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Scandinavian Journal of Clinical and Laboratory Investigation


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Sources of biological and methodological variation in salivary cortisol and their impact on measurement among healthy adults: A review
se Marie Hansen a; Anne Helene Garde a; Roger Persson a a National Research Centre for the Working Environment, Copenhagen, Denmark First Published:2008

To cite this Article Hansen, se Marie, Garde, Anne Helene and Persson, Roger(2008)'Sources of biological and methodological

variation in salivary cortisol and their impact on measurement among healthy adults: A review',Scandinavian Journal of Clinical and Laboratory Investigation,68:6,448 458
To link to this Article: DOI: 10.1080/00365510701819127 URL: http://dx.doi.org/10.1080/00365510701819127

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The Scandinavian Journal of Clinical & Laboratory Investigation, Vol. 68, No. 6, October 2008, 448458

REVIEW ARTICLE

Sources of biological and methodological variation in salivary cortisol and their impact on measurement among healthy adults: A review
se Marie Hansen, Anne Helene Garde and Roger Persson A
National Research Centre for the Working Environment, Copenhagen, Denmark Salivary cortisol is often used in occupational field studies when measuring stress reactions. For purposes of precision and accuracy in measurement, and interpretation of results, it is crucial to know the sources of variability that exert systematic influence on sampling. Variability can be both biological and methodological in origin, and failure to identify its sources may induce erroneous interpretations of Type I and Type II. This review aims to increase our knowledge and provide an overview of the biological and methodological variations of relevance for field measurements of salivary cortisol. It is concluded that: (i) time of sampling has to be carefully registered and included in the statistical analysis; (ii) samples have to be collected at the same time of year in longitudinal designs; (iii) food intake has to be avoided in at least the 2 h before sampling; (iv) vigorous exercise has to be avoided in at least the 2 h, preferably longer, before saliva is collected for measurement of cortisol; (v) variation in results obtained by different laboratory techniques emphasizes use of the same, or otherwise made comparable, laboratory techniques; (vi) concentration of cortisol is dependent on the material of the tampon; (vii) despite the absence of hard evidence, it is recommended that information be collected and results possibly statistically controlled for alcohol consumption, medication, such as oral contraceptives, and treatment for mental diseases; (viii) saliva samples can be stored at 220 C for at least 1 year; (ix) cross-comparisons of absolute concentrations across studies might be difficult and therefore the establishment of reference intervals for the population studied and method used is recommended. Keywords: Cortisol; field studies; reference intervals; saliva; sampling; storage

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Introduction Throughout medical history, measurement of hormones and other physiological parameters have been used in clinical settings with the purpose of detecting and monitoring progress of disease. During the past three or four decades, however, hormones and other physiological effect markers have been increasingly used in occupational settings for purposes of assessing the effects of psychosocial circumstances and occupational stress (for reviews see [14]). Accordingly, the focus has partly drifted from detecting and monitoring disease to including detecting and monitoring precursors of disease and risk factors for poor health in otherwise healthy subjects before medical manifests. Because the majority of the workforce is in good health, differences in hormonal and other physiological parameters are often expected to be less pronounced than the differences that typically render clinical interest. This has led to an increased need for knowledge concerning the many non-work-related factors that potentially obscure the interpretation of results by affecting

hormone concentrations. Unless biological and methodological variation is taken into proper consideration when designing a study, the variability may randomly or systematically affect subsequent analysis and the interpretation of results. To increase general understanding, and to facilitate reflections and interpretations concerning biological variations, we attempt briefly to summarize the existing knowledge in a systematic but, in a sense, personalized form. Our focus, however, is limited to salivary cortisol, which nowadays is perhaps the most widely employed stress biomarker in occupational health research. It is also a method and hormone which we have worked with almost on a daily basis during the past decade. Salivary cortisol Known cortisol-related diseases, or affective conditions, include Cushings syndrome [5], depression and chronic fatigue syndrome [6,7]. However, salivary cortisol has also increasingly been used in study of the responsiveness of the hypothalamic pituitary adrenal

se Marie Hansen, National Research Centre for the Working Environment, DK-2100 Copenhagen , Denmark. Tel: +45 39 16 52 55. *Correspondence author. A Fax: +45 39 16 52 01. Email: aamh@nrcwe.dk (Received 9 November 2007; accepted 20 November 2007) ISSN 0036-5513 print/ISSN 1502-7686 online # 2008 Informa UK Ltd (Informa Healthcare, Taylor & Francis AS). DOI: 10.1080/00365510701819127 http://www.informaworld.com

Analytical and biological variation (HPA) axis in occupational stress studies and employed in both field studies and experimental studies [812]. The reason for the increasing spread of salivary cortisol in occupational settings is that it is a simple, non-invasive, harm-free and pain-free measure that allows the longitudinal study of HPA-axis activity without substantial interference with the subjects normal habits and environment. When carrying out our ordinary and daily work routine, changes in hormone concentrations and physiological measures reflect our adaptive efforts to the work environment. To some degree, this means that the measures obtained will reflect environmental exposure at work (e.g. physical activity level, heat, cold and sound) as well as how we perceive the work task and our relationships with work colleagues, management and others that we encounter at work. But the measures obtained will also reflect normal cyclic biological variations (e.g. diurnal and seasonal variations), effects of lifestyle factors, as well as performance of the selected analytical methods and errors. The magnitude of variations, however, can be estimated, statistically modelled and attributed to variations within the individual (intra-individual variation) as well as between individuals (interindividual variation) [1318]. Measurement of salivary cortisol has been found to be an excellent indicator of unbound concentrations of cortisol in serum [1921]. Studies equally shows that the correlation between mean salivary cortisol and mean serum cortisol is approximately r50.6, with a mean cortisol concentration in serum 1020 times higher than measured in saliva. Furthermore, a close correspondence in circadian fluctuations has been reported for cortisol in saliva and plasma [22]. However, it is not just the total concentrations of cortisol that have rendered interest; a number of derived measures thought to better describe the dynamics of the stress response have been invented and put to use. The two most common examples are the awakening response (ACR) and recovery. The ACR is sometimes called reactivity, and recovery is sometimes referred to as fall-during-theday. The ACR is typically defined as the difference in concentration of cortisol between the first and second saliva samples in the morning, i.e. the difference between concentrations at awakening and concentrations obtained 15 to 60 minutes later. Recovery is typically defined as the difference between the highest concentration of cortisol in morning samples and an evening sample. Another derivate measure is the area under the curve, which is used as a proxy for the total concentration during a predefined time period. As with any measurement, measurement of salivary cortisol includes variation that comes from the performance of the analytical method. In the assessment of psycho-physiological effects related to

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the working environment, the quality of an analytical method must be described clearly and in sufficient detail for other laboratories to be able to repeat the measurements; the method should also be evaluated for precision, accuracy, linearity and robustness [23]. Documentation of statistical and analytical control of measurement results must be included by internal quality control (e.g. use of control charts) [24] and, finally, analysis of certified reference materials and external quality control are recommended (e.g. participation in interlaboratory comparisons) [25]. It is further recommended that the measured results are reported together with an estimated uncertainty so that the user can evaluate the reliability of the results [23]. One way of securing the quality of an analytical method is by accreditation, which is granted to laboratories that can demonstrate technical competence, impartiality and the existence of a welldocumented quality system. Because salivary cortisol is used increasingly as a supplement to self-reports assessing occupational stress, there are many good reasons for scrutinizing possibilities and constraints of its use in field studies (for review see [26]). An underlying rationale for the present review is that failure to identify sources of variability may lead to observed changes being erroneously interpreted as effects (Type I error, or false positives) or as absence of effects (Type II error, or false negatives). Hence, to be able to attribute salivary cortisol correctly as an effect of the work environment (physical and psychosocial), it is necessary to know about normal biological variation (e.g. diurnal variation, within-subject variation and between-subject variation) as well as methodological variation (laboratory techniques, material specifics, etc.). The purpose of the present paper is to provide a literary review of current knowledge on these sources of variation in relation to the use of saliva in occupational field studies. Specifically, the present review addresses the impact of:

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Biological variation that is theoretically independent of individual choices (i.e. diurnal salivary cortisol profiles, within-subject and betweensubject variation, seasonal variation and effects on age and gender). Biological variation that is theoretically dependent on individual choices (i.e. lifestyle choices, diet, medication, smoking, alcohol and physical activity). Methodological variation relating to sampling and storage procedures (i.e. cotton versus polyester tampons and freezing of samples at different temperatures).

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. M. Hansen et al. A healthy subjects carrying out their routine work. Recovery (decrease from 20 min after awakening to 1800 h) was estimated to be 80 % (CI: 51; 109 %), while reactivity (increase from awakening to 20 min after awakening) was estimated to be 82 % (CI: 2179; 345 %) [32]. Previous studies have demonstrated an association between derived measures of salivary cortisol and psychosocial variables, stress and health [4,3337]. However, results have been disparate and there are no generally agreed norms for the absolute concentrations of free cortisol in saliva either immediately post-awakening (range 4.7 18.5 nmol/L) or 30 min post-awakening (range 8.6 21.9 nmol/L) [30]. Another concern is the stability of the ACR. Previous studies have shown that approximately 20 % of participants show an inverted ACR. Hansen et al. reported that 18 % of a study group showed negative ACRs [32]. Wust et al. found that, over 2 days, a mean of 23.2 % of a study group were classified as non-responders [38]. Non-responders were defined as having v2.5 nmol increase from basal level (corresponding to one secretory episode) within the first 45 min post-awakening. Eek et al. [39] found that among 142 participants (75 F and 67 M) monitored during 3 work days and 1 day during the weekend, the daily prevalence of negative ACRs varied between 19 % on a work day and 38 % on the day off. Most participants exhibited one or more negative ACRs occasionally during the 4 days, and most a mixture of positive and negative responses. The difference between positive and negative responses could not be explained by selfreported awakening time, subjective stress or sleep disturbance [39]. In addition, in previous studies certain proportions of participants have not followed the expected diurnal rhythm when saliva samples have been collected at fixed time-points. In such studies, the expected diurnal pattern is a negative slope from the first sample (not collected immediately upon awakening); hence, deviations from this negative slope have been noted as flat cycles [40]. A study covering 2 days of saliva sampling showed that 51 % of the participants had the typical decline during both days; 17 % did not show the diurnal pattern on both days; and 31 % had a different pattern during the 2 days [40]. A replication of this study, including four different studies, concluded that at least 10 % had no significant diurnal cycle [41]. The reason for these inconsistent patterns is not known. Previously, some studies have aimed to identify certain traits or specific demographic characteristics of individuals with flat cycles [42] or non-response [40]. However, with the recent findings of Eek et al. [39], non-responding appears to be related to state rather than trait, but it cannot

Methodological variation relating to applied laboratory techniques (i.e. analytical method, analytical precision of the measurement and long-term stability of measurements).

Methods Literature search In a first step, an online search of the NCBI PubMed database was conducted (National Library of Medicine, National Institutes of Health, Bethesda, MD, USA http://www.ncbi.nlm.nih.gov/PubMed) covering the time period up to August 2007. Search terms were selected with reference to relevant PubMed terms and key words such as salivary, cortisol, measurement, healthy adults, biological variation, seasonal variation, analytical variation, reference interval, age, physical activity, diet, etc. In a second step, a supplementary literature searching method was applied which included the use of personal reference lists and the related studies search option in PubMed. Studies were included in this review only if the topic was covered by the aim and were written in English. Because the PubMed search revealed that only a few studies had analytical and biological variation in salivary cortisol as their main focus, much information was found by scrutinizing the manuscripts where salivary cortisol was used as a measure of the physiological response to stressors. We do not consider the review to be wholly exhaustive, but rather to have captured central articles that help illuminate the research question.

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Results and discussion Biological variation independently of individual choices Diurnal variation Cortisol secretion in saliva exhibits a distinct diurnal variation, with the highest concentration in the morning soon after awakening and lowest in the evening and night [2729]. Reactivity and recovery The ACR is a discrete and distinctive part of the cortisol circadian cycle. In most healthy adults, salivary free-cortisol concentrations increase by between 50 % and 160 % in the first 30 min immediately post-awakening (approximate average increase of 9 nmol/L, range 415 nmol/L, estimated to be equivalent to about three secretory episodes) [30,31]. Hansen et al. estimated confidence intervals for reactivity and recovery of cortisol in saliva in

Analytical and biological variation be excluded that it is related to adherence to time of sampling (see later). Seasonal variation Hormones and other physiological response variables measured in biological fluids often exhibit a multifrequency time structure with significant circadian or seasonal periodicity. In a study by King et al. [43], a seasonal effect of quarterly cortisol concentrations was observed among 147 healthy adults between 22 and 70 years of age (75 men and 72 women). The participants were originally enrolled in the Seasonal Variation in Cholesterol Study (SEASONS) [43]. The highest salivary cortisol levels were observed in winter (6 November to 4 February) and fall (6 August to 6 November), compared to spring (5 February to 6 May) and summer (7 May to 5 August). Apart from the seasonal effect, King et al. [43] also observed a slight sequence effect, which they attributed to habituation. In a more recent study of our own, in which we employed a monthto-month resolution and measurement of the diurnal pattern in healthy adults between 32 and 61 years of age (16 F and 7 M), we found that the diurnal patterns of salivary cortisol were similar across the months (interaction pw0.4). However, the overall concentrations exhibited a seasonal variation. On closer examination, the effect of month indicated that the highest concentrations were observed in February, March and April, with the lowest during July and August. Pairwise comparisons confirmed that the measured concentrations in February, March and April all deviated from those in July and August (pv0.001) unpublished data. A seasonal variation in salivary cortisol concentrations was thus traceable in an occupationally active population. Seasonal variation has also been observed for cortisol in urine, although the months with the highest excretion were December and January [18]. It was speculated that the variation might be related to the time of daybreak. In December and January, the sun rises above the horizon markedly later than during the rest of the year in Denmark (at approximately 0830 h). It has been hypothesized that the mechanism mediating the metabolism of cortisol is influenced by daylight. In summary, data from several studies have shown that seasonal fluctuation might be a potential effect modifier in longitudinal studies, and has to be taken into account when field studies or interventions are being designed and evaluated.
1 2

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Within-subject and between-subject variation Among 28 healthy individuals (12 M, 16 F) who volunteered to provide 5 non-consecutive first-morning saliva samples over a 2-week period, CVi1 was found to be 6.3 %, CVg2 to be 20.5 % and index of individuality to be 0.36 [44]. A recent study in which 27 men and women collected saliva samples 4 times a day, once per month during the course of one year, showed CVi to be 57.4 % (unpublished data). The variation was adjusted for diurnal and seasonal variation. Ockenfels et al. [45] have presented between-subject variation for unemployed and employed subjects as SDs to values measured at six different times of the day. On awakening, betweensubject SD was reported to be 61 % and at 1800 h 86 %. Hansen et al. presented SD at awakening to be 16 % and 51 % at 1800 h [32]. A recent study among 27 men and women showed CVg to be 20.5 % (unpublished data). The variation was adjusted for diurnal variation. However, the discrepancy may have been due to the fact that the data in the studies by Hansen et al. [18] and in our own study (unpublished data) were logarithmically transformed and that the diurnal variation and sampling time were included in the model. The measured group levels in the study by Ockenfels et al. [45] differed slightly in the awakening values, but not in the samples collected at 1800 h compared to the study of Hansen et al. The importance of providing data on biological variations has been discussed in detail earlier [46,47]. Withinsubject variation can be used to set analytical goals for methods used for monitoring subjects in clinical settings. It has been suggested that a desirable performance can be defined as CVav0.5 CVi and an optimum performance as CVav0.25 CVi [50]. In our own study (unpublished data), CVa/CVi wasv0.17, indicating that the analytical variation in the method employed was adequate for use in measurements of healthy subjects.

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Gender and age Basal level of salivary cortisol has been found to increase in older age groups of healthy subjects [49], whereas no age effects have been found in a healthy working population [32,38]. Nicolson et al. [49] state that age differences are more likely to be found when samples include subjects older than 70 years. Potential age differences therefore do not appear relevant when basal levels are being studied in a

*CVi5within subject variation *CVg5between subject variation

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. M. Hansen et al. A occurred depending on the proportion of protein in the meal, i.e. increasing after the high-protein meal, but not the low-protein meal [53]. Furthermore, the HPA axis among subjects with abdominal obesity is more sensitive to meals containing high lipoprotein meals compared to that of a reference group [54]. Another study has shown that cortisol was associated with self-reported habitual carbohydrate intake and dietary lipids [57]. However, acute intake of glucose did not affect concentrations of cortisol [58]. In a recent field study, concentrations of cortisol were increased in subjects up to 2 h after eating a meal. The effects of meals were the same regardless of protein content. Since stress reactions may result in changed behaviour, such as more intake of coffee, it is relevant to study the extent to which coffee affects the excretion of cortisol in saliva. In a double-blind, crossover trial conducted over 4 weeks in a laboratory setting among 48 men and 48 women, cortisol response was measured after caffeine challenge with controlled levels of caffeine intake. In each week, subjects abstained for 5 days from dietary caffeine and instead took capsules totalling 0 mg, 300 mg and 600 mg/day in 3 divided doses. Caffeine decreased cortisol secretion acutely to the initial 0900 h caffeine dose, but raised it again after the second caffeine dose taken at 1300 h. Cortisol levels declined to control levels during the evening sampling period. The conclusion was that cortisol responses to caffeine are reduced, but not eliminated, in healthy young men and women who consume caffeine on a daily basis [57]. Daily consumption of breakfast cereal was associated with lower cortisol levels [58]. Owing to the acute effects of intake of a meal on concentrations of cortisol, it is recommended that intake of food be avoided in at least the 2 h prior to sampling. Medication In field studies of work environmental stress, there are several types of medication with potential effects on salivary cortisol that may be of interest, e.g. medication of mental diseases, oral contraceptives and medication containing hydrocortisone, e.g. for asthma and allergies. Only a few studies deal with confounding from medication of mental diseases, and their effects on concentrations of cortisol are divergent. Extract of Ginkgo biloba, used for cognitive deficits, has been found to inhibit salivary cortisol to some stress stimuli [59]. Hypericum extract, used as medication for somatization, fatigue and depression, has been related to increased activity of glucocorticoids in the brain [60]. Phosphatidylserine used for stress dampening has

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working population. However, age may be important when looking at increases in cortisol. Seeman et al. [50] reported among younger adults that it was the men who exhibited greater increases, whereas among the older adults it was the women. Several studies have reported no gender differences in basal levels of cortisol [43,49,51]. In one study, no gender difference was observed in salivary cortisol concentrations (19 to 55 years of age) on awakening for men and women in low chronic stress groups [51]. However, two studies observed different ACRs among men and women. The increase in cortisol levels after awakening was greater for women with chronic overload compared to men [51]. Correspondingly, Hansen et al. found a significantly higher concentration of salivary cortisol in women 60 min after awakening compared to men [32]. It is concluded that gender is an important determinant and potential confounder when examining ACRs. In regard to details concerning womens unique menstruation periods, no differences were found between profiles obtained during either the follicular or luteal menstrual cycle phase. Hence, the data of Hansen et al. suggest that the morning cortisol response is influenced by the awakening time but not by the menstrual cycle phase. Moreover, health status and age appear to have an impact on this marker of adrenocortical activity. Wake-up time, health status and age should therefore be controlled for in future studies measuring cortisol responses to awakening [52]. To summarize, in certain circumstances both gender and age may influence concentrations of salivary cortisol.

Biological variation dependent on individual choice (lifestyle) Lifestyle factors may have different effects on concentrations of cortisol depending on the time perspective. Acute effects may differ from long term effects. The acute effects are more relevant to sample protocol, whereas the long-term effects may be more relevant to interpretation of the results. In this review, we try to distinguish between these two perspectives. Diet A meal in the middle of the day may be a convenient physiological challenge to the hypothalamic-pituitaryadrenal (HPA) axis. In healthy women in a standardized non-clinical setting, salivary cortisol was measured before and after a protein-rich meal (32 % energy as protein) on one day and again after a lowprotein meal (5 % energy as protein) on another. An acute meal-dependent increase in salivary cortisol

Analytical and biological variation been shown previously to dampen the ACTH and cortisol response to physical stress [61]. Oral contraceptives are a more widely spread type of medication with potential effects on concentrations of cortisol in a generally healthy working population. The effect of a low oestrogen oral contraceptive was studied among 23 healthy women. Both plasma free and salivary cortisol were higher among the women in the pill group compared to a reference group [62]. However, in other more recent studies with lower doses of hormones, no influence of oral contraceptives was found on salivary cortisol [38,63,64]. In summary, when salivary cortisol is used as a measure for the bodily reaction to work stress, it is advisable to collect information on medication, such as oral contraceptives and treatment for mental diseases. Yet it is recognized that the firm evidence for doing so is not provided in the current literature.
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or more in a study group consisting of 37 (19 to 24 years of age) smokers [74]. This was confirmed in a recent laboratory study, where plasma cortisol concentrations increased within 20 min of a cigarette being smoked [75]. Consequently, we recommend asking participants to refrain from smoking before saliva is collected for measurement of cortisol and to provide information on smoking status. Physical activity Heavy short-term physical activity may increase cortisol in saliva [29]. Physical activity, when it is not exhaustive, usually leads to elevated blood levels of hormones of stress and activity [76]. During vigorous physical activity (i.e. running a marathon) serum cortisol has been reported to increase acutely [77]. This has been confirmed for salivary cortisol in a study of a working population, where 90 min of vigorous exercise caused an increase in concentrations of cortisol within the following 2 h compared to an evening with normal physical activity. In a study of our own, no carry-over effect on ACR the following morning was observed (unpublished data). More long-term effects of physical activity on serum cortisol were studied among 27 patients. Exercise on a bicycle ergonometer did not affect serum cortisol when exercising for 30 min 3 times a week for 3 months with a load of 5060 % of peak oxygen consumption [78]. In another study of our own that concerned the association between high stress and energy and higher general level of salivary cortisol, it was observed that the association between stress and energy and evening salivary cortisol was affected by physical activity at leisure time, specifically the amount of vigorous physical activity. Salivary cortisol in the evening was found to be higher in physically active subjects. No such effect was observed for physical activity at work, at home, or during transportation to and from work (unpublished data). In summary, it is advisable to record periods of physical exercise in field studies and to ask participants to refrain from vigorous physical activity in at least the 2 h, preferably longer, before saliva is collected for measurement of cortisol.

Alcohol Chronic heavy drinking may be associated with high salivary cortisol concentrations [65,66]. Reduced cortisol reactivity in heavier drinkers has been found to be consistent with reports that individuals at risk of alcoholism are hypo-responsive to physical and psychological stress [67]. However, in a study of our own, concentrations of cortisol were not affected within the 2 h after consumption of moderate quantities of alcohol (10 cl) (unpublished data). Although in contrast to one study [71], this is in line with a study by Gianoulakis et al. [72]. Therefore, it does not appear to be necessary to restrict moderate alcohol intake on days of saliva collection. Smoking A review has found that basal cortisol concentrations are higher in habitual smokers than in non-smokers [70], and this has been confirmed in two recent studies [71,72]. One study was based on the Whitehall II cohort and included 3103 men and 1128 women [70]. In the other study it was found that smoking cessation was accompanied by an abrupt decrease in salivary cortisol [71]. These findings contrast with other studies in which no effects of habitual smoking on concentrations of free cortisol were found [32,38,52], or lower levels of plasma cortisol among current smokers [73]. Smoking also has an acute effect on cortisol concentrations. In a study of 10 smokers and 10 non-smokers (19 to 25 years of age) with sampling every 20 min over a 12-h period, it was found that normal cigarette smoking acutely increased free cortisol levels. Mean salivary cortisol levels were significantly elevated after two cigarettes

Methodological variation relating to sampling and storage Collecting saliva for salivary cortisol: cotton versus polyester Influence of the material of the tampon in the Salivette used for sampling has been found previously for steroid components [80]. In a recent study, it was found that cotton tampons reduce the measured concentration of cortisol to 62 % of that found for

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. M. Hansen et al. A stored on Salivette tampons was twice that for samples stored in separate vials after centrifugation. In conclusion, centrifuged saliva samples for analysis of cortisol may be stored at 5 C for up to 3 months, or at 220 C or 280 C for at least one year. However, long-term storage at room temperature cannot be recommended. Repeated cycles of freezing and thawing did not appear to affect the concentrations of cortisol [84,85].

polyester tampons. Furthermore, the cotton tampons detained more saliva than polyester. Recovery of saliva from polyester tampons was 95.8 (1.1) % and from cotton tampons 54.7 (2.3) %. This is extremely relevant, since a huge problem in sampling saliva is small volumes. It has been suggested that additives be used to raise the saliva production. A widely used additive is citric acid [81], but this interferes with many immunoassays, and it is stressed not to use SalivetteH with citric acid in order to increase saliva production, when using immunoassays without testing the effect first.

Methodological variation relating to laboratory techniques Variation in the applied laboratory techniques Several different analytical methods are presently employed for measuring cortisol in saliva: liquid chromatography with laser-induced fluorimetric detection [86], liquid chromatography tandem mass spectrometry (LC-MS-MS) [87], radioimmunoassay (RIA) [88,89], enzyme immunoassay (EIA) [88] and an immunological method with time-resolved fluorometric end-point measurement [90]. In order to compare results from studies using different techniques, the methods must fulfil several requirements. It has been stated that there is likely to be a high degree of overlap between what is good practice in a routine situation, and what is good practice in a nonroutine situation. Hence, we suggest that methods used in research and development fulfil the requirements for routine analysis stated in the guide on accreditation [23]: The analytical method should be described clearly and in sufficient detail to allow other laboratories to repeat the measurements. Precision, range, robustness and other relevant performance parameters are prerequisites. Statistical control of measurement results and possible bias must be documented by method evaluation, internal quality control (e.g. use of control charts), analysis of certified reference materials and external quality control (e.g. participation in proficiency-testing schemes) [91,92]. Furthermore, measurement results should be reported together with an uncertainty, which allows the user to evaluate the reliability of the results [23]. This is accommodated by following the recommendations given by the ISO [91], Eurachem [93] and by Kristiansen & Christensen [94]. Most of the statements are performance parameters that can be achieved by the individual laboratory, while external quality control requires a proficiency-testing scheme, preferably international. A laboratory comparison of measurement of cortisol in saliva was performed in 2002 [95]. Results were reported for three groups of methods with a minimum of three laboratories per group: RIA-Spectia, HPLC-MSMS

Compliance with time of sampling An important factor of compliance is time of sampling. Some studies have used electronic devices to track when participants actually accessed the cotton swab, or tampon. In one study, it was observed that 74 % of the participants accessed the tampon according to the study protocol, whereas 26 % failed to access the tampon at the proper time in the case of at least one out of six samples. Of this latter group of non-compliants, 55 % failed to take the second morning sampling correctly after 30 min. Participants who were not informed that their sampling was being tracked were significantly less compliant than informed participants [82]. In another study examining participant adherence, it was found that 71 % of participants who were unaware they were being monitored correctly followed the protocol. Their self-reported compliance was 93 %. Among the individuals who were aware of being monitored, the objective compliance was 90 %, consistent with the self-reported compliance of 93 % [83]. In both studies, the non-adherent participants had significantly lower morning cortisol values than the adherent participants.

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Long-term storage of cortisol in saliva collected on SalivetteH In research projects, samples are often required to be stored for longer periods of time. A study on longterm storage found no effects on cortisol concentrations after storage of saliva at 5 C for up to 3 months or at 220 C and 280 C for up to one year. In contrast, concentrations of cortisol were found to decrease by 9.2 % (95 % confidence interval (CI): 3.8 %; 14.3 %) per month in samples stored at room temperature. Repeated freezing and thawing of samples up to four times before analysis did not affect the measured concentrations of cortisol. The coefficient of residual variation (CVresid) for samples

Analytical and biological variation and other methods. The results were compared to the consensus of laboratories in the same group. In general, recovery of the used methods in the laboratory comparison was 108 %, while the mean standard deviation at a concentration of 10.0 nmol/L was 3.5 nmol/L. The laboratory comparison showed that the variation between laboratories was high, thus emphasizing the need for a proficiency testing scheme for cortisol in saliva. A recent study compared measurement of cortisol in saliva on LC-MS/MS with immunoassay results. Mean concentrations using LC-MS/MS were 1.8 nmol/L and using a Roche Cobas Cortisol immune assay from (Roche Diagnostics) from 4.4 nmol/L [96], which was in line with the laboratory comparison by Garde et al. [95]. In summary, the wide range of method variation emphasizes the need for a proficiency testing scheme and a certified reference material for cortisol in saliva, e.g. for calibration of kits.
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saliva samples collected 20 min after awakening (05470747 h), and LOD to 10.3 nmol/L for late afternoon samples (17001900 h) [32]. Vogeser et al. [96] found mean late-night salivary cortisol of 5.0 nmol/L for healthy individuals; the absolute range was 1.416.7 nmol/L, and the 95th percentile was 8.9 nmol/L. Recommendations Based on the present review, we believe that a few recommendations about occupational field studies will help ensure a practical protocol and will increase the quality of future studies. Governing thoughts behind the recommendations are that they should: (a) provide as few restraints and inconvenience for the participants as possible, (b) serve to help reduce unnecessary variability in the study design, and (c) provide suggestions for dealing with variability in cases where influences are unavoidable. (i) To attend the diurnal variation in salivary cortisol concentrations, the time of sampling should be carefully registered and, if necessary, included in the statistical analysis, or samples should be obtained at the same time of day. (ii) To prevent bias being introduced in longitudinal studies by seasonal variations, saliva samples should be collected at the same time of the year. (iii) To prevent bias from ordinary metabolic functions of cortisol, food intake should be avoided for at least 2 h before sampling. (iv) To avoid bias from non-work-related physical activity vigorous exercise should be avoided in at least the 2 h, preferably longer, before saliva specimens are collected. (v) To avoid bias from variation caused by using different laboratory techniques, it is recommended that the same, or otherwise made comparable, laboratory techniques are used. (vi) To avoid bias from the material in the tampon for collecting saliva specimens, it is recommended to use of the same material throughout the study or studies. (vii) To avoid bias from storage, it is recommended that saliva samples are stored at 220C at least, as the changes in concentrations at this temperature will be negligible for one year. If there is a need to store longer, an industrial freezer is recommended (280C). (viii) Despite the absence of hard evidence, it is recommended to collect information regarding alcohol consumption and medication (e.g. oral

Reference intervals In occupational health studies, the study groups usually comprise healthy subjects carrying out their work. Sampling is often planned in the most practical way, with a minimum of inconvenience for the participants, e.g. sampling of blood in the morning at the work site just after work starts or self-sampling of saliva or urine. Optimal use of reference intervals requires that the population on which the reference interval is based is representative of the study group in question. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommends estimating the reference interval on at least 120 subjects. Since it may be costly and difficult to get group sizes of that order of magnitude for all topics in question, new methods for estimating reference intervals for small sample sizes are needed. Previous papers by Bjerner [97] and Hansen [98] provide new tools for estimating reference intervals in small sample sizes. Hansen et al. estimated reference intervals by a variance component model and compared to reference intervals calculated by use of recommendation from IFCC. Where comparable, the IFCC calculated reference intervals that had a wider range compared to the variance component models. The study provides a tool that enables occupational health researchers to calculate reference intervals for specific groups, i.e. smokers versus non-smokers, etc. The last method was applied to estimate reference interval for cortisol in saliva for samples collected at awakening, 20 min after awakening and at 1800 h. Reference intervals were estimated to be from 3.6 to 35.1 nmol/L for samples at the time of awakening (05270727 h), 7.639.4 nmol/L for peak level in

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. M. Hansen et al. A contraceptives or medication for treatment for mental diseases) This information could be used in statistical analysis or to evaluate whether a differential distribution of these potential determinants exists between comparison groups. Because of points (v) and (vi), cross-comparisons of absolute concentrations across studies might be difficult, and establishment of reference intervals for the population studied and method used is recommended.
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