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Diagnosing hyper- and hypocortisolism using saliva samples: pitfalls and how to avoid them
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine.ORCID iD: 0000-0003-2110-4602
2023 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Biokemisk diagnostik av hyper- och hypokortisolism med salivprover : fallgropar och hur man undviker dem (Swedish)
Abstract [en]

Background: Cushing's syndrome (CS) is caused by high cortisol secretion whereas insufficient cortisol secretion is called adrenal insufficiency (AI). Both are rare diseases with substantial diagnostic delay, and high morbidity and mortality even though effective treatment is available. This thesis aims to improve diagnostic tests for CS and AI using analyses of cortisol and its inactive metabolite cortisone in saliva samples.

Methods: Papers 1 and 2 are based on a reference cohort including 155 individuals and 22 patients with CS. Salivary samples were collected at late-night (23:00 hours ± 15 minutes) and after a 1-mg overnight dexamethasone suppression test (DST). In Paper 1, reference intervals for salivary cortisol and cortisone analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were established for late-night and post-DST samples. Diagnostic accuracy for CS was calculated using the established reference intervals. Potential effects of age, comorbidities, season, and sampling time point were also studied. In Paper 2, different analytical methods for measurement of salivary cortisol (3 LC-MS/MS and 3 immunoassays) and salivary cortisone (3 LC-MS/MS assays) were compared regarding reference intervals and diagnostic accuracies for CS. Paper 3 elucidated the potential effect of liquorice consumption, blood contamination, and topical hydrocortisone handling prior to sampling on salivary cortisol and cortisone. Paper 4 investigated whether salivary cortisol and cortisone are less affected than plasma cortisol by estrogen-containing oral contraceptive (OCs) in women undergoing a short Synacthen test (SST) by comparing the response in women with (n=41) and without OCs (n=46).

Results: Paper 1 established reference intervals for salivary cortisol and cortisone at 23:00 hours and after DST. Using the upper reference limits as cut-offs, the diagnostic tests rendered high diagnostic accuracy for CS using salivary cortisol (sensitivity 90–95 %, specificity 96 %). There was no seasonal variation and no significant difference between samples collected at 22:00 vs 23:00 hours. Salivary cortisone showed a higher diagnostic accuracy for CS (sensitivity 100 % and specificity 94–95 %) and was less affected by other comorbidities compared to salivary cortisol. Paper 2 showed very high agreement between the three LC-MS/MS methods and that measuring salivary cortisol with immunoassays resulted in higher cortisol concentrations than with LC-MS/MS. However, using the newly established reference limits for each method, all had high diagnostic accuracy for CS. Late-night salivary cortisone analyzed with the LC-MS/MS methods and salivary cortisol analyzed with the Roche immunoassay showed the highest diagnostic accuracies. Paper 3 showed that liquorice consumption increased late-night salivary cortisol, which was sustained for up to 6 days, whereas no effect was seen on salivary cortisone. Salivary cortisol, but not cortisone, was increased by contamination of saliva with ≥0.5 % blood, which could be revealed by a clearly visible red discoloration of the saliva. Handling of topical hydrocortisone before saliva sampling affected salivary cortisol to a much higher degree than salivary cortisone. Paper 4 showed that women using OCs have considerably higher plasma cortisol levels during an SST, whereas salivary cortisol and salivary cortisone were lower compared to controls. However, the lower reference limits were not significantly different for salivary measurands, with salivary cortisone slightly more robust, opting for a common cut-off to exclude AI regardless of OCs.

Conclusion: Using the reference intervals calculated for several clinically used analytical methods showed high diagnostic accuracy for CS, with cortisone showing the highest accuracy. Analyzing salivary cortisone was not affected by liquorice consumption or blood contamination. Salivary cortisone was least affected by OCs during an SST. In summary, salivary cortisone is very useful in the diagnostic work-up for CS and AI.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2023. , p. 72
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2237
Keywords [en]
Cushing's syndrome, Adrenal Insufficiency, Salivary cortisol, Salivary cortisone, Reference intervals, Diagnostic accuracy, Error sources
National Category
Endocrinology and Diabetes
Research subject
Internal Medicine; Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-208137ISBN: 978-91-8070-045-0 (print)ISBN: 978-91-8070-046-7 (electronic)OAI: oai:DiVA.org:umu-208137DiVA, id: diva2:1756240
Public defence
2023-06-09, Lärosal A, målpunkt T9, Norrlands universitetssjukhus, Daniel Naezéns väg, Umeå, 09:00 (Swedish)
Opponent
Supervisors
Funder
Region VästerbottenUmeå University, 370549500Available from: 2023-05-17 Created: 2023-05-11 Last updated: 2024-03-25Bibliographically approved
List of papers
1. Reference intervals of salivary cortisol and cortisone and their diagnostic accuracy in Cushing's syndrome
Open this publication in new window or tab >>Reference intervals of salivary cortisol and cortisone and their diagnostic accuracy in Cushing's syndrome
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2020 (English)In: European Journal of Endocrinology, ISSN 0804-4643, E-ISSN 1479-683X, Vol. 182, no 6, p. 569-582Article in journal (Refereed) Published
Abstract [en]

Objective: The challenge of diagnosing Cushing's syndrome (CS) calls for high precision biochemical screening. This study aimed to establish robust reference intervals for, and compare the diagnostic accuracy of, salivary cortisol and cortisone in late-night samples and after a low-dose (1 mg) dexamethasone suppression test (DST).

Design and methods: Saliva samples were collected at 08:00 and 23:00 h, and at 08:00 h, after a DST, from 22 patients with CS and from 155 adult reference subjects. We also collected samples at 20:00 and 22:00 h from 78 of the reference subjects. Salivary cortisol and cortisone were analysed with liquid chromatography-tandem mass spectrometry. The reference intervals were calculated as the 2.5th and 97.5th percentiles of the reference population measurements. Diagnostic accuracies of different tests were compared, based on areas under the receiver-operating characteristic curves.

Results: The upper reference limits of salivary cortisol and cortisone at 23:00 h were 3.6 nmol/L and 13.5 nmol/L, respectively. Using these reference limits, CS was detected with a sensitivity (95% CI) of 90% (70-99%) and specificity of 96% (91-98%) for cortisol, and a 100% (84-100%) sensitivity and 95% (90-98%) specificity for cortisone. After DST, cortisol and cortisone upper reference limits were 0.79 nmol/L and 3.5 nmol/L, respectively. CS was detected with 95% (75-100%) sensitivity and 96% (92-99%) specificity with cortisol, and 100% (83-100%) sensitivity and 94% (89-97%) specificity with cortisone. No differences in salivary cortisol or cortisone levels were found between samples collected at 22:00 and 23:00 h.

Conclusion: Salivary cortisol and cortisone in late-night samples and after DST showed high accuracy for diagnosing CS, salivary cortisone being slightly, but significantly better.

Place, publisher, year, edition, pages
Bioscientifica, 2020
National Category
Endocrinology and Diabetes Analytical Chemistry
Identifiers
urn:nbn:se:umu:diva-180275 (URN)10.1530/EJE-19-0872 (DOI)000626969000002 ()32213657 (PubMedID)2-s2.0-85084187749 (Scopus ID)
Available from: 2021-02-16 Created: 2021-02-16 Last updated: 2024-03-25Bibliographically approved
2. Salivary cortisol and cortisone in diagnosis of Cushing's syndrome: a comparison of six different analytical methods
Open this publication in new window or tab >>Salivary cortisol and cortisone in diagnosis of Cushing's syndrome: a comparison of six different analytical methods
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2023 (English)In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 61, no 10, p. 1780-1791Article in journal (Refereed) Published
Abstract [en]

Objectives: Salivary cortisol and cortisone at late night and after dexamethasone suppression test (DST) are increasingly used for screening of Cushing’s syndrome (CS). We aimed to establish reference intervals for salivary cortisol and cortisone with three liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques and for salivary cortisol with three immunoassays (IAs), and evaluate their diagnostic accuracy for CS.

Methods: Salivary samples at 08:00 h, 23:00 h and 08:00 h after a 1-mg DST were collected from a reference population (n=155) and patients with CS (n=22). Sample aliquots were analyzed by three LC-MS/MS and three IA methods. After establishing reference intervals, the upper reference limit (URL) for each method was used to calculate sensitivity and specificity for CS. Diagnostic accuracy was evaluated by comparing ROC curves.

Results: URLs for salivary cortisol at 23:00 h were similar for the LC-MS/MS methods (3.4–3.9 nmol/L), but varied between IAs: Roche (5.8 nmol/L), Salimetrics (4.3 nmol/L), Cisbio (21.6 nmol/L). Corresponding URLs after DST were 0.7–1.0, and 2.4, 4.0 and 5.4 nmol/L, respectively. Salivary cortisone URLs were 13.5–16.6 nmol/L at 23:00 h and 3.0–3.5 nmol/L at 08:00 h after DST. All methods had ROC AUCs ≥0.96.

Conclusions: We present robust reference intervals for salivary cortisol and cortisone at 08:00 h, 23:00 h and 08:00 h after DST for several clinically used methods. The similarities between LC-MS/MS methods allows for direct comparison of absolute values. Diagnostic accuracy for CS was high for all salivary cortisol and cortisone LC-MS/MS methods and salivary cortisol IAs evaluated.

Place, publisher, year, edition, pages
Walter de Gruyter, 2023
Keywords
Cushing's syndrome, immunoassay, LC-MS/MS, method comparison, salivary cortisol, salivary cortisone
National Category
Analytical Chemistry Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-206793 (URN)10.1515/cclm-2023-0141 (DOI)000964106600001 ()37013440 (PubMedID)2-s2.0-85151863068 (Scopus ID)
Funder
Region Västerbotten
Available from: 2023-04-24 Created: 2023-04-24 Last updated: 2024-03-26Bibliographically approved
3. Confounding effects of liquorice, hydrocortisone, and blood contamination on salivary cortisol but not cortisone
Open this publication in new window or tab >>Confounding effects of liquorice, hydrocortisone, and blood contamination on salivary cortisol but not cortisone
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2023 (English)In: Endocrine Connections, E-ISSN 2049-3614, Vol. 12, no 1, article id e220324Article in journal (Refereed) Published
Abstract [en]

Objective: To determine the effects of liquorice consumption, topical hydrocortisone, and blood contamination on salivary cortisol and cortisone concentrations.

Design and methods: Thirty healthy volunteers were randomized to a low, medium, or high dose of liquorice. Late-night saliva samples were collected using a Salivette® collection device at baseline, during 1 week of daily liquorice consumption, and during 4 weeks' washout. Saliva sampling was also performed before and after the application of topical hydrocortisone on the skin. Furthermore, in a subgroup (n  = 16), saliva and venous blood were collected from each individual and mixed to achieve graded blood contamination in saliva. Salivary cortisol and cortisone were analyzed with liquid chromatography-tandem mass spectrometry.

Results: Significant increases in salivary cortisol concentrations were observed during medium- (+49%) and high-dose (+97%) liquorice intake, which returned to baseline 4 days after liquorice withdrawal. Topical hydrocortisone on fingers holding the collection swab increased salivary cortisol concentrations >1000-fold with concomitant pronounced elevation of the cortisol:cortisone ratio. Salivary cortisol increased significantly after contamination with blood ≥0.5%. Visual examination could safely detect these samples. Salivary cortisone concentrations were unaffected by liquorice consumption and blood contamination, and only marginally affected by topical hydrocortisone.

Conclusion: Liquorice, topical hydrocortisone, and blood contamination may all cause elevated salivary cortisol concentrations. Improved sampling instructions and visual examination of the sample may minimize these risks. Salivary cortisone is essentially unaffected by the different preanalytical confounders and may be used as a first-line screening test for Cushing's syndrome.

Place, publisher, year, edition, pages
Bioscientifica, 2023
Keywords
Cushing’s syndrome, salivary cortisol, salivary cortisone, liquorice, sample contamination
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-208130 (URN)10.1530/ec-22-0324 (DOI)000971893300001 ()36383173 (PubMedID)2-s2.0-85151875249 (Scopus ID)
Funder
Region Västerbotten
Available from: 2023-05-10 Created: 2023-05-10 Last updated: 2024-03-26Bibliographically approved
4. Comparison of plasma cortisol-, salivary cortisol- and salivary cortisone-response to the short Synacthen test in women using oral contraceptives
Open this publication in new window or tab >>Comparison of plasma cortisol-, salivary cortisol- and salivary cortisone-response to the short Synacthen test in women using oral contraceptives
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(English)Manuscript (preprint) (Other academic)
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-208129 (URN)
Available from: 2023-05-10 Created: 2023-05-10 Last updated: 2024-03-25

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