Heat inactivation of SARS-CoV 2 enabled the measurement of salivary cortisol during COVID-19 pandemic.

BACKGROUND AND AIM: Salivary cortisol has become an essential tool in the management of cortisol-related disease. In 2020 the sudden outbreak of COVID-19 pandemic caused several concerns about the use of saliva, due to the risk of contamination, and a European consensus further discourage using salivary cortisol. To decrease infectious risk, we handled specimens by applying a heat treatment to inactivate viral particles, further evaluating the impact of the COVID-19 pandemic on the use of salivary cortisol in clinical practice. MATERIAL AND METHODS: Saliva samples were exposed for 10 min at 70 °C, then cortisol was measured using LC-MS/MS. The number of salivary cortisol examinations from 2013 to 2022 was extracted from the local electronic database: those performed in 2019, 2020, and 2021 were analyzed and compared with the historical data. RESULTS: During 2020 we observed a decrease of 408 (-20%) examinations (p = 0.05) compared to 2019; especially in salivary cortisol daily rhythm and salivary cortisol/cortisone ratio (respectively reduction of 47% and 88%, p = 0.003 and p = 0.001). Analyzing year 2021 compared with 2020 we reported an increase of 420 examinations (+20%, p = 0.01), with a complete recovery of salivary cortisol measurement (considering 2019: p = 0.71). Major differences were observed between morning salivary cortisol (-20%, p = 0.017), LNSC (-21%, p = 0.012) and salivary cortisol rhythm (-22%, p = 0.056). No Sars-Cov2 infections related to working exposure were reported among laboratory's employers. CONCLUSIONS: We speculate that the adoption of an appropriate technique to inactivate viral particles in saliva specimens allowed the safety maintenance of salivary collections, also during the Sars-CoV-2 outbreak.

Toward Sustainable and Healthy Fish Products-The Role of Feeding and Preservation Techniques.

Fish is a fundamental component of the human diet, and in the near future the proportion of aquatic foods originating from aquaculture production is expected to increase to over 56%. The sustainable growth of the aquaculture sector involves the use of new sustainable raw materials as substitutes for traditional fishmeal and fish oil ingredients, but it is crucial that the substitution maintains the nutritional value of the fish meat. In addition, the preservation of the nutritional value should be a mandatory requirement of new technologies that extend the shelf life of fish. In this context, we evaluated the impact of a newly formulated feed and three preservation treatments (brine, pulsed electric field (PEF), and PEF plus brine) on the fatty acid composition and protein and lipid digestibility of sea bass fillets. In non-digested fillets, although slightly reduced by the newly formulated feed (standard = 2.49 ± 0.14; newly formulated = 2.03 ± 0.10) the n-3/n-6 PUFA ratio indicated good nutritional value. The preservation treatments did not modify the fatty acid content and profile of non-digested fillets. Conversely, protein and lipid digestibility were not affected by the different diets but were significantly reduced by brine, with or without PEF, while PEF alone had no effect. Overall, our results indicated that the newly formulated feed containing 50% less fishmeal is a good compromise between the sustainability and nutritional value of cultivated seabass, and PEF is a promising preservation technology deserving of further study.

Association of autoantibodies targeting endothelin type-A receptors with no-reflow in ST-elevation myocardial infarction.

BACKGROUND AND AIMS: No-reflow (NR), where the coronary artery is patent after treatment of ST-elevation myocardial infarction (STEMI) but tissue perfusion is not restored, is associated with worse outcomes. We aimed to investigate the relationship between autoantibodies activating endothelin-1 receptor type A (ETAR-AAs) and NR after primary percutaneous coronary intervention (PPCI) in STEMI. METHODS: We studied 50 patients (age 59 ± 11 years, 40 males) with STEMI who underwent PPCI within 6 h after the onset of symptoms. Blood samples were obtained from all patients within 12 h following PPCI for ETAR-AA level measurement. The seropositive threshold was provided by the manufacturer (>10 U/ml). NR was assessed by cardiac magnetic resonance imaging (MVO, microvascular obstruction). As a control group, 40 healthy subjects matched for age and sex were recruited from the general population. RESULTS: MVO was observed in 24 patients (48%). The prevalence of MVO was higher in patients with ETAR-AAs seropositivity (72% vs. 38%, p = 0.03). ETAR-AAs were higher in patients with MVO (8.9 U/mL (interquartile range [IQR] 6.8-16.2 U/mL) vs. 5.7 U/mL [IQR 4.3-7.7 U/mL], p = 0.003). ETAR-AAs seropositivity was independently associated with MVO (OR 3.2, 95% CI 1.3-7.1; p = 0.03). We identified ≥6.74 U/mL as the best cut-off for prediction of MVO (sensitivity 79%; specificity 65%; NPV 71%; PPV 74%; accuracy 72%). CONCLUSIONS: The ETAR-AAs seropositivity is associated with NR in STEMI patients. These findings may open up new options in the management of myocardial infarction even if confirmation in a larger trial is needed.

Cleaning the Label of Cured Meat; Effect of the Replacement of Nitrates/Nitrites on Nutrients Bioaccessibility, Peptides Formation, and Cellular Toxicity of In Vitro Digested Salami.

Curing salts composed of mixtures of nitrates and nitrites are preservatives widely used in processed meats. Despite many desirable technological effects, their use in meat products has been linked to methemoglobinemia and the formation of nitrosamines. Therefore, an increasing "anti-nitrite feeling" has grown among meat consumers, who search for clean label products. In this view, the use of natural compounds as alternatives represents a challenge for the meat industry. Processing (including formulation and fermentation) induces chemical or physical changes of food matrix that can modify the bioaccessibility of nutrients and the formation of peptides, impacting on the real nutritional value of food. In this study we investigated the effect of nitrate/nitrite replacement with a combination of polyphenols, ascorbate, and nitrate-reducing microbial starter cultures on the bioaccessibility of fatty acids, the hydrolysis of proteins and the release of bioactive peptides after in vitro digestion. Moreover, digested salami formulations were investigated for their impacts on cell proliferation and genotoxicity in the human intestinal cellular model (HT-29 cell line). The results indicated that a replacement of synthetic nitrates/nitrites with natural additives can represent a promising strategy to develop innovative "clean label" salamis without negatively affecting their nutritional value.

Insight on Glucose and Fructose Absorption and Relevance in the Enterocyte Milieu.

Although epidemiological studies indicate a strong correlation between high sugar intake and metabolic diseases, the biological mechanisms underlying this link are still controversial. To further examine the modification and crosstalk occurring in enterocyte metabolism during sugar absorption, in this study we evaluate the diffusion and intestinal metabolism of glucose, fructose and sucrose, which were supplemented in equimolar concentration to Caco-2 cells grown on polyester membrane inserts. At different time points after supplementation, changes in metabolite concentration were evaluated in the apical and basolateral chambers by nuclear magnetic resonance (NMR) and gas-chromatography (GC). Sucrose was only minimally hydrolyzed by Caco-2 cells. Upon supplementation, we observed a faster uptake of fructose than glucose, the pentose sugar being also faster catabolized. Monosaccharide absorption was concomitant to the synthesis/transport of other metabolites, which occurred differently in glucose and fructose supplemented cells. Our results confirm the prominent role of intestinal cells in fructose metabolism and clearance after absorption, representing a further step forward in the understanding of the role of dietary sugars. Future research, including targeted analysis on specific transporters/enzymes and the use of labeled substrates, will be helpful to confirm the present results and their interpretation.

Impact of a Shorter Brine Soaking Time on Nutrient Bioaccessibility and Peptide Formation in 30-Months-Ripened Parmigiano Reggiano Cheese.

Reducing the salt content in food is an important nutritional strategy for decreasing the risk of diet-related diseases. This strategy is particularly effective when applied to highly appreciated food having good nutritional characteristics, if it does not impact either upon sensory or nutritional properties of the final product. This work aimed at evaluating if the reduction of salt content by decreasing the brine soaking time modifies fatty acid and protein bioaccessibility and bioactive peptide formation in a 30-month-ripened Parmigiano Reggiano cheese (PRC). Hence, conventional and hyposodic PRC underwent in vitro static gastrointestinal digestion, and fatty acid and protein bioaccessibility were assessed. The release of peptide sequences during digestion was followed by LC-HRMS, and bioactive peptides were identified using a bioinformatic approach. At the end of digestion, fatty acid and protein bioaccessibility were similar in conventional and hyposodic PRC, but most of the bioactive peptides, mainly the ACE-inhibitors, were present in higher concentrations in the low-salt cheese. Considering that the sensory profiles were already evaluated as remarkably similar in conventional and hyposodic PRC, our results confirmed that shortening brine soaking time represents a promising strategy to reduce salt content in PRC.

Comparison of Cortisol, Androstenedione and Metanephrines to Assess Selectivity and Lateralization of Adrenal Vein Sampling in Primary Aldosteronism.

Success of adrenal vein sampling (AVS) is verified by the selectivity index (SI), i.e., by a step-up of cortisol levels between the adrenal vein and the infrarenal inferior vena cava samples, beyond a given cut-off. We tested the hypothesis that androstenedione, metanephrine, and normetanephrine, which have higher gradients than cortisol, could increase the rate of AVS studies judged to be bilaterally successful and usable for the clinical decision making. We prospectively compared within-patient, head-to-head, the selectivity index of androstenedione (SIA), metanephrine (SIM), and normetanephrine (SINM), and cortisol (SIC) in consecutive hypertensive patients with primary aldosteronism submitted to AVS. Main outcome measures were rate of bilateral success, SI values, and identification of unilateral PA. We recruited 136 patients (55 + 10 years, 35% women). Compared to the SIC, the SIA values were 3.5-fold higher bilaterally, and the SIM values were 7-fold and 4.4-fold higher on the right and the left side, respectively. With the SIA and the SIM the rate of bilaterally successful AVS increased by 14% and 15%, respectively without impairing the identification of unilateral PA. We concluded that androstenedione and metanephrine outperformed cortisol for ascertaining AVS success, thus increasing the AVS studies useable for the clinical decision making.

Low-dose short synacthen test with salivary cortisol in patients with suspected central adrenal insufficiency.

CONTEXT: The low-dose short synacthen test (LDSST) is recommended for patients with suspected central adrenal insufficiency (AI) if their basal serum cortisol (F) levels are not indicative of an intact hypothalamic-pituitary-adrenal (HPA) axis. OBJECTIVE: To evaluate diagnostic threshold for salivary F before and 30 min after administering 1 µg of synacthen, performed before 09:30 h. DESIGN: A cross-sectional study from 2014 to 2020. SETTING: A tertiary referral university hospital. PATIENTS: In this study, 174 patients with suspected AI, 37 with central AI and 137 adrenal sufficient (AS), were included. MAIN OUTCOME MEASURE: The diagnostic accuracy (sensitivity (SE), specificity (SP)) of serum and salivary F levels measured, respectively, by chemiluminescence immunoassay and liquid chromatography-tandem mass spectrometry. RESULTS: Low basal serum or salivary F levels could predict AI. For the LDSST, the best ROC-calculated threshold for serum F to differentiate AI from AS was 427 nmol/L (SE 79%, SP 89%), serum F > 500 nmol/L reached SP 100%. A salivary F peak > 12.1 nmol/L after administering synacthen reached SE 95% and SP 84% for diagnosing central AI, indicating a conclusive reduction in the likelihood of AI. This ROC-calculated threshold for salivary F was similar to the 2.5th percentile of patients with a normal HPA axis, so it was considered sufficient to exclude AI. Considering AS those patients with salivary F > 12.1 nmol/L after LDSST, we could avoid unnecessary glucocorticoid treatment: 99/150 subjects (66%) had an inadequate serum F peak after synacthen, but salivary F was >12.1 nmol/L in 79 cases, who could, therefore, be considered AS. CONCLUSIONS: Salivary F levels > 12.1 nmol/L after synacthen administration can indicate an intact HPA axis in patients with an incomplete serum F response, avoiding the need to start glucocorticoid replacement treatment.

Coenzyme Q10 Phytosome Formulation Improves CoQ10 Bioavailability and Mitochondrial Functionality in Cultured Cells.

Coenzyme Q10 (CoQ10) is a lipid-soluble molecule with a dual role: it transfers electrons in the mitochondrial transport chain by promoting the transmembrane potential exploited by the ATPase to synthesize ATP and, in its reduced form, is a membrane antioxidant. Since the high CoQ10 hydrophobicity hinders its bioavailability, several formulations have been developed to facilitate its cellular uptake. In this work, we studied the bioenergetic and antioxidant effects in I407 and H9c2 cells of a CoQ10 phytosome formulation (UBIQSOME®, UBQ). We investigated the cellular and mitochondrial content of CoQ10 and its redox state after incubation with UBQ. We studied different bioenergetic parameters, such as oxygen consumption, ATP content and mitochondrial potential. Moreover, we evaluated the effects of CoQ10 incubation on oxidative stress, membrane lipid peroxidation and ferroptosis and highlighted the connection between the intracellular concentration of CoQ10 and its antioxidant potency. Finally, we focused on the cellular mechanism that regulates UBQ internalization. We showed that the cell lines used in this work share the same uptake mechanism for UBQ, although the intestinal cell line was less efficient. Given the limitations of an in vitro model, the latter result supports that intestinal absorption is a critical step for the oral administration of Coenzyme Q10 formulations.

High sodium intake, glomerular hyperfiltration, and protein catabolism in patients with essential hypertension.

AIMS: A blood pressure (BP)-independent metabolic shift towards a catabolic state upon high sodium (Na+) diet, ultimately favouring body fluid preservation, has recently been described in pre-clinical controlled settings. We sought to investigate the real-life impact of high Na+ intake on measures of renal Na+/water handling and metabolic signatures, as surrogates for cardiovascular risk, in hypertensive patients. METHODS AND RESULTS: We analysed clinical and biochemical data from 766 consecutive patients with essential hypertension, collected at the time of screening for secondary causes. The systematic screening protocol included 24 h urine (24 h-u-) collection on usual diet and avoidance of renin-angiotensin-aldosterone system-confounding medications. Urinary 24 h-Na+ excretion, used to define classes of Na+ intake (low ≤2.3 g/day; medium 2.3-5 g/day; high >5 g/day), was an independent predictor of glomerular filtration rate after correction for age, sex, BP, BMI, aldosterone, and potassium excretion [P = 0.001; low: 94.1 (69.9-118.8) vs. high: 127.5 (108.3-147.8) mL/min/1.73 m2]. Renal Na+ and water handling diverged, with higher fractional excretion of Na+ and lower fractional excretion of water in those with evidence of high Na+ intake [FENa: low 0.39% (0.30-0.47) vs. high 0.81% (0.73-0.98), P < 0.001; FEwater: low 1.13% (0.73-1.72) vs. high 0.89% (0.69-1.12), P = 0.015]. Despite higher FENa, these patients showed higher absolute 24 h Na+ reabsorption and higher associated tubular energy expenditure, estimated by tubular Na+/ATP stoichiometry, accordingly [Δhigh-low = 18 (12-24) kcal/day, P < 0.001]. At non-targeted liquid chromatography/mass spectrometry plasma metabolomics in an unselected subcohort (n = 67), metabolites which were more abundant in high versus low Na+ intake (P < 0.05) mostly entailed intermediates or end products of protein catabolism/urea cycle. CONCLUSION: When exposed to high Na+ intake, kidneys dissociate Na+ and water handling. In hypertensive patients, this comes at the cost of higher glomerular filtration rate, increased tubular energy expenditure, and protein catabolism from endogenous (muscle) or excess exogenous (dietary) sources. Glomerular hyperfiltration and the metabolic shift may have broad implications on global cardiovascular risk independent of BP.

Including Relative Adrenal Insufficiency in Definition and Classification of Acute-on-Chronic Liver Failure.

BACKGROUND & AIMS: Relative adrenal insufficiency (RAI) is defined by insufficient production of cortisol relative to organ demand. RAI is observed frequently in hospitalized patients with cirrhosis, but there is disagreement over the clinical effects of RAI in these patients. We evaluated the prevalence and the clinical effects of RAI in hospitalized patients with cirrhosis. METHODS: We performed a prospective study of 160 patients admitted to a hospital in Italy for acute decompensation of cirrhosis from May 2011 through September 2016. Patients were followed up until death, liver transplantation, or a maximum of 90 days. Serum and salivary levels of cortisol were measured before and after a 1-hour Short Synacthen Test. A diagnosis of RAI was given to patients with an increase in serum cortisol of less than 9 µg/dL, after Synacthen administration, in patients with baseline serum levels of cortisol less than 35 µg/dL. We collected blood samples before the Synacthen test and analyzed them for blood cell counts, liver and renal function, levels of C-reactive protein, and lipid profiles (total cholesterol, high-density lipoprotein cholesterol, apolipoprotein-A1). RESULTS: A diagnosis of RAI was made for 78 patients (49%). Age (odds ratio [OR], 0.95; P = .030), number of leukocytes (OR, 3.10; P = .006), and levels of high-density lipoprotein cholesterol (OR, 0.30; P = .039) were associated independently with RAI. Patients with RAI had a significantly higher risk of developing bacterial infections (hazard ratio [HR], 1.60; P = .038), sepsis (HR, 2.95; P = .001), septic shock (HR, 4.94; P = .038), new organ failures (HR, 2.45; P = .014), and acute-on-chronic liver failure (HR, 2.27; P = .037) than patients without RAI. RAI was associated independently with death within 90 days of diagnosis (subdistribution HR, 4.83; P = .001). Patients with RAI and mild renal dysfunction or hepatic encephalopathy had no significant difference in cumulative incidence of 28-day mortality vs patients with acute-on-chronic liver failure grade 1 (25% vs 22%). CONCLUSIONS: We found RAI to occur in almost half of patients admitted to a hospital for acute decompensation of cirrhosis. RAI was associated with a deficit of substrates for steroidogenesis and an increase in markers of inflammation. Patients with RAI have a high risk of developing sepsis, septic shock, organ failure, and death within 90 days. RAI has similar prognostic value to nonrenal organ failures.

Interference of lipemia in samples for routine coagulation testing using a Sysmex CS-5100 coagulometer.

INTRODUCTION: Lipemia in samples can cause analytical errors in coagulation tests using photometric assays. To define the level of this interference, some studies assessed lipemic interferences by in vitro 'spiking' of different types of lipids obtaining interesting information, but spiked samples do not represent a real-world situation as natively lipemic samples do. METHODS: A total of 101 samples flagged as 'lipemic' by a Sysmex CS-5100 coagulometer were analyzed for PT, aPTT, fibrinogen Clauss assay, antithrombin activity, D-dimer concentration, before and after a double high-speed centrifugation procedure to reduce lipemic interference. We evaluated using Bland-Altman test if high-speed centrifugation and retesting are justified, considering that's a resource-consuming procedure; when a statistically significant difference was found, quality specification for imprecision was considered and compared to the observed delta. RESULTS: Statistically significant differences were found for PT, antithrombin activity and fibrinogen. Considering the Bland-Altman plot, fibrinogen results were split into two groups, and statistically significant difference was confirmed only for samples >2 g/L. CONCLUSIONS: For PT and antithrombin activity a mean percentual difference between the two determinations lower than within-subject biologic variation and one of the Fraser's quality specifications can be considered as a confounding 'noise' factor that is neither analytically nor clinically relevant. If the instrument determines a result on the first run, for PT, aPTT, D-dimer concentration and antithrombin activity tests, the double plasma high-speed centrifugation is unnecessary. It is instead necessary if fibrinogen >2 g/L or if the instrument cannot determine a result on the first run.

Improved salivary cortisol rhythm with dual-release hydrocortisone

Introduction and Aim: The purpose of replacement therapy in adrenal insufficiency (AI) is mimicking endogenous cortisol levels as closely as possible: dual release hydrocortisone (DR-HC) has been introduced to replicate the circadian cortisol rhythm. Multiple daily saliva collections could be used to assess the cortisol rhythm during real life: our aim was to study the salivary cortisol profile in AI. Materials and Methods: We prospectively evaluated, in an observational study, 18 adult outpatients with AI (11 primary and 7 secondary AI), switched from conventional treatment (conv-HC, 25 mg/day) to the same dose of DR-HC. We collected six samples of saliva in a day, measuring cortisol (F) and cortisone (E) with LC-MS/MS. Forty-three matched healthy subjects served as controls. Results: F levels were similar in the morning (and higher than controls) in patients treated with conv-HC or DR-HC; otherwise F levels and exposure were lower in the afternoon and evening in patients with DR-HC, achieving a cortisol profile closer to healthy controls. Daily cortisol exposure, measured with area under the curve, was lower with DR-HC. Morning F and E presented sensitivity and specificity >90% to diagnose AI (respectively threshold of 3 and 9.45 nmol/L). Total cholesterol and HbA1c levels reduced with DR-HC. Conclusions: Salivary cortisol daily curve could be used as a new tool to assess the cortisol profiles in patients treated with conv-HC and DR-HC. A lower daily cortisol exposure was achieved with DR-HC (despite the same HC dose), especially in the afternoon-evening.

Validation model of a laboratory-developed method for the ISO15189 accreditation: The example of salivary cortisol determination.

BACKGROUND: The ISO 15189:2012 describes the requirements for quality and competence, which are specific for all sectors of a medical laboratory. Laboratory-developed methods, standard methods used outside their intended scope and validated methods subsequently modified shall be validated. The main objective of validation of an examination procedure (EP) is to demonstrate its fitness-for-purpose. The aim of the present study is to illustrate a model to validate laboratory-developed methods in agreement with the ISO 15189:2012 requirement, through the example of salivary cortisol (sF) measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: The proposed validation model for a laboratory developed method includes 6 steps: 1) analysis of available scientific documentation pertinent to specific EP; 2) evaluation of EP intended use; 3) identification of performance characteristics to evaluate; 4) definition of experimental procedure; 5) identification of acceptability criterion for results evaluation; 6) preparation of a validation plan; 7) production of a validation certificate. RESULTS: sF is an EP sampled during the accreditation audit and resulted in compliance with the Standard. A structured procedure to certify that the achieved performance specifications are appropriate for the purpose of the test is therefore necessary. CONCLUSIONS: A high competence is required for laboratory professionals in order to guarantee that the validated quality specifications have a positive impact on patients' management. The ISO 15189 accreditation is an important tool that allows demonstrating the value of a medical laboratory in the clinical context.

Metyrapone treatment in Cushing's syndrome: a real-life study.

INTRODUCTION AND AIM: Medical treatment is increasingly used in patients with Cushing's syndrome (CS). Metyrapone (MET) is an inhibitor of 11ß-hydroxylase: retrospective studies reported a decrease of cortisol secretion in 50% of cases. We evaluated the effectiveness of MET in an observational study, considering the normalization of urinary-free cortisol (UFC) and late-night salivary cortisol (LNSC) levels. MATERIALS AND METHODS: We enrolled 31 patients with CS, treated with MET for at least 1 month (16 for primary treatment and 15 after surgical failure). A planned dose-titration regimen considering baseline UFC levels was adopted; MET dose was uptitrated until UFC normalization, surgery, or side effect occurrence. UFC and LNSC levels were routinely measured by liquid chromatography-tandem mass spectrometry. RESULTS: Patients were treated with a median dose of 1000 mg for 9 months. UFC and LNSC decreased quickly after the first month of treatment (-67 and -57% from baseline), with sustained UFC normalization up to 12 and 24 months (in 13 and 6 patients, respectively). UFC and LNSC normalized later (after 3-6 months) in patients with severe hypercortisolism (>5-fold baseline UFC). Regarding the last visit, 70 and 37% of patients normalized UFC and LNSC, respectively. Body weight reduction (-4 kg) was observed after UFC normalization. Severe side effects were not reported, half of the female patients complained of hirsutism, and blood pressure was not increased. CONCLUSIONS: MET therapy is a rapid-onset, long-term effective, and safe medical treatment in CS patients, achieving UFC normalization (in 70% of patients) more than cortisol rhythm recovery (in 37% of subjects).

Measurement uncertainty in laboratory reports: A tool for improving the interpretation of test results.

BACKGROUND: Measurement uncertainty (MU) estimation has been introduced by ISO 15189 for the accreditation of clinical laboratories. Although MU reporting is not required, its inclusion in medical reports is of potential assistance to physicians in results interpretation. METHODS: MU reporting was evaluated with respect to different test purposes, namely comparison with reference intervals (RI), patient monitoring or comparison with clinical decision limits. Clinical Biochemistry, Hematology, Coagulation and Clinical Immunology measurands were used as examples. Assuming Gaussian RI distribution, the probability of retesting due to MU was determined by simulations. Significant MU variations were compared against the reference change value (RCV) and clinical decision limits. RESULTS: Three potential scenarios emerged for RI. For 12 measurands, depending on the MU interval, a potential change in results interpretation was found only for Sodium and S-Protein. On considering only the results within RI, simulations confirmed that up to 8.6% of MU intervals encompassed the RI limits, thus potentially leading to retesting. For tests used in patient monitoring, significant MU variations were comparable to those calculated by RCV, with the exception of CEA. For tests results evaluated with respect to clinical decision limits, on including MU, the clinical interpretation may be improved (e.g. for tPSA). CONCLUSION: The findings made in the present study, which considers real MU data and hypothetical results obtained for a series of measurands, support the concept that MU may aid the physician's interpretation thus ensuring reliable clinical decision making.

What information on measurement uncertainty should be communicated to clinicians, and how?

The communication of laboratory results to physicians and the quality of reports represent fundamental requirements of the post-analytical phase in order to assure the right interpretation and utilization of laboratory information. Accordingly, the International Standard for clinical laboratories accreditation (ISO 15189) requires that "laboratory reports shall include the information necessary for the interpretation of the examination results". Measurement uncertainty (MU) is an inherent property of any quantitative measurement result which express the lack of knowledge of the true value and quantify the uncertainty of a result, incorporating the factors known to influence it. Even if the MU is not included in the report attributes of ISO 15189 and cannot be considered a post-analytical requirement, it is suggested as an information which should facilitate an appropriate interpretation of quantitative results (quantity values). Therefore, MU has two intended uses: for laboratory professionals, it gives information about the quality of measurements, providing evidence of the compliance with analytical performance characteristics; for physicians (and patients) it may help in interpretation of measurement results, especially when values are compared with reference intervals or clinical decision limits, providing objective information. Here we describe the way that MU should be added to laboratory reports in order to facilitate the interpretation of laboratory results and connecting efforts performed within laboratory to provide more accurate and reliable results with a more objective tool for their interpretation by physicians.

Daily salivary cortisol and cortisone rhythm in patients with adrenal incidentaloma.

BACKGROUND AND AIM: Impaired cortisol rhythm is a characteristic feature of Cushing's Syndrome, nevertheless late night salivary cortisol (LNSC) is not suitable to detect subclinical hypercortisolism in patients with adrenal incidentaloma (AI). We studied daily salivary cortisol (F) and cortisone (E) rhythm in patients with AI. MATERIALS AND METHODS: Six saliva samples were collected from awakening to night in 106 patients with AI and 40 controls. F and E were measured with LC-MS/MS and daily F exposure was calculated with the area under the curve (AUC). RESULTS: Patients with serum cortisol after dexamethasone suppression test (DST) > 50 nmol/L showed higher morning F (15.5 ± 14.5 vs. 8.6 ± 5.5 nmol/L, p = 0.001), suppressed corticotropin levels (76 vs. 35%, p < 0.001) and increased daily F exposure (3795 ± 1716 vs. 2898 ± 1478, p = 0.012), especially in the morning (2035 ± 1267 vs. 1365 ± 777, p = 0.003), otherwise LNSC levels were similar. Salivary E and AUC levels were higher in patients with DST > 50 nmol/L. AUC was not correlated with urinary cortisol levels or adenoma size. F and E levels were similar among patients with unilateral or bilateral adenoma, or considering the presence of hypertension, dyslipidemia, diabetes, or cardiovascular events. CONCLUSION: Daily cortisol exposure, evaluated with AUC from multiple saliva collections, is increased in AI patients with serum cortisol > 50 nmol/L after DST, especially in the morning, leading to reduced corticotropin levels. Cortisol rhythm is preserved in patients with AI, remarking that LNSC is not a screening test for subclinical hypercortisolism.

Diagnostic accuracy of increased urinary cortisol/cortisone ratio to differentiate ACTH-dependent Cushing's syndrome.

BACKGROUND AND AIM: Differential diagnosis between Cushing's Disease (CD) and Ectopic ACTH Syndrome (EAS) may be a pitfall for endocrinologists. The increasing use in clinical practice of chromatography and mass spectrometry improves the measurement of urinary free cortisol (UFF) and cortisone (UFE). We have recently observed that cortisol to cortisone ratio (FEr) was higher in a small series of EAS; in this study we collected a larger number of ACTH-dependent Cushing's Syndrome (CS) to study the role of FEr to characterize the source of corticotropin secretion. MATERIALS AND METHODS: High-pressure liquid chromatography with UV detection (HPLC-UV, n=35) or liquid chromatography-tandem mass spectrometry (LC-MS/MS, n=72) were used to measure UFF, UFE and FEr in 83 patients with CD and 24 with EAS. RESULTS: UFF, UFE and FEr levels were higher in EAS than in CD (UFF: 6671 vs 549 nmol/24 hours; UFE: 2069 vs 464 nmol/24 hours; FEr: 4.13 vs 0.97; all P<.001). FEr >1.15 (the best ROC-based threshold) was able to distinguish CD from EAS with 75% sensitivity (SE) and 75% specificity (SP), AUC 0.811; results were similar between HPLC-UV (SE 73%, SP 79%, AUC 0.708) and LC-MS/MS (SE 77%, SP 73%, AUC 0.834; P=.727). The diagnostic accuracy of FEr was similar to that of CRH test or high-dose dexamethasone suppression test (respectively P=.171 and P=.683), also combined. Finally, FEr was able to increase the number of correct diagnosis in patients with discordant dynamic tests. CONCLUSIONS: Urinary FEr >1.15 was able to suggest EAS, with a diagnostic accuracy similar to that of other dynamic tests proposed to study ACTH-dependent CS.