Essential Endocrinology and Diabetes. Richard I. G. Holt
advanced by methods to aid diagnosis, and monitor and assess treatment. Investigation in endocrinology and diabetes remains centred on laboratory assays that determine the concentration of hormones and metabolites, usually in blood. In addition to clinical biochemistry (also called chemical pathology), molecular genetics and cytogenetics are routine investigations to provide personalized genetic diagnoses that predict the course of some endocrine disorders (e.g. multiple endocrine neoplasia; Chapter 10).
Outside of the laboratory, clinical investigation draws heavily upon expertise in radiology and nuclear medicine. Some investigations are highly specific (e.g. visual fields for pituitary tumours or retinal screening for diabetes) and these are covered in later topic‐specific chapters.
Pre‐analytical requirements for accurate endocrine testing
Before any sample is measured, it is important that the endocrine testing has been performed using standard protocols to reduce the risk of misleading laboratory results. This involves ensuring that the correct test is performed with the patient properly prepared. It is important that clear instructions are given to the patient about steps that need to be taken prior to the test, e.g. fasting (Box 4.1). Care must be taken with sample collection, transport and handling in the laboratory.
Laboratory assay platforms
Hormones (and other metabolites) are most commonly measured by immunoassay, although increasingly mass spectrometry is used. Immunoassays, introduced in the 1960s, are sufficiently sensitive, precise and hormone specific for routine application in clinical biochemistry. Bioassays, which measure physiological responses induced by a stimulus, are near obsolete in clinical practice.
Immunoassays
Immunoassay is a broad term for one of two different techniques: true immunoassay and immunometric assay. Both forms are based on the premise that the hormone to be measured is antigenic and can be bound by specific antibodies to form an antibody–antigen complex. Both forms of immunoassay also employ a label to generate a quantitative signal. Historically a radioactive isotope [e.g. iodine‐125 (I125)] was used, but now non‐radioactive methods, commonly using a fluorescent tracer, are employed. Both assays also rely on comparison of the patient sample with known concentrations of a reference compound.
To set up a calibration or standard curve for the immunoassay, a constant amount of antibody is added to a series of tubes with increasing, known amounts of a reference preparation; the example in Figure 4.1 is growth hormone (GH). This reaction is reversible with the antigen and antibody continuously associating and dissociating; however, after incubation, equilibrium is reached when tubes with more GH generate more bound complex. Measurement of the amount of bound complex (e.g. in terms of fluorescence or radioactivity) can thus be related to the quantity of GH that was originally added. This allows a calibration curve to be plotted, against which the same process can now deduce the GH concentration in a patient sample.
Box 4.1 Pre‐analytical requirements for accurate endocrine testing
Patient preparation:
Prior fasting may be requiredGlucoseLipidsCalcium
Dietary restrictions may be neededOral glucose tolerance testAdequate carbohydrate intake required the day before5‐Hydroxyindoleacetic acid (5‐HIAA)Avocados, bananas, pineapples, plums, walnuts, tomatoes, kiwi fruit and aubergine (eggplant) should be avoided
Exercise restrictions may be neededOral glucose tolerance test
Prior medication may be needed or should be avoidedOvernight dexamethasone suppression testDexamethasone is taken at midnight before the testSynacthen testExogenous steroids should be withheld for at least 8‐h prior to the test
Attendance at a particular time0900 for serum cortisol and testosterone0800–1000 for aldosterone–renin ratio
Posture/stressAmbulant for 30 min for plasma aldosteroneRested for 30 min for plasma catecholamines
Other investigationsAvoidance of rectal examination prior to PSA measurement.
Sample Collection
Blood samples
Avoid use of ‘drip arm’ to avoid dilution or contamination
Avoid prolonged use of tourniquetVenous stasis can affect measurement of calcium and protein.
Ensure efficient techniqueStress of venepuncture can increase prolactin and other stress hormonesAvoid haemolysis
Ensure correct tube typeLithium heparin: most hormonesMay interfere with ACTH assaysFluoride oxalate: glucoseMay interfere with IGFBP‐3 assaysEDTATests requiring DNA isolationImproves stability of peptides but can interfere with some enzyme testsAvoid risk of contamination by ensuring correct ‘order of draw’plain → citrate → Li heparin → EDTA → fluoride.
Take an adequate volume of sample
Ensure samples are properly labelled
Urine
24‐h collections may be difficult and require the patients to understand a full collection is needed
Ensure correct preservative is usedHydrochloric acid for catecholaminesGlycerol for gonadotrophins
Sample Handling
Minimize delay between sample collection and separation of plasma from cellsAnalytes may decreaseGlucose and ACTHAnalytes may increaseVasopressinSamples may require transport on ice
Ensure efficient centrifugationNeed to remove cellular component without inducing haemolysis
Minimize the number of freeze‐thaw cycles
After thawing, ensure adequate mixing prior to analysis
5‐HIAA, 5‐Hydroxyindoleacetic acid; PSA, prostatic specific antigen; ACTH, Adrenocorticotropic hormone; IGFBP‐3, insulin‐like growth factor binding protein‐3; EDTA, Ethylenediaminetetraacetic acid
Immunometric assays – the sandwich assays
In the immunometric assay (shown for GH in Figure 4.2), a constant amount of antibody is added to each tube with increasing, known amounts of reference preparation. After incubation, the amount of GH bound to the antibody is detected by adding an excess of a second labelled antibody to all tubes. The second antibody is directed against a different antigenic site on GH from the first antibody to form a triple complex sandwiching GH between the two antibodies. Any unbound antibody is removed, leaving the amount of triple complex to be determined by quantifying the bound label (e.g. fluorescence or radioactivity). This emission is plotted for increasing, known amounts of reference compound to generate a calibration curve (Figure 4.2). In practice, five to eight concentrations of hormone standard are used to generate a precise calibration curve, against which patient samples can be interpolated. The immunometric assay is suitable only when the hormone to be measured permits discrete binding of two antibodies. This would not work for small hormones such as thyroxine (T4) or tri‐iodothyronine (T3), for which the competitive‐binding immunoassay system must be