Interferences in immunoassay: An estimate based on ‘real-world’ experience

Dear Editor,

With the implementation of total laboratory automation, immunometric assays are becoming increasingly integrated with other branches of laboratory medicine, especially within clinical chemistry services. However, these assays present specific analytical challenges, especially interferences due to heterophilic antibodies, biotin or autoantibodies. ¹ The estimated frequency of such interferences varies substantially (e.g. from 0.03% to 4%).^1–8 The wide range in reported prevalence of immunoassay interferences may reflect: the interference classification criteria used; the type of analyte; the type of assay; the number of samples examined (large studies, carried out on thousands of cases,^1,2,6,7 report prevalences of 0.03% to 0.5%); and the presence or relative quantity of blocking agents in the assay. ⁶ Strategies like removing the Fc fragment of the capture antibody, and adding non-specific murine immunoglobulin to buffer, are now widely used by immunoassay manufacturers. ⁹

A retrospective evaluation was carried out of thyroid function tests (TFT) (fT3, fT4, TSH) measured over 3 years (2020–2022), using the UniCel DxI 800 Immunoassay System (Beckman Coulter, Inc., Brea, CA, USA) according to the manufacturer’s instructions. The data collected between 2020 and 2022 on the suspected and verified interferences are summarized. During this period, 368,159 individual measurements for all three TFT were performed (corresponding to 220,453 requests). The observed prevalences of ‘abnormal’ results (i.e. outwith the respective reference interval) were 19.6% for TSH, 12.3% for fT4 and 12.1% for fT3. An estimated 61,168 individual TFT were therefore examined before validation (corresponding to 53,129 requests with at least one parameter outside the reference range). Six hundred and seventy-eight samples were selected for further investigation for analytical interference on the basis of results that were inconsistent or discrepant or not in keeping with the clinical information provided. Additional investigations included a linearity check with dilution; an incubation with HBT; and a test with a different method. Significant interference (result after additional testing different by at least 30% compared to the initial value) was detected in 28 cases, with the revised results congruent with other analytical and clinical information. Interferences were mainly due to the presence of heterophilic antibodies, or other low-affinity antibodies (e.g. directed against the analyte or test reagents) or biotin supplementation.

Estimated prevalence of interferences was 0.046% of the 61,168 individual parameters examined (0.053% of the 53,129 requests), a figure compatible with the results of Ward, Bjerner and Kim,^4,6,7 although not with those of Ismail. ²

Estimates of the prevalence of interferences are highly variable. In this study, interfering factors were detected in spite of routine precautions (e.g. the blocking agents) already implemented by the manufacturer during the production of reagents. Notwithstanding some possible caveats, our estimate reflects ‘real-world’ experience during routine validation, and as such represents a reasonable approximation of interferences that cause clearly inconsistent results in immunoassay and have a real impact on daily work. Despite the relatively low percentage, the absolute number of cases in which interference was detected was not trivial and underlines the importance of careful validation by laboratory professionals with specific skills (supported, where possible, by self-verification and clinical decision support tools ¹⁰ ).