Determination of cortisol cut-off limits and steroid dynamics in the ACTH stimulation test: a comparative analysis using Roche Elecsys Cortisol II immunoassay and LC-MS/MS

Abstract

Purpose: Measurement of cortisol concentrations is method dependent. The study aimed to establish assay-specific cut-off limits for cortisol after adrenocorticotropic hormone (ACTH) stimulation, comparing Roche Elecsys Cortisol II immunoassay to liquid chromatography-mass spectrometry (LC-MS/MS), and to assess the impact of patient characteristics, estrogen containing oral contraceptives as well as relation to other adrenocortical steroid hormone dynamics.

Methods: One hundred healthy participants underwent a 250 μg ACTH-test, with plasma samples analyzed using ElecsysCortI, ElecsysCortII, and LC-MS/MS. Cortisone, corticosterone, 17-OH-progesterone, dehydroepiandrosterone sulfate (DHEAS), androstenedione, and testosterone were additionally analyzed with LC-MS/MS. Cut-off limit for a normal cortisol response to the ACTH-test was defined as: 2.5th percentile-1.96 × SE.

Results: ElecsysCort II measured cortisol concentrations 21% (95% CI: 19-22%) lower than ElecsysCort I. Cut-off limits for cortisol 30 and 60 min after ACTH were 426 and 485 nmol/L (ElecsysCort II) and 411 and 470 nmol/L (LC-MS/MS). Cut-offs were unaffected by gender, or body-composition. The ACTH-test resulted in significantly increased adrenocortical steroid hormones, except for decreased cortisone concentrations (both sexes), and decreased testosterone in men (1.9 nmol/L, 95% CI: 1.3-2.5). Testosterone was increased in women (0.07 nmol/L, 95% CI: 0.02-0.13).

Conclusion: ElecsysCort II has high analytical performance and yields significantly lower cortisol concentrations than prior polyclonal immunoassays. This clinically relevant difference underscores the necessity for revised cut-off limits for improved diagnostic precision. Suggested 30-minute cortisol cutoff limits are 411 nmol/L (LC-MS/MS) and 426 nmol/L (ElecsysCort II). Adrenocortical steroids increased upon ACTH stimulation, except for cortisone in both sexes and testosterone in men, both of which decreased.

Keywords: Adrenal insufficiency; Adrenocorticotropic hormone test; Cortisol; Immunoassay; LC-MS/MS; Steroids.

Fig. 1

The plots show the relationship between a Roche Elecsys Cortisol II and I, and b Roche Elecsys Cortisol II and LC-MS/MS for all P-cortisol measurements from the 100 participants before and after the ACTH test. The dashed line is the equivalence between methods. The black line is the line of best fit

Fig. 2

Bland–Altman plots. The first row (a–c) shows the difference between ElecsysCort I and II at baseline, 30, and 60 min after an ACTH test. The second row (d–f) shows the difference between ElecsysCort II and LC-MS/MS. For each plot the thick black line is the bias line (mean difference of the methods), and the dashed lines are the upper and lower limits of agreement (LOA, mean difference ± 1.96 × SD). A difference of 0 represents the best fit. Men; black triangles. Women; gray dots. Women on OC; white dots. OC oral contraceptives

Fig. 3

Histogram a illustrates the distribution of the P-cortisol concentrations from the 100 participants (women on oral contraceptives are not included) 30 min post ACTH stimulation measured with both ElecsysCort I and ElecsysCort II. The ranges are staggered. Histogram b illustrates the distribution of P-cortisol concentrations from the 100 participants 30 min post ACTH stimulation measured with ElecsysCort II and LC-MS/MS