More sensitivity is always better: Measuring sub-clinical levels of serum thyroglobulin on a µLC–MS/MS system

Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for thyroglobulin (Tg) are resistant to autoantibody (TgAb) interference, recent studies have demonstrated approximately 40% of TgAb-positive individuals with recurrent thyroid cancer have Tg concentrations below the lower limit of quantification (LLOQ) of the LC-MS/MS assays described to date (i.e., <0.5 ng/mL), resulting in false-negative findings during post-thyroidectomy monitoring. To reduce false negative results due to insufficient analytical sensitivity, a new Tg assay was developed on a commercially available LC-MS/MS system operating at microliter/minute flow-rates (i.e., µLC-MS/MS) to maximize the analytical sensitivity and achieve a LLOQ of 0.02 ng/mL. When applied to the measurement of TgAb-negative and TgAb-positive patient serum samples previously measuring below the LLOQ of current immunometric and LC-MS/MS assays (LLOQ, 0.1-0.2 ng/mL), concentrations were measurable by µLC-MS/MS in 66% and 44% of samples, respectively - possibly explaining the persistence of TgAb in those patients. Patients with low Tg concentrations measured by µLC-MS/MS (<0.1 ng/mL) also exhibited elevation in their Tg concentrations upon hormone stimulation, indicating the detected Tg was produced from remnant thyroid tissue and would be suitable as a tissue biomarker. Forty-eight TgAb-positive patient specimens with undetectable Tg by both conventional LC-MS/MS (<0.15 ng/mL) and immunometric (<0.1 ng/mL) assays demonstrated measureable Tg concentrations by the new µLC-MS/MS assay in approximately one-third of the specimens, despite all patients being disease free at the time of collection, suggesting interference-free monitoring of low Tg levels may be feasible prior to the on-set of recurrent disease using a sensitive LC-MS/MS assay.