Phantoms in the Assay Tube: Heterophile Antibody Interferences in Serum Thyroglobulin Assays

Abstract

Serum thyroglobulin (Tg) measurement is a major means of detecting thyroid cancer recurrence. Unlike anti-Tg autoantibody interferences, heterophile antibody (HAB) immunoassay interferences are not well recognized by laboratorians or clinicians as a Tg assay problem. When HAB interferences occur, they usually result in false positive test results. With the current trend to treat some thyroid cancer patients with radioiodine on the basis of an elevated serum Tg result alone, this has the potential to result in unwarranted therapy. We evaluated the prevalence of HAB interference in a commonly used automated immunoassay in 1106 consecutive specimens with Tg values greater than 1 ng/ml. All Tg measurements were repeated after sample incubation in heterophile-blocking tubes (HBT). Results, which showed a more than 3 SD percentage difference from the original result, were considered to suffer from HAB interference. All possible interferences were confirmed by dilution testing. After HBT treatment, Tg levels dropped to less than 1 ng/ml in 32 specimens (P < 0.0000001), 20 of which fell to less than 0.1 ng/ml (P < 0.00002). Of these 20, 17 were anti-Tg autoantibody negative, and all 32 showed a fall of greater than 3 SD percentage (>56.91%) compared with the original result. There were also two samples that showed a significant increase of greater than 56.91% after HBT treatment. HAB interference is relatively prevalent (1.5-3%) in a commonly used automated Tg assay and can lead to clinically significant artifacts. It is currently unknown, but possible, that other immunometric Tg assays suffer from similar problems. Unless a Tg assay is confirmed to be free of HAB interference or uses additional blocking steps, as ours now does, HAB interference should be suspected if Tg results do not fit the clinical picture.

Heterophile antibodies may falsely increase or decrease thyroglobulin measurement in patients with differentiated thyroid carcinoma

“…HAbs can form a bridge between the capture and detection antibody, leading to a false positive result in absence of analyte, or to a falsely higher result in presence of analyte (5). Preissner and co-workers reported 32 false positive or falsely increased serum Tg values from 1106 patients with serum TgG1.00 ng/mL (2%) (6). However, HAb may also bind to the capture antibody preventing the binding of analyte and/or detection antibody causing false negative or falsely low results (5).…”

Development, characterization and clinical validation of new sensitive immunofluorometric assay for the measurement of serum thyroglobulin

“…Interference caused by heterophilic antibodies (HAMA) is a potential problem for all assays employing IMA methodology and mouse monoclonal antibody reagents (11). HAMA interference arises when antibodies that recognize murine proteins are present in the patient serum and interact with the capture and/ or labeled monoclonal antibody reagents.…”

“…HAMA is a potential problem for all immunometric assays, since most of them are based on monoclonal antibodies produced in mice. In the case of Tg assays, as in most other immunometric assays, the presence of HAMA can lead to false-positive (most of the time), or false-negative results (11,12). The potential presence of HAMA is still a challenge, but accumulated knowledge has paved the way to deal adequately with the phenomenon most of the time (13).…”

Molecular technology and the recombinant TSH have changed diagnostics of thyroid carcinoma with positive I-131 whole body scan but low serum thyroglobulin

“…Unlike anti-Tg autoantibody interferences, heterophile antibody (HAB) immunoassay interferences are not well recognized by laboratorians or clinicians as a Tg assay problem. When HAB interferences occur, they usually result in false positive test results (Preissner et al, 2003). With the current trend to treat some thyroid cancer patients with radioiodine on the basis of an elevated serum Tg result alone, this has the potential to result in unwarranted therapy.…”