Scandinavian Journal of Clinical & Laboratory Investigation, 2014; Early Online: 1–4


The effect of storage conditions on salivary cortisol concentrations using an Enzyme Immunoassay

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ANJANA A. NALLA, GERDA THOMSEN, GITTE M. KNUDSEN & VIBE G. FROKJAER Neurobiology Research Unit and Center for Integrated Molecular Brain Imaging (CIMBI), Copenhagen University Hospital, Rigshospitalet, Section 9201, Copenhagen, Denmark Abstract Saliva samples are easy to collect and are applicable for home-sampling, e.g. when studying HPA-axis dynamics to characterize diurnal cortisol profiles and the cortisol awakening response. However, the storing and transport conditions might be critical in the home-sampling approach. Here, we tested the stability of saliva cortisol in samples stored at different temperatures and after repeated thawing-freezing cycles when measured with an Enzyme Immuno Assay (EIA). Thirteen healthy volunteers, six women and seven men, mean age 31 (range 26–49) years collected saliva either in the morning hours (08:00–10:00 h) or before lunch (11:00–12:00 h). Storage at six different conditions were tested: Storage at ⫺ 18°C, ⫺ 4°C, 4°C and room temperature for 72 h. One condition tested was at ⫺ 18°C for 72 h and then kept in an envelope for 72 h with a freezing element in room temperature surroundings where after it was stored at ⫺ 80°C. The last tube was stored directly at ⫺ 80°C and served as the ‘gold standard’. The saliva samples were assayed using Salivary Cortisol Diagnostic EIA. Differences in cortisol measurements between each of the five conditions and the ‘gold standard’ (⫺ 80°C) were evaluated by one-sample t-test. No significant differences were observed. This indicates that an EIA method can be used reliably when measuring salivary cortisol samples obtained by home-sampling including a postal delivery. Key Words: Cortisol, Enzyme Immuno Assay, HPA-axis, saliva, storage, stability

Introduction The stability of cortisol under home-sampling storage and transport conditions is critical for the outcome of cortisol determinations. Validating easy-to-handle procedures to characterize HPA-axis output over the diurnal rhythm or in response to a challenge such as awakening or psychosocial stress is important for studies addressing HPA-axis physiology and pathophysiology. Dysbalances in HPA axis with blunted or excessive cortisol response to stimulation or at baseline is involved in the pathophysiology of a wide spectrum of disorders [1–7]. Particularly in longitudinal studies and when addressing the HPA-axis dynamics in a natural setting, home-sampling is needed as opposed to methods that introduce stress, e.g. invasive methods [8]. Another advantage of saliva sampling as opposed to blood sampling is that the saliva cortisol represents the free non-protein-bound fraction of circulating cortisol and thus reflects more closely the relevant biologically active fraction. In the blood,

1–15% of cortisol is in its unbound or biologically active form. The remaining cortisol is bound to serum proteins [9]. Salivary cortisol levels are unaffected by salivary flow rate or salivary enzymes [9]. The salivary cortisol concentrations are 50–60% of the serum cortisol concentrations [10]. In the literature, some discrepancy exists with regard to cortisol stability in saliva samples with repeated freezing and thawing. It has been reported that cortisol concentrations decrease in average around 10% when salivary samples undergo five repeated freezing and thawing cycles [11]. On the contrary, other studies report that temperature did not affect the concentration of cortisol at 5°C, ⫺ 20°C and ⫺ 80°C up to three months and after four freezing thawing cycles [12], and that cortisol concentration did not differ between non-frozen saliva samples and saliva samples frozen at ⫺ 70°C [13]. Storage at room temperature is generally not recommended for steroid hormones due to stability

Correspondence: Vibe G. Frokjaer, Neurobiology Research Unit and Center for Integrated Molecular Brain Imaging, Rigshospital, Copenhagen University Hospital, Section 9201, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Tel: ⫹ 45 3545 6711. Fax: ⫹ 45 3545 6713. E-mail: [email protected] (Received 17 December 2013 ; accepted 27 October 2014 ) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2014.985252


A. A. Nalla et al.

problems [14] and bacterial growth [15]. It is known that mucins are produced in saliva when it is not stored immediately at ⫺ 80°C, which is reported to decrease the cortisol binding in Radio Immuno Assay kits [16]. Thus cortisol concentrations in saliva measured with an EIA may depend on storage conditions after collection. Here we tested whether storage conditions would bias cortisol measurements relative to samples stored immediately at ⫺ 80°C. The conditions tested were: ⫺ 18°C, ⫺ 4°C and 4°C for 72 h and room temperature for 72 h, and finally storage at ⫺ 80°C.

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Methods Ethics statement The study was approved by the Ethics Committee of Copenhagen and Frederiksberg under the protocol ID: (KF) 01 2006-20 and all experiments were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.

were collected over one week. Salivette® tubes were centrifuged at 4°C at 1000 g for 5 min and all the collected tubes were centrifuged immediately after collection. The volume from each volunteer varied from 0.5–1 mL per collection tube. All the samples were assayed using two enzyme immunoassay kits within one month from saliva sample collection. Stability of cortisol on storage temperature, freezing and thawing Saliva samples taken from the same individual was divided into six Eppendorph tubes that were treated differently, immediately after above-mentioned centrifugation. Four of these tubes were stored at following temperatures: ⫺ 18°C, ⫺ 4°C and 4°C for 72 h and room temperature for 72 h. One tube underwent the following procedure; storage at ⫺ 18°C for 72 h followed by storage in an envelope at room temperature for 72 h, and finally stored at ⫺ 80°C. Thus it underwent two freezing and thawing cycles before analyzed. The last tube was stored directly at ⫺ 80°C and served as the ‘gold standard’ for comparison.

Subjects Thirteen healthy volunteers (six women and seven men, mean age ⫽ 31, range: 26–49 years) participated in this study. None of the volunteers had a history of chronic somatic illnesses such as autoimmune disease, diabetes, cancer, metabolic disturbances, or obesity. None of the volunteers had a history of neurological or psychiatric disorders or alcohol abuse, and they all were drug free. None of the women were pregnant or were in their postmenopausal period. Saliva sample collection Saliva samples were taken at different time-points over the day to secure substantial variation of cortisol concentrations. Saliva samples from 13 volunteers were collected with Salivette® tubes, for cortisol measurement with interference-free oral swab without citric preparation (Sarstedt, Germany) [17]. This is a widely used saliva collection procedure due to the convenience in handling and laboratory processing compared to crude saliva collected by passive drool, where swabs are made from non-toxic inert polymer which is guaranteed for consistency across all lots, making it ideal for longitudinal and multi-participant group studies [18]. Volunteers were instructed to rinse the mouth with water 10 min prior to the saliva sample collection. As sequential aliquoting do not influence hormone concentration in saliva [19], three tubes from each volunteer were collected at the same time point with 5 min interval the same day, either in the morning hours (08:00– 10:00 h) or before lunch (11:00–12:00 h). Samples

Determination of cortisol concentration with EIA The saliva samples were assayed in duplicate using a commonly used commercially available competitive Cortisol EIA kit (Salivary ER Cortisol Diagnostic EIA, batch number 1-3112, (Salimetrics, Philadelphia, USA) [20] according to the manufacturer’s instruction. The majority of the duplicate determinations of absorbency had a Coefficient of variation (CV) less than 10%. The concentration of the samples was calculated from the standard curve obtained from the same plate. All samples from the same individual were run on the same plate. According to the manufacturer, the interassay precision for the kit is 9–11%. The standard curve is a four parameter sigmoid minus curve in the concentration range of 0.33–82.77 nmol/L. The specificity for cortisol is approximately 100% and the cross reactivity for related steroid compounds vary from 0.004–0.6%. A high and a low cortisol standard supplied with the assay kit were processed with all measurements. Standard curves exceeded the sample concentrations at all times. Statistics The cortisol concentrations measured in the saliva samples was calculated using GraphPad (version 4) and R-studio (version 2), utilizing a parameter logistic curve-fitting program. Measurements from samples stored at different conditions were compared by one-sample t-test as the measured concentrations were considered normally distributed. All data are presented as means ⫾ SD. A p-value ⬍ 0.05 was considered statistically significant.

Cortisol stability


Table I. Descriptive statistics. Storage conditions Moments Average ⫾ SD Range CV Difference in concentration vs. ⫺ 80°C (⫾ SD) One-sample t-testa

⫺ 80°C

⫺ 18°C

⫺ 4°C




6.24 ⫾ 4.94 [0.83–16.31] 0–13.9%

6.06 ⫾ 5.59 [0.45–19.27] 0–12.8% ⫺ 11% (⫾ 23)

5.95 ⫾ 4.69 [0.96–15.74] 0–19.7% ⫺ 3% (⫾ 15)

5.30 ⫾ 4.73 [0.61–15.34] 0–10% ⫺ 14% (⫾ 25)

5.91 ⫾ 4.46 [0.96–14.58] 0–15.9% ⫹ 1% (⫾ 20)

5.70 ⫾ 4.29 [0.73–12.1] 0–16.3% ⫺ 6% (⫾ 28)

(p ⬍ 0.08)

(p ⬍ 0.53)

(p ⬍ 0.06)

(p ⬍ 0.94)

(p ⬍ 0.43)

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All concentrations are given in nmol/L. n ⫽ 13. Statistics are given in values. aDifferences were tested using one-sample t-test to show if the % changes differ from zero.

Results For technical reasons, five out of the total 78 determinations were in single determination. Seven samples had a CV ⬎ 10% varying from (12.1–19.7%). The CV of the remaining double determinations (n ⫽ 66) was below 10%. All 78 observations entered the primary analyses. One-sample t-test was repeated when excluding the seven samples where CV ⬎ 10% to test if excluding these would affect the outcome of statistical evaluation. Apart from slight variations in the obtained p-values, the outcome did not change. The double determinations for the controls given in the assay kits varied with 7% for the high control and 12% for the low control. Mean salivary cortisol concentration was 5.93 (⫾ 4.65) nmol/L, range: 0.45–19.28 nmol/L. Salivary cortisol concentrations of the total subjects (n ⫽ 13) for each storage condition and CV on double determinations are shown in Table I. The cortisol concentrations for each subject

Figure 1. Salivary cortisol concentrations in nmol/L for each subject (n ⫽ 13) for each storage condition are shown.

(n ⫽ 13) for each storage condition are shown in Figure 1. Difference in concentration vs. ⫺ 80°C (cortisol concentration measured after storage condition/cortisol concentration of the ‘gold standard’ at ⫺ 80°C ⫻ 100%) is shown in Figure 2. The descriptive statistics for the recovery is given in Table I. The findings show that the concentrations changed during storage with the highest average on ⫺ 14% and lowest on 1% (Table I). No significant differences were observed between cortisol concentration measurements for samples stored at the five different conditions as compared to the gold standard (⫺ 80°C) using one-sample t-test, p ⬍ 0.06 (Table I).

Discussion and conclusion The purpose of this study was to compare the impact of different storage conditions on salivary cortisol concentrations obtained by one of the highest sensitive immunoassays available. We found that storage at ⫺ 18°C, ⫺ 4°C, 4°C, RT and 3-step generated similar results as when compared to direct storing at ⫺ 80°C. Thus, storing

Figure 2. Measured salivary cortisol concentrations for total subjects (n ⫽ 13) where % difference for each storage conditions vs. ⫺ 80°C is shown. The data for the subjects (n ⫽ 13) is illustrated for each subject at each storage condition. The line for mean indicate the average of percentage for each storage condition. The horizontal dotted line at 100% is given at the right side of the figure.

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A. A. Nalla et al.

the samples in various conditions including at up to 20°C for 72 h and after two repeated freezingthawing cycles, did not have any significant impact on the values of salivary cortisol in our sample of 13 subjects. We observed a trend towards lower cortisol values when samples were stored at 4°C but not at worse conditions, i.e. room temperature for up to 72 h. Therefore, this observation may represent a spurious finding in the context of a very sensitive analysis thresholding p-levels at 0.05 uncorrected for multiple comparisons. The present data must be interpreted under the following methodological limitations. First, that the power to detect minor differences was limited with a study sample of 13. However, our findings are supported by observations on the stability of cortisol in saliva samples when measured with a RIA [12,13,15]. Second, we worked with saliva harvested from Salivette tubes by centrifugation. Thus our results may deviate from saliva storage within the tubes. To the extent that tube material might influence saliva or non-centrifugated saliva might react differently to storage our results do not generalize directly to home-sampling conditions. In conclusion our data support that storage of saliva at up to room temperature in up to 3 days or conditions simulating a postal trip may give rise to noise, but importantly do not bias subsequent cortisol measurements with sensitive EIA methods in a manner detectable with a sample size of 13.

Acknowledgements We thank Anetta Claussen and Mette Søgaard Hansen for their skilled assistance in the laboratory. We thank Anders Bue Marcussen for methodological discussions, William Frank Hurst for methodological instructions and Amarnadh Nalla for statistical support. The Dagmar Marshalls Foundation, Danish Medical Research Council, The Health Science Faculty, University of Copenhagen, and the Lundbeck Foundation are thanked for financial support. Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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The effect of storage conditions on salivary cortisol concentrations using an enzyme immunoassay.

Saliva samples are easy to collect and are applicable for home-sampling, e.g. when studying HPA-axis dynamics to characterize diurnal cortisol profile...
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