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CLN’s improviNg heaLthCare through Laboratory mediCiNe series 10 CliniCal laboratory news July 2011 Thyroglobulin 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 postoperative 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 tumorspecific, 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, secondgeneration IMAs are still prone to TgAb and HAMA interferences. 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 (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). Between-method variability—whether caused by TgAb interferences or patientrelated 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 1). 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 concentra- figure 1 factors that influence Tg reference intervals schematic diagram of how the tg reference range is influenced by the degree of surgery: lobectomy (center panel) or near-total thyroidectomy (right panel), tsH status (on abscissa), and assay functional sensitivity (first- versus second-generation). Adapted from reference 6. tion 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 firstgeneration 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 1 and Table 1). 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 rhTSHstimulated 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 table 1 characteristics of current Tg assays from 0.05–0.1 µg/L (9). These more sensitive assays show that there is a strong 10fold difference between basal and rhTSHstimulated 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). 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 Assay # 1 2 3 4 5 6 7 8 9 Assay Name Access Tg Tg-Plus TgIRMA Immulite 2000 Tg TgRIA Elecsys Tg Tg Wallac Delfia Tg Manufacturer Beckman Coulter, US BRAHMS diagnostica, Germany CISbio-Schering, Germany Siemens, US Genesis Diagnostics, UK University Southern California, US Roche, Germany Sanofi Pasteur, France Perkin-Elmer, Finland Assay Type non-competitive ICMA competitive IRMA competitive IRMA competitive ICMA non-competitive ELISA competitive RIA competitive IECMA competitive IRMA competitive IFMA Capture Antibody 4xMab pab (rabbit) 4xMab Mab pab (rabbit) pab (rabbit) Mab 4xMab Mab Tracer ap-Mab 125-iMab 125-iMab (sheep) ap-pab (rabbit) 125-itg Mab 125-iMab eu-Mab Sample Size µL 40 100 100 50 50 200 20 100 50 1:1 CRM-457 Standardization Functional yes no (factor of 2) yes yes no yes yes no no Sensitivity ng/mL (µg/L)*# Reference 0.1 [5,1] 0.4* 0.7 0.9 na 0.5 1.0 na na Range ng/mL (µg/L)* 3–32 [3] 2–34 [3] 2.1–43 [3] 1.6–60 2.0–50 3.0-40 1.4–78 1.5–50 1.7–35 *expressed relative to 1:1 CrM-457 standardization; # defined by naCb guidelines (6); [ ] literature citation, no manufacturer data CliniCal laboratory news July 2011 11
Page 1 and 2: news brief Forecast: robust Market
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