Carol Evans

Measurement of urinary free cortisol by tandem mass spectrometry and comparison with results obtained by gas chromatography-mass spectrometry and two commercial immunoassays

Abstract Background Determination of urinary free cortisol (UFC) is an important adjunct for the assessment of adrenal function. In this study, we have analysed cortisol concentrations in urine samples by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and two immunoassays. The results were compared with GC-MS. The interference of cortisol ring-A metabolites in immunoassays was also assessed. Methods The GC-MS technique involved solvent extraction, LH-20 clean-up and derivatization. Only solid-phase extraction procedure was used for LC-MS/MS. The samples were analysed in positive electro-spray ionization mode, monitoring the transitions for cortisol and deuterated-cortisol at m/z 363.3 > 121.2 and m/z 365.3 > 122.2, respectively. Immunoassays were performed according to the manufacturers instructions. Results When compared with GC-MS results both immunoassays (Coat-A-Count; approximately 1.9-fold, Centaur; approximately 1.6-fold) overestimated UFC concentrations. Cortisol ring-A dihydro- and tetrahydrometabolites contribute significantly to this overestimation. There was no interference by these metabolites in either GC-MS or LC-MS/MS methods. The sensitivity of the LC-MS/MS procedure was 2 nmol/L and the intra- and inter-assay variations were <5% in each quality-control sample. The comparison of the UFC results achieved by assaying the study samples with GC-MS and LC-MS/MS indicated that the agreement between the two methods was excellent (LC-MS/MS = 1.0036GC-MS – 0.0841; r 2 = 0.9937). Conclusions The interference of cortisol ring-A metabolites in immunoassays contribute to overestimation of UFC concentrations. The LC-MS/MS procedure had the sensitivity, specificity, linearity, precision and accuracy for the determination of UFC concentrations. The method is suitable for routine use provided that method-dependant reference values are established.

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

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.

IRIS2: a working infrared multi-object spectrograph and camera

IRIS2 is a near-infrared imager and spectrograph based on a HAWAII1 HgCdTe detector. It provides wide-field (7.7’×7.7’) imaging capabilities at 0.4486”/pixel sampling, long-slit spectroscopy at λ/Δλ≈2400 in each of the J, H and K passbands, and the ability to do multi-object spectroscopy in up to three masks. These multi-slit masks are laser cut, and have been manufactured for both traditional multiple slit work (≈20-40 objects in a 3’×7.4’ field-of-view), multiple slit work in narrow-band filters (≈100 objects in a 5’×7.4’ field-of-view), and micro-hole spectroscopy in narrow-band filters allowing the observation of ≈200 objects in a 5’×7.4’ field.

A luminescent bioassay for thyroid blocking antibodies

Thyroid blocking antibodies (TBAb) have a role in the development of hypothyroidism and in the neonate are responsible for transient hypothyroidism. Specific measurement of TBAb requires a bioassay, but current methods are lengthy and cumbersome. We describe a rapid luciferase‐based method for the detection of TBAb using the lulu* cell line which is suitable for the provision of a clinical service

Causes of discordance between thyroglobulin antibody assays.

Background Anti-thyroglobulin (Anti-Tg) assays show poor concordance. Methods We have investigated concordance and the causes of discordance between Abbott, Roche and Immulite Anti-Tg assays in 606 patients followed up for differentiated thyroid cancer (DTC). The reference range (RR) or lower reporting limit (LRL) was used to classify samples as negative or positive. Results Anti-Tg prevalence ranged between 6% and 55% depending on the method and cut-off. Concordance was 45% using LRL and 75% using RR. Specimens between the RR and LRL using the Immulite and Roche assays were identified that were positive by the Abbott assay and showed poor recovery of Tg in the Tg assay. This suggests misclassification using the RR. Anti-Tg International Reference Preparation (IRP) concentrations measured by the Roche and Abbott methods agreed well but patient samples did not. This is likely to be due to the heterogeneity of Anti-Tg. The Immulite assay appeared less sensitive than the Abbott and Roche based on investigations using the IRP and the low prevalence of Anti-Tg in the DTC patients (6–8%). Interference by Tg (>1000 μg/L) in the Roche assay was also identified as a cause of assay discordance. Conclusions Anti-Tg is used as a tumour marker for DTC and to predict interference in Tg assays themselves and hence inform clinicians of reported Tg concentrations. We have identified several causes of Anti-Tg assay discordance. This includes variation in assay sensitivity and interference from Tg, the heterogeneity of Anti-Tg and the use of different cut-offs to classify samples as antibody-positive or -negative.

Is the current threshold level for screening for congenital hypothyroidism too high? An audit of the clinical evaluation, confirmatory diagnostic tests and treatment of infants with increased blood spot thyroid-stimulating hormone concentrations identified on newborn blood spot screening in Wales

The UK Newborn Screening Programme has set standards for the identification, investigation and early management of children with congenital hypothyroidism. (1)

Development of a rapid assay for the analysis of serum cortisol and its implementation into a routine service laboratory

Background LC-MS/MS is rapidly becoming the technology of choice for measuring steroid hormones. We have developed a rapid LC-MS/MS assay for the routine analysis of serum cortisol. We have used this assay to investigate the effects of gender and exogenous steroid interference on the immunoassay measurement of serum cortisol. Methods Zinc sulphate (40 µL) was added to 20 µL of sample. This was vortexed for 10 s followed by the addition of 100 µL of internal standard in methanol. Following mixing and centrifugation, 10 µL of sample was injected into an Acquity LC system coupled to a Quattro Premier tandem mass spectrometer. Serum samples (n = 149) were analysed by LC-MS/MS and two commercial immunoassays. Results were then compared for all samples and for gender differences. A further set of serum samples (n = 171) was analysed by the LC-MS/MS assay and a GC-MS assay. Results Cortisol had a retention time of 0.98 min and the assay had an injection-to-injection time of 2.6 min per sample. Mean recovery was 99% and mean CV was 8%. The immunoassays gave comparisons of: Roche = 1.23 × LC-MS/MS −1.12 nmol/L and Abbott = 0.94 × LC-MS/MS + 11.97. The comparison with GC-MS showed LC-MS/MS = 1.11 × GC-MS – 22.90. Discussion We have developed an LC-MS/MS assay for serum cortisol analysis that is suitable for routine clinical use and has been in use in our laboratory for 12 months. The availability of this assay will give more reliable results in patients receiving exogenous steroid therapy.

The effect of serum matrix and gender on cortisol measurement by commonly used immunoassays

Background Considerable intermethod bias has been observed between cortisol immunoassays, with some also displaying a gender difference. Cortisol immunoassay performance is affected by serum matrix effects such as changes in steroid binding proteins and presence of interfering steroids which can be altered in various clinical settings. This study investigates cortisol immunoassay bias in pregnancy, renal failure and intensive care patients. Methods Serum remaining after routine analysis from pregnant patients, patients on the intensive care unit and patients with renal failure were obtained prior to disposal and used to create 20 anonymous samples per group. A male and female serum pool was prepared and spiked with cortisol. Samples were aliquoted and distributed to four hospitals for cortisol analysis by immunoassays from four different manufacturers. Cortisol was also measured by an isotope dilution-gas chromatography–mass spectrometry method for comparison of assay bias. Results Differences in cortisol immunoassay bias were observed across the different patient groups. A negative bias compared to pooled serum samples was observed for pregnancy serum, whilst a more positive bias was seen in renal failure and intensive care patients. Variation in bias was greatest in renal failure with the Roche E170 the most affected and the Abbott architect the least (interquartile ranges 44% and 14%, respectively). Conclusions Cortisol immunoassay bias may be affected by gender and differences in serum matrix from patients with various clinical conditions. Users of cortisol assays should be aware of differing matrix effects on their assay and the relevance of these for the interpretation of clinical results.

Measuring cortisol in serum, urine and saliva – are our assays good enough?:

Cortisol is a steroid hormone produced in response to stress. It is essential for maintaining health and wellbeing and leads to significant morbidity when deficient or present in excess. It is lipophilic and is transported bound to cortisol-binding globulin (CBG) and albumin; a small fraction (∼10%) of total serum cortisol is unbound and biologically active. Serum cortisol assays measure total cortisol and their results can be misleading in patients with altered serum protein concentrations. Automated immunoassays are used to measure cortisol but lack specificity and show significant inter-assay differences. Liquid chromatography – tandem mass spectrometry (LC-MS/MS) offers improved specificity and sensitivity; however, cortisol cut-offs used in the short Synacthen and Dexamethasone suppression tests are yet to be validated for these assays. Urine free cortisol is used to screen for Cushing’s syndrome. Unbound cortisol is excreted unchanged in the urine and 24-h urine free cortisol correlates well with mean serum-free cortisol in conditions of cortisol excess. Urine free cortisol is measured predominantly by immunoassay or LC-MS/MS. Salivary cortisol also reflects changes in unbound serum cortisol and offers a reliable alternative to measuring free cortisol in serum. LC-MS/MS is the method of choice for measuring salivary cortisol; however, its use is limited by the lack of a single, validated reference range and poorly standardized assays. This review examines the methods available for measuring cortisol in serum, urine and saliva, explores cortisol in disease and considers the difficulties of measuring cortisol in acutely unwell patients and in neonates.

Adult testosterone and calculated free testosterone reference ranges by tandem mass spectrometry

Background Testosterone is measured for the investigation of female hyperandrogenism and male hypogonadism. Liquid chromatographytandem mass spectrometry (tandem MS) is becoming the method of choice but comprehensive reference ranges are lacking. Methods Testosterone was measured by tandem MS on 90 healthy women, 67 young healthy men and pregnant women (59 first trimester and 60 second trimester). Results The male, male calculated free, first trimester and second trimester testosterone reference ranges (derived using the antilog of mean ± 1.96 SD of log transformed data) were 10.6-31.9, 0.23-0.63, 0.6-4.9 and 0.9-4.9 nmol/L, respectively. The female testosterone upper reference range limit, derived non-parametrically from the 97.5th centile, was < 1.7 nmol/L. Conclusions We have derived tandem MS testosterone reference ranges to support clinical services.