Thyroglobulin Testing Updates and Unanswered Questions

Thyroglobulin (Tg) testing is still recommended for monitoring recurrence of thyroid cancer following thyroidectomy or ablation, but thyroglobulin autoantibody (TgAb) interference may occur in as many as 25% of patients. A 2015 Scientific Short considered thyroglobulin (Tg) testing by mass spectrometry versus immunoassay, as well as cost savings realized by using an appropriate reflex ordering option (1). At that time, 89% of test orders at one reference lab utilized the reflex ordering option. Analysis of 2017 – 2023 data from the same lab shows similar results, with 82% of the Tg testing being ordered as a reflex order choice and only 18% ordered directly by mass spectrometry. Of those reflex tests, 10% had TgAb results that required Tg to be measured using mass spectrometry.

How have these assays changed since 2015? Most manufacturers now have 2nd generation ultrasensitive Tg immunoassays, with a functional sensitivity ≤ 0.1 ng/mL. Unfortunately, harmonization is still lacking as two widely used ultrasensitive assays showed comparable clinical performance only when assay-specific cut-offs were used (2). Improvements in mass spectrometry assays (LC-MS/MS) have also been published, but it is unclear if they are in clinical use. The original published methods had lower limit of quantitation (LLOQ) in the range of 0.4 – 2.6 ng/mL, but improvements have resulted in methods with LLOQs of 0.02 ng/mL (3) and 0.15 ng/mL (4), thus rivaling some ultrasensitive Tg immunoassays. The lower LLOQ was achieved using a smaller column diameter and microliter/min chromatographic flow rates (3). To make it more feasible for other labs to implement LC-MS/MS testing for Tg, a standard operating procedure was published and reference materials are now freely available (4). However, it remains to be seen whether additional labs will adopt this, especially with the possibility of FDA oversight of lab developed tests in the near future.

With a lower LLOQ for ultrasensitive immunoassays compared to most clinically available mass spectrometry methods, some recent studies have explored whether Tg can ever be detected in TgAb positive samples. One study determined optimized cut-offs for TgAb positive versus TgAb negative subjects using an ultrasensitive immunoassay; 87% of TgAb positive patients with recurrent disease or distant metastases were correctly detected (5). Another study compared Tg in patients with low titer TgAb (<10 U/mL) by three methods, showing equivalent performance of immunoassay and LC-MS/MS (6). Thus, the antibody titer may make a difference in detection by immunoassay.

Recently, a new immunometric method with automated specimen pretreatment (acid and detergent) to inactivate the TbAb and dissociate the Tg-TgAb complexes has been reported. This method has an LLOQ of 0.03 ng/mL and was shown to perform as well as LC-MS/MS (LLOQ of 0.4 ng/mL) in patients with TgAb (7). Studies comparing this to the recently published LC-MS/MS methods with lower LLOQ are still needed. Also, to our knowledge, this instrument is not FDA-approved.

It will be interesting to see how these new methods and future clinical outcome studies affect testing strategies in the next 5 – 10 years. Should we keep pursuing lower and lower LLOQs? Will methods that effectively remove antibodies become widely available? Will they also remove heterophilic antibody interference? There may also be patients with low Tg concentrations (<LLOQ) despite having evidence of structural disease. Physiological reasons could include tumors secreting less Tg, or secreting Tg variants, or increased clearance of circulating Tg in the presence of TgAb (8). Will there always be some patients with residual disease but low levels of circulating Tg?

References

1. Powers, J.  Screen with Reflex to Better Test Utilization: Mass Spectrometry or Immunoassay for Tg Testing? The Academy of Diagnostics & Laboratory Medicine's Scientific Shorts. October 27, 2015. Available from: https://www.myadlm.org/science-and-research/scientific-shorts/2015/screen-with-reflex-to-better-test-utilization

2.  Trimboli, P et al. Clin Endocrinol 2018, 88, 295-302.

3.  Shuford, CM et al. Clin Mass Spectrom 2020, 15, 29-35.

4.  Shi, J et al.  J Mass Spectrom Adv Clin Lab 2022, 26, 28-33.

5.  Giovanella, L et al. Clin Chem Lab Med 2019, 57:1185-91.

6.  Dekker BL et al. Eur Thyroid J 2022, 11, e220173.

7.  Kitamura, Y et al. Clin Biochem 2023, 118, 110598.

8.  Barbesino G et al. J Endocrine Soc 2023, 7, 1-7.