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The immuassay handbook parte74

  1. 1. 695© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00057-9 The Adrenal Cortex Les Perry1 (Leslie.Perry@croydonhealth.nhs.uk) Sami Medbak2 1 This edition. 2 Previous editions. Normal Adrenocortical Function There are two adrenal glands, one above each kidney, hence also known as the suprarenal glands. Each gland is composed of an outer adrenal cortex and an inner adrenal medulla. Histologically, the cortex is composed of three layers and secretes steroid hormones. The outer layer, zona glomerulosa, predominantly secretes the mineralo- corticoid hormone, aldosterone. The middle zone, zona fasciculata has finger-like protrusions toward the central adrenal medulla and secretes glucocorticoids, cortisol being the principal glucorticosteroid. Finally, the inner- most adrenal cortex layer is the zona reticularis which is responsible for the secretion of adrenal androgens, princi- pally dehydroepiandrostenedione (DHEA) and its sulfated metabolite (DHEAS) but also some androstenedione and testosterone. Cortisol and aldosterone are the most important physiological products of the adrenal cortex. Like most steroids cortisol is not very water soluble so is transported around the body bound to a protein, an α2-globulin, known as cortisol-binding globulin (CBG) or transcortin. In blood, ~92% cortisol is bound to CBG and ~8% is the non-protein-bound “free” hormone, which is the biologically active component. Note, in saliva, the measured “free fraction” is ~5%, and the difference from blood is that the salivary glands can convert cortisol to cor- tisone. Cortisol is mainly metabolized in the liver into a number of inactive compounds. Unbound cortisol is fil- tered by the kidney with the majority being reabsorbed by the tubules. Small amounts of free cortisol are excreted in urine; hence, a 24h urine free cortisol (UFC) collection produces an integrated measure of serum cortisol, evening out the variations in cortisol production over the 24h period. The major problem with this test is the potential for an inadequate 24h collection. Corticosteroids exert their cellular effects through specific receptors in the cell cytosol, producing vital and widespread effects on various tissues in the body includ- ing actions on carbohydrate, protein and fat metabo- lism, water metabolism, hematopoiesis and hemostasis. They affect the gastrointestinal, cardiovascular, skeletal, neuromuscular, and immunological systems and possess anti-inflammatory action. Cortisol secretion is controlled by the anterior pituitary hormone, adrenocorticotropic hormone (ACTH). ACTH is in turn controlled by the hypothalamic peptide corticotrophin-releasing hormone (CRH). CRH is released from the median eminence of the hypothalamus into the hypophyseal portal blood and acts on the corticotrophs of the anterior pituitary to cause the synthesis and release of ACTH. Vasopressin, from the posterior pituitary, acts in a synergistic way with CRH to promote ACTH secretion. Three major factors are involved in the control of the hypothalamic–pituitary–adrenocortical (HPA) axis: circa- dian rhythm, negative feedback, and stress mechanisms. Cortisol secretion in man is periodic, with peak secretion around 0700–0800h and its nadir around 0200–0400h at night. Cortisol exerts a negative feedback effect whereby high concentrations of serum cortisol suppress CRH and ACTH production at both the hypothalamus and the ante- rior pituitary, and, consequently, cortisol secretion is reduced (see Fig. 1). Conversely, CRH and ACTH production is increased when serum cortisol levels become too low. Stress is the third control mechanism and overrides the other two. Thus during any stressful condition (such as illness, acci- dents, operations, or psychological stress), the HPA axis is activated, leading to increased secretion of ACTH and cortisol, which can reach extremely high concentrations. Clinical Disorders The main disorders of the adrenal cortex are those relating to abnormal cortisol (~30 cases per million per year) and aldosterone (~10 cases per million per year) secretion. Rarely, abnormalities in androgen secretion are encountered. C H A P T E R 9.3 Hypothalamus Anterior Pituitary Adrenal Cortex Cortisol ACTH CRH Stress + stimulates - negative feedback FIGURE 1 Interactions within the HPA axis. (The color version of this figure may be viewed at www.immunoassayhandbook.com).
  2. 2. 696 The Immunoassay Handbook HYPERCORTISOLEMIA The clinical condition that results from the excessive secre- tion of cortisol is termed Cushing’s syndrome. Tradi- tionally, the causes of Cushing’s syndrome are divided into: G ACTH dependent—the hypercortisolism is due to hypersecretion of ACTH, such as pituitary tumors (also referred to as Cushing’s disease), and ectopic ACTH syndrome (where ACTH is secreted by non-endocrine tumors, such as lung cancer). G ACTH independent—where hypercortisolism is pres- ent, yet ACTH concentrations are very low or even undetectable due to an adrenal tumor (adenoma or carcinoma) or to exogenous administration of glucocorticoids. Pseudo-Cushing is a clinical condition that has all the clinical and most of the biochemical features of hypercor- tisolism but is obviated after treating the predisposing cause. The pathophysiology has yet to be fully established but the two commonest causes occur in patients with severe depression or with alcohol abuse. Cushing’s syndrome is more common in women (4F:1M), and the chronic excess of cortisol that character- izes the illness leads to a number of symptoms and signs including obesity (mainly of the face and trunk), easy bruising, purple abdominal striae, hirsutism, acne and greasy skin, hypertension, muscular weakness, menstrual disturbances, depression, and osteoporosis. The diagnosis of Cushing’s syndrome depends on: G demonstrating the presence of excessive cortisol secretion; G establishing the cause of the hypercortisolemia. This diagnostic process often involves difficult and pro- longed investigations, including the measurement of serum cortisol and plasma ACTH, first under basal condi- tions (circadian rhythm studies), and also after a number of provocative dynamic tests of both stimulation and sup- pression, such as the CRH stimulation test, dexametha- sone suppression test (DST), and venous catheter studies. A number of radiological imaging procedures are also required. This analytical process may appear simple, but the syndrome has an element of cyclicity making diagnosis more difficult. It is therefore essential that such patients are cared for in medical centers with special expertise in dealing with this condition. Treatment of Cushing’s syndrome depends on the cause. Hypercortisolemia is most effectively controlled with surgery but can also be managed using medical therapy (metyrapone, ketoconazole) or radiotherapy. In primary hyperaldosteronism, which is usually due to an adrenal adenoma and known as Conn’s syndrome, there are high levels of aldosterone in plasma, often result- ing in hypokalemic alkalosis with muscle weakness, polyuria and polydipsia, and high blood pressure. HYPOCORTISOLEMIA Hypocortisolemia is caused by adrenocortical insufficiency which is either primary (disease of the adrenal cortex) or secondary (due to pituitary or hypothalamic lesions). A life-threatening acute form of primary adrenocortical insufficiency may occur following operations or overwhelm- ing systemic infections with hemorrhage. However, it is more commonly chronic and is termed Addison’s disease. The commonest cause of Addison’s disease in the western world is autoimmunity. Other causative factors include sar- coidosis, tuberculosis, amyloidosis, hemochromatosis, and secondary malignant deposits. Patients with Addison’s disease suffer from generalized tiredness, anorexia and nausea, pigmentation or white patches in the skin, weight loss, dizziness and hypotension, and slow recovery from illness. Most of these features are due to the deficiency of cortisol. Excessive skin pigmenta- tion, which may be the earliest sign of the disease and is characteristic of this condition, is thought to be due to the high levels of plasma ACTH (or its related peptides). In secondary hypoadrenalism, there is no skin pigmentation and little change in blood pressure. Diagnosis of Addison’s disease relies on the demonstra- tion of low or undetectable serum cortisol levels that fail to rise following stimulation with ACTH (synacthen; tetra- cosactrin). The basal plasma ACTH concentrations in these patients are typically very elevated. In contrast, patients with secondary adrenocortical insufficiency have low or undetectable serum cortisol with an inappropriately low concentration of plasma ACTH. Treatment of adrenocortical insufficiency is with hydro- cortisone or prednisolone (a synthetic glucocorticoid) given in dosage forms so as to mimic the normal circadian rhythm of serum cortisol. A typical dose would be 20mg hydrocortisone in the morning and 10mg in the evening, although in some patients, glucocorticoids given three times a day is a more appropriate treatment. Another, rare disease of the adrenal cortex resulting in hypocortisolemia is congenital adrenal hyperplasia. This is a family of congenital enzymatic defects in the pathway of cortisol synthesis. The commonest deficiency (~92% of all causes) is that of 21-hydroxylase enzyme leading to hypocor- tisolemia with elevated ACTH levels; the latter leads to bilateral adrenal enlargement (hyperplasia). There is a con- comitant deficiency of aldosterone in over 60% of cases resulting in salt loss. The enzyme block, coupled with excess ACTH, leads to increased androgen secretion by the adre- nals resulting in virilization. The diagnosis relies on finding: 1. Low serum cortisol. 2. Elevated levels of 17α-hydroxyprogesterone (a pre- cursor of cortisol before the enzyme block) either basally or following stimulation with ACTH. Androstenedione and ACTH levels are also raised. Early diagnosis and treatment with cortisol (and fludro- cortisone, a synthetic mineralocorticoid) can be life saving. Analytes CORTISOL Cortisol is the most important steroid hormone. It is pro- duced by the adrenal cortex from low-density lipoprotein cholesterol by several enzymatic reactions (see Fig. 2).
  3. 3. 697CHAPTER 9.3 The Adrenal Cortex Function Cortisol is an essential hormone and has a wide variety of effects on most tissues in the body (see NORMAL ADRENO- CORTICAL FUNCTION). It is particularly important through its gluconeogenic action, in coping with situations of men- tal or physical stress such as infections and operations. Most of the cortisol circulates bound to protein in plasma with small amounts circulating in the free, biologi- cally active form. Predominantly, immunoassays measure the total cortisol in serum so concentrations can seem high in situations where concentrations of CBG are raised such as pregnancy and estrogen therapy. Some commercial ana- lyzer reagents have been developed to measure the “free” cortisol fraction only. These assays have been developed for the measurement of cortisol in saliva samples. Reference Interval The quoted reference intervals will vary with different manufacturers’ analyzers and reagents, due to the calibra- tion of the method. Generally, the 0900h range is Serum: 160–600nmol/L at 0900h. : <50nmol/L at 2400h (asleep). Saliva: 10–27nmol/L at 0900h. Clinical Applications The measurement of cortisol in serum is used in the diag- nosis of disorders of the HPA axis. In contrast to other endocrine disorders where assay of single or basal samples is sufficient for diagnosis, dynamic tests (stimulation and/ or suppression tests) are almost always required in the diagnosis of disorders of the HPA axis. Cushing’s syndrome As stated above, hypercortisolemia together with the loss of the normal circadian rhythm of cortisol secretion and disturbed feedback of the HPA axis are the cardinal bio- chemical features of Cushing’s syndrome. Tests to con- firm the diagnosis are based on these principles. It is important to realize that the validation of the published test criteria employed has been for specific assays, and Cholesterol (LDL) Pregnenolone 17OHP5 DHEA Progesterone 17OHP4 A4 Testosterone 11-DOC 11-deoxycortisol Estrone Estradiol Corticosterone Cortisol Aldosterone Key: Major pathway for cortisol synthesis Minor pathways 17OHP5 : 17-Hydroxypregnenolone 17OHP4 : 17-Hydroxyprogesterone 11-DOC : 11-Deoxycorticosterone A4 : Androstenedione DHEA : Dehydroepiandrosterone FIGURE 2 Adrenal steroid biosynthetic pathways.
  4. 4. 698 The Immunoassay Handbook thus, test responses should ideally be validated on the local assay used before the results can be interpreted in particu- lar patients. In Cushing’s syndrome, where there is an excessive secretion of cortisol, the following diagnostic strategy is recommended: 1. Screening tests (as outpatient) for overproduction of cortisol by either: G An overnight (1mg single dose) DST: patients with Cushing’s syndrome fail to suppress their serum cortisol to <50nmol/L following the inges- tion of dexamethasone, a synthetic glucocorticoid. G Or a 24h urinary free cortisol measurement: expected to be elevated in patients with Cushing’s syndrome. G Night time salivary cortisol can be used as a screening test for Cushing’s syndrome. Salivary cortisol concentrations follow the serum circa- dian rhythm pattern. This simple, noninvasive test can be performed at home and offers advan- tages as it is possible to monitor patients over time—a useful feature for a syndrome that is rec- ognized to be clinically active and then “cycle” into a nonactive phase. In the event of an abnormal screening result or if a nor- mal result was seen on screening, but the clinical suspi- cion of Cushing’s syndrome is very high, then the patient needs to have in-patient investigation tests to confirm overproduction of cortisol. 2. Confirm overproduction of cortisol and establish the cause of Cushing’s syndrome by performing in this order the following tests: a) Measure plasma ACTH concentration: if ACTH is undetectable, an adrenal tumor is indicated; detectable ACTH suggests a pituitary or ectopic source. b) Measure serum cortisol to assess for a circadian rhythm: rhythm of cortisol is lost in Cushing’s syndrome, i.e., in normal subjects serum cortisol levels at midnight are <50nmol/L provided the patient is asleep. c) CRH test: 100µg human CRH-41 injected intravenously and serum cortisol (and ACTH) measured. An exaggerated response (rise in cor- tisol to above 600nmol/L) indicates pituitary disease. In contrast, patients with the ectopic ACTH syndrome do not respond (see Fig. 3). This is carried out prior to the low-dose DST. Originally, the CRH test was done after the low- dose dexamethasone suppression, but on occa- sion, some patients had a prolonged suppressive effect of the low-dose DST leading to misinter- pretation of the CRH test. d) Perform a low-dose DST: 2mg orally every 6h for 48h; normal subjects suppress their serum cortisol to <50nmol/L; patients with Cushing’s syndrome do not (Fig. 4). e) Venous catheter studies: see ADRENOCORTICO- TROPIC HORMONE, CLINICAL APPLICATIONS. The high-dose DST and the insulin-induced hypoglyce- mia test are rarely used to investigate patients with sus- pected Cushing’s syndrome. The high-dose DST consists of 2mg dexamethasone orally 6 hourly for 48h; 50% or more suppression of serum cortisol indicates pituitary dis- ease. This test is now rarely used due to the improvements in imaging techniques. The insulin-induced hypoglycemia test was used for excluding depression as the cause of hypercortisolemia: patients with Cushing’s syndrome show no cortisol rise during hypoglycemia. Addison’s disease 1. Morning (0900h) serum cortisol: low or undetectable. 2. ACTH (tetracosactrin; synacthen) stimulation test: short and long synacthen tests. In healthy individu- als, serum cortisol levels rise to >500nmol/L (or an increment of >200nmol/L over the basal sample) or more 30–60min after 0.25mg synacthen is given intramuscularly. No cortisol rise is seen in Addi- son’s disease. A delayed rise (6–24h) is seen in sec- ondary hypoadrenocorticism (due to pituitary or hypothalamic causes). FIGURE 3 Examples of cortisol response in CRH test. FIGURE 4 Examples of responses in DST.
  5. 5. 699CHAPTER 9.3 The Adrenal Cortex Limitations G Serum cortisol levels are affected by changes in the concentrations of CBG; cortisol concentrations can seem high in situations where concentrations of CBG are raised such as pregnancy and estrogen therapy. G Cortisol measurements can be affected by one or more of the synthetic or natural steroids and so it is unreliable in patients taking prednisolone, a commonly prescribed glucocorticoid. Some immunoassay methods for corti- sol cross-react significantly with 11-deoxycortisol, the immediate precursor of cortisol. This compound is excessively elevated in patients with Cushing’s syn- drome on metyrapone treatment (metyrapone inhibits the conversion of 11-deoxycortisol to cortisol). This is a serious drawback because this cross-reactivity results in apparently normal or even elevated cortisol levels in patients who have been rendered hypoadrenal. Such assays must therefore be used with great care. G Some methods for cortisol measurement that use non- isotopic immunoassays have a serious problem of non- specific interference, often associated with reagents used to denature cortisol-binding proteins before assay. UKNEQAS (2010) identified a possible male and female difference in results returned by users of this EQA scheme dependent on which analyzer was used. When male specimens were dispatched, a Gaussian dis- tribution was observed. However, for the female speci- men pools, there is a bimodal result distribution. The higher peak was with the Roche Elecsys analyzer. However, when this was followed up with a mass spec- trometry method, the Roche analyzer demonstrated good agreement with MS, while the other assays under- estimated the cortisol concentrations. This gender dif- ference may be due to the effect of different blocking agents used or due to a protein problem in the com- mercial assays. It is important to remember therefore that the normal cortisol response to a specific provoca- tive test should ideally be validated on the local assay used before the results can be interpreted in particular patients. This is best illustrated with the short synac- then test. G A national audit of the short synacthen test in the UK has shown that although the preanalytical procedures were similar in most laboratories, there is a require- ment to recognize the effect that method bias may have on the reference intervals and consequently on the diagnosis of adrenal insufficiency. There is a need to develop consensus guidelines, which can aid both clini- cians and laboratories, and the impact of MS may help to minimize the method-related bias and generate har- monization of cortisol reference intervals during a short synacthen test. G Many of the automated, non-extraction immunoassays for UFC are modifications of the serum cortisol assay. There are many structurally similar compounds and metabolites of cortisol in urine which can result in an overestimate of the true 24h UFC result (look at the 24h UFC reference intervals quoted in the kit inserts of the major analyzers). Many laboratories (and manu- facturers) try to minimize this nonspecificity by incor- porating an organic solvent extraction step prior to immunoassay. Increasingly though, LCMS is used to measure UFC, which overcomes the previous prob- lem for immunoassays of cross-reactivity of other structurally similar compounds. Assay Technology The commonly used methods for cortisol measurement are all automated robotic instruments and include Seimens ACS™: 180, Abbott Architect, Siemens IMMULITE™, DELFIA™, Roche Diagnostics Modular, and Tosoh. Serum cortisol by isotopic immunoassays has virtually dis- appeared in routine service laboratories. Very recently, there has been interest in major service laboratories to move to tandem mass spectrometry as the preferred ana- lytical technique for serum cortisol to obviate the well- recognized specificity problems of immunoassay. Saliva cortisol assays in general use modified serum cortisol radioimmunoassay, enzyme-linked immunoassay, or tan- dem mass spectrometry. Desirable Assay Performance Characteristics Knowledge of the degree of cross-reactivity with other ste- roids especially 11-deoxycortisol, prednisolone, and dexa- methasone is vital. Current automated assays for serum cortisol can reproducibly measure down to 10nmol/L. An analyzer should generate a result within 60min (for urgent cases, e.g., some patients in intensive care units with occult adrenocortical insufficiency). Saliva cortisol assays have different reference intervals and diagnostic cutoff values because of the different assays and the reader should refer to the laboratory method derived values. The same will apply to the 24h UFC sample. Types of Sample Serum, plasma, or saliva. Ideally, specimen collection should be done at 0800–1000h since reference intervals are usually established for this time of day. A 24h urine collection can be used, but the patient must be given writ- ten instructions on the sample collection protocol. Frequency of Use Very common. ADRENOCORTICOTROPIC HORMONE ACTH (corticotropin) is a 39 amino acid peptide with a relative molecular mass of 4500 (4.5kDa) that is derived from a larger (31kDa) precursor, pro-opiomelanocortin, which is secreted by the corticotroph cells of the anterior pituitary. ACTH exists in the circulation in different molecular forms, some of which may be inactive biologically and undetected by immunoassays. Function ACTH is secreted by the anterior pituitary to stimulate the adrenal cortex directly, to synthesize and secrete glucocor- ticoids, the most important of which is cortisol. ACTH in
  6. 6. 700 The Immunoassay Handbook turn is secreted in response to CRH and vasopressin from the hypothalamus. Serum cortisol exerts a negative feed- back effect on the hypothalamus and pituitary to control the level of ACTH in the circulation. The bioactivity of ACTH resides in its first 24 amino-terminal amino acids. Reference Interval 10–60ng/L at 0900h (2.2–13.2pmol/L). Clinical Applications The measurement of plasma ACTH simultaneously with serum cortisol is recommended to help to deter- mine the etiology of Cushing’s syndrome, primary and secondary adrenal insufficiency, and rarer disorders such as adrenoleukodystrophy and Nelson’s syndrome. Cushing’s syndrome G Basal ACTH: measurement of ACTH at 0900h helps to establish the cause of Cushing’s syndrome. Periph- eral plasma ACTH levels are consistently <10ng/L if the underlying pathology is an adrenal tumor (both adenomas and carcinomas) because these tumors secrete cortisol independently of ACTH and lead to suppression of plasma ACTH levels through the nega- tive feedback mechanism. In contrast, if plasma ACTH concentrations are either detectable (>15ng/L) or high, then a diagnosis of adrenal tumor is excluded, and the underlying pathology is most likely to be either a pitu- itary or non-pituitary (ectopic) tumor such as lung can- cer. Excessively elevated levels (>300ng/L) are highly indicative of an ectopic source of ACTH. G CRH test: used to differentiate the diagnosis of ACTH-dependent Cushing’s syndrome. Pituitary ACTH-secreting adenomas retain their response to CRH, while ectopic ACTH tumors lack CRH recep- tors and therefore do not respond to CRH. Globally, there have been many protocols used including the type of CRH used and sampling time points, conse- quently, there is no unified agreement when interpret- ing the ACTH response to this test. The test involves the intravenous administration of 100µg (human or ovine) CRH-41 and the measurement of serum cortisol and plasma ACTH thereafter. Patients with active pituitary disease usually respond by elevating their ACTH levels to above 100ng/L, whereas patients with the ectopic ACTH syndrome do not show a response. G Venous catheter studies: ACTH measurements dur- ing venous catheter studies are considered the “gold standard” test to differentiate between Cushing’s dis- ease and an ectopic ACTH-secreting source. Thus, if a pituitary cause is suspected, the inferior petrosal sinuses (veins that drain the pituitary) can be cannulated and blood obtained for ACTH measurements before and after an injection of CRH. This procedure helps in the diagnosis of pituitary-dependent Cushing’s disease and in localizing the tumor within the pituitary itself. A central (inferior petrosal) to peripheral plasma ACTH gradient of 2:1 or greater pre-CRH or a gradient 3:1 or greater post-CRH is consistent with Cushing’s disease. Plasma ACTH measurement also helps in the follow- up of patients treated for Cushing’s disease (see Fig. 5). If the cause of Cushing’s syndrome is suspected to be an ectopic (non-pituitary) tumor, then a whole-body catheter Time after CRH Sampling site Baseline 3–5 min 8–10 min 1. Right low internal jugular X X 2. Left low internal jugular X X 1+2 peripheral X X 3. Right high internal jugular X X 4. Left high internal jugular X X 3+4 Peripheral X X 5. Right inferior petrosal sinus 6. Left inferior petrosal sinus 5+6 Peripheral FIGURE 5 Venous sampling for ACTH with CRH administration in ACTH-dependent Cushing’s syndrome. The peripheral sample is collected simultaneously to the respective jugular or petrosal sinus sample for ACTH and cortisol. Samples marked “X” are not required.
  7. 7. 701CHAPTER 9.3 The Adrenal Cortex study may need to be done where different veins in the chest and abdomen are cannulated and blood drawn for ACTH measurement. High concentrations of ACTH will be demonstrable in the veins draining the tumor and thus aid in tumor localization. This procedure is rarely used these days due to the significant improvement in imaging techniques. See MANAGEMENT OF PATIENTS WITH CUSH- ING’S SYNDROME. Nelson’s syndrome Nelson’s syndrome: is a rare but serious ACTH-secret- ing pituitary tumor that arises as a complication of bilateral adrenalectomy, a surgical procedure employed in difficult cases of Cushing’s syndrome where the primary source cannot be identified, but there is a need to control the effects of the hypercortisolism. These cases on the grounds of probability are usually pituitary Cushing’s disease. Post- adrenalectomy, plasma ACTH measurement pre- and 2h post-hydrocortisone are extremely valuable to evaluate the aggressiveness of the Nelson’s tumor; the more aggressive the tumor the less it responds to hydrocortisone. Adrenocortical insufficiency G Plasma ACTH levels are very useful in discerning the etiology of adrenocortical failure. ACTH levels are elevated (usually >150ng/L but sometimes grossly so [>300ng/L]) in primary adrenocortical insufficiency (Addison’s disease), whereas in secondary adrenocorti- cal insufficiency (due to pituitary or hypothalamic causes), ACTH concentrations are inappropriately low or undetectable, with a concurrent low serum cortisol. G CRH test: this test occasionally will help to differenti- ate the pituitary from hypothalamic causes of adreno- cortical insufficiency. Thus, patients with hypothalamic lesions will have raised ACTH levels after CRH injec- tion, whereas patients with pituitary disease show no response. Limitations G ACTH is an unstable peptide in plasma due to degra- dation by endogenous peptidases. Therefore, process- ing of the blood specimen must be done quickly so you will require a blood collection tube with antico- agulant (e.g., EDTA or lithium–heparin), a refriger- ated (4°C) centrifuge and solid CO2 to freeze the plasma immediately, once it has been transferred to a clean labeled tube. It is recommended that the entire process of venepuncture to freezing the separated plasma sample should be done within 20min to avoid a spuriously low result. G The assay is useful for the differential diagnosis but not the diagnosis of Cushing’s syndrome where only cortisol measurement is required. G The absolute concentrations of ACTH are not very useful in distinguishing pituitary from ectopic causes of Cushing’s syndrome. In the majority of such cases, the ACTH concentrations lie between 30 and 200ng/L. Thus, other ways to differentiate these two entities are often required. G Not all of the ACTH assays available are sensitive enough to detect levels in all healthy individuals and are therefore not useful in differentiating low normal levels from the suppressed levels recorded in patients with adrenal tumors. Assay Technology Early assays used for the measurement of ACTH were competitive radioimmunoassays that used a plasma extrac- tion procedure, such as the use of Vycor glass to adsorb the peptide. These extraction assays were time-consuming, labor intensive and had low sample throughput. They have been superseded with non-isotopic immunometric assay methods that have the required sensitivity and are rapid to perform. Specificity can be a problem as some immuno- metric assays are so specific for ACTH 1–39 that they do not detect the larger forms of ACTH or its smaller frag- ments. These forms may be the only ones circulating in patients with the ectopic ACTH syndrome and would therefore be reported as undetectable instead of grossly elevated. This could lead to disastrous errors in diagnosis. It is therefore mandatory to check for the specificities of ACTH assays in relation to their clinical performance before they can be used for patient samples. Most smaller laboratories send specimens to specialized centers for ACTH measurement. The popular methods used include Siemens IMMULITE® and Roche Modular analyzer assays. As previously mentioned under serum cor- tisol, it is important to remember that there may be differ- ences in plasma ACTH results from different commercial analyzer manufacturers due to the lack of an international calibrant. Desirable Assay Performance Characteristics Clinical assay sensitivity of 5–10ng/L is necessary. Detailed knowledge of assay specificity is mandatory to avoid errors in diagnosis. Type of Sample Plasma: blood is collected into plastic tubes containing heparin or EDTA; glass tubes must not be used for blood collection as ACTH will be adsorbed to glass and lost. Owing to the instability of the peptide, blood needs to be centrifuged at 4°C immediately after venepuncture and plasma decanted, flash frozen, and stored at −20°C until assay (stable for 6 months). Ideally, sample collection should be performed at 0800–1000h since reference ranges are usually established for this time of day. Frequency of Use Uncommon. Management of Patients with Cushing’s Syndrome PREADMISSION G Diagnosis of Cushing’s syndrome suspected on clinical grounds.
  8. 8. 702 The Immunoassay Handbook G Diagnosis of Cushing’s syndrome highly likely bio- chemically due to failure to suppress serum cortisol by low-dose DST or by elevated urinary free cortisol concentration. Ideally, obtain a plasma sample for ACTH. ADMIT TO ENDOCRINE WARD (E.G., ON MONDAY) Monday Admit patient to ward by noon. Routine chemistry (plasma potassium, bicarbonate, and glucose in particular), hema- tology, and chest X-ray. Blood sample for plasma ACTH. Take blood at midnight (while patient asleep). Tuesday Circadian rhythm study: samples for cortisol at 0900, 1800h, and midnight (while asleep). CRH test: give 100µg hCRH intravenously and measure cortisol and ACTH. Wednesday Finish circadian rhythm study: samples for cortisol at 0900h. This sample is also the start of the low-dose DST: 0.5mg dexamethasone orally 6 hourly for 48h. Friday Take blood at 0900h for serum cortisol. This sample ends the low-dose DST. By Following Monday All results of tests are available and provide provisional diagnoses: G No suppression on low-dose DST=Cushing’s syn- drome. If pseudo-Cushing due to depression is sus- pected, perform an insulin hypoglycemia test. G Plasma ACTH undetectable: diagnostic of adrenal tumors. Proceed to radioimaging of abdomen and adrenalectomy. G Plasma ACTH detectable but not very grossly elevated: pituitary or ectopic ACTH-producing tumor. a. Serum cortisol rises during CRH test and shows inadequate suppression during low-dose DST=pitu- itary tumor. Proceed to pituitary radioimaging, petrosal sinus catheter study, and hypophysectomy. b. Serum cortisol does not rise during CRH test and does not suppress on low-dose DST=ectopic ACTH-producing tumor. Plasma ACTH may be grossly elevated (greater than 300ng/L) plus hypo- kalemia, alkalosis, and hyperglycemia. Proceed to radioimaging studies (and venous catheters) of chest and abdomen to localize tumor followed by its surgi- cal removal. c. If results of CRH and high-dose DST are discrep- ant, i.e., do not point to a uniform diagnosis as in (a) or (b), it becomes imperative to proceed to petrosal catheter study since pituitary disease is much more common than ectopic ACTH-producing lesions. If petrosal catheterization is not possible, patient needs to be referred to a specialist center capable of per- forming the procedure. Further Reading Assie, G., Bahurel, H., Coste, J., Silvera, S., Kujas, M., Dugue, M.A., Karray, F., Dousset, B., Bertherat, J., Legmann, P. and Bertagna, X. Corticotroph tumor progression after adrenalectomy in Cushing’s disease: a reappraisal of Nelson’s syndrome. J. Clin. Endocrinol. Metab. 92, 172–179 (2007). Beko, G., Varga, I., Glaz, E., Sereg, M., Feldman, K., Toth, M., Racz, K. and Patocs, A. Cutoff values of midnight salivary cortisol for the diagnosis of overt hypercortisolism are highly influenced by methods. Clin. Chim. Acta 41, 364–367 (2010). Clark, P.M., Neylon, I., Raggatt, P.R., Sheppard, M.C. and Stewart, P.M. Defining the normal cortisol response to the short Synacthen test: implications for the investiga- tion of hypothalamic–pituitary disorders. Clin. Endocrinol. 49, 287–292 (1998). Carroll, T.B. and Findling, J.W. The diagnosis of Cushing’s syndrome. Rev. Endocr. Metab. Disord. 11, 147–153 (2010). Carroll, T., Raff, H. and Findling, J.W. Late-night salivary cortisol for the diagno- sis of Cushing syndrome: a meta-analysis. Endocr. Pract. 15, 335–342 (2009). Chatha, K.K., Middle, J.G. and Kilpatrick, E.S. National UK audit of the short synacthen test. Ann. Clin. Biochem. 47, 158–164 (2010). De Brabandere, V.I., Thienpont, L.M., De Stockl, D. and Leenheer, A.P. Three routine methods for serum cortisol evaluated by comparison with an isotope dilution gas chromatography–mass spectrometry method. Clin. Chem. 41, 1781–1783 (1995). Elamin, M.B., Murad, M.H., Mullan, R., Erickson, D., Harris, K., Nadeem, S., Ennis, R., Erwin, P.J. and Montori, V.M. Accuracy of diagnostic tests for Cushing’s syndrome: a systematic review and metaanalyses. J. Clin. Endocrinol. Metab. 93, 1553–1562 (2008). Erturk, E., Jaffe, C.A. and Barkan, A.L. Evaluation of the integrity of the hypotha- lamic–pituitary–adrenal axis by insulin hypoglycemia test. J. Clin. Endocrinol. Metab. 83, 2350–2354 (1998). Grossman, A.B., Howlett, T.A., Perry, L., Coy, H., Savage, M.O., Lavender, P., Rees, L.H. and Besser, G.M. Corticotropin-releasing hormone in the differen- tial diagnosis of Cushing’s syndrome: a comparison with the dexamethasone suppression test. Clin. Endocrinol. 29, 167–178 (1988). Horrocks, P.M., Jones, A.F., Ratcliffe, W.A., Holder, G., White, A., Holder, R., Ratcliffe, J.G. and London, D.R. Patterns of ACTH and cortisol pulsatility over twenty-four hours in normal males and females. Clin. Endocrinol. 32, 127–134 (1990). Hurel, S.J., Thompson, C.J., Watson, M.J., Baylis, P.H. and Kendall-Taylor, P. The short synacthen and insulin stress tests in the assessment of the hypotha- lamic–pituitary–adrenal axis. Clin. Endocrinol. 44, 141–146 (1996). Lamberts, S.W.J., Bruining, H.A. and De Jong, F.H. Corticosteroid therapy in severe illness. N. Engl. J. Med. 337, 1285–1293 (1997). Mayenknecht, J., Diederich, S., Bahr, V., Plockinger, U. and Oelkers, W. Comparison of low and high-dose corticotropin stimulation tests in patients with pituitary disease. J. Clin. Endocrinol. Metab. 83, 1558–1562 (1998). Meinardi, J.R., Wolffenbuttel, B.H. and Dullaart, R.P. Cyclic Cushing’s syndrome: a clinical challenge. Eur. J. Endocrinol. 157, 245–254 (2007). Mukherjee, J.J., Jacome de Castro, J., Kaltsas, G., Afshar, F., Grossman, A.B., Wass, J.A.H. and Besser, G.M. A comparison of the insulin tolerance/glucagon test with the short ACTH stimulation test in the assessment of the hypothal- amo–pituitary–adrenal axis in the early post-operative period after hypophysec- tomy. Clin. Endocrinol. 47, 51–60 (1997). Neary, N. and Nieman, L. Adrenal insufficiency: etiology, diagnosis and treatment. Curr. Opin. Endocrinol. Diabetes Obes. 17, 217–223 (2010). Newell-Price, J., Trainer, P.J., Perry, L.A., Wass, J.A.H., Grossman, A.B. and Besser, G.M. A sleeping midnight cortisol has 100% sensitivity for the diagno- sis of Cushing’s syndrome. Clin. Endocrinol. 43, 545–550 (1995). Newell-Price, J., Morris, D.G., Drake, W.M., Korbonits, M., Monson, J.P., Besser, G.M. and Grossman, A.B. Optimal response criteria for the human CRH test in the differential diagnosis of ACTH-dependent Cushing’s syndrome. J. Clin. Endocrinol. Metab. 87, 1640–1645 (2002). Nieman, L.K., Biller, B.M., Findling, J.W., Newell-Price, J., Savage, M.O., Stewart, P.M. and Montori, V.M. The diagnosis of Cushing’s syndrome: an Endocrine Society Clinical Practice Guideline. J. Clin. Endocrinol. Metab. 93, 1526–1540 (2008). Nunes, M.L., Vattaut, S., Corcuff, J.B., Rault, A., Loiseau, H., Gatta, B., Valli, N., Letenneur, L. and Tabarin, A. Late-night salivary cortisol for diagnosis of overt and subclinical Cushing’s syndrome in hospitalized and ambulatory patients. J. Clin. Endocrinol. Metab. 94, 456–462 (2009). Oldfield, E.H., Doppman, J.L., Nieman, L.K., Chrousos, G.P., Miller, D.L., Katz, D.A., Cutler, Jr., G.B. and Loriaux, D.L. Petrosal sinus sampling with and without corticotropin-releasing hormone for the differential diagnosis of Cushing’s syndrome. N. Engl. J. Med. 325, 897–905 (1991). Orth, D.M. Differential diagnosis of Cushing’s syndrome. N. Engl. J. Med. 325, 957–959 (1991). Owen, L.J., Halsall, D.J. and Keevil, B.G. 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