Thyroglobulin in the Washout Fluid of Lymph-Node Biopsy: What Is Its Role in the Follow-Up of Differentiated Thyroid Carcinoma?

Abstract

Background:

The clinical evaluation of enlarged local lymph nodes (LNs) is difficult at the beginning and throughout the follow-up of differentiated thyroid carcinoma (DTC). Although the examination of samples collected from LNs by fine-needle aspiration biopsy cytology (FNAB-C) is extremely specific for the diagnosis of metastases, its sensitivity is low, especially in paucicellular samples.

Summary:

The measurement of thyroglobulin (Tg) in the fine-needle aspiration biopsy (FNAB) washout fluid (FNAB-Tg) increases the diagnostic performance of cytology to up to 100% sensitivity and specificity. However, the application of FNAB-Tg is currently hindered by the absence of methodological standardization, a lack of definite cutoff points, and the ongoing debate regarding its accuracy in nonthyroidectomized patients, those with elevated serum Tg, and those with circulating anti-Tg antibodies.

Conclusion:

FNAB-Tg improves the diagnostic performance of FNAB-C in LN metastases, even when the latter is unable to diagnose the metastases. For that reason, FNAB-Tg should be included in the monitoring of DTC.

Introduction

The incidence of thyroid cancer has increased worldwide in the past few years (1 –5). An estimated 60,220 new cases of thyroid cancer will be diagnosed in 2013 in the United States, with three out of four cases occurring in females (6). The incidence rate of thyroid cancer has been dramatically increasing since the mid-1990s, and it is the fastest-increasing cancer in both men and women (6), mainly due to the papillary histotype (1 –3,7).

Up to 30% of the patients with papillary thyroid carcinoma (PTC) exhibit recurrent or persistent metastasis to the neck lymph nodes (LNs) (8,9). The data from centers where sentinel LN biopsy or routine cervical LN dissection are performed show that occult metastases may occur in up to 90% of PTC patients (10 –12).

The clinical evaluation of enlarged local LNs is difficult at the beginning and throughout the follow-up of patients with differentiated thyroid carcinoma (DTC) because inflammatory lymphadenopathies are extremely frequent. Furthermore, neck LN metastases from a multiplicity of extrathyroidal malignancies are a relatively common finding (13).

Therefore, several tools have been proposed to facilitate the accurate identification of metastatic LNs before or after primary surgery. The monitoring of DTC after thyroidectomy and radioiodine ablation is based on the measurement of baseline and thyrotropin (TSH)-stimulated serum thyroglobulin levels, whole-body scans (WBS) using radioactive iodine (RAI), neck ultrasound (US) (13), and fine-needle aspiration biopsy (FNAB) with cytological analysis of the LNs. Additionally, staging of the neck LNs is mandatory in the management of thyroid cancer before thyroidectomy, which subjects patients with metastatic DTC to more radical surgery. Staging is based on the neck US, and in suspected cases, the metastatic nature of LNs is confirmed by cytological analysis of the FNAB (14).

Although the specificity of serum Tg (sTg) to detect recurrent disease is high, it is not the ideal measurement for metastases affecting small LNs. In addition, it is not able to establish the location of the neoplastic focus and is highly affected by the presence of serum antithyroglobulin antibodies (TgAb), which are found in 25–30% of patients with DTC (15,16). In turn, the results of WBS might be affected by the tumor size, RAI dose, and radiotracer uptake. For instance, LN metastases might not take up iodine in 20–40% of cases, even when high ¹³¹I doses are applied (17,18).

The role of US has become increasingly important in the follow-up of patients with DTC after initial treatment (19). The sensitivity of US in detecting occult masses and nonpalpable neck LNs is well documented. However, despite the establishment of predictive criteria for malignancy, their precision is not sufficient to distinguish between benign (inflammatory or reactive) and metastatic or suspected LNs, even when pooled together (20). In addition, proper identification might only be achieved by a skilled and experienced operator.

The cytological analysis of FNAB samples (FNAB-C) from LNs has been widely used to confirm suspect findings on US, mainly in patients with undetectable sTg due to suppressed TSH or with a noniodine-concentrating metastasis (19). Nevertheless, the sensitivity of this method is far from ideal, as it varies from 75% to 85% (21,22), with a rate of false-negative results of 6% to 8% (21,23) and a rate of up to 20% of nonrepresentative samples or samples with inadequate cellularity, which are a function of the cytopathologists' skill and experience (24,25).

To improve the diagnostic performance of FNAB-C, several authors have suggested measuring Tg in the needle washout fluid (FNAB-Tg). This method was initially suggested in 1992 by Pacini et al. (21), who showed that high FNAB-Tg in the LNs of individuals subjected to thyroidectomy and radioablation was due to thyroid carcinoma metastasis, whereas undetectable FNAB-Tg indicated inflammatory lymphadenopathy or lymphadenopathy of nonthyroid origin. In that study, the sensitivity of FNAB-Tg was 100%, whereas the sensitivity of cytology was only 85.7%. In 1993, Lee et al. (26) reported that the combination of FNAB-C and FNAB-Tg detected a larger number of metastatic LNs than either technique alone and reached 100% sensitivity and specificity.

Several authors have performed FNAB-Tg and found that its sensitivity varied from 81.4% to 100% and its specificity from 85% to 100% (13,21,22,27 –39). In spite of those results, controversy remains on some issues, including the scarcity of studies validating the alleged benefit of FNAB-Tg over FNAB-C alone and the variation of the cutoff points used, which range from 0.9 to 39 ng/mL as a function of the method used in the measurement of Tg (22,28 –30), in addition to divergences related to its diagnostic performance in patients not yet subjected to thyroidectomy. Moreover, the best method of FNAB-Tg has not yet been standardized, and thus, all published studies on this topic exhibit significant differences in the techniques used to wash needles and to measure Tg, as well as the units used (ng/FNAB, ng/mL) and the interpretation of the FNAB-Tg values. In consequence, the proper role of FNAB-Tg in the follow-up of patients with DTC is still a subject of debate.

Thus, the aims of this article are to summarize the published evidence on the use of FNAB-Tg in the follow-up of individuals with DTC and to discuss the following topics: (i) the accuracy of FNAB-Tg in the detection of neck metastases in individuals subjected to or awaiting total thyroidectomy; (ii) the diagnostic benefit of FNAB-Tg compared to FNAB-C alone in DTC recurrence in cervical LNs; (iii) the methods reported in the literature to establish the cutoff points of FNAB-Tg, including a comparison of their diagnostic performance to detect malignancy; and (iv) the differences among the techniques used to measure FNAB-Tg, including an assessment of the possible influence of technical variations on its diagnostic accuracy.

Discussion

The European consensus for the management of patients with DTC recommends screening the patients 6–12 months after the initial treatment by means of neck US and recombinant TSH-stimulated Tg (14).

The selection of patients for FNAB must be grounded on the architecture of the investigated LN on US. The most reliable indicators of malignancy are the presence of heterogeneous hyperechogenicity compatible with microcalcifications, loss of hilum, cystic alterations, and peripheral vascularization (40). All such ultrasonographic findings exhibit strong correlations with malignant findings on postoperative histological analysis (19).

Some authors (14,40,41) suggest that FNAB should be performed only in cases when the smallest diameter of the LN is >5 mm, as this criterion is associated with a satisfactory balance between the sensitivity and specificity of cytology alone for detection of metastatic PTC (61% and 96% respectively) (19).

Because Pacini et al. (21) reported approximately 20 years ago that high Tg concentrations are detectable in LNs with thyroid cancer metastases, several studies have shown that the sensitivity of FNAB-Tg to detect DTC neck LN metastases is higher than the sensitivity of FNAB-C (13,21,22,27 –39). Frasoldati et al. (22) assessed the role of FNAB-Tg in the detection of LN metastases in patients with DTC before thyroidectomy and found a sensitivity and specificity of 78.6% and 100% respectively. In addition, the sensitivity of combined FNAB-C and FNAB-Tg to assess neck LNs was better compared to WBS and sTg measurement. In a later study on patients with neck-recurrent DTC, those same authors showed that the diagnostic sensitivity increased from 84.8% to 95.6% when FNAB-Tg was added to cytology (42).

In 2003, Cignarelli et al. (27) showed that FNAB-Tg might also contribute to diagnosis in paucicellular samples from cystic metastases. The diagnosis in two out of the six patients with PTC cystic metastases in the neck LNs included in the study was established by FNAB-Tg but not by cytology alone.

Bournaud et al. (36) assessed 114 consecutive patients with thyroid cancer and 16 controls. The sensitivity of FNAB-Tg was 94.2% and its specificity 97.8%. As for FNAB-C, although its specificity was 100%, its sensitivity was 71%, mostly due to inadequate samples.

Salmaslhoğlu et al. (35) compared the final histological diagnosis of 255 LNs with the results of preoperative FNAB-C and FNAB-Tg. FNAB-C failed to detect 17% of the metastatic LNs compared with 1% by FNAB-Tg. The specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of FNAB-Tg were significantly higher compared to FNAB-C.

The cystic degeneration of PTC metastatic deposits is not uncommon (43,44), and FNAB of LNs in those cases, even with ultrasonographic guidance, might only show colloidal material, cell remnants, and macrophages, but no identifiable tumor cells. Such samples are usually classified as “nondiagnostic” or “inadequate” (45 –47). In turn, FNAB-Tg has shown high sensitivity in the detection of cystic metastases when cytology is inconclusive. For instance, Cunha et al. (30) assessed 83 enlarged neck LNs from 67 patients with DTC and found that the sensitivity of FNAB-Tg was 100%, whereas cytology alone failed to diagnose 9 out of the 17 patients with recurrent disease. FNAB-C alone identified only one out of six metastatic LNs with a cystic component (16.7% sensitivity).

Uruno et al. (31) studied 129 enlarged LNs from 111 patients with DTC, and found that the sensitivity of FNAB-Tg and cytology was 81.4% and 78% respectively, and that the sensitivity of FNAB-Tg for detection of cystic or mixed PTC metastases was 100%. On the whole, 36.4% of the cases classified as benign and 37.5% of the cases classified as inadequate on cytological examination were classified as malignant based on FNAB-Tg.

Snozek et al. (28) assessed 122 FNAB samples from 88 patients with a history of DTC and thyroidectomy. They found that the Tg level was ≤1 ng/mL in 50 out of the 52 benign samples (96.2%) and >1 mg/mL in all 70 malignant samples. Five (4.9%) of the 103 samples with diagnostic cytology had discordant Tg results, and in four of these samples, FNAB-Tg was concordant with the final diagnosis. As 18 out of 19 (94.7%) cases classified as nondiagnostic or with absent cytology were correctly diagnosed by FNAB-Tg, the authors suggested replacing FNAB-C with FNAB-Tg in the assessment of neck LN metastases.

The European consensus regarding the management of patients with DTC (14) and the Brazilian Consensus on Thyroid Nodules and Differentiated Thyroid Cancer (48) currently recommend combining FNAB-Tg with the cytological assessment of the FNAB samples to confirm suspected LN metastases. The American Thyroid Association (49), however, made no mention of that method in its guidelines, most likely because thyroid specialists in the United States have less experience with FNAB-Tg. The available data on the diagnostic value of FNAB-Tb were collected using several types of Tg assays, variable amounts of washout fluid, and different cutoff points to define the negative results (19). The lack of consensus regarding the recommendation of FNAB-Tg among the various international guidelines for the management of DTC is most likely due to the lack of international standards for the performance and interpretation of the technique.

Factors that might affect the accuracy of Tg assays

Measuring Tg poses one of the major challenges to laboratories due to interfering factors that might alter the test results, the lack of methodological standards, inadequate functional sensitivity, and variability in the specificity of the commercially available antibody kits. All of these factors affect the accuracy of the results and are associated with large interassay variation, which makes comparison among studies difficult.

The methods available to measure Tg include immunoassays, such as immunometric assay (IMA) and radioimmunoassay (RIA), in addition to enzyme-linked immunosorbent assay (ELISA), which is seldom used. The older competition-based measurement methods using radioactive iodine tracers have progressively been replaced by the more specific and sensitive IMA, which comprises immunoradiometric (IRMA) and immunochemiluminescence assays (ICMA) (47). The IMA method allows Tg levels <0.1 ng/mL to be detected, and its technical advantages include shorter incubation periods and more stable reagent antibodies (50). Historically, high between-method variability in the Tg methods has been reported (51). Interassay precision must always be maximized so that the interpretation of results is not compromised by changes in the reagents or the equipment calibration over the course of the patients' follow-up (51).

Coefficients of interassay variation of 48% for sTg values of approximately 5.0 ng/mL and 80–120% for undetectable Tg values have been reported (52). Such discrepancies are partially accounted for by the heterogeneity of human Tg and the differences in the epitope specificity of the antibodies used as reagents in the various immunoassays (53). In particular, thyroid carcinomas might secrete one predominant or various Tg isoforms, which are detected with variable intensity as a function of the antibodies and assay methods used (54).

Various guidelines and consensus statements recommend the use of kits that comply with the European standards for manufacturing Tg assays, for example CRM 457 (55,56), which recommends the use of purified Tg obtained from human thyroid as the antigenic stimulus (57). Unfortunately, because many assays do not comply with that consensus, although it reduces the variability in the methods available to measure Tg, it cannot eliminate it completely (51,54).

Additionally, the functional sensitivity is of fundamental importance in the assessment of the performance of Tg measurement kits. Functional sensitivity is defined as the lowest measured Tg concentration with a coefficient of interassay variation ≤20% (58), and it represents the lowest clinically relevant value detected by the test. The functional sensitivity has gradually improved since the earliest methods for Tg measurement, from 5–10 ng/mL with the older RIAs to 0.5–0.9 ng/mL with the first-generation IMAs, and to 0.05–0.1 ng/mL with the “ultrasensitive” IMAs (59).

The functional sensitivity of a method directly influences the cutoff points. The original study by Pacini et al. (21) showed that the cutoff point, defined as the mean±two standard deviations (SD) of the FNAB-Tg value of patients with the final diagnosis of benign or malignant nonthyroid disease, was 21.7 ng/FNAB. Such a high cutoff point might be explained by the low functional sensitivity of the method used to measure Tg, which was 3 ng/mL. The greater functional sensitivity of the currently available assays accounts for the progressive reduction of the suggested diagnostic thresholds. Thus, some authors suggest that the FNAB-Tg cutoff point might be adapted to the method of Tg measurement used by each individual center (36).

Several published studies focus on methodological factors that might influence the Tg values found in the FNAB washout fluid, such as the volume and type of fluid used, procedures for sample collection, and the interference of TgAb and sTg with the FNAB-Tg values.

Needle washing

Needle washout fluid

The medium used to wash the needle used in FNAB varies among studies, from 0.9% saline solution to Tg-free serum, the standard “zero” provided in the Tg assay kit (Table 1). Saline solution is the most widely accessible and least expensive option and is therefore the most commonly used (28 –30,32 –35). To compare these two types of washout fluid used in FNAB, Frasoldati et al. (22) used different needles to collect two consecutive samples from the same 10 metastatic and 10 nonmalignant LNs. One needle was washed with 1.0 mL of saline solution, and the other was washed with 1.0 mL of Tg-free solution. The FNAB-Tg concentration did not differ between washouts as a function of the rinsing method.

Table 1.

Review of the Different Methods of Measuring Thyroglobulin by Fine-Needle Aspiration Biopsy

	Numbers of patients/lymph nodes	Tg measurement method (kit)	Needle (gauge)	Volume and type of washing fluid	Unit ^a
Boi et al. (13)	73/–	Immunoradiometric (CIS Bio International) or chemiluminescent immunometric (Immulite 2000—automatic immunoassay analyzer, Siemens)	22–25	Tg-free serum 0.5 mL	ng/mL
Pacine et al. (21)	35/–	Immunoradiometric (Sorin)	21	Tg-free serum^b 0.5 mL	ng/FNAB
Frasoldati et al. (22)	130/216	Immunoradiometric (Radim)	22–23 spinal needle	Tg-free serum or saline solution 1 mL	ng/mL
Snozek et al. (28)	88/122	Automatic immunoassay analyzer DXI (Beckman Coulter) or Immulite 2000 automatic immunoassay analyzer (Siemens)	25	Saline solution 0.5–1 mL	ng/mL
Borel et al. (29)	34/53	Immunoradiometric (CIS Bio International)	22 BD spinal^® or 23 BD Microlance 3 ^®	Saline solution 1 mL	ng/mL
Cunha et al. (30)	67/83	Chemiluminescent immunometric (Immulite 2000, Euro/DPC Ltd.)	22 or 25	Saline solution 1 mL	ng/mL
Uruno et al. (31)	111/129	—	22	Saline solution 0.5 mL	ng/mL
Kim et al. (32)	168/168	Immunoradiometric (CIS Bio International)	23	Saline solution 1 mL	ng/mL
Jeon et al. (33)	47/76	—	21–23	Saline solution 1 mL	ng/mL
Sohn et al. (34)	92/95	Immunoradiometric (CIS Bio International)	23	Saline solution 1 mL	ng/mL
Salmashõglu et al. (35)	225/255	Immunoradiometric (CIS Bio International)	—	Saline solution 3 mL	ng/mL
Bornaud et al. (36)	89/122	Immunoradiometric (CIS Bio International)	27	Tg-free serum 1 mL	ng/FNAB
Sigstad et al. (37)	145/256	Immunofluorometric	25 or 27	Tg-free serum or saline solution 0.5 mL	ng/mL
Biscolla et al. (38)	32/44	Immunofluorometric (Delphia)	—	Saline solution 1 mL	ng/mL
Zanella et al. (39)	43/43	Electrochemiluminescence (Modular E-170, Roche)	22–25	Saline solution 1 mL	ng/mL
Giovanella et al. (61)	108/156	Immunoradiometric DYNO test Tg-plus (BRAHMS Diagnostica GmbH)	—	Saline solution 1 mL	ng/mL
Kim et al. (62)	68/91	Immunoradiometric (CIS Bio International)	21–23	Saline solution 0.5 mL	ng/mL

Unit used to express the results of the measurement of thyroglobulin in the washout fluid of fine-needle aspiration biopsy (FNAB-Tg).

Tg-free serum, standard “zero” given by the Tg kit company.

FNAB, fine-needle aspiration biopsy.

However, the studies by Baskin (15) and Snozek et al. (28) pointed out the possibility of a matrix effect when saline solution is used. The matrix effect is a type of interference that alters the reactivity of Tg with the assay antibodies due to the medium components. Snozek et al. (28) performed a recovery test after exogenous Tg overload and found that the Tg values increased by approximately 25% (varying from 100% to 140% of the expected values) when the FNAB-Tg needle was washed with saline solution. In fact, the nature of the buffer used to wash the needle might influence the protein conformation, thereby affecting the binding of antibodies, which was observed in the case of Tg when saline solution was used as a buffer (28,29,31,37). Such a matrix effect might account for the high Tg levels found by some authors in benign LNs (37). Nonspecific interactions might be the cause of such interference in the measurement of Tg, as observed by Baskin (15).

Therefore, although the amount of solution included in the kits is limited and its cost higher, it is preferable to saline solution, as this procedure avoids creating a bias in the measurement of Tg in the FNAB washout fluid (36). Nevertheless, when saline solution is used for practical reasons, ruling out the presence of a matrix effect is recommended (60). To validate the use of saline solution in their study, Borel et al. (29) measured the immunoreactivity of Tg in samples of Tg-free solution, saline solution, and saline solution supplemented with 70 g/L of human serum albumin. The results showed that the immunoreactivity of Tg did not reach the assay detection threshold in any of the study samples.

Volume of washout fluid

In various studies, the volume of fluid used to rinse the FNAB needle varies from 0.5 to 3.0 mL (Table 1), and 1.0 mL is the amount most widely used (22,29,30,32 –34,36,42). That is also the amount recommended in the consensus published by Leenhardt et al. (60). Some authors suggest washing the needle several times and collecting the full amount of washout fluid in a single tube. According to Borel et al. (29), three rinses adding up to 1.0 mL of fluid suffice to collect more than 97% of the Tg present in the needle.

Sample collection

Several test tubes are commercially available, and their use in FNAB-Tg must be validated. According to Giovanella et al. (61), the different types of tubes used to store the washout fluid might be a cause of bias in the measurement of FNAB-Tg. Following US-guided FNAB of 156 LNs from 108 patients subjected to thyroidectomy and radioablation for DTC, those authors washed the needles using 1.0 mL of saline solution, which they divided homogeneously into three different types of test tubes: the plain serum tube, the serum separator tube, and the lithium heparin tube. The use of the serum separator or lithium heparin tubes was associated with significantly reduced FNAB-Tg compared to the plain serum tube. In addition, the measurement of FNAB-Tg from the plain serum tube and serum separator tubes had 100% sensitivity, whereas two false-negative results occurred in the samples collected in lithium heparin tubes (98% sensitivity). Thus, the serum separator tubes and the lithium heparin tubes may decrease the measured FNA-Tg concentration, and the collection of washout fluids into plain serum tubes is recommended.

Interference by circulating thyroglobulin

Only a few studies have reported on the diagnostic value of FNAB-Tg for the LN staging of PTC before thyroidectomy (22,32,36,37,62), and their results tend to disagree.

Although Frasoldati et al. (22) found 100% specificity in the FNAB-Tg of patients awaiting thyroidectomy, and Cunha et al. (30) found 100% PPV in patients before surgery, false-positive results have been reported in patients with high sTg (15,21,32). Indeed, Tg as high as 88.8 ng/mL has been found in benign LNs from patients awaiting surgery for thyroid cancer (32,36) and in patients without any thyroid disease, which could lead to unnecessary surgery for many of those patients (31).

According to Sigstad et al. (37), thyroid manipulation or palpation in patients who conserve all or part of the gland might lead to Tg escaping into the lymph, resulting in false-positive LN FNAB-Tg results. The FNAB-Tg must also be avoided in the examination of the LNs close to the thyroid (e.g., paratracheal, pretracheal, and prelaryngeal LNs) before thyroidectomy, when the risk for the biopsy needle to run through the thyroid is high, resulting in elevated FNAB-Tg independently of the presence of LN malignancy (31). Other possible explanations for the false-positive results include contamination of the LN by circulating sTg and artifacts caused by the method used for Tg detection because of nonspecific binding of the antibodies to various medium components. Such artifacts, described above as matrix effects, depend on the antibodies and the media that are used.

The data obtained by Borel et al. (29) do not support the hypothesis of sTg contamination of the LNs. According to them, even if the sample collected by FNAB was composed of blood alone, then as a function of the dilution, the rate of contamination of FNAB-Tg would be 0.2 times the sTg value, which in that study varied from 0.003% to 0.012% and was thus patently insignificant. To strengthen their argument, they further showed that FNAB-Tg was undetectable in negative controls who were not subjected to previous thyroidectomy and thus had detectable sTg. Finally, they measured FNAB albumin and showed that the contamination of FNAB-Tg by serum proteins was very low. They concluded that sTg might interfere significantly with the FNAB-Tg values only in patients with high sTg, in whom the measurement of FNAB-Tg is actually not needed because they usually exhibit evident metastases. Other authors, including Bournaud et al. (36) and Kim et al. (62), were also unable to identify any influence of the presence of the thyroid gland on the FNAB-Tg values, and found that this test has high sensitivity, specificity, and accuracy in the staging of LNs before thyroidectomy.

In a large-scale study conducted by Kim et al. (32), FNAB-Tg performed before surgery showed less sensitivity and accuracy relative to five assessed cutoff points compared to the measurement after thyroidectomy. According to Sohn et al. (34), it is not yet clear whether the interference of sTg on low or medium FNAB-Tg levels is negligible because the patients assessed by Borel et al. exhibited high FNAB-Tg. Thus, the solution to this issue requires more thorough analysis.

Several cutoff points have been suggested for FNAB-Tg before thyroidectomy. According to some authors, who believe in the hypothesis of contamination by circulating Tg, different diagnostic thresholds ought to be established for patients who have undergone thyroidectomy and those awaiting thyroidectomy, whereas others recommend the adoption of a single cutoff point for both categories of patients. According to Borel et al. (29), any detectable level of FNAB-Tg in the neck LNs of patients without elevated sTg is associated with the presence of thyroid cells. That hypothesis is strengthened by the recent result that single FNAB-Tg cutoff points as low as 0.93 ng/mL correctly identified the LNs with DTC metastases (36).

Interference by antithyroglobulin antibodies

Doubtlessly, interference from circulating TgAb is the most serious technical complication that hinders the use of sTg as a marker of DTC recurrence (13,16).

As the methods used to measure FNAB-Tg and sTg are the same, the possible interference of TgAb with FNAB-Tg values was taken into consideration (22,42) until Boi et al. (13) showed the absence of any significant interaction between the TgAb and the Tg level measured in the neck LNs of patients with DTC, even in those with TgAb detectable inside the LNs, by either active synthesis or blood contamination. Although those authors reported that the presence of TgAb in the samples could result in underestimation of the FNAB-Tg level, such interference actually exerted a small effect on the diagnostic performance of the test because the FNAB-Tg level in the washout fluid that contained TgAb was much higher than the selected cutoff point in all the evaluated cases (13).

Approximately two years earlier, Baskin (15) reported on the lack of difference in the sensitivity and specificity for the detection of LN metastases between patients with and without serum TgAb. However, the small number of cases assessed in that study precluded any strong conclusion.

The reduced interference of TgAb with FNAB-Tg results compared to the interference exerted on the sTg measurements could be explained by the dilution of samples (most likely being 1:50 or higher) (37); elevated Tg concentrations in the washout fluid, which overcome the interference by saturating the TgAb-binding sites; or the absence or scarcity of TgAb inside the LNs (13).

In 2007, Sigstad et al. (37) studied the role of possible TgAb interference and found that those antibodies were absent in the FNAB washout fluid of individuals with positive serum TgAb and no or low FNAB-Tg. The absence of TgAb inside the LNs was also reported by other authors (13,29,37,63).

According to Boi et al. (13), although one cannot rule out the possibility that the presence of TgAb in the FNAB washout fluid will sometimes hinder the detection of Tg in metastatic LNs, this phenomenon was not observed in their large case series, and thus its frequency might be very low.

In turn, Cappelli et al. (64) recently observed a shift from undetectable to detectable FNAB-Tg in suspected LN metastases on US or WBS following recombinant human (rh)TSH stimulation in two patients with detectable serum TgAb. Those authors believe that in such patients, the presence of TgAb fully prevented the measurement of Tg in the FNAB washout fluid and that the Tg in the LNs increased to levels sufficient to saturate all the TgAb-bindings sites, and thus became detectable, only after stimulation with rhTSH, a possibility that had been suggested previously. As the presence of TgAb in the FNAB washout fluid of the LNs was not assessed because it is not recommended by most authors and because of the lack of histological data, that hypothesis could not be confirmed.

Zanella et al. (39) assessed the influence of serum TSH on the FNAB-Tg levels of patients without evidence of LN metastases on cytology who were undergoing levothyroxine-suppressive therapy (TSH=0.07 mIU/mL) or were hypothyroid in order to measure a stimulated sTg (TSH=82.2 mIU/mL). According to those authors, there was no significant difference in the median FNAB-Tg value between groups (3.3 ng/mL vs. 3.8 ng/mL respectively), which suggests a lack of interference of serum TSH on the FNAB-Tg measurements.

Cutoff points for measuring thyroglobulin in the washout fluid of fine-needle aspiration biopsy

Although the diagnostic performance of FNAB-Tg is well established, its cutoff points remain controversial. A wide variety of possible diagnostic thresholds ranging from 0.9 to 39.3 ng/mL have been described in the literature (Table 2), and it is not yet clear whether the same cutoff point ought to be used before and after thyroidectomy and for patients with high sTg. The major hindrances to the definition of one specific cutoff point for FNAB-Tg are the variety of available measuring kits and the different techniques and volumes of fluid used to wash the FNAB syringes. In some studies, the cutoff point was defined as the mean±2 SD of the FNAB-Tg level of patients with negative cytology (15,21,27). Other authors established the cutoff point based on the comparison of the FNAB-Tg and sTg levels, whereby the LNs were considered metastatic when the former was higher than the latter (31,33,37,62), which rules out the possibility of contamination by the circulating Tg. Cunha et al. (30) defined the cutoff point as the functional sensitivity of the Tg assay. In other studies, the cutoff point was established based on the area under the receiver operating characteristic (ROC) curve that best represented the relationship between the sensitivity and specificity of the analysis (32,34 –36). Boi et al. (13) used the highest FNAB-Tg value found in benign LNs that showed ultrasonographic regression in 6–12 months as the cutoff point.

Table 2.

Diagnostic Performances of FNAB-Tg and FNAB-C and the Cutoff Points of FNAB-Tg

	Analytic sensitivity/functional sensitivity of the FNAB-Tg measurement kit	Method to establish the FNAB-Tg cutoff point	FNAB-Tg cutoff point	FNAB-C sensitivity/specificity	FNAB-Tg sensitivity/specificity	Gold standard ^a
Boi et al. (13)	0.2 (IRMA) or 0.5 ng/mL (ICMA)/–	Greatest FNAB-Tg value in benign LNs^b	1.7 ng/mL (T) or 36 ng/mL (NT)	—	100%/100%	Histopathology or reactive FNAB-C+ultrasonographic regression in 6–12 months
Pacine et al. (21)	3 ng/mL/–	Mean±2 SD^c	21.7 ng/FNAB	85.7%/–	100%/–	Histopathology
Frasoldati et al. (22)	0.25 ng/mL/–	97.5th percentile of LNs with benign FNAB-C^d	1.1 ng/mL (T) or 39.3 ng/mL (NT)	—	84.0%/95.4%	Histopathology
Snozek et al. (28)	0.06 or 0.2 ng/mL/0.1 or 0.9 ng/mL	ROC curve	1 ng/mL	—	100%/96.2%	Histopathology. Neck US or clinical evidence after more than one year follow-up
Borel et al. (29)	–/1 ng/FNAB	—	High: >10 ng/mLIntermediate: 1–10 ng/mLUndetectable: <1 ng/mL	—	—	—
Cunha et al. (30)	0.5 or 0.2 ng/mL/0.9 ng/mL	Kit's functional sensitivity	0.9 ng/mL	55% (T) and 57% (NT)/100%(T)	100% (T and NT)/100% (T)	Histopathology
Uruno et al. (31)	—	sTg	—	78%/–	81%/–	Histopathology
Kim et al. (32)	0.2 ng/mL/0.7 ng/mL	Assessed five cutoff points through ROC curve	sTg, mean±2 SD (32 ng/mL) or 10 ng/mL	77.3%/98%	90.8%/89.8%	Histopathology and US follow-up
Jeon et al. (33)	—	Based on Uruno et al.	sTg or 36 ng/mL (when sTg is unavailable)	80.4%/100%	94.6%/90%	Histopathology
Sohn et al. (34)	0.2 ng/mL/0.7 ng/mL	Assessed six cutoff points through ROC curve	5 ng/mL	40.5%/100%	69%/83%	Histopathology
Salmashõglu et al. (35)	—	ROC curve	28.5 ng/mL	95%/40%	100%/96%	Histopathology
Bornaud et al. (36)	0.2 ng/mL/0.7 ng/mL	Assessed seven cutoff points through ROC curve	0.93 ng/FNAB	71.1%/73%	94.2%/97.8%	Histopathology or WBS+disappearance/no progression on US
Sigstad et al. (37)	0.1 ng/mL/–	sTg	sTg	75%/50% and 100% (NT)	100%/100% and 83% (NT)	Histopathology
Biscolla et al. (38)	1 ng/ml/–	—	Positive:>50 ng/mLNegative: <1 ng/mL	86%/100%	—	Histopathology, periodic US
Zanella et al. (39)	1 ng/mL/–	Based on Kim et al. and Baskin	10 ng/mL	—	100%/100%	Histopathology or negative FNAB-C+neck US follow-up; and stimulated sTg for at least one year
Giovanella et al. (61)	—	—	1 ng/mL	—	98% or 100%/100%	Histopathology, clinical examination, sTg, and neck US
Kim et al. (62)	0.2 ng/mL/0.7 ng/mL	sTg	sTg	96.4%/68.9% (NT) and 100%/90% (T)	95%/90.9% (NT) and 80%/100% (T)	Histopathology

Methods considered gold standards to establish the sensitivity and specificity of FNAB-Tg and FNAB-C.

LNs were classified as benign when they exhibited reactive FNAB-C associated with ultrasonographic regression in 6–12 months or when they had benign histology.

Mean±2 SD of the FNAB-Tg values of benign LNs from patients with history of differentiated thyroid carcinoma (DTC).

97.5th percentile of the FNAB-Tg level of patients with negative FNAB-C.

FNAB, fine-needle aspiration biopsy; FNAB-Tg, measurement of Tg in the washout fluid of FNAB; FNAB-C, cytological assessment of samples collected by FNAB; Tg, thyroglobulin; US, ultrasound; WBS, whole-body scan; NT, patients awaiting thyroidectomy; T, patients submitted to thyroidectomy; LN, lymph node; IRMA, immunoradiometric assays; ICMA, immunochemiluminescence assays.

Due to ethical reasons, the patients with benign cytology are not usually subjected to surgery. Thus, in some studies, the cutoff point was based on the FNAB-C results. Nevertheless, cytology could not be used as a gold standard to define the cutoff point of FNAB-Tg because the aim of the studies was to compare the diagnostic performance of both procedures (36).

The use of the sTg value as the cutoff point for FNAB-Tg might be difficult in patients awaiting surgery because most consensus statements do not recommend measurement of sTg in such patients. In addition, the blood samples for sTg measurements and FNAB-Tg must be collected simultaneously because the sTg values vary over the course of days to weeks and might thus distort the data. It is also worth keeping in mind that in the presence of TgAb, the sTg level might be higher than the level actually measured by most available assays (13,15).

Some authors (13,15,32) express FNAB-Tg results as ng/mL (or μg/L), while others (21,27,29,36) use the unit ng/punction (Table 2). According to some authors, the unit ng/punction better represents the test, as it reflects the dilution of the Tg left in the needle, whereas ng/mL might mislead the reader into believing that FNAB-Tg reflects the true concentration of Tg in the LN. Conversely, some authors assert that ng/mL allows for the comparison of the FNAB-Tg and sTg levels, which is important for decision making regarding surgical excision of the LNs when the FNAB-Tg cutoff point is defined as the patient's sTg level.

Such differences in the reporting of values, the lack of standardization of the commercially available Tg kits, the heterogeneity of control groups or “negative” patients, and the fact that some authors studied patients subjected to thyroidectomy while others focused on patients awaiting surgery make it difficult to compare published data.

Due to their belief in the possible interference of sTg with FNAB-Tg, some authors suggest establishing different diagnostic thresholds for patients before and after thyroidectomy (13,22,35). Frasoldati et al. (22) defined the FNAB-Tg cutoff point as the 97.5th percentile of the FNAB-Tg levels of LNs with negative FNAB-C. In the patients awaiting surgery, that value was 39.3 ng/mL, compared with 1.1 ng/mL among those already subjected to thyroidectomy. The sensitivity and specificity, calculated based on patients with histological confirmation, were 84.0% and 95.4% respectively. Boi et al. (13) defined as the cutoff point the highest FNAB-Tg value found in benign LNs (on histological examination or a combination of reactive FNAB-C and ultrasonographic regression in 6–12 months), which was 36 ng/mL among the patients awaiting surgery and 1.7 ng/mL among the patients subjected to thyroidectomy, with sensitivity and specificity of 100%.

Other studies applied a single threshold to FNAB-Tg independently of the status of patients regarding thyroidectomy (30,31 –33,36,37,62). Cunha et al. (30) selected the functional sensitivity of the method they used to measure Tg (0.9 ng/mL) as the cutoff point of FNAB-Tg, which resulted in 100% sensitivity in both groups and 100% specificity in the group of patients subjected to surgery. To rule out the possibility of contamination by circulating Tg, other studies (31,33,37,62) used the sTg level as the cutoff point for FNAB-Tg. In the study by Kim et al. (62), the sensitivity and specificity were calculated based on the patients with histological confirmation, and the values found were 95% and 90.9% respectively before surgery, and 80% and 100% after surgery. Bournaud et al. (36) and Kim et al. (32) suggest using a unique and simple threshold. This suggestion agrees with the current guidelines, which do not recommend the measurement of sTg in patients with thyroid cancer who are awaiting surgery. In those two studies, the cutoff point was established based on ROC curve analysis.

Independently of the values found, the sensitivity levels reported by the various authors have been similar (84–100% when different cutoff points are established for measurements before and after surgery vs. 81–100% when a single cutoff point is used). Nevertheless, one might expect a lower number of false-positive results in the assessment after surgery due to the lack of thyroid tissue and sTg suppression (32).

The issues related to the cutoff point were recently addressed by Kim et al. (32), Bournaud et al. (36), and Sohn et al. (34), who each measured Tg in suspicious LNs using the same ultrasensitive immunoradiometric assay based on monoclonal antibodies. Kim et al. (32) studied 168 patients with PTC and neck LNs considered to be potentially malignant on US before and after surgery. The FNAB-Tg results were interpreted based on five different cutoff points (1 ng/mL, 10 ng/mL, 100 ng/mL, the patient's sTg level, and mean±2 SD of the FNAB-Tg level found in LNs shown not to be metastatic on histological examination, which was equal to 32 ng/mL) and compared to the final diagnosis as established by histological examination, TSH stimulated-Tg measurement, and US follow-up. The authors suggested using the sTg level, 10 ng/mL, or mean±2 SD of the results of negative patients as the cutoff point for FNAB-Tg, as these criteria exhibited high sensitivity and accuracy in addition to similar diagnostic performance. Using these cutoff points, the sensitivity varied from 88.2% to 90.8%, specificity from 84.2% to 89.8%, PPV from 93.9% to 95.6%, NPV from 74.4% to 80.0%, and accuracy from 88.0% to 90.5%.

According to these authors, dissection of the neck LNs must be strongly recommended when the FNAB-Tg is ≥100 ng/mL, as no false-positive results have yet been reported at such levels, and merely suggested when Tg is >10 ng/mL, Tg is greater than sTg, or Tg is greater than the mean±2 SD of the level of negative patients. Nevertheless, when the FNAB-Tg is <1 ng/mL with a negative FNAB-C, a follow-up study should be considered, and when the Tg ranges from 1 to 10 ng/mL, the decision about LN dissection must be made based on the clinical and US findings. All five studied cutoff points were more sensitive and accurate in the assessment after than before thyroidectomy.

In the study by Bournaud et al. (36), 114 patients with thyroid cancer before surgery (n=13) or throughout follow-up (n=93) and 16 controls were subjected to FNAB-Tg and FNAB-C. The negative control group comprised 16 individuals with nonthyroid cancers, who exhibited 16 enlarged LNs suspected of metastasis on US performed during follow-up. The sensitivity and specificity of FNAB-Tg and FNAB-C were established for different cutoff points within a range of 0.24–32.60 ng/FNAB by means of ROC curve analysis. The LNs were classified as benign or malignant based on their histological features or, in the patients not subjected to surgery, on the scintigraphy findings and 12-month minimum follow-up. The most adequate cutoff point for diagnosis of LN metastases of thyroid cancer in that study was 0.93 ng/FNAB, which was slightly above the functional sensitivity of the method used to measure Tg. Using that cutoff point, sensitivity was 94.2%, specificity 97.8%, PPV 98.0%, and NPP 93.7%. The use of the method's functional sensitivity as the cutoff point, as suggested by Cunha et al. (30), which in the study by Bournaud et al. was 0.69 ng/mL (36), would have resulted in a similar sensitivity but lower specificity compared to the cutoff point of 0.93 ng/FNAB. Previous studies that used low cutoff points in the assessment of FNAB-Tg also reported excellent performance, with few false-positive or -negative results (13,15,28,30). The adoption of a higher cutoff point in the study by Bournaud et al. (36), as suggested by some authors, could have resulted in the absence of false-positive results but would have increased the number of false-negative samples, leading to a significant reduction of the sensitivity (86.5% for the cutoff point of 4.95 ng/FNAB). In that series, the sensitivity and specificity of FNAB-Tg were similar between the 29 samples of the controls or patients awaiting surgery and the 93 samples collected after thyroidectomy.

To compare the frequency of metastatic and nonmetastatic LNs diagnosed by means of FNAB-C and FNAB-Tg in a group of patients in whom the relationship between the FNAB-Tg level and malignancy is not yet well established, Sohn et al. (34) studied 691 consecutive patients with neck LNs bearing traits on US compatible with metastases of PTC. A total of 95 LNs with histological confirmation were included in the study, corresponding to patients with FNAB-Tg values of 0.2–100 ng/mL, because >100 ng/mL, no false-negative results have been reported. The diagnostic performances of multiple Tg levels (0.7 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, and 50 ng/ml) were evaluated, and the best diagnostic performance was recorded using 5.0 ng/mL: the area under the curve (AUC) was 0.76 [CI 0.6731–0.8476] and the sensitivity, specificity, accuracy, PPV, and NPV were 69.0%, 83.0%, 76.8%, 76.3%, and 77.2% respectively. However, that cutoff point did not exhibit a significant difference in the sensitivity, specificity, accuracy, PPV, NPV, or AUC compared to 10.0 and 20.0 ng/mL. The cutoff points 0.7, 1.0, and 50 ng/mL were not recommended due to their low sensitivity and AUC. Among the 78 cases with negative cytology, 25 (32%) were confirmed to be metastases on the final diagnosis and exhibited FNAB-Tg varying from 0.22 to 88.34 ng/mL. Although seven cases were classified as negative with all six analyzed FNAB-Tg cutoff points, 13 cases (52%) were identified as positive when a cutoff point of 10 ng/mL was applied, 15 cases (60%) with the cutoff point 5 ng/mL, 18 cases (72%) with the cutoff point 0.7 ng/mL, 18 cases (72%) with the cutoff point 1.0 ng/mL, and eight cases (32%) with all six cutoff points.

A large prospective study was recently published in which 255 neck LNs of 225 patients with neck LN metastases or PTC recurrence (35) were assessed by FNAB-C and FNAB-Tg. The final diagnosis, based on histological examination, was compared to the FNAB-C and FNAB-Tg values before surgery. When the FNAB-Tg cutoff point was established as 1 ng/mL, 200 metastatic LNs were correctly diagnosed, but the method failed to diagnose 55 LNs. The sensitivity, specificity, and diagnostic accuracy of FNAB-Tg (cutoff point=1 ng/mL) for diagnosis of metastases of thyroid carcinoma before surgery were 100%, 0%, and 78% respectively. The PPV and NPV were 78% and 0% respectively. The cutoff point of FNAB-Tg with the best sensitivity and specificity on ROC curve analysis was 28.5 ng/mL, as 253 metastatic LNs were correctly diagnosed and only two were misdiagnosed using that cutoff point. The sensitivity, specificity, and accuracy of FNAB-Tg (cutoff point=28.5 ng/mL) for diagnosis of metastases of thyroid carcinoma before surgery were 100%, 96%, and 99% respectively. The PPV and NPV were 99% and 100% respectively.

Summary

FNAB-Tg is simple to perform, is low cost, and improves the diagnostic performance of cytology in the early detection of LN metastases of DTC. This technique allows diagnosis even in cases of paucicellular samples (metastatic deposits with degeneration and a cystic component) and cases in which cytology fails to provide a diagnosis, and it is not affected by TgAb, is reliable in the case of very small lesions, and exhibits high diagnostic accuracy.

Because the current Tg methods are highly variable, they cannot be used interchangeably to manage patients with DTC (51). Therefore, it is recommended that the measurements of sTg and FNAB-Tg levels be systematically performed using the same method and assay, and that they should be included in the clinical laboratory report in the patient's medical record.

In the case of patients treated with ¹³¹I, the interval between treatment and FNAB-Tg must be sufficient (more than three months) to allow the definitive destruction of the metastatic LN (57), otherwise FNAB-Tg might produce false-positive results. It is also not recommended to measure FNAB-Tg in masses found in the thyroid bed, because this test is unable to distinguish thyroid tissue remnants from cancer recurrence.

The diagnostic performance of FNAB-Tg is not affected by the presence of thyroid tissue. Therefore, it might be used for screening LN metastases in patients subjected to thyroidectomy, as well as to perform LN staging in patients with DTC who have not yet undergone initial surgery.

Conclusion

Measuring Tg in the FNAB washout fluid exhibits some advantages over cytology. The combination of FNAB-Tg and FNAB-C is recommended for the staging and follow-up of patients with DTC and suspected neck LNs.

FNAB-Tg measurements must be performed using the same methods that are used for the measurement of sTg levels. Washing the needle with the Tg-free serum provided in the Tg assay kit is preferable to washing with normal saline solution, and the use of 1 mL volume of washout fluid, which should be stored in plain serum tubes, is recommended. Although the cutoff point for the FNAB-Tg values has not yet been definitively established, values >10 ng/mL are recommended for identifying neck LN dissection, as they showed high sensitivity and accuracy for the diagnosis of DTC metastases in the neck LNs in several studies.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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