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Many of the most common tests used to diagnose endocrinological disorders are antibody-based immunoassays, although there is a shift toward developing liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays that provide excellent clinical specificity and often superior sensitivity (Table 51-1). Radioimmunoassays (RIAs) pioneered development of immunoassays and further refinement into mass spectrometry methods for quantifying peptide hormones but are not routinely used in clinical laboratories any longer.1,2,3,4

TABLE 51-1Testing Methodologies

Selection of a particular assay to measure a specific biochemical analyte depends on several factors, including the biological variability of the analyte, the analytical specificity (ie, the ability of the assay to distinguish the analyte of interest from similar compounds, hormones, partial peptides, etc.), minimal bias from interfering substances (ie, heterophile antibodies, hemolysis, lipemia) and the precision of the assay (intra-assay and inter-assay variability). Age- and Tanner stage–specific normative data are becoming increasingly important for proper interpretation of pediatric endocrine assays. Reference intervals should be specific to the assay, methodology, instrument platform, and clinical utility due to the general lack of standardization or harmonization of endocrine assays.

Clinicians should be aware of the particular performance characteristics, as well as the limitations, of the specific assays used in their local and clinical reference laboratories. Open and frequent communication between clinicians and the laboratory director or pathologist can aid in determining the proper laboratory tests to order, providing input on send-out testing needs, interpretation of results that do not correlate with the clinical picture, and understanding of the limitations of particular assays.



In competitive immunoassays, the unlabeled analyte in the patient’s blood competes with a labeled antigen for a limited number of antibody-binding sites specific for that particular analyte. The antibody solution is commonly diluted to a concentration that would bind approximately half the labeled antigen if there were no analyte present in the patient’s specimen.5 The reaction is calibrated at each laboratory using standards with known antigen concentrations.3,5 Immunoassays can use chemiluminescence, fluorescence, or radioactivity to quantitate the reaction.6,7


Less labeled antigen measured in the immunoassay means a greater concentration of analyte present in the specimen; that is, they are inversely correlated. This process produces a dose–response or enzyme activity curve that is converted into clinically interpretable concentrations of hormones by mathematical hardware or software.5

Examples of antigen label/immunoassays include radioactive compound/radioimmunoassay, enzymes/enzyme-linked immunosorbent assay (ELISA), and fluorophores/fluoroimmunoassay.


In ELISA, ...

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