Table 2.

Summary of published studies on the use of second-line tests in the differential diagnosis of pseudo-Cushing’s states and Cushing’s syndrome.

First author, year (reference)	Patients, n°	Second-line test	Protocols	Assays	Cut-offs for interpretation	SE (%)	SP (%)	Remarks
Papanicolaou DA, 1998 (62)	PCS 23 CS 240 (CD 198, EAS 27, ACS 15)	Midnight serum cortisol	Serum was obtained at 12:00 h using an indwelling venous catheter placed at 22:00. Patients were fasting from 21:00 h.	RIA – Fluorescence polarization immunoassay	Cortisol 207 nmol/L (7.5 μg/dL)	96	100	Hospital admission is required. In a subset of CD patients, the cortisol diurnal rhythm is preserved. The midnight serum cortisol test may be falsely negative in patients with mild or quiescent CD.
Alwani RA, 2014 (63)	PCS 20 CD 53	Midnight serum cortisol	Serum was obtained at 12:00 h in wakeful patients, using an indwelling venous catheter placed one day in advance.	Chemiluminescence immunoassay	Cortisol 243 nmol/L (8.8 μg/dL)	98	95
Gatta B, 2007 (64)	PCS 14 CD 17 (mild)	Midnight serum cortisol	Serum was obtained at 12:00 husing an indwelling venous catheter placed at least 6 h before blood sampling. All patients had at least one night of habituation to the hospital environment.	RIA – Chemiluminescence immunoassay	Cortisol 256 nmol/L (9.3 μg/dL)	100	100
Alwani RA, 2014 (63)	PCS 20 CD 53	Cortisol midnight:morning ratio	Serum was obtained at 09:00 h and 12:00 h in wakeful patients, using an indwelling venous catheter placed one day in advance.	Chemiluminescence immunoassay	Ratio 0.67	87	100
Yanovski JA, 1993 (65)	PCS 19 CS 39 (CD 35, ACS 2, EAS 2)	DST-CRH test	Serial blood samples for ACTH and cortisol were obtained at time −15′, −10′, −5′, 0′, 5′, 15′, 30′, 45′, 60′ after administering 1 μg/kg of oCRH as an i.v. bolus at 08:00 and within 2 h of administering the last 0.5 mg Dexa in the 2 mg/day suppression test (0.5 mg Dexa every 6 h for 48 h starting at 12:00).	RIA	Cortisol at 15′ 38 nmol/L (1.4 μg/dL)	100	100	Expensive and cumbersome. Hospital admission is required. Lack of standardization of protocols and assays. Inter-individual variation in Dexa metabolism. Normal pituitary corticotrophs may retain a degree of responsiveness to pharmacological doses of exogenous CRH while suppressed with Dexa.
Martin NM, 2006 (66)	PCS 3 CS 12 (CD 8, ACS 4) CS-E 16	DST-CRH test	Patients received 0.5 mg Dexa orally every 6 h for 48 h, starting at 09:00. 48 h after the first Dexa dose a blood sample for serum cortisol was taken and a ninth dose of 0.5 mg Dexa was given. 2 h after the ninth dose, a blood sample was taken and an i.v. bolus of 100 μg hCRH was done. A final blood sample was collected 15′ after hCRH injection.	Chemiluminescence immunoassay	Cortisol at 15′ 50 nmol/L (1.8 μg/dL)	100	88
Gatta B, 2007 (64)	PCS 14 CD 17 (mild)	DST-CRH test	Serial blood samples for ACTH and cortisol were obtained at time −15′, −10′, −5′, 0′, 5′, 15′, 30′, 45′, 60′ after administration of 100 μg hCRH as an i.v. bolus at 08:00 and within 2 h from the administration of the last 0.5 mg Dexa tablet in the 2 mg/day suppression test (0.5 mg Dexa every 6 h for 48 h starting at 12:00). oCRH was used in 6 CD patients.	RIA – Chemiluminescence immunoassay	Cortisol at 15′ 110 nmol/L (4 μg/dL) ACTH at 15′ 3.5 pmol/L (16 pg/mL)	100 100	86 85
Erickson D, 2007 (67)	PCS 30 CD 21	DST-CRH test	Subjects initiated 0.5 mg Dexa orally every 6 h for 8 doses (first dose at 12:00). oCRH in a dose of 1 μg/kg (maximum 100 μg) was administered i.v. 2 h after completion of the last dose of Dexa.	Chemiluminescence immunoassay	Cortisol at 15′ 70 nmol/L (2.5 μg/dL) ACTH at 15′ 5.9 pmol/L (27 pg/mL)	90 95	90 97
Alwani RA, 2014 (63)	PCS 20 CD 53	DST-CRH test	Patients received 0.5 mg Dexa orally every 6 h for 48 h, starting at 12:00. This was followed by i.v. administration of 1 μg/kg hCRH 2 h after the last Dexa tablet. Serum cortisol and ACTH were measured at time −15′, −1′, 5′, 15′, 30′, 45′, 60′ after CRH administration.	Chemiluminescence immunoassay	Cortisol at 15′ 87 nmol/L (3.2 μg/dL)	94	100
Pecori-Giraldi F, 2007 (70)	PCS 23 CS 32 (CD 29, ACS 3)	DST-CRH test	Serial blood samples for ACTH and cortisol were obtained at time −30′, 0′, 15′, 30′, 45′, 60′, 90′ and 120′ after administration of 100 µg oCRH injected as an i.v. bolus within 2 h (08:00) of administering the last 0.5 mg Dexa tablet in the 2 mg/day suppression test (0.5 mg Dexa every 6 h for 48 h starting at 12:00).	Immunometric assay	Cortisol at 15′ 38 nmol/L (1.4 μg/dL)	100	62.5
Martin NM, 2006 (66)	PCS 3 CS 12 (CD 8, ACS 4) CS-E 16	LDDST	0.5 mg Dexa orally, every 6 h for 48 h, starting at 09:00.	Chemiluminescence immunoassay	Cortisol 50 nmol/L (1.8 μg/dL)	100	88	Inter-individual variation in Dexa metabolism.
Gatta B, 2007 (64)	PCS 14 CD 17 (mild)	LDDST	0.5 mg Dexa orally, every 6 h for 48 h starting at 12:00.	RIA – Chemiluminescence immunoassay	Cortisol 55 nmol/L (2.0 μg/dL)	94	86
Alwani RA, 2014 (63)	20 PCS 53 CD	LDDST	0.5 mg Dexa orally, every 6 h for 48 h, starting at 12:00.	Chemiluminescence immunoassay	Cortisol 50 nmol/L (1.8 μg/dL)	94	84
Yanovski JA, 1993 (65)	PCS 19 CS 39 (CD 35, ACS 2, EAS 2)	CRH stimulation test	Serial blood samples for ACTH and cortisol were obtained at time −15′, −10′, −5′, 0′, 5′, 15′, 30′, 45′, 60′ after administering 1 μg/kg oCRH as an i.v. bolus at 08:00.	RIA	Sum of post-CRH cortisol levels >3450 nmol/L (125 μg/dL)	64	100	Lack of standardization of protocols and assays. Excellent diagnostic performance with combined ACTH and cortisol analysis. Test interpretation based entirely on absolute cut-off values, rather than on their increment (deeply dependent on assay methodology).
Arnaldi G, 2009 (69)	PCS 26 CS 58 (CD 51, EAS 7) CT 31	CRH stimulation test	An i.v. bolus of 100 μg hCRH was injected over 30 s at 08:30. Blood samples were collected at time −15′, 0′, 15′, 30′, 45′, 60′, 90′ and 120′. Basal serum cortisol and basal plasma ACTH were the means of the two baseline values (−15 and 0 min) before CRH administration.	Chemiluminescent immunometric assay	Basal serum cortisol >331 nmol/L (12 μg/dL) and peak plasma ACTH >12 pmol/L (54 pg/mL) Peak serum cortisol >580 nmol/L (21 μg/dL) and peak plasma ACTH >10 pmol/L (45 pg/mL)	91.3 94.8	98.2 91.2
Tirabassi G, 2011 (74)	PCS 18 CD 30 CT 12	CRH stimulation test	An i.v. bolus of 100 μg hCRH was injected over 30 s at 08:30. Blood samples were collected at time −15′, 0′, 15′, 30′, 45′, 60′, 90′ and 120′. Basal serum cortisol and basal plasma ACTH were the means of the two baseline values (−15 and 0 min) before CRH administration.	Chemiluminescent immunometric assay	Basal serum cortisol >331 nmol/L (12 μg/dL) and peak plasma ACTH >12 pmol/L (54 pg/mL) Peak serum cortisol >580 nmol/L (21 μg/dL) and peak plasma ACTH >10 pmol/L (45 pg/mL)	96.6 90	100 83.3
Moro M, 2000 (71)	PCS 30 CD 76 SO 36 CT 31 PCS 30 CD 20 (mild)	DDAVP	Serial blood samples for ACTH and cortisol were obtained from an indwelling venous catheter at time −30′, 0′, 15′, 30′, 45′, 60′, 90′ and 120′ after administering 10 μg DDAVP i.v.	Two-site IRMA	∆-ACTH ≥6 pmol/L within 30′ after DDAVP injection	86.8 90	90.7 96.7	Less expensive than CRH stimulation test. Excellent diagnostic performance with combined ACTH and cortisol analysis. Useful in cases of mild hypercortisolism given its independence from UFC, 1-mg DST and midnight serum cortisol. Lack of standardization of ACTH and cortisol assays.
Pecori-Giraldi F, 2007 (70)	PCS 23 CS 32 (CD 29, ACS 3)	DDAVP	Serial blood samples were obtained from an indwelling venous catheter 30′ before the test, at 0′ and 10′, 20′, 30′, 45′, 60′, 90′ and 120′ after administration of 10 µg DDAVP i.v.	Immunometric assay	∆-ACTH ≥6 pmol/L within 30′ after DDAVP injection	81.5	90
Rollin G, 2015 (72)	CD 68 PCS 75	DDAVP	Serial blood samples for ACTH and cortisol measurements were obtained from an indwelling venous catheter 15′ before the test, at 0′, 15′, 30′, 45′ and 60′ after administration of 10 µg DDAVP iv	Chemiluminescent immunometric assay	Peak ACTH of 15.8 pmol/L or ∆-ACTH ≥8.1 pmol/L 15′–30′ following DDAVP injection	90.8 88	94.6 96.4
Tirabassi G, 2010 (73)	PCS 28 CD 52 CT 31	DDAVP	Serial blood samples were obtained from an indwelling venous catheter at time −15′, 0′ 10′, 20′, 30′, 45′, 60′, 90′ and 120′ after administering 10 µg DDAVP i.v. Basal serum cortisol and basal plasma ACTH were the means of the two baseline values (−15 and 0 min) before DDAVP administration.	Chemiluminescent immunometric assay	∆-ACTH >6 pmol/L within 30′ after DDAVP injection Basal serum cortisol >331 nmol/L (12 μg/dL) and ∆-ACTH >4 pmol/L (18 pg/mL)	75 90.3	89.8 91.5
Tirabassi G, 2011 (74)	PCS 18 CD 30 CT 12	DDAVP	Serial blood samples were obtained from an indwelling venous catheter at time −15′, 0′ 10′, 20′, 30′, 45′, 60′, 90′ and 120′ after administering 10 µg DDAVP i.v. Basal serum cortisol and basal plasma ACTH were the means of the two baseline values (−15 and 0 min) before DDAVP administration.	Chemiluminescent immunometric assay	Basal serum cortisol >331 nmol/L (12 μg/dL) and ∆-ACTH >4 pmol/L (18 pg/mL)	96.6	100

ACS, adrenal Cushing’s syndrome; CD, Cushing’s disease; CS, Cushing’s syndrome; CS-E, Cushing’s syndrome excluded; CT, controls; Dexa, dexamethasone; Dexa-CRH test, dexamethasone-suppressed corticotropin-releasing hormone test; EAS, ectopic ACTH syndrome; hCRH, human-sequence CRH; LDDST, low-dose dexamethasone suppression test; oCRH, ovine-sequence CRH; PCS, pseudo-Cushing’s state; SE, sensitivity; SO, simple obesity; SP, specificity; UFC, urinary free cortisol.