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studies (unpublished data) frequent washing with shampoo, and the use of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.