Advancements in Thyroglobulin Quantitation in the Presence of Anti-thyroglobulin Antibodies [Hot Topic]

Alicia Algeciras-Schimnich, Ph.D.
Alicia Algeciras-Schimnich, Ph.D.

The main use of serum thyroglobulin measurement is to follow patients with differentiated follicular cell-derived thyroid carcinoma who have had thyroidectomy. Because thyroglobulin is thyroid-specific, thyroglobulin levels should be undetectable or very low. The presence of anti-thyroglobulin autoantibodies, which occur in 15 to 30 percent of thyroid cancer patients, could lead to inaccurate quantitation of thyroglobulin. Recently, trypsin digestion of serum proteins has allowed accurate quantification via mass spectrometry of thyroglobulin in specimens with antibody interferences. Dr. Algeciras-Schimnich describes this new assay.

Presenter and Credentials:
Alicia Algeciras-Schimnich, Ph.D., Chair of the Division of Clinical Biochemistry at Mayo Clinic in Rochester, Minn. and Associate Professor of Laboratory Medicine and Pathology in the College of Medicine.

Transcript

Our speaker for this program is Dr. Alicia Algeciras-Schimnich, Chair of the Division of Clinical Biochemistry at Mayo Clinic in Rochester, Minnesota. Dr. Algeciras-Schimnich is also Associate Professor of Laboratory Medicine and Pathology in the College of Medicine.Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Today our topic is Advancements in Thyroglobulin Quantitation in the Presence of Anti-thyroglobulin Antibodies.

Thank you for the introduction.

I have nothing to disclosure.

As you view this presentation, consider the following points. The presence of antithyroglobulin antibodies might interfere with the quantitation of thyroglobulin in immunometric assays. Measurement of antithyroglobulin antibodies in conjunction with thyroglobulin is necessary to evaluate potential interference. Mass spectrometry thyroglobulin assays are not susceptible to antithyroglobulin antibodies interference and allow for accurate thyroglobulin quantitation.

Thyroid cancer is the most common malignant tumor of the endocrine system. Thyroid cancer could be divided into poorly differentiated and differentiated thyroid carcinoma. The poorly differentiated medullary and anaplastic thyroid cancers account for 3% of thyroid cancer cases and tend to be very aggressive and usually have a poor prognosis. Papillary and follicular thyroid cancers are differentiated tumors derived from the follicular cells of the thyroid and account for approximately 97% of thyroid cancer cases. These are highly treatable and curable cancers. In these cancers, measurement of thyroglobulin levels is considered a standard of practice for patient follow-up.

The treatment of differentiated thyroid cancer consists of total removal of the thyroid gland followed by radioactive iodine (131I) treatment to destroy any remaining healthy thyroid tissue, as well as microscopic areas of thyroid cancer that were not removed during surgery. Because of the good prognosis of the majority of patients with differentiated thyroid cancer, measurement of thyroglobulin has emerged as a noninvasive and cost-effective follow-up tool to monitor recurrence and persistent disease.

Thyroglobulin (Tg) is a 660,000 molecular weight glycoprotein produced exclusively by the follicular cells of the thyroid. It is secreted into the follicular lumen, where it serves as the precursor of, and storage reservoir for, thyroxin or T4 and triiodothyronine or 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, such as in the case of thyroiditis and autoimmune hypothyroidism, in the cases of nodular goiter, or rapid disordered growth of thyroid tissue, as may be observed in Graves disease or follicular cell-derived thyroid neoplasms.

Measurement of thyroglobulin 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, thyroglobulin levels might overlap with the levels seen in normal individuals.

The primary use of serum thyroglobulin is in the follow-up of patients with differentiated thyroid cancer following total thyroidectomy and radioactive iodine ablation. These individuals should have unstimulated and simulated serum thyroglobulin concentrations of equal or less than 2 ng/mL. Patients with higher levels should be investigated for persistent or recurring disease. Athyrotic thyroid cancer patients with unstimulated or stimulated serum thyroglobulin concentrations greater than 10 ng/mL are likely to have evidence of persistent or recurrent disease.

For patients with small thyroidal remnants there are currently no universally accepted cutoff levels for thyroglobulin. It has been suggested that thyroglobulin levels should not exceed approximately 0.5 ng/mL per gram of remnant tissue in patients with suppressed TSH, or approximately 1 ng/mL if TSH is not suppressed.

Most laboratories perform thyroglobulin measurements by automated immunometric assay methods. In these assays, the clinical utility of thyroglobulin testing can be negatively affected by various analytical issues. Interference caused by antithyroglobulin antibodies remains the most serious problem limiting the clinical utility of thyroglobulin testing by immunoassay. Antithyroglobulin antibodies are detected in up to 30% of patients with differentiated thyroid cancer, compared with the 10% incidence reported for the general population. Thyroglobulin antibody interference is characterized by undetectable or falsely low thyroglobulin concentration using immunometric assays. Due to this problem various guidelines, including those from the National Academy of Clinical Biochemistry and the American Thyroid Association, stress that antithyroglobulin autoantibody measurement should be performed in all samples tested for thyroglobulin. Failure to detect thyroglobulin antibody interference in the presence of an undetectable thyroglobulin concentration could greatly impact patient management as disease recurrence might go undiagnosed.

In a typical immunometric thyroglobulin assay, thyroglobulin present in the patient serum is sandwiched between a capture and a detection antibody. The signal generated by the detection antibody is directly proportional to the concentration of thyroglobulin in the sample.

In the presence of thyroglobulin antibodies binding of thyroglobulin can be prevented by blocking the access of the capture and/or the binding antibody to their respective epitopes on thyroglobulin. This will result in falsely low or false-negative thyroglobulin concentrations. Alternatively, the thyroglobulin antibody in the patient’s serum might bind an epitope different from the binding site of the capture and/or detection antibody and in these cases thyroglobulin quantitation will not be affected. The most likely scenario is that due to the heterogeneity of thyroglobulin antibody in patients some antibodies will interfere with binding of thyroglobulin to the capture and detection antibodies, while others will not, making it difficult to determine whether the thyroglobulin result is unaffected, falsely low, or falsely negative.

There are a number of considerations to keep in mind regarding thyroglobulin antibody assays. All FDA-approved thyroglobulin assays are aimed for the aid in the diagnosis if 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 thyroglobulin assays. Values within the normal range could still cause interference in the thyroglobulin immunoassays.

The poor numerical agreement between the thyroglobulin antibody assays in any given sample makes it very difficult to have a standardized thyroglobulin antibody concentration that is likely to cause interference in the thyroglobulin assay. Ideally, each laboratory should establish the value of thyroglobulin antibody that interferes with their respective thyroglobulin assay.

Recently, measurement of serum thyroglobulin by mass spectrometry has become available in clinical laboratories. Thyroglobulin measurement by mass spectrometry overcomes thyroglobulin antibody interference by trypsin digestion of thyroglobulin and thyroglobulin antibodies in the patient’s serum and quantitation of specific thyroglobulin peptides.

The mass spectrometry work-flow for thyroglobulin measurement involves selective precipitation of large proteins by ammonium sulfate. The precipitated proteins undergo trypsin digestion to generate predictable fragments of thyroglobulin. The thyroglobulin fragment of interest is then immunopurified by the use of antipeptide antibodies. Finally, these fragments are analyzed by liquid chromatography-tandem mass spectrometry and thyroglobulin quantified by the use of a standard curve that uses a synthetic version of the measured peptides.

The thyroglobulin mass spectrometry assay performed at Mayo Medical Laboratories is standardized against the Certified Reference Material CRM457. The assay has a limit of detection and limit of quantitation of 0.5 ng/mL.

Total intra- and interassay imprecision in serum samples is less than 10% and the measurable range of the assay is between 0.5 and 250 ng/mL.

To determine how well the results of the thyroglobulin mass spec assay correlated with those from the Beckman Access thyroglobulin immunoassay, the reference material was tested as unknown. These graphs show the expected thyroglobulin concentration in the X axis and the measured thyroglobulin concentration on the y axis. For the mass spec assay, the observed slope was 1.02  whereas the Beckman assay showed a posivite bias with a slope of 1.18. These findings indicate that the thyroglobulin concentrations by the immunoassay will be approximately 18% higher than the mass spectrometry assay.

Method comparison of thyroglobulin antibody-negative samples with a thyroglobulin concentration between 1 and 138 ng/mL by the immunoassay, show good correlation. However, the concentrations of thyroglobulin by the mass spec assay were approximately 20% lower than the immunoassay which is in agreement with the reference material data shown previously.

In thyroglobulin antibody-positive samples, which have a detectable thyroglobulin concentration by the immunoassay, the assays show a good correlation. However, the slope of the method comparison was 1.3 which is consistent with a systematic under recovery of thyroglobulin in the immunoassay compared to the mass spectrometry assay in the presence of antithyroglobulin antibodies.

To further determine the performance of the thyroglobulin mass spec assay in thyroglobulin antibody-positive samples, 105 samples that were thyroglobulin antibody positive and had an undetectable thyroglobulin by the immunoassay were analyzed. In this group of samples the mass spectrometry showed detectable thyroglobulin in 20 out of 105 samples (or approximately 20% of the cases). These results demonstrate that the presence of thyroglobulin antibodies is not by default indicative of interference in the thyroglobulin immunoassay. However, by measuring thyroglobulin by mass spectrometry in thyroglobulin antibody-positive samples, accurate quantitation could be achieved.

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

Thyroglobulin by immunoassay:in this case, thyroglobulin and thyroglobulin antibodies will be measured using the Beckman Access assays.

Thyroglobulin by mass spectrometry:in this case there will be no assessment of the thyroglobulin antibody levels.

The third option is thyroglobulin reflex to mass spec or immunoassay. In this case, testing begins with analysis of the thyroglobulin antibody by immunoassay. If the sample is antibody negative, thyroglobulin is measured by immunoassay. If the sample is antibody positive, thyroglobulin is measured by mass spectrometry. This test, we believe, is the most efficient and cost effective way to ensure accurate quantitation of thyroglobulin in thyroglobulin antibody-positive patients.

To conclude, serum thyroglobulin can be measured accurately by LC MS/MS regardless of the thyroglobulin antibody status. The Beckman Access immunoassay may underestimate the true thyroglobulin concentration in antibody-positive samples with detectable thyroglobulin. In thyroglobulin antibody-positive samples with undetectable thyroglobulin by immunoassay, 80% of cases also had an undetectable thyroglobulin by mass spectrometry. The other 20% of cases contained low levels of thyroglobulin by the mass spectrometry assay. There is no relationship between the thyroglobulin antibody concentrations and the failure of the Beckman immunoassay to detect thyroglobulin. The use of the thyroglobulin reflex assay allows for assessment of the thyroglobulin antibody status and triage of thyroglobulin measurement by the most accurate method.

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