Jonathan Middle

Dehydroepiandrostenedione sulphate interferes in many direct immunoassays for testosterone

Background: In response to a report that the Abbott Architect direct testosterone immunoassay was affected by dehydroepiandrostenedione sulphate (DHEAS) cross-reactivity, this study investigated the effect of DHEAS on other testosterone methods using a UK National External Quality Assessment Scheme (UK NEQAS) for steroid hormones special distribution. Methods: Separate male and female matrix pools of normal human serum were prepared and divided into three portions. Two portions from each matrix were spiked with 10.07 and 20.14. μmol/L DHEAS, respectively. Aliquots of these pools were distributed to participants in UK NEQAS for male and female testosterone at distribution 319 (July 2006). Median testosterone results for each method group were examined for an increase in testosterone concentration. Results: In the female matrix, the effect on Abbott Architect was confirmed and shown also for Roche Elecsys, Roche E170 Modular and Beckman Access (includes some Beckman DxI users). In the male matrix, the effect on Abbott Architect, Roche Elecsys and Roche E170 Modular was similar in magnitude to that in the female matrix, but was smaller for Beckman Access/DxI. However, the effect on Immulite 2000 (includes some Immulite 2500 users) was greater in the male matrix. Indications of interference in other methods, used by only a few laboratories (notably Tosoh AIA and Wallac Delfia), were also observed. Conclusions: DHEAS interference in testosterone assays is a feature of many routine testosterone methods, and occurs in both male and female matrices.

Measurement of serum testosterone in women; what should we do?

All immunoassays for female serum testosterone give falsely high results in some samples. The effect is variable and cannot be predicted for any given sample. Inaccurate calibration or interference by cross-reacting substances is almost certainly the cause of the problem, but for many immunoassays, the exact nature of the interferent is not known. Some of the interference can be removed by employing an extraction step prior to immunoassay. The advent of fast simple and sensitive liquid chromatography tandem mass spectrometry methods offers an exciting alternative to immunoassay for serum testosterone measurement. It is recommended that all high serum testosterone concentrations in women are checked, before reporting, by a method which is accurate (i.e. minimal bias to isotope dilution gas chromatography mass spectrometry [ID-GCMS] method) and is not subject to interference. Action should also be taken by assay users, manufacturers, regulators and professional bodies to ensure accurate standardization and comparability of assays.

Oestradiol assays: fitness for purpose?

In this review we discuss the analytical inadequacies of oestradiol assays in relation to the clinical requirements for performing them, and make recommendations for their improvement. The measurement of oestradiol can be requested in a number of clinical scenarios (precocious puberty, infertility, assisted conception, hormone replacement therapy). The very wide dynamic range of oestradiol concentrations is a huge challenge for routine assays, which they are unlikely to meet on theoretical as well as practical grounds. The EQA performance of oestradiol assays in terms of trueness, comparability, recovery and analytical sensitivity leaves much to be desired and indicates that calibration is compromised by poor analytical specificity. To make oestradiol assays fit for purpose requires concerted action by all stakeholders to define analytical quality specifications for the various clinical scenarios involved, and then to encourage concerted action by the diagnostic industry to use the steroid reference measurement system to improve specificity, trueness and traceability.

National UK audit of the Short Synacthen® Test

We present the first national audit of the Short Synacthen Test (SST), identifying the clinical, analytical and interpretative procedures adopted by 89 laboratories. Background The SST has replaced the insulin stress test as the first-line test to assess adrenal insufficiency and has received considerable attention regarding its sensitivity and specificity. Concerns regarding this test include the bias of cortisol methods, cut-off values used, contraindications and the limitations of the test in diagnosing recent, mild secondary adrenal insufficiency. The audit took into consideration the protocols used by laboratories, the advice provided prior and after the SST and the analytical bias of the methods used. Methods A web-based questionnaire using Microsoft FrontPageTM was prepared to collect data from laboratories and provided drop-down lists and other form-field elements to capture additional comments. The resultant data were exported to Microsoft ExcelTM for data clean-up and analysis. Results The workloads were highly variable; however, most laboratories were in general agreement to the indications, contraindications, timing and reference ranges. In contrast, there was variability in the bias of the cortisol methods, which had not been translated to the cut-off values used by the majority of laboratories. Conclusions The audit has shown that though the preanalytical procedures were similar in most laboratories, there is a requirement to recognize the effect that method bias may have on the reference ranges and consequently on the diagnosis of adrenal insufficiency. There is a need to develop consensus guidelines, which can aid both clinicians and laboratories.

Standardization of steroid hormone assays

In this article, I will present my views on steroid hormone assay standardization, illustrated with examples from the UK National External Quality Assessment Schemes (UK NEQAS) data for oestradiol, although all the problems indicated also occur with progesterone, testosterone (especially in the female range), and, to some extent, cortisol. I will indicate where, in my opinion, effort should be directed to improve what seems to be an intractable unsatisfactory situation.

Evaluation of the Elecsys NTproBNP assay

FOBT should therefore be accurate and sensitive enough to achieve a high analytic and diagnostic accuracy for cancers and precancerous colorectal neoplasia. In our opinion the guaiac-based tests, even socalled ‘reference’ or ‘sensitive’ tests, do not meet these requirements: they are problematic considering the preanalytical, analytical, and postanalytical points of view (especially the interpretation of spurious colour) and the high false-positive and false-negative rates. The only screening that may reach the necessary diagnostic sensitivity and speci¢city with clinically relevant positive and negative predictive values is immunochemical testing without any special dietary adjustment or specimen preparation, including delay of testing. Such screening is, in our opinion, more cost-e¡ective than guaiac testing or a combined guaiac and immunochemical approach because it cuts down on unnecessary and expensive retesting and follow-up procedures.

Comment on: Standardization of steroid tests and implications for the endocrine community:

able to use PBS-based calibrators which simplifies the process. The combined oxalate and citrate assay has now been in use for over a year (as of December 2017) with acceptable EQA performance and reproducibility for both oxalate and citrate. In addition to this, we routinely monitor retention times and peak height to look for any unexpected changes which may affect assay performance; we have found our assay to be very robust over this time and we continue to use it for routine stone screens. In conclusion, we would encourage the use of our complete published method on an entry level instrument which should produce more favourable results. As we have stressed in the manuscript, we believe that sample specific issues exist with the enzymatic method for oxalate measurement and would actively encourage development of more LC-MS/MS assays for both oxalate and citrate.