title: Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies.
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.
authors: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA. kushnmm@aruplab.com
authors: Mark M
authors: MM
authors: Kushnir
authors: Alan L
authors: AL
authors: Rockwood
authors: William L
authors: WL
authors: Roberts
authors: Dev
authors: D
authors: Abraham
authors: Andrew N
authors: AN
authors: Hoofnagle
authors: A Wayne
authors: AW
authors: Meikle
mesh_terms: D000293:Adolescent; D001323:Autoantibodies / Q000378:metabolism*; D001774:Blood Chemical Analysis / Q000379:methods* / Q000592:standards; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid; D005189:False Positive Reactions; D005260:Female; D006801:Humans; D007223:Infant; D057230:Limit of Detection; D008297:Male; D010949:Plasma / Q000737:chemistry*; D053719:Tandem Mass Spectrometry; D013954:Thyroglobulin / Q000097:blood*
chemical_list: D001323:Autoantibodies; C006606:anti-thyroglobulin; D013954:Thyroglobulin
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