MSACL 2025 Abstract

Validation of a Clinical LC-MS/MS Assay for Thyroglobulin Measurement in Serum

Magdalena Bolek, Chris Hodgkins, Cameron Wood, Mark Ridgwell, Laura Buckton, Maria Charan, Basia Hiley, Lucy Ding, Doug Chesher, Peter Ward
Royal North Shore Hospital, Sydney, Australia

Magdalena Bolek, PhD (Presenter) NSW Health Pathology

Presenter Bio: Magda is a Medical Laboratory Scientist at the Royal North Shore Hospital. She gained experience in Biomedical Research working as Postdoctoral Research Scientist in the higher education sector. Strong analytical, problem-solving and project management skills. Experienced in LC/MS/MS operation and method development, clinical and biomedical research, with good knowledge of research ethics, quality control and quality system regulations for clinical laboratory and clinical laboratory standards.

Relevant Financial Disclosures (within past 24 months, reported on Jun 29, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Serum thyroglobulin (Tg) determination is important in clinical management of patients with differentiated thyroid cancer (DTC), to monitor residual or recurrent disease. Tg is typically measured by automated immunoassays. However, thyroglobulin autoantibodies (TgAb), which are present in 20–30% of patients with DTC, can interfere with antibody-based analyses, giving rise to falsely low, or negative Tg measurements [1]. This interference varies between patients and Tg assays, and only loosely correlates with TgAb concentrations [1,2].

Mass spectrometry-based quantification of Tg [3-5] eliminates antibody interferences and has become the preferred method, despite technical challenges. This study describes the adaptation and validation of an LC-MS/MS method for Tg measurement in human serum.

METHODS:
The methodology was based on a previously published method [5], with some modifications. Calibration samples were prepared in Tg(-), TgAb(-) human serum spiked with human Tg (Tg II CalSet, Cobas, Roche). The method was compared to Elecsys Tg II assay (Cobas e601, Roche) using patient samples (n=70) and three levels of QCs prepared from pooled patient serum (0.3, 1.1, and 7 ng/mL). Analyte recovery was evaluated by spiking the internal standard before and after key extraction steps and compared to neat standard in the elution buffer.

RESULTS:
The LC-MS/MS assay achieved a lower limit of quantification of 0.05 ng/mL (CV <20%) and showed a good agreement with the immunoassay in TgAb(-) samples (n=41). The imprecision of low, mid, and high QC samples was 6.8, 7.3 and 5.4 % CV respectively (based on n=66 from at least 20 days). Linearity of the calibration curve between 0.05 and 12.5 ng/mL was excellent, with an average correlation co-efficient (r2) of 0.999, and average slope of 0.952 (intra-day %CV = 7.5). Improved recovery was achieved by decreasing denaturation time to 30 min and increasing the digestion time to 1 h.

CONCLUSION:
The adapted method has acceptable performance characteristics and suggests that accurate and sensitive mass-spectrometry-based quantification of serum thyroglobulin can be achieved in clinical laboratories with access to adequate LC-MS instrumentation and laboratory infrastructure. Future work will evaluate the usefulness of Tg-MS analyses for clinical decision making and the impact of automation on assay performance.

REFERENCES:
[1] C.A. Spencer, Clinical review: Clinical utility of thyroglobulin antibody (TgAb) measurements for patients with differentiated thyroid cancers (DTC), J. Clin. Endocrinol. Metab. 96 3615–3627 (2011).
[2] C. Evans, S. Tennant, P. Perros, Thyroglobulin in differentiated thyroid cancer, Clin. Chim. Acta 444:310-317 (2015).
[3] Clarke NJ, Zhang Y, Reitz RE. A novel mass spectrometry-based assay for the accurate measurement of thyroglobulin from patient samples containing antithyroglobulin autoantibodies. J Investig Med. 2012; 60:1157–1163.
[4] Netzel BC, Grebe SK, Algeciras-Schimnich A. Usefulness of a thyroglobulin liquid chromatography-tandem mass spectrometry assay for evaluation of suspected heterophile interference. Clin Chem (2014) 60:1016 –1018.
[5] Shi J, Phipps W, et al., A distributable LC-MS/MS method for the measurement of serum thyroglobulin. JMSACL, 2022, 26: 28-33.