Prospective Evaluation of Late-Night Salivary Cortisol and Cortisone by EIA and LC-MS/MS in Suspected Cushing Syndrome

Abstract

Context: Late-night salivary cortisol (LNSC) measured by enzyme immunoassay (EIA-F) is a first-line screening test for Cushing syndrome (CS) with a reported sensitivity and specificity of >90%. However, liquid chromatography-tandem mass spectrometry, validated to measure salivary cortisol (LCMS-F) and cortisone (LCMS-E), has been proposed to be superior diagnostically.

Objective setting and main outcome measures: Prospectively evaluate the diagnostic performance of EIA-F, LCMS-F, and LCMS-E in 1453 consecutive late-night saliva samples from 705 patients with suspected CS.

Design: Patients grouped by the presence or absence of at least one elevated salivary steroid result and then subdivided by diagnosis.

Results: We identified 283 patients with at least one elevated salivary result; 45 had an established diagnosis of neoplastic hypercortisolism (CS) for which EIA-F had a very high sensitivity (97.5%). LCMS-F and LCMS-E had lower sensitivity but higher specificity than EIA-F. EIA-F had poor sensitivity (31.3%) for adrenocorticotropic hormone (ACTH)-independent CS (5 patients with at least 1 and 11 without any elevated salivary result). In patients with Cushing disease (CD), most nonelevated LCMS-F results were in patients with persistent/recurrent CD; their EIA-F levels were lower than in patients with newly diagnosed CD.

Conclusions: Since the majority of patients with ≥1 elevated late-night salivary cortisol or cortisone result did not have CS, a single elevated level has poor specificity and positive predictive value. LNSC measured by EIA is a sensitive test for ACTH-dependent Cushing syndrome but not for ACTH-independent CS. We suggest that neither LCMS-F nor LCMS-E improves the sensitivity of late-night EIA-F for CS.

Keywords: Cushing disease; adrenal Cushing syndrome; assay performance; diagnosis; ectopic ACTH.

Figure 1.

Flow chart showing late-night salivary samples and associated patients in the study.

Figure 2.

Late-night salivary enzyme immunoassay cortisol (EIA-Cortisol), liquid chromatography-tandem mass spectrometry (LCMS) cortisol and cortisone, and the ratio of LCMS cortisol to cortisone in 35 patients with Cushing disease compared with the 121 patients randomly chosen with all normal salivary steroid results and without the diagnosis of Cushing syndrome of any type. Horizontal line is the median; box indicates 25^th to 75^th percentile, whisker indicates 10^th and 90^th percentiles, and outliers are indicated by circles. P values are from Mann-Whitney Rank Sum tests.

Figure 3.

Number of Cushing disease patients (N = 35) with late-night EIA-Cortisol and LC-MS/MS cortisol and cortisone equal to or below (Normal) or above (Abnormal) the reference range for that assay [26]. P values are from chi-square analysis.

Figure 4.

Number of Cushing disease patients with de novo Cushing disease (N = 12) and patients with recurrent/persistent Cushing disease (N = 23) with late-night EIA-Cortisol and LC-MS/MS cortisol and cortisone equal to or below (Normal) or above (Abnormal) the reference range for that assay [26]. P values indicate comparison of LC-MS/MS cortisol vs other 2 analytes.

Figure 5.

Correlation of salivary steroid measurements vs salivary transferrin concentrations in 39 patients from Table 6. There were no significant correlations found.