TABLE II. Methods available for measurement of Urine Free Cortisol
Manufacturer	Test Principle	Reference Ranges	Senstivity*	Sample Size
Beckman Coulter	RIA (manual) 3 h	13.8-71.4 μ g/24h	0.3 μ g/dL (LOD)	50 mL
Roche	Competitive, chemilumimnesence 18 min	36-137 μ g/24h	≤ 0.036 μ g/dL (LOD) ≤ 0.308 μ g/dL (LOQ)	600 μ L (pre-extraction)
Siemens Centaur	Competitive, chemilumimnesence 18 min	20.9-292.3 μ g/24h	0.20 μ g/dL (LOD)	20 μ L
Abbott Architect	Chemiluminescent Microparticle Immunoassay (CMIA) 30 min	4.3-176 μ g/24h	≤ 0.8 μ g/dL (LOD) ≤ 1.0 μ g/dL(LOQ)	70 μ L
DiaSorin LIASON	Competitive, Solid Phase Antigen Linked Technique (SPALT), chemiluminesence 10 min	12.8-82.5 μ g/24h	<0.15 μ g/dL (LOD) <0.5 μ g/dL (LOQ)	50 μ L
	The manufacturer recommends that each laboratory establishes and validates its own extraction method and reference ranges.
Vitros	Competitive EIA lumimnesence 30 min Note: Turbidity can cause erroneous result.	1.3-25.3 μ g/24h	0.11 μ g/dL (LOD) 0.16 μ g/dL (LOQ)	250 μ L (pre-extraction)
LC-MS/MS (4)	Liquid chromatography-tandem mass spectrometry, MRM 3 min instrument analysis time ≤ 60 min, including sample prep	3.5-45 μ g/24h ( http://www.mayomedicallaboratories.com , accessed on 02-28-11)	0.20 μ g/dL (LOQ)	2 mL (pre-extraction)
*Sensitivity is either expressed as analytical sensitivity (LOD) or functional sensitivity (LOQ). LOD is defined as the concentration corresponding to th e mean signal of the blank + 2SD; LOQ corresponds to the minimum concentration of analyte where the %CV of cortisol measurements is within 20%.

ACTH

ACTH ACTH measurements, while subject to the same circadian variability as cortisol (actually it is the variability of the ACTH that is directly responsible for the variability of the cortisol), are not subject to the effects of CBG. Values of ACTH > 100 pg/ml in the setting of possible adrenal insufficiency are usually suggestive of primary adrenal insufficiency, while values >500 pg/ml are diagnostic. Low concentrations of plasma ACTH are not diagnostic, except for the undetectable levels observed in patients with cortisol producing adrenal adenomas. Plasma ACTH concentration is also low in patients taking exogenous steroids.

Salivary measurement of ACTH has not been reported. Frequent blood sampling for ACTH is as useful for assessment of hypercortisolism as cortisol (see above).

All currently available methods for ACTH measurements are based on the immunoassay principles. Concentration of ACTH in plasma is very low (<100 pg/mL), thus radiotracer methods have mostly been used historically due to their higher sensitivity. Assays used for ACTH measurements are generally based on the “sandwich” principle, where two antibodies that recognize different ACTH epitopes are utilized. One antibody (capture) detects one specific site on ACTH molecule, while the second, labeled antibody detects a different ACTH epitope. Such a design is termed “immuno-metric” assay. The major difference between the design of radiolabeled and non-radiolabeled assays is therefore, in the label design and of, course, the fact that non-radiolabeled immunometric design is more amenable to automation. Assays that use radiolabel are referred to immunoradiometric assays (IRMAs), where I is usually attached to the ACTH specific antibody. The non-radiolabeled assay design utilize either enzyme or chemilumiescent labels. Automated immunoassays can be either one-step, where all the reagents are added at the same time or sequential where the plasma specimen is incubated first with the capture antibody, followed by a wash of unbound reagents and incubation with the detection antibody. Since the sequential assays incorporate a wash step, they are generally less prone to the interferences due to a hook effect.

It should also be kept in mind that ACTH is heat labile, and if not collected and preserved on ice, proteolysis can reduce the plasma concentration.

TABLE IV. Methods available for measurement of ACTH
Manufacturer	Test Principle	Reference Ranges	Senstivity*	Sample Size
Biomerica (IBL)	ELISA (manual) 4.5 h	7.9-66.1 pg/mL	0.46 pg/mL	400 μ L EDTA plasma
ALPCO Diagnostics	Two-site immunoradiometric assay (IRMA) (manual)	4.1-41 pg/mL	3.0 pg/mL	200 μ L EDTA plasma
Beckman Coulter	Two-site immunoradiometric assay (IRMA) (manual) 3h	<50 pg/mL	1.2 pg/mL	150 μ L EDTA plasma
DiaSorin	RIA, double antibody (manual) 3 h		15 pg/mL	100 μ L EDTA plasma
	IRMA (manual) 20 h		≤ 1.5 pg/mL	400 μ L EDTA plasma
DiaSorin LIASON	1-step Immunochemiluminometric assay 20 min		≤ 1.6 pg/mL	150 μ L EDTA plasma
Siemens Immulite	Two-site sequential immunochemiluminometric assay 1 h	<46 pg/mL	5.0 pg/mL	75 μ L EDTA plasma
Roche	Two-site immunochemiluminometic assay 18 min	7.2-63.3 pg/mL	1.0 pg/mL	50 μ L EDTA plasma
*All manufacturers listed above report analytical sensitivity (LOD) for ACTH, defined as the concentration corresponding to the mean signal of the blank + 2SD

Miscellaneous Non-Stimulated Measurements

Cortisol Binding Globulin (CBG)

This is not a common test. Esoterix inc.CA ( http://www.esoterix.com/prodserv/test_menu/index.shtml , accessed on 02-28-11) performs the test with 0.5ml of serum with direct RIA in pretreated diluted serum.

11-deoxycortisol (Compound S)

This is the immediate precursor of cortisol and is typically increased when ACTH is elevated or in 11 beta-hydroxylase deficiency

The method for 11-deoxycortisol measurement is now available by LC-MS/MS technology and is offered by the major reference laboratories such as Mayo Clinic, Quest Diagnostic and ARUP.

Anti-adrenal Antibodies

The use of anti-adrenal antibodies has been suggested as useful in detecting early evidence of adrenal insufficiency, before cortisol values are decreased even in response to stimuli. There are no standardized assays for anti-adrenal antibodies and these tests are only available in highly specialized laboratories. Furthermore, there is no known correlation between antibody titer and physiology.

Corticotropin Releasing Hormone (CRH)

Serum concentration of CRF is markedly elevated in pregnancy, presumably due to the production of CRF by the placenta. High levels are associated with high levels of CRH binding protein. Although mentioned as useful in the diagnosis of ectopic CRH syndromes, little data is available in this regard.

CRH testingis not commonly done and is available from Quest Diagnostics laboratory. The principle of the method is RIA, requiring minimum of 1 mL plasma.

DYNAMIC TESTING

Glucocorticoid Deficiency

Despite that more than 35 years have elapsed since the initial description of the use of the insulin tolerance test (ITT) to diagnose adrenocortical deficiency (6), and more than 200 scientific publications in this area, clinicians today still argue as to which is the most sensitive and specific test to diagnose adrenocorticol deficiency. The ITT is still regarded as the gold standard upon which to compare all other tests of HPA axis function. Unfortunately, this test has a considerable spectrum of intra-individual and inter-individual variation (7, 8). Therefore when comparing other tests to the "gold standard", if the standard is not reliable, how can one determine the effectiveness of the other forms of testing? The problem lies in the ability of a single laboratory to know what the values are for their tests. Therefore ranges from an ITT test response in normal subjects performed in one laboratory may not be normal for another laboratory. Taking this into account there are some general guidelines that are available for treating patients with suspected adrenal insufficiency.

a. High dose ACTH stimulation Test

WHEN TO USE THIS TEST: Patients acute ill in the hospital or clinic who present with signs and symptoms suggestive of primary adrenal insufficiency. Patients who are thermodynamically unstable should be resuscitated with crystalloid and given dexamethasone prior to testing if the diagnosis of primary adrenal insufficiency is being considered.

PROCEDURE: An intravenous line is placed 30 minutes before the test for rapid phlebotomy and to eliminate a temporary rise in cortisol associated with a needle stick. The i.v. line is to be kept open with 0.9% NaCl at rate of 50 ml/hr. Blood is drawn at 0' for ACTH (2 ml in a lavander top tube on ice) and cortisol (2 ml in a red top tube). Cotrosyn, 0.25 mg is administered as an IV bolus over 2 minutes. The Cotrosyn comes as a lyophilized powder which should be reconstituted with 1 ml of 0.9% NaCl. 30' after the injection blood is obtained from the iv line (2 ml) for cortisol. The same is repeated at 60' (2 ml) for cortisol.

SPECIAL CONSIDERATIONS: The test can be performed at any time of the day. If the patient is receiving hydrocortisone or cortisone acetate, the medication should be held for at least 12 hours prior to testing (if possible). Although the test can be performed while the patient is receiving dexamethasone, there is some cross-reactivity in some assays and cortisol levels may not be accurate. Each laboratory should determine for itself, the effect of dexamethasone on their assay. Patients with known sensitivity to Cotrosyn or its preservatives should not have it administered.

CONTRAINDICATIONS: Hypersensitivity to cosyntropin or any component of the formulation

WARNINGS / PRECAUTIONS: Use with caution in patients with pre-existing allergic disease or a history of allergic reactions to corticotropin. Class C in pregnancy.

ADVERSE REACTIONS 1% to 10%: Cardiovascular: Flushing Central nervous system: Mild fever Dermatologic: Pruritus Gastrointestinal: Chronic pancreatitis

<1%: Hypersensitivity reactions

DRUG INTERACTIONS: Decreased effect: May decrease effect of anticholinesterases in patients with myasthenia gravis; nondepolarizing neuromuscular blockers, phenytoin and barbiturates may decrease effect of cosyntropin

INTERPRETATION OF RESULTS: Baseline cortisol values <5 µg/dl and ACTH concentrations >100 pg/ml are usually diagnostic of primary adrenal insufficiency. The normal peak cortisol value post stimulation should be an increment no less than 7µg/dl and a maximal level >20 µg/dl at 30'. Since 37% of subjects had a peak response to Cotrosyn at 30' and 63% had a peak response at 60', both time points are analyzed in all patients and if either the 30' or 60' sample reaches the criteria as noted above, the test is considered normal (9).

Serum aldostserone can be measured in 0', 30' and 60' blood samples as ACTH stimulation of the adrenal cortex will also stimulate aldosterone. It has been suggested that a normal aldosterone response to ACTH in the presence of a suboptimal cortisol response is diagnostic of secondary adrenal insufficiency (10).

b. Low dose ACTH stimulation Test

WHEN TO USE THIS TEST: Patients with subtle signs of adrenal insufficiency or patients who have been treated with glucocorticoids and determination of adrenal reserve is necessary. Patients who have autoimmune disease and may have early adrenocortical insufficiency may be best assessed with this test.

PROCEDURE: An intravenous line is placed 30 minutes before the test for rapid phlebotomy and to eliminate a temporary rise in cortisol associated with a needle stick. The i.v. line is to be kept open with 0.9% NaCl at rate of 50 ml/hr. Blood is drawn at 0' for ACTH (2 ml in a lavander top tube on ice) and cortisol (2 ml in a red top tube).

Cotrosyn, 1 µg is administered as an IV bolus over 2 minutes. The injection material was prepared according to the method of Dickstein as follows: The Cotrosyn was diluted with 50 ml of sterile saline to a stock concentration of 5 µg/ml. Aliquots of 0.2 ml were aliquoted into sterile plastic tubes and kept at 4oC for a maximum of 4 months (11). Immediately prior to testing 0.8 ml of saline is added to the tube (final dilution 1 µg/ml) and 1 ml is injected into the patient. 30' after the injection blood is obtained from the iv line (2 ml) for cortisol. The same is repeated at 60' (2 ml) for cortisol.

SPECIAL CONSIDERATIONS: Same as for high dose ACTH stimulation test, see above.

INTERPRETATION OF RESULTS: This test was originally developed to be more sensitive for diagnosing secondary adrenal insufficiency because of it was more of a "physiologic" dose. It is much better at diagnosis secondary adrenal insufficiency than the high dose, although it is not at all recommended in acute or recent hypopituitarism when the intact adrenal glands can still respond to normally to any dose of ACTH. Although probably not useful for the initial purpose of secondary adrenal insufficiency, it may be more sensitive at distinguishing more mild forms of primary adrenal insufficiency (12). Furthermore, this low dose test was helpful in identifying mild adrenal suppression in asthmatic children being treated with inhaled steroids (13). As noted above, each laboratory should establish their normal values, however in general, a stimulated value at 30' or 60' greater than 20 µg/dl would be considered normal.

c. Insulin tolerance testing (ITT)

WHEN TO USE THIS TEST: Patients in whom pituitary or hypothalamic disease may result in impaired corticotroph (or somatotroph) activity. Patients following pituitary surgery or pituitary radiation can be tested at any time, unlike the ACTH stimulation tests described above which are not useful in the acute setting. A random serum cortisol should be drawn prior to scheduling the test, as if the value is > 20 µg/dl, the test may not be helpful. This test, performed in our outpatient clinic, while relatively safe, requires a trained endocrine registered nurse to be present with the patient during the course of the test.

PROCEDURE: A 50 ml vial of 50% Dextrose should be at the patient's bedside in a syringe ready for injection before beginning the procedure.

An intravenous line is placed 30 minutes before the test for rapid phlebotomy, to eliminate a temporary rise in cortisol associated with a needle stick, and in order to have i.v. access for 50% Dextrose in the event of severe hypoglycemia. The i.v. line is to be kept open with 0.9% NaCl at rate of 50 ml/hr. Blood is drawn at 0' for cortisol (2 ml in a red top tube) and glucose (1 ml in a gray top tube). Blood glucose is also checked at the bedside with a glucose monitor.

Regular (short acting) insulin is administered as an IV bolus at a dose of 0.1 units/kg. Blood is sampled for cortisol and glucose as noted above at 10', 15', 30', 45', 60', 90' and 120'. Bedside nurse should monitor blood sugar more frequently if glucose drops below 60 mg/dl on the glucometer or if the patient complains of neuroglycopenic symptoms, such as fatigue, diaphoresis, hunger, lightheadedness or nausea. The test should continue until the blood sugar drops below 40 mg/dl.

In patients with diabetes on insulin consideration should be given that they may be insulin resistant. In which case, larger doses of insulin may be given. We usually begin with a single bolus of 0.1U/kg and then re-bolus with insulin depending on the response to the initial dose (either give the same dose again if there was some response but insufficient, or doubling the dose if there was only minimal response to blood glucose, or giving half the dose if the hypoglycemic response was close to the desired goal). This can be repeated several times until adequate hypoglycemia is reached.

Once the goal response of a glucose < 40 mg/dl is reached, patients can be fed a meal such as crackers and orange juice. Blood glucose should be checked at 5', 10' and 15' minutes post feeding. If there is no increase in glucose or a clinical response within 5 minutes intravenous glucose should be administered. If no response, then repeat bolus of glucose is suggested. If no response or iv access is lost, glucagons 1 mg im can be given.

SPECIAL CONSIDERATIONS: The test can be performed at any time of the day, although due to the need for patients to be fasting it is most conveniently done in the morning. If the patient is receiving hydrocortisone or cortisone acetate, the medication should be held for at least 12 hours prior to testing (if possible). Unlike the ACTH stimulation tests, the ITT cannot be performed while the patient is receiving dexamethasone, due to suppression of the hypothalamic pathways necessary to respond to hypoglycemia.

In general ITT is not recommended in patients with uncontrolled seizure disorder or significant coronary artery disease.

In order to determine if the level of dysfunction is at the hypothalamus or at the pituitary this test is sometimes used in addition to the CRH stimulation test. When the ITT fails to stimulate cortisol, but the CRH test does stimulate it is likely that the patient is having hypothalamic dysfunction.

INTERPRETATION OF RESULTS: Serum cortisol should increase within 30 minutes of the hypoglycemic response to > 20 µg/dl. If the serum cortisol at baseline is =18 the test may not be diagnostic as there are no standard values for subjects who start the test with high serum cortisol. Although the response of cortisol is more reproducible than that of growth hormone in the ITT, intra-subject differences have been reported (8, 14-15).

b. Metyrapone testing

WHEN TO USE THIS TEST: This test is perhaps the most sensitive to determine whether the HPA axis is intact. Although the metyrapone is not generally available from your neighborhood pharmacy, it can be obtained by calling Novartis Pharmaceutical Corp. at 1-800-988-7768 on weekdays. Metyrapone blocks 11-beta hydroxylase and results in the inhibition of conversion of 11-deoxycortisol to cortisol. Serum levels of cortisol decrease and concentration of 11-deoxycortisol increases, however 11-deoxycortisol does not down regulate ACTH. Therefore in a normally functioning HPA axis there is an increase in 11-deoxycortisol. This metabolite can be directly measured in the serum or measured in the urine as 17-OH corticosteroids. This test will be abnormal in either primary adrenal deficiency or ACTH deficiency.

PROCEDURE: For assessment of adrenal or pituitary insufficiency the test can be performed as an overnight test. Metyrapone is given orally (30 mg/kg body weight, or two grams for <70 kg, 2.5 grams for 70 to 90 kg, and three grams for >90 kg body weight) at midnight with a glass of milk or a small snack (16). Serum 11-deoxycortisol and cortisol are measured at 8 AM the next morning; plasma ACTH can also be measured (17).

SPECIAL CONSIDERATIONS: The concurrent use of glucocorticoids will interfere with the test. Any medications that the patient is taking which increase the P450 enzymes will increase the metabolism and clearance of the metyrapone such as rifampin and phenobarbital and phenytoin (18). Similarly hypothyroidism or hyperthyroidism will effect clearance of metyrapone and the adrenal responsiveness. Therefore thyroid function tests should be measured prior to performing this test. Measurement of 11-deoxycortisol, like cortisol itself is dependent on CBG and drugs such as estrogens and oral contraceptives will falsely increase the concentrations of 11-deoxycortisol (19).

PREGNANCY IMPLICATIONS - Use during pregnancy only if clearly needed. Subnormal response may occur in pregnant women and the fetal pituitary may be affected. Excretion in breast milk is unknown; use with caution.

LACTATION - Excretion in breast milk unknown/use caution

ADVERSE REACTIONS - Frequency not defined. Central nervous system: Headache, dizziness, sedation. Dermatologic: Allergic rash. Gastrointestinal: Nausea, vomiting, abdominal discomfort or pain. Hematologic: Rarely, decreased white blood cell count or bone marrow suppression.

INTERPRETATION OF RESULTS: 8 AM serum 11-deoxycortisol concentrations should be >7 µg/dL with serum cortisol less than 5 µg/dL (138 nmol/L), confirming adequate metyrapone blockade. The plasma ACTH concentration at 8 AM should exceed 75 pg/mL (17 pmol/L), confirming that any increases in serum 11-deoxycortisol concentrations are ACTH-dependent, thereby separating primary from secondary adrenal insufficiency (20, 21).

Glucocorticoid Excess

Measurement of endogenous cortisol production in response to exogenous dexamethasone suppression was the first provocative test and still remains among the most useful tests used for the evaluation of excess cortisol. Dexamethasone, due to its high affinity to the glucocorticoid receptor is a potent inhibitor of ACTH synthesis and release. In addition, most of the radioimmunoassays for cortisol (both urine and serum) utilize an antibody that does not cross-react with dexamethasone. Therefore the combination of being able to use relatively low doses and at the same time not interfere with the measurement of cortisol make dexamethasone suppression useful for establishing the presence of a perturbation in the pituitary - adrenal axis and for diagnosing the etiology of hypercortisolism.

At least five different tests have been described using dexamethasone, which differ in the dose and timing of dexamethasone treatment and differ in whether there is measurement of urine or serum cortisol or 17-OH-corticostseroids (Table V). Although the endocrine basis for the tests are in general the same, none are perfect. Confirming the diagnosis of patients with suspected hypercortisolism requires several tests for accurate diagnosis.

All these tests require significant patient participation as the patients are required to self-administer the dexamethasone at inconvenient hours of the day (11PM) or up to 4 times a day. Sampling requires either collection of urine for 24 hours or coming to the physician's office at 8 AM for multiple blood sampling. Drugs that induce hepatic cytochrome P-450 enzymes, such as barbiturates, phenytoin, rifampin and aminoglutethimide, increase the metabolism of dexamethasone and other steroids. Measurement of serum dexamethasone a few hours after the last dose will help determine if there is abnormal metabolism. All these caveats are in addition to the other problems associated with measurement of cortisol as noted above, including the variable diurnal variation as well as interference with concurrent administration of glucocorticoids, estrogen or other medication that increase cortisol binding globulin. A popular screening test for confirming hypercortisolism is the overnight 1mg dexamethasone. A single dose of 1 mg is administered (or 0.3 mg/Kg for children (22) at 11PM and blood is obtained by 8AM the following morning. The dexamethasone dose is given prior to the diurnal rise in endogenous ACTH release and therefore suppresses the early AM cortisol. A normal response would be a serum cortisol concentration < 5 µg/dl, if >10 µg/dl the likelihood of hypercortisolism is high. The other dexamethasone suppression tests are reviewed in Table VIII. Patients with corticotroph macroadenomas or very active tumors, may have urine free cortisol in excess of 1000 µg/dl which will require higher doses of dexamethasone to confirm suppressiblity and/or rule out ectopic ACTH production (23).

WHEN TO USE THIS TEST: This test is one of the most sensitive to determine if there is an abnormality in the HPA axis and for diagnosing the etiology of hypercortisolism, especially when used in conjunction with dexamethasone (see below). Although the CRH is expensive ($300), when one considers the cost of multiple urine collections and analyses of cortisol as well as the cost of a single MRI of the pituitary (which generally exceeds $1500), CRH is at least cost effective when one considers the overall expense in the evaluation of these patients.

PROCEDURE: An intravenous line is placed 30 minutes before the test for rapid phlebotomy and to eliminate a temporary rise in cortisol associated with a needle stick. Blood is drawn at -15' and 0' for cortisol and ACTH (2 ml in a lavender top tube on ice). CRF is then injected IV at a dose of 1 µg/Kg up to a maximum of 200 µg. Blood is obtained at 15, 30 60, 90 120, 180 and 210 minutes for cortisol and ACTH (2 ml in a lavender top tube on ice).

SPECIAL CONSIDERATIONS: The test can be performed at any time of the day, although the initial studies describing the test have been done in the morning.

Side effects the patient may experience are: 1) slight nausea, 2) metallic taste, 3) urgency to urinate, 4) a change in blood pressure (either increase or decrease), 5) a change in heart rate, 6) headaches, 7) abdominal discomfort, 8) facial flushing, and 9) lightheadiness. These side effects are mild and last for only few minutes. One patient reported chest pain during the infusion that was associated with a drop in blood pressure. In general the test is very well tolerated.

INTERPRETATION OF RESULTS: The mean ACTH concentrations at 15 and 30 min after CRH should increase by at least 35% above the mean basal value at -15 and 0 min in patients with Cushing's disease, but not in patients with ectopic ACTH secretion. This measure gave the best sensitivity (93%) and specificity (100%) (24, 25). The best cortisol criterion was a mean increase at 30 and 45 min of 20% or more above mean basal values, which gave a sensitivity of 91% and a specificity of 88%. It should be noted that the criterion for Cushing's disease is based on the presence of hypercortisolism. The CRF test will not adequately differentiate subjects with pseudoCushings and those with true pituitary dependent Cushing's disease.

WHEN TO USE THIS TEST: Several investigators have found that modifications of the CRH stimulation test can increase further the sensitivity and specificity in the diagnosis of the etiology of Cushing's disease. While the simultaneous use of vasopressin can augment the response to CRH, dexamethasone can be used to suppress all but pathologic responses to CRH stimulation (26). While without dexamethasone the sensitivity and specificity of the CRH test is 65 and 100%, respectively, while with dexamethasone the CRH test is 100% sensitive and specific. In our clinic we screen all patients with elevated urine free cortisol and detectable ACTH with a dexamethasone suppressed CRF test.

PROCEDURE: Dexamethasone, 0.5 mg is self-administered po by the patient every 6 hours for 2 days, at 6AM, 12 Noon, 6 PM and 12 midnight. On the morning of the 3rd day an additional dose of dexamethasone is given at 6AM. The patient arrives at the testing center by 8AM and an intravenous line is placed 30 minutes before the test for rapid phlebotomy and to eliminate a temporary rise in cortisol associated with a needle stick. Blood is drawn at -15' and 0' for cortisol and ACTH (2 ml in a lavender top tube on ice). CRF is then injected IV at a dose of 1 µg/Kg up to a maximum of 200 µg. Blood is obtained at 15, 30 60, 90 120, 180 and 210 minutes for cortisol and ACTH (2 ml in a lavender top tube on ice).

SPECIAL CONSIDERATIONS: The test can be performed at any time of the day, although it is usually done in the morning.

Side effects the patient may experience are: 1) slight nausea, 2) metallic taste, 3) urgency to urinate, 4) a change in blood pressure (either increase or decrease), 5) a change in heart rate, 6) headaches, 7) abdominal discomfort, 8) facial flushing, and 9) lightheadiness. These side effects are mild and last for only few minutes. One patient reported chest pain during the infusion that was associated with a drop in blood pressure. In general the test is very well tolerated.

INTERPRETATION OF RESULTS: A normal response would be a plasma cortisol concentration less than 1.3 µg/dl measured 15 minutes after the administration of CRH. Values of cortisol greater than 1.3 µg/dl correctly identified all cases of Cushing's syndrome and all cases of pseudo-Cushing's states (100% specificity, sensitivity, and diagnostic accuracy). While this is a general recommendation, each laboratory should confirm based on the sensitivity of cortisol assay. Furthermore it is important to confirm the serum level of dexamethasone at the time of the blood draw to assure patient compliance with the dexamethasone regimen. Patients with ectopic ACTH production will have nonsuppressed cortisol and ACTH levels that are not stimulated by CRH.

WHEN TO USE THIS TEST: Once the diagnosis of Cushing's disease has been made based on endocrinologic testing, the final step in the evaluation of such patients should be an MRI of the pituitary to confirm the presence of a pituitary mass. Unfortunately, MRI imaging of the pituitary as a primary diagnostic tool is distinctly unhelpful due to the fact that 10% of all normal individuals may have slight abnormalities of their pituitary and that in many subjects with Cushing's disease, the tumor may be too small to be imaged with MRI scans. However, subjecting a patient to surgical pituitary exploration in the absence of a demonstrable mass is likely to result in an unsuccessful surgery. Furthermore, if previous dexamethasone and/or CRH testing is equivocal, then IPSS should be performed to further confirm the pituitary as the source of the ACTH (27). Although this test is less reliable in lateralizing the ACTH source (i.e. left versus right), than it is in confirming that the ACTH is central in origin it can rule out ectopic ACTH production by a tumor (although ectopic CRF secreting tumors would be difficult to distinguish from true Cushings' disease based on IPSS). Simultaneous measurement of prolactin in the central samples can normalize the data if there is any difference in the location of the catheters (28).

PROCEDURE: This test is done in conjunction with a skilled interventional neuroradiologist. It is important that the endocrinologist is personally present in the room during the procedure so that assurance can be made that the proper blood tests were drawn at the specified times. The patient is brought to the angiogram suite without sedation. A large bore IV line is placed in an antecubatal fossa (to be certain there is access to peripheral blood sampling and CRF injection). Catheters (5 French) are placed in the femoral veins and threaded under fluoroscopic guidance to the inferior petrosal sinus. Injection of IV contrast confirms proper placement of the catheters.

Patients are on constant, pulse, blood pressure and oxygenation monitors during the course of the procedure. Test tubes are prechilled in ice and labeled so that during the rapid sampling period, blood can be placed in the tubes without delay.

We routinely obtain 4 baseline measurements at -15, -10, -5 and at 0 minutes. This allows for practice allowing proper coordination between the radiologists drawing blood from the IPSS and the individual drawing blood from the brachial vein. Appropriate amounts of blood should be removed to discard the dead space of the catheter (this varies depending on the size of the catheter used). 2 ml of blood is obtained in lavender top vacutainer tubes on ice for measurement of cortisol (on peripheral samples); ACTH and prolactin (on central samples).

At 0' CRF is then injected as described above for the peripheral CRF test. Blood is then sampled from both central and peripheral lines at 2', 5' 10' and 15'. After the 15' time point and right before the IPSS catheters are removed, repeat fluoroscopic localization of the catheters should be performed to confirm that there was no displacement during the sampling. However, sampling on peripheral blood may continue as described in the CRF test discussed above.

SPECIAL CONSIDERATIONS: The test can be performed at any time of the day, although it is usually done in the morning.

Side effects the patient may experience are: 1) slight nausea, 2) metallic taste, 3) urgency to urinate, 4) a change in blood pressure (either increase or decrease), 5) a change in heart rate, 6) headaches, 7) abdominal discomfort, 8) facial flushing, and 9) lightheadiness. These side effects are mild and last for only few minutes.

Patients greater than 300 pounds in weight may not be able to be supported by the standard fluoroscopic table. Furthermore such large patients may have an abdominal pannus that precludes reasonable access to the femoral veins. In such instances we have performed the IPSS in patients via catheters placed in the antecubtal vein and had the patient immobilized in the sitting position.

At our institution we have performed over 50 IPSS without complication. However there are reports in the literature that a stroke may occur (29). We recommend that the catheters remain in the petrosal sinus for no more than 30 minutes to minimize this possibility.

Freeze/thawing can decrease the immunoassayable ACTH (see above), therefore we recommend that the samples be brought to the endocrine lab and analyzed with 24 hours with the plasma separated on ice during this time. If analysis is not possible with 24 hours, the samples should be aliquoted and frozen to minimize the amount of freeze/thawing.

INTERPRETATION OF RESULTS: Plasma ACTH values are normalized to the prolactin value in order to correct for possible different localization of the catheters, or movement of the catheters during the study. The post CRF ACTH/Prolactin value of the central catheters should be >2.1 fold the ACTH/Prolactin value of the peripheral sample. In most cases of pituitary dependent Cushings, the increase is > 5.0 fold. Lateralization would mean that the ratio of the left to right side is >2.0. Often times the ratio criteria can be met without the need for CRF stimulation, however the diagnostic accuracy increases from 86% to 90% with CRF (30).