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63 Salivary cortisol can be measured by immunoassay or liquid chromatography�Ctandem mass spectrometry (LC/MS-MS). Given the lower sensitivity of LC/MS-MS (as it may not measure cortisol metabolites/precursors), immunoassay remains the preferred methodology for screening of Cushing��s syndrome.67�C69 Educating the patient on optimal timing of specimen collection while taking into account their normal sleep�Cwake cycle, as well as avoiding exciting or stressful experiences on the evening of the test, are important for an accurate salivary cortisol result. False-positive results can also be seen, due to contamination of the saliva sample with widely available nonprescription topical hydrocortisone creams and ointments. However, overall, ease of collection, stability at room temperature, and a greater than 90% sensitivity and specificity Natural Product Library make it a highly useful test, especially in outpatients, children, and in assessment of cyclic Cushing��s syndrome.67�C69 Midnight plasma cortisol levels also can be used for screening. A single sleeping midnight plasma cortisol level of less than 1.8 ��g/dL (50 nmol/L) excludes active Cushing��s syndrome.70 A midnight plasma cortisol level, drawn while the patient is resting but not fully asleep, of more than 7.5 ��g/dL (207 nmol/L) is a more specific cutoff, but can miss up to 7% cases of mild Cushing��s syndrome.71,72 If the diagnosis remains unclear, dexamethasone-suppressed CRH stimulation (Dex-CRH) test73,74 or the desmopressin test75,76 can be performed to distinguish further between CD and pseudo-Cushing��s Aldosterone states. GDC-0973 price However, the exact diagnostic accuracy of these tests and the optimal cutoffs for diagnosis need further evaluation.77 Moreover, similarly to other dexamethasone tests, variable absorption and metabolism of dexamethasone, especially in the setting of concomitant use of other commonly prescribed medications, can affect the accuracy of the test.78 Recently, a prospective study of 73 patients with clinical features of hypercortisolism and an abnormal DST or UFC result reported a positive predictive value of 100% and a negative predictive value of 90% for the Dex-CRH test (sensitivity 94%, specificity 100%) using a 15-minute post-CRH cortisol cutoff of 3.2 ��g/dL (87 nmol/L).79 Establishing the cause of Cushing��s syndrome Once the diagnosis of Cushing��s syndrome has been established, the next step is to differentiate between the three causes. Measurement of plasma ACTH levels is the initial step in the differential diagnosis. A two-site immunoradiometric assay is preferred over radioimmunoassay, because it better discriminates low or suppressed ACTH levels.80 To avoid falsely low results, ACTH levels should be measured on multiple occasions, and samples should be collected in prechilled ethylenediaminetetraacetic acid tubes, transported in an ice bath, and processed immediately.1 Values