Serum thyroglobulin (Tg) measurement is used in the post-operative monitoring for differentiated thyroid cancer (DTC) recurrence. Following total thyroidectomy, serum Tg should remain undetectable, and any measurable Tg indicates residual or recurrent disease. In addition, Tg measurement in fine-needle aspirate biopsy (FNAB) saline washings is used for the evaluation of potential DTC metastases to lymph nodes. Despite the development of highly sensitive Tg assays, serum Tg monitoring does not successfully detect all DTC recurrence. There are several known limitations in the measurement of Tg. Most notably, antibodies to Tg (TgAb), which develop in up to 30% of carcinoma patients, are likely to falsely suppress thyroglobulin results by immunoassay. Interference from heterophile antibodies, which can cause both false positive and false negative results, are also known limitations of immunoassays for Tg. Mass spectrometry (MS) assays were developed to address antibody interference and have been reported as not subject to TgAb or heterophile antibody interference. Analytical sensitivity has historically been a limitation in some Tg-MS assays, but this has been overcome in recent years. The Tg-MS assay at our institution has a lowest reportable concentration of 0.2 ng/mL, versus 0.1 ng/mL for our immunoassay. Despite the availability of sensitive Tg-MS assays, recurrent DTC with unexplained undetectable serum Tg is a persistent occurrence. The objective of this study was to determine whether post-thyroidectomy patients with undetectable serum Tg and cytology-confirmed lymph-node DTC metastases have detectable Tg in FNAB washing and whether the failure to detect serum Tg in this patient population is a result of TgAb interference.
FNAB washing (n=30) from patients with cytology-confirmed DTC lymph node metastases but with an undetectable serum Tg by immunoassay were collected and assayed for Tg and TgAb. Serum Tg and TgAb concentrations were obtained within three months (mean: 14 days) prior to FNAB. In a subset of patients (n=8) a residual serum sample was available for additional investigations. Tg was measured on serum and FNAB washing using the Beckman Access Thyroglobulin (Tg2) immunoassay (Tg-IA) on a DxI analyzer, and an in-house developed LC-MS/MS Tg assay (Tg-MS). TgAb in serum was measured using the Beckman Access Thyroglobulin Antibody II assay on a DxI analyzer. Spike-recovery experiments were performed in a subset of samples (n=8) by adding the patient’s Tg-positive FNAB or certified reference material (BCR 457) to matched patient serum. A control serum (TgAb negative, Tg negative) was included for all spiked recovery experiments. Spiked serum was assayed using Tg-IA and percent recovery calculated as (expected – measured)/expected *100. Recovery (percent difference) between immunoassay and MS methods was calculated for FNA samples, and mean recovery for antibody positive versus antibody negative patients was compared by Student’s t-test.
Of 30 patients with cytology-confirmed DTC lymph node metastases and an undetectable serum Tg, 26 (87%) had detectable Tg in the FNAB washings by Tg-IA, and 25 (83%) were also Tg-positive by Tg-MS. Fifty-eight percent of these patients (15/26) had detectable TgAb in serum, while 42% (11/26) did not. In 8 samples with residual serum (4 TgAb negative and 4 TgAb positive), measurement of serum Tg by a sensitive MS method only detected Tg in one patient (Tg-MS= 0.27 ng/mL). Interestingly, this one discrepant patient was not TgAb positive, and exhibited Tg recovery of >90% in spike-recovery experiments. Spike-recovery experiments performed on the TgAb positive samples showed an interference for the immunoassay as expected, with 4/4 patients exhibiting <90% recovery (range: 27-87%) of Tg-positive FNA or Tg reference material; while TgAb negative serum exhibited no signs of interference (recovery between 94-111% for Tg-positive FNAB or Tg reference material). No evidence of TgAb interference in FNA washings was detected, as there was no significant difference in recovery between antibody positive and antibody negative patients’ Tg in FNA measured by Tg-MS versus Tg-IA.
This study investigated a subset of post-thyroidectomy patients with DTC lymph-node metastases and undetectable serum Tg by immunoassay and MS methods. Tg was detectable by both MS and immunoassay methods in the majority of FNAB washing samples from patients with cytology-confirmed DTC lymph node metastases and Tg-negative serum. The absence of detectable serum Tg in these patients does not appear to be completely explained by the presence of TgAb, as a subset of patients were TgAb negative and showed >90% recovery of Tg on spike-recovery experiments. These findings imply that in a subset of patients with DTC lymph node metastases, Tg may not be secreted into the circulation, and the lack of measurable serum Tg is not related to the analytical limitations of the assays. Additional studies and testing in a larger number of patients are needed to further prove this concept.