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 used.
Figure 4.1 The basics of immunoassay are shown for growth hormone (GH; see text for details). For clarity, in Figures only small numbers of hormone molecules and antibodies are shown; in practice, numbers are in the order of 108–1013.
Immunoassays – the competitive‐binding assays
In the competitive‐binding immunoassay (shown for T4 in Figure 4.3), constant amounts of antibody and labelled antigen are added to each tube. A ‘zero’ tube is set up that contains labelled T4, as well as a tube that also includes a known amount of unlabelled standard T4. Incubation allows the antigen–antibody complex to form. Since the zero tube contains twice as much labelled T4 as antibody, half of the labelled hormone will be bound and the other half will remain free (i.e. in excess). In the other tube, unlabelled and labelled T4 compete for the limited opportunity to bind antibody. The total antibody‐bound T4 is separated (e.g. by precipitation) and the label measured (e.g. by fluorescence or radioactivity). There will be less signal from the second tube because of competition from the unlabelled T4; the decrease will be a function of the amount of unlabelled T4 added, i.e. the signal decreases as the amount of unlabelled T4 increases, allowing the construction of a calibration curve (Figure 4.3). For clinical use, standard T4 is replaced by the patient sample, with all other assay conditions kept the same. As for immunometric assays, a five to eight‐point calibration curve offers sufficient precision for patient samples to be interpolated.
Figure 4.2 The basics of an immunometric assay for growth hormone (GH; also see text). As in Figure 4.1, in practice, large numbers of molecules are present for each reagent and the incubation of the first and second antibodies is usually simultaneous. Because the hormone is bound between the two antibodies in the triple complex (
