Multicenter performance evaluation of a second generation cortisol assay

Michael Vogeser 1 , Jürgen Kratzsch 2 , Yoon Ju Bae 2 , Mathias Bruegel 3 , Uta Ceglarek 2 , Tom Fiers 4 , Alexander Gaudl 2 , Hedwig Kurka 5 , Christoph Milczynski 6 , Cristina Prat Knoll 7 , Anna C. Suhr 3 , Daniel Teupser 3 , Ingrid Zahn 6 ,  and Richard E. Ostlund Jr. 8
  • 1 Institute of Laboratory Medicine, Hospital of the University of Munich (LMU), Marchioninistr. 15, 81377 Munich, Germany, Phone: +49 89 4400-73221
  • 2 Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany
  • 3 Institute of Laboratory Medicine, Hospital of the University of Munich (LMU), Munich, Germany
  • 4 University Hospital Ghent, Klinische Biologie, Ghent, Belgium
  • 5 Roche Diagnostics GmbH, Penzberg, Germany
  • 6 Limbach Laboratory, Medical Care Center, Heidelberg, Germany
  • 7 Roche Diagnostics GmbH, Mannheim, Germany
  • 8 Division of Endocrinology, Metabolism and Lipid Research, Washington University, St Louis, MO, USA
Michael Vogeser, Jürgen Kratzsch, Yoon Ju Bae, Mathias Bruegel, Uta Ceglarek, Tom Fiers, Alexander Gaudl, Hedwig Kurka, Christoph Milczynski, Cristina Prat Knoll, Anna C. Suhr, Daniel Teupser, Ingrid Zahn and Richard E. Ostlund Jr.

Abstract

Background:

Untreated disorders of the adrenocortical system, such as Cushing’s or Addison’s disease, can be fatal, and accurate quantification of a patient’s cortisol levels is vital for diagnosis. The objective of this study was to assess the analytical performance of a new fully-automated Elecsys® Cortisol II assay (second generation) to measure cortisol levels in serum and saliva.

Methods:

Four European investigational sites assessed the intermediate precision and reproducibility of the Cortisol II assay (Roche Diagnostics) under routine conditions. Method comparisons of the Cortisol II assay vs. liquid chromatography-tandem mass spectrometry (LC-MS/MS), the gold standard for cortisol measurement, were performed. Cortisol reference ranges from three US sites were determined using samples from self-reported healthy individuals.

Results:

The coefficients of variation (CVs) for repeatability, intermediate precision, and reproducibility for serum samples were ≤2.6%, ≤5.8%, and ≤9.5%, respectively, and for saliva were ≤4.4% and ≤10.9%, and ≤11.4%, respectively. Agreement between the Cortisol II assay and LC-MS/MS in serum samples was close, with a slope of 1.02 and an intercept of 4.473 nmol/L. Reference range samples were collected from healthy individuals (n=300) and serum morning cortisol concentrations (5–95th percentile) were 166.1–507 nmol/L and afternoon concentrations were 73.8–291 nmol/L. Morning, afternoon, and midnight saliva concentrations (95th percentile) were 20.3, 6.94, and 7.56 nmol/L, respectively.

Conclusions:

The Cortisol II assay had good precision over the entire measuring range and had excellent agreement with LC-MS/MS. This test was found suitable for routine diagnostic application and will be valuable for the diagnosis of adrenocortical diseases.

Introduction

Diseases of the adreno-cortisol system – such as Cushing’s disease and Addison’s disease – are rather rare conditions [1], [2], [3]; life-threatening on the one hand but treatable on the other [4]. Consequently, a straightforward diagnostic approach is of essential importance, and accurate quantification of cortisol levels plays a key role in the diagnosis of patients with suspected disease [5]. Cortisol levels are often measured from a patient’s serum or plasma, which reflects total cortisol, both free and bound. However, in patients with liver disease, those receiving estrogen treatment or with critical illness, total serum cortisol levels may be difficult to interpret because of the variation in binding proteins. Recently, quantification of cortisol in saliva has become more widely used since it is considered of equal diagnostic relevance as serum but can be obtained more easily and non-invasively [6], [7]. Salivary cortisol is free (i.e. not bound) and is useful in patients with serum results that are challenging to interpret [8], [9]. Very low salivary cortisol concentrations are typically found in healthy individuals (<8 nmol/L at midnight) [10], [11]. Therefore, accurate test results in the lower cortisol ranges are needed because this is where reproducibility is currently reduced but where diagnostic test decisions are often made.

Automated cortisol immunoassays are commercially available, which have acceptable precision and are widely used not only because of their high performance but also because of their speed, simplicity, and cost-effectiveness [12]. One major limitation of immunoassays is their specificity with interference of structurally similar compounds, for example endogenous steroids such as cortisone, or other steroid drugs such as prednisolone or dexamethasone [13], [14]. In addition, different immunoassays show high inter-assay variation [12], [15].

Gas chromatography-tandem mass spectrometry (GC-MS/MS) is traditionally used as a reference method for serum cortisol quantification. The introduction of LC-MS/MS allowed the use of isotope dilution mass spectrometry also in routine testing; however, the application of this technology is still very demanding and limited to very few laboratories at present [16], [17], [18], [19], [20] with The Reference Institute for Bioanalytics (RfB; Bonn, Germany) reporting only 2.7% (14/513) laboratories using LC-MS/MS in their last survey [21]. Consequently, immunoassays are still the mainstay of cortisol testing worldwide [22].

The first generation Elecsys® Cortisol assay (Roche Diagnostics) was introduced in April 2000 to measure cortisol levels in serum, plasma, urine, and saliva and has been used for the quantification of cortisol in clinical laboratories worldwide [23], [24], [25], [26]. In order to improve the specificity of cortisol measurement, a novel second-generation cortisol assay (Elecsys® Cortisol II assay; Roche Diagnostics) has been developed. Poly-clonal cortisol-specific antibodies in the first generation assay were switched to more specific monoclonal antibodies in the new assay. Both the first- and second-generation assays measure the total cortisol fraction in serum and the free cortisol in saliva [22], [23], [27]. Furthermore, the new test generation is traceable to a GC-MS/MS-based reference method according to the concept of an unbroken chain of traceability in clinical laboratory testing. The objective of this study was to assess the analytical performance of the Cortisol II assay under field conditions in a multicenter performance evaluation study.

Materials and methods

Study sites and instruments

The technical evaluation of the Cortisol II assay was performed between June and November 2014 at four European investigational sites: three in Germany (Munich, Heidelberg, and Leipzig) and one in Belgium (Ghent). All sites used cobas e 411 analyzers for the Cortisol II assay experiments presented in this study. In addition to a cobas e 411 system, the Leipzig site utilized a cobas e 601 analyzer.

Assay design

The Elecsys® Cortisol II assay is the second generation assay (Roche Diagnostics GmbH, Mannheim, Germany) and can be used to quantify cortisol levels in serum, plasma (Li-heparin, K2-K3-EDTA), or saliva. Briefly, the sample is incubated with a biotinylated cortisol antibody along with a ruthenium complex labeled cortisol derivative to form an immune complex. Streptavidin-coated microparticles are added and the complex becomes bound to the solid phase via a biotin/streptavidin interaction. Once aspirated, the microparticles are magnetically captured onto the surface of an electrode within a measuring cell, a voltage is applied with an electrochemiluminescence signal as the readout. The sample volume required is 10 μL, the assay measuring range is 1.5–1750.0 nmol/L, and the entire procedure takes a total of 18 min [27]. The assay is calibrated using the Cortisol CalSet (Roche Diagnostics) and has been standardized to the Institute for Reference Materials and Measurements (IRMM; Geel, Belgium)/IFCC-451 panel (ID-GC/MS) for serum [28].

Ethics statement

The investigation and sample collection sites adhered to the International Conference on Harmonization guideline for Good Clinical Practice and conducted the study in accordance with the Declaration of Helsinki (revised version). The study was approved by the Local Ethics Committees of the participating sites. All individuals participating in the reference range study signed an informed consent agreement.

Sample storage and handling

The control samples for the precision experiments were stored at –80°C at Roche Diagnostics GmbH before being shipped to the study sites. At the sites, the samples were stored at –20 or –80°C. For the method comparison experiments, leftover, anonymized serum or plasma and saliva samples from daily routine were used. These were stored at –20°C until measurements, for a maximum of 12 weeks [29].

For the reference range assessments, serum or plasma was collected using a 3.5-mL Vacutainer® Serum Separation Tube (SST). A serum sample was preferred, but alternatively, Li-heparin-plasma/K2,-K3-EDTA plasma was used. Saliva was obtained using a cotton swab Salivette® device (Sarstedt, Germany). Samples were processed within 24 h of donation. For the samples obtained at midnight, these were stored at 2–8°C and delivered to the collection sites in the morning hours, no later than 24 h after taken. After collection, all samples were stored at 2–8°C for a maximum of 3 days then frozen at –70°C at the collection facilities. The handling of the saliva samples was in line with a previous study into the long-term stability of saliva cortisol [29]. At the end of the collection process, frozen specimens were shipped on dry ice to the Roche Diagnostics Indianapolis site in a thermally insulated container and were received at the destination in a frozen state. Good Laboratory Practices and national regulations for the shipping of samples were strictly followed. Upon receipt, the samples were stored at –80°C before being shipped to the study testing site. The samples at the study testing sites were stored at –70°C until testing occurred. Samples that were not handled, stored, or shipped according to this procedure were excluded from the analysis.

Cross-reactivity

The cross-reactivity of the Cortisol II and Cortisol (generation 1) assays was determined using two human serum samples, one of which was spiked with potential cross-reactant compounds. The cortisol concentration of the samples was approximately 165.5 nmol/L (60 μg/L) and 551.7 nmol/L (200 μg/L) (conversion factor μg/L×2.7586=nmol/L). The spiked and non-spiked samples were tested in duplicate and the difference from the non-spiked sample represented the analyte concentration simulated by cross-reaction. The percentage of cross-reaction was calculated using the following formula:

Cross-reactivity (%)=100× simulated cortisol concentrationspiked cross-reactant concentration

The following cross-reactants were assessed: 6-α-methylprednisolone (0.1 mg/L), cortisone (10 mg/L), dexamethasone (10 mg/L), prednisolone (0.1 mg/L), and prednisone (10 mg/L). The concentrations used to test the cross-reactivity were chosen based on those previously used to test the cross-reactivity of the first generation Cortisol assay [26]. These tests, in part, followed the Clinical and Laboratory Standards Institute Evaluation Protocol (CLSI EP) 07-A2 Guideline [30].

Precision and accuracy

Assay precision was evaluated using quality control samples which were provided by Roche Diagnostics. These were the serum control samples Preci Control Universal 1 and 2 (PC U1 and PC U2). In addition, five lots of human sample pools (HSP 01–05) spiked with cortisol were used: HSP 01 was a saliva control and HSP 02–05 were serum controls. The concentration of the control samples were determined by Roche and were defined as the target concentration. All precision experiments were performed in compliance with the CLSI EP 05-A3 guideline [31]. A minimum of 30 specimens (same matrix as the precision samples) were included in every run between the precision determinations to simulate normal laboratory conditions.

Repeatability (intra-assay) and intermediate (inter-assay) precision experiments were performed in a four-fold determination on the same day over 21 days. The resulting mean concentration and the coefficients of variation (CVs,%) were calculated. Reproducibility was evaluated at each site with five human sample pools (HSP 01–05), two QC materials (PCU 1 and 2) on a five-fold determination over 5 days. This resulted in 175 analyses per site, using three different lots of reagent from all four sites.

The reference serum samples (HM 3/13A and HM 3/13B) from the RfB proficiency testing program were analyzed at each of the four European sites. The RfB runs a reference measurement procedure, which is done according to an International Organization for Standardization (ISO) standard by accredited laboratories only listed by the Joint Committee of Traceability in Laboratory Medicine (JCTLM), and is based on GC-MS. The assigned values for HM3/13A and HM3/13B were obtained by the RfB using this reference measurement procedure. The samples were tested in a three-fold determination on three separate days.

Method comparison

Leftover, anonymized serum or plasma and saliva samples from daily routine were used for the method comparison experiments. The Cortisol II assay was compared with the Cortisol assay (generation 1) on either the cobas e 411 or the cobas e 601 systems at the four study sites. The Cortisol II assay was also compared with in-house isotope dilution LC-MS/MS assays. The LC-MS/MS methods are summarized in Table 1. The general use of cortisol of LC-MS/MS in endocrine testing has also been published elsewhere [17].

Table 1:

LC-MS/MS methods used for method comparison at the three study sites.

GhentLeipzigMunich
InstrumentLC Shimadzu

MS ABSciex Triple Quad 5500
Serum: QTRAP® 6500

Saliva: QTRAP® 5500
Water Acquity UPLC

Waters Xevo TQ-S
Ionization modeAPCI+APCI+ESI+
Mass transitionCortisol: 363.2/121.1

Cortisol-d4: 367.2/121.1
Cortisol: 363/121

Cortisol-d4: 367/121
Cortisol 363/121

Cortisol-d3 366/121
Sample preparationLiquid/liquid extraction (serum and saliva)Protein precipitation

plus on-line SPE

Serum: 100 μL

Saliva: 50 μL
Protein precipitation plus on-line SPE

Sample volume: 100 μL
CalibratorsIn-houseSerum: MassChrom® Steroids (Chromsystems, Munich, Germany)

Saliva: in-house
In-house
QC samplesNIST Reference material 971

UK NEQAS for Steroid Hormones
Serum: MassChrom® Steroids (Chromsystems, Munich, Germany) and in-house pooled control

Saliva: in-house
Serum pools (in-house)

PC Saliva 1 (Roche)

Generic QCs (in-house)
Intermediate precision8%Serum: 2.9%–4.9%

Saliva: 4.5%–5.1%
Serum pools: 1.3%–4.9%

PC Saliva 1: 3.6% Generic QCs: 5.8%–6.3%
ReferenceFiers et al. [32]

Janssens et al. [33]
Serum: Ceglarek et al. [34], Gaudl et al. [35]

Saliva: Bae et al. [36]
Suhr et al. [37]
ValidationCLSI C57 [38] and C62-A [39] guidelinesAccording to the FDA guidance [40], EU guideline [41], and ICH guideline [42]Based on the EMA guideline [41]

ACPI, atmospheric pressure chemical ionization; ESI, electrospray ionization; NIST, National Institute of Standards and Technology; NEQAS, National External Quality Assessment Scheme; QC, quality control; SPE, solid phase extraction; TQ-S, tandem quadrupole mass spectrometer; UPLC, ultra performance liquid chromatography.

Reference range assessment

The reference range samples were collected from healthy individuals at three US sites between June and December 2014. The collection sites were Medical Research Center (Spartanburg, SC, USA), Premier Research (Austin, TX, USA), and Quest Research Institute (Bingham Farms, MI, USA). Testing was performed between October and December 2014 at the Washington University School of Medicine, Core Laboratory for Clinical Studies (St. Louis, MO, USA).

Key inclusion criteria were age ≥21 years, self-reported healthy status, no infectious disease or symptoms in the prior 8 weeks, and a minimum volume of 1 mL each of serum/plasma or saliva was required. Individuals were excluded if they were pregnant, lactating, using oral contraceptives, receiving medication with cortisone/cortisol or hormone replacement therapy, or unable to read or understand and sign the informed consent/assent form.

In total, a minimum of 120 evaluable individuals were required to be recruited in order to calculate the 5th to 95th/2.5th to 97.5th (serum/plasma) and the 95th/97.5th (saliva) percentiles. For determination of the Cortisol II assay reference ranges, the population was required to have a distribution of race/ethnicities of 40% African American/40% Caucasian/20% Hispanic, in a female to male ratio of approximately 1:1. Morning samples (n=150 minimum required) were collected between 6 and 10 a.m. independent of waking time. Afternoon samples (n=150 minimum required) were collected between 4 and 8 p.m. in order to have a closer view of circadian rhythm of cortisol, decreasing over the period a.m.-p.m.-midnight. Midnight saliva samples were allowed to be collected at 12 p.m.±30 min at a fixed time independent from the bedtime.

Data management and statistical analysis

The output from the Cortisol II assay was directly captured on the Windows-based computer-aided evaluation (WinCAEv), a Code of Federal Regulations (CFR) 21 Part 11 compliant electronic data capture software [43]. The WinCAEv software was run from a laptop attached to the cobas e 411 and cobas e 601 analyzers. Repeatability, intermediate precision, and reproducibility were calculated using the statistical programs SAS (version 9.3) and R (version 3.0.1, additive package VCA version 1.0.6), in compliance with the CLSI EP05-A3 Guideline [31].

An outlier analysis was performed on the precision samples at the end of the study: the outliers results were defined according to the recommendations of the CLSI EP05-A3 guidelines [31]. Based on these, it was permitted to reject at most two results due to an outlier, which was equal to one part in a 21-day study. This was applied to each precision sample and each 21-day experiment. The recovery of the target value for each sample based on the median of all determinations was calculated for accuracy experiments. Passing-Bablok regression analysis and Pearson correlation coefficients were used to assess the correlation of the method comparison studies in compliance with CLSI EP09-A3 [44].

The reference range analysis was performed using SAS software version 9.3 and R version 3.0.1. For serum and plasma samples, the 2.5th, 5th, 95th, and 95th percentiles were calculated. The 95th and 97th percentiles were reported for saliva samples. The limit of quantitation (LoQ) and the limit of detection (LoD) of the Cortisol II assay were calculated based on the CLSI EP17-A2 Guideline [45]. It is important that the LoD and LoQ were the same for both the serum/plasma and saliva samples: for standardization of the assay, both serum/plasma and saliva samples were used to ensure no matrix effect. The LoD and LoQ results of the assay were not recorded as part of the study.

Results

Cross-reactivity

To assess the specificity of the new Cortisol II assay vs. the first generation one, compounds with similar structures to cortisol were measured on both assays. The new Cortisol II assay monoclonal antibody had an improved cross-reactivity profile compared with the Cortisol assay (generation 1) for 6-α-methylprednisolone, dexamethasone, and prednisolone (Table 2). Cross-reactivity levels were higher with the second generation assay vs. the first in the measurement of cortisone and prednisone, but both were still very low (≤6.58%).

Table 2:

Cross-reactivity of the first and second generation cortisol assay antibodies.

SubstanceConcentration, mg/LaCortisol (generation 1)Cortisol II
Cross-reactivity,%Cross-reactivity,%
6-α-Methylprednisolone0.138912
Cortisone100.306.58
Dexamethasone100.08ND
Prednisolone0.11717.98
Prednisone100.282.23

aConcentration of substance added; ND, non-detectable.

Precision and accuracy

Across the four European sites, the repeatability CVs for the HSP 01–05 samples (excluding outliers) ranged from 0.8% to 4.4% (Table 3). For the PC U1 and PC U2 samples, the repeatability CVs were between 1.3% and 2.2%.

Table 3:

Precision results of the Cortisol II assay.

SampleTypenTarget conc., nmol/LaMean measured conc., nmol/LbRepeatability CV,%cIntermediate precision CV,%cReproducibility CV,%
HSP 01Saliva4148.438.442.8–4.45.0–10.911.4
HSP 02Serum41696.099.71.1–2.22.3–4.26.8
HSP 03Serum418489.04820.8–2.32.1–4.87.5
HSP 04Serum418982.09661.1–2.52.7–5.69.5
HSP 05Serum336d1605.016111.8–2.62.5–5.85.8
PC U1Serum4183043101.4–2.21.9–4.14.6
PC U2Serum4207347361.3–2.22.0–4.85.1

aThe target concentration was provided by the manufacturer; bmean concentration measured by the sites; crange of CVs observed across the four study sites; ddata from one site not included in this analysis due to samples initially being tested without dilution – retesting not possible due to stability date being exceeded. Conversion of units: μg/dL×27.59=nmol/L; nmol/L×0.0362=μg/dL.

The intermediate precision CVs for the HSP 02–05 serum samples ranged from 2.1% to 5.8%, but were higher for the saliva sample (HSP 01, 5.0%–10.9%). The intermediate precision CVs for the two PC control samples were between 1.9% and 4.8%. Reproducibility CVs across the four laboratories ranged from 5.8% to 9.5% for the HSP 02–05 serum samples, and 11.4% for the saliva HSP 01 sample. The reproducibility CVs were 4.6%–5.1% for the PC U1 and PC U2 control samples, respectively.

A test of the accuracy of the assay was performed by comparison with GC-MS, based on proficiency testing samples specified by the RfB. Deviation from the GC-MS specified target concentrations of the proficiency testing serum cortisol samples were ≤10% (Table 4). CVs for the Cortisol II assay with the HM3/13A and HM3/13B GC-MS reference serum samples were –4.5% and –3.7%, respectively.

Table 4:

Accuracy results of the Cortisol II assay.

RfB sampleSample typeTarget, nmol/LAccuracy per center range,%CV of accuracy results across all centers,%
HM 3/13ASerum667–8.3 to +1.4–0.9
HM 3/13BSerum365–5.9 to +0.1–0.7

Method comparisons

For the method comparison studies, the serum samples covered a measuring range of 1.7–1735.0 nmol/L and the saliva samples from 1.5 to 209.5 nmol/L. The Passing-Bablok regression analysis for the Cortisol II assay and the Cortisol assay (generation 1) with serum samples (n=541) resulted in a slope of 0.76 [95% confidence intervals (CI), 0.74–0.77] and an intercept of 10.3 nmol/L (95% CI, 6.09–15.53) (Figure 1A). The correlation coefficient was 0.968. Bland-Altman analysis showed a combined mean bias of –91.11 nmol/L (±2 SD, –238.91 to 56.69) across all sites (Figure 1B). For saliva samples (n=404), the slope agreement for Cortisol II vs. the Cortisol assay (generation 1) was 1.21 (95% CI, 1.17–1.25) and the intercept was –5.5 nmol/L (95% CI, –5.97 to –5.01), with a correlation coefficient of 0.992 (Figure 1C). The Bland-Altman difference plots of the saliva samples on the Cortisol II vs. Cortisol (generation 1) assays revealed a mean bias of –2.72 nmol/L (±2 SD, –9.94 to 4.51; Figure 1D).

Figure 1:
Figure 1:

Method comparison results and Bland-Altman plots of the Cortisol assay vs. the Cortisol II assay in serum or plasma samples (A) and (B), and saliva samples (C) and (D).

Citation: Clinical Chemistry and Laboratory Medicine (CCLM) 55, 6; 10.1515/cclm-2016-0400

The Cortisol II assay was also compared with LC-MS/MS, the current gold standard for cortisol measurement. For serum samples (n=405), the agreement between the Cortisol II assay and LC-MS/MS was high, with a slope of 1.02 (95% CI, 1.00–1.04), an intercept of 4.473 nmol/L (95% CI, 0.94–9.45), and a correlation coefficient of 0.9863 (Figure 2A). The mean bias for serum samples measured on the Cortisol II assay compared with LC-MS/MS was 14.55 nmol/L (±2 SD, –50.84 to 79.93; Figure 2B). The relationship between the Cortisol II assay vs. LC-MS/MS for saliva samples (n=253) was given with a slope of 1.134 (95% CI, 1.10–1.16) and an intercept of 0.825 nmol/L (95% CI, 0.67–1.06) and a correlation coefficient of 0.9930 (Figure 2C). For these comparisons, the mean bias was 2.56 nmol/L (±2 SD, –5.54 to 10.65; Figure 2D).

Figure 2:
Figure 2:

Method comparison results and Bland-Altman plots of the Cortisol II assay vs. LC-MS/MS in serum or plasma samples (A) and (B) and saliva samples (C) and (D).

Citation: Clinical Chemistry and Laboratory Medicine (CCLM) 55, 6; 10.1515/cclm-2016-0400

Reference ranges

To calculate the references ranges, a total of 894 saliva and 596 serum samples were collected from 300 self-reported healthy individuals and cortisol levels were measured using the Cortisol II assay (Table 5). Serum morning cortisol concentrations (5th–95th percentile) were 166.1–507 nmol/L and afternoon concentrations were 73.8–291 nmol/L. Morning, afternoon, and midnight saliva cortisol concentrations (95th percentile) were 20.3, 6.94, and 7.56 nmol/L, respectively. For midnight salivary cortisol, the median values were below the measuring range of the assay [LoD (3.0 nmol/L) and LoQ (1.5 nmol/L)] and the majority of individuals levels were below the LoD (81.6%) and the LoQ (61.5%). No statistically significant difference was observed between males and females for the serum or saliva samples.

Table 5:

Cortisol II assay reference range values from serum or plasma and saliva of healthy individuals.

nMale/female, nMedian, nmol/L5th–95th percentile, nmol/L2.5th–97.5th percentile, nmol/Ln (%) below LOQan (%) below LODa
Serum or plasma a.m.296144/152302.7166.1–507.3132.9–537.000
Serum or plasma p.m.300146/154160.673.8–291.568.2–327.000
95th percentile, nmol/L97.5th percentile, nmol/L
Saliva a.m.297144/1538.020.324.124 (8.1)5 (1.7)
Saliva p.m.298146/1522.76.949.65171 (57.4)75 (25.2)
Saliva midnight299146/153<MR7.5611.3244 (81.6)184 (61.5)

A.m., 06:00–10:00; p.m., 16:00–22:00; midnight, 00:00 ±30 min; MR, measuring range; athe lower limit of the reference range is defined by the LoQ (3.0 nmol/L) and by the LoD (1.5 nmol/L).

Discussion

We here report the results of a performance evaluation study of a novel serum and salivary cortisol assay and, since serum and saliva cortisol measurements are key in the work-up of suspected adrenocortical diseases, these results are of clinical significance. The particular strength of this study was its multi-center and multi-lot design that aimed to simulate the long-term diagnostic application of this test in endocrine care. A main finding was that the between-laboratory and between-production lot reproducibility was roughly 10% in the typical concentration range of salivary cortisol and roughly 6%–10% for serum cortisol. According to the concept of critical difference, this allows to distinguish a true cortisol concentration of a diagnostic sample of 11.3 nmol/L (upper limit of the normal range observed in this study for midnight salivary cortisol) from 14.7 nmol/L with >95% probability.

Despite being the gold standard, LC-MS/MS is still quite demanding and the complexity of the entire system makes routine measurement a very substantial challenge and it is still limited to very few laboratories [16], [17], [18], [19], [20], [21]. An immunoassay offers a simpler approach in contrast. Compared to the first generation Cortisol assay, we observed improved agreement of results with LC-MS/MS, which currently represents the gold standard for cortisol measurement in the clinical laboratory. This improvement may be due to both the application of a more specific monoclonal antibody and the specification of calibrator concentrations which were now based on a GC-MS reference method [28]. Close agreement with LC-MS/MS was observed for a large series of serum and saliva samples, as well as good recovery of the RfB proficiency testing samples, demonstrated high accuracy of the study’s assay.

Daytime serum and saliva cortisol concentrations were studied in a large cohort of self-reported healthy adults to address the need for assay-specific cut-offs. The cut-offs varied depending on the time of day and whether serum or saliva was measured (Table 5). Of note, the majority of the saliva midnight samples were below the LoQ (3.0 nmol/L) and the LoD (1.5 nmol/L) of the assay compared to the morning and afternoon samples. However, the LoD and LoQ of the assay was not investigated as part of this study and was the manufacturer’s in-house data. It is important to state that the daytime data should not be used in a diagnostic approach in patients with suspected Cushing’s syndrome. These patients typically have normal serum cortisol concentration in the morning and the most important method to exclude Cushing’s syndrome is the dexamethasone suppression test. Alternatively, late-night cortisol secretion can be tested to exclude autonomous cortisol secretion in Cushing’s syndrome due to excess adrenocorticotropic hormone synthesis by hypophyseal adenoma or extra-hypophyseal tumors as a paraneoplastic syndrome or excess cortisol secretion by adrenal adenomas. In this respect, the midnight salivary cortisol concentrations observed with the Cortisol II assay in the 299 healthy individuals are of substantial relevance in the context of this study. In this population, the 97.5th percentile was 11.3 nmol/L. This is slightly higher than reported for the first generation Cortisol assay (8.9 nmol/L) [25]. However, it is still recommended that each laboratory should determine and apply their own assay specific cut-offs in the diagnostic work-up of adrenocortical diseases when salivary cortisol is used since no conclusive system of between assay standardization of this emerging analyte in endocrine care has been achieved so far [46]. This is in contrast to serum cortisol measurement, which has a rather close between-assay agreement, as was observed in proficiency testing programs [47].

In summary, for the Cortisol II assay, we observed a degree of between-laboratory and between-production lot reproducibility and agreement with several assays of higher metrological order that we consider compatible with the diagnostic use of this assay. The Cortisol II assay will be beneficial to endocrinologists in assessing patients with adreno-cortisol disorders.

Acknowledgments

The authors would like to thank all of the patients, technicians, and support staff at their respective institutions for participating in the study. The following contributors performed the reference range sample collection: Charles M. Fogarty of Spartanburg Medical Research, Spartanburg, SC, USA; Steven Daniels of Premier Research International, Austin, TX, USA; and Eric Knipple of Quest Research Institute, Bingham Farms, MI, USA. The sample collection was supported by the Contract Research Organization Aptiv Solutions/ICON plc, US. Medical writing support was provided by Kim Brown of Roche Diagnostics International Ltd, Rotkreuz, Switzerland.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: This study was sponsored by Roche Diagnostics GmbH, Germany. All investigator sites received research funding from Roche Diagnostics. MV received travel expenses from Roche as part of a speaker’s agreement.

Employment or leadership: C.P.K. (Study Management) and H.K. (Biostastistics) are both employees of Roche Diagnostics, Germany.

Honorarium: None declared.

Competing interests: All investigator sites received research funding from Roche Diagnostics. M.V. received travel expenses from Roche as part of a speaker’s agreement. J.K., Y.J.B., M.B., U.G., T.F., A.G., C.M., A.C.S., D.T., A.C.S., I.Z., and R.O. have no other conflicts of interest to declare.

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  • 1.

    Lindholm J, Juul S, Jorgensen JO, Astrup J, Bjerre P, Feldt-Rasmussen U, et al. Incidence and late prognosis of cushing’s syndrome: a population-based study. J Clin Endocrinol Metab 2001;86:117–23.

    • PubMed
    • Export Citation
  • 2.

    Kong MF, Jeffcoate W. Eighty-six cases of Addison’s disease. Clin Endocrinol (Oxf) 1994;41:757–61.

    • Crossref
    • PubMed
    • Export Citation
  • 3.

    Laureti S, Vecchi L, Santeusanio F, Falorni A. Is the prevalence of Addison’s disease underestimated? J Clin Endocrinol Metab 1999;84:1762.

    • PubMed
    • Export Citation
  • 4.

    Oelkers W. Clinical diagnosis of hyper- and hypocortisolism. Noise Health 2000;2:39–48.

    • PubMed
    • Export Citation
  • 5.

    Gatti R, Antonelli G, Prearo M, Spinella P, Cappellin E, De Palo EF. Cortisol assays and diagnostic laboratory procedures in human biological fluids. Clin Biochem 2009;42:1205–17.

    • Crossref
    • PubMed
    • Export Citation
  • 6.

    Inder WJ, Dimeski G, Russell A. Measurement of salivary cortisol in 2012 – laboratory techniques and clinical indications. Clin Endocrinol (Oxf) 2012;77:645–51.

    • Crossref
    • PubMed
    • Export Citation
  • 7.

    Raff H. Utility of salivary cortisol measurements in Cushing’s syndrome and adrenal insufficiency. J Clin Endocrinol Metab 2009;94:3647–55.

    • Crossref
    • PubMed
    • Export Citation
  • 8.

    Poll EM, Kreitschmann-Andermahr I, Langejuergen Y, Stanzel S, Gilsbach JM, Gressner A, et al. Saliva collection method affects predictability of serum cortisol. Clin Chim Acta 2007;382:15–9.

    • Crossref
    • PubMed
    • Export Citation
  • 9.

    Vining RF, McGinley RA, Maksvytis JJ, Ho KY. Salivary cortisol: a better measure of adrenal cortical function than serum cortisol. Ann Clin Biochem 1983;20(Pt 6):329–35.

    • Crossref
    • PubMed
    • Export Citation
  • 10.

    Papanicolaou DA, Mullen N, Kyrou I, Nieman LK. Nighttime salivary cortisol: a useful test for the diagnosis of Cushing’s syndrome. J Clin Endocrinol Metab 2002;87:4515–21.

    • Crossref
    • Export Citation
  • 11.

    Yaneva M, Mosnier-Pudar H, Dugue MA, Grabar S, Fulla Y, Bertagna X. Midnight salivary cortisol for the initial diagnosis of Cushing’s syndrome of various causes. J Clin Endocrinol Metab 2004;89:3345–51.

    • Crossref
    • PubMed
    • Export Citation
  • 12.

    Briegel J, Sprung CL, Annane D, Singer M, Keh D, Moreno R, et al. Multicenter comparison of cortisol as measured by different methods in samples of patients with septic shock. Intensive Care Med 2009;35:2151–6.

    • Crossref
    • PubMed
    • Export Citation
  • 13.

    Klee GG. Interferences in hormone immunoassays. Clin Lab Med 2004;24:1–18.

    • Crossref
    • PubMed
    • Export Citation
  • 14.

    Krasowski MD, Drees D, Morris CS, Maakestad J, Blau JL, Ekins S. Cross-reactivity of steroid hormone immunoassays: clinical significance and two-dimensional molecular similarity prediction. BMC Clin Pathol 2014;14:33.

    • Crossref
    • PubMed
    • Export Citation
  • 15.

    El-Farhan N, Pickett A, Ducroq D, Bailey C, Mitchem K, Morgan N, et al. Method-specific serum cortisol responses to the adrenocorticotrophin test: comparison of gas chromatography-mass spectrometry and five automated immunoassays. Clin Endocrinol (Oxf) 2013;78:673–80.

    • Crossref
    • PubMed
    • Export Citation
  • 16.

    Leung KS, Fong BM. LC-MS/MS in the routine clinical laboratory: has its time come? Anal Bioanal Chem 2014;406:2289–301.

    • Crossref
    • PubMed
    • Export Citation
  • 17.

    Grebe SK, Singh RJ. LC-MS/MS in the clinical laboratory – where to from here? Clin Biochem Rev 2011;32:5–31.

    • PubMed
    • Export Citation
  • 18.

    Vogeser M, Seger C. Mass spectrometry methods in clinical diagnostics – state of the art and perspectives. Trends Analyt Chem 2016;84:1–4.

    • Crossref
    • Export Citation
  • 19.

    Annesley TM, Cooks RG, Herold DA, Hoofnagle AN. Clinical mass spectrometry-achieving prominence in laboratory medicine. Clin Chem 2016;62:1–3.

    • Crossref
    • PubMed
    • Export Citation
  • 20.

    Annesley T, Diamandis E, Bachmann L, Hanash S, Hart B, Javahery R, et al. A spectrum of views on clinical mass spectrometry. Clin Chem 2016;62:30–6.

    • Crossref
    • PubMed
    • Export Citation
  • 21.

    Reference Institute for Bioanalytics (RfB) – External Quality Assessment (EQA) Survey. www.rfb.bio.

  • 22.

    Turpeinen U, Hamalainen E. Determination of cortisol in serum, saliva and urine. Best Pract Res Clin Endocrinol Metab 2013;27:795–801.

    • Crossref
    • PubMed
    • Export Citation
  • 23.

    Roche Diagnostics. Cortisol: Method Sheet. https://pim-eservices.roche.com 2010. Accessed: 16 July 2015.

  • 24.

    Chiu SK, Collier CP, Clark AF, Wynn-Edwards KE. Salivary cortisol on ROCHE Elecsys immunoassay system: pilot biological variation studies. Clin Biochem 2003;36:211–4.

    • Crossref
    • PubMed
    • Export Citation
  • 25.

    Vogeser M, Durner J, Seliger E, Auernhammer C. Measurement of late-night salivary cortisol with an automated immunoassay system. Clin Chem Lab Med 2006;44:1441–5.

    • PubMed
    • Export Citation
  • 26.

    van Aken MO, Romijn JA, Miltenburg JA, Lentjes EG. Automated measurement of salivary cortisol. Clin Chem 2003;49:1408–9.

    • Crossref
    • PubMed
    • Export Citation
  • 27.

    Roche Diagnostics. Cortisol II: Method Sheet. https://pim-eservices.roche.com 2015. Accessed: 16 July 2015.

  • 28.

    Thienpont LM, De Leenheer AP, Siekmann L, Kristiansen N, Linsinger T, Schimmel H, et al. The characterisation of cortisol concentrations in a serum reference panel (IRMM/IFCC No. 451). EUR 19007 EN. IRMM/IFCC Information, Reference Materials. Brussels, Belgium. 1999.

  • 29.

    Garde AH, Hansen AM. Long-term stability of salivary cortisol. Scand J Clin Lab Invest 2005;65:433–6.

    • Crossref
    • PubMed
    • Export Citation
  • 30.

    Clinical and Laboratory Standards Institute. Interference testing in clinical chemistry; approved guideline – second edition. EP07-A2 2005;25:27.

  • 31.

    Clinical and Laboratory Standards Institute. Evaluation of precision of quantitative measurement methods: approved guideline – third edition. EP05-A3 2014;34:13.

  • 32.

    Fiers T, Delanghe J, T’Sjoen G, Van Caenegem E, Wierckx K, Kaufman JM. A critical evaluation of salivary testosterone as a method for the assessment of serum testosterone. Steroids 2014;86:5–9.

    • Crossref
    • PubMed
    • Export Citation
  • 33.

    Janssens H, Clays E, Fiers T, Verstraete AG, de Bacquer D, Braeckman L. Hair cortisol in relation to job stress and depressive symptoms. Occup Med (Lond). 2016; pii: kqw114. [Epub ahead of print].

  • 34.

    Ceglarek U, Kortz L, Leichtle A, Fiedler GM, Kratzsch J, Thiery J. Rapid quantification of steroid patterns in human serum by on-line solid phase extraction combined with liquid chromatography-triple quadrupole linear ion trap mass spectrometry. Clin Chim Acta 2009;401:114–8.

    • Crossref
    • PubMed
    • Export Citation
  • 35.

    Gaudl A, Kratzsch J, Bae YJ, Kiess W, Thiery J, Ceglarek U. Liquid chromatography quadrupole linear ion trap mass spectrometry for quantitative steroid hormone analysis in plasma, urine, saliva and hair. J Chrom A 2016;1464:64–71.

    • Crossref
    • Export Citation
  • 36.

    Ju Bae Y, Gaudl A, Jaeger S, Stadelmann S, Hiemisch A, Kiess W, et al. Immunoassay or LC-MS/MS for the measurement of salivary cortisol in children? Clin Chem Lab Med 2016;54:811–22.

    • PubMed
    • Export Citation
  • 37.

    Suhr AC, Vogeser M, Grimm SH. Isotope inversion experiment evaluating the suitability of calibration in surrogate matrix for quantification via LC-MS/MS-Exemplary application for a steroid multi-method. J Pharm Biomed Anal 2016;124:309–18.

    • Crossref
    • PubMed
    • Export Citation
  • 38.

    Clinical and Laboratory Standards Institute. Mass spectrometry for androgen and estrogen measurements in serum, 1st ed. C57 2015;35:4.

  • 39.

    Clinical and Laboratory Standards Institute. Liquid chromatography-mass spectrometry methods; Approved Guideline. C62-A 2014;34:16.

  • 40.

    U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine. Guidance for Industry: Bioanalytical Method Evaluation. Available at: http://www.birminghamquality.org.uk/%5b1%5dSteroidAnnualReview_2011.pdf. Accessed: 23 June 2015.

  • 41.

    Official Journal of the European Communities. Commission Decision of 12 August 2002; Implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Available at: http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32002D0657&from=EN. Accessed: 23 June 2015.

  • 42.

    ICH harmonized tripartite guideline validation of analytical procedures: Text and methodology Q2(R1). Available at: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf. Accessed: 20 Aug 2015.

  • 43.

    Kunst A, Busse Grawitz A, Engeldinger W, Koch W, Luthe H, Stockmann W. Wincaev – a new program supporting evaluations of reagents and analysers. Clin Chim Acta 2005;355:S361.

  • 44.

    Clinical and Laboratory Standards Institute. Measurement procedure comparison and bias estimation using patient samples; approved guideline – third edition. EP09-A3 2013 33:11.

  • 45.

    Clinical and Laboratory Standards Institute. Evaluation of detection capability for clinical laboratory measurement procedures; approved guideline – second edition. EP17-A2 2012;32:8.

  • 46.

    Guignat L, Bertherat J. The diagnosis of Cushing’s syndrome: an Endocrine Society Clinical Practice Guideline: commentary from a European perspective. Eur J Endocrinol 2010;163:9–13.

    • Crossref
    • Export Citation
  • 47.

    UK NEQAS for Steroid Hormones. Review for UK NEQAS Clinical Chemistry Specialist Advisory Group for Endocrinology and Immunoassay 2011. Available at: http://www.birminghamquality.org.uk/%5b1%5dSteroidAnnualReview_2011.pdf. Accessed: 26 Apr 2016.

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1.

    Lindholm J, Juul S, Jorgensen JO, Astrup J, Bjerre P, Feldt-Rasmussen U, et al. Incidence and late prognosis of cushing’s syndrome: a population-based study. J Clin Endocrinol Metab 2001;86:117–23.

    • PubMed
    • Export Citation
  • 2.

    Kong MF, Jeffcoate W. Eighty-six cases of Addison’s disease. Clin Endocrinol (Oxf) 1994;41:757–61.

    • Crossref
    • PubMed
    • Export Citation
  • 3.

    Laureti S, Vecchi L, Santeusanio F, Falorni A. Is the prevalence of Addison’s disease underestimated? J Clin Endocrinol Metab 1999;84:1762.

    • PubMed
    • Export Citation
  • 4.

    Oelkers W. Clinical diagnosis of hyper- and hypocortisolism. Noise Health 2000;2:39–48.

    • PubMed
    • Export Citation
  • 5.

    Gatti R, Antonelli G, Prearo M, Spinella P, Cappellin E, De Palo EF. Cortisol assays and diagnostic laboratory procedures in human biological fluids. Clin Biochem 2009;42:1205–17.

    • Crossref
    • PubMed
    • Export Citation
  • 6.

    Inder WJ, Dimeski G, Russell A. Measurement of salivary cortisol in 2012 – laboratory techniques and clinical indications. Clin Endocrinol (Oxf) 2012;77:645–51.

    • Crossref
    • PubMed
    • Export Citation
  • 7.

    Raff H. Utility of salivary cortisol measurements in Cushing’s syndrome and adrenal insufficiency. J Clin Endocrinol Metab 2009;94:3647–55.

    • Crossref
    • PubMed
    • Export Citation
  • 8.

    Poll EM, Kreitschmann-Andermahr I, Langejuergen Y, Stanzel S, Gilsbach JM, Gressner A, et al. Saliva collection method affects predictability of serum cortisol. Clin Chim Acta 2007;382:15–9.

    • Crossref
    • PubMed
    • Export Citation
  • 9.

    Vining RF, McGinley RA, Maksvytis JJ, Ho KY. Salivary cortisol: a better measure of adrenal cortical function than serum cortisol. Ann Clin Biochem 1983;20(Pt 6):329–35.

    • Crossref
    • PubMed
    • Export Citation
  • 10.

    Papanicolaou DA, Mullen N, Kyrou I, Nieman LK. Nighttime salivary cortisol: a useful test for the diagnosis of Cushing’s syndrome. J Clin Endocrinol Metab 2002;87:4515–21.

    • Crossref
    • Export Citation
  • 11.

    Yaneva M, Mosnier-Pudar H, Dugue MA, Grabar S, Fulla Y, Bertagna X. Midnight salivary cortisol for the initial diagnosis of Cushing’s syndrome of various causes. J Clin Endocrinol Metab 2004;89:3345–51.

    • Crossref
    • PubMed
    • Export Citation
  • 12.

    Briegel J, Sprung CL, Annane D, Singer M, Keh D, Moreno R, et al. Multicenter comparison of cortisol as measured by different methods in samples of patients with septic shock. Intensive Care Med 2009;35:2151–6.

    • Crossref
    • PubMed
    • Export Citation
  • 13.

    Klee GG. Interferences in hormone immunoassays. Clin Lab Med 2004;24:1–18.

    • Crossref
    • PubMed
    • Export Citation
  • 14.

    Krasowski MD, Drees D, Morris CS, Maakestad J, Blau JL, Ekins S. Cross-reactivity of steroid hormone immunoassays: clinical significance and two-dimensional molecular similarity prediction. BMC Clin Pathol 2014;14:33.

    • Crossref
    • PubMed
    • Export Citation
  • 15.

    El-Farhan N, Pickett A, Ducroq D, Bailey C, Mitchem K, Morgan N, et al. Method-specific serum cortisol responses to the adrenocorticotrophin test: comparison of gas chromatography-mass spectrometry and five automated immunoassays. Clin Endocrinol (Oxf) 2013;78:673–80.

    • Crossref
    • PubMed
    • Export Citation
  • 16.

    Leung KS, Fong BM. LC-MS/MS in the routine clinical laboratory: has its time come? Anal Bioanal Chem 2014;406:2289–301.

    • Crossref
    • PubMed
    • Export Citation
  • 17.

    Grebe SK, Singh RJ. LC-MS/MS in the clinical laboratory – where to from here? Clin Biochem Rev 2011;32:5–31.

    • PubMed
    • Export Citation
  • 18.

    Vogeser M, Seger C. Mass spectrometry methods in clinical diagnostics – state of the art and perspectives. Trends Analyt Chem 2016;84:1–4.

    • Crossref
    • Export Citation
  • 19.

    Annesley TM, Cooks RG, Herold DA, Hoofnagle AN. Clinical mass spectrometry-achieving prominence in laboratory medicine. Clin Chem 2016;62:1–3.

    • Crossref
    • PubMed
    • Export Citation
  • 20.

    Annesley T, Diamandis E, Bachmann L, Hanash S, Hart B, Javahery R, et al. A spectrum of views on clinical mass spectrometry. Clin Chem 2016;62:30–6.

    • Crossref
    • PubMed
    • Export Citation
  • 21.

    Reference Institute for Bioanalytics (RfB) – External Quality Assessment (EQA) Survey. www.rfb.bio.

  • 22.

    Turpeinen U, Hamalainen E. Determination of cortisol in serum, saliva and urine. Best Pract Res Clin Endocrinol Metab 2013;27:795–801.

    • Crossref
    • PubMed
    • Export Citation
  • 23.

    Roche Diagnostics. Cortisol: Method Sheet. https://pim-eservices.roche.com 2010. Accessed: 16 July 2015.

  • 24.

    Chiu SK, Collier CP, Clark AF, Wynn-Edwards KE. Salivary cortisol on ROCHE Elecsys immunoassay system: pilot biological variation studies. Clin Biochem 2003;36:211–4.

    • Crossref
    • PubMed
    • Export Citation
  • 25.

    Vogeser M, Durner J, Seliger E, Auernhammer C. Measurement of late-night salivary cortisol with an automated immunoassay system. Clin Chem Lab Med 2006;44:1441–5.

    • PubMed
    • Export Citation
  • 26.

    van Aken MO, Romijn JA, Miltenburg JA, Lentjes EG. Automated measurement of salivary cortisol. Clin Chem 2003;49:1408–9.

    • Crossref
    • PubMed
    • Export Citation
  • 27.

    Roche Diagnostics. Cortisol II: Method Sheet. https://pim-eservices.roche.com 2015. Accessed: 16 July 2015.

  • 28.

    Thienpont LM, De Leenheer AP, Siekmann L, Kristiansen N, Linsinger T, Schimmel H, et al. The characterisation of cortisol concentrations in a serum reference panel (IRMM/IFCC No. 451). EUR 19007 EN. IRMM/IFCC Information, Reference Materials. Brussels, Belgium. 1999.

  • 29.

    Garde AH, Hansen AM. Long-term stability of salivary cortisol. Scand J Clin Lab Invest 2005;65:433–6.

    • Crossref
    • PubMed
    • Export Citation
  • 30.

    Clinical and Laboratory Standards Institute. Interference testing in clinical chemistry; approved guideline – second edition. EP07-A2 2005;25:27.

  • 31.

    Clinical and Laboratory Standards Institute. Evaluation of precision of quantitative measurement methods: approved guideline – third edition. EP05-A3 2014;34:13.

  • 32.

    Fiers T, Delanghe J, T’Sjoen G, Van Caenegem E, Wierckx K, Kaufman JM. A critical evaluation of salivary testosterone as a method for the assessment of serum testosterone. Steroids 2014;86:5–9.

    • Crossref
    • PubMed
    • Export Citation
  • 33.

    Janssens H, Clays E, Fiers T, Verstraete AG, de Bacquer D, Braeckman L. Hair cortisol in relation to job stress and depressive symptoms. Occup Med (Lond). 2016; pii: kqw114. [Epub ahead of print].

  • 34.

    Ceglarek U, Kortz L, Leichtle A, Fiedler GM, Kratzsch J, Thiery J. Rapid quantification of steroid patterns in human serum by on-line solid phase extraction combined with liquid chromatography-triple quadrupole linear ion trap mass spectrometry. Clin Chim Acta 2009;401:114–8.

    • Crossref
    • PubMed
    • Export Citation
  • 35.

    Gaudl A, Kratzsch J, Bae YJ, Kiess W, Thiery J, Ceglarek U. Liquid chromatography quadrupole linear ion trap mass spectrometry for quantitative steroid hormone analysis in plasma, urine, saliva and hair. J Chrom A 2016;1464:64–71.

    • Crossref
    • Export Citation
  • 36.

    Ju Bae Y, Gaudl A, Jaeger S, Stadelmann S, Hiemisch A, Kiess W, et al. Immunoassay or LC-MS/MS for the measurement of salivary cortisol in children? Clin Chem Lab Med 2016;54:811–22.

    • PubMed
    • Export Citation
  • 37.

    Suhr AC, Vogeser M, Grimm SH. Isotope inversion experiment evaluating the suitability of calibration in surrogate matrix for quantification via LC-MS/MS-Exemplary application for a steroid multi-method. J Pharm Biomed Anal 2016;124:309–18.

    • Crossref
    • PubMed
    • Export Citation
  • 38.

    Clinical and Laboratory Standards Institute. Mass spectrometry for androgen and estrogen measurements in serum, 1st ed. C57 2015;35:4.

  • 39.

    Clinical and Laboratory Standards Institute. Liquid chromatography-mass spectrometry methods; Approved Guideline. C62-A 2014;34:16.

  • 40.

    U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine. Guidance for Industry: Bioanalytical Method Evaluation. Available at: http://www.birminghamquality.org.uk/%5b1%5dSteroidAnnualReview_2011.pdf. Accessed: 23 June 2015.

  • 41.

    Official Journal of the European Communities. Commission Decision of 12 August 2002; Implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Available at: http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32002D0657&from=EN. Accessed: 23 June 2015.

  • 42.

    ICH harmonized tripartite guideline validation of analytical procedures: Text and methodology Q2(R1). Available at: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf. Accessed: 20 Aug 2015.

  • 43.

    Kunst A, Busse Grawitz A, Engeldinger W, Koch W, Luthe H, Stockmann W. Wincaev – a new program supporting evaluations of reagents and analysers. Clin Chim Acta 2005;355:S361.

  • 44.

    Clinical and Laboratory Standards Institute. Measurement procedure comparison and bias estimation using patient samples; approved guideline – third edition. EP09-A3 2013 33:11.

  • 45.

    Clinical and Laboratory Standards Institute. Evaluation of detection capability for clinical laboratory measurement procedures; approved guideline – second edition. EP17-A2 2012;32:8.

  • 46.

    Guignat L, Bertherat J. The diagnosis of Cushing’s syndrome: an Endocrine Society Clinical Practice Guideline: commentary from a European perspective. Eur J Endocrinol 2010;163:9–13.

    • Crossref
    • Export Citation
  • 47.

    UK NEQAS for Steroid Hormones. Review for UK NEQAS Clinical Chemistry Specialist Advisory Group for Endocrinology and Immunoassay 2011. Available at: http://www.birminghamquality.org.uk/%5b1%5dSteroidAnnualReview_2011.pdf. Accessed: 26 Apr 2016.

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Clinical Chemistry and Laboratory Medicine ( CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. CCLM welcomes contributions on the progress in fundamental and applied research and cutting-edge clinical laboratory medicine. It is one of the leading journals in the field, with an impact factor of over three. CCLM is the official journal of nine national clinical societies and associated with EFLM.

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  • View in gallery

    Method comparison results and Bland-Altman plots of the Cortisol assay vs. the Cortisol II assay in serum or plasma samples (A) and (B), and saliva samples (C) and (D).

  • View in gallery

    Method comparison results and Bland-Altman plots of the Cortisol II assay vs. LC-MS/MS in serum or plasma samples (A) and (B) and saliva samples (C) and (D).