Hormone Immunoassay Interference: A 2021 Update

Abstract

Immunoassays are powerful qualitative and quantitative analytical techniques. Since the first description of an immunoassay method in 1959, advances have been made in assay designs and analytical characteristics, opening the door for their widespread implementation in clinical laboratories. Clinical endocrinology is closely linked to laboratory medicine because hormone quantification is important for the diagnosis, treatment, and prognosis of endocrine disorders. Several interferences in immunoassays have been identified through the years; although some are no longer encountered in daily practice, cross-reaction, heterophile antibodies, biotin, and anti-analyte antibodies still cause problems. Newer interferences are also emerging with the development of new therapies. The interfering substance may be exogenous (e.g., a drug or substance absorbed by the patient) or endogenous (e.g., antibodies produced by the patient), and the bias caused by interference can be positive or negative. The consequences of interference can be deleterious when clinicians consider erroneous results to establish a diagnosis, leading to unnecessary explorations or inappropriate treatments. Clinical laboratories and manufacturers continue to investigate methods for the detection, elimination, and prevention of interferences. However, no system is completely devoid of such incidents. In this review, we focus on the analytical interferences encountered in daily practice and possible solutions for their detection or elimination.

Keywords: Biotin; Heterophile antibodies; Hormone; Immunoassay; Interference.

Fig. 1

Two types of immunoassays. (A) Competitive immunoassay. The sample is incubated with an anti-cortisol antibody (Ab) (for example), and a tracer (cortisol labeled with a measurable signal: enzyme, fluorescent, or luminescent compound isotope) is added. Competition occurs between the cortisol in the sample and the labeled cortisol for the Ab-binding sites. These cortisol molecules bound to the Ab are captured on a solid phase. Unlike sandwich assays, the higher the cortisol concentration in the sample, the lower the signal-labeled cortisol bound to the antibody and finally linked to the solid phase. (B) Sandwich immunoassay. TSH (for example) is “sandwiched” between two different antibodies: one is labeled with a signal to be measured (luminescent or fluorescent compound, enzyme, isotope) and the other one, named the “capture antibody,” will allow the separation of the immune complexes on a solid phase. The higher the TSH concentration, the higher the signal linked to the solid phase will be. Abbreviations: Ab, antibody; Ag, antigen; TSH, thyroid-stimulating hormone.

Fig. 2

The hook effect in sandwich immunoassays. The excess Ag saturates both capture and labeled Abs; no or little amount of the Ab–Ag–Ab complex is formed, leading to false low results. Abbreviations: Ab, antibody; Ag, antigen.

Fig. 3

Mechanisms of Ab interference in immunoassays. (A) In sandwich immunoassays, the Ab can (1) cross-link the capture and detection Abs (false positive), and (2, 3) prevent the formation of the Ab–Ag–Ab complex by blocking the Abs or the analyte (false negative). (B) In competitive immunoassays, the Ab can block the capture Ab or the analyte giving false high results. Abbreviations: Ab, antibody; Ag, antigen.