Serum thyroglobulin (Tg) monitoring of patients with differentiated thyroid cancer using sensitive (second-generation) immunometric assays can be disrupted by false-negative and false-positive serum thyroglobulin autoantibody misclassifications

Abstract

Context: Reliable thyroglobulin (Tg) autoantibody (TgAb) detection before Tg testing for differentiated thyroid cancer (DTC) is critical when TgAb status (positive/negative) is used to authenticate sensitive second-generation immunometric assay ((2G)IMA) measurements as free from TgAb interference and when reflexing "TgAb-positive" sera to TgAb-resistant, but less sensitive, Tg methodologies (radioimmunoassay [RIA] or liquid chromatography-tandem mass spectrometry [LC-MS/MS]).

Objective: The purpose of this study was to assess how different Kronus (K) vs Roche (R) TgAb method cutoffs for "positivity" influence false-negative vs false-positive serum TgAb misclassifications that may reduce the clinical utility of reflex Tg testing.

Methods: Serum Tg(2G)IMA, TgRIA, and TgLC-MS/MS measurements for 52 TgAb-positive and 37 TgAb-negative patients with persistent/recurrent DTC were compared. A total of 1426 DTC sera with TgRIA of ≥ 1.0 μg/L had false-negative and false-positive TgAb frequencies determined using low Tg(2G)IMA/TgRIA ratios (<75%) to indicate TgAb interference.

Results: TgAb-negative patients with disease displayed Tg(2G)IMA, TgRIA, and TgLC-MS/MS serum discordances (% coefficient of variation = 24 ± 20%, range, 0%-100%). Of the TgAb-positive patients with disease, 98% had undetectable/lower Tg(2G)IMA vs either TgRIA or TgLC-MS/MS (P < .01), whereas 8 of 52 (15%) had undetectable Tg(2G)IMA + TgLC-MS/MS associated with TgRIA of ≥ 1.0 μg/L. Receiver operating characteristic curve analysis reported more sensitivity for TgAb method K vs R (81.9% vs 69.1%, P < .001), but receiver operating characteristic curve cutoffs (>0.6 kIU/L [K] vs >40 kIU/L [R]) had unacceptably high false-negative frequencies (22%-32%), whereas false positives approximated 12%. Functional sensitivity cutoffs minimized false negatives (13.5% [K] vs 21.3% [R], P < .01) and severe interferences (Tg(2G)IMA, <0.10 μg/L) (0.7% [K] vs 2.4% [R], P < .05) but false positives approximated 23%.

Conclusions: Reliable detection of interfering TgAbs is method and cutoff dependent. No cutoff eliminated both false-negative and false-positive TgAb misclassifications. Functional sensitivity cutoffs were optimal for minimizing false negatives but have inherent imprecision (20% coefficient of variation) that, exacerbated by TgAb biologic variability during DTC monitoring, could cause TgAb status to fluctuate for patients with low TgAb concentrations, prompting unnecessary Tg method changes and disrupting Tg monitoring. Laboratories using reflexing should limit Tg method changes by considering a patient's Tg + TgAb testing history in addition to current TgAb status before Tg method selection.

Figure 1.

Comparative data for Tg measurements made by ^2GIMA, RIA, and LC-MS/MS for the group A sera from patients with persistent/recurrent DTC. Sera with Tg values below detectability are shown in the shaded areas, indicating the respective sensitivity limits of the methods: <0.10 μg/L (FS) for Tg^2GIMA, <0.5 μg/L (LOQ) for TgLC-MS/MS, and < 0.5 μg/L (FS) for TgRIA. A, 37 patients with disease who had no TgAb detected by either method K or method R (below FS limits). The solid red symbols indicate 5 patients displaying >30% CV between-method discordances. B, Serum Tg values for 52 TgAb-positive patients with disease. The squares show the method comparisons for 12 sera with Tg below the LOQ of the LC-MS/MS. A distinction is made between sera with unequivocally undetectable TgLC-MS/MS values (no peak = solid red squares) and sera with marginally detectable TgLC-MS/MS values in the 0.3 to 0.5 μg/L range (open red squares) (6, 9, 40).

Figure 2.

A, Relationship between TgRIA (abscissa) and Tg^2GIMA (ordinate) for the 367 group B sera that were classified as TgAb negative according to the FS cutoff of both methods K and R. B, Relationship between TgRIA (abscissa) and Tg^2GIMA (ordinate) for the 834 group B sera that were classified as TgAb-positive according to the FS cutoff of both methods K and R. The table below shows the median TgAb for methods K and R, and the percentage of severe interferences (Tg^2GIMA of <0.10 μg/L) seen when TgAb-positive group B sera were analyzed according to TgRIA values (1.0–2.5, 2.6–5.0, 5.1–10, or >10 μg/L).

Figure 3.

Top panels: distribution of TgAb concentrations found for the 607 sequential DTC sera (group C) received for routine Tg + TgAb testing. Method K (left panel) and method R (right panel) data are analyzed as subgroups with cutoff values covering the range from low to very high values, with focus on the LOD, FS, MCO, and ROC curve cutoffs. Bottom panels: method K (left panel) and method R (right panel) data for the 1426 group B DTC sera with TgRIA of ≥1.0 μg/L (n = 1426). The black bars show the group frequencies for false-negative misclassifications, expressed as a percentage of the total number of sera classified as “negative” by that cutoff. A false-negative TgAb classification was defined as a serum with a TgAb below the cutoff that had a Tg^2GIMA/TgRIA ratio of <75%, suggesting the presence of interfering TgAb. The yellow bars show the group frequencies for false-positive misclassifications, expressed as a percentage of the total number of sera classified as “positive” by that cutoff. A false-positive TgAb misclassification was defined as a serum with a TgAb value more than or equal to the cutoff that had a Tg^2GIMA/TgRIA ratio of ≥75%, suggesting the absence of interfering TgAb. The red bars show the frequencies for severe interference, defined as an undetectable Tg^2GIMA (<0.10 μg/L) associated with an unequivocally detectable TgRIA (≥1.0 μg/L). Blue bars show the frequencies for sera with inappropriately low Tg^2GIMA (<0.30 μg/L).

Figure 4.

Performances of TgAb methods K and R analyzed by ROC curve analyses of group B sera data for cutoffs covering the entire range of concentrations of TgAb methods K and R. AUC, area under the curve.