Serum Cortisol: An Up-To-Date Assessment of Routine Assay Performance.

BACKGROUND: Accurate serum cortisol quantification is required for the correct diagnosis and management of adrenal pathologies. Presently, most laboratories use immunoassay to measure serum cortisol with proficiency schemes demonstrating a wide dispersion of results. Here, we investigate the effects of sex, matrix, and antibody specificity on serum cortisol quantification in 6 routine assays. METHODS: Surplus serum was obtained before disposal and the following cohorts were created: males, nonpregnant females, pregnant patients, and patients prescribed either metyrapone or prednisolone. Samples were anonymized and distributed to collaborating laboratories for cortisol analysis by 6 routine assays. Cortisol was also measured in all samples using an LC-MS/MS candidate reference measurement procedure (cRMP); cortisol-binding globulin (CBG) was measured in the nonpregnant and pregnant female cohorts. RESULTS: Considerable inter- and intraassay variation was observed across the male and nonpregnant female cohorts relative to the cRMP. Four immunoassays underrecovered cortisol in the pregnancy cohort, and CBG was found to be significantly higher in this cohort than in the nonpregnant females. In the metyrapone and prednisolone cohorts, all immunoassays overestimated cortisol. The first generation Roche E170 and Siemens Centaur XP were particularly prone to overestimation. In all cohorts the routine LC-MS/MS assay aligned extremely well with the cRMP. CONCLUSIONS: Despite the clinical importance of serum cortisol, the performance of routine immunoassays remains highly variable. Accurate quantification is compromised by both matrix effects and antibody specificity. Underpinning this study with a cRMP has highlighted the deficiencies in standardization across routine cortisol immunoassays.

Heterophilic antibody interference affecting multiple hormone assays: Is it due to rheumatoid factor?

Assay interference with heterophilic antibodies has been well described in literature. Rheumatoid factor is known to cause similar interference leading to falsely elevated hormone levels when measured by immunometric methods like enzyme-linked immunosorbent assay (ELISA) or multiplex immunoasays (MIA). We report a case of a 60-year-old male patient with a history of rheumatoid arthritis referred to our endocrine clinic for investigation of hypogonadism and was found to have high serum levels of LH, FSH, SHBG, Prolactin, HCG and TSH. We suspected assay interference and further tests were performed. We used Heteroblock tubes and PEG precipitation to eliminate the interference and the hormone levels post treatment were in the normal range. We believe the interference was caused by high serum levels of rheumatoid factor. Although he was treated with thyroxine for 3 years, we believe he may have been treated inappropriately as his Free T4 level was always normal despite high TSH due to assay interference. Our case illustrates the phenomenon of heterophilic antibody interference likely due to high levels of rheumatoid factor. It is essential for clinicians and endocrinologists in particular to be aware of this possibility when making treatment decisions in these groups of patients.

Analysis of 17 α-hydroxyprogesterone in bloodspots by liquid chromatography tandem mass spectrometry.

BACKGROUND: Monitoring of treatment for patients diagnosed with congenital adrenal hyperplasia (CAH) can be performed by measuring the concentration of 17α-hydroxyprogesterone (17OHP) in bloodspots collected on filter papers. A method is described here for measuring 17OHP by liquid chromatography tandem mass spectrometry (LCMSMS). METHODS: 17OHP was extracted by liquid-liquid extraction and analysed by LCMSMS. The method was validated for sensitivity, specificity, linearity, recovery, ion suppression, precision and bias. RESULTS: The standard curve was linear from 0 to 400 nmol/L. Intra-assay %CVs were <10 and inter-assay %CVs were <15 over the range 10-200 nmol/L. Limit of quantitation was 6 nmol/L. No ion suppression was detected. The only interfering compound detected was deoxycorticosterone, an intermediate steroid with the same molecular weight as 17α-hydroxyprogesterone. The method was more accurate and precise than an existing radioimmunoassay. There was poor correlation between the two assays. CONCLUSIONS: We have developed a sensitive and specific assay suitable for quantitation of 17OHP in bloodspots. This method performs better than radioimmunoassay and allows smaller samples to be used.

Method-specific serum cortisol responses to the adrenocorticotrophin test: comparison of gas chromatography-mass spectrometry and five automated immunoassays.

OBJECTIVE: The serum cortisol response to the adrenocorticotrophin (ACTH) test is known to vary significantly by assay, but lower reference limits (LRL) for this response have not been established by the reference gas chromatography-mass spectrometry (GC-MS) method or modern immunoassays. We aimed to compare the normal cortisol response to ACTH stimulation using GC-MS with five widely used immunoassays. DESIGN, PATIENTS AND MEASUREMENTS: An ACTH test (250 µg iv ACTH1-24 ) was undertaken in 165 healthy volunteers (age, 20-66 years; 105 women, 24 of whom were taking an oestrogen-containing oral contraceptive pill [OCP]). Serum cortisol was measured using GC-MS, Advia Centaur (Siemens), Architect (Abbott), Modular Analytics E170 (Roche), Immulite 2000 (Siemens) and Access (Beckman) automated immunoassays. The estimated LRL for the 30 min cortisol response to ACTH was derived from the 2·5th percentile of log-transformed concentrations. RESULTS: The GC-MS-measured cortisol response was normally distributed in males but not females, with no significant gender difference in baseline or post-ACTH cortisol concentration. Immunoassays were positively biased relative to GC-MS, except in samples from women on the OCP, who showed a consistent negative bias. The LRL for cortisol was method-specific [GC-MS: 420 nm; Architect: 430 nm; Centaur: 446 nm; Access 459 nm; Immulite (2000) 474 nm] and, for the E170, also gender-specific (female: 524 nm; male 574 nm). A separate LRL is necessary for women on the OCP. CONCLUSIONS: Normal cortisol responses to the ACTH test are influenced significantly by assay and oestrogen treatment. We recommend the use of separate reference limits in premenopausal women on the OCP and warn users that cortisol measurements in this subgroup are subject to assay interference.

Cross-reactivity of 12 recombinant insulin preparations in the Beckman Unicel DxI 800 insulin assay.

BACKGROUND: Insulin analogues used in the treatment of insulin-dependent diabetes may result in hypoglycaemia; however, these preparations show variable cross-reactivity in many insulin assays. In order to investigate a suspected insulin overdose, it is therefore essential to characterize the cross-reactivity of these preparations in insulin assays. METHODS: The cross-reactivity of 12 commercial insulin preparations was assessed on the Beckman DxI analyser, by comparison with the insulin calibration curve corresponding to the midpoint of the dose-response curve. This was achieved by manually plotting the relative light units generated by the analyser for each preparation. RESULTS: All 12 insulin preparations examined showed significant cross-reactivity with the Beckman DxI insulin assay, with 11 of the insulin preparations demonstrating cross-reactivity of 83% or greater. The lowest cross-reactivity was observed with detemir with a value of 47.6%. CONCLUSION: When investigating hypoglycaemia it is important to be aware of the cross-reactivity of the recombinant insulin analogues in insulin assays. This study has established the cross-reactivity of 12 preparations in the Beckman DxI insulin assay.

Effect of hemolysis on insulin determination by the Beckman Coulter Unicell DXI 800 immunoassay analyzer.

OBJECTIVES: To evaluate the effect of hemolysis on the measurement of insulin using the Beckman Coulter Unicell DXI 800 immunoassay analyzer. DESIGN AND METHODS: Insulin concentrations were measured using the Beckman Coulter Unicell DXI 800. Interference studies were conducted by mixing plasma of known insulin concentrations with hemolysates of increasing hemoglobin concentration. Hemolysates were prepared by osmotic shock. A change of >10% from baseline results was taken as evidence of significant interference. RESULTS: Hemolysates mediated a concentration-dependent reduction in insulin concentration. CONCLUSIONS: A concentration-dependent negative bias in the measurement of insulin is associated with increasing degrees of hemolysis on the Beckman Coulter Unicell DXI 800.

Hormone replacement therapy (oestradiol-only preparations): can the laboratory recommend a concentration of plasma oestradiol to protect against osteoporosis?

Target concentrations of plasma oestradiol to protect against osteoporosis have now been published in at least one textbook and are increasingly discussed by clinicians. A survey of the literature revealed that many reports do not give details of oestradiol assay methods, despite the fact that there is a wide range of biases. Plasma oestradiol measurements made after ingestion of equine oestrogen preparations are not valid, and for oral oestradiol and oestradiol valerate preparations a single untimed sample does not give a reliable estimate of the average exposure over 24 h. Oestradiol measurements in plasma from women using patch, gel or implant preparations could be useful provided that consideration is given to the time of sampling and to the assay method used, although target levels have not been established. Further studies are required in this area.