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.

Measurement of aldosterone in blood.

A detailed method for measuring aldosterone in serum/plasma is described. Aldosterone is first extracted into dichloromethane to improve the specificity of the assay. A radioimmunoassay is performed on the dried extract using an antibody raised in rabbits to aldosterone conjugated at position C3, and iodinated aldosterone. Details are given for optimizing conditions for the second antibody, raised to rabbit immunoglobulins, and carrier protein used in the separation of the antibody bound fraction from the free aldosterone at the end of equilibration. Details are provided in additional notes, for steps in the assay that could be problematic.

Measurement of plasma renin activity.

Measurement of angiotensin 1, released in plasma by the action of endogenous enzyme renin on endogenous substrate, angiotensinogen is described. Details are given for the generation of angiotensin 1 from plasma using controlled conditions of pH and temperature. A radioimmunoassay to quantify the generated material is then described using anti-angiotensin 1 antibody and iodinated angiotensin 1 as label. Separation of the antibody-bound fraction from the free is achieved using dextran-coated charcoal. Problems of cryoactivation of prorenin and the labile nature of angiotensin 1 are highlighted. Additional notes describe steps in the assay that are critical.

Testosterone measurement by isotope-dilution liquid chromatography-tandem mass spectrometry: validation of a method for routine clinical practice.

BACKGROUND: Immunoassay is unsatisfactory for measuring the testosterone concentrations typically found in women. Bench-top tandem mass spectrometers are a viable alternative technology for measurements in the clinical laboratory. METHODS: We used stable-isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS/MS) to measure testosterone in plasma and serum. The sample volume was 50 muL in duplicate; preparation and analysis were carried out in a single tube, and a batch of 192 tubes was analyzed in 17.5 h. RESULTS: Intra- and interassay imprecision was <15% in the range 0.3-49 nmol/L. Recovery of testosterone added to samples at concentrations of 0.625-20 nmol/L was 96% (CV = 12%; n = 26). Six samples were serially diluted with double charcoal-stripped serum to demonstrate linearity. Correlation (r(2)) with isotope-dilution gas chromatography-mass spectrometry for 20 pools of clinical samples (range, 0.5-38.5 nmol/L) was 0.99. Correlations with our extraction RIA were 0.97 for clinical samples from men (range, 8-46.3 nmol/L) and 0.66 for samples from women (range, 0.7-3.0 nmol/L), but were 0.35 for male samples containing <3 nmol/L testosterone and 0.77 for female samples containing >8 nmol/L. Various steroids added to double charcoal-stripped serum showed no interference at the retention time of the testosterone peak. CONCLUSIONS: The ID/LC-MS/MS method has improved accuracy compared with immunoassay. The low sample volume and simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.

Limitations of 17-hydroxyprogesterone in investigating neonatal hyponatraemia.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is a possible cause of hyponatraemia in the neonate. Elevated 17-hydroxyprogesterone (17-OHP) is considered diagnostic of the condition, although there have been reports of anomalous high concentrations, up to 110 nmol/L, in premature, sick infants subsequently shown to have normal adrenocortical function. We describe a case of a 6-week-old girl with a chest infection and hyponatraemia whose plasma 17-OHP concentration was 300 nmol/L, well within the range associated with 21-hydroxylase deficiency. However, there was no genital ambiguity and plasma cortisol was also significantly elevated, raising the possibility of generalized adrenal hyperstimulation rather than CAH. The patient was treated with antibiotic and saline infusions but no steroids. CAH was subsequently excluded by normal 17-OHP and cortisol responses to Synacthen stimulation. In sickness, an increased plasma 17-OHP concentration may not be synonymous with 21-hydroxylase deficiency, even when grossly raised. Simultaneous measurement of plasma cortisol could aid interpretation and avoid potential misdiagnosis, especially in male infants.