Lymph Node Metastases of Differentiated Thyroid Carcinoma: Does Serum Anti-Tg Antibodies or TSH Level Influence Tg measurement in Fine Needle Aspiration Washouts?

Introduction: Thyroglobulin evaluation in the washout of fine-needle aspiration (FNA-Tg) is an accurate diagnostic method of lymph node metastases (LNM) of differentiated thyroid carcinoma (DTC). Serum anti-thyroglobulin antibodies (AATg) may cause falsely low serum Tg values, but their effect on FNA-Tg has not been well established. There are also concerns about the possibility that suppressed TSH results in false-negative FNA-Tg. Our objectives were to evaluate the effect of serum AATg and TSH level on FNA-Tg of LNM of DTC and to determine the presence of AATg on the washout of fine needle aspiration (FNA-AATg). Methods: Retrospective analysis of patients who underwent FNA-Tg assay in LNM of DTC. The sample was divided in two groups according to the presence of serum AATg at the time of FNA-Tg evaluation (Group 1: positive AATg, n =47; Group 2: negative AATg, n =50). Results: There was no significant difference in the FNA-Tg between the two groups ( with FNA-Tg value ( p =0.732). There were no differences in the median FNA-Tg measurements between those on levothyroxine suppressive therapy and those on substitutive therapy ( p =0.800). Conclusion: FNA-Tg assay appears to be a good diagnostic tool even in patients with positive serum AATg and those under suppressive levothyroxine therapy.

The ultrasonographic diagnosis of cervical LN metastases from DTC is challenging due to its similarity with inflammatory adenopathies or nonthyroidal cancer metastases [1,3,6]. The diagnosis of suspicious LNs is confirmed through fine needle aspiration (FNA) cytology guided by ultrasound (US) and/or measurement of thyroglobulin (Tg) in the washout fluid of the fine needle aspiration (FNA-Tg) [1,2]. Since inadequate cellularity or unsatisfying sampling precludes diagnosis in up to 20% of specimens with FNA [1,[3][4][5], measurement of FNA-Tg has been used as a powerful tool for the diagnosis of LNM, especially in cystic LNM, inadequate cytological evaluation, and divergent cytology and US findings [1,2,4].
Serum anti-Tg antibodies (AATg) are present in about 25% of the patients with thyroid cancer and may lower serum Tg levels and even make them undetectable (5). Nevertheless, there is still some debate on the role of AATg in lowering FNA-Tg levels and its influence in the diagnostic accuracy of FNA-Tg in suspicious LNs [1][2][3][5][6][7][8]. Only a few studies evaluated the presence of AATg in the washout of the fine needle aspiration (FNA-AATg) so, there is little information about there real presence in LNM [1,9].  (sphere) when just two axes were available.

Cytological Evaluation
Cytological evaluation was performed by experienced pathologists. They were categorized accordingly to the Bethesda System for Reporting Thyroid Cytopathology [10].
Definitions FNA-Tg was considered positive and indicative of LNM if higher than 10 ng/mL [11], antibody positivity was defined as a AATg exceeding 280 U/mL and levothyroxine suppressive therapy when serum TSH was below 0.5 µIU/mL. ; p = 0.311).
The clinical, biochemical and US characteristics of the patients of both groups are presented in Table 1.

Thyroglobulin Evaluation on FNA Washout Fluid
There was no significant difference in the Tg value in the FNA washout fluid between the two groups (p = 0.069), although it was lower in Group 1 (median 1428 ng/mL; interquartile range 11696 ng/mL) than in Group 2 (median 4842 ng/mL; interquartile range 29799 ng/mL). FNA-Tg did not seem to differ significantly between patients with positive or negative FNA-AATg (median of 8059 ng/mL vs 6199 ng/mL, p = 0.735).
In table 2, we present the characteristics of of the FNA washout fluid of patients with positive FNA-AATg. All, but one patient, had possible blood contamination.

TSH Interference on FNA-Tg Evaluation
There were no significant differences in the median FNA-Tg measurements between patients on levothyroxine suppressive therapy (median TSH <0.02 µIU/mL) and those on substitutive levothyroxine (median TSH 2.97 µIU/mL) (median Tg 2563 ng/mL, interquartile range 29789 ng/mL vs median Tg 4534 ng/mL, interquartile range 27046 ng/mL; p = 0.820), although there was a tendency for a lower FNA-Tg in the group with suppressive therapy.

Discussion
As previously suggested by various authors [1,6] our study confirms that FNA-Tg is an important tool for the diagnosis of LN DTC metastases. It allows the identification of approximately more 10.3% LN DTC metastases than cytological evaluation.
The optimal cutoff point for the FNA-Tg value has not yet been definitively established and different authors use distinct values [3,4,9,11]. We considered >10 ng/mL as a positive value for FNA-Tg as there is evidence that this cutoff has the highest sensitivity and accuracy for the diagnosis of LN DTC metastases in several studies and is used in the majority of published reports [7,9]. The interpretation of any slightly to moderately elevated FNA-Tg level should also take in consideration the serum Tg level because it may represent blood contamination. In the majority of cases of DTC-LNN metastases, FNA-Tg is usually several times higher than serum Tg [12].
Since serum AATg may interfere with the evaluation of serum Tg by immunometric assays, it is still a matter of debate whether it also interferes with FNA-Tg levels and decreases the ability to diagnose LN DTC metastases [1,3,[5][6][7][8]. In our work, the presence of serum AATg seemed to reduce the FNA-Tg value, but not to affect its discriminatory capacity.
Also, the volume of punctured adenopathies does not seem to be influencing the outcome of FNA-Tg, We have shown that the presence of FNA-AATg is rare and might be the result of blood contamination rather than active production of AATg in the LN [9].
Since, levothyroxine suppressive therapy lowers serum Tg, the concern that it could also reduce the FNA-Tg has been raised. In our study, as well as in others previously reported, levothyroxine suppressive therapy does not seem to reduce the diagnostic accuracy of FNA-Tg [1,9].
Our study has some limitations such as its retrospective nature, the fact that best FNA-Tg cutoff value is not currently well established, the absence of histological confirmation of some DTC metastases, the absence of FNA-Tg dilution on the highest values and the absence of analytical confirmation of blood contamination.
In conclusion, Tg assay in FNA washout fluid is an excellent diagnostic tool of DTC LNM, which is not affected by positive serum AATg or by levothyroxine suppressive therapy.

Declarations
Funding: There were no funding sources to this study.

Conflicts of interest:
The authors declare that they have no conflicts of interest.

Data availability:
The data that support the findings of this study is available upon request from the corresponding author. Due to privacy restrictions it is not publicly available.

Statement of Ethics:
This study was a conducted in accordance with the Declaration of Helsinki. Confidentiality was kept throughout the study and the authors have followed the protocols of their center on the publication of data.