Strengths and limitations
The possible uncertainty when extracting and measuring cortisol in hair samples has been minimised in this study since the applied method has previously been tested in several studies and proven valid15,19,25. Measurements of cortisol in hair instead of in saliva, blood, and urine have advantages, e.g. the measurements are non-invasive and hair samples can be stored at room temperature. There is also evidence for a high level of intra-individual stability in hair cortisol concentrations11.
Several confounding factors should be discussed when interpreting cortisol levels, especially since previous research has shown conflicting results. Age and sex and to a weaker degree use of oral contraceptives, hair washing frequency and hair treatment may influence hair cortisol levels26. However, in our previous studies (unpublished data) frequent washing with shampoo, and the use of hair spray, gel and wax did not affect HCC levels, in line with a prior report27. On the other hand, the use of chemicals in hair treatments including; bleaching, dying, straightening, or permanent waves may interfere with the cortisol concentrations27-28, although results are conflicting11, 29. Our use of radioimmuno- assay minimises the risks of confounding caused by treated, e.g. coloured, hair.
To our knowledge the highest possible physiological HCC level is unknown, and no clinical standards or reference values have so far been presented. An international interlaboratory process is ongoing to establish benchmark reference values30. The biological samples of HCC that could be considered extreme values in this study were replicated and analysed on two independent occasions giving practically identical results. The cortisol values were logarithmically transformed in the statistical analyses to reduce the variation possibly caused by extreme values.
A strength of this study was that the hair was cut into pieces as part of the analytical process in the laboratory to ensure that all analysed hair samples were from the latest month. Another strength is that pulverised hair was used in our analyses. Recent research has concluded that pulverising hair prior to hormone extraction is crucial31.
In addition, the study is of a well characterised population. The attrition was mainly explained by language difficulties and therefore, we assume that it has negligible impact on data.