Impact of repeat aspiration timing on the second diagnosis of thyroid nodules initially diagnosed as atypia of undetermined significance

Authors

Abstract

AimAtypia of undetermined significance (AUS) is a term used by pathologists when both cytologic changes cannot be definitively associated with malignancy and malignancy also cannot be definitively excluded. In patients with AUS, the next step is repeating fine needle aspiration (FNA). The aim of this study was to evaluate the relationship between the waiting period before repeating FNA and second FNA diagnosis in patients whose initial FNA was reported as AUS.
Methods176 patients whose initial thyroid FNAs were reported as AUS and who underwent repeat FNAs from the same lobe and had diagnostic results between 01.01.2015-01.01.2025 were rewieved.
ResultsSecond FNA was performed within 30 days in 25 (14.20%) patients, 60 days in 97 (55.11%) patients, and 90 days in 118 (67.05%) patients. Abnormalities were found in 63 patients (35.80%) during second FNAs. The rates were 44.00% if repeated within 30 days, 34.44% after 30 days, 30.38% after 60 days, and 25.86% after 90 days (p > 0.05). Abnormality rates decreased with longer waiting periods. True histopathologic abnormalities were detected in 40% of patients with under 90 days between abnormal aspirations and in 66.67% with over 90 days (p>0.05). True histopathologic anormality rates increased with longer waiting periods. However, these differences were not statistically significant.
ConclusionFor patients strongly suspected of malignancy, repeat FNA does not need to be delayed. Physicians may still choose to wait up to 90 days for repeat FNAs, except when suspicion of malignancy is high.

Keywords

Introduction

Studies showed that there was a significant increase in thyroid cancers in the Black Sea region of Türkiye due to radioactive fallout from the Chernobyl nuclear power plant disaster in 1986.¹ Iodine deficiency presented in the same region is also recognized as a risk factor for thyroid cancer.^2,3
Nodules are the most common thyroid disease.⁴ Thyroid nodules are reported in 19-68% of general population with ultrasonographic examination. However, malignancy is detected in 7-15%.⁵ The American College of Radiology (ACR) recommends radiologists to use the Thyroid Imaging Reporting and Data System (TIRADS) during the evaluation of thyroid nodules. ACR-TIRADS categorizes thyroid nodules into five groups based on malignancy risk. According to this system, mildly suspicious (TIRADS 3), moderately suspicious (TIRADS 4), and highly suspicious (TIRADS 5) nodules should be referred for fine needle aspiration (FNA) if their diameters are 25 mm, 15 mm, and 10 mm or larger, respectively.⁶
The Bethesda System (TBS) offers a standardized way for pathologists to report cytologic findings and for physicians to guide patient care. Introduced in 2010, its latest edition came out in 2023 and organizes thyroid nodule findings into six categories: nondiagnostic, benign, atypia of undetermined significance (AUS), follicular neoplasia, suspicious for malignancy, and malignant. FNA is recommended for nondiagnostic cases, follow-up is advised for benign cases, molecular tests and surgery are recommended for cases classified as follicular neoplasm or suspicious for malignancy, and surgery is advised for malignant cases. More complex individualized approach is required for patients whose FNA is reported as AUS.⁷
AUS is a term used by pathologists when both cytologic changes cannot be definitively associated with malignancy and malignancy also cannot be definitively excluded. Malignancy risk of AUS cases is approximately 13-30%. Nonmalignant factors such as bleeding during FNA, preparation related artifacts, thyroiditis or cystic degeneration, can complicate cytologic evaluations and lead to an AUS diagnosis by mimicking nuclear changes characteristic of papillary thyroid carcinoma, which is the most common thyroid malignancy.⁷
In patients with AUS, potential management options include molecular tests, repeat FNA, and diagnostic surgery or active surveillance.^7,8 If molecular tests are negative, the malignancy risk decreases to 3-5%. However, molecular tests are expensive and not widely accessible. When the same nodule is reevaluated, patient management is based on the new FNA diagnosis. The likelihood of a repeat AUS diagnosis is 10-30%. Given the relatively high incidence of thyroid cancer in Türkiye and the limited access to molecular tests, the most common approach for recurrent AUS cases is surgery.⁷
We can reduce the impact of external factors within our control, such as allowing adequate time for thyroid recovery prior to repeat FNA. Additionally, this may help alleviate the emotional, labor, and time, and financial-related challenges associated with repeated but overstated AUS diagnoses. This study aimed to evaluate the relationship between the waiting time before repeat FNA and the second FNA diagnosis in patients whose initial thyroid FNA was reported as AUS.

Materials and Methods

Thyroid FNAs reported as AUS at the pathology laboratory of Harran University Hospital between 01.01.2015 and 01.01.2025, were identified by retrospective reviewing the pathology report archive. After the AUS diagnosis, patients who underwent repeat FNA from the same thyroid lobe and had diagnostic results were included in the study.
The first FNA report was used to determine the patient's age. Autoantibody levels present in the hospital system and measured at any time up to the date of the second FNA were evaluated. A value of 500 U/mL or higher for anti-thyroglobulin (anti-TG) and 60 U/mL or higher for anti-thyroid peroxidase (anti-TPO) were considered elevated according to the reference values of the hospital. Patients with elevated anti-TG or anti-TPO levels detected at least once were classified as autoantibody positive. If the nodule location was specified as the right/left lobe and isthmus junction in the report, the nodule was considered to be in the right/left lobe. The widest diameter of the nodule was recorded based on the first FNA report. The time between the two FNAs was calculated in days.
Patients whose second FNA diagnosis was reported as benign were classified as normal, and patients with other diagnostic results were classified as abnormal. Patients who underwent lobectomy from the same lobe or total thyroidectomy after the second abnormal FNA were identified. Patients whose surgical diagnoses were reported as benign conditions such as follicular nodular disease or chronic lymphocytic thyroiditis, were classified as normal, and patients with at least one low-risk or malignant condition, such as noninvasive follicular thyroid neoplasm with papillary-like nuclear features, suspicious for papillary thyroid carcinoma or papillary thyroid carcinoma, were classified as abnormal.
*Ethical ApprovalThis study was approved by the Ethics Committee of Harran University (Date: 2025-03-24, No: HRÜ/25.06.19).
Statistical Analysis
Data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 26.0. Descriptive statistics including minimum, maximum, mean, numbers and percentages were calculated. For the analysis of relationships between categorical variables, the Chi-square test or Fisher's exact test was employed. P-values of <0.05 were considered statistically significant.
Reporting Guidelines
This study is reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.

Results

A total of 4731 thyroid FNAs were evaluated in the laboratory within the specified time frame, and 558 (11.79%) of these were diagnosed as AUS. Of these, only 176 underwent a second FNA with diagnostic results. The results of the second FNAs were categorized as benign in 113 patients, AUS in 52 patients, follicular neoplasia in two patients, and suspicious for malignancy in nine patients. The detailed data are provided in Supplementary Table 1 and Table 2. Surgery was performed on 23 patients with abnormal second FNA results. In patients with a waiting period of 30 days or less between two abnormal FNAs, excision results were normal in 3 (75.0%). In patients with more than 30 days, 10 had normal excision results (52.63%) (p = 0.404). In patients with 60 days or less, excision results were normal in 9 (52.94%). In patients with more than 60 days, 4 had normal excision results (66.67%) (p = 0.463). In patients with 90 days or less, excision results were normal in 12 (60.0%). In patients with more than 90 days, 2 had abnormal excision results (66.67%) (p = 0.398).

Discussion

AUS may be used as an evasive diagnosis, particularly by pathologists who are inexperienced or hesitant in making definitive interpretations. To minimize such misuse, TBS recommends maintaining the AUS diagnosis rate below 10%.⁷ In our study, the average 10-year AUS rate in our pathology laboratory was 11.79%, which is only slightly above the recommended threshold.
A review of the literature revealed a consistent female predominance in most thyroid-related diseases, including thyroid dysfunction,⁴ thyroid nodules,^4,9 thyroiditis,^4,10,11 and thyroid cancer.^10,12 This trend has been attributed to factors such as sex hormones, differences in immune response, and genetic predisposition.¹² The female predominance observed in our study supported the association between sex and the development of thyroid nodules.
Chronic lymphocytic thyroiditis (CLT) is the second most common thyroid disorder after thyroid nodules and is the leading cause of hypothyroidism in regions without iodine deficiency.⁴ In patients with CLT, anti-TPO antibodies are positive in over 90% of cases, and anti-TG antibodies are positive in approximately 50-80% of cases.¹⁰ Parenchymal inflammation associated with CLT contributes to nodule formation, and thyroid nodules are detected in 20-30% of affected individuals.¹⁰ In our study, all patients presented with thyroid nodules, and autoantibody positivity was identified in 28.7% of cases. This rate is significantly higher than the global prevalence of CLT, which is estimated to be approximately 7.5%.¹⁰ This finding supports the hypothesis that CLT is a contributing risk factor for the development of thyroid nodules. In CLT, characteristic histopathologic features include lymphocytic infiltration, follicular cell destruction, fibrosis, and oncocytic (Hurthle cell) changes.10 When lymphocyte density in cytologic samples is low, or when autoantibody testing is either omitted or returns negative results, these histologic alterations may be misinterpreted, potentially leading to an overdiagnosis of AUS on FNA.
In our study, CLT-related histopathological features were identified in six patients who underwent thyroid surgery following two consecutive abnormal FNA results. Interestingly, two of these patients had negative autoantibody levels. This observation reinforced the hypothesis that elevated autoantibody levels are not necessarily required for the initiation of thyroid tissue damage.¹¹
It is a well established fact that the right thyroid lobe is anatomically larger than the left, a condition attributed to genetic factors involved in thyroid development. It is believed that this size asymmetry may contribute to the higher prevalence of both benign and malignant thyroid lesions in the right lobe.^13,14 In our study, the predominance of thyroid nodules in the right lobe, with their rare occurrence in the isthmus, supported this hypothesis.
In our study, the majority of patients had thyroid nodules measuring 2.5 cm or less in diameter. This may be attributed to the fact that individuals with larger nodules often opt for surgery due to cosmetic or functional reasons, such as dysphagia, dyspnea, chronic cough, a sensation of obstruction, or significant compression of surrounding structures. In such cases, patients are frequently referred directly to surgery without undergoing FNA.¹⁵
Most patients in our study underwent repeat FNA after more than 30 days, less than 60 days after the initial procedure. When the same nodule is reevaluated with FNA, the probability of a repeat AUS diagnosis is only 10-30%.7 In our study, when other cytologic abnormalities were observed in addition to recurrent AUS, the abnormality detection rate in repeat FNAs was close to this rate at 35.80%.
Studies have shown that older age is a predictive factor for thyroid malignancies (9,16). Although there were more patients aged over 45 years in our study population, the rates of abnormality in second FNAs in our study patients aged 45 years and under was slightly higher. However, this relationship was not statically significant (p = 0.702). Aditionally, cytologic abnormalities do not always reflect histopathologic malignancy.
Although thyroid malignancies are more common in women, the risk of a nodule detected in men being malignant is higher.9 In the study by Al-Hakami et al., the risk of malignancy was determined as 17.72% in women and 58.38% in men.¹⁶ It is usual to find recurrent cytologic abnormalities in a group at high risk of malignancy, and in our study, the rates of abnormality in second FNAs were higher in males. However, this relationship observed between sex and abnormal repeat FNA results was not statistically significant (p = 0.102).
In our study, the rates of abnormalities in second FNAs were higher in patients with autoantibody positivity but this relationship observed between autoantibody positivity and abnormal repeat FNA results was not statistically significant (p = 0.479). However, it is also known that the incidence of thyroid cancer increases in CLT.¹⁰ Therefore, not every change seen should be interpreted as secondary to thyroiditis.
We found that the abnormality rates in second FNAs were more common in the isthmus, left lobe, and right lobe, respectively. This relationship observed between nodule location and abnormal repeat FNA results was not statistically significant (p = 0.098). Contrarily, studies showed that thyroid cancers were located in the right lobe, left lobe, and isthmus, in order of frequency.¹⁷ The recurrence of the abnormality in the second FNA may not always reflect the final diagnosis. In adition, although the right side of the patient is often preferred in FNA, the procedure may be challenging when sampling is performed from the left lobe and the isthmus, which is already small. The increase in abnormalities may be due to changes caused by the processing difficulties.
In the present study, the rates of abnormalities in second FNAs were slightly higher in nodules < 25 mm. Even though this relationship observed between nodule diameter and abnormal repeat results was not statistically significant (p = 0.342), different studies have shown that the risk of malignancy increases as nodule diameter decreases.¹⁸ The higher risk of malignancy in this group may explain the recurrent abnormalities detected on FNAs. However, there are also studies that argue that increased nodule size is a predictor of thyroid malignancy.⁹
Kahramanca et al. examined patients with inadequate or AUS initial FNAs, the rate of adequate or AUS second FNA results did not vary with the timing of repeat FNAs and they indicated that the wait of 1 month for a repeat FNA was not needed.¹⁹ Deniwar et al. reported that the diagnostic rate in patients whose initial FNA results were nondiagnostic was 84.38% when the FNA was repeated within 3 months and 90.95% when the FNA was performed after more than 3 months. Although completing the traditional 3-month waiting period resulted in a slight increase in diagnosticity, this relationship was not statistically significant.²⁰ In our study, the abnormality rates in patients whose FNAs were repeated within 30, 60, or 90 days were higher than those in patients whose FNAs were repeated after more than 30, 60, or 90 days. Furthermore, the abnormality rate in the second FNA was 44.00% when repeated within 30 days, 34.44% when repeated after 30 days, 30.38% when repeated after 60 days, and 25.86% when repeated after 90 days. Abnormality rates decreased with longer waiting periods. However, the p-value was not less than 0.05 for any of these cut-off values.
Based on such studies, the 3-month waiting period was presented as a weak recommendation with low-quality evidence in the American Thyroid Association guidelines. A 3-month waiting period was recommended at the National Cancer Institute Thyroid Fine Needle Aspiration Science Conference. However, it was stated that this period could be shortened in patients at high risk.²¹ The TBS was also not convinced that early repeat FNA altered the evaluation results and did not specify a time limit. The high abnormality rates in patients whose FNA was repeated early may be due to insufficient time for the thyroid to heal in some patients. However, physicians who consistently follow the 3-month waiting period may choose to perform early FNA in patients with a high clinical risk of malignancy, which could also explain the higher abnormality rates seen with early repeat procedures.
Thyroid lobectomy or total thyroidectomy was performed in our 23 patients after two abnormal FNAs. Histopathologic evaluation of these patients was normal in 56.52% and abnormal in 43.48%. This abnormality rate, which we obtained with the histopathologic correlation of some of our patients with recurrent abnormalities in FNAs, is higher than the 10- 30% malignancy risk determined for AUS diagnosis.7 This finding supported the increase in the risk of malignancy in recurrent abnormal FNA results. Although true histopathologic abnormalities were detected in 40% of patients with a waiting period of less than 90 days between two abnormal aspirations, true histopathologic abnormalities were found in 66.67% of patients with a waiting period longer than 90 days (p = 0.398). This situation may support the idea that repeating FNA before 90 days may cause false positivity. Statistically significant results may be obtained in future studies conducted with larger case numbers.

Limitations

Since, only the patients who had initially AUS diagnosis then underwent repeat FNA from the same thyroid lobe and had diagnostic results were included in the study, the sample size decreased considerably. Therefore, the primary limitation of this study was the small sample size. Furthermore, since surgery was performed on only 23 patients after two abnormal FNAs, cytopathologic and histopathologic correlations were limited.

Conclusion

No statistically significant relationship was found between the waiting period before repeat FNA and the second FNA diagnosis. In patients with a clinically high suspicion of malignancy, there is no need to wait for a repeat FNA. However, recurrent cytologic abnormality rates decreased, and true histopathologic positivity increased with longer waiting times. Physicians may still prefer delaying repeat FNA up to 90 days, except in cases with high suspicion of malignancy.

Declarations

Abbreviations

ACR, American College of Radiology;
AUS, atypia of undetermined significance;
CLT, chronic lymphocytic thyroiditis;
FNA, fine-needle aspiration;
SPSS, Statistical Package for the Social Sciences;
STROBE, Strengthening the Reporting of Observational Studies in Epidemiology;
TBS, The Bethesda System;
TIRADS, Thyroid Imaging Reporting and Data System.

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About This Article

Received:

February 21, 2026

Accepted:

April 24, 2026

Published Online:

May 1, 2026