Education Resource from the Society for Endocrinology
D J Halsall
Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, UK
Summer School 11-14 July 2006
The Møller Centre, Storeys Way, Cambridge, UK
The ability to measure circulating hormones has been vital to the development of endocrinology as a specialty. Since the 1960’s highly sensitive (able to measure low concentrations) and specific (free from cross-reactivity) immunoassays have been the major workhorse for hormone analysis. Robust and highly specific sandwich assays for hormones present in serum at picomolar concentrations are in routine use, and there is the future promise of micro-volume multiplex assays with attomolar sensitivity1. Most endocrine laboratories now routinely rely on automated immunoassay platforms to provide rapid and robust high throughput immunoassay results2. The majority of these systems use heterogenous chemiluminescent assays to provide the necessary analytical sensitivity, and to reduce assay times. However perceived demand for ever faster turnaround times and the wider application of these tests in less well defined clinical situations has vastly increased workload. Whilst expectations of hormone immunoassay results have increased, many assays are now at performance limits, and for some assays quality has been compromised in favour of cost and rapidity3.
It is the responsibility of the laboratory to ensure that the assays chosen have suitable analytical sensitivity, imprecision (reproducibility) and analytical specificity, and that these levels of performance are verified on a regular basis. This is usually achieved by periodic assessment of dummy samples (IQC or Internal Quality Control). As well as being aware of the analytical limitations of a particular assay the endocrinologist must also be aware of the clinical sensitivity (ability to rule out) and clinical specificity (ability to rule in) of the test in the clinical context in which it is being used, taking into account the limitations of using population based reference ranges. This is a two-way process as the acceptable assay performance is dictated by clinical requirements and vice versa.
As higher demands are placed on immunoassay results, assay standardization becomes a major issue (bias between different assay methods)4. Different assay platforms must generate similar results if data from the literature is to be transferable; this is essential when diagnosing rare conditions or for use in ‘pre-clinical’ screening situations. For relatively simple hormones such as steroids, which have a defined molecular composition, the pure compound is often available so, at least in theory, different assays could be harmonised. For heterogenous compounds such as the peptide hormones this is a far greater challenge as reference materials are difficult to produce, and may not be representative of the biologically active compounds present in vivo. Standardized reference materials are becoming available for some peptide hormones such as growth hormone. Most laboratories attempt to achieve some degree of compatibility using EQA schemes (external quality assurance) such as UKNEQAS in the UK5. Typically dummy samples are distributed across the country for analysis by different accredited laboratories. Results from these schemes are invaluable in detecting inconsistencies arising from both individual laboratories and different assay methods, but can be rather alarming considering the faith that is put in the results of some of these assays in the clinic. The clinician should also be aware of rare complications that can confound immunoassay results such as interference from endogenous antibodies6.
Greater accessibility of other analytical methods, particularly Mass Spectrometry augurs well for the future of hormone analysis7. However whether such methods will prove rapid and robust enough to replace immunoassay for routine hormone analysis remains to be seen.
1. Kusnezow W, Syagailo YV, Rueffer S, Baudenstiel N, Gauer C, Hoheisel JD,
Wild D, Goychuk I. Optimal design of microarray immunoassays to compensate
for kinetic limitations- theory and experiment. Mol Cell Proteomics. 2006
May 30; [Epub ahead of print]
2. Wheeler MJ. Automated immunoassay analysers. Ann Clin Biochem. 2001 May;38:217-29.
3. Herold DA, Fitzgerald RL. Immunoassays for testosterone in women: better than a guess? Clin Chem. 2003;49:1250-1.
4. Stenman UH. Immunoassay standardization: is it possible, who is responsible, who is capable? Clin Chem. 2001;47:815-20.
5. http://www.ukneqas.org.uk/
6. Jones AM, Honour JW. Unusual results from immunoassays and the role of the clinical endocrinologist. Clin Endocrinol (Oxf). 2006;64(3):234-44.
7. Guo T, Taylor RL, Singh RJ, Soldin SJ. Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry. Clin Chim Acta. 2006 Apr 3; [Epub ahead of print]
The opinions expressed in this paper are those of the speaker and do not necessarily reflect the views of the Society
Revised:
24-Aug-2006