Cross-reactivity of steroid hormone immunoassays: clinical significance and two-dimensional molecular similarity prediction

Abstract

BackgroundImmunoassays are widely used in clinical laboratories for measurement of plasma/serum concentrations of steroid hormones such as cortisol and testosterone. Immunoassays can be performed on a variety of standard clinical chemistry analyzers, thus allowing even small clinical laboratories to do analysis on-site. One limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid of the assay. Interfering molecules include structurally related endogenous compounds and their metabolites as well as drugs such as anabolic steroids and synthetic glucocorticoids.MethodsCross-reactivity of a structurally diverse set of compounds were determined for the Roche Diagnostics Elecsys assays for cortisol, dehydroepiandrosterone (DHEA) sulfate, estradiol, progesterone, and testosterone. These data were compared and contrasted to package insert data and published cross-reactivity studies for other marketed steroid hormone immunoassays. Cross-reactivity was computationally predicted using the technique of two-dimensional molecular similarity.ResultsThe Roche Elecsys Cortisol and Testosterone II assays showed a wider range of cross-reactivity than the DHEA sulfate, Estradiol II, and Progesterone II assays. 6-Methylprednisolone and prednisolone showed high cross-reactivity for the cortisol assay, with high likelihood of clinically significant effect for patients administered these drugs. In addition, 21-deoxycortisol likely produces clinically relevant cross-reactivity for cortisol in patients with 21-hydroxylase deficiency, while 11-deoxycortisol may produce clinically relevant cross-reactivity in 11β-hydroxylase deficiency or following metyrapone challenge. Several anabolic steroids may produce clinically significant false positives on the testosterone assay, although interpretation is limited by sparse pharmacokinetic data for some of these drugs. Norethindrone therapy may impact immunoassay measurement of testosterone in women. Using two-dimensional similarity calculations, all compounds with high cross-reactivity also showed a high degree of similarity to the target molecule of the immunoassay.ConclusionsCompounds producing cross-reactivity in steroid hormone immunoassays generally have a high degree of structural similarity to the target hormone. Clinically significant interactions can occur with structurally similar drugs (e.g., prednisolone and cortisol immunoassays; methyltestosterone and testosterone immunoassays) or with endogenous compounds such as 21-deoxycortisol that can accumulate to very high concentrations in certain disease conditions. Simple similarity calculations can help triage compounds for future testing of assay cross-reactivity.

Multicenter performance evaluation of a second generation cortisol assay

“…One major limitation of immunoassays is their specificity with interference of structurally similar compounds, for example endogenous steroids such as cortisone, or other steroid drugs such as prednisolone or dexamethasone [13,14]. In addition, different immunoassays show high interassay variation [12,15].…”

Sex hormone measurements using mass spectrometry and sensitive extraction radioimmunoassay and risk of estrogen receptor negative and positive breast cancer: Case control study in UK Collaborative Cancer Trial of Ovarian Cancer Screening (UKCTOCS)

“…The most commonly used methods are direct immunoassays, which tend to overestimate concentrations and suffer from crossreactivity with other steroids (18). In the last few years, assays with an organic extraction step and mass spectrometry are emerging as the 'gold standard' (19). Mass spectrometry has improved specificity and has a wide analytical range compared to the immunoassays and allows the measurement of multiple hormones during a single run (20).…”

Theoretical Studies of Cortisol-Imprinted Prepolymerization Mixtures: Structural Insights into Improving the Selectivity of Affinity Sensors

“…14,15 Moreover, as recently reviewed by Krasowski et al, commercially available cortisol affinity assays still have cross-reactivity of more than 100% with the analogs, especially prednisolone. 9 An important finding from this study is the increasing H-bond strength and binding energy with increasing number of double bonds around the carbonyl group at C 3 , which could be used as a basic clue to design a specific imprinted polymers.…”

“…The major limitation of currently available corticosteroid immunoassays is cross-reactivity with structurally similar steroid hormones. 9 One potential alternate to address the cross-reactivity issue is designing a synthetic receptor with tailor-made recognition sites that are able to bind preferentially to specific target molecules. This will eliminate cross-reactivity to similarly structured compounds.…”