the interaction between antibodies and their antigens. In practice, five to eight calibration points are used to construct the curve.
Analytical methods linked to mass spectrometry
In some situations, immunoassays are unreliable or unavailable, commonly because antibodies lack sufficient specificity, or there are difficulties with measurements at low concentrations (e.g. serum testosterone in women). This leads to differences in measurements across different assay platforms that inhibit the development of internationally agreed standards for diagnosis and care. For some steroid or peptide hormones, or metabolic intermediaries, mass spectrometry (MS) is becoming increasing helpful. It is applied either by itself or, for increased ability to resolve and measure substances, in tandem (MS/MS) or downstream of liquid chromatography (LC/MS) or gas chromatography (GC/MS). These approaches provide definitive identification of the relevant hormone or compound according to its chemical and physical characteristics, e.g. particularly useful for the unequivocal detection of performance‐enhancing agents in sport.
Figure 4.3 The basics of an immunoassay for thyroxine (T4; also see text). As in Figure 4.1, in practice, large numbers of molecules are present for each reagent. Under the conditions shown, the competition between equal amounts of labelled and unlabelled T4 in Tube 2 will be such that, on average, 50% of the antibody binding sites will be occupied by labelled T4. Because of competition between labelled and unlabelled hormone for a limited amount of antibody, this type of immunoassay is sometimes called a ‘competitive‐binding’ assay. After removing unbound label (as in Figure 4.2 legend), the fluorescent or radioactive bound fraction is quantified and a calibration curve constructed. In practice, five to eight calibration points are used to construct the curve.
GC allows separation of vaporized molecules according to their chemical structure. For a sample loaded on a GC column, different components exit the column and pass to the mass spectrometer at different times. MS ionizes compounds to charge them, after which the spectrometer measures mass and charge during passage through an electromagnetic field. This gives a characteristic mass‐to‐charge ratio for any one substance. As with immunoassays, patient samples can be judged against the performance of precisely known standards. LC/MS is similar to GC/MS; however, the initial separation is performed in the liquid rather than the gaseous phase.
Enzymatic assays
Some metabolites are assayed enzymatically, frequently using dye substrates that are catalyzed to products that are coloured or fluoresce. By incorporating known standards, the amount of colour or fluorescence can be used for precise quantification. For example, glycated haemoglobin (HbA1c), a measure of long‐term diabetes control (Chapter 11) can be measured in an enzymatic assay as well as by immunoassay and chromatography/MS approaches. Serum glucose can be measured by oxidation to generate a product that interacts with a dye to generate colour or fluorescence in an enzymatic assay.
Reference ranges
Typical adult reference ranges are listed for a number of hormones in Table 4.1. Whenever possible, hormones are measured in molar units (e.g. pmol/L) or mass units (e.g. ng/L). However, this is not possible for complex hormones such as the glycoproteins thyroid‐stimulating hormone (TSH), luteinizing hormone (LH) and follicle‐stimulating hormone (FSH), because they circulate in a variety of slightly different forms (‘microheterogeneity’). In this scenario, international reference preparations are agreed, with potency expressed in ‘units’ (U) and their subdivisions [e.g. milliunits (mU)]. Potency is assigned after large collaborative trials involving many laboratories worldwide using a range of assay platforms and physical analytical techniques. Patient results are then expressed relative to the reference data.
Static and dynamic testing
Most endocrinology testing is ‘static’; hormones and metabolites are measured as they circulate at any one time. However, rhythmical, pulsatile or variable hormone secretion makes interpretation of single random samples meaningless or misleading (Chapter 1). For some hormones, such as GH, a clinical impression can be gained from a series of six to eight measurements during the course of a day. Alternatively, dynamic testing may be necessary where, based on understanding normal physiology, responses are measured following a stimulus. This might be metabolic, such as insulin‐induced hypoglycaemia to study the expected rise in serum GH and cortisol (Chapter 5), or the administration of glucose during a glucose tolerance test to diagnose diabetes (Chapter 11). Alternatively, the stimulus might be hormonal, such as injecting adrenocorticotrophic hormone (ACTH; the anterior pituitary hormone) to measure secretion of cortisol (the adrenocortical hormone). In this sense, fasting measurements, as required for serum lipids or commonly for glucose, could be viewed as dynamic, where fasting is the stimulus.
Table 4.1 Endocrine reference ranges
| Adult reference hormone | Range | Units | Range | Unit |
|---|---|---|---|---|
| 17‐Hydroxyprogesterone (male) | 0.18–9.1 | nmol/L | 5.9–300 | ng/dL |
| 17‐Hydroxyprogesterone (female) | 0.6–3.0 | nmol/L | 20–99 | ng/dL |
| Adrenocorticotrophic hormone (ACTH, 9 AM) | 0–8.8 | pmol/L | 0–40 | ng/L |
| Aldosterone (AM; out of bed for 2 h; seated 5–15 min)a | 100–500 | pmol/L | 3.6–18.1 | ng/dL |
| Androstenedione (adult male and female) | 2.1–9.4 | nmol/L | 60–270 | ng/dL |
| Anti‐Müllerian hormone (to indicate poor ovarian reserve)b | >7 | pmol/L | >1 | ng/mL |
| Chromogranin A (fasting) | 0–5.2 | nmol/L | 0–250 |
