Utility of dilution tests in investigating interference in the free thyroxine assay

Abstract

Interference, for instance due to the presence of heterophilic antibodies, is a well-known pitfall in diagnostic immunoassays. Although these antibodies were reported to be present in 30%-40% of the general population, falsely elevated or decreased results due to interference are uncommon (estimated at 0.05%-0.5%) and unpredictable [1,2]. Interference can be present in only one immunoassay for a specific patient, even when the same analyzer is used for multiple tests on the same patient. Consequently, interference is often discovered by chance, based on discrepancies between the laboratory results and the patient's clinical condition, or when inconsistent results are found for related tests in the same sample, for instance between thyroid stimulating hormone (TSH) and free thyroxine (fT4). Detection of interference therefore relies on careful evaluation of test results by both clinicians and laboratory specialists.When interference is suspected, common methods to further investigate the sample are serial dilution experiments, polyethyleneglycol (PEG) precipitation, heterophilic blocking tubes, or performing repeat measurements on a different analytical platform. Although interferences are not always detected by all methods, dilution and PEG precipitation are easy to perform. Blocking tubes are often unavailable in most laboratories and have a limited shelf life. Repeating the analysis on a different platform often performs well for interference detection, but almost always requires shipment of the sample and is therefore time consuming and costly.Although dilution experiments and PEG precipitation can be performed for most immunoassays, they are often considered inappropriate to investigate interference in the fT4 assay. In fact, the manufacturer's instructions often preclude diluted testing of fT4, since modern fT4 assays are optimized for minimal disturbance of the endogenous equilibrium of free/bound thyroxine, also in patients with variable concentrations of binding proteins. Therefore, these assays are robust toward dilution and/or sequestration of thyroxine by assay ingredients and dilution is not helpful if the test result is above the analytical range. Nevertheless, we will show here that dilution is in fact an appropriate and simple method to investigate interference in the fT4 assay.All TSH and fT4 measurements were performed on Beckman-Coulter UniCel DxI800 immunoassay systems (Beckman-Coulter, Brea, CA, USA). More specifically, fT4 is measured using a one-step chemiluminescent immunoassay with 30 μL of sample in a total reaction volume of 190 μL (i.e. a 6.3× standard dilution of the sample). Interference in the fT4 assay was suspected for 12 patients, who all had increased fT4 results (25-36 pmol/L) with discrepantly high or high normal TSH levels. Discrepancies could not be explained by the patients' clinical condition and/or medication use, leading us to further investigate potential analytical interference within the sample. TSH results were not considered suspicious based on previous results, clin...

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Falso hipertiroidismo por interferencia en inmunoanálisis

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