ENDOCRINE APPENDIX

MULTIPLE-SPECIMEN TESTING

The procedures listed below are for timed, multiple-specimen tests only. All specimens are from the same patient and will be submitted to the laboratory simultaneously. Each specimen must be clearly labeled with the patient's name and the date and time of collection. Multiple-specimen labels (sequence labels) can be obtained from LabCorp. See below for ordering and labeling instructions. Only one test request form accompanies the specimens; do not submit a separate test request form for each specimen. The test request form is completed with all patient information, including any medications administered and the number of specimens submitted. The test request form and all specimens should be submitted in one container (box or plastic specimen transport bag). Each result will be identified on the report by the corresponding specimen sequence numbers, associated times, or draw site(s) supplied. See the Comprehensive List of Procedures for additional individual test information.

Labeling Multiple-Specimen Submissions

Labels (Multispecimen Panel) can be ordered from LabCorp using item #3572885801. These labels, orange in color, are available in a pack containing five sheets with a total of 600 labels. Sequence labels will be applied at the top of the specimen tube, just below the tube's top or cap (see following diagram for correct orientation). This places the label away from other labels on the tube and should prevent it from being covered up by bar codes, accession labels, etc. Room is available on the label so that a sequence number (eg, #1, #2, etc), collection time (eg, 8 AM, 9 AM), time description (eg, fasting, 30 minutes, etc), or the description of a draw site (eg, in renal vein sampling, an abbreviation such as IVC) may be written.

ENDOCRINE DYNAMIC TESTING

Some of these tests involve significant risk and should be performed only by qualified personnel who are familiar with the tests and who have taken adequate precautions to protect the safety of the patients involved. Every effort has been made to ensure accuracy in these recommendations, but clinicians must use their judgment and refer to specific pharmaceutical resources to determine appropriate drug dosages for their patients. In addition, many of the tests require special containers and/or handling. Please refer to individual test descriptions for appropriate specimen requirements.

ACTH Stimulation Test, 1-Hour

Screen for Adrenal Insufficiency

The ACTH stimulation test measures the functional integrity of the adrenal glands and their sensitivity to ACTH stimulation.^1,2,3,4,5 Individuals with primary adrenal insufficiency fail to produce cortisol levels >18 μg/dL after ACTH stimulation. The test also indirectly assesses hypothalamic and pituitary function. When endogenous ACTH production is impaired by pituitary or hypothalamic dysfunction, the adrenal gland loses its capacity to respond to exogenous stimulation. It should be noted that there is a short period (up to 3 months) after the onset of pituitary/hypothalamic dysfunction during which the adrenals continue to respond to exogenous ACTH. Recent pituitary surgery or other debilitation of the pituitary/hypothalamic axis can produce misleading results. Generally, peak cortisol values >18 μg/dL at any point during the ACTH stimulation indicate adequate adrenal-pituitary-hypothalamic function. Peak cortisol results between 13 and 17 μg/dL are indeterminate and often become normal when ACTH stimulation testing is repeated. While the standard or low-dose ACTH stimulation test can be diagnostic of adrenal insufficiency when the response is subnormal, some patients with normal results may exhibit adrenal insufficiency in severely stressful situations (eg, surgery or trauma).⁶

Some investigators recommend measuring serum aldosterone levels along with cortisol. If the cortisol response is insufficient, the aldosterone levels can help localize the deficiency. The aldosterone response in a cosyntropin test is blunted or absent in patients with primary adrenal insufficiency. In secondary or tertiary adrenal insufficiency, aldosterone response is normal (an increase of two times baseline) because the renin-angiotensin axis is not affected by decreased endogenous ACTH.

Protocol: Draw blood for baseline cortisol with or without aldosterone. Inject cosyntropin 250 μg (see note below) I.M. or I.V. (if I.V., dilute cosyntropin in 2-5 mL of sterile saline and inject over 2 minutes). Draw blood for cortisol (with or without aldosterone) at 30 and 60 minutes after injection.

Orderable Tests: 028498 Cortisol x 3, 053272 Aldosterone x 3

Note: The standard, high-dose ACTH stimulation test involves the administration of 250 μg of cosyntropin. The resultant concentration is super-physiologic (ie, in vast excess to endogenous concentrations of ACTH). Stimulation studies with a more physiologic concentration (1 μg) of cosyntropin have been described.⁷ This low-dose ACTH stimulation protocol may be more sensitive than the traditional 250 μg test. Cortisol cutoff levels used for the low-dose test are the same as those used for the traditional test. Aldosterone levels during the low-dose test have not been documented. Use of low-dose ACTH has not been documented for testing for CAH.

Screen for Congenital Adrenal Hyperplasia (CAH)

The ACTH stimulation test has been used as a screen for specific adrenal enzymatic deficiencies in individuals with CAH.^8,9 The ratio of baseline precursor to product steroid can often be used to localize the enzyme dysfunction in severe CAH cases even without exogenous cosyntropin stimulation. In children with milder defects, the enzymatic defect can often be localized by substantially disproportionate fold increases in precursor and product steroid levels after ACTH stimulation.

Protocol: Draw blood for baseline steroid(s). Inject cosyntropin 250 μg I.M. or I.V. (if I.V., dilute cosyntropin in 2-5 mL of sterile saline and inject over 2 minutes). Draw blood for stimulated steroid(s) 60 minutes after injection.^8,9 See following Tables 1, 2, and 3 for expected values for various steroids at baseline and after ACTH stimulation.^8,9

Orderable Tests: Panels that include baseline and stimulated tests: 140761 Cortisol, 140751 Aldosterone, 140758 Androstenedione, 140765 11-Deoxycortisol, 140768 DHEA, 140771 17-OH Pregnenolone, 140776 17-OH Progesterone, 140787 Pregnenolone.

Table 1. Male Reference Intervals for Steroids, Baseline, and After ACTH Stimulation^8,9

Table 2. Female Reference Intervals for Steroids, Baseline, and After ACTH Stimulation^8,9

Table 3. Steroid Precursor/Product Ratios in Healthy Individuals⁹

ACTH Stimulation Test, 48-Hour

Rationale: The prolonged cosyntropin-stimulation (Rose) test has been used to differentiate primary adrenal insufficiency from adrenal insufficiency caused by pituitary or hypothalamic failure.^10,11 In normal individuals, urinary 17-hydroxycorticosteroid excretion increases 2-5 times baseline (days 1 and 2) after ACTH infusion. In primary adrenal insufficiency, no change is seen in cortisol or 17-hydroxycorticosteroid concentrations after prolonged ACTH stimulation. In secondary or tertiary adrenal insufficiency, an incremental increase occurs during the course of the infusion. The adrenal gland that has undergone atrophy because of insufficient ACTH stimulation is capable of functioning with longer stimulation. Protocol:¹¹ Beginning at 9 AM, obtain 24-hour urine for 17-hydroxycorticosteroids (17-OHCS) and creatinine. Collect 24-hour as on day 1. Beginning at 9 AM, start I.V. and give 250 μg synthetic ACTH (cosyntropin) in 250 mL normal saline over 8 hours. This dose of ACTH is infused every 8 hours for 48 hours. Alternatively, 40 IU of depot formulation of purified bovine ACTH in gelatin can be injected intramuscularly every 12 hours for 48 hours. Repeat 24-hour urine as on days 1 and 2.

Days 4 and 5: Collect 24-hour urine as on previous days.

Orderable Tests: See Comprehensive List of Procedures section for information regarding individual tests.

Note: Patients may eat a regular diet and remain ambulatory during the test. In those individuals strongly suspected of having primary adrenal insufficiency, dexamethasone, 20 μg/kg/day can be given to prevent adrenal crisis. This will not interfere with the test. Cosyntropin is preferable to bovine ACTH due to lower risk of adverse allergic reaction.¹¹

Aldosterone Suppression Test (Oral Salt Load)

Rationale: In healthy individuals, aldosterone production is inversely correlated with salt intake.^12,13 This test is based on the fact that aldosterone levels should be suppressed in individuals given a high salt diet. A urine aldosterone level >14 μg/24 hours after 3 days of a high salt diet is consistent with hyperaldosteronism.¹³

Protocol: The patient should consume a high salt diet supplemented with sodium chloride tablets (12 g/day) for 3 days. Starting on the morning of the third day, a 24-hour urine should be collected (no preservative) for measurement of sodium and aldosterone excretion. Urine sodium levels >200 mEq/24 hours to indicate adequate salt loading for proper test interpretation.¹²

Orderable Tests: See Comprehensive List of Procedures section for individual test information.

Note: Increasing dietary sodium chloride in patients with severe hypertension should be avoided. Potassium chloride replacement should be considered due to increased potassium excretion associated with a high salt diet.¹²

Calcium-Pentagastrin Stimulation Test

Rationale: Calcium and pentagastrin stimulate the secretion of calcitonin by the parafollicular cells of the thyroid gland.¹⁴ This secretion is increased in patients with medullary thyroid carcinoma (MTC) relative to healthy individuals. Stimulation with the combination of calcium and pentagastrin produces better sensitivity and specificity for the diagnosis of MTC than basal calcitonin measurement or use of calcium or pentagastrin alone. The reference intervals in pg/mL established at baseline and after calcium/pentagastrin stimulation have been established by the assay manufacturer (Nichols Institute Diagnostics):

Protocol: The patient should fast overnight prior to the stimulation procedure. The patient should remain supine during the procedure. A baseline serum sample should be collected for calcitonin measurement. Calcium (2 mg/kg) should be administered intravenously over 60 seconds followed by pentagastrin (0.5 μg/kg) over 5 seconds. Samples should then be collected for calcitonin at 1, 2, 5, and 10 minutes.

Orderable Tests: 026807 Calcitonin x 5 (baseline, 1-, 2-, 5- and 10-minute)

Captopril Test for Renovascular Hypertension

Rationale: The administration of captopril, an angiotensin-converting enzyme inhibitor, produces an exaggerated rise in plasma renin activity (PRA) in patients with renovascular hypertension relative to patients with essential hypertension.^15,16 Postcaptopril PRA results meeting the following criteria suggest renovascular hypertension. All three criteria must be met.¹⁶

1. PRA >12 ng/mL/hour
2. Increase in PRA of at least 10 ng/mL/hour relative to baseline
3. Increase in PRA ≥150%, or of 400% if the baseline PRA is <3 ng/mL/hour

Protocol: The patient should be taken off antihypertensive medications and have normal salt intake for several days prior to test. The patient should remain seated throughout the test. An EDTA plasma sample should be drawn for renin activity (PRA). Administer 50 mg captopril orally. A second plasma sample for PRA should be drawn 1 hour after captopril dosing.¹⁵

Orderable Tests: 053686 Renin Activity x 2 (baseline, 1-hour)

Note: The captopril test has to been shown to produce false-positive results when used to screen patients with low risk of renovascular hypertension and high baseline PRA values.¹⁶

Clonidine Suppression Test

Rationale: This test has been used to diagnose pheochromocytoma and those paragangliomas that may secrete epinephrine, norepinephrine, or both. Such tumors may cause paroxysmal or persistent hypertension. The test is useful in the investigation of hypertensive patients, especially younger individuals, particularly when hypertension is paroxysmal, suggesting pheochromocytoma.¹⁷ Several definitions of a normal plasma catecholamine response to clonidine have been postulated: a minimum plasma norepinephrine level ≤500 pg/mL¹⁸ ≥50% norepinephrine decline from baseline, and a norepinephrine level ≤500 pg/mL¹⁹ a minimum plasma total catecholamine (norepinephrine + epinephrine) concentration ≤500 pg/mL²⁰

Sjoberg et al²¹ have concluded that minimal suppression occurs 2-3 hours after clonidine administration with the greatest diagnostic accuracy (92%) obtained when the normal response is defined as a level of total plasma catecholamine ≤500 pg/mL. Taylor et al²² have demonstrated an increase in false-positive results by using the 50% reduction criteria. This is especially true if the baseline results are within the established reference interval. Plasma levels are useful if elevated, especially during or immediately following an episode of hypertension, but false-negative results occur when the specimen is drawn during an uneventful period. Normotensive pheochromocytoma has been reported.²³ False-positive results are common. Epinephrine secretion increases in response to cold and hypoglycemia.

Protocol: The patient should fast overnight and abstain from smoking. Thirty minutes after the insertion of the indwelling catheter, blood is drawn for the baseline catecholamine determination. Clonidine hydrochloride (0.3 mg) is given orally, and repeat specimens for plasma catecholamines are collected 2 and 3 hours later.²¹ Collection of a fourth tube at 4 hours is optional.

Note: Several medications have been shown to prevent clonidine suppression, thus rendering false-positive results. These include beta-adrenergic blockers, tricyclic antidepressants, and thiazide diuretics. If possible, these drugs should be discontinued 48 hours before collection. The alpha-adrenergic blocking agents do not interfere with clonidine suppression. Drugs that may affect plasma norepinephrine levels include alpha- and beta-adrenergic blockers, vasodilators, clonidine, bromocriptine, theophylline, phenothiazine, tricyclic antidepressants, labetalol, calcium channel blockers, converting enzyme inhibitors, bromocriptine, chlorpromazine, haloperidol, and cocaine.

Walnuts, bananas, and interfering medications should be avoided for a week prior to specimen collection. An indwelling heparinized catheter is recommended, as venipuncture can cause an increase in the substances for which testing is being performed. The patient should remain recumbent during the entire collection procedure.

Orderable Tests: 123133 Plasma Catecholamines x 3 (baseline, 2-, and 3-hour); 123158 Plasma Catecholamines x 4 (baseline, 2-, 3-, and 4-hour)

Note: For each collection time interval, draw blood into lavender top (EDTA) tubes. Invert tubes to allow preservatives to mix thoroughly. Centrifuge and transfer the plasma to labeled plastic transport tubes (4 mL each; 2 mL minimum). Freeze immediately and ship frozen. The time between blood collection and the preparation of plasma is critical; if the time exceeds 1 hour, catecholamine values increase (when blood is refrigerated) or decrease (when kept at room temperature).²⁴

Corticotropin-Releasing Hormone (CRH) Stimulation Test

Rationale: The CRH stimulation test has been used as a diagnostic test in both adrenal insufficiency and Cushing syndrome. In 95% of normal subjects, baseline ACTH increases two- to fourfold within 30-60 minutes of CRH administration.^2,25,26,27 Plasma cortisol typically peaks at >20 μg/dL within the same period.

Indications

Adrenal Insufficiency. Patients with adrenal insufficiency exhibit one of three patterns of response to CRH stimulation depending on the cause.² Individuals with primary adrenal insufficiency have high baseline ACTH (which increases in response to CRH), with low cortisol levels before and after CRH. Individuals with secondary (pituitary) adrenal insufficiency exhibit low baseline ACTH levels that do not respond to CRH. Cortisol levels in these patients are not affected by CRH. Patients with tertiary (hypothalamic) disease (ie, CRH deficiency) typically exhibit low baseline ACTH levels and an exaggerated and prolonged response to CRH. Serum cortisol levels in these patients typically do not reach 20 μg/dL.

Cushing Syndrome. The CRH stimulation test has been used to identify the source of excess ACTH in ACTH-dependent Cushing syndrome.¹¹ Pituitary tumors tend to be sensitive to CRH stimulation while ectopic tumors do not usually respond. Patients with hypercortisolism due to oversecretion of ACTH by the pituitary (Cushing disease) generally respond to CRH with a >20% rise in cortisol (average of 30- and 45-minute value relative to average of minus 15- and minus 1-minute value) and a >35% increase in ACTH (average of 15- and 30-minute value relative to average of minus 5- and minus 1-minute value). In most cases, patients with primary adrenal hypercortisolism or ectopic ACTH syndrome do not respond to CRH.

Protocol: Patient should fast for at least 4 hours prior to the test.²⁶ Ovine* or human CRH at 1.0 μg/kg body weight is injected intravenously as a bolus over 30 seconds. Blood samples should be collected at 15 minutes and 1 minute before CRH administration and at 15, 30, 45, 60, 90, and 120 minutes after for measurements of cortisol and ACTH.

*Ovine CRH is not FDA-approved (6/00). While earlier studies suggested that ovine CRH produced superior stimulation compared to human CRH,²⁶ more recent data indicate that the two perform comparably.²⁷

Orderable Tests: 210823 Cortisol x 8 (minus 15-, minus 1-, 15-, 30-, 45-, 60-, 90-, 120-minute); 267708 ACTH x 8 (minus 15-, minus 1-, 15-, 30-, 45-, 60-, 90-, 120-minute)

Note: The CRH stimulation test can be performed at any time of day. The ACTH increment is similar morning and evening, but peak values are higher in the morning. Cortisol levels peak at similar values in the morning and evening.

C-Peptide Suppression Test

Rationale: This test has been used in cases in which the 72-hour diagnostic fast is inconclusive.^28,29 Production of C-peptide by normal subjects is generally suppressed during hypoglycemia. The degree of C-peptide suppression is diminished in patients with insulinoma. Normative data, based on age and body mass index (BMI), are calculated as weight (kg) divided by the square of height in meters.²⁹ The extent of suppression (as a percentage) of the 60-minute C-peptide concentration relative to the baseline sample should be ≥ the values tabulated below.

Protocol: After an overnight fast, insulin (0.125 units/kg) should be infused over a period of 60 minutes. Blood should be collected for glucose and C-peptide measurements at baseline and 60 minutes.²⁹

Orderable Tests: 239822 Glucose x 2 (baseline, 1-hour) 143302 C-Peptide x 2 (baseline, 1-hour)

Note: Intravenous access should be maintained, and glucose should be available for infusion.

Dexamethasone Suppression Tests

Rationale: Dexamethasone is a synthetic glucocorticoid that is 64 times more potent than cortisol.^1,25,30 At the low doses administered in dexamethasone suppression tests, it does not interfere with assays of cortisol and its metabolites. Administration of dexamethasone suppresses the release of ACTH by negative feedback in individuals with a normal hypothalamic-pituitary-adrenal axis. As a result, adrenal cortisol levels fall to very low levels in normal individuals. Using modern immunochemiluminometric methods of measurement, the plasma cortisol level after dexamethasone suppression should be <1.8 μg/dL in healthy individuals.^25,30

Indications

Diagnosing Hypercortisolism. Patients with hypercortisolism lose normal negative feedback control and as a result have higher cortisol levels after dexamethasone administration.^25,30 The overnight and 48-hour low-dose dexamethasone tests have been found to be more specific and sensitive than 24-hour urinary free cortisol in identifying patients with hypercortisolism.

Identifying Cause of Hypercortisolism. In the presence of documented hypercortisolism, consistently undetectable plasma ACTH strongly suggests the presence of a functioning adrenal tumor.²⁵ When ACTH levels are not consistent with an adrenal tumor, pituitary and ectopic ACTH production can be differentiated with the 48-hour high-dose dexamethasone test. This test is generally used to distinguish Cushing disease (excess ACTH from the pituitary) from ectopic ACTH secretion.

Glucocorticoid-Remediable Aldosteronism (GRA). Aldosterone levels in patients with GRA are controlled by ACTH levels.³¹ Measurement of plasma aldosterone levels after the 48-hour low-dose dexamethasone test (see below) can differentiate individuals with GRA from patients with other causes of primary aldosteronism.

Low-Dose Dexamethasone Test, Overnight

This simple test takes advantage of the principle that the sensitivity of the pituitary is greatest at night. Dexamethasone is administered orally (1.0 mg; 20 μg/kg in children) between 11 PM and midnight.²⁵ Serum cortisol is collected at 8-9 AM the next morning. A cortisol level <1.8 μg/dL essentially excludes Cushing syndrome.

Low-Dose Dexamethasone Test, 48-Hour

Serum cortisol is collected at 8-9 AM. Dexamethasone is administered (0.5 mg; 10 μg/kg in children) immediately after the cortisol is drawn and again every 6 hours for 48 hours.²⁵ A second plasma cortisol is drawn at 9 AM, 6 hours after the last dexamethasone dose. Serum cortisol concentrations <1.8 μg/dL essentially exclude Cushing syndrome. This test can be used to diagnose GRA by measuring plasma aldosterone levels 6 hours after the last dexamethasone dose.³¹ Aldosterone levels <4 ng/dL distinguishes GRA from other forms of primary aldosteronism with high sensitivity and specificity.

High-Dose Dexamethasone Test, 48-Hour

Serum cortisol is collected at 9 AM. Dexamethasone is administered (2.0 mg; 50 μg/kg in children) immediately after the cortisol is drawn and again every 6 hours for 48 hours.²⁵ A second plasma cortisol is drawn at 9 AM, 6 hours after the last dexamethasone dose. Patients with functional adrenal adenomas show no suppression of cortisol levels in the 48-hour sample relative to the initial (baseline) sample. Seventy-eight percent of patients with pituitary source of excess ACTH showed >50% suppression of plasma cortisol while only 11% of patients with an ectopic source of excess ACTH had a >50% suppression.

Comprehensive, 6-Day, Low-/High-Dose Dexamethasone Test

This protocol incorporates the low- and high-dose dexamethasone tests in succession.¹ The 24-hour urine-free cortisol and/or 17-hydroxycorticosteroid (17-OHCS) measurement that is included in this protocol can help verify the diagnosis. Collect baseline serum cortisol and ACTH. Begin 24-hour urine collection for free cortisol and/or 17-OHCS. Complete 24-hour urine collection. The patient should start taking dexamethasone (0.5 mg, 10 μg/kg in children) orally at 9 AM and again every 6 hours for 48 hours. Continue dexamethasone dosing from day 2. Begin second 24-hour urine collection for free cortisol and/or 17-OHCS. Collect serum for AM cortisol level. Complete 24-hour urine collection started on day 3. Starting at 9 AM, increase the dose of the dexamethasone to 2.0 mg (50 μg/kg in children). Continue taking 2.0 mg dose of dexamethasone every 6 hours for 48 hours. Continue dexamethasone dosing from day 4. Begin third 24-hour urine collection for free cortisol and/or 17-OHCS. Collect serum for AM cortisol and ACTH. Complete third 24-hour urine collection.

Orderable Tests: See Comprehensive List of Procedures section for individual test information.

Patients with normal cortisol metabolism or pseudo-Cushing syndrome have urinary free cortisol levels on the second collection (day 3) <20 μg/24 hours, and plasma cortisol levels <1.8 μg/dL on day 4.¹¹ Patients who do not exhibit normal suppression during the low-dose phase of the test can be further characterized during the second, high-dose phase. The high-dose dexamethasone phase has been used to distinguish Cushing disease (excess secretion of ACTH from the pituitary) from other causes of Cushing syndrome. In Cushing disease, the third 24-hour urine collection (day 5) generally produces at a significant decrease in cortisol and 17-OHCS from baseline (day 1). Lesser degrees of suppression suggest a nonpituitary cause of Cushing syndrome (eg, adrenal adenoma or carcinoma, ectopic ACTH production). Seventy-eight percent of patients with pituitary source of excess ACTH exhibited at least a 50% suppression of plasma cortisol while only 11% of patients with ectopic ACTH production had 50% suppression.

Diagnostic Fast (up to 72 Hours)

Rationale: This test has been used to diagnose insulin hypersecretion syndromes such as insulinoma.^28,32 Failure to produce hypoglycemia (glucose <45 mg/dL) by the end of a 72-hour fast effectively excludes the diagnosis of hypoglycemic disorder.³² When a normal subject becomes hypoglycemic as the result of a prolonged fast, insulin levels generally drop below 6 microU/mL, C-peptide levels drop below 200 pmol/L and proinsulin levels drop below 5 pmol/L. Most patients with insulinoma will become hypoglycemic during this test and will have beta-cell polypeptides greater than the cutoffs listed above.

Further information can be gained by administering glucagon to the hypoglycemic patient. Patients with insulin-mediated hypoglycemia will have increases in glucose >25 mg/dL, while patients with other causes of hypoglycemia will have lower increments.

Protocol (Phase 1): During the fast, the patient is allowed to take in only calorie-free and caffeine-free fluids. Nonessential medication should be withheld, and the patient should be encouraged to be active when awake. Glucose levels should be measured every 6 hours until the level drops below 60 mg/dL. Samples should then be collected hourly for glucose, insulin, C-peptide, and proinsulin levels. The fast should be concluded when the glucose level drops below 45 mg/dL and the patient exhibits symptoms of hypoglycemia or after a maximum of 72 hours.

Protocol (Phase 2): Perform this phase of the test only if the patient becomes hypoglycemic during phase 1. At the end of phase 1, inject 1 mg of glucagon intravenously and measure glucose levels at 10, 20, and 30 minutes.

Orderable Tests: See Comprehensive List of Procedures section for individual test information.

Note: The patient should be hospitalized during this protocol and intravenous access should be maintained. Glucose should be available for infusion.

Glucose Tolerance Testing for Diabetes Mellitus

Rationale: The American Diabetes Association (ADA) adopted the following criteria for diagnosing diabetes mellitus in 1997^41,42: Symptoms of diabetes plus casual plasma glucose concentration >200 mg/dL. Casual is defined as any time of day without regard to time since the last meal. The classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight loss. Fasting plasma glucose (FPG) >126 g/dL. Fasting is defined as no caloric intake for at least 8 hours. 2-hour postload glucose (2hPG) >200 mg/dL during an oral glucose tolerance test (OGTT). The OGTT is not recommended for routine clinical use. The OGTT test should be performed using a glucose load containing the equivalent of 75 g of anhydrous glucose dissolved in water.

In the absence of unequivocal hyperglycemia with acute metabolic decompensation, these criteria should be confirmed by repeat testing on a different day. These standards do not include different criteria for pediatric subjects.⁴¹ Note that the ADA recommends that the OGTT be reserved for research studies, and for patients whose status remains equivocal after repeated fasting glucose measurements. It is not to be used routinely to diagnose diabetes mellitus.⁴² There is an intermediate group of subjects whose glucose levels, although not meeting criteria for diabetes, are nevertheless too high to be considered normal. This group is considered to have impaired fasting glucose (IFG). Using the 1997 categories of FPG values allows three classes of patients to be defined. FPG <110 mg/dL = normal fasting glucose FPG ≥110 and <126 mg/dL = IFG FPG ≥126 mg/dL = provisional diagnosis of diabetes

The corresponding categories when the OGTT is used are: 2hPG <140 mg/dL = normal glucose tolerance2hPG ≥140 and <200 mg/dL = impaired glucose tolerance (IGT)2hPG ≥200 mg/dL = provisional diagnosis of diabetes

The ADA recommended criteria are for diagnosis of diabetes mellitus and are not treatment criteria or goals of therapy. No change is made in the American Diabetes Association's recommendations of FPG <120 mg/dL and Hb A_1c <7 % as treatment goals.

Oral Glucose Tolerance Test (OGTT) Protocol

Standard patient preparation conditions and procedure for OGTT recommended by the American Diabetes Association include⁴¹: a minimal carbohydrate intake of 150 g/day for 3 days prior to the OGTT 8- to 16-hour fast ambulatory patient avoid exercise and emotional stress during the OGTT give the glucose load as glucose only, not a mix of mono- and disaccharides test between 7 AM and noon standard load is 75 g glucose collect sample for glucose measurement at 2 hours

Orderable Tests: 002022 Glucose, 2-Hour Postprandial (1 sample)

Glucose Tolerance Testing for Gestational Diabetes Mellitus (GDM)

Rationale: GDM risk assessment should be carried out at the first prenatal visit. Women at very high risk for GDM should be screened for diabetes as soon as possible after the confirmation of pregnancy. Criteria for very high risk are: severe obesity, prior history of GDM or delivery of large-for-gestational-age infant, presence of glycosuria, diagnosis of polycystic ovarian syndrome (PCOS), and strong family history of type 2 diabetes. Screening/diagnosis at this stage of pregnancy should use standard diagnostic testing (see Glucose Tolerance Testing for Diabetes Mellitus, Rationale). All women of higher than low risk of GDM, including those above not found to have diabetes early in pregnancy, should undergo GDM testing at 24-28 weeks of gestation. Low risk status, which does not require GDM screening, is defined as women with all of the following characteristics: age <25 years, weight normal before pregnancy, not member of an ethnic/racial group with a high prevalence of diabetes, including Hispanic-American, Native American, Asian-American, African-American, and Pacific Islander, no known diabetes in first-degree relatives, no history of abnormal glucose tolerance, and no history of abnormal obstetrical outcome.^[50] The American College of Obstetricians and Gynecologists (ACOG) suggests that the prevalence of GDM in high-risk populations is so high that pregnant women can be considered to have a positive screen and should proceed directly to diagnostic testing.^[43]

Protocol: Two approaches may be followed for GDM screening:

1. Two-step approach:

1. Perform initial screening by measuring plasma or serum glucose 1 hour after a 50-g oral glucose load (the patient need not be fasting). A glucose threshold after 50-g load of >139 mg/dL identifies approximately 80% of women with GDM, while the sensitivity is further increased to approximately 90% by a threshold of >129 mg/dL.
2. Perform a diagnostic 100-g OGTT on a separate day in women who exceed the chosen threshold on 50-g screening.

2. One-step approach (may be preferred in clinics with high prevalence of of GDM): Perform a diagnostic 100-g
OGTT in all women to be tested at 24-28 weeks.^[50]

The 100-g OGTT test should be performed in the morning after an overnight fast of at least 8 hours but not more than 14 hours and after at least 3 days of unrestricted diet (<150-g carbohydrate per day) and physical activity. The subject should remain seated and should not smoke throughout the test. A diagnosis of GDM requires that at least two of the plasma glucose values must meet or exceed the concentrations listed in the Table 1.^[50]

OGTT (ADA, Clinical Practice Recommendations 2008)

These diagnostic values are the basis of the reference intervals now used by LabCorp.

Table 1. Diagnosis of GDM With a 100 g Oral Glucose Load⁴⁴

Orderable Tests: 102004 Glucose Tolerance, 100 g (baseline, 1-, 2-, and 3-hour)

OGGT (World Health Organization)

Using the 75 g oral glucose load has been recommended by the World Health Organization (WHO).⁴⁴ The WHO protocol identifies a greater number of pregnancies with maternal or perinatal complications associated with high plasma glucose. While the American Diabetes Association considers it premature to recommend any change from the current regimen because it is so widely accepted and practiced in the U.S., some obstetricians do use the 75 g oral glucose load. Recommended limits for this test are given in Table 2.

Table 2. Diagnosis of GDM With a 75 g Oral Glucose Load⁴⁴

Orderable Tests: See Comprehensive List of Procedures section for individual test information.

Gonadotropin-Releasing Hormone (GnRH) Stimulation Test

Rationale: This test has been used in the diagnosis of central precocious puberty (CPP) and in the differentiation of CPP from other causes of precocious puberty.^33,34 The test has also been used to monitor the effectiveness of GnRH analogue therapy.³⁵ In early childhood, the hypothalamic-pituitary-gonadal axis is inhibited, delaying the onset of reproductive maturity. Puberty occurs as increased secretion of GnRH by the hypothalamus produces an increase in pituitary secretion of gonadotropins. Gonadotropin response, especially that of luteinizing hormone (LH), to exogenous GnRH is markedly enhanced after the onset of puberty. A pubertal response has been defined as an LH level after GnRH stimulation >8 IU/L.³⁴

Protocol: GnRH (100 μg) is administered intravenously. A sample for serum LH should be collected at 40 minutes after GnRH administration.³⁴

Orderable Tests: See Comprehensive List of Procedures section for individual test information.

Growth Hormone Stimulation Tests

Rationale: Growth hormone (GH) is secreted in a pulsatile manner and is cleared rapidly, resulting in dramatic fluctuations in GH levels.^36,37,38 For this reason, random GH levels are generally not useful in establishing GH deficiency. A number of physiologic and pharmacologic stimuli can be used to provoke GH release. Several growth hormone stimulation protocols are described below. These tests are best performed in the morning after an overnight fast.³⁸ Patients should be confirmed as euthyroid before these protocols are initiated.

GH Stimulation in Children

The exercise stimulation test is often used as an initial screen for GH deficiency, but combinations of other tests have been advocated by various institutions. A subnormal response from a single provocative test is not diagnostic for GH deficiency and should be confirmed with a second provocative test. These tests produce an increase in plasma GH to >7 ng/mL in individuals with appropriate GH production.^36,37,38 Some institutions use 10 ng/mL as a cutoff for normal GH response.^36,38

The insulin-induced hypoglycemia and the GHRH tests provide additional information beyond establishing GH deficiency. The insulin-induced hypoglycemia test allows the assessment of the entire hypothalamic-pituitary-adrenal (HPA) axis. A normal GH response (>7 ng/mL) to GHRH in a patient proven to be GH deficient by previous stimulation testing suggests that the GH deficiency is due to insufficient GHRH production by the hypothalamus.

Due to low baseline levels of GH, prepubertal children should be “primed” prior to performing the stimulation tests by one of the following³⁸: 5 mg Premarin® P.O. the night before and the morning of the test, or 50-100 μg/day of ethinyl estradiol for 3 consecutive days prior to testing, or 100 mg/day depot testosterone for 3 consecutive days prior to testing

GH Stimulation in Adults

The insulin-induced hypoglycemia test is considered to be the test of choice for diagnosing GH deficiency in adults.³⁹ Since this test involves some risk to the patient (see note below), the GHRH + arginine test has been proposed as an equally sensitive alternative.⁴⁰ Most normal adults will produce a GH concentration >5 ng/mL after either of these stimulation protocols. Severe GH deficiency has been defined as the inability to produce GH levels >3 ng/mL during these tests.^39,40

Arginine Stimulation Protocol

Arginine hydrochloride, 0.5 g/kg body weight, is infused I.V. over 30 minutes. Exercise (10-15 minutes) may be added to potentiate the response. Collect GH samples at baseline, 30, 60, 90, and 120 minutes.³⁶

Note: Arginine should be administered with caution in patients with liver or renal disease. The patient can walk around during the test.

Orderable Tests: 038836 Growth Hormone x 5 (baseline, 30-, 60-, 90-, and 120-minute)

Clonidine Stimulation Protocol

Clonidine is administered at a dose of 0.15 mg/m². Collect GH samples at baseline, 30, 60, 90, and 120 minutes.³⁷

Note: Patients can complain of tiredness and postural hypotension can occur.

Orderable Tests: 038836 Growth Hormone x 5 (baseline, 30-, 60-, 90-, and 120-minute)

Exercise Stimulation Protocol

The subject is asked to exercise vigorously for 20 minutes (ie, calisthenics or running up and down stairs). Collect a sample for GH immediately after the exercise.³⁶

Orderable Tests: See Comprehensive List of Procedures section for individual test information.

Growth Hormone Releasing Hormone (GHRH) Stimulation Protocol

GHRH at 1.0 μg/kg body weight is administered by I.V. bolus. Collect GH samples at baseline, 15, 30, 45, 60, 90, and 120 minutes.³⁸

Note: Estrogen priming does not enhance GH response to GHRH.³⁷ Patients may experience flushing and may describe a metallic taste in their mouth.

Orderable Tests: 038869 Growth Hormone x 7 (baseline, 15-, 30-, 45-, 60-, 90-, and 120-minute)

Insulin-Induced Hypoglycemia Protocol

The patient should remain recumbent for 30 minutes prior to the initiation and during the test.³⁶ Regular insulin, 0.10 unit/kg body weight, is administered by I.V. injection. Collect samples at baseline, 30, 60, and 90 minutes for glucose, GH, and cortisol determination. Glucose levels should be followed using bedside glucose monitoring. Adequate pituitary stimulation is evident when the patient becomes symptomatic (sweating or tremor), and/or when the glucose level drops below 45 mg/dL within 30 minutes. Additional insulin can be administered at 30 minutes if these criteria are not met. In this case, an additional (120-minute) sample should be collected. Cortisol levels at peak >20 μg/dL suggest that the HPA axis is intact. GH and ACTH levels reflect hypothalamic and pituitary functionality respectively. Orderable Tests:045997 Growth Hormone x 4 (baseline, 30-, 60-, and 90-minute)038836 Growth Hormone x 5 (baseline, 30-, 60-, 90-, and 120-minute)026948 Cortisol x 4 (baseline, 30-, 60-, and 90-minute)039222 Cortisol x 5 (baseline, 30-, 60-, 90-, and 120-minute)

Note: This test is not without risk and a physician should be present. I.V. glucose should be available in case of severe hypoglycemia. The test should not be performed in patients with seizure disorder or cardiovascular disease.

Levodopa Stimulation Protocol

Levodopa should be administered orally at a dose of 10 mg/kg in children or 500 mg in adults. Collect GH samples at 30, 60, 90, and 120 minutes.³⁷

Note: Patients may be given water throughout the test but should remain recumbent. Nausea and vomiting may occur.

Orderable Tests: 038836 Growth Hormone x 5 (baseline, 30-, 60-, 90-, and 120-minute)

Growth Hormone Suppression Test

Rationale: Up to 10% of individuals with GH excess (acromegaly) have random GH values in the normal range. GH excess can be established by measuring GH response to a glucose load. The GH levels in normal subjects drop below 2.0 ng/mL after the oral administration of glucose. Patients with acromegaly fail to show this suppression and sometimes show a paradoxical increase in GH level. The test can produce false results in nonacromegalic patients with liver disease, uremia, or heroin addiction.³⁶

Protocol: The test should be performed after an overnight fast with the patient maintained at bedrest. The patient should drink a solution of 100 g glucose. Collect GH samples at baseline, 60, and 120 minutes.³⁶

Orderable Tests: 038844 Growth Hormone x 3 (baseline, 60-, and 120-minute)

Metyrapone Stimulation (Overnight) Test

Rationale: Metyrapone selectively inhibits the adrenal enzyme 11-β-hydroxylase which converts 11-deoxycortisol (compound S, 11-S) to cortisol.^2,11,25,46 As cortisol levels fall, the pituitary secretes ACTH, which in turn stimulates the adrenal to increase 11-S production. Because 11-S does not have glucocorticoid activity, it does not inhibit pituitary secretion of ACTH. In individuals with a normal hypothalamic-pituitary-adrenal (HPA) axis, metyrapone stimulation results in 11-S levels >70 ng/mL. Adequate metyrapone activity is verified by measuring cortisol. Cortisol levels >5 μg/dL indicate that 11-β-hydroxylase has not been adequately inhibited for proper interpretation of the test.

Indications

Adrenal Insufficiency. This test has been used to assess the entire HPA axis but will not differentiate primary from secondary adrenal insufficiency. Adrenal insufficiency is diagnosed if the 11-S level does not exceed 70 ng/mL and the cortisol level is <5 μg/dL²

Cushing Syndrome. The metyrapone stimulation test has been used to localize the cause of secondary hypercortisolism. Metyrapone produces a supranormal increase in 11-S in patients with pituitary-based hypercortisolism (Cushing disease). Some patients with ectopic ACTH-secreting tumors often show little or no increase in 11-S because the tumors are not sensitive to low cortisol levels. However, since as many as 60% of patients with ectopic ACTH-producing tumors have been found to respond, many institutions no longer use the metyrapone test in the differential diagnosis of Cushing syndrome.^25,47

Protocol: At 11 PM, the patient should take metyrapone (30 mg/kg, maximum 3 g) orally, with a snack to prevent gastric irritation. On the following morning, at 8 AM, blood should be drawn for cortisol and 11-deoxycortisol.²

Orderable Tests: See Comprehensive List of Procedures section for individual test information.

Note: Due to the risk of acute adrenal insufficiency precipitated by metyrapone ingestion, this test should be conducted on an inpatient basis. At the conclusion of the test, a prophylactic dose of glucocorticoid should be administered to individuals with suspected hypopituitarism.²

Approximately 4% of individuals will produce inadequate inhibition (postmetyrapone cortisol >5 μg/dL) due to enhanced clearance of metyrapone. A number of drugs that induce hepatic cytochrome P-450, including phenytoin, phenobarbital, rifampin, and glucocorticoids, accelerate the clearance of metyrapone.¹¹

Secretin Stimulation Test

Rationale: This test has been used to help diagnose gastrinoma.⁴⁸ The administration of secretin produces a marked increase in gastrin (increase >200 ng/L) in most patients with gastrinoma. Normal subjects and patients with ordinary peptic ulcers, achlorhydria or isolated retained antrum typically exhibit minimal changes in gastrin levels on administration of secretin.

Protocol: Secretin (2 μg/kg body weight) is administered intravenously. Samples for serum gastrin should be collected at

-10, -1, 2, 5, 10, 15, 20, and 30 minutes.⁴⁸

Orderable Tests: 211268 Gastrin x 8 (minus 10-, minus 1-, 2-, 5-, 10-, 15-, 20-, and 30-minute)

Note: Approximately 10% of patients with gastrinoma fail to produce diagnostic elevation in gastrin levels upon secretin administration.⁴⁸

Tolbutamide Tolerance Test

Rationale: This test has been used to establish fasting hypoglycemia and insulin hypersecretion syndromes.^32,49 Tolbutamide stimulates the pancreas to produce insulin. This causes the plasma glucose levels of healthy individuals to drop approximately 50% within 30 minutes and return to baseline within 2 hours. The peak insulin concentration at 2 minutes does not usually exceed 150 μIU/mL.⁴⁹

Indications

Establishing Hypoglycemia. The average of the glucose values at 120, 150, and 180 minutes (G_120-180) can be used to identify patients with fasting hypoglycemia. G_120-180 values <55 for lean and <62 for obese subjects are consistent with fasting hypoglycemia. Low G_120-180 values can be caused by insulin hypersecretion (ie, insulinoma) but can be secondary to other conditions (eg, liver disease, renal failure, or malnutrition).^23,49

Diagnosing Insulinoma. The insulin secretory response is typically exaggerated in patients with hyperinsulinism such as insulinoma. While a number of conditions can result in low G_120-180 values, only patients with insulinoma exhibit exaggerated plasma insulin concentrations. Insulin levels >150 μIU/mL at 60 minutes are consistent with insulinoma.³²

Protocol: Patient should fast overnight prior to the test. Fasting glucose should be measured prior to initiating test (see note below). Tolbutamide, 1 g (25-40 mg/kg), should be administered as an I.V. bolus over 2 minutes. Blood should be collected for glucose and insulin measurements immediately prior to the injection and at 0, 2, 30, 60, 90, 120, 150, and 180 minutes.

Orderable Tests: 014319 Insulin x 8 (0-, 2-, 30-, 60-, 90-, 120-, 150-, and 180-minute)

Note: The tolbutamide suppression test should not be performed in patients with evidence of hypoglycemia (glucose levels <60 mg/dL) prior to the initiation of the test.³²

Water Deprivation Test (Overnight)

Rationale: This test has been used as an aid in the diagnosis of polyuric disorders.³⁶ Individuals with diabetes insipidus (DI), due to either hypothalamic (HDI) or nephrogenic disease (NDI), cannot conserve free water. When these patients are deprived of water for extended periods they will lose weight due to dehydration to a greater extent than healthy controls. This water loss is accompanied by hypernatremia and an increase in plasma osmolality. Normal individuals and patients with psychogenic polydipsia will not lose >3% of their body weight during the water deprivation test and will maintain serum sodium and osmolality levels within normal limits. The urine osmolality of patients with DI remains low, often less than that of plasma whereas subjects that do not have DI will produce concentrated urine (osmolality typically between 300 and 400 mOsm/kg). Some patients with psychogenic polydipsia fail to produce concentrated urine unless the deprivation is prolonged. Patients with NDI show high ADH levels as the plasma osmolality exceeds 300 mOsm/kg while patients with HDI have low or normal levels.

Administration of ADH in the second phase of the test can be used to identify the cause of DI. ADH administration to patients with HDI will cause an increase in urine osmolality >10% and will fail to produce this level of urine concentration in patients with NDI.³⁶

Protocol (Phase 1): The test should be initiated at 10 PM at which time serum and urine specimens are collected for the determination of sodium and osmolality. The patient should also be weighed at this time. No oral intake is allowed until the test is terminated. At 6 AM, the following morning, the patient should be weighed again. Weight should be measured and urine should be collected hourly for measurement of volume and determination of osmolality. Once urine osmolality becomes stable (a change <30 mOsm/kg for 2 consecutive hours) specimens are collected for serum sodium, osmolality, and ADH levels.³⁶

Protocol (Phase 2): Five units of aqueous vasopressin (ADH) is given subcutaneously, and urine osmolality is measured 1hour later. The test can then be terminated.³⁶

Orderable Tests: See Comprehensive List of Procedures section for individual test information.

Note: This test should not be performed if the patient's initial serum osmolality is >295 mOsm/kg. The test should be halted if the patient's loses >5% of their body weight.³⁶

Water Loading Test

Rationale: This test has been used to help diagnose the syndrome of inappropriate antidiuretic hormone (SIADH).³⁶ SIADH is a syndrome where the autonomous production of ADH interferes with the ability of the kidneys to excrete a water load normally. This results in a dilutional hyponatremia and low plasma osmolality. The water loading test can be useful in equivocal cases were SIADH is suspected and the patient has mild hyponatremia or a low normal plasma sodium level. When the water loading test is administered to healthy individuals, plasma osmolality will drop by >5 mOsm/kg and urine will become diluted (osmolality will drop to <100 mOsm/kg). Ninety percent of the water load is typically excreted within 4 hours by healthy individuals. Patients with SIADH excrete <90% of the water load within 4 hours and do not produce urine osmolality <100 mOsm/kg.

Protocol: The test is initiated 2 hours after the patient has eaten a light breakfast. Serum and urine osmolalities are measured. The patient is given water to drink (20 mL/kg) over a 15- to 30-minute period. Lightly salted crackers may be given with the water if needed. The patient should remain recumbent during the test. Collect samples for plasma and urine osmolality at the beginning of the water loading and hourly for the next 4 hours. Total urine output over the 4 hours should be measured.³⁶

Orderable Tests: See Comprehensive List of Procedures section for individual test information.

Note: This test should not be performed in patients who are already significantly hyponatremic because of the danger that the water load will worsen the hyponatremia. Normal results exclude SIADH but abnormal results can occur in patients with glucocorticoid deficiency, hypothyroidism, and renal disease.³⁶

Footnotes