Introduction: Methodological differences in the immunoassay measurement of cortisol are apparent when reviewing External Quality Assessment scheme data. A bimodal distribution is often observed, particularly for samples from female subjects. It has been postulated that some immunoassays are inefficient at removing cortisol from its binding proteins and therefore have the potential to under-recover from female samples. We investigated the results obtained from two immunoassays and compared them to a liquid chromatography tandem mass spectrometry assay (LC-MS/MS).
Methods: 155 routine serum samples for cortisol analysis were analysed by three methods and results analysed for males and females separately. The two immunoassays utilised were the Roche E170 immunoassay and the Abbott Architect immunoassay. The LC-MS/MS assay used 20 µL serum to which 40 µL zinc sulphate was added. Following a brief vortex 100 µL internal standard (D4 cortisol) was added. The samples were vortexed and centrifuged then subsequently analysed on a Waters Acquity LC and Quattro Premier tandem mass spectrometer. The calibration of the LC-MS/MS assay was aligned to reference materials 192 & 193.
Results: The Abbott immunoassay gave a good overall comparison to LC-MS/MS; LC-MS/MS=1.06×Abbott−7 nmol/L. The Roche immunoassay tended to over-estimate the cortisol; LC-MS/MS=0.81 Roche+19. The Roche immunoassay gave a more consistent comparison to the LC-MS/MS assay between males and females than the Abbott assay. This is consistent with data from the UK NEQAS for Steroid Hormones. The comparison between the Abbott assay was different in males and females.
Discussion: Users of immunoassays need to be aware of the different results that can be seen depending on the immunoassay used. Many routine assays underestimate cortisol in female subjects and this may affect result interpretation. This may be particularly important when using a generic cut-off e.g. for synacthen testing.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.