Measurement Of Late-night Salivary Cortisol Research Articles

THU011 Cushing’s Disease Diagnosis Challenge With Standard Pituitary MRI: A Clinical Case Report

Abstract Disclosure: E. Kasiri: None. N. Shah: None. Introduction: Cushing’s disease (CD); the most common cause of endogenous Cushing’s syndrome (CS) is mainly caused by an adrenocorticotropin (ACTH)-secreting pituitary adenomas. Although MRI remains the preferred imaging to detect these adenomas, still about 50% of microadenomas may be missed using standard 1.5-Tesla MRI and on the other hand higher resolution MRI with 3T magnets is not widely used. Here we present a case of Cushing’s disease with equivocal 1.5T MRI which made a next step of management rather challenging. Case presentation: A 57-year-old female with history of hypertension, type 2 diabetes mellitus and obesity (BMI: 34.41 kg/m2) presented to our endocrinology clinic to establish care. Her main complaint was difficulty losing weight. She denied taking any steroids or oral contraceptive pill. Vitals showed BP: 124/85 mm Hg, HR: 100 bpm and T: 98.3 °F. Physical exam was significant for moon facies, buffalo hump and abdominal obesity. She was screened for Cushing syndrome with 1mg overnight low dose dexamethasone test which resulted in abnormal cortisol response with a level of 2.1mcg/dl (<1.8 mcg/dl). A 24hr urine free cortisol: 7.3 mcg/dl (<45 mcg/dl) and two late night salivary cortisol measurements resulted in 0.388 and 0.229 mcg/dl (> 3 times upper limit of normal). Other labs showed elevated baseline ACTH :67.4 pg/ ml (7.2 - 63.3 pg/mL), cortisol :19.4 mcg/dl (4.5-23 mcg/dl) and normal DHEAS:1864.1ng/ml (650-3400 ng/mL). We decided to distinguish ectopic vs pituitary source of hypercortisolism by an 8 mg (high dose) dexamethasone suppression test which showed suppressed ACTH <1.5 pg/ml, Cortisol :2.6 mcg/dl (>50% suppression from baseline) suggesting pituitary origin. The standard pituitary 1.5T MRI with and without contrast as next step resulted in questionable microadenoma in the right aspect of pituitary gland and was equivocal. Within the multidisciplinary team decision was made to perform 3T pituitary MRI rather than invasive inferior petrosal sinus sampling (IPSS) which confirmed the pituitary microadenoma of 8.1 mm size with no mass effect and patient is scheduled for transsphenoidal pituitary surgery without needing further testing. Discussion: 3T MRI, with higher magnetic field than 1.5T allows to improve image quality and help with tumor detection in Cushing’s disease. It reportedly has higher sensitivity (80%) and specificity (∼100%) compared to 1.5T MRI which has overall sensitivity and specificity of 71% and 87% respectively. However, it is not the first to order by the clinicians when evaluating the ACTH secreting pituitary adenoma. In the case of equivocal 1.5 T MRI usually the next step is IPSS which is invasive. With availability of Corticotropin Releasing Hormone in the USA being difficult, limited studies of using DDAVP and limitations in finding tertiary centers to perform IPSS, a 3T MRI would be more feasible as a next step to identify a pituitary adenoma. Presentation: Thursday, June 15, 2023

Approach to the Patient: Diagnosis of Cushing Syndrome.

Cushing syndrome results from supraphysiological exposure to glucocorticoids and is associated with significant morbidity and mortality. The pathogenesis includes administration of corticosteroids (exogenous Cushing syndrome) or autonomous cortisol overproduction, whether or not ACTH-dependent (endogenous Cushing syndrome). An early diagnosis of Cushing syndrome is warranted; however, in clinical practice, it is very challenging partly because of resemblance with other common conditions (ie, pseudo-Cushing syndrome). Initial workup should start with excluding local and systemic corticosteroid use. First-line screening tests including the 1-mg dexamethasone suppression test, 24-hour urinary free cortisol excretion, and late-night salivary cortisol measurement should be performed to screen for endogenous Cushing syndrome. Scalp-hair cortisol/cortisone analysis helps in the assessment of long-term glucocorticoid exposure as well as in detection of transient periods of hypercortisolism as observed in cyclical Cushing syndrome. Interpretation of results can be difficult because of individual patient characteristics and hence requires awareness of test limitations. Once endogenous Cushing syndrome is established, measurement of plasma ACTH concentrations differentiates between ACTH-dependent (80%-85%) or ACTH-independent (15%-20%) causes. Further assessment with different imaging modalities and dynamic biochemical testing including bilateral inferior petrosal sinus sampling helps further pinpoint the cause of Cushing's syndrome. In this issue of "Approach to the patient," the diagnostic workup of Cushing syndrome is discussed with answering the questions when to screen, how to screen, and how to differentiate the different causes. In this respect, the latest developments in biochemical and imaging techniques are discussed as well.

Late-night salivary cortisol and cortisone should be the initial screening test for Cushing's syndrome.

Endogenous Cushing’s syndrome (CS) poses considerable diagnostic challenges. Although late-night salivary cortisol (LNSC) is recommended as a first-line screening investigation, it remains the least widely used test in many countries. The combined measurement of LNSC and late-night salivary cortisone (LNS cortisone) has shown to further improve diagnostic accuracy. We present a retrospective study in a tertiary referral centre comparing LNSC, LNS cortisone, overnight dexamethasone suppression test, low-dose dexamethasone suppression test and 24-h urinary free cortisol results of patients investigated for CS. Patients were categorised into those who had CS (21 patients) and those who did not (33 patients). LNSC had a sensitivity of 95% and a specificity of 91%. LNS cortisone had a specificity of 100% and a sensitivity of 86%. With an optimal cut-off for LNS cortisone of >14.5 nmol/L the sensitivity was 95.2%, and the specificity was 100% with an area under the curve of 0.997, for diagnosing CS. Saliva collection is non-invasive and can be carried out at home. We therefore advocate simultaneous measurement of LNSC and LNS cortisone as the first-line screening test to evaluate patients with suspected CS.

Morbidity and mortality in Cushing's syndrome

Endogenous Cushing's syndrome is arare endocrine disorder that is fatal if left untreated. It can be distinguished into adrenocorticotropic hormone (ACTH)-dependent (central and ectopic Cushing's syndrome) and ACTH-independent subtypes (unilateral or bilateral adrenal adenomas). The clinical presentation of patients includes typical stigmata of cortisol excess with physical symptoms of catabolic metabolism (myopathy, striae, parchment skin, osteoporosis) and components of metabolic syndrome (diabetes mellitus, obesity, arterial hypertension, hypercholesterolemia). Biochemical diagnosis is performed in three steps: 1.Confirmation of the diagnosis by 1‑mg dexamethasone suppression test, 24‑h urine free cortisol, and measurement of late-night salivary cortisol. 2.Differentiation of ACTH-dependent Cushing's syndrome from ACTH-independent adrenal Cushing's syndrome by measurement of plasma ACTH. 3.Further subtyping by corticotropin-releasing hormone (CRH) test, inferior petrosal sinus sampling, and imaging modalities. Therapeutic decisions are made on an interdisciplinary basis. First-line therapy for all subtypes is surgery when possible; additional options for all forms include drug therapy and bilateral adrenalectomy. Despite adequate treatment, Cushing's syndrome is associated with increased long-term morbidity and mortality. Interdisciplinary and multimodal therapy management is necessary in the long term to positively influence mortality and reduced quality of life.

Distinct Late-Night Salivary Cortisol Cut-Off Values for the Diagnosis of Hypercortisolism.

Due to high morbidity and mortality of untreated hypercortisolism, a prompt diagnosis is essential. Measurement of late-night salivary cortisol provides a simple and non-invasive method. However, thresholds and reference ranges differ among studies. The goal of this study was to define a threshold of late-night salivary cortisol for the diagnosis of hypercortisolism based on the used assay. Moreover, the influence of different aetiologies of hypercortisolism and individual comorbidities were investigated. Prospective analyses of 217 patients, including 36 patients with proven hypercortisolism were carried out. A sum of 149 patients with suspicion of hypercortisolism but negative endocrine testing and 32 patients with hypercortisolism in remission served as control group. Late-night salivary cortisol was measured using an automated chemiluminescence immunoassay. Cut-off values were calculated by ROC analysis. The calculated cut-off value for the diagnosis of hypercortisolism was 10.1 nmol/l (sensitivity 94%; specificity 84%). Only slightly lower thresholds were obtained in patients with suspected hypercortisolism due to weight gain/obesity (9.1 nmol/l), hypertension or adrenal tumours (both 9.8 nmol/l) or pituitary adenomas (9.5 nmol/l). The late-night salivary cortisol threshold to distinguish between Cushing's disease and Cushing's disease in remission was 9.2 nmol/l. The cut-off value for the diagnosis of ectopic ACTH-production was 109.0 nmol/l (sensitivity 50%, specificity 92%). Late-night salivary cortisol is a convenient and reliable parameter for the diagnosis of hypercortisolism. Except for ectopic ACTH-production, thresholds considering different indications for evaluation of hypercortisolism were only slightly different. Therefore, they might only be useful if late-night salivary cortisol results near the established cut-off value are present.

Toward a Diagnostic Score in Cushing's Syndrome.

Cushing's syndrome (CS) is a classical rare disease: it is often suspected in patients who do not have the disease; at the same time, it takes a mean of 3 years to diagnose CS in affected individuals. The main reason is the extreme rarity (1–3/million/year) in combination with the lack of a single lead symptom. CS has to be suspected when a combination of signs and symptoms is present, which together make up the characteristic phenotype of cortisol excess. Unusual fat distribution affecting the face, neck, and trunk; skin changes including plethora, acne, hirsutism, livid striae, and easy bruising; and signs of protein catabolism such as thinned and vulnerable skin, osteoporotic fractures, and proximal myopathy indicate the need for biochemical screening for CS. In contrast, common symptoms like hypertension, weight gain, or diabetes also occur quite frequently in the general population and per se do not justify biochemical testing. First-line screening tests include urinary free cortisol excretion, dexamethasone suppression testing, and late-night salivary cortisol measurements. All three tests have overall reasonable sensitivity and specificity, and first-line testing should be selected on the basis of the physiologic conditions of the patient, drug intake, and available laboratory quality control measures. Two normal test results usually exclude the presence of CS. Other tests and laboratory parameters like the high-dose dexamethasone suppression test, plasma ACTH, the CRH test, and the bilateral inferior petrosal sinus sampling are not part of the initial biochemical screening. As a general rule, biochemical screening should only be performed if the pre-test probability for CS is reasonably high. This article provides an overview about the current standard in the diagnosis of CS starting with clinical scores and screenings, the clinical signs, relevant differential diagnoses, the first-line biochemical screening, and ending with a few exceptional cases.

A commentary on Diagnosing Cushing's disease in the context of renal failure.

The diagnosis of endogenous hypercortisolism (Cushing's syndrome) is extremely challenging. Chronic kidney disease (CKD) increases the activity of the hypothalamic-pituitary-adrenal axis making the diagnosis of Cushing's syndrome even more challenging. This is particularly so since urine free cortisol (UFC) testing is not useful in CKD. The case report by Stroud et al. in this issue of the European Journal of Endocrinology highlights this problem by finding normal UFC in a patient with pituitary ACTH-dependent Cushing's syndrome. Elevated late-night salivary cortisol (LNSC) testing was diagnostic and pituitary adenomectomy was curative. LNSC measurement is the diagnostic test of choice in patients with suspected Cushing's syndrome, particularly in the presence of CKD..

Bedtime Salivary Cortisol and Cortisone by LC-MS/MS in Healthy Adult Subjects: Evaluation of Sampling Time.

The measurement of late-night salivary cortisol is a mainstay in the diagnosis of Cushing syndrome. Furthermore, the measurement of salivary cortisol is useful in assessing the cortisol awakening response. Because the salivary glands express 11-β-hydroxysteroid dehydrogenase, the measurement of salivary cortisone may improve the performance of salivary corticosteroid measurements. We measured salivary cortisol by enzyme immunoassay (EIA) and salivary cortisol and cortisone by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in only 50 µL of saliva sampled from 54 healthy subjects (aged 20 to 64 years). We allowed patients to sample at their normal bedtime (2025 to 2400 hours) to answer a common question as to whether sampling at the normal bedtime is equivalent to the standard required sampling at 2300 to 2400 hours. We found that the salivary cortisol and cortisone results by LC-MS/MS correlated well with salivary cortisol measured with the US Food and Drug Administration-cleared EIA. Furthermore, the upper limit of normal of salivary cortisol by EIA for bedtime samples was lower than the previously published upper limit of normal with sampling required at 2300 to 2400 hours. There were no significant effects of age or sex on any of the salivary steroid measurements. We conclude that (i) salivary cortisol and cortisone can be reliably measured by LC-MS/MS in small volumes of saliva and (ii) that patients can be evaluated using saliva sampled at their normal bedtime, rather than being required to stay awake until 2300 to 2400 hours.

Specificity of late-night salivary cortisol measured by automated electrochemiluminescence immunoassay for Cushing's disease in an obese population.

Data about the specificity of late-night salivary cortisol (LNSC) in obese subjects are still conflicting. Therefore, with this study, we aimed to evaluate the specificity of LNSC measurement in an obese cohort with or without type 2 diabetes mellitus (T2DM) using an automated electrochemiluminescence immunoassay (ECLIA). A total number of 157 patients involving 40 healthy subjects (HS) with BMI < 25kg/m2, 83 obese subjects (OS) with BMI ≥ 35kg/m2, and 34 histopathologically proven Cushing's disease (CD) were included. All patients underwent LNSC testing. Salivary cortisol was measured at 11p.m. for all groups using an ECLIA. Reference range was established using values of LNSCs of HS and ROC curves were used to determine diagnostic cutoffs. In the HS group, mean LNSC was 4.7nmol/l (SD ± 3.1), while the OS group had a mean value of 10.9nmol/l (SD ± 7.5) and the CD group of 19.9nmol/l (SD ± 15.4). All groups differed significantly (p < 0.001). The ROC analysis of CD against HS alone showed a sensitivity of 85.3% and a specificity of 87.5% with a cut-off value of 8.3nmol/l. The ROC analysis between OS and CD showed a maximum sensitivity of 67.6% and specificity of 78.3% for a cut-off value of 12.3nmol/l. Taken both (HS and OS) groups together against the CD group, ROC analysis showed a maximum sensitivity of 67.6% and specificity of 85.4% for a cut-off value of 12.3nmol/l. No correlation was found between BMI, T2DM, and LNSC for all groups. In our obese cohort, we found that LNSC assayed by ECLIA had a low specificity in the diagnosis of CD.

Prolonged hypothalamic-pituitary-adrenal axis activation after acute coronary syndrome in the GENESIS-PRAXY cohort.

Background Glucocorticoid excess has been linked with cardiovascular disease. Little is known about the long-term cortisol response in patients after acute coronary syndrome. Design The objective of this study was to describe the distribution of salivary cortisol in the post-acute phase of acute coronary syndrome and to describe the association of late-night salivary cortisol with cardiovascular risk factors. Methods We used late-night salivary cortisol measurements post-discharge to estimate hypothalamic-pituitary-adrenal axis activity in 309 patients aged 18-55 years enrolled in the GENESIS-PRAXY study from January 2009-April 2013. We evaluated hypothalamic-pituitary-adrenal axis activity and its association with hypertension, dyslipidemia, diabetes, smoking, family history, prior acute coronary syndrome, psychiatric diseases, acute coronary syndrome severity, as well as mortality and rate of rehospitalization at 12 months. Results Persistently elevated late-night salivary cortisol>2.92 nmol/l was seen in 99 (32.0%) patients: within the range of what may be seen in Cushing's disease. Elevated late-night salivary cortisol was associated with previous acute coronary syndrome (13.3% vs 24.2%, p = 0.02), peripheral vascular disease (3.8% vs 13.1%, p = 0.002), and smoking (32.9% vs 46.5% p = 0.02). Elevated late-night salivary cortisol was associated with higher hemoglobin A1c values (5.6 ± 3.0 vs 6.1 ± 2.9, p = 0.008) and lower high density lipoprotein values (0.94 ± 0.53 vs 0.86 ± 0.50, p = 0.01). There were no differences in psychiatric symptom scores, acute coronary syndrome severity or mortality, and rate of rehospitalization at 12 months. Conclusions Many patients post-acute coronary syndrome have prolonged, marked activation of the hypothalamic-pituitary-adrenal axis. Late-night salivary cortisol co-associates with several cardiovascular risk factors. Further studies are needed to confirm the exact role of hypothalamic-pituitary-adrenal axis activity in the pathophysiology of cardiovascular disease.

Late night salivary cortisol measurement may help in excluding Cushing's syndrome in patients with chronic kidney disease

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Evolution, Global Warming, Smart Phones, and Late-Night Salivary Cortisol

Recently, I was asked to see a young woman for a second opinion regarding the possible diagnosis of Cushing syndrome. She had already been evaluated by another endocrinologist who had dismissed the diagnosis because the patient’s urine free cortisol was normal. When I asked the patient whether a measurement of late-night salivary cortisol (LNSC) had been secured, she replied, “The endocrinologist I saw doesn’t believe in salivary cortisol.” Having heard this on other occasions, I smiled and replied, “There are people who do not believe in evolution or global warming, but the scientific evidence for these phenomena is very persuasive.” Similarly, the scientific evidence now for the diagnostic utility of LNSC in the evaluation of patients with suspected endogenous hyper-cortisolism is very strong.

Urine free cortisol in the diagnosis of Cushing's syndrome: is it worth doing and, if so, how?

Urine free cortisol in the diagnosis of Cushing's syndrome: is it worth doing and, if so, how?

Accuracy of repeated measurements of late‐night salivary cortisol to screen for early‐stage recurrence of Cushing's disease following pituitary surgery

The performance of late-night salivary cortisol (LNSC) to accurately screen for postoperative recurrence of Cushing's disease (CD) at an early stage is unknown. The aim of this study was to compare the accuracy of multiple sampling strategies to suggest the optimal number of LNSC samples needed for diagnosing post-surgical recurrences of CD at an early stage. Retrospective analysis in a single centre. Thirty-six patients in surgical remission of CD had successive measurements of LNSC, defined as 'sequences', using a locally modified RIA assay as part of long-term follow-up (69·2 ± 10·6 months). Patients underwent an extensive biochemical evaluation within 3 months before or after a sequence of saliva sampling and were classified as being in remission or in early-stage recurrence. The accuracy of three diagnostic strategies combining two, three or four LNSC results from a sequence was estimated using areas under the ROC curves (AUC), sensitivity, specificity and predictive values. Forty-four sequences of LNSC measurements were available. Fifty-two percent of sequences were performed during early-stage recurrence. The intrasequence variability of LNSC was higher during recurrence than during remission (medians of SDs: 2·1 vs 0·5 nm; P < 0·0001). AUCs from ROC curves ranged from 0·93 to 0·96 depending on the strategy. For 90% sensitivities, the best specificities (92·9% and 90·9%) were achieved by strategies taking into account three or four measurements summarized either by their mean or their maximum value. Increase in LNSC concentration is an early abnormality during post-surgical recurrence of CD. However, due to a major within-patient variability of LNSC from 1 day to another, a screening strategy using three or four samples collected on successive days may be recommended to detect early-stage recurrence of CD with a high accuracy.

The role of late-night salivary cortisol measurement in the diagnosis of subclinical hypercortisolism in patients with adrenal incidentalomas

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Measurement of Late-Night Salivary Cortisol and Cortisone by LC-MS/MS to Assess Preanalytical Sample Contamination with Topical Hydrocortisone

To the Editor: Measurement of increased late-night salivary cortisol (LNSC)1 is now a mainstay in the diagnosis of endogenous Cushing syndrome (1). Patients typically sample their saliva at home and are carefully instructed to avoid using potential contaminants, such as topical creams or ointments containing hydrocortisone (authentic cortisol). Using these creams or ointments can cause preanalytical contamination of the saliva sample that cannot be distinguished from endogenous cortisol as measured by either immunoassay or liquid chromatography–tandem mass spectrometry (LC-MS/MS). With endogenous cortisol production, the cortisone concentration in the saliva is usually higher than the cortisol concentration, and typically the cortisol-to-cortisone ratio is <1, owing to the transfer of cortisone from the plasma and the conversion of cortisol to cortisone by 11-β-hydroxysteroid dehydrogenase type 2 (11-β-HSD2) in the salivary gland (2, 3). Contamination of the saliva with topical or oral hydrocortisone (cortisol) during sample collection would not be expected to increase salivary cortisone …

Salivary cortisol and the diagnosis of Cushing’s syndrome: a coming of age

The diagnosis and differential diagnosis of endogenous hypercortisolism—the Cushing’s syndrome—is one of the most challenging problems in clinical endocrinology. Screening the large proportion of the population with one or more of the signs and symptoms of Cushing’s syndrome requires a simple, inexpensive, and reliable test [1]. As has been demonstrated in multiple studies, the measurement of an increased late-night salivary cortisol (LNSC) clearly fits the bill because of its high sensitivity and specificity for the diagnosis of Cushing’s syndrome [2]. The theory behind the test and its execution has been exhaustively reviewed and is now widely accepted [3, 4]. The study by Belaya et al. [5] appearing in this journal confirms prior studies that the measurement of LNSC can be performed by a widely available platform immunoassay. Considering the simplicity of obtaining an LNSC sample and the availability of the measurement by platform immunoassay, there should be no barrier to any clinician obtaining this screening test even with a modest degree of suspicion of Cushing’s syndrome. The study of Belaya et al. brings up a few caveats and issues that should be considered when doing the test.

Diagnostic performance of late-night salivary cortisol measured by automated electrochemiluminescence immunoassay in obese and overweight patients referred to exclude Cushing’s syndrome

This study estimates diagnostic performance of late-night salivary cortisol (LNSC) as measured by automated electrochemiluminescence immunoassay (ECLIA), evaluates the clinical implication of two consecutive LNSC measurements, and compares its accuracy with enzyme-linked immunosorbent assay (ELISA) and serum cortisol after low-dose dexamethasone suppression test (DST) in obese and overweight patients referred for suspected Cushing's syndrome (CS). One hundred twenty three consecutive obese and overweight referred patients and 98 healthy volunteers provided two saliva samples collected at 23:00 using a Salivette (Sarstedt, Germany), assayed by ECLIA (Cobas e601) and ELISA. The patients underwent DST and were further evaluated until CS was pathologically confirmed (n=45) or excluded. Diagnostic performance of LNSC was evaluated by receiver operating characteristic (ROC) analysis. The total areas under the curve (AUC) were calculated to compare the different tests. We found that a cut-off value of 9.4nmol/l can differentiate CS among obese and overweight patients with sensitivity of 84.4% (95% CI 71.2-92.2), specificity of 92.3% (95% CI 84.2-96.4), and diagnostic odds ratio of 65.1 (95% CI 20.4-207.6). No difference was found between AUCs from the first, second, and the mean from the two LNSC measurements (ECLIA), LNSC (ELISA), or DST. The single LNSC (ECLIA) and DST improved the sensitivity and specificity for concordant results up to 100 and 97.4%, respectively. In conclusion, due to its automation and its comparable diagnostic performance, ECLIA is preferable as a first-line LNSC screening test for CS. The initial use of single LNSC followed by DST provides better diagnostic performance for concordant results.

Late‐night salivary cortisol for diagnosis of Cushing’s syndrome by liquid chromatography/tandem mass spectrometry assay

Late-night salivary cortisol (LNSC) measurements have been increasingly used by physicians as an initial diagnostic test for evaluation of patients with clinical suspicion of Cushing's syndrome (CS). Published studies include various numbers of cases, controls and importantly, various assay methods (vast majority various immunoassays), as well as various methods to generate cut-points. The retrospective study evaluated the diagnostic utility of LNSC measurements in 249 patients evaluated for possibility of CS because of various clinical conditions using liquid chromatography/tandem mass spectrometry method (LC-MS/MS). CS was confirmed in 47 patients (18·9%) and excluded in 202 (81·1%) patients at the time of analysis. Late-night salivary cortisol was abnormal or >2·8 nmol/l in 35 of 47 patients with CS; sensitivity of 74·5% and elevated in 20 of 202 patients who were found not to have CS; specificity 90·1%. Using receiver-operator characteristic statistics for calculation of the most optimal sensitivity and specificity, the cut-off based on this data was LNSC > 2·1 nmol/l with sensitivity of 83·0% and specificity of 84·2%. Analysis of data at one referral institution showed somewhat limited sensitivity of LNSC for diagnosis of CS using current reference ranges.

Reproducibility and performance of one or two samples of salivary cortisol in the diagnosis of Cushing’s syndrome using an automated immunoassay system

The purpose of this article is to evaluate the variability and reproducibility of late night salivary cortisol (LNSC) using electrochemiluminescence immunoassay (ECLIA) and compare the accuracy of one or two samples in diagnosis of Cushing's syndrome (CS). We prospectively included 64 healthy volunteers (HV), 35 patients with clinically suspected CS (S), and 26 patients with confirmed CS. Nine patients in the CS group had 24-h urinary free cortisol (UFC) less than two times the upper limit of normal (mild CS). UFC and two consecutive LNSC (LNSC1, LNSC2) were collected at home. All patients in the S group had normal UFC and low-dose dexamethasone suppression test. No differences were found between the HV and S groups in UFC, LNSC1, and LNSC2. Intra-individual variability between the two samples of LNSC was 22% in HV (1.6-91%), 32% in the S group (1.6-144%), and 51% (1.6-156%) in the CS group. Variability was higher in CS patients than those in the HV (P<0.001) and S groups (P=0.05). The AUC of LNSC1 was 0.945 (IC 95% 0.880-1.004); when considering the highest LNSC, the AUC was 0.980 (IC 95% 0.954-1.007) (P<0.01). We found 23% of discordant LNSC in the S group and 11% in the CS group. Three patients with CS had only one elevated LNSC, all of them with mild CS. Our results suggest that LNSC is variable, and reproducibility is affected in both CS and S patients. We found significant improvements in the diagnostic accuracy of the LNSC measurement by obtaining two samples.