Session: SUN 459-496-Thyroid Neoplasia & Case Reports
Poster Board SUN-496
Tg was enriched from serum samples using rabbit polyclonal anti-Tg antibody and protein precipitation; unrelated proteins were partially depleted. Internal standard was added to the Tg-containing fraction, proteins were then denatured, reduced, and digested with trypsin. A Tg-specific tryptic peptide was purified by immunoaffinity enrichment and analyzed by 2D-LC-MS/MS. Instrument cycle time was 6.5 min per sample.
The lower limit of quantification was 0.5 ng/mL (0.76 fmol/mL). Total imprecision of triplicate measurements in serum samples over five days was less than 10%. Comparison with a commercial IA using serum samples free of Tg-AAb (n=73) showed Deming regression equation IA= 1.00*LC-MS/MS-2.35, r=0.982, Sy,x=9.52. In a set of Tg-AAb positive samples tested negative for Tg using IA (n=71), concentrations determined by LC-MS/MS method were at or above 0.5 ng/mL in 23% of samples (median 1.2, range 0.7-11 ng/mL). The method was also compared with an LC-MS/MS method of another laboratory using a set of Tg-AAb negative (n=21) and Tg-AAb positive (n=29) samples. For Tg-AAb negative samples Deming regression equation was LC-MS/MS1=1.17*LC-MS/MS2 -1.81, r=0.951, Sy,x=8.14; for the Tg-AAb positive samples, LC-MS/MS1=1.23*LC-MS/MS2 + 0.15, r=0.917, Sy,x=0.475.
The method has acceptable performance characteristics for use in clinical diagnostic applications. The most substantial disagreement between the methods was observed in Tg-AAb positive samples with concentration below 2 ng/mL (determined with LC-MS/MS), which was likely caused by interference of Tg-AAb with the IA.
Nothing to Disclose: MMK, ALR, ANH, AWM
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