MASS SPECTROMETRY BASED METHOD FOR ACCURATE MEASUREMENT OF THYROGLOBULIN IN THE PRESENCE OF ANTI-THYROGLOBULIN AUTOANTIBODIES

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 459-496-Thyroid Neoplasia & Case Reports
Clinical
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-496
Mark M Kushnir*1, Alan L Rockwood2, Andrew N Hoofnagle3 and A Wayne Meikle4
1ARUP Laboratories, Salt Lake City, UT, 2University of Utah, Salt Lake City, UT, 3University of Washington, Seattle, WA, 4Univ of UT Med Sch, Salt Lake City, UT
Measurement of thyroglobulin (Tg) in serum and plasma is used to monitor patients after treatment for differentiated thyroid carcinoma (DTC). Difficulty in using Tg as a biomarker of the recurrence of DTC is related to the presence in many patients of endogenous anti-Tg autoantibodies (Tg-AAb), which can interfere with immunoassays (IA) and cause false-negative results.

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

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT
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