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. 2013 Jun;59(6):982-90.
doi: 10.1373/clinchem.2012.195594. Epub 2013 Feb 8.

Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies

Mark M Kushnir  1 , Alan L Rockwood, William L Roberts, Dev Abraham, Andrew N Hoofnagle, A Wayne Meikle
Affiliations

Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies

Mark M Kushnir et al. Clin Chem. 2013 Jun.

Abstract

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.

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Figures

Figure 1
Figure 1
Chromatogram of patient sample containing 5 ng/mL of thyroglobulin. Mass transitions of the VIFDANAPVAVR peptide m/z 636.36/1059.56 (A), 636.36/912.49 (B), 636.36/541.35 (C); and the internal standard m/z 639.34/1065.56 (D), 639.34/918.48 (E), 639.34/547.34 (F).
Figure 2
Figure 2
Results of the LC-MS/MS method comparison with Access™ analyzer (Beckman Coulter) using Tg-AAb negative (A, B) and Tg-AAb positive samples (C). A: concentrations 0–350 ng/mL IA=0.99*LC-MS/MS-3.1, r=0.980, Sy,x=14.8; B: concentrations <60 ng/mL. IA=0.94*LC-MS/MS-3.7, r=0.946, Sy,x=4.2; C: IA=0.53*LC-MS/MS −0.1, r=0.586, Sy,x = 1.1.
Figure 3
Figure 3
Results of comparison of the evaluated LC-MS/MS method with LC-MS/MS method of the University of Washington (12) using (A) Tg-AAb negative (n=21), (B) Tg-AAb positive samples (n=29), and (C) comparison with Access™ analyzer (Beckman Coulter) using Tg-AAb positive samples (samples corresponding to the results shown on pane B). A: LC-MS/MSUW=1.17* LC-MS/MSeval. −1.81, r=0.951, Sy,x=8.14; B: LC-MS/MSUW=1.23*LC-MS/MS eval. + 0.15, r=0.917, Sy,x=0.475.)
Figure 4
Figure 4
Distribution of concentrations of thyroglobulin in healthy children and adults.
See this image and copyright information in PMC

Comment in

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