Standardised, Metabolite-Specific Assays with Validated Decision Limits: Utopian Ideal or Achievable Goal?

This issue of Clinical Biochemist Reviews is dedicated to the study of assays commonly used in the endocrine area. We have also introduced a new feature called Analytical Commentaries. Articles in this section will summarise recent assay development and critique current assays but are not as comprehensive as a review article. Future unsolicited contributions to this section of the journal are welcome.

We explore the development and current limitations of three assays that are commonly used in the endocrine area: HbA_1c, 25 hydroxyvitamin D (25OHD) and total testosterone. It is pertinent to compare HbA_1c with 25OHD and total testosterone as HbA_1c assays have come a long way in the last decade and standardisation has assisted new assay development for this analyte and reduced inter-method bias. So much progress has been achieved that HbA_1c assays have achieved improvements in precision and inter-assay agreement that have surpassed most other assays in the endocrine area. Readers perusing Goodall’s comprehensive review in this issue of the history of the efforts at standardisation of HbA_1c assays will be impressed by the development of not one but two definitive methods for the quantification of HbA_1c. Futhermore, despite many potential glycated moieties, only the major fraction, N-terminal valine glycated hexapeptide protein, is quantified in these methods. In comparison, testosterone assays and 25OHD assays suffer from significant inter-method bias and problems with metabolite recognition as outlined in the articles by Sacks and Wootton respectively in this issue. The way forward to achieving standardisation has been illuminated by the progress towards HbA_1c standardisation and global standardisation of testosterone and 25OHD assays could be achieved using a similar strategy. But what do laboratories and users of laboratories do in the interim period?

25OHD assay development has been reviewed by Wootton in this issue and is appropriate as RMIT University is in the process of establishing a reference method for 25OHD supported by the Australasian Association of Clinical Biochemists (AACB). This should help to improve standardisation of 25OHD assays in the region in the short term whilst we await standardisation by assay manufacturers. In the immediate term, I believe application of assay-specific decision limits are needed.¹ But will this resolve the problems regarding equity in recognition of the two 25OHD metabolites, 25OHD₂ and 25OHD₃ that Wootton outlines in his article and Morris identifies in his comprehensive review of the area of vitamin D? I don’t think this is likely and assay manufacturers will need to consider whether separate assays are required for measurement of each 25OHD metabolite. Only then may we be able to apply single decision limits to assay results for 25OHD. As Morris and Wootton both emphasise, there may be other reasons to measure 25OHD apart from calcium homeostasis and skeletal health and application of a single decision limit may still not be achievable.

Testosterone assays suffer from similar issues relating to standardisation, metabolite recognition and application of appropriate decision limits. In fact, only recently, another reference/definitive method for total testosterone was published and this mass spectrometric method may improve standardisation in this area.² Testosterone is a prehormone that requires metabolic activation by a hydroxylation step in androgen sensitive tissues. However, testosterone values usually correlate well with dihydrotestosterone values except in the presence of a rare disorder of the 5-alpha reductase gene or a drug induced change in enzyme activity. Testosterone circulates highly bound to sex hormone binding globulin but weakly bound to albumin. In fact the analogy with thyroid hormone measurements is apt as it is the free testosterone and the weakly-albumin bound fraction of testosterone that is of interest. Whilst free thyroid hormone assays are readily available, free testosterone assays are relatively uncommon and suffer from limitations of assay insensitivity and bias.³ These issues are important as improvements in total and free testosterone assay sensitivity and specificity are needed to meet the clinical needs of assay users. This is especially pertinent as testosterone assays are being increasingly used to help define androgen insensitivity in women as well as men.⁴ The other pre- and post-analytical issues related to testosterone assays are addressed by Coates in this issue.

Are standardisation, metabolite-specific assays and well validated clinical decision limits achievable for most assays in the endocrine area? It is certainly possible and achievable for HbA_1c. Achievement of similar improvements in quality for 25OHD and testosterone assays should help to limit the guesswork in the provision of laboratory results.⁵

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