Abstract
Background
The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to overcome interference from thyroglobulin autoantibodies (TgAb); however, it has not yet been implemented in Korea. This study aimed to confirm the accuracy of LC-MS/MS compared to conventional methods and to identify its advantages in patients with thyroid carcinoma (TC).
Methods
A total of 206 TC and 18 Hashimoto’s thyroiditis samples were collected. TgAb-positive (TgAb-P) was defined as TgAb >60 U/mL. Tg testing was performed using LC-MS/MS, immunoradiometric assay (Tg-IRMA), and chemiluminescence microparticle immunoassay (Tg-CMIA). The interference of TgAb in LC-MS/MS and CMIA methods was evaluated through an in vitro TgAb spiking experiment.
Results
The frequency of TgAb-P in TC samples was 76.2%. Correlations between assays were as follows: Tg measurements made by LC-MS/MS (Tg-MS) and Tg-IRMA (R=0.93), Tg-MS and Tg-CMIA (R=0.96), and Tg-CMIA and Tg-IRMA (R=0.99), and it was lower in TgAb-P than TgAb-negative group. Clinical factors (total thyroidectomy, thyroid lobectomy, and Hashimoto’s thyroiditis) did not affect these correlations. In TgAb spiking experiments, Tg-CMIA showed false negatives in TgAb-P, whereas Tg-MS did not. Among 21 TC cases with highly suspicious disease recurrence but Tg-IRMA <1 ng/mL, Tg-MS detected Tg ≥0.5 ng/mL in six samples. However, there was no consistent pattern of recurrence or TgAb trends.
Conclusion
Correlations between assays were lower in TgAb-P cases. The spike test results show Tg-MS is less prone to false negatives in TgAb-P cases. Tg-MS may improve Tg detection in TgAb-P cases. However, we could not identify a distinct patient group with shared clinical features that would benefit from Tg-MS.
Keywords: Thyroglobulin, Mass spectrometry, Immunoradiometric assay, Chemiluminescence microparticle immunoassay, Autoantibody, Clinical utility
INTRODUCTION
Thyroglobulin (Tg) is a large iodoglycoprotein that serves as the primary substrate for the synthesis of thyroid hormones. Because Tg is synthesized only in thyroid follicular cells, it is a useful marker for the presence of thyroid tissue [1]. The primary clinical use of serum Tg is as a tumor marker for differentiated thyroid carcinoma (DTC), which is derived from the follicular epithelial cells [2-5] and comprises papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC). A thyroidectomy is the initial standard treatment for patients with this condition [4]. Postoperative serum Tg plays a crucial role in assessing the persistence of disease or thyroid remnant and predicting potential future disease recurrence, particularly in patients who have undergone a total thyroidectomy followed by adjuvant radioactive iodine (RAI) therapy [6,7].
Various methods, including the immunoradiometric assay (IRMA), chemiluminescence microparticle immunoassay (CMIA), and radioimmunoassay (RIA), are used to measure serum Tg. IRMA and CMIA are used more often than RIA due to shorter incubation times, lower functional sensitivities (≤0.1 ng/mL), and stability of labeled antibody reagents [8]. However, current Tg assays are susceptible to interference from thyroglobulin autoantibodies (TgAb), which are present in approximately 25% of DTC patients [9,10]. Endogenous TgAb can mask epitopes used by reagent antibodies when measuring serum Tg by an IRMA or CMIA. Consequently, serum Tg in patients with TgAb is often underestimated, leading to false-negative results, and masking disease recurrence [11-14]. While an RIA is generally less prone to TbAb interference than an IRMA or a CMIA, it may still occasionally yield both false-positives and false-negatives [8,14,15]. The Community Bureau of Reference of the Commission of the European Communities developed the certified reference material (CRM)-457 international standard; however, interlaboratory variability between Tg assays remains unresolved [16].
To overcome such interference, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as an analytic technique for quantifying peptides and proteins in biological samples [17-19]. In brief, Tg is digested in trypsin, and antibodies to those peptides are quantified using LC-MS/MS. However, the clinical significance of LC-MS/MS compared with an IRMA is not well established. The aim of this study was to evaluate the accuracy of LC-MS/MS, a method performed for the first time in Korea, in comparison with conventional methods such as IRMA and CMIA. Furthermore, we analyzed the effectiveness of LC-MS/MS, IRMA, and CMIA for measuring Tg in various clinical settings and assessed the potential advantages of LC-MS/MS in patients with thyroid carcinoma (TC).
METHODS
Patients and samples
The patients enrolled in this study were divided into two groups. For the first group, we randomly selected 98 retrospectively collected residual samples from TC patients to evaluate the accuracy of Tg measurements made by LC-MS/MS (Tg-MS) between October 2019 and December 2021 (period 1). The primary purpose of collecting this group was to confirm the accuracy of the LC-MS/MS test compared to Tg-IRMA and Tg-CMIA. All samples in this group were tested using LC-MS/MS, IRMA, and CMIA methods. All measurements were performed on the same samples collected at the same time point and then compared. Of the 98 samples, Tg-IRMA results were obtained for all 98 samples, Tg-MS results for 92 samples, and Tg-CMIA results for 96 samples.
In the second group, we prospectively enrolled 108 TC samples from patients who underwent a total thyroidectomy followed by RAI therapy and were positive for TgAb (TgAb-P), defined as TgAb >60 U/mL. These well-characterized samples were collected between May 2022 and December 2022 (period 2). The main purpose of collecting this group was to evaluate the clinical advantage of the LC-MS/MS method in TgAb-P samples, as TgAb positivity is known to interfere with Tg measurement. All samples yielded Tg-MS and Tg-IRMA results; however, CMIA was not tested in this group. All measurements were performed on the same samples collected at the same time point and then compared. Patients visiting the outpatient clinic of the Samsung Medical Center, Seoul, Korea, for a regular check-up supplied blood samples and provided informed consent to participate in the study. Their electronic medical records, pathology reports, imaging, and previous blood test results, including Tg and TgAb levels, were reviewed.
During the study period, 18 Hashimoto’s thyroiditis samples were gathered to analyze the accuracy of Tg-MS in the TgAb-P samples. Among the 18 samples, four were collected during period 1, and 14 during period 2.
There were no differences in treatment, follow-up, or Tg measurement methods between the two periods. The only difference between the two groups was the method of enrollment. Therefore, we combined the groups for analysis to provide a broader representation of clinical characteristics.
This study underwent a review and was approved by the Institutional Review Board of the Samsung Medical Center. For samples in period 1, the need for informed consent was waived by the committee because the group involved residual samples (IRB number 2021-06-198). For samples in period 2, informed consent was obtained (IRB number 2022-04-161).
Tg and TgAb measurement assays
Three assays were selected for Tg measurements. In a protocol we developed for Tg-MS measurements, 80 μL of eluted samples were injected into an LC-MS/MS system consisting of an Agilent 1290 infinity liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled with a Qtrap 5500+ mass spectrometer (ABSciex, Framingham, MA, USA). For high-performance liquid chromatography columns, a Zorbax 300 SB-C8 (Agilent Technologies) and Poroshell 120 EC-C18 (Agilent Technologies) were used. Tryptic Tg-specific peptides (FSPDDSAGASALLR) and IS were quantified in multiple-reaction monitoring mode with a 5-point linear calibration curve using Access Thyroglobulin Calibrators (Beckman Coulter, Pasadena, CA, USA). The lower limit of quantitation (LLoQ) of Tg-MS was <0.5 ng/mL.
Tg-IRMA was conducted using the BRAMHS Tg plus assay (Thermo Fisher Scientific, Waltham, MA, USA) and a Gamma 10 counter (Shinjin Medics Inc., Seoul, Korea). The assay was calibrated by CRM-457 and measurement values were doubled before analysis, following the manufacturer’s instructions. CMIA (Tg-CMIA) was performed using Architect Thyroglobulin on an Architect i2000SR immunoassay analyzer (Abbott Laboratories, Illinois, CA, USA). The LLoQs of Tg-IRMA and Tg-CMIA were <0.2 and <0.14 ng/mL, respectively. TgAb was measured using an IRMA on the RIA-MAT 280 assay apparatus (Stratec, Birkenfeld, Germany). The cut-off for serum TgAb was 60 IU/mL.
In vitro TgAb spiking experiments
To evaluate the effect of TgAb in samples, two spiking experiments were performed in vitro. The mixtures were measured using both LC-MS/MS and CMIA, and the percent difference was calculated from (test–control)/control−100 (%).
In the first in vitro study, eight pairs of mixtures were manufactured by mixing Tg-rich TgAb-negative (TgAb-N) samples and athyrotic TgAb-P samples (test) or pooled athyrotic TgAb-N plasma (control) in a 1:1 ratio. The TgAb concentration ranged from 125 to 426 IU/mL.
In the second in vitro study, five mixtures with varying TgAb concentrations were prepared to evaluate the correlation between interference and TgAb concentrations. An Access Calibrator S5 (500 ng/mL) was mixed with athyrotic TgAb-P samples (test) or pooled athyrotic TgAb-N plasma (control). The final TgAb concentrations were set at 0, 200, 400, and 800 IU/mL. TgAb-P samples included those from TC and Hashimoto’s thyroiditis patients to minimize bias caused by patient-specific TgAb factors.
Assessment of clinical significance
We categorized samples into four response groups based on 2015 American Thyroid Association (ATA) guidelines: excellent, indeterminate, biochemically incomplete, and structurally incomplete [4]. A structurally incomplete response was defined as the persistence of disease and/or identification of new locoregional or distant metastases. Disease recurrence was confirmed pathologically or through computed tomography, magnetic resonance imaging, 18F-fluorodeoxyglucose positron emission tomography, and/or an RAI scan. To examine the relationship between the changing trend of TgAb and the detection rate of Tg-MS, we divided patients into three groups: those with a decrease in TgAb compared with the previous result, those with an increase in TgAb of less than 50% compared with the previous result, and those with an increase in TgAb of more than 50%. The previous result was obtained within 6 and 12 months before the day Tg-MS was tested.
The clinical significance of Tg-MS was evaluated in samples from patients who underwent a total thyroidectomy and/or RAI. Currently, the IRMA method is used to measure Tg in our institution. We therefore focused on comparing Tg-IRMA and Tg- MS to assess the clinical significance of Tg-MS. Detailed clinical information was provided for samples exhibiting discrepancies between imaging studies and Tg-IRMA levels. According to current ATA guidelines, non-stimulated Tg levels of 0.2≤ Tg <1 ng/mL and Tg ≥1 ng/mL are considered cut-offs for indeterminate response and biochemical incomplete response, respectively [4,20,21]. We therefore assessed clinical information in samples showing structural recurrence with Tg-IRMA <1 ng/mL or no evidence of tumor recurrence in imaging studies with Tg-IRMA <1 ng/mL but Tg-MS ≥1 ng/mL.
Statistical analysis
Continuous variables were presented as means±standard deviations or medians and interquartile ranges. Categorical variables were presented as numbers and percentages. Regression analysis was conducted to compare the levels of serum Tg, and the Pearson product-moment correlation coefficient (R) was calculated to determine the relationships among the results of the assays. The LLoQ value in each assay was excluded when calculating the R-value. The statistical analysis was performed in SPSS version 27.0 (IBM, Chicago, IL, USA), and Excel 2019 (Microsoft, Redmond, WA, USA).
RESULTS
Baseline characteristics
Clinicopathological characteristics of TC patients are presented in Table 1. A total of 206 samples from 191 patients were included. All 206 samples had Tg-IRMA results, 200 samples had Tg-MS results, and 96 samples had Tg-CMIA results. The median TgAb level among all samples was 104.3 U/mL (range, 5.2 to 43,058.0). There were 49 TgAb-N samples (23.8%), with a median TgAb level of 27.7 U/mL (range, 5.2 to 58.4). The number of TgAb-P samples was 157 (76.2%), with median TgAb level of 143 U/mL (range, 60.1 to 43,058.0). Among them, 164 (79.6%) underwent total thyroidectomy and/or followed by RAI therapy, and 42 (20.4%) underwent a less-than-total thyroidectomy. Among the samples, 154 (94.2%) were diagnosed with PTC, and 55 (26.7%) exhibited structurally incomplete responses at the time of the draw.
Table 1.
Baseline Characteristics of Thyroid Carcinoma Patients
| Characteristic | Value |
|---|---|
| Total samples (patients) | 206 (191) |
| Age at time of diagnosis, yr | 45.8±13.0 |
| Sex | |
| Female | 163 (79.1) |
| Male | 43 (20.9) |
| Thyroglobulin, LC-MS/MS, ng/mLa | 7.4 (0.5–22,771.0) |
| Thyroglobulin, IRMA, ng/mLa | 0.4 (0.2–25,990.0) |
| Thyroglobulin, CMIA, ng/mLa | 10.0 (0.2–801.19) |
| Thyroglobulin antibody, U/mL | 104.3 (5.2–43,058.0) |
| Thyroglobulin antibody <60 U/mL | 49 (23.8) |
| Thyroglobulin antibody ≥60 U/mL | 157 (76.2) |
| Treatment | |
| Less than total thyroidectomy | 42 (20.4) |
| Total thyroidectomy without RAI therapy | 8 (3.9) |
| Total thyroidectomy with RAI therapy | 156 (75.7) |
| Subtype | |
| Papillary thyroid carcinoma | 154 (94.2) |
| Follicular thyroid carcinoma | 8 (3.9) |
| Poorly differentiated thyroid carcinoma | 4 (1.9) |
| T stageb | |
| T1 | 127 (64.1) |
| T2 | 19 (9.6) |
| T3 | 35 (17.7) |
| T4 | 17 (8.6) |
| N stageb | |
| N0/Nx | 51 (25.8) |
| N1a | 54 (27.3) |
| N1b | 93 (47.0) |
| M stage | |
| M0 | 192 (93.2) |
| M1 | 14 (6.8) |
| Disease status at the time of drawc | |
| Excellent response | 22 (10.7) |
| Indeterminate response | 118 (57.3) |
| Biochemical incomplete response | 11 (5.3) |
| Structural incomplete response | 55 (26.7) |
| Follow-up duration from diagnosis to draw, mo (median, IQR) | 68.5 (29.0–122.0) |
Values are expressed as mean±standard deviation, number (%), or median (range) unless otherwise indicated.
LC-MS/MS, liquid chromatography-tandem mass spectrometry; IRMA, immunoradiometric assay; CMIA, chemiluminescence microparticle immunoassay; RAI, radioactive iodine; T, tumor; N, node; M, metastasis.
Of 206 samples, all 206 samples had thyroglobulin results using the IRMA method, 200 had thyroglobulin results using the LC-MS/MS method, and 96 had thyroglobulin results using the CMIA method;
Eight samples were excluded due to information about the initial stage was unavailable;
Disease status was assessed based on the dynamic risk stratification system outlined in the 2015 American Thyroid Association guidelines.
Supplemental Table S1 lists the clinical characteristics of patients with Hashimoto’s thyroiditis. A total of 18 samples were included, with a median TgAb level of 526.2 IU/mL (range, 62.4 to 34,104.6).
Comparison of three assays
Correlations between Tg-MS and Tg-IRMA, Tg-MS and Tg-CMIA, and Tg-CMIA and Tg-IRMA were evaluated in TgAb-P and TgAb-N samples, respectively (Fig. 1). A strong correlation was shown between Tg-MS and Tg-IRMA (R=0.93), Tg-MS and Tg-CMIA (R=0.99), and Tg-CMIA and Tg-IRMA (R=0.99). In a comparison of the three assays, a correlation was particularly strong in TgAb-N samples compared with TgAb-P samples.
Fig. 1.
Comparison between thyroglobulin (Tg) measured by liquid chromatography-tandem mass spectrometry (Tg-MS) and Tg measured by immunoradiometric assay (Tg-IRMA), Tg-MS and Tg measured by chemiluminescence microparticle immunoassay (Tg-CMIA), and Tg-IRMA and Tg-CMIA. (A) Between Tg-MS and Tg-IRMA in all samples. (B) Between Tg-MS and Tg-IRMA in samples with thyroglobulin antibody (TgAb) ≤60 U/mL. (C) Between Tg-MS and Tg-IRMA in samples with TgAb >60 U/mL. (D) Between Tg-MS and Tg- CMIA in all samples. (E) Between Tg-MS and Tg-CMIA in samples with TgAb ≤60 U/mL. (F) Between Tg-MS and Tg-CMIA in samples with TgAb >60 U/mL. (G) Between Tg-CMIA and Tg-IRMA in all sample. (H) Between Tg-CMIA and Tg-IRMA in samples with TgAb ≤60 U/mL. (I) Between Tg-CMIA and Tg-IRMA in samples with TgAb >60 U/mL.
When comparing Tg-MS with Tg-IRMA based on clinical situations, the results demonstrated largely close agreement (Supplemental Fig. S1). The closest agreement was observed in TC patients with TgAb-N (R=0.99), while a lesser fit was seen in TC patients with TgAb-P (R=0.92), lobectomy patients (R= 0.95), and the Hashimoto’s thyroiditis group (R=0.98). Similar results were obtained between Tg-MS and Tg-CMIA (Supplemental Fig. S2), and between Tg-CMIA and Tg-IRMA (Supplemental Fig. S3). We did not calculate the correlation in Hashimoto’s thyroiditis group in those two comparisons due to the small sample size.
Assessment of discrepancies between three assays
The overall concordance between Tg-MS, Tg-IRMA, and Tg-CMIA is presented in Supplemental Table S2. Of the 224 samples, 218 had both Tg-MS and Tg-IRMA results, and 94 had both Tg-MS and Tg-CMIA results. Tg-MS identified an additional 12 or 13 samples compared with Tg-IRMA, depending on the positivity cut-off values of 0.2 or 0.5 ng/mL, respectively. Tg-MS identified an additional one or four samples compared with Tg-CMIA, depending on the positivity cut-off values of 0.14 or 0.5 ng/mL. Supplemental Table S3 presents the data for TgAb >60 and ≤60 U/mL separately. Among the TgAb >60 U/mL samples, Tg-MS identified 12 cases that were not detected by Tg-IRMA, using a positivity cut-off value of 0.5 ng/mL. The TgAb levels in these cases ranged from 103.0 to 7,241.0 U/mL. Tg-MS identified three cases compared to Tg-CMIA. The TgAb levels in these cases ranged from 68.9 to 1,027.7 U/mL.
Supplemental Fig. S4 depicts the absolute Tg levels measured by three assays at various Tg and TgAb levels for individual samples. In samples with Tg-MS ≤2 ng/mL, most showed negative biases in Tg-IRMA and Tg-CMIA compared with Tg-MS (Supplemental Fig. S4A). For samples with Tg-MS >2 and ≤ 20 ng/mL, the negative bias was less prominent in samples with TgAb levels <150 U/mL. In this group, all samples with TgAb ≥150 U/mL showed a negative bias in Tg-IRMA and Tg-CMIA compared with Tg-MS (Supplemental Fig. S4B). In samples with Tg-MS >10 and ≤50 ng/mL, a negative bias was predominant in samples with TgAb ≥1,000 IU/mL (Supplemental Fig. S4C). No significant bias was noted in samples with Tg-MS >20 ng/mL; however, samples with TgAb >1,000 IU/mL were not included in this group (Supplemental Fig. S4D).
An in vitro TgAb spiking test was conducted to identify TgAb interference (Fig. 2). In the CMIA method, TgAb-P mixtures consistently displayed a negative bias compared with controls. In contrast, LC-MS/MS did not produce a consistent bias (Fig. 2A). The median percentage differences of CMIA and LC-MS/MS were −29.4% (range, −48.8% to −7.9 %) and 3.7% (range, −9.1% to 23.2%), respectively.
Fig. 2.
Results of the in vitro thyroglobulin antibody (TgAb) spiking test. (A) Mixing TgAb-negative and thyroglobulin (Tg)-positive samples. (B) TgAb spiking to Tg with varying TgAb concentrations. CMIA, chemiluminescence microparticle immunoassay; N, negative; P, positive; LC-MS/MS, liquid chromatography-tandem mass spectrometry.
Although CMIA exhibited a consistent negative bias in the presence of TgAb, there was no TgAb concentration-dependent bias (Fig. 2B). The median percentage differences for CMIA were −41.9%, −44.0%, and −45.2% for TgAb concentrations of 200, 400, and 800 IU/mL, respectively. The magnitude of the negative bias varied according to the source of TgAb. However, spiking TgAb did not result in a constant bias when measured with LC-MS/MS. The median percentage differences for LCMS/MS were 10.1%, 2.6%, and 7.6% for TgAb concentrations of 200, 400, and 800 IU/mL, respectively.
Detailed clinical information in samples with a discordant imaging study, Tg-MS, and Tg-IRMA
Table 2 lists the clinical characteristics of samples with Tg-IRMA <1 ng/mL that had suspicious or pathologically confirmed structural recurrence. In 10 of the 21 samples, Tg was undetectable by both Tg-MS and Tg-IRMA, even though seven had distant metastasis. Among the 21 patients, Tg-MS detected Tg in an additional six samples (samples 11 to 16) compared with Tg-IRMA. The recurrence patterns varied among these six patients: three had local lymph node recurrence, two had lung metastasis, and one had concurrent local lymph node recurrence and lung metastasis. The changing pattern of TgAb also varied in those six patients.
Table 2.
Detailed Clinical Information of Thyroid Carcinoma Patients with the Presence or Suspicion of Structural Recurrence and Tg-IRMA <1 ng/mL
| Sex/age, yr | Tg-MS, ng/mL | Tg-IRMA, ng/mL | Tg-CMIA, ng/mL | TgAb, U/mL | TgAba | Biopsy | Lesions of recurrence | |
|---|---|---|---|---|---|---|---|---|
| 1 | M/23 | <0.5 | <0.20 | 12.1 | 0 | No | Lung, bone | |
| 2 | F/46 | <0.5 | <0.20 | 36.6 | 0 | No | Lung | |
| 3 | F/52 | <0.5 | <0.20 | 93.7 | 0 | Yes | Local LN | |
| 4 | F/51 | <0.5 | <0.20 | <0.14 | 95.4 | 0 | Yes | Local LN |
| 5 | F/55 | <0.5 | <0.20 | 249.4 | 0 | Yes | Local LN | |
| 6 | M/59 | <0.5 | <0.20 | 490.1 | 0 | No | Local LN, lung | |
| 7 | F/59 | <0.5 | <0.20 | 509.4 | 2 | No | Lung | |
| 8 | M/50 | <0.5 | <0.20 | 747.4 | 1 | No | Lung | |
| 9 | F/72 | <0.5 | <0.20 | <0.14 | 1,341.4 | 1 | Yes | Local LN, lung |
| 10 | F/60 | <0.5 | <0.20 | 1,642.8 | 1 | No | Lung | |
| 11 | F/54 | 0.5 | <0.20 | 108.5 | 1 | Yes | Local LN | |
| 12 | M/28 | 0.61 | <0.20 | 1,060.8 | 1 | No | Lung | |
| 13 | F/46 | 1.04 | <0.20 | 103.0 | 0 | No | Local LN | |
| 14 | F/69 | 1.24 | <0.20 | 4,648.0 | 1 | Yes | Local LN, lung | |
| 15 | F/29 | 2.42 | <0.20 | 1,027.0 | 0 | No | Lung | |
| 16 | F/48 | 3.08 | <0.20 | 2,842.7 | 0 | No | Local LN | |
| 17 | F/26 | <0.5 | 0.38 | 368.1 | 2 | No | Local LN | |
| 18 | F/46 | <0.5 | 0.40 | 1,139.5 | 0 | Yes | Local LN | |
| 19 | F/42 | 0.67 | 0.60 | 0.27 | 111.0 | 1 | Yes | Local LN |
| 20 | F/40 | <0.5 | 0.64 | 43,058.0 | 0 | No | Lung | |
| 21 | M/63 | 3.38 | 0.76 | 107.8 | 0 | Yes | Lung |
Tg-IRMA, thyroglobulin measured by immunoradiometric assay; Tg-MS, thyroglobulin measured by liquid chromatography-tandem mass spectrometry; Tg-CMIA, thyroglobulin measured by chemiluminescence microparticle immunoassay; TgAb, thyroglobulin antibody; LN, lymph node.
The TgAb trend is compared to the previous result obtained with 6 and 12 months before the day Tg-MS was tested: 0 (TgAb was measured ≤60 U/mL or decreased), 1 (TgAb increased by less than 50%), and 2 (TgAb increased by more than 50%).
Five samples from patients in whom disease recurrence was not clinically suspicious were found to have Tg-MS ≥1 ng/mL with Tg-IRMA <1 ng/mL (Table 3). Among them, Tg-MS detected Tg in four samples in which it was undetectable by Tg-IRMA (samples 1 to 4).
Table 3.
Detailed Clinical Information of Thyroid Carcinoma Patients with No Evidence of Tumor Recurrence on Imaging and Tg-IRMA <1 ng/mL but Tg-MS ≥1 ng/mL
| Sex/age, yr | Tg-MS, ng/mL | Tg-IRMA, ng/mL | TgAb, U/mL | TgAba | Clinical information | |
|---|---|---|---|---|---|---|
| 1 | F/20 | 1.03 | 0.20 | 109.4 | 0 | No evidence of recurrence |
| 2 | F/34 | 1.71 | 0.20 | 142.2 | 1 | Benign-looking nodule in the operative bed |
| 3 | F/59 | 1.78 | 0.20 | 820.3 | 0 | Benign-looking tiny two lung nodules |
| 4 | F/42 | 3.72 | 0.20 | 1,099.0 | 0 | No evidence of recurrence |
| 5 | F/30 | 1.84 | 0.90 | 13.6 | 0 | Benign-looking tiny lung nodule |
Tg-IRMA, thyroglobulin measured by immunoradiometric assay; Tg-MS, thyroglobulin measured by liquid chromatography-tandem mass spectrometry; TgAb, thyroglobulin antibody.
The TgAb trend is compared to the previous result obtained with 6 and 12 months before the day Tg-MS was tested: 0 (TgAb was measured ≤60 U/mL or decreased), 1 (TgAb increased by less than 50%), 2 (TgAb increased by more than 50%).
During the study period, an interesting trend in Tg and TgAb was observed in a single patient (Fig. 3). This patient, a 66-year-old male diagnosed with classic PTC (T3bN1bM0) underwent a total thyroidectomy with both modified radical neck dissection followed by high-dose RAI therapy. Local lymph node metastasis and lung metastasis occurred during follow-up. Before the draw of an enrolled sample, the Tg trend slowly increased, even after the third round of RAI therapy, and during that time, TgAb was negative in this patient. At visit 8, which was 6 months after visit 7, TgAb rose from 5.2 to 812.3 IU/mL. A sample drawn for Tg-MS produced a measurement of 12.4 ng/mL whereas for Tg-IRMA it was 1.5 ng/mL.
Fig. 3.
An interesting case that showed rapidly changing thyroglobulin antibody (TgAb) levels. Tg-IRMA, thyroglobulin measured by immunoradiometric assay; Tg-MS, thyroglobulin measured by liquid chromatography-tandem mass spectrometry; RAI, radioactive iodine.
DISCUSSION
This study evaluated the correlation between Tg-MS and current immunometric assays (Tg-IRMA and Tg-CMIA) in various clinical situations. We performed a spike test and the results showed that the LC-MS/MS method was less prone to negative bias. Furthermore, we described the detailed clinical information in samples with discrepancies in the results of imaging studies and Tg assays to identify potential candidates for the Tg-MS assay.
We observed close agreement between Tg-MS and immunometric assays, and this agreement was closer in the TgAb-negative group (R=0.99 in Tg-MS and Tg-IRMA, R=0.99 in Tg-MS and Tg-CMIA, and R=0.99 in Tg-CMIA and Tg-IRMA) than in the TgAb-P group (R=0.93 in Tg-MS and Tg-IRMA, R=0.96 in Tg-MS and Tg-CMIA, and R=0.99 in Tg-CMIA and Tg-IRMA). The correlation did not indicate a significant difference across various clinical conditions, such as TC patients with a total thyroidectomy or lobectomy and those with Hashimoto’s thyroiditis. The correlation was weaker in all TgAb-P situations regardless of the clinical circumstance. Kushnir et al. [22] reported close agreement between Tg levels measured by LC-MS/ MS and an immunometric method but did not provide clinical information about the participants. This study confirmed that clinical conditions may not cause discrepancies between Tg-MS and immunometric methods.
Accurate measurement of Tg is important during the follow-up of TC patients. Several studies have confirmed a high postoperative Tg reflex in the persistence of disease and predicted potential future disease recurrence [6,23,24]. Underestimation by Tg-IRMA or Tg-CMIA can lead to undertreatment of active disease. In a previous report, the presence of TgAb, which was found in approximately 25% of TC patients [25], was associated with false-negative results for Tg in immunometric methods [12,14,26]. Netzel et al. [14] reported finding a false low bias in immunometric assays, whereas LC-MS/MS did not show such a bias. Consequently, underestimation was observed in immunometric assays compared with LC-MS/MS [14]. Consistent results were obtained in this study. A negative bias was observed in immunometric assays compared with LC-MS/MS. Spike test results also support a negative bias in Tg-CMIA whereas Tg-MS did not. This bias was predominant at Tg ≤2 ng/mL or TgAb >1,000 IU/mL, which is consistent with a previous report. Kushnir et al. [22] reported finding weak agreement between LC-MS/MS and immunometric assays as predominantly seen in Tg <2 ng/mL within the TgAb-P group. Also, Netzel et al. [14] reported marked negative biases according to increasing TgAb levels.
Because mass spectrometry can detect specific peptides in complex mixtures, we assumed that the detection of tryptic digests of Tg by LC-MS/MS can overcome the limitations of current immunoassay methods. If Tg cannot be detected by immunometric method in TgAb-P samples due to potential interference between Tg and TgAb, LC-MS/MS can theoretically overcome this problem [18]. In this study, 175 of 224 samples were TgAb-P (157 samples from TC patients and 18 samples from patients with Hashimoto’s thyroiditis), and 173 of 175 samples were tested by Tg-MS. Of the 173 samples, 99 were Tg-IRMA <0.5 ng/mL. Of these, only 12 (12/99, 12.1%) were detected at Tg-MS ≥0.5 ng/mL, and 87 samples (87/99, 87.9%) were reported as <0.5 ng/mL. Tg-MS detected Tg in an additional 12.1% of the TgAb-P samples with Tg-IRMA <0.5 ng/mL compared with Tg-IRMA. This result is consistent with a previous study. Netzel et al. [14] reported that 6% to 20% of samples were detected by LC-MS/MS compared with immunometric methods. Contrary to theoretical expectations, the LC-MS/MS method demonstrated a lower-than-expected rate of additional Tg detection in TgAb-P samples. Future studies will need to explain this phenomenon.
We also attempted to identify who can be a candidate for Tg-MS tests. In this study, Tg-IRMA levels of <1 ng/mL were observed in 21 patients with structural recurrence. Among these, six patients showed discrepant results between Tg-MS and Tg-IRMA. The TgAb levels in these patients ranged from 103.0 to 4,648.0 IU/mL. However, due to the small sample size, we were unable to identify a clear TgAb cut-off value associated with the additional detection of Tg by LC-MS/MS in patients with suspected recurrence. In addition, no common clinical characteristics were identified among these patients. Persistent or increased serum TgAb levels are associated with recurrence or persistent disease, and changing patterns are also associated with recurrence rates [25,27-29]. We therefore hypothesized that a rapidly increasing TgAb trend can affect the detection rate in Tg-MS. However, we were unable to identify a clear relationship between the trends in TgAb changes and the Tg-MS detection rate in those patients. Furthermore, there was no clear pattern in recurrent lesions. Only one interesting case was found during the study period. A rapid increase in TgAb was detected during the regular follow-up, and a sample for Tg-MS was drawn at that time. Tg-MS and Tg-IRMA showed a large discrepancy. Afterward, TgAb levels normalized within one year without any intervention. However, we could not obtain longitudinal results by Tg-MS because no samples were available for testing by Tg-MS. Based on this finding, LC-MS/MS appears to measure Tg more accurately than conventional methods in certain situations, such as when TgAb levels change rapidly. However, further studies are needed to confirm this finding.
As we could not identify specific clinical situations in which Tg-MS is more useful than current immunometric assays, using multiple assays to detect Tg appears to be beneficial in assessing a patient’s disease status, particularly when TgAb is present. Regular imaging should be considered in initial high-risk patients with consistently lower Tg levels. While LC-MS/MS is less prone to interference in TgAb-P circumstances, it still has some pitfalls. The Tg-MS has a higher LLoQ (<0.5 ng/mL) than current immunometric assays (<0.2 and <0.14 ng/mL in Tg-IRMA and Tg-CMIA, respectively). Conventional LC-MS/MS can yield false-negative results for patients with Tg between 0.14 and 0.5 ng/mL. Thus, LC-MS/MS had lower sensitivity compared to immunoassays in general situations. Furthermore, false low measurements also can occur in the LC-MS/MS method [30]. In this study, we observed that 9.3% (10/107) and 12.5% (3/24) of samples showed lower Tg-MS results compared to Tg-IRMA and Tg-CMIA, respectively. For these reasons, LC-MS/MS can be a good choice when assessing TC patients with high TgAb levels, but it may not be perfect.
This study has several limitations. First, the method of patient enrollment may have introduced selection bias. The prospectively recruited group included only patients with TgAb-positive samples and did not have Tg-CMIA results. Second, a relatively small sample size was enrolled. Third, we were unable to obtain longitudinal results by Tg-MS. We identified five patients with no structural recurrence but a Tg-MS ≥1 ng/mL in the Tg-IRMA <1 ng/mL cohort. Long-term follow-up results for those patients will be essential to provide insights into the clinical utility of Tg-MS. Fourth, we did not assess the cost-effectiveness of Tg-MS, and such an evaluation will be necessary in future studies.
In conclusion, the values obtained using LC-MS/MS showed better correlation in TgAb-N samples than TgAb-P samples. LC-MS/MS detected an additional 12.1% among TgAb-P samples when using a positivity cut-off value of 0.5 ng/mL. Thus, Tg detection in TgAb-P samples was improved by using the LC-MS/MS alongside conventional methods. However, false low measurements can also occur with LC-MS/MS. In patients with a high clinical suspicion of recurrence, it is important to evaluate Tg using multiple assay methods, including Tg-MS, and to interpret the results within the clinical context.
Footnotes
CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.
ACKNOWLEDGMENTS
The authors thank Jisook Park and Hyeonju Oh for their contributions to the experimental design and technical assistance.
AUTHOR CONTRIBUTIONS
Conception or design: S.Y.L., S.W.K. Acquisition, analysis, or interpretation of data: H.P., E.Y., S.M.K., T.H.K., J.H.C. Drafting the work or revising: H.P., E.Y. Final approval of the manuscript: S.Y.L., S.W.K.
Supplementary Material
Baseline Characteristics of Hashimoto’s Thyroiditis Patients
Concordance among Tg-MS, Tg-IRMA, and Tg-CMIA
Concordance among Tg-MS, Tg-IRMA, and Tg-CMIA in Samples with TgAb >60 and ≤60 U/mL
Comparison between thyroglobulin measured by liquid chromatography-tandem mass spectrometry (Tg-MS) and thyroglobulin measured by immunoradiometric assay (Tg-IRMA) according to the clinical situations. (A) All thyroid carcinoma (TC) samples, (B) TC samples from patients who underwent total thyroidectomy and thyroglobulin antibody (TgAb) ≤60 U/mL, (C) TC samples from who underwent total thyroidectomy and TgAb >60 U/mL, (D) TC samples from who underwent lobectomy, (E) Hashimoto’s thyroiditis.
Comparison between thyroglobulin measured by liquid chromatography-tandem mass spectrometry (Tg-MS) and thyroglobulin measured by chemiluminescence microparticle immunoassay (Tg-CMIA) according to the clinical situations. (A) All thyroid carcinoma (TC) samples, (B) TC samples from patients who underwent total thyroidectomy and thyroglobulin antibody (TgAb) ≤60 U/mL, (C) TC samples from who underwent total thyroidectomy and TgAb >60 U/mL, (D) TC samples from who underwent lobectomy, (E) Hashimoto’s thyroiditis.
Comparison between thyroglobulin measured by chemiluminescence microparticle immunoassay (Tg-CMIA) and thyroglobulin measured by immunoradiometric assay (Tg-IRMA) according to the clinical situations. (A) All thyroid carcinoma (TC) samples, (B) TC samples from patients who underwent total thyroidectomy and thyroglobulin antibody (TgAb) ≤60 U/mL, (C) TC samples from who underwent total thyroidectomy and TgAb >60 U/mL, (D) TC samples from who underwent lobectomy, (E) Hashimoto’s thyroiditis.
Comparison of thyroglobulin (Tg) results from thyroglobulin measured by liquid chromatography-tandem mass spectrometry (Tg-MS), thyroglobulin measured by immunoradiometric assay (Tg-IRMA), and thyroglobulin measured by chemiluminescence microparticle immunoassay (Tg-CMIA) at various Tg and thyroglobulin antibody (TgAb) levels in each sample (A) Tg-MS ≤2 ng/mL, (B) Tg-MS >2 and ≤10 ng/mL, (C) Tg-MS >10 and ≤50 ng/ml, and (D) Tg-MS >50 ng/mL.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Baseline Characteristics of Hashimoto’s Thyroiditis Patients
Concordance among Tg-MS, Tg-IRMA, and Tg-CMIA
Concordance among Tg-MS, Tg-IRMA, and Tg-CMIA in Samples with TgAb >60 and ≤60 U/mL
Comparison between thyroglobulin measured by liquid chromatography-tandem mass spectrometry (Tg-MS) and thyroglobulin measured by immunoradiometric assay (Tg-IRMA) according to the clinical situations. (A) All thyroid carcinoma (TC) samples, (B) TC samples from patients who underwent total thyroidectomy and thyroglobulin antibody (TgAb) ≤60 U/mL, (C) TC samples from who underwent total thyroidectomy and TgAb >60 U/mL, (D) TC samples from who underwent lobectomy, (E) Hashimoto’s thyroiditis.
Comparison between thyroglobulin measured by liquid chromatography-tandem mass spectrometry (Tg-MS) and thyroglobulin measured by chemiluminescence microparticle immunoassay (Tg-CMIA) according to the clinical situations. (A) All thyroid carcinoma (TC) samples, (B) TC samples from patients who underwent total thyroidectomy and thyroglobulin antibody (TgAb) ≤60 U/mL, (C) TC samples from who underwent total thyroidectomy and TgAb >60 U/mL, (D) TC samples from who underwent lobectomy, (E) Hashimoto’s thyroiditis.
Comparison between thyroglobulin measured by chemiluminescence microparticle immunoassay (Tg-CMIA) and thyroglobulin measured by immunoradiometric assay (Tg-IRMA) according to the clinical situations. (A) All thyroid carcinoma (TC) samples, (B) TC samples from patients who underwent total thyroidectomy and thyroglobulin antibody (TgAb) ≤60 U/mL, (C) TC samples from who underwent total thyroidectomy and TgAb >60 U/mL, (D) TC samples from who underwent lobectomy, (E) Hashimoto’s thyroiditis.
Comparison of thyroglobulin (Tg) results from thyroglobulin measured by liquid chromatography-tandem mass spectrometry (Tg-MS), thyroglobulin measured by immunoradiometric assay (Tg-IRMA), and thyroglobulin measured by chemiluminescence microparticle immunoassay (Tg-CMIA) at various Tg and thyroglobulin antibody (TgAb) levels in each sample (A) Tg-MS ≤2 ng/mL, (B) Tg-MS >2 and ≤10 ng/mL, (C) Tg-MS >10 and ≤50 ng/ml, and (D) Tg-MS >50 ng/mL.