July 2011: Volume 37, Number 7
Solutions to Analytical Pitfalls in Differentiated Thyroid Cancer Monitoring
By Carole Spencer, MT, PhD, FACB
Differentiated thyroid cancer (DTC), the most common endocrine malignancy, is a highly curable disease with 5-year survival rates in excess of 95%. Treatment for DTC typically involves thyroidectomy followed by, in certain cases, radioactive iodine ablation, as well as thyroid-stimulating hormone (TSH) suppression. Serum thyroglobulin (Tg) measurement is a cornerstone of post-operative monitoring and long-term surveillance of DTC patients.
A radioimmunoassay (RIA) for measuring serum Tg measurement was first developed in the 1970s, but since then, several different immunometric assay (IMA) methods have been introduced and gained in popularity over time. In concert with these changes, clinicians now use serum Tg primarily for DTC monitoring, whereas in the past they also relied on it to investigate non-neoplastic pathologies such as hyperthyroidism, thyroiditis, and goiter.
Despite the fact that serum Tg measurement is a well-accepted test that has been in common use for 4 decades, it still is subject to a variety of analytical challenges, including insensitivity, wide between-method variability, and Tg autoantibody (TgAb) interference, among others. This review will contrast the clinical utility of Tg testing in DTC with the technical limitations related to the test and provide laboratorians with suggested remedies for these analytical challenges.
The Role of Tg in DTC
Following thyroidectomy, DTC patients need life-long surveillance to monitor for tumor recurrence. An estimated 10% experience recurrence during the first decade after surgery, and an additional 5% have late recurrences that may develop decades after the initial treatment. Serum Tg measurement and periodic cervical ultrasound are the main tools for long-term surveillance (1).
The reason serum Tg measurement has such value as a tumor-marker for DTC is that Tg protein is synthesized uniquely in thyroid follicular cells. Even though the pre-operative Tg concentration is not informative as a biomarker for DTC, preoperative values can provide a gauge of the tumor’s efficiency for Tg secretion and validate the utility of postoperative Tg monitoring. Postoperative Tg measurement is likely to be most sensitive when small tumors are associated with a high preoperative Tg concentration, as compared to large tumors being associated with low preoperative serum Tg values. The latter suggests the tumor is not capable of secreting appreciable amounts of Tg.
Staging and risk-stratification are critical for determining both the frequency of Tg monitoring and the need for additional imaging modalities (CT, MRI, PET) or radioiodine treatment. Most Tg testing is done with serum. However, in recent years it has become common to measure Tg in saline wash-outs of the needles used to biopsy suspicious lymph nodes (2).
Because Tg is thyroid- but not tumor-specific, patient-related factors influence the interpretation of serum Tg concentrations. Examples include the mass of remaining thyroid tissue after thyroidectomy, recent injury, and the TSH status of the patient.
Tg Assay Limitations
The technical pitfalls of Tg measurement include between-method variability, inappropriate reference ranges, suboptimal functional sensitivity (FS), hook effects, and human anti-mouse antibody (HAMA), as well as TgAb interferences. Table 1 summarizes the characteristics of nine current Tg assays, which include several different IMA methods, an enzyme-linked immunosorbent assay, and an RIA. First-generation IMA methods have two particularly serious limitations, including insensitivity and TgAb interference. Second-generation IMA methods have a ten-fold higher FS that facilitates monitoring of basal Tg levels and may eliminate the need for expensive and inconvenient recombinant human TSH (rhTSH) stimulation. However, second-generation IMAs are still prone to TgAb and HAMA interferences.
Click here for Table 1
Method and Biological Variability
Although a Tg reference preparation (CRM-457) has been available for at least 15 years, method-related variabilities in Tg measurement persist (3). Some disparities reflect TgAb interference, which will be discussed more fully later in this review. However, even in the absence of interfering TgAb, the between-method coefficient of variation is about 30%, more than twice the within-person biologic variability (Figure 1a) (3–5). Complicating matters, serum Tg obtained from DTC patients is very heterogeneous, and different from the glandular Tg preparations used as standards. Abnormalities in the post-translational maturation of Tg protein involving glycosylation, phosphorylation, sulfation, and iodination cause this heterogeneity. Indeed, some tumors secrete immature, poorly iodinated, and/or conformationally abnormal Tg molecules that are detected with different specificities by different assays depending on the monoclonal antibody reagents they use (3).
Laboratory Comparison of Mean Tg Values
Sera from single donations were or measured by nine different Tg assays in 22 different laboratories. See Table 1 for identification of assay methods.
a. Serum containing no TgAb detected by any of three TgAb assays: Kronus/RSR, Siemens Immulite and Beckman Access.
b. Serum containing a low level of TgAb detected by Kronus/RSR, but judged negative according to the manufacturer-recommended cut-off for the Beckman Access and Immulite TgAb methods.
UD = undetectable.
Data is taken from the UK NEQAS Thyroglobulin Surveys (www.birminghamquality.org.uk) and used with permission from Finlay MacKenzie, program director.
Between-method variability—whether caused by TgAb interferences or patient-related Tg heterogeneity compounded by differences in assay sensitivity and specificity—require serial Tg monitoring for each patient using the same method over time. This is a challenge because DTC patients need lifelong Tg monitoring and may move, change physicians, and/or insurance plans that result in a change in contract laboratory. When a change in Tg method is necessary, it is critical to re-baseline the patient’s Tg level to prevent disrupting patient care. Unfortunately, most laboratories are not able to do this because archived unused specimens usually are not available.
Setting the Reference Range
Biochemical test results are typically reported relative to a reference range established from measurements made on individuals without conditions likely to affect the test. Any Tg assay reference range established using normal euthyroid subjects will be influenced by the rigor used to exclude individuals with thyroid pathologies such as goiter and thyroiditis. Regardless of these factors, Tg reference ranges established for normal euthyroid subjects have little relevance when interpreting serum Tg concentrations in thyroidectomized DTC patients. In these patients, it is better to interpret serum Tg levels relative to the degree of surgery (lobectomy versus near-total thyroidectomy), the TSH status of the patient, and the technical benchmarks of the assay used (Figure 2).
Click here for Figure 2
Another factor in interpreting postoperative Tg values is that Tg is thyroid- but not tumor-specific, so the serum Tg concentration represents the contribution from normal and any residual tumor tissue. For example, the typical 1–2 g normal thyroid remnant left after thyroidectomy contributes 1–2 µg/L Tg to the serum concentration in the absence of TSH stimulation. Additionally, some tumors are not efficient Tg secretors. In extreme cases, tumors may not secrete a detectable Tg concentration or may secrete abnormal Tg isoforms that are not detected by the monoclonal antibodies employed as IMA reagents. Lastly, there typically is a 10 to 20-fold difference between Tg measured in the absence versus the presence of TSH stimulation with either rhTSH or the high endogenous TSH associated with thyroid hormone withdrawal.
The Benefits of Functional Sensitivity
A realistic determination of Tg assay sensitivity is critical for the effective management of DTC patients following thyroidectomy, when very little Tg-producing tissue is left. Current guidelines recommend using FS as a means of determining Tg assay sensitivity (6). FS is a clinically relevant parameter based on low-end, between-run precision. The guidelines define it as the Tg concentration that can be measured with 20% coefficient of variation determined from multiple measurements of a human serum pool containing a low Tg level made across a clinically-relevant time-span (6–12 months) and employing at least two calibrator lots and two reagent lots. The guidelines committee developed this definition to realistically represent the sensitivity of the test used in clinical practice, and to replace descriptive terms like “ultrasensitive” and “supersensitive” that manufacturers favor for marketing.
First- Versus Second-Generation FS
RIA methodology is only capable of first-generation FS ranging from 0.5–1.0 µg/L (3). Laboratorians hoped that replacing RIA with IMA methodology would improve FS by an order of magnitude, as was the case in the 1980s for TSH. Unfortunately, most current Tg IMA methods still only have first-generation FS, with a detection limit only marginally below the lower reference limit for control subjects with intact thyroid glands (Figure 2 and Table 1, above).
First-generation assays are too insensitive clinically to use for basal Tg monitoring without TSH stimulation. As a result, it is customary to measure serum Tg after rhTSH stimulation, a maneuver analogous to the use of thyrotropin-releasing hormone stimulation to overcome the insensitivity of the TSH assays used before the 1990s (7). By consensus, a 72-hour rhTSH-stimulated Tg above 2.0 µg/L is considered a risk factor for disease (7). In reality, this fixed rhTSH-Tg cutoff has a low positive predictive value for disease of about 50%. Furthermore, depending on the method used, a Tg value of 2.0 µg/L could be reported as being anywhere between 1.5 and 3.2 µg/L using different methods (8).
More recently, second-generation IMAs have become available with FS ranging from 0.05–0.1 µg/L (9). These more sensitive assays show that there is a strong 10-fold difference between basal and rhTSH-stimulated Tg values, thereby obviating the need for expensive and inconvenient rhTSH-stimulated Tg testing for most patients (Figure 3) (8). Second generation IMAs also facilitate the monitoring of basal Tg trends that improve positive and negative predictive values for assessing risk for disease, as compared with the rhTSH-Tg cutoff value of 2.0 µg/L (1,10,11).
Click here for Figure 3
Notably, when TgAb is present, rhTSH stimulation offers no diagnostic benefit. This is because rhTSH-Tg responses are paradoxically blunted or absent in the presence of TgAb, possibly because of increased metabolic clearance of Tg-TgAb complexes (9).
The Hook Effect
A high-dose hook effect can occur when using IMA to measure exceedingly high Tg concentrations characteristic of patients with metastatic disease. These high Tg levels overwhelm the assay’s reagent binding capacities, which yields inappropriately low values. Although adoption of a two-step assay design has reduced this problem, a hook effect still can occur when measuring saline wash-outs of the needles used to biopsy metastatic lymph nodes. In such cases, Tg levels often exceed 10,000 µg/L. Because of these challenges, laboratories need to use linearity studies to check the high range for hooking and the potential for carry-over contamination of specimens.
Unfortunately, the Tg IMA methodology favored by most laboratories is more prone to interferences from both human anti-mouse antibody (HAMA) and TgAb than is Tg RIA methodology (3,12,13). HAMA interference usually results in a falsely high serum Tg that may prompt unnecessary imaging or radioiodine treatment for presumed disease. In contrast, TgAb interference causes falsely low or undetectable serum Tg that can have more serious consequences because it can mask the presence of disease. Whereas contemporary IMA methods routinely include blocker reagents to minimize HAMA interference to around 0.5%, currently there are no effective measures to overcome TgAb interference encountered with Tg IMA methodology (8).
HAMA in the specimen can interact with one of the monoclonal antibody reagents to create a false signal that simulates the presence of a high antigen (Tg) concentration (12). Rarely, HAMA can block the participation of the antibody reagents and cause a falsely low Tg value (13). Physicians should suspect HAMA when the serum Tg level appears inappropriate in the context of the patient’s clinical status, or fails to respond appropriately to changes in TSH. An example would be when Tg levels rise <1.5-fold after rhTSH (8). HAMA interference can be investigated by re-testing using RIA methodology that is not influenced by HAMA.
The Importance of Direct TgAb Measurement
Guidelines recommend measuring TgAb in all specimens prior to Tg testing because the qualitative TgAb status (positive or negative) determines the risk for interference with Tg measurement, and the quantitative TgAb concentration can serve as a surrogate tumor marker. TgAb should be measured directly by immunoassay rather than by exogenous Tg recoveries because the latter are unreliable for detecting interfering TgAb (1,3,6).
Direct TgAb measurement also can be a surrogate tumor marker indicative of DTC recurrence in certain patients. The immune system is sensitive to circulating Tg antigen, so for the approximately 20% of DTC patients with detectable TgAb, the TgAb concentration can be used to monitor changes in tumor mass in preference to measuring Tg by IMA (14). Specifically, when the antigenic stimulus is removed by thyroidectomy, TgAb concentrations typically decline by approximately 50% within the first year and eventually disappear within a median of 3 years when patients are rendered disease-free (14). Conversely, TgAb concentrations rise in response to increased antigen concentrations following second surgeries, fine needle biopsy, or radioiodine therapy as well as with recurrence. Serial monitoring of TgAb concentrations can overcome the problem of unreliable Tg IMA measurements.
TgAb Interference in IMA
TgAb interference with Tg IMA measurements can cause Tg underestimation and the reporting of falsely low or undetectable values that can mask the presence of disease (3,6). Although RIA methods tend to be more resistant to TgAb interference, they too can produce falsely low or high values, depending on the characteristics of the assay reagents and the endogenous TgAb in the specimen. Therefore, reliable TgAb detection is critical for authenticating all Tg measurements. Although the propensity for interference is related to the TgAb concentration, high TgAb levels do not necessarily produce interference, and in some cases, low TgAb concentrations may profoundly interfere (3).
Manufacturer-recommended assay cutoffs for detecting TgAb are typically set in the detectable range and relate to the diagnosis of autoimmune thyroid disease, not the detection of interfering TgAb. Inappropriate cutoffs, together with differences in assay sensitivity and specificity, cause some specimens to be classified as TgAb-positive by one method and TgAb-negative by another (5). Laboratorians should be aware that any TgAb detected above the analytic sensitivity limit has the potential to interfere with Tg measurement. Notably, these between-method disparities exist despite the methods’ purported standardization with the same international reference preparation (WHO 1st IRP 65/93). TgAb method-related disparities are difficult to eliminate because they reflect patient-related TgAb heterogeneity compounded by differences in assay sensitivity and specificity (5).
Labs on the Front Lines
Given the importance of life-long postoperative monitoring of DTC patients, labs have a vital responsibility to ensure that Tg measurements are as accurate as possible, and that they keep abreast of and address the analytical limitations of their Tg assay methods. Ongoing dialogue with endocrinologists and oncologists likewise is essential, so these clinicians can be well-informed of any method changes and confer readily with laboratorians about any discrepant results.
- Cooper DS, Doherty GM, Haugen BR, Kloos RT, et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2009;19:1–48.
- Snozek CL, Chambers EP, Reading CC, Sebo TJ, et al. Serum thyroglobulin, high-resolution ultrasound, and lymph node thyroglobulin in diagnosis of differentiated thyroid carcinoma nodal metastases. J Clin Endocrinol Metab 2007;1992:4278–4281.
- Spencer CA, Bergoglio LM, Kazarosyan M, Fatemi S, et al. Clinical impact of thyroglobulin (Tg) and Tg autoantibody method differences on the management of patients with differentiated thyroid carcinomas. J Clin Endocrinol Metab 2005;1990:5566–5575.
- Jensen E, Petersen PH, Blaabjerg O, Hegedüs L. Biological variation of thyroid autoantibodies and thyroglobulin. Clin Chem Lab Med 2007;45:1058–1064.
- Spencer C, Petrovic I, Fatemi S. Current thyroglobulin autoantibody (TgAb) assays often fail to detect interfering TgAb that can result in the reporting of falsely low/undetectable serum Tg IMA values for patients with differentiated thyroid cancer. J Clin Endocrinol Metab 96:1283–91, 2011.
- Baloch Z, Carayon P, Conte-Devolx B, Demers LM, et al. Laboratory medicine practice guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid 2003;13:3–126.
- Mazzaferri EL, Robbins RJ, Spencer CA, Braverman LE, et al. A consensus report of the role of serum thyroglobulin as a monitoring method for low-risk patients with papillary thyroid carcinoma. J Clin Endocrinol Metab 2003;88:1433–1441.
- Spencer CA, Fatemi S, Singer P, Nicoloff JT, et al. Serum basal thyroglobulin measured by a 2nd generation assay correlates with the recombinant human tsh-stimulated thyroglobulin response in patients treated for differentiated thyroid cancer. Thyroid 2010;20:587–95.
- Spencer CA, Lopresti JS. Measuring thyroglobulin and thyroglobulin autoantibody in patients with differentiated thyroid cancer. Nat Clin Pract Endocrinol Metab 2008;4:223–233.
- Tuttle RM, Leboeuf R. Follow up approaches in thyroid cancer: a risk adapted paradigm. Endocrinol Metab Clin North Am 2008;37:419–435.
- Giovanella L, Ceriani L, Suriano S, Ghelfo A, et al. Thyroglobulin measurement before rhTSH-aided (131)I ablation in detecting metastases from differentiated thyroid carcinoma. Clin Endocrinol (Oxf) 2008;69:659–663.
- Preissner CM, O’Kane DJ, Singh RJ, Morris JC, et al. Phantoms in the assay tube: heterophile antibody interferences in serum thyroglobulin assays. J Clin Endocrinol Metab 2003; 88:3069–3074.
- Giovanella L, Ghelfo A. Undetectable serum thyroglobulin due to negative interference of heterophile antibodies in relapsing thyroid carcinoma. Clin Chem 2007;53:1871–1872.
- Chiovato L, Latrofa F, Braverman LE, Pacini F, et al. Disappearance of humoral thyroid autoimmunity after complete removal of thyroid antigens. Ann Intern Med 2003;139:346–351.
Carole Spencer, MT, PhD, FACB, is a professor of medicine in the Department of Medicine at the University of Southern California. Email: firstname.lastname@example.org