"Different ways by which hormones can be assayed"

Hormones can be assayed using various methods, primarily immunoassays and mass spectrometry techniques.

The most commonly employed methods in clinical laboratories are immunoassays, which include both competitive and non-competitive (sandwich) immunoassays.

• Competitive immunoassays involve competition between the hormone in the sample and a labelled hormone analogue to bind to antibody sites. They are more prone to interference, particularly from heterophile antibodies, especially when analyte concentrations are low Ekinci et al. 2026.
• Non-competitive (sandwich) immunoassays use two antibodies binding different epitopes on the hormone, and are widely used due to high specificity and sensitivity. Both Beckman Coulter UniCel DxI 800 and Roche Cobas e601 autoanalyzers utilize sandwich immunoassays with chemiluminescence and electrochemiluminescence detection respectively Ekinci et al. 2026.

Immunoassays often exploit labels such as chemiluminescent or electrochemiluminescent substrates to generate signals proportional to hormone concentration Ekinci et al. 2026.

However, immunoassays are susceptible to interference from endogenous antibodies such as heterophile antibodies, human anti-animal antibodies (HAAAs), and rheumatoid factor, which can lead to falsely elevated or decreased hormone levels, particularly in pediatric populations during puberty Ekinci et al. 2026.

To address specificity and interference issues, advanced mass spectrometry methods are emerging. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides direct quantification of intact hormones with improved specificity and reduced inter-assay variability, exemplified by its use in standardized parathyroid hormone assays Jørgensen et al. 2026.

LC-MS/MS is currently considered a reference method for steroid hormone measurements, especially when immunoassay results are inconsistent or suspected of interference Ekinci et al. 2026.

In terms of clinical assay selection and practice:

• Immunoassays remain clinically acceptable and widely available for routine hormone measurements including total testosterone, luteinizing hormone (LH), and others, provided that interference is considered and managed Corona et al. 2026 Corona et al. 2026.
• Sampling conditions such as fasting and timing (e.g., morning measurement for testosterone) are important to ensure reproducibility and diagnostic accuracy Corona et al. 2026 Corona et al. 2026.
• When interference is suspected, laboratories may employ heterophile blocking tubes (HBTs) to reduce antibody interference, or perform confirmatory testing on alternative immunoassay platforms, dilution protocols, or mass spectrometry Ekinci et al. 2026.

However, HBTs do not always effectively exclude interference and may have limitations depending on the assay design and reagents used Ekinci et al. 2026.

Summary of hormone assay methods:

• Competitive immunoassays
• Non-competitive (sandwich) immunoassays with chemiluminescence or electrochemiluminescence detection
• Application of heterophile blocking reagents or tubes to mitigate antibody interference
• Alternative immunoassay platforms for cross-validation
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS), especially for steroid hormones or when high specificity is needed

Emerging advances in mass spectrometry and harmonization strategies aim to improve assay standardization and clinical interpretation of hormone measurements in the future Jørgensen et al. 2026.