tube (cortisol) 1 x brown gel tube (cortisol)

4 An increment above 200 nmol/l may also be considered as normal. The increment in cortisol concentration after Synacthen is an unreliable index of adrenal function, but may occasionally merit consideration (All Wales Clinical Biochemistry Audit Group Standards for the Performance and Interpretation of the Short Synacthen Test in investigating suspected Adrenocortical Insufficiency, 2005; Grinspoom & Biller, 1994; Dorin et al, 2003). The increment should not be used in critically ill patients (Arafah, BM, 2006) References Arafah BM (2006); REVIEW: Hypothalamic Pituitary Adrenal Function during Critical Illness: Limitations of current assessment methods. JCEM 91, Dorin RL, Qualls CR, Crapo LM (2003): Diagnosis of adrenal insufficiency. Ann Intern Med 139, Grinspoon SK & Biller BM (1994): Laboratory Assessment of Adrenal Insufficiency. JCEM 79,

5 Cortisol Day Curve Indications: Establishment of the correct dose and distribution through the day of the replacement dose of hydrocortisone (n.b. this has no value in patients taking prednisolone). Some hepatic enzyme inducers such as Rifampicin, Phenobarbitone and Phenytoin will increase clearance of hydrocortisone and may lead to problems with maintenance therapy. Samples are taken immediately prior to a medication dose and then approximately 1 hour post dose when cortisol levels are documented to have peaked (Howlett TA, 1997) Contra-indications None Requirements Procedure IV cannula Patient s replacement medication (hydrocortisone/cortisone acetate) Brown top serum tubes PATIENT PREPARATION TEST Where possible consider stopping Oestrogen containing medication 6 weeks prior to the test, otherwise document details of this medication with the test request Time Procedure Samples On patient Take sample for cortisol 1 x brown top serum tube Arrival (7.30- Pre dose sample 1 8am) 08:00 Patient should take normal morning dose of replacement medication 08:30 Insert IV cannula 09:00 Take sample for cortisol 1 x brown top serum tube Post dose Sample 1 12:30 Take sample for cortisol 1 x brown top serum tube Pre dose sample 2 14:00 Patient should take afternoon Please note, this time may dose of replacement vary between patients medication 15:00 Take sample for Cortisol 1 x brown top serum tube Post Dose Sample 2 17:30 Take sample for cortisol 1 x brown top serum tube Pre dose sample 3 18:00 Patient should take evening dose of medication 19:00 Take sample for Cortisol 1 x brown top serum tube Post dose sample 3 5

6 Interpretation of results 09:00 cortisol should be in range of nmol/l. The 12:30 and 17:30 value should be >100 nmol/l. References Howlett TA (!997): An assessment of optimum hydrocortisone replacement therapy. Clin Endocrinol (Oxf) 46;

7 Overnight Dexamethasone Suppression Screening test for Cushing s Syndrome Principle In normal subjects, dexamethasone suppresses ACTH and therefore cortisol secretion. In Cushing s syndrome, there is incomplete suppression. Individuals suffering from depression, obesity or systemic illness may not suppress using this screening test and further tests may be indicated. Indications This should be used as a first line screening test for all subjects suspected of having Cushing s syndrome. Contraindications Patients on enzyme inducing drugs e.g. anti-convulsants may rapidly metabolise dexamethasone. Oestrogens (e.g. pregnancy, HRT or OCP) may induce cortisol binding protein and artefactually increase total cortisol levels. Urine collection for 24 hr urinary free cortisol must not occur during or on the day following this test. Side Effects None Requirements 1 mg dexamethasone (available as 2 x 0.5mg tablets) 1 brown top serum tube Procedure Patient preparation Where possible consider stopping Oestrogen containing medication 6 weeks prior to the test, otherwise document details of this medication with the test request. Give 1mg dexamethasone tablet to be taken orally, at At 9am the following morning, collect 5mls of blood into a brown gel tube for serum cortisol. Interpretation of results A normal response is shown by suppression of 0900 h cortisol to < 50 nmol/l. 7

8 Normal subjects rarely (2%) fail to suppress with overnight dexamethasone unless they are depressed (10-50%), obese (10%) or systemically unwell (10-20%) (Crapo, 1979; Wood et al, 1997) It is recommended that the results of this test are confirmed with a second test either collection of Urine free cortisol (at least two collections) or a low dose 48 hour suppression test (see next section) (Nieman et al, 2008) References Crapo L (1979): Cushing s Syndrome: a review of diagnostic tests. Metabolism 28, Nieman LK,. Biller BMK, Findling JW, Newell-Price J, Martin O. Savage MO, Stewart PM & Montori VM (2008):The Diagnosis of Cushing s Syndrome: An Endocrine Society Clinical Practice Guideline JCEM 93, Raff H & Findling JW (2003): A physiologic approach to Diagnosis of Cushing Syndrome. Ann Intern Med 138, Wood P, Barth JH, Freedman DB, Perry L & Sheridan B (1997): Evidence for the low dose dexamethasone suppression test to screen for Cushing s Syndrome recommendations for a protocol for UK Biochemistry laboratories. Ann Clin Biochem 34,

9 Low dose 48 hour Dexamethasone Suppression Test Screening test for Cushing s Syndrome Indications An alternative Screening test for Cushing's syndrome. Can be used if the result of the overnight suppression test contradicts other investigations. This test offers similar sensitivity and specificity to the overnight dexamethasone test (Raff & Findling, 2003) and may be used to confirm the results of the overnight test. The addition of ACTH measurements may also provide clues to the differential diagnosis. Contraindications Patients on enzyme inducing drugs e.g. anti-convulsants may rapidly metabolise dexamethasone. Oestrogens (e.g. pregnancy, HRT or OCP) may induce cortisol binding protein and artificially increase total cortisol levels. Care in diabetes mellitus and patients who are psychologically unstable. Side Effects None Requirements A total of eight doses of dexamethasone should be written up (9am, then 3pm, 9pm, 3am, 9am, 3pm, 9pm, 3am must adhere to the 6-hourly dosing frequency, especially important not to omit or delay the 3am dose) Adult dose 0.5mg, children 20µg/kg. 2 x brown serum tubes for cortisol 2 x Lithium Heparin Green top plasma tubes for ACTH which must be sent on iced slurry. Procedure Patient Preparation: Where possible, stop all oral oestrogen therapy 6 weeks prior to test. Patients on sex steroid implants might generate results that are difficult to interpret. Measuring SHBG and CBG might be helpful in this circumstance. The test: At take basal samples for cortisol (brown top serum tubes) and ACTH (lithium heparin tube send directly to the lab on ice) Give the patient 0.5mg Dexamethasone at 9am, 3pm, 9pm and on the following day at 3am, 9am, 3pm, 9pm and 3am. At 9am on day 2 (T+48 hrs) take a further sample for cortisol (brown top serum tube) and ACTH (lithium heparin tube send directly to the lab on ice) 9

10 Interpretation of results If the 9am cortisol value on day 2 is less than 50nmol/l the patient has shown suppression (Neiman et al, 2008; Raff & Findling, 2003; Wood et al, 1997) Patients with Cushing s syndrome, from whatever cause, lose the normal negative feedback control by circulating glucocorticoids on ACTH release and thus exhibit detectable plasma ACTH and cortisol concentrations after dexamethasone administration. References Nieman LK,. Biller BMK, Findling JW, Newell-Price J, Martin O. Savage MO, Stewart PM & Montori VM (2008):The Diagnosis of Cushing s Syndrome: An Endocrine Society Clinical Practice Guideline JCEM 93, Raff H & Findling JW (2003): A physiologic approach to Diagnosis of Cushing Syndrome. Ann Intern Med 138, Wood P, Barth JH, Freedman DB, Perry L & Sheridan B (1997): Evidence for the low dose dexamethasone suppression test to screen for Cushing s Syndrome recommendations for a protocol for UK Biochemistry laboratories. Ann Clin Biochem 34,

11 Short Synacthen Test with 17-hydroxyprogesterone Principle Adrenal glucocorticoid secretion is controlled by adrenocorticotrophic hormone (ACTH) released by the anterior pituitary. In subjects with an enzyme deficiency in the steroid synthetic pathway (leading to congenital adrenal hyperplasia), there is excessive secretion of the precursor steroids before the defective enzyme. The commonest form of CAH is due to deficiency of 21-hydroxylase and in these subjects increased secretion of 17 OH-progesterone can be detected. This secretion is exaggerated in response to stimulation with Synacthen which forms the basis of this test. Indications This test is usually performed for the investigation of late onset congenital adrenal hyperplasia (CAH) or suspected heterozygosity of CAH in children and adults. Contraindications The Synacthen test gives unreliable results within 2 weeks of pituitary surgery. Test is usually not required if an early morning 17-OHP, taken during the follicular phase, is < 6 nmol/l. (Armengaud et al, 2009; Azziz et al, 1999) Side Effects There are rare reports of hypersensitivity reactions to Synacthen particularly in patients with a history of allergic disorders. Requirements Procedure 2 brown top tubes 250 microgram Synacthen (1 vial) This test should be performed preferably in the morning between 0800 and 0900 hours but can be performed later in the day. Minutes Procedure Sample 0 Take sample for Cortisol and 17 OHP and 2 x brown gel tubes then administer 250µg Synacthen i/m (note time of injection on form) 30 Take sample for Cortisol and 17 OHP 2 x brown gel tubes Label with patients name and time of sample 60 Take sample for Cortisol and 17 OHP 2 x brown gel tubes Label with patients name and time of sample 11

12 Interpretation of results 17 OHP at 60 mins Interpretation (nmol/l) < 30 Normal response rules out homozygosity for CAH. There may some carriers of CYP21 mutations within this group (Bachega et al, 2002) Results suggestive of late onset CAH Genotyping may be required to distinguish heterozygotes from homozygotes (Bidet et al, 2009; Pang, 1997) > 50 Diagnostic of late onset CAH (homozygotes) (Merke & Bornstein, 2005; White & spencer, 2000) References Armengaud JB, Charkaluk ML, Trivin C, Tardy V, Bréart G, Brauner R, Chalumeau M. (2009) Precocious pubarche: distinguishing late-onset congenital adrenal hyperplasia from premature adrenarche. J Clin Endocrinol Metab. 94: Azziz R, Hincapie LA, Knochenhauer ES, Dewailly D, Fox L, Boots LR. (1999) Screening for 21-hydroxylase-deficient nonclassic adrenal hyperplasia among hyperandrogenic women: a prospective study. Fertil Steril. 72: Bachega TA, Brenlha EM, Billerbeck AE, Marcondes JA, Madureira G, Arnhold IJ, Mendonca BB (2002):. Variable ACTH-stimulated 17-hydroxyprogesterone values in 21-hydroxylase deficiency carriers are not related to the different CYP21 gene mutations. JCEM. 87(2): Bidet M, Bellanné-Chantelot C, Galand-Portier MB, Tardy V, Billaud L, Laborde K, Coussieu C, Morel Y, Vaury C, Golmard JL, Claustre A, Mornet E, Chakhtoura Z, Mowszowicz I, Bachelot A, Touraine P, Kuttenn F (2009): Clinical and molecular characterization of a cohort of 161 unrelated women with nonclassical congenital adrenal hyperplasia due to 21-hydroxylase deficiency and 330 family members. JCEM 94: Merke DP, Bornstein SR (2005): Congenital adrenal hyperplasia. Lancet 365: Pang S (1997): Congenital adrenal hyperplasia. Endocrinol Metab Clin North Am. 26: White PC, Speiser PW (2000): Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev. 21:

13 Thyrotropin Releasing Hormone (TRH) Test Indication To assess TSH reserve. Differential diagnosis of pituitary and hypothalamic causes of TSH deficiency. This test is no longer required for the diagnosis of hyperthyroidism as the sensitivity of TSH assays is sufficient to make the diagnosis. Contraindications Test is rarely used in patients who are receiving thyroxine as they will need to be off thyroxine for 3 weeks prior to this test. Side Effects Patients should be warned that they may have transient side effects after the injection such as a metallic taste in the mouth, flushing and mild nausea, and should be on a recliner or bed. Requirements 200 µg TRH in 2mls saline (for injection) i.v. cannula 3 x brown top tubes Procedure Patient preparation No specific preparation required but patient s must not be taking thyroxine (see contraindications). Fasting is not necessary. Test Site indwelling cannula. Take 5mls of blood into a brown top tube for TSH and FT4 analysis. Inject TRH slowly i.v. over 2 minutes. Flush butterfly with heparin/saline. Take samples for TSH at t = 20 mins and 60 mins. Interpretation The normal result is a TSH rise to >5 mu/l with the 20 min value exceeding the 60 min value. If the 60 min sample exceeds the 30 min value then this usually indicates primary hypothalamic disease. In hyperthyroidism, the TSH remains suppressed and in hypothyroidism there is an exaggerated response. With the current sensitive TSH assays basal levels are now adequate and dynamic testing is not usually needed to diagnose hyperthyroidism. 13

14 Arginine Stimulation Test for Growth Hormone Principle Arginine stimulates growth hormone secretion in healthy individuals but response will be impaired in hypopituitarism. Indications 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 or has diabetes mellitus 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 <5 µg/l), and 2nd confirmatory test is needed before commencing GH treatment, especially if overweight or ITT contraindicated such as after traumatic brain injury (Molitch et al, 2011) Side effects and Precautions Some adolescents may need sex hormone priming before this test. Please check with the requesting doctor. Arginine can cause nausea and some irritation at the infusion site and the patient should be made aware of this. Arginine can cause vasospasm so sampling may be difficult if only one cannula is used. For this reason large veins should be selected. Requirements Ensure the L-arginine hydrochloride 10% in mls (0.5g/kg max dose 30 g) normal saline is prescribed and ordered from pharmacy prior to the patient's admission. 6 brown top serum tubes. Procedure Patient preparation If the patient is on growth hormone replacement, this should be stopped for one month before testing. Fast the patient overnight before the test (water is allowed). Weigh the patient and document accurately in the medical notes. 14

15 The test 1. Insert a reliable cannula and proceed as follows: Minutes Procedure Samples -30 Take blood for Growth Hormone 1 x Brown top serum tube Label this sample as BASELINE Begin infusion of L-Arginine over 30 minutes 0 (end of infusion) Take sample for growth hormone Label this sample POST INFUSION +30 Take sample for growth hormone Label this sample 30 MINS +45 Take sample for growth hormone Label this sample 45 MINS +60 Take sample for growth hormone Label this sample 60 MINS +90 Take sample for growth hormone Label this sample 90 MINS +120 Take sample for growth hormone Label this sample 120 MINS 1 x Brown top serum tube 1 x Brown top serum tube 1 x Brown top serum tube 1 x Brown top serum tube 1 x Brown top serum tube 1 x Brown top serum tube Interpretation of results Adults: GH should rise to at least 5.8 µg/l. GH levels of <3 µg/l suggest severe growth hormone deficiency References Molitch ME, Clemmons DR, Malozowski S, Merriam GR and Vance ML (2011): Evaluation and Treatment of Adult Growth Hormone Deficiency: An Endocrine Society Clinical Practice Guideline. JCEM 96,

16 Glucagon Stimulation Test for Growth hormone Principle Glucagon increases blood glucose which causes insulin release and indirectly stimulates GH and ACTH release through provocation of the hypothalamic-pituitary axis. Indications Assessment of growth hormone reserve Contraindications Phaeochromocytoma or insulinoma (may provoke an attack) Starvation >48 hours or glycogen storage diseases (inability to mobilise glycogen may result in hypoglycaemia) Severe hypocortisolaemia (09:00h level <100 nmol/l) Thyroxine deficiency may reduce GH and cortisol response. This test is unreliable in patients with Diabetes Mellitus Side Effects Glucagon may cause nausea, vomiting and abdominal pain Requirements 6 yellow top fluoride oxalate tubes 6 brown top serum tubes Procedure Patient Preparation Systemic steroids should be stopped 24 hours before the test. Fast from midnight. Calculate glucagon dose: adults: 1 mg, (1.5mg if >90kg), Test Minutes Procedure Samples -30 Insert an indwelling cannula 0 Take basal samples for glucose and GH 1 x brown top serum (GH) 1 x yellow top fluoride oxalate (glucose) 0 Give the glucagon im 90 Take samples for glucose and GH. 120 Take samples for glucose and GH x brown top serum (GH) 1 x yellow top fluoride oxalate (glucose) 1 x brown top serum (GH) 1 x yellow top fluoride oxalate (glucose)

17 150 Take samples for glucose and GH. 180 Take samples for glucose and GH. 210 Take samples for glucose and GH. 240 Take samples for glucose and GH. 1 x brown top serum (GH) 1 x yellow top fluoride oxalate (glucose) 1 x brown top serum (GH) 1 x yellow top fluoride oxalate (glucose) 1 x brown top serum (GH) 1 x yellow top fluoride oxalate (glucose) 1 x brown top serum (GH) 1 x yellow top fluoride oxalate (glucose) Interpretation of results Adults: GH should rise to at least 5.8 µg/l. GH levels of <3 µg/l suggest severe growth hormone deficiency Molitch ME, Clemmons DR, Malozowski S, Merriam GR and Vance ML (2011): Evaluation and Treatment of Adult Growth Hormone Deficiency: An Endocrine Society Clinical Practice Guideline. JCEM 96, Yuen KCJ (2011): Glucagon Stimulation Testing in Assessing for Adult Growth Hormone Deficiency: Current Status and Future Perspectives ISRN Endocrinology Vol 2011,

18 Oral Glucose Tolerance test (OGTT) with Growth Hormone Diagnosis of Acromegaly Principle GH secretion is part of the counter-regulatory defence against hypoglycaemia and physiological GH secretion is inhibited by hyperglycaemia. In acromegaly, or gigantism, GH secretion is autonomous and does not suppress and may paradoxically rise with hyperglycaemia. Indications This is the gold standard investigation to establish the biochemical diagnosis of acromegaly or gigantism. This test is also used to assess response to medical/surgical treatment of acromegaly. The test should be carried out in those patients who have demonstrated an elevated level of IGF-1 and are exhibiting clinical features of acromegaly (Giustina et al, 2010; Lugo et al, 2012; Melmed et al, 2009) Side Effects Some subjects feel nauseated and may have vaso-vagal symptoms during this test. Requirements POLYCAL (contains 75g of glucose) plus special dilution beaker supplied by Biochemistry 5 x Yellow top fluoride oxalate vacutainers, 5 x serum brown top tubes, 1x extra brown top serum tube for IGF-1 analysis. Indwelling cannula. Procedure Patient Preparation The patient should fast overnight (10-14 hours) and should rest throughout the test Test Minutes Procedure 0 Insert cannula and take samples for growth hormone, glucose and IGF-1 Give patient Polycal solution to drink over 5 minutes followed by 100 mls water 30 Take samples for Growth Hormone and Glucose 60 Take samples for Growth Hormone and Glucose 90 Take samples for Growth Hormone and Glucose 120 Take samples for Growth Hormone and Glucose Sample 1 x yellow top fluoride oxalate (glucose) 1 x brown top serum (growth hormone) 1 x brown top serum (IGF-1) 1 x yellow top fluoride oxalate (glucose) 1 x brown top serum (growth hormone) 1 x yellow top fluoride oxalate (glucose) 1 x brown top serum (growth hormone) 1 x yellow top fluoride oxalate (glucose) 1 x brown top serum (growth hormone) 1 x yellow top fluoride oxalate (glucose) 1 x brown top serum (growth hormone) 18

19 Label all samples with patients name and time of sample and send to the laboratory together. Interpretation of results Normal subjects will exhibit suppression of GH to <0.7 µg/l, However, the consensus statement of 2009 suggests that suppression should be less than 0.3 µg/l (Melmed et al, 2009) Failure of suppression or a paradoxical rise in GH suggests acromegaly. NB paradoxical rise in GH may occur during GTT during normal adolescence. GH may fail to suppress due to chronic renal failure, liver failure, active hepatitis, anorexia nervosa, malnutrition, hyperthyroidism, diabetes and adolescence. References Giustina A, Chanson P, Bronstein MD, Klibanski A, Lamberts S, Casanueva FF, Trainer P, Ghigo E, Ho K, and Melmed S (2010): A Consensus on Criteria for Cure of Acromegaly JCEM 95, Lugo G, Pena L and Cordido F (2012): Clinical Manifestations and Diagnosis of Acromegaly Int J Endo, EPub February 2012 Melmed S, Colao A, Barkan A, Molitch M, Grossman AB, Kleinberg D, Clemmons D, Chanson P, Laws E, Schlechte J, Vance ML, Ho K, Giustina A; Acromegaly Consensus Group (2009): Guidelines for acromegaly management: an update J Clin Endocrinol Metab. 94(5),

20 GnRH Stimulation Test for LH and FSH Principle GnRH (gonadotropin releasing hormone) is a decapeptide secreted by the hypothalamus which stimulates the production and secretion of LH and FSH by the anterior pituitary Indications Investigation of pubertal disorders/suspected hypogonadotrophin hypogonadism Contraindications Avoid HCG injections prior to the test and do not perform following priming for an arginine test. Side effects GnRH may rarely cause nausea, headache and abdominal pain. Requirements GnRH bolus (2.5µg/kg up to a maximum of 100 µg) order as Gonadorelin-LHRH from pharmacy 3 brown top serum tubes for LH/FSH Procedure No specific preparation is required. Insert a reliable cannula and take baseline samples for LH, FSH (1 brown top tube), oestradiol or testosterone and SHBG (plain clotted tube) Give GnRH intravenously Take a blood sample at 30 minutes (brown top) for LH and FSH. Take a further sample at 60 minutes (brown top) for LH and FSH Remember: Clearly label samples with patient details and times Use the specific DFT protocol request forms Send all samples together to lab Interpretation of results Normal Response - Adults: LH FSH peak response 2-3x baseline peak response 2-3x baseline An inadequate response may be an early indication of hypopituitarism or delayed puberty. Gonadotrophic deficiency is diagnosed on the basal levels rather than the dynamic response. 20

21 Oral Glucose Tolerance Test Principle In normal individuals pancreatic insulin secretion maintains blood glucose within a tight concentration range following an oral glucose load. Failure of insulin secretion, or resistance to insulin action, will result in an elevation in blood glucose. Indications The diagnosis of diabetes is made on the basis of repeatedly elevated fasting plasma glucose. The use of the oral glucose tolerance test is to clarify borderline elevations in fasting plasma glucose and is used for the diagnosis of gestational diabetes. Contra-indications This test is only necessary if fasting and/or random glucose measurements are equivocal i.e mmol/l. This test should NOT be performed in patients who fulfil the diagnostic criteria for diabetes mellitus as follows: Two diagnostic glucose results on separate occasions; either fasting plasma glucose 7.0 mmol/l or random plasma glucose of 11.1 mmol/l. One diagnostic glucose result and clinical symptoms of diabetes e.g. polydipsia, polyuria, ketonuria and rapid weight loss. The test should not be carried out on patients who are under physical stress e.g. post surgery, trauma or infection or extreme psychological stress as these may give misleading results. The test should not be carried out on patients with hypokalaemic periodic paralysis. Side Effects Some subjects feel nauseated and may have vasovagal symptoms during this test. Requirements POLYCAL (contains 75g of glucose) plus special dilution beaker supplied by Biochemistry 2 X Yellow top fluoride oxalate blood tubes Procedure Patient Preparation Patients should be advised to eat a normal carbohydrate diet (>150g daily) for at least 3 days prior to the test and undertake normal physical activity. Patients must have fasted from 9pm on the previous evening but may drink small volumes of plain water. Smoking and physical exercise should NOT be allowed in the morning prior to, and during, the test. 21

22 Test This test should be performed in the morning. Patients should remain at rest during the test and should not be allowed to smoke. Polycal (113ml) should be diluted to 200ml with water. Minutes Procedure Samples 0 Take sample for glucose 1 x Yellow Fluoride Oxalate (glucose). 0 Give patient Polycal to drink over 5 minutes followed by a further 100ml of water 120 Take sample for glucose 1 x Yellow Fluoride oxalate (glucose) Ensure both samples are labelled with the patients name and time of sample. Send both samples to Biochemistry together. Interpretation of results According to WHO guidelines for diagnosis of Diabetes and intermediate hyperglycaemia (reviewed 2006): Plasma Glucose (mmol/l) 0 minute 120 minute Non Diabetic <6.1 and <7.8 Impaired fasting glucose and <7.8 Impaired glucose tolerance <7.0 and Diabetes 7.0 or greater or 11.1 or greater Please refer to specific guidelines for Antenatal patients as the cut off values are different. 22

23 Prolonged Oral Glucose Tolerance Test Indications This test is an extension of the standard oral glucose tolerance test in cases of suspected reactive hypoglycaemia. The test should be carried out in patients who complain of feeling faint or have abdominal discomfort after a meal. This test should not be carried out following a prolonged fast. The test may also be used to support investigations into patients with suspected Insulinoma (Kar et al, 2006; Iida et al, 2010). Contra-indications None Side Effects None Requirements POLYCAL (contains 75g of glucose) plus special dilution beaker supplied by Biochemistry 10 x Yellow top (Fluoride oxalate) tubes Procedure Patient Preparation Fast from midnight (sips of water are permitted) Smoking should be avoided on the day of the test Any regular medication should be taken as normal Test Record any symptoms of hypoglycaemia in the patient notes and monitor blood glucose at each time point using Point of care glucose testing. Minutes Procedure Samples 0 Take sample for glucose 2ml Yellow tube (fluoride oxalate) 0 Give patient Polycal to drink over 5 minutes followed by a further 100ml of water. Ensure that the patient washes their hands after this 30 Take sample for lab glucose &POCT glucose 2ml Yellow tube (fluoride oxalate) 60 Take sample for lab glucose &POCT glucose 2ml Yellow tube (fluoride oxalate) 90 Take sample for lab glucose &POCT glucose 2ml Yellow tube (fluoride oxalate) 120 Take sample for lab glucose &POCT glucose 2ml Yellow tube (fluoride oxalate) 150 Take sample for lab glucose &POCT glucose 2ml Yellow tube (fluoride oxalate) 180 Take sample for lab glucose &POCT glucose 2ml Yellow tube (fluoride oxalate) 240 Take sample for lab glucose &POCT glucose 2ml Yellow tube (fluoride oxalate) 270 Take sample for lab glucose &POCT glucose 2ml Yellow tube (fluoride oxalate) 300 Take sample for lab glucose &POCT glucose 2ml Yellow tube (fluoride oxalate) NB: Label all tubes with patient details and time of sample 23

24 If blood glucose drops below 3mmol/l at any time point, collect samples for Insulin and C peptide (orange top tubes - keep on ice). Send the samples to the laboratory immediately and inform them that the samples will be arriving. Interpretation of results A laboratory glucose result <3 mmol/l is consistent with reactive hypoglycaemia and requires follow up. References Kar P, Price P, Sawers S, Bhattacharya S, Reznek RH & Grossman AB (2006): Insulinomas may present with normoglycaemia after prolonged fasting but Glucose stimulated hypoglycaemia. JCEM 91, Iida K, Ohara T, Hino Y, Nobuhara M, Ishida J & Chihara K (2010): Glucose-Responsive Insulinoma in a patient with Post Prandial Hypoglycaemia in the morning. Inter Med 49,

25 72 hour Prolonged Fast Principle Prolonged fasting is a sensitive procedure for detection of endogenous hyperinsulinism (sensitivity >90 %) and is routinely employed as the initial test to detect inappropriately elevated insulin secretion as the cause for recurrent hypoglycemia. Indications For the diagnosis of Insulinoma. Requirements Yellow Top fluoride oxalate sample tubes for glucose (at least 18 tubes) Orange top sample tubes for insulin, C-peptide and ketones Iced slurry for transportation of samples (Insulin and C peptide). 50ml 50% dextrose available for immediate administration for hypoglycaemia Point of care glucose testing device Patient preparation Procedure Patient is to have a light breakfast at 6am Calorie free, caffeine free beverages only may be taken. Prescribed medication can be continued. Smoking is not permitted during the test. Encourage the patient to remain physically active during waking hours, but do not allow them to leave the ward. Monitor blood glucose levels (POCT) at 4 hourly intervals, starting with the first sample at 9am on Day 1, and record them on the chart provided. At each time point take one yellow top tube for laboratory glucose analysis and forward this to the lab. Record any clinical symptoms of hypoglycaemia on the chart provided. If the bedside blood glucose is found to be <3mmol/L take two orange top tubes for Insulin, C-peptide and ketones and a brown top tube for a sulphonylurea screen. Place the orange top tubes on ice and send to the laboratory immediately after collection as urgent samples (DO NOT PLACE IN PNEUMATIC TUBE!). Ensure they are labelled with the time of collection and send together with the corresponding glucose sample. If the laboratory glucose level is found to be <2.2 mmol/l or the patient is exhibiting severe symptoms, stop the test If the patient becomes severely unwell, take samples for glucose, insulin, C peptide and ketones as described above and reverse the hypoglycaemia NB Insulin and c-peptide samples will only be analysed when laboratory glucose <2.5mmol/L. If there are no symptoms during the fast, finish with minutes of exercise eg brisk walk 25

26 Interpretation The diagnosis of insulinoma rests on the demonstration of hypoglycaemia by laboratory plasma glucose <2.2mmol/L with concurrent serum insulin level >5mU/L, in the presence of a negative sulphonylurea screen (1). Hirshberg et al (2000) demonstrated that 95% of patients being investigated for Insulinoma had a fast terminated by 48 hours and the other 5% should have had the fast terminated at this time point had it not been for inconsistencies with the assessment of hypoglycaemic symptoms. There are several other reports confirming that Insulinoma cases will terminate within 48 hours (Service et al, 1991; Dizon et al, 1999). References Dizon AM et al (1999): Neuroglycopenic and other symptoms in patients with Insulinomas. Am J Med 106, Hirshberg B, et al (2000): Forty eight hour fast: The Diagnostic test for Insulinoma JCEM 85 (9), Service FJ et al (1991): Functioning Insulinoma incidence, recurrence and long-term survival of patients: A 60 year study. Mayo Clin Proc 66, Vaidakis D et al (2010): Pancreatic Insulinoma: current issues and trends. Hepatobiliary Pancreat. Dis Int 9 (3);

27 Hypoglycaemia monitoring chart for 72 hour fast Name: DoB: Hospital number: RXR. Time started fast (last calorific intake):. Time POCT Glucose Signs/Symptoms Lab glucose Insulin/Cpep Required? 27

28 Lactose Tolerance Test Principle Generalised disease of the intestinal wall or genetic deficiency may lead to selective disaccharidase deficiency (namely lactase in this instance). A preparation of lactose is given and glucose is measured. This provides an indication of enzyme activity. Indications Assessment of disaccharidase activity (specifically lactase). Contraindications for this test This test should not be performed in those patients with periodic hypokalaemic paralysis. Side effects Some patients may have nausea during the test. Patient Preparation The patient should have fasted for hours prior to the test, and should have been on a normal diet for at least 72 hours prior to the test. Patients should not smoke. The patient must not have had any unaccustomed exercise prior to the test. The patient must not be suffering from any overt infection or trauma. Requirements Lactose 50g in water (obtained from pharmacy) Fluoride oxalate blood collection tubes (5 tubes). Procedure This test should be performed in the morning. Patients should remain at rest during the test and should not be allowed to smoke. Lactose should be dissolved in a glass of water. Confirm that he patient has fasted and take a baseline sample for glucose (1 x yellow top tube) Give patient lactose solution to drink over 5 minutes followed by a further 100ml of water Take further samples for glucose at 30, 60, 90 and 120 mins after patient has finished the lactose drink Ensure all samples are labelled with the patients name and time of sample. Send all samples to Biochemistry together. Interpretation A rise in blood glucose of more than 1.7 mmol/l is normal (Henderson & Rinker in TietzTextbook of Clinical Chemistry, 1999). A rise of less than 1.1 mmol/l is abnormal and indicative of lactase deficiency. Symptoms occurring during the test are a good indication of lactose intolerance. References Tietz Textbook of Clinical Chemistry, 3 rd Edition (1999) Ed: Burtis, CA & Ashwood ER Matter R, Ferraz decampos Mazo D & Carrilho FJ (2012): Clin and Exp Gastro 5,

29 Water Deprivation Test This test is potentially very dangerous and must be undertaken with great care. Patients unable to conserve water may become critically dehydrated within a few hours of water restriction This test must ONLY be carried out following referral by a Consultant Endocrinologist or a member of the Endocrinology team Important: Inform the Biochemistry laboratory at BGH, at least 1 day in advance of performing this procedure so that samples can be processed efficiently. Principle Water restriction in the normal individual results in secretion of AVP from the posterior pituitary in order to reabsorb water from the distal renal tubules and concentrate the urine. Failure of this mechanism results in a rise in plasma osmolality due to water loss, and a dilute urine of low osmolality. The two causes are a. A failure of AVP secretion (cranial DI) b. Insensitivity of the renal tubules to AVP (nephrogenic DI) The cause may be distinguished by the administration of DDAVP (synthetic AVP). Indications Used in the differential diagnosis of polyuria. Distinguishing between Cranial Diabetes Insipidus (CDI), Nephrogenic Diabetes Insipidus (NDI) and psychogenic polydipsia (PP) as a cause for polyuria. Polyuria is generally considered if the urine output is >2.5l/day with persistent urine osmolalities of <300msom/kg (Barth, Butler and Hammond (2001) in Biochemical Investigations in Laboratory Medicine) Contraindications Do not proceed to this test until a thirsted (from midnight) early morning urine has been obtained for measurement of osmolality. If there is evidence of the kidney s ability to concentrate the urine e.g. spot urine osmolality >750mOsm/kg this test is not required. Exclude other causes of polydipsia and polyuria: Diabetes Mellitus Hypoadrenalism Hypercalcaemia Hypokalaemia Hypothyroidism Urinary Infections Chronic kidney disease Therapy with Carbamazepine, Chlorpropamide, Lithium Therapy 29

30 Precautions Patients should not have any access to any food or drink throughout the test and must be closely monitored throughout the test to ensure this. Side Effects Patients with true diabetes insipidus may become severely water depleted during water deprivation and MUST be carefully monitored (by weighing and quantifying urine output regularly) throughout the test. Requirements Accurate scales for weighing the patient Universal urine pots for urine osmolality Measuring jug for measuring urine volume Brown top serum tubes for serum osmolality Procedure Patient Preparation: Patient should attend at 7am on the day of the test. 1. If the patient is on DDAVP, this is discontinued 24hrs before the test. 2. If indicated give normal steroid and/or thyroid hormone replacement before the test 3. Tea, coffee, alcohol and tobacco are specifically excluded after midnight on the day of the test and during the test because they directly stimulate (vagus) the secretion of AVP independently of the osmoreceptors. 4. Patient is allowed to drink freely until the start of the test i.e h 5. A light breakfast is permitted before test commences e.g h The Test (Part 1): 1. Print out the Water Deprivation test template. 2. At 07.00h (after a light breakfast if required) the patient should empty their bladder and this urine should be discarded h commence fluid restriction, weigh the patient and calculate 97% of their weight. Begin the fluid balance chart. Take urine and serum samples for osmolality 4. At the times indicated in the table below, weigh the patient and record the volumes of urine passed. At times where urine osmolality is indicated transfer 10mls of the sample into a plain urine container. At the times where serum osmolality is indicated, collect 5ml of blood into a brown top tube. Label each sample with the time and send to the laboratory using a separate form for each batch (urine and serum) of samples. Do not save any samples on the ward as osmolality measurements are required promptly. 5. Use the water deprivation chart provided to monitor the patient throughout. 30

31 Time Weigh Urine volume Urine Osm Serum Osm YES YES YES YES YES + + Urine volume: monitor hourly from Urine osmolality: samples at 08.00, 11.00, and Serum Osmolality: samples at 08.30, 11.30, Review the results at each collection. If urine osmolality rises above 750mOsm/kg the test can be stopped as this is sufficient evidence of a concentrating ability. Part 2: Await contact from the laboratory before proceeding to this stage. 1. If urine osmolality is <750 mosm/kg or if urine osmolality failed to rise by more than 30 mosm/kg over 3 successive urine samples, then administer 2µg DDAVP (0.3µg in Children) i.m. at 16.00h and allow food and fluids. Do not allow patient to drink >500ml for 8 hours after DDAVP administration as the patient will be at risk of developing profound hyponatraemia 2. Check urine volume and osmolality at hourly intervals for until There is no point in measuring serum osmolality as the patient is now eating and drinking freely (remember: do not allow excessive drinking) Interpretation of results INDICATIONS FOR STOPPING THE TEST: Weight loss is >3% of initial weight Serum osmolality rises to >300 mosmol/kg Post-Dehydration Osmolality (mosm/kg) Post DDAVP osmolality (mosm/kg) Diagnosis Serum Urine Urine >750 >750 Normal >293 <300 <300 Nephrogenic DI >293 <300 >750 Cranial DI 31

32 < <750 Primary Polydipsia or partial nephrogenic DI EQUIVOCAL RESULTS These may be due to partial DI or the patient drinking during the test. In these cases the test can be repeated after fasting the patient from midnight the night before the test. Elderly patients may not achieve maximal concentration of their urine and therefore results should be interpreted on a case by case basis. If results are equivocal and there remains clinical suspicion of DI then proceed to hypertonic saline infusion test. SENSITIVITY AND SPECIFICITY When correctly performed, the water deprivation test has a sensitivity and specificity of 95% for diagnosing and differentiating severe cranial DI and nephrogenic DI. The incidence of false positive and false negative results for PP or partial CDI/NDI is 30-40% (investigate further). 32

33 Water Deprivation Test Template Patient Name: Hospital No: Today s Date: Sex: DOB: Perform test under strict supervision to ensure the patient has no access to fluids The test must be stopped if weight loss exceeds 3%, or if serum osmolality rises above 300 mosm/kg Weight patient: kg Calculate weight minus 3%: kg Time Weight Urine volume Urine Osm Serum Osm Loss >3% Yes/No > 300 Yes/No Loss >3% Yes/No > 300 Yes/No Loss >3% Yes/No Loss >3% Yes/No > 300 Yes/No Loss >3% Yes/No If the urine osmolality is >750 mosm/kg STOP the test If urine osmolality is <750mOSM/kg or has failed to rise by >30mOSM/kg over 3 successive urines then administer 2µg DDAVP i/m and fill in the table on the next page. 33

34 Water Deprivation Test Part 2: Distinguishing between nephrogenic DI and cranial DI Allow food and minimal fluids DO NOT allow excessive drinking Time DDAVP administered: Time after DDAVP Clock time Urine volume (mls) Urine Osm (mosm/kg) + 1hr : +2hr : +3hr : +4hr : 34