A re-investigation of saliva collection procedures that highlights the risk of potential positive interference in cortisol immunoassay
Introduction
The measurement of cortisol in saliva is a widely accepted and convenient procedure to assess adrenocortical activity in children and adults, both in research and clinical settings [1], [2], [3], [4], [5]. Saliva collection can be difficult in young children, however, where saliva volumes may be low, and sample collection problematic. Some investigators have used citric acid to increase saliva flow and volume [6], but this may increase cortisol results [7], [8]. Another common technique in both adult and paediatric studies is the use of cotton swabs placed in the mouth to absorb saliva. The swabs, which resemble dental rolls, may then be squeezed in a syringe by the syringe plunger so that saliva drips into a collection pot. The saliva may also be centrifuged from the swab into the bottom of a collection chamber in a purpose made device [‘Salivette’ (Sarstedt, Numbrucht, FRG)]. Cotton swabs have been found to affect the results of some salivary chemical measurements and polyester swabs, which are likely to contain fewer interferants, were introduced as an alternative [9]. An additional advantage of swab-collected saliva is that the process of extracting the saliva essentially filters it, giving a non-viscous, clear sample that is free of cellular and other oral debris that is more pleasant and technically easier to handle. There is evidence, however, that use of these swabs may affect steroid measurement [10]. We set out to investigate the effect of different methods of saliva collection in adult volunteers on cortisol measured in an ‘in-house’ direct radioimmunoassay.
Section snippets
Experimental
Ethical approval was obtained from the Lothian Region Ethics Committee. Sixteen healthy adult volunteers were recruited. Subjects were aged between 21 and 40. Seven were male. None was on steroid therapy. Details of oral contraceptive, caffeine and tobacco use were not obtained. All samples were taken during the day, with most obtained mid morning. Subjects had not eaten or drunk for at least 1 h before sampling, and not cleaned their teeth within 2 h of sampling.
Simultaneous saliva collection
Results
Results in the individuals are displayed in Fig. 1 and results are summarised in Table 1.
Data were close to a normal distribution, with a slight left skew in the cotton, polyester swab and citric acid results. Cortisol measured in saliva collected using citric acid was not significantly different from that measured in plain saliva (p = 0.997). However, cortisol in saliva collected using cotton and polyester swabs resulted in variable and significant (p < 0.01 ANOVA and KW) over-estimation of
Discussion
There was a significant increase in the cortisol value in the cotton and polyester swabs, compared with straight saliva. This discrepancy was more pronounced at lower cortisol concentrations. The discrepancy was presumed to be due to cross-reactivity of swab constituents with the assay antibody. A similar effect has been noted with variable over-estimation of testosterone [12] and 17-hydroxyprogesterone [13] by RIA after Salivette collection. Constituents of cotton swabs have also been found to
Acknowledgement
Dr. Susan Kidd was supported by the Cunningham Trust.
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