Measurement of thyroglobulin by liquid chromatography tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies.
BACKGROUND Measurement of serum thyroglobulin (Tg) is used to monitor patients after treatment for differentiated thyroid carcinoma (TC) Difficulty in using Tg as a biomarker of the recurrence of TC in many patients stems from the presence of endogenous anti Tg autoantibodies (Tg AAbs) which can interfere with immunoassays (IAs) and cause false negative results. METHODS We enriched Tg from serum samples using rabbit polyclonal anti Tg antiserum and protein precipitation. Unrelated proteins were partially depleted in the process. Enriched proteins were then denatured, reduced, and digested with trypsin after the addition of a winged internal standard peptide. A Tg specific tryptic peptide was purified by immunoaffinity extraction and analyzed by 2 dimensional LC MS/MS. Instrument cycle time was 6.5 min per sample. RESULTS The lower limit of quantification was 0.5 ng/mL (0.76 fmol/mL dimer) Total imprecision of triplicate measurements in serum samples over 5 days was <10% Comparison with a commercial IA using serum samples free of Tg AAb (n 73) showed Deming regression, IA 1.00 LC MS/MS 2.35, r 0.982, standard error of the estimate (S(y|x) 9.52. In a set of Tg AAb positive samples that tested negative for Tg using IA (n 71) concentrations determined by LC MS/MS were =0.5 ng/mL in 23% of samples (median 1.2, range 0.7 11 ng/mL) CONCLUSIONS The introduced method has acceptable performance characteristics for use in clinical diagnostic applications. The most substantial disagreement between methods was observed in Tg AAb positive samples with concentrations <2 ng/mL (determined with LC MS/MS) The affinity assisted enrichment strategy used for Tg in this method should be applicable to other biomarkers that have endogenous autoantibodies.
Clinical chemistry