Endocrine Abstracts

Background: Serum thyroglobulin (Tg) measurements are one of the main pillars of thyroid cancer follow-up, because of the high organ and disease specificity of Tg in athyrotic patients. However, 20–25% of patients have detectable serum anti-Tg auto-antibodies (TgAB), which might cause false low Tg measurements in immunometric assays (IMA). This can lead to significant challenges in patient management. Measurement of Tg by liquid chromatography-tandem mass spectrometry (LC–MS/MS) after tryptic digestion can in theory overcome this problem, as TgAB and Tg are both digested equally, and Tg-specific tryptic fragments can be detected selectively. Proof of concept for such a LC–MS/MS Tg assay was shown for TgAB-negative patients in 2008 (Clin Chem 54:1796–1804). We extended these observations to TgAB-positive patients.

Methods: Samples were depleted of albumin and other low/mid molecular weight proteins, trypsin digested, and purified by solid phase extraction after addition of synthetic non-radioactive isotopic peptide internal standard (IS). The extracts were analyzed by LC–MS/MS for a highly ionizable proteotypic Tg fragment and its corresponding IS. Purified Tg, matched to the international Tg reference preparation, was used for calibration.

Results: The assay’s limit of detection was 2 ng/mL. Inter-assay imprecision (CV) was 4–12% (Tg range: 3.8–150 ng/ml). Method agreement with the Beckman Coulter Tg IMA in 119 TgAB-negative patients with detectable Tg showed a slope of 0.75, intercept: +3.8, R-squared: 0.87. By contrast, in 20 TgAB-positive patients with detectable Tg, the slope was 1.77, consistent with under-recovery of Tg in the IMA. Consistent with these observations, a comparison of Tg recovery between LC–MS/MS and IMA, following addition of high TgAB concentrations to Tg samples, showed under-recovery in the IMA.

Conclusion: Tryptic digest-based LC–MS/MS assays can measure Tg accurately in the presence of TgAB that lead to false low IMA measurements.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.