ABSTRACT Objectives The presence of thyroglobulin (Tg) in needle washouts of fine needle aspiration biopsy (Tg-FNAB) in neck lymph nodes (LNs) suspected of metastasis has become a cornerstone in the follow-up of patients with papillary thyroid carcinoma (PTC). However, there are limited data regarding the measurement of anti-Tg antibodies in these washouts (TgAb-FNAB), and it is not clear whether these antibodies interfere with the assessment of Tg-FNAB or whether there are other factors that would more consistently justify the finding of low Tg-FNAB in metastatic LNs. Materials and methods We investigated 232 FNAB samples obtained from suspicious neck LNs of 144 PTC patients. These samples were divided according to the patient’s serum TgAb status: sTgAb- (n = 203 samples) and sTgAb+ (n = 29). The TgAb-FNAB levels were measured using two different assays. Tg-FNAB was also measured using two assays when low levels (< 10 ng/mL) were identified in the first assay of the metastatic LNs from the sTgAb+ samples. Results The TgAb-FNAB results were negative in both assays in all samples. Low levels of Tg-FNAB were identified in 11/16 of the metastatic LNs of the sTgAb+ patients and 16/63 of the sTgAb- patients (p < 0.05) using assay 1. The measurement of the Tg-FNAB levels using assay 2 indicated additional metastases in 5 LNs of the sTgAb+ patients. Conclusions Factors other than the presence of TgAb-FNAB may contribute to the higher number of metastatic LNs with undetectable Tg-FNAB in the sTgAb+ group. In addition, the measurement of Tg-FNAB using different assays was useful to enhance the diagnosis of metastatic LNs, particularly when cytological and Tg-FNAB results are discordant.