Brian Keevil (Presenter)
Authorship: Brian Keevil.
Performance of immunoassay methods used to measure serum cortisol can be highly variable. We have developed a candidate reference method for serum cortisol which we have used to evaluate the major immunoassay diagnostic kits on the market today. Results show that prednisolone and metyrapone all interfere significantly with the diagnostic kits but not LC-MS/MS, and binding proteins can also have a significant effect. This work supports the view that different reference ranges may be needed for some of these methods and cut offs for diagnosing and monitoring hypo and hyperadrenalism need to be carefully evaluated. LC-MS/MS suffers less from interference and should be the method of choice for measuring serum cortisol.
The major stress hormone cortisol is one of the more commonly requested endocrine tests and is used in the evaluation of the hypopituitary adrenal axis. Routine analysis of cortisol in serum samples is generally performed using immunoassay methods (IA) on large clinical analyser platforms, although LC-MS/MS has been the preferred method in this institution for the past 3 years. Performance of the IA methods can be highly variable because of the different antibodies used by each manufacturer and method interfernce can arise from structural analogues and the effect of binding proteins.
A candidate reference method (cRMP) for serum cortisol was developed which was used to evaluate the major IA diagnostic kits on the market today and also compared against a routine LC-MS/MS method. Surplus serum samples from males , females, pregnant females, patients taking metyrapone and prednisolone were anonymised and sent to collaborating laboratories for analysis by six different commercially available routine cortisol methods.
Prednisolone and metyrapone all interfere significantly with the diagnostic IA kits but not LC-MS/MS. Inter-assay variation was observed across the male and female cohorts relative to the cRMP, but intra assay variation across these cohorts was similar. Four immunoassays under recovered cortisol in the pregnanct cohort, CBG was found to be significantly higher (p=<0.0001) than in the normal females. The routine LC-MS/Ms assay agreed well with the cRMP.
Failure to strip cortisol effectively from binding proteins in many of the IA methods can have a significant effect , leading to differences in reported results. Problems with result interpretation can arise, particularly if assay specific reference ranges are not used. This work supports the view that different reference ranges may be needed for some of these methods and cut offs for diagnosing and monitoring hypo and hyperadrenalism need to be carefully evaluated. LC-MS/MS suffers less from interference and should be the method of choice for measuring serum cortisol.
References & Acknowledgements:
Monaghan PJ , Keevil BG , Stewart PM , Trainer PJ. Case for the Wider Adoption of Mass Spectrometry-Based Adrenal Steroid Testing, and Beyond. J Clin Endocrinol Metab
Owen LJ, Adaway JE, Davies S, Neale S, El-Farhan N, Ducroq D, Evans C, Rees DA, MacKenzie F, Keevil BG. Development of a rapid assay for the analysis of serum cortisol and its implementation into a routine service laboratory. Ann Clin Biochem. 2013 Jul;50(Pt 4):345-52.
Hawley JM, Owen LJ, MacKenzie F, Mussell C, Cowen S, Keevil BG Candidate Reference Measurement Procedure for the Quantification of Total Serum Cortisol with LC-MS/MS. Clin Chem. 2016 Jan;62(1):262-9.
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