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Cited by 9Pituitary Gland Disorders and TreatmentsAdrenal Hormones and DisordersClinical Laboratory Practices and Quality Control

Salivary cortisol and cortisone in diagnosis of Cushing's syndrome: a comparison of six different analytical methods

Open access
Bäcklund, Nils, Brattsand, Göran, Lundstedt, Staffan, Aardal, Elisabeth, Bartuseviciene, Inga, Berinder, Katarina, Höybye, Charlotte, Burman, Pia +8 more·2023·Clinical Chemistry and Laboratory Medicine (CCLM)
DOI 10.1515/cclm-2023-0141
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Abstract

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.

AI snapshot

Written from the paper’s full text.

TL;DR

This study established reference intervals for salivary cortisol and cortisone using three LC-MS/MS methods and three immunoassays, then compared diagnostic accuracy for Cushing’s syndrome (CS). All evaluated methods showed high diagnostic accuracy, with LC-MS/MS methods agreeing closely enough to allow direct comparison of absolute values.

Key findings

  • Late-night salivary cortisol upper reference limits (URLs) were similar across LC-MS/MS methods (3.4–3.9 nmol/L) but varied across immunoassays: Roche 5.8 nmol/L, Salimetrics 4.3 nmol/L, Cisbio 21.6 nmol/L. After 1-mg DST, cortisol URLs were 0.7–1.0 nmol/L for LC-MS/MS and 2.4, 4.0, and 5.4 nmol/L for the three immunoassays, 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 for LC-MS/MS methods.
  • Diagnostic accuracy was high for all methods: all ROC AUCs were 0.964–0.996. At 23:00 h, LC-MS/MS cortisol sensitivity was 86–94% with specificity 95–96%; immunoassay cortisol sensitivity was 79–95% with specificity 93–96%. After DST, LC-MS/MS cortisol sensitivity was 90–95% with specificity 95–97%; immunoassay cortisol sensitivity was 81–95% with specificity 94–95%. Salivary cortisone after DST showed 100% sensitivity and 94–95% specificity.
  • LC-MS/MS methods correlated strongly: LC-MS/MS 2 vs. LC-MS/MS 1 had R=0.97 for cortisol and R=0.99 for cortisone; LC-MS/MS 3 vs. LC-MS/MS 1 had R=0.99 for both. Immunoassay cortisol correlations vs. LC-MS/MS 1 were Roche R=0.93, Salimetrics R=0.95, and Cisbio R=0.94.

Methods

  • Prospective reference population of 175 adults (20–84 years, 68% female) recruited March 2015–April 2016; 20 were excluded from reference interval calculations, leaving 155. Concurrently, 24 patients with strongly suspected CS were included at five Swedish university hospitals; 2 were excluded because CS could not be confirmed, leaving 22 CS patients (22–82 years, 77% female; Cushing’s disease n=12, ectopic ACTH-producing tumor n=5, adrenal tumor n=4).
  • Saliva was collected at 08:00 h, 23:00 h, and 08:00 h after a 1-mg overnight DST. Aliquots were analyzed by three LC-MS/MS methods and three immunoassays (Roche, Salimetrics, Cisbio).
  • Reference intervals were calculated as 2.5th and 97.5th percentiles after Dixon outlier exclusion, with bootstrap 95% CIs. The 97.5th percentile URL for each method was used as the diagnostic cut-off for sensitivity and specificity. ROC AUCs were compared pairwise against LC-MS/MS 1 using DeLong’s test, including only samples with valid data from all six methods (at 23:00 h, n=153 reference individuals and n=17 CS patients; after DST, n=147 reference individuals and n=16 CS patients).

Limitations

  • The paper states long and variable storage duration at −80 °C for up to 5 years, though prior stability data and local tests supported stability. It also states that the study did not evaluate whether variations in cortisol-binding globulin concentration or genetic variations affecting CBG–cortisol affinity influence salivary cortisol and cortisone.
  • The paper states that reference individuals were also used to calculate diagnostic accuracy for CS patients, which may introduce an advantage for the used reference interval. It also notes missing data, which were considered random and unlikely to introduce bias.
  • Method-specific caveats stated: the Cisbio immunoassay had a substantially higher URL because high reference-cohort values were not identified as outliers by Dixon criteria; the Salimetrics immunoassay has known 19% cross-reactivity for dexamethasone, which could be clinically significant after DST. Evident from the text: the CS cohort was small (n=22), and diagnostic accuracy comparisons used even smaller CS subsets (n=17 at 23:00 h and n=16 after DST).

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Integrity signals

Registry

Crossref · 10.1515/cclm-2023-0141

Cites

49 of 49 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 43 cited sources checked · 0 supported · 0 flagged

  • Findingown data
    § Method comparison

    The three LC-MS/MS methods showed high agreement for salivary cortisol and cortisone, with Passing-Bablok slopes near 1 and intercepts near 0.

    “The slopes and intercepts comparing the three LC-MS/MS methods were close to 1 and 0, respectively, indicating a high degree of agreement between the methods for both salivary cortisol and cortisone.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Method comparison

    Bland-Altman plots showed a constant percentage difference between methods across the concentration range relevant to Cushing's syndrome diagnosis.

    “Bland-Altman plots showed a constant percentual difference between methods over the concentration range of interest in CS diagnosis (Figures 3 and 4).”
    Quote in the paperSubstantial claim
  • Findingown data
    § Diagnostic accuracy for Cushing's syndrome

    Salivary cortisol at 08:00 after dexamethasone suppression measured by immunoassay had sensitivity 81-95% and specificity 94-95% for Cushing's syndrome.

    “Corresponding sensitivity and specificity for CS with the IA methods were 81-95% and 94-95%, respectively.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Abstract

    Every analytical method evaluated had a ROC AUC of at least 0.96 for Cushing's syndrome.

    “All methods had ROC AUCs ≥0.96.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Diagnostic accuracy for Cushing's syndrome

    Salivary cortisone at 23:00 measured by LC-MS/MS had sensitivity 81-100% and specificity 95-97% for Cushing's syndrome.

    “Salivary cortisone at 23:00 h (LC-MS/MS methods) showed a sensitivity and specificity for CS of 81-100% and 95-97%, respectively (Table 2).”
    Quote in the paperSubstantial claim
  • May combine two claims. 30
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 14

  • May combine two claims.

    Findingown data
    § Abstract

    For salivary cortisol after the 1-mg dexamethasone suppression test, the upper reference limits were 0.7-1.0 nmol/L for the LC-MS/MS methods and 2.4, 4.0, and 5.4 nmol/L for Roche, Salimetrics, and Cisbio, respectively.

    “Corresponding URLs after DST were 0.7-1.0, and 2.4, 4.0 and 5.4 nmol/L, respectively.”
    • Also stated in: § Reference intervals for salivary cortisol and cortisone, and cortisol:cortisone ratio · plain
      “After DST, URLs for salivary cortisol were 0.7-1.0nmol/L (LC-MS/MS methods) and 2.4-5.4 nmol/L (IA methods), and for salivary cortisone 3.0-3.5 nmol/L (LC-MS/MS methods).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Method comparison

    The immunoassays gave higher absolute salivary cortisol concentrations than LC-MS/MS, especially Cisbio.

    “The IAs exhibited higher absolute concentrations of salivary cortisol, especially Cisbio (Figure 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Abstract

    For salivary cortisol at 23:00, the upper reference limits were similar across the three LC-MS/MS methods (3.4-3.9 nmol/L) but differed among the three immunoassays: Roche 5.8 nmol/L, Salimetrics 4.3 nmol/L, and Cisbio 21.6 nmol/L.

    “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).”
    • Also stated in: § Reference intervals for salivary cortisol and cortisone, and cortisol:cortisone ratio · plain
      “URLs for salivary cortisol at 23:00 h were 3.4-3.9nmol/L (LC-MS/MS methods) and 4.3-21.6nmol/L (IA methods).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Method comparison

    LC-MS/MS 2 correlated with LC-MS/MS 1 with R=0.97 for cortisol and R=0.99 for cortisone.

    “LC-MS/MS 2 correlated with LC-MS/MS 1 (R=0.97 for cortisol and R=0.99 for cortisone), while LC-MS/MS 3 correlated with LC-MS/MS 1 (R=0.99 for both cortisol and cortisone).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Reference intervals for salivary cortisol and cortisone, and cortisol:cortisone ratio

    The 97.5th percentile for the salivary cortisol:cortisone ratio was 1.06 for LC-MS/MS 1 (n=540), 0.78 for LC-MS/MS 2 (n=517), and 0.91 for LC-MS/MS 3 (n=517).

    “The 97.5th percentile for the saliva cortisol:cortisone ratio was 1.06 for LC-MS/MS 1 (n=540 samples), 0.78 for LC-MS/MS 2 (n=517 samples) and 0.91 for LC-MS/MS 3 (n=517 samples).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Diagnostic accuracy for Cushing's syndrome

    All ROC AUCs were high (0.964-0.996) and similar at 23:00 and after dexamethasone suppression.

    “All ROC AUCs were high (0.964-0.996) and similar both for samples at 23:00 h and 08:00 h after DST; with slightly, but significantly higher ROC AUCs for all cortisone analysis methods (0.985-0.986) as well as for Roche cortisol at 23:00 h (0.980) compared to the LC-MS/MS 1 LNSC (0.975) (Table 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Comparison of diagnostic accuracy for CS between different methods

    After DST, every assay had a very high ROC AUC (>0.96), and no assay differed significantly from the others.

    “All methods showed very high ROC AUCs (>0.96) with no significant differences between analytical methods after DST.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning
    § Comparison of diagnostic accuracy for CS between different methods

    For late-night salivary cortisol, the Cisbio assay had a higher ROC AUC than the Roche assay, though not significantly above LC-MS/MS 1.

    “LNSC with the Cisbio IA method showed a higher ROC AUC than with the Roche IA, but not significantly higher than LC-MS/MS 1: this was due to higher ROC AUC correlation between Roche IA and LC-MS/MS 1 than between Cisbio IA and LC-MS/MS 1.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingnone given
    § Reference intervals

    Reference ranges for unsuppressed 08:00 h saliva cortisol and cortisone were considerably wider than at other time points.

    “As expected, reference ranges were considerably wider at this time point.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Method comparisons

    Among the three LC-MS/MS assays, R-values and slopes were near 1, indicating very strong correlation.

    “Comparisons of the three LC-MS/MS methods showed both R-values and slopes close to 1, indicating very high correlation and the narrow limit of agreements (3-10%) in Bland-Altman plots allowing for close to direct comparison between methods, both for salivary cortisol and cortisone.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Method comparisons

    Each assay compared with LC-MS/MS 1 had strong correlations, with high R-values.

    “The analyses of each method against LC-MS/MS 1 showed excellent correlations with high R-values, but the slope in regression analysis indicated higher cortisol values for the IAs, presumably due to cross-reactivity of the primary antibody to different cortisol metabolites in saliva [23].”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Comparison of diagnostic accuracy for CS between different methods

    These samples were kept in the cohort, which led to a much higher upper reference limit for the Cisbio assay than for the other assays.

    “Thus, they were retained in the cohort and consequently resulted in a substantially higher URL than for the other methods.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Conclusions

    The six salivary cortisol assays, three of which also measured salivary cortisone, agreed closely, particularly the three LC-MS/MS methods.

    “Six different salivary cortisol methods including three that analyzed salivary cortisone showed a high level of analytical agreement, especially between the three LC-MS/MS methods.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation
    § Population

    In most Cushing's syndrome cases, the diagnosis was confirmed by histological examination of the ACTH- or cortisol-producing tumor.

    “In a majority of the cases, the diagnosis was confirmed by histological examination of the ACTH-or cortisol producing tumor [2].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Bäcklund, N, Brattsand, G, Israelsson, M, Ragnarsson, O, Burman, P, Edén Engström, B, et al.. Reference intervals of salivary cortisol and cortisone and their diagnostic accuracy in Cushing’s syndrome. Eur J Endocrinol 2020;182:569–82. https://doi.org/10.1530/eje-19-0872. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductcitation
    § Laboratory analysis methods for salivary cortisol and cortisone LC-MS/MS methods:

    The three LC-MS/MS methods were optimized to chromatographically separate cortisol from cortisone and to avoid interference from prednisolone, betamethasone, dexamethasone, and 20α- and 20β-dihydrocortisone.

    “All three methods were optimized to chromatographically separate cortisol from cortisone, and to avoid interference from prednisolone, betamethasone, dexamethasone, and 20α-and 20β-dihydrocortisone [30].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] Israelsson, M, Brattsand, R, Brattsand, G. 20α- and 20β-dihydrocortisone may interfere in LC-MS/MS determination of cortisol in saliva and urine. Ann Clin Biochem 2018;55:341–7. https://doi.org/10.1177/0004563217724178. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Introduction

    LC-MS/MS techniques are more expensive and time-consuming than immunoassays.

    “LC-MS/MS techniques are more expensive and time consuming, but carry advantages of high sensitivity and specificity and the possibility of simultaneous measurement of salivary cortisol and cortisone [8,9].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [8] Taylor, AE, Keevil, B, Huhtaniemi, IT. Mass spectrometry and immunoassay: how to measure steroid hormones today and tomorrow. Eur J Endocrinol 2015;173:D1–12. https://doi.org/10.1530/eje-15-0338. source

      Not checked in the free run.

    • cites [9] Raff, H, Homar, PJ, Burns, EA. Comparison of two methods for measuring salivary cortisol. Clin Chem 2002;48:207–8. https://doi.org/10.1093/clinchem/48.1.207. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Reference intervals

    Serum cortisol below 50 nmol/L after DST makes Cushing's syndrome very unlikely, while a value above 138 nmol/L makes it likely.

    “It is widely accepted that serum cortisol levels <50 nmol/L after DST makes CS diagnosis very unlikely, whereas a value >138 nmol/L makes CS likely [38].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Ceccato, F, Boscaro, M. Cushing’s syndrome: screening and diagnosis. High Blood Pres Cardiovasc Prev 2016;23:209–15. https://doi.org/10.1007/s40292-016-0153-4. source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data
    § Laboratory analysis methods for salivary cortisol and cortisone LC-MS/MS methods:

    Roche Cortisol II was validated for salivary analysis and traceable to the IRMM/IFCC-451 test panel.

    “Roche Cortisol II, an electrochemiluminescence assay (ECLIA) was run on Cobas e602 (Roche, Basel, Switzerland) originally intended for use in plasma/serum but validated for salivary analysis, traceable to IRMM/IFCC-451 test panel with isotope dilution-gas chromatography/ mass spectrometry.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    An abnormally high salivary cortisol:cortisone ratio indicates contamination, such as with topical hydrocortisone.

    “An aberrantly high cortisol:cortisone ratio in saliva indicates contamination e.g., with topical hydrocortisone [2,4,[15][16][17].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2,4,[15][16][17]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given
    § Laboratory analysis methods for salivary cortisol and cortisone LC-MS/MS methods:

    The Salimetrics assay is designed for salivary cortisol determination and its standards are traceable to the National Institute of Standards and Technology.

    “Salimetrics Expanded Range High Sensitivity Salivary Cortisol Enzyme Immunoassay (Salimetrics LLC, Carlsbad, CA) is designed for the determination of cortisol in saliva.Standards are traceable to National Institute of Standards and Technology.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § Introduction

    Diagnostic accuracy of biochemical tests depends on well-established reference intervals, irrespective of analytical method.

    “Irrespective of analytical method, diagnostic accuracy of biochemical tests is dependent on well-established reference intervals [25][26][27][28][29].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25][26][27][28][29]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Laboratory analysis methods for salivary cortisol and cortisone LC-MS/MS methods:

    The Cisbio RIA was validated for cortisol analysis in saliva.

    “RIA CORT-CT2 (Cisbio Bioassays, Codolet, France), a competitive radioimmunoassay validated for cortisol analysis in saliva, was employed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation
    § Reference intervals

    The late-night salivary cortisol upper reference limits for the LC-MS/MS and immunoassay methods in this study were comparable to those in previous smaller studies, with a somewhat higher value for the Cisbio immunoassay.

    “The LNSC URLs for the LC-MS/ MS and IA methods included in our study were comparable with previous smaller studies [13,[35][36][37], with a somewhat higher value for the Cisbio IA [29].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [13,[35][36][37]

      Not checked in the free run.

    • cites [29] Petersenn, S. Biochemical diagnosis of Cushing’s disease: screening and confirmatory testing. Best Pract Res Clin Endocrinol Metabol 2021;35:101519. https://doi.org/10.1016/j.beem.2021.101519. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Introduction

    Immunoassays have advantages including small sample volumes, high automation, quick turnover, and relatively low cost.

    “IAs have advantages such as small sample volumes, high degree of automation, quick turnover times and a relatively low cost, but are often limited by cross-reactivities, matrix effects and high measurement uncertainty at low concentrations [6,7].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [6] Garde, AH, Hansen, ÅM, Nikolajsen, TB. An inter-laboratory comparison for determination of cortisol in saliva. Accred Qual Assur 2003;8:16–20. https://doi.org/10.1007/s00769-002-0548-x. source

      Not checked in the free run.

    • cites [7] Dodd, AJ, Ducroq, DH, Neale, SM, Wise, MP, Mitchem, KL, Armston, A, et al.. The effect of serum matrix and gender on cortisol measurement by commonly used immunoassays. Ann Clin Biochem 2014;51:379–85. https://doi.org/10.1177/0004563213514567. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductown data
    § Population

    Cushing's syndrome diagnoses were made by endocrinologists at tertiary endocrine centers based on clinical signs and symptoms combined with all or some of abnormal dexamethasone suppression test, 24-h urinary free cortisol, midnight serum cortisol, and grading from mild to severe disease.

    “The CS diagnoses were made by endocrinologists at tertiary endocrine centers based on clinical signs and symptoms combined with all or some of: Abnormal DST, 24-h urinary free cortisol and midnight serum cortisol, and a grading from mild to severe CS.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data
    § Sample collection and handling

    Of 578 collected samples, results were missing for 29 (LC-MS/MS 2), 25 (LC-MS/MS 3), 7 (Roche and Salimetrics), and 26 (Cisbio) because of low sample volume, pipetting error, or incorrect labelling.

    “Of 578 collected samples, results were missing in 29 (LC-MS/MS 2), 25 (LC-MS/MS 3), 7 (Roche and Salimetrics) and 26 (Cisbio) due to low sample volume, pipetting error or incorrect labelling.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation
    § Reference intervals

    The authors and others have shown that salivary cortisol and cortisone after DST have high diagnostic accuracy for Cushing's syndrome, but many methods lack an established diagnostic cut-off.

    “We and others have shown salivary cortisol and cortisone after DST to have high diagnostic accuracy for CS, but many methods lack an established cut-off for diagnosis [2-5, 41, 42].”
    Quote in the paperSubstantial claim0 of 6 sources checked
    • cites [2] Bäcklund, N, Brattsand, G, Israelsson, M, Ragnarsson, O, Burman, P, Edén Engström, B, et al.. Reference intervals of salivary cortisol and cortisone and their diagnostic accuracy in Cushing’s syndrome. Eur J Endocrinol 2020;182:569–82. https://doi.org/10.1530/eje-19-0872. source

      Not checked in the free run.

    • cites [3] Ueland, GÅ, Methlie, P, Kellmann, R, Bjørgaas, M, Åsvold, BO, Thorstensen, K, et al.. Simultaneous assay of cortisol and dexamethasone improved diagnostic accuracy of the dexamethasone suppression test. Eur J Endocrinol 2017;176:705–13. https://doi.org/10.1530/eje-17-0078. source

      Not checked in the free run.

    • cites [4] Ng, CM, Lam, TK, Au Yeung, YC, Choi, CH, Iu, YP, Shek, CC, et al.. Clinical utility of late-night and post-overnight dexamethasone suppression salivary cortisone for the investigation of Cushing’s syndrome. Hong Kong Med J 2017;23:570–8. https://doi.org/10.12809/hkmj176240. source

      Not checked in the free run.

    • cites [5] Manetti, L, Rossi, G, Grasso, L, Raffaelli, V, Scattina, I, Del Sarto, S, et al.. Usefulness of salivary cortisol in the diagnosis of hypercortisolism: comparison with serum and urinary cortisol. Eur J Endocrinol 2013;168:315–21. https://doi.org/10.1530/eje-12-0685. source

      Not checked in the free run.

    • cites [41] Laudat, MH, Cerdas, S, Fournier, C, Guiban, D, Guilhaume, B, Luton, JP. Salivary cortisol measurement: a practical approach to assess pituitary-adrenal function. J Clin Endocrinol Metab 1988;66:343–8. https://doi.org/10.1210/jcem-66-2-343. source

      Not checked in the free run.

    • cites [42] Berndt, V, Dahlqvist, P, de Verdier, J, Ryberg, H, Ragnarsson, O. The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism. Front Endocrinol 2022;13:1028804. https://doi.org/10.3389/fendo.2022.1028804. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation
    § Introduction

    LC-MS/MS techniques have high sensitivity and specificity and can measure salivary cortisol and cortisone simultaneously.

    “LC-MS/MS techniques are more expensive and time consuming, but carry advantages of high sensitivity and specificity and the possibility of simultaneous measurement of salivary cortisol and cortisone [8,9].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [8] Taylor, AE, Keevil, B, Huhtaniemi, IT. Mass spectrometry and immunoassay: how to measure steroid hormones today and tomorrow. Eur J Endocrinol 2015;173:D1–12. https://doi.org/10.1530/eje-15-0338. source

      Not checked in the free run.

    • cites [9] Raff, H, Homar, PJ, Burns, EA. Comparison of two methods for measuring salivary cortisol. Clin Chem 2002;48:207–8. https://doi.org/10.1093/clinchem/48.1.207. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation
    § Introduction

    A large fraction of free cortisol passing through the salivary glands is converted to cortisone by 11β-hydroxysteroid dehydrogenase type 2, giving a salivary cortisol:cortisone ratio of about 1:5 compared with 5:1 in blood.

    “A large proportion of the free cortisol passing through the salivary glands is metabolized into cortisone by the enzyme 11β-hydroxysteroid dehydrogenase type 2, resulting in a cortisol:cortisone ratio in saliva of about 1:5 compared to 5:1 in blood [2,[10][11][12][13][14].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2,[10][11][12][13][14]

      Not checked in the free run.

  • May combine two claims.

    Study design and conductcitation
    § Comparison of diagnostic accuracy for CS between different methods

    The study's large reference population, with age and sex distributions and rates of smoking and common diseases that reflect the population, is a strength that supports external validity and clinical usefulness.

    “A strength in our study is the large reference population with representative age and gender distributions, and proportions of smokers and patients with common diseases, giving a high external validity and results useful for clinical practice [2].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] Bäcklund, N, Brattsand, G, Israelsson, M, Ragnarsson, O, Burman, P, Edén Engström, B, et al.. Reference intervals of salivary cortisol and cortisone and their diagnostic accuracy in Cushing’s syndrome. Eur J Endocrinol 2020;182:569–82. https://doi.org/10.1530/eje-19-0872. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation
    § Reference intervals

    Salivary cortisol and cortisone measured in the morning are unlikely to add much to Cushing's syndrome diagnosis, though they might help screen for adrenal insufficiency.

    “Morning salivary cortisol and cortisone are not expected to contribute significantly to CS diagnosis, but may be useful for screening of adrenal insufficiency [45].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] Debono, M, Elder, C, Lewis, J, Fearnside, J, Caunt, S, Dixon, S, et al.. Home waking salivary cortisone to screen for adrenal insufficiency. NEJM Evid 2022;2(2):EVIDoa2200182. https://doi.org/10.1056/EVIDoa2200182. source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data
    § Comparison of diagnostic accuracy for CS between different methods

    There was enough sample volume to run six different assays, which made a direct method comparison possible.

    “Our reference cohort is larger than many previous cohorts, enabling calculation of 95% CIs for the reference intervals, and sample volumes sufficient for six different methods, allowing a direct comparison of methods, which to our knowledge has not been performed previously.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductnone given
    § Comparison of diagnostic accuracy for CS between different methods

    Reference and Cushing's syndrome samples were collected prospectively under one protocol and kept at -80 °C until analysis, which prevented drift in the assays.

    “Reference samples and CS samples were prospectively collected according to the same protocol and stored in -80 °C until analyses avoiding drifts in the analytical methods.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given
    § Comparison of diagnostic accuracy for CS between different methods

    Some data were missing, but the missingness was random and therefore should not bias the results.

    “The study did have some missing data, which was however random, and should therefore not introduce bias.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The missing data were random and so should not bias the findings.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation
    § Comparison of diagnostic accuracy for CS between different methods

    The study did not examine whether changes in cortisol binding globulin levels or genetic differences in its cortisol affinity affect salivary cortisol and cortisone.

    “This study does not evaluate whether variations in cortisol binding globulin (CBG) concentration or genetic variations affecting CBG-cortisol affinity may influence salivary cortisol and cortisone concentrations [50].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [50] Meyer, EJ, Nenke, MA, Rankin, W, Lewis, JG, Torpy, DJ. Corticosteroid-binding globulin: a review of basic and clinical advances. Horm Metab Res 2016;48:359–71. https://doi.org/10.1055/s-0042-108071. source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data
    § Comparison of diagnostic accuracy for CS between different methods

    The reference cohort was larger than many earlier ones, which allowed 95% confidence intervals to be calculated for the reference intervals.

    “Our reference cohort is larger than many previous cohorts, enabling calculation of 95% CIs for the reference intervals, and sample volumes sufficient for six different methods, allowing a direct comparison of methods, which to our knowledge has not been performed previously.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductreasoning
    § Comparison of diagnostic accuracy for CS between different methods

    The same individuals used to derive reference intervals also contributed to the diagnostic accuracy analysis for Cushing's syndrome, which could favor the reference interval used.

    “Furthermore, the individuals used to calculate the reference intervals were also used to calculate diagnostic accuracy for CS patients, which may introduce an advantage for the used reference interval.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation
    § Comparison of diagnostic accuracy for CS between different methods

    Following CLSI guidance, outliers in the reference samples were identified with the Dixon method.

    “In accordance with CLSI recommendations, we have used the Dixon method to identify outliers in the reference samples [31].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Horowitz, GL, Altaie, S, Boyd, JC, Ceriotti, F, Garg, U, Horn, P, et al.. Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline–third edition. C28-A3c ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2010.

      Not checked in the free run.

38 of 38

  • The paper relies on this without support.

    No direct comparison of diagnostic accuracy for Cushing's syndrome using salivary cortisol and cortisone across different analytical methods had been performed.

    “Direct comparison of diagnostic accuracy for CS using salivary cortisol and cortisone with different analysis methods has, to our knowledge, not been performed.”

  • The paper relies on this without support.

    Salivary cortisol and cortisone measured late at night and after dexamethasone suppression are increasingly used for Cushing's syndrome screening.

    “Salivary cortisol and cortisone at late night and after dexamethasone suppression test (DST) are increasingly used for screening of Cushing's syndrome (CS).”

  • The paper relies on this without support.

    An overnight 1-mg dexamethasone suppression test using serum cortisol is a recommended screen for Cushing's syndrome, including when evaluating adrenal incidentalomas.

    “Overnight 1-mg DST with analysis of serum cortisol is a recommended screening test for CS, including endocrine evaluation of adrenal incidentalomas.”

  • The paper relies on this without support.

    Post-DST salivary cortisol at 08:00 h is seldom used in practice, unlike blood sampling.

    “Although patient friendly, cost-effective and simple, salivary cortisol at 08:00 h after DST is rarely used compared with blood samples.”

  • The paper relies on this without support.

    No earlier study had directly compared LC-MS/MS methods for salivary cortisol and cortisone.

    “This is the first study directly comparing LC-MS/MS salivary cortisol and cortisone analysis methods, allowing comparisons of absolute values, reference intervals and diagnostic accuracy.”

  • The paper relies on this without support.

    The missing data were random and so should not bias the findings.

    “The study did have some missing data, which was however random, and should therefore not introduce bias.”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

  • Listed Cushing's syndrome etiologies account for only 21 of 22 patients

    Minor

    The listed etiologies total 21 patients (12 + 5 + 4), but the text says 22 patients were included. One patient's etiology is unaccounted for.

    Reported 22 · recomputed 21

1 of 1

Cites

Flagged first, then by citations

2 of the 51 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. Horowitz, GL, Altaie, S, Boyd, JC, Ceriotti, F, Garg, U, Horn, P, et al.. Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline–third edition. C28-A3c ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2010.

Cited by

Sorted by citations

Related

Authors
Bäcklund, N., Brattsand, G., Lundstedt, S., Aardal, E., Bartuseviciene, I., Berinder, K., Höybye, C., Burman, P., Edén Engström, B., Isaksson, A., Blomgren, A., Ragnarsson, O., Rüetschi, U., Wahlberg, J., Olsson, T., & Dahlqvist, P.
Affiliations
  • Umeå University (SE)
  • Linköping University (SE)
  • Karolinska University Hospital (SE)
  • Skåne University Hospital (SE)
  • Department of Medical Sciences, Endocrinology and Mineral Metabolism , Uppsala University , Uppsala , Sweden
  • Lund University (SE)
  • Institute of Medicine, Sahlgrenska Academy , University of Gothenburg , Gothenburg , Sweden
  • Sahlgrenska University Hospital (SE)
  • Örebro University (SE)
Corresponding author
Bäcklund, Nils
ORCID
Venue
Clinical Chemistry and Laboratory Medicine (CCLM)
Publisher
Walter de Gruyter GmbH
Published
2023-04-04
Volume
61
Issue
10
Pages
1780-1791
ISSN
  • 1434-6621 (ISSN-L)
  • 1437-4331
DOI
10.1515/cclm-2023-0141
OpenAlex
W4362522534
Semantic Scholar
594ba987dd7612031129930a26df2417ebf7c766
Cited by
9
References
51
Citations by year
2026: 3 · 2025: 2 · 2024: 4
FWCI
1.84
Type
journal article
Language
en
Open access
Licence
other-oa
Locations
  • Clinical Chemistry and Laboratory Medicine (CCLM) (publishedVersion)
  • PubMed (publishedVersion)
  • DiVA at Umeå University (Umeå University) (submittedVersion, other-oa)
  • Lund University Publications (Lund University) (submittedVersion)
Funders
  • Västerbotten Läns Landsting
  • Private donation to Umeå University for research on pituitary disease
Topics
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Pituitary Gland Disorders and Treatments
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
  • Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
Keywords
Cushing's syndrome, salivary cortisol, salivary cortisone, dexamethasone suppression test, reference values, LC-MS/MS, immunoassay, diagnostic accuracy, method comparison
MeSH terms
Chromatography, Liquid, Cortisone, Cushing Syndrome, Humans, Hydrocortisone, Saliva, Tandem Mass Spectrometry

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

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