Use of Mass Spectrometry to Improve Thyroglobulin Quantitation [Communiqué]

Background

Thyroid cancer is the most common malignancy of the endocrine system. The American Cancer Society estimates that approximately 62,450 new cases of thyroid cancer will be diagnosed in 2015 in the United States. Thyroid follicular epithelial cell-derived cancers are divided into 3 categories: papillary, follicular, and anaplastic cancers. Papillary and follicular cancers are considered differentiated cancers and treatment approaches are similar. Surgery is the primary mode of therapy for patients with differentiated thyroid cancer followed, in some cases, by radioactive iodine ablation to destroy any remaining healthy thyroid tissue, as well as microscopic areas of thyroid cancer that were not removed during surgery. Serum thyroglobulin concentrations are used to monitor patients with differentiated thyroid cancer for persistent or recurrent disease after initial therapy.

Thyroglobulin (Tg) is a 660,000 molecular weight glycoprotein produced exclusively by the follicular epithelial cells of the thyroid. It serves as the precursor of, and storage reservoir for, thyroxine (T4) and triiodothyronine (T3). Small amounts of intact thyroglobulin are secreted alongside T4 and T3 and are detectable in the serum of normal individuals with levels roughly paralleling thyroid size. The concentration of serum thyroglobulin increases following follicular destruction due to inflammation. This may include thyroiditis and autoimmune hypothyroidism, nodular goiter, or rapid disordered growth of thyroid tissue as may be observed in Graves disease or follicular cell-derived thyroid neoplasms.

Thyroglobulin as a Tumor Marker

Measurement of Tg is not recommended for the screening or initial diagnosis of thyroid cancer due to the significant overlap in concentrations with benign thyroid diseases. In addition, in patients with small cancers, Tg levels might overlap with the levels seen in normal individuals.

The primary use of serum Tg is in the follow-up of patients with differentiated thyroid cancer following treatment. Because Tg is highly organ-specific, serum Tg concentrations should be undetectable, or very low, after the thyroid gland is removed during surgical treatment for thyroid cancer. Serial Tg measurements should be performed using the same assay. Use of an assay with a functional sensitivity of 0.1 to 0.2 ng/mL is preferable. If unstimulated (on thyroxine) serum Tg measurements are <0.1 to 0.2 ng/mL, the risk of disease is <1%. Patients with higher Tg levels, who have no demonstrable remnant thyroid tissue, might require additional testing, such as stimulated Tg measurements and neck ultrasound. A stimulated Tg >2 ng/mL is considered suspicious of possible residual or recurrent disease.

Serum Tg measurement might be misleading in the presence of antithyroglobulin autoantibodies (TgAb), which occur in up to 30% of patients with differentiated thyroid cancer. The presence of TgAb may lead to false-low results in Tg immunometric assays (most commonly used assays) or false-high results in Tg competitive radioimmunoassays. Although less common, the presence of heterophile antibodies (HAB) could also result in unreliable Tg measurements. HABs are capable of interacting with the antibodies used in immunoassays, usually resulting in false-high measurements. This can lead to erroneously high Tg results in 0.1% to 3% of patients.

Assessment of TgAb interference in the Tg immunoassays is compounded by the fact that interference is variable among patients and Tg assays, and it does not correlate with TgAb concentrations. Consequently, in TgAb-positive specimens it is difficult to predict the degree of interference and provide an accurate Tg result. Laboratories, however, should alert the ordering physicians that TgAb are present. Some cases with interference might also be missed, because any given TgAb assay will only detect a subset of interfering TgAb. Current professional guidelines recommend measurement of serum Tg every 6 to 12 months by an immunometric assay that is calibrated against the Certified Reference Material (BCR 457) and TgAb concentrations should be quantitatively assessed with every measurement of serum Tg. If TgAb are detected, the laboratory report should alert the ordering provider to the possibility of false-low Tg results.

When selecting a TgAb assay for assessment of Tg immunoassay interference, the following should  be considered.

  • All FDA-approved Tg assays are optimized as an  aid in the diagnosis of thyroid autoimmune disease.
  • The assay manufacturer-provided reference intervals are specific for the evaluation of thyroid autoimmune disease and are not optimized for the detection of interference in Tg assays. There is no consensus as to the minimal level of antibody levels that are likely to cause interference in the Tg assay, so values within the normal range could still cause interference in  the Tg immunoassays.
  • The poor numerical agreement among the TgAb assays in any given specimen makes it very difficult to have a standardized TgAb concentration that is likely to cause interference in the Tg assay. Serial TgAb measurements should be performed using the same assay.

Thyroglobulin Mass Spectrometry Assays

In recent years, measurement of serum Tg by mass spectrometry (Tg-MS) has become available in clinical laboratories. Tg measurement by mass spectrometry overcomes TgAb interference by trypsin digestion of Tg  and TgAb in the patient’s serum and quantitation of  specific Tg peptides.

Mass spectrometry workflow for Tg measurement involves selective precipitation of large proteins. The precipitated proteins undergo trypsin digestion to generate predictable fragments of Tg. Then, the Tg fragment of interest is immunopurified by the use of antipeptide antibodies. Finally, these fragments are analyzed by liquid chromatography-tandem mass spectrometry and Tg quantified by the use of a standard curve that uses a synthetic version of the measured peptides. Published studies on Tg mass spectrometry assays have shown good comparability (R2>0.95, slope 0.8–1.2) to immunoassays in TgAb-negative specimens. In TgAb-positive specimens, a positive bias (slope 1.3–1.5) is consistent with 50% to 60% under-recovery of Tg in the immunoassays.

The Tg mass spectrometry assay performed at Mayo Medical Laboratories (TGMS / Thyroglobulin Mass Spectrometry, Serum) is standardized against the Certified Reference Material BCR 457. The assay has a lower limit of quantitation of 0.5 ng/mL. Comparison of the Mayo Medical Laboratories Tg by mass spectrometry and Tg immunoassay showed good correlation and a slope of 0.8 in TgAb-negative specimens. (Figure 1)

In TgAb-positive specimens, with detectable Tg concentration by the immunoassay, the assays show a good correlation (Figure 2) and a slope of 1.3 consistent with a systematic under-recovery of thyroglobulin in the immunoassay compared to the mass spectrometry assay in the presence of TgAb.

In the evaluation of a set of TgAb-positive specimens with undetectable Tg by immunoassay, the Tg mass spectrometry assay showed detectable thyroglobulin in 20 of 105 specimens (or approximately 20% of the cases). These results demonstrate that the presence of TgAb is not by default indicative of interference in the Tg immunoassay. However, by measuring Tg by mass spectrometry in TgAb-positive specimens, accurate quantitation could be achieved in cases where interference is present.

One of the current disadvantages of Tg mass spectrometry assays when compared with sensitive Tg immunoassays is the functional sensitivity. The newest Tg immunoassays have functional sensitivities as low as 0.1 ng/mL, whereas functional sensitivities of Tg mass spectrometry assays are between 0.5 and 1.0 ng/mL. As such, patients with Tg between 0.1 and 0.5 ng/mL might yield false-negative results by Tg mass spectrometry. To ensure that the most sensitive Tg assay is used, a reflexive testing strategy has been suggested. This consists of an initial immunoassay screen for TgAb. If the specimen is TgAb negative, Tg is measured by immunoassay. If the specimen is TgAb positive, Tg is measured by mass spectrometry. This reflexive strategy might be the most efficient and cost-effective way to ensure accurate Tg quantitation in TgAb-positive patients.

Mayo Medical Laboratories Ordering Options

There are 3 options for ordering thyroglobulin through Mayo Medical Laboratories.

  1. Thyroglobulin by immunoassay: Thyroglobulin and thyroglobulin antibodies will be measured using the Beckman Access assays. (HTG2 / Thyroglobulin, Tumor Marker, Serum)
  2. Thyroglobulin by mass spectrometry: Thyroglobulin  is measured using the mass spectrometry assay  and there is no assessment of the thyroglobulin antibody levels. (TGMS / Thyroglobulin Mass Spectrometry, Serum)
  3. Thyroglobulin reflex to mass spectrometry or immunoassay. In this assay, testing begins with analysis of the thyroglobulin antibody by immunoassay. If the specimen is antibody negative, thyroglobulin is measured by immunoassay. If the specimen is antibody positive, thyroglobulin is measured by mass spectrometry. (HTGR / Thyroglobulin, Tumor Marker Reflex to LC-MS/MS or Immunoassay)

Considerations in Thyroglobulin Measurement

  • The presence of antithyroglobulin autoantibodies might interfere with quantitation of thyroglobulin in immunoassays
  • Measurement of antithyroglobulin autoantibodies in conjunction with thyroglobulin is necessary to evaluate potential interference
  • Mass spectrometry thyroglobulin assays are not susceptible to antithyroglobulin autoantibodies interference and allow for accurate thyroglobulin quantitation

Conclusion

Serum thyroglobulin concentrations are used to monitor patients with differentiated thyroid cancer, but may be misleading in the presence of antithyroglobulin autoantibodies and heterophile antibodies. Mayo Medical Laboratories has developed an assay to address this issue by testing first for the presence of thyroglobulin antibodies by immunoassay and, in antibody-positive specimens, measuring thyroglobulin by a mass spectrometry assay  that overcomes the influence of thyroglobulin and heterophile antibodies.

Authored by Alicia Algeciras-Schimnich, PhD, DABCC

 

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This is an archived feature article of the Communiqué, which was previously a peer-review-style print publication. Specific author(s) for this article, when applicable, are listed above.