Thyroid nodules are a common clinical finding, with palpable nodules detected in approximately 4-7% of the adult population and a substantially higher prevalence of subclinical nodules identified incidentally on high-resolution ultrasonography, occurring in up to 50-60% of individuals by later adulthood (1,2). Although the overwhelming majority of thyroid nodules are benign, a small but clinically important proportion, estimated at 7-15% depending on age, sex, and radiation exposure history, harbour malignancy, making accurate preoperative risk stratification essential to avoid both unnecessary surgery for benign disease and delayed treatment of thyroid cancer (3).

Fine needle aspiration cytology (FNAC) has become the cornerstone investigation in the initial evaluation of thyroid nodules, owing to its minimally invasive nature, low complication rate, cost-effectiveness, and high diagnostic yield when combined with ultrasound guidance (4). Since its widespread adoption from the 1980s onward, FNAC has substantially reduced the proportion of patients undergoing diagnostic surgery for ultimately benign nodules, while reliably identifying those who require surgical intervention (5). Ultrasound guidance, in particular, has further improved sampling adequacy and diagnostic accuracy compared with palpation-guided aspiration, particularly for small, posteriorly located, or partially cystic nodules (6).

A major limitation historically affecting the clinical utility of thyroid FNAC was inconsistent and non-standardised cytological reporting terminology across institutions, which hindered communication between cytopathologists and clinicians and complicated comparison of outcomes across studies (7). This was substantially addressed by the introduction of the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) in 2007, subsequently updated in 2017 and again in 2023, which stratifies cytological findings into six diagnostic categories, each associated with an estimated risk of malignancy and a recommended clinical management pathway (8,9). The six categories range from nondiagnostic or unsatisfactory (category I) through benign (category II), atypia of undetermined significance or follicular lesion of undetermined significance (category III), follicular neoplasm or suspicious for follicular neoplasm (category IV), suspicious for malignancy (category V), to malignant (category VI) (9).

Despite the widespread adoption of TBSRTC, the indeterminate categories, particularly category III and IV, remain a persistent diagnostic challenge, with reported malignancy rates varying considerably across studies, from approximately 6% to over 30% for category III and 10% to 40% for category IV, reflecting differences in institutional thresholds for cytological atypia, sampling technique, and the underlying population's pretest probability of malignancy (10,11). This diagnostic uncertainty has prompted increasing interest in ancillary techniques, including molecular testing for BRAF, RAS, and RET/PTC alterations, as well as core needle biopsy, to refine risk stratification for indeterminate nodules and reduce diagnostic surgery rates for what ultimately prove to be benign lesions (12,13).

Numerous studies have evaluated the diagnostic performance of FNAC against the gold standard of histopathological examination following surgical excision, generally reporting high sensitivity and specificity, although considerable institutional variation exists, influenced by operator experience, sample adequacy, and the proportion of indeterminate cytology within a given cohort (14,15). Given this variability and the ongoing clinical importance of accurately characterising thyroid nodules, continued evaluation of FNAC diagnostic accuracy within individual institutional settings, alongside category-specific risk-of-malignancy estimation, remains clinically valuable. This study therefore aimed to evaluate the diagnostic utility of FNAC in the assessment of thyroid nodules by correlating Bethesda cytological categories with final histopathological diagnosis, and to determine the sensitivity, specificity, and predictive values of FNAC, along with the category-specific risk of malignancy, within our patient cohort.

Study Design and Setting This cross-sectional cytohistological correlation study was conducted in the Department of Pathology in collaboration with the Department of Surgery at [Institution Name] over a two-year period from January 2022 to December 2023. Ethical approval was obtained from the Institutional Ethics Review Committee (Approval No. [XXX]), and written informed consent was obtained from all participants prior to FNAC and surgical procedures. Patient Selection All patients presenting with a clinically palpable or radiologically detected thyroid nodule who underwent ultrasound-guided FNAC followed by subsequent thyroid surgery (lobectomy, near-total thyroidectomy, or total thyroidectomy) at the study institution during the study period were considered for inclusion. Inclusion criteria required availability of an adequate cytology smear, a documented Bethesda category, and a corresponding histopathological report following surgical excision. Patients with non-diagnostic cytology who did not undergo repeat FNAC or proceed to surgery, those with incomplete clinical records, and patients who underwent surgery for reasons unrelated to nodule characterisation without paired histopathology were excluded. A total of 232 patients were initially screened, of whom 210 met all inclusion criteria and were included in the final analysis. FNAC Procedure and Cytological Reporting Ultrasound-guided FNAC was performed using a 23-25 gauge needle with a 10 mL syringe under aseptic conditions by experienced radiologists or cytopathologists, with at least two to three passes obtained per nodule to ensure adequate cellularity. Aspirated material was smeared onto glass slides, with a portion air-dried for Giemsa staining and a portion fixed in 95% ethanol for Papanicolaou staining; selected cases also underwent liquid-based cytology preparation. All smears were independently reviewed by two experienced cytopathologists blinded to the final histopathological outcome, with discordant cases resolved by consensus review. Cytological findings were categorised according to the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC, 2017 update) into six categories: I (nondiagnostic/unsatisfactory), II (benign), III (atypia of undetermined significance/follicular lesion of undetermined significance, AUS/FLUS), IV (follicular neoplasm/suspicious for follicular neoplasm, FN/SFN), V (suspicious for malignancy), and VI (malignant). Histopathological Correlation Following surgical excision, specimens were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin, with sections stained with haematoxylin and eosin examined by a senior histopathologist blinded to the cytological diagnosis. The final histopathological diagnosis was considered the reference (gold) standard against which the cytological diagnosis was correlated. For diagnostic accuracy calculation, category II was classified as cytologically benign and categories V and VI as cytologically malignant; categories I, III, and IV (indeterminate) were excluded from the primary accuracy calculation and analysed separately for category-specific risk of malignancy, consistent with standard methodology in comparable studies. Statistical Analysis Data were entered and analysed using SPSS version 27.0 (IBM Corp., Armonk, NY). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall diagnostic accuracy of FNAC were calculated using standard 2x2 contingency table methodology for the definitive cytological categories (II, V, VI). Category-specific risk of malignancy was calculated for all six Bethesda categories as the proportion of histologically malignant cases within each category. Categorical variables were expressed as frequencies and percentages and compared using the chi-square test. A p-value of less than 0.05 was considered statistically significant.

Demographic and Clinical Characteristics

A total of 210 patients (172 female, 38 male; female-to-male ratio 4.5:1) with a mean age of 41.3 ± 13.7 years (range 18-76 years) were included. The majority of nodules were solitary (148/210, 70.5%), with the remainder occurring in the context of multinodular goitre (62/210, 29.5%). Mean nodule size on ultrasound was 2.6 ± 1.3 cm. Table 1 summarises the baseline demographic and clinical characteristics of the study cohort.

Table 1. Baseline demographic and clinical characteristics (N=210)

SD, standard deviation.

Distribution of Bethesda Cytological Categories

Cytological smears were distributed across all six Bethesda categories, with category II (benign) accounting for the largest proportion of cases (56.2%). Category I (nondiagnostic) accounted for 6.7% of aspirates; all of these underwent either repeat FNAC or proceeded directly to surgery based on clinical and sonographic suspicion, which accounts for their inclusion in the histopathological correlation despite being nondiagnostic on initial cytology. Table 2 summarises the distribution of Bethesda categories and the corresponding final histopathological diagnosis for each category, from which the category-specific risk of malignancy (ROM) was derived.

Table 2. Distribution of Bethesda categories with corresponding histopathological correlation and risk of malignancy

AUS/FLUS, atypia of undetermined significance/follicular lesion of undetermined significance; FN/SFN, follicular neoplasm/suspicious for follicular neoplasm. Risk of malignancy = (malignant on histopathology / total in category) x 100.

The risk of malignancy increased progressively across Bethesda categories, from 2.5% in category II to 96.8% in category VI, broadly consistent with the implied risk stratification of the Bethesda system, although the observed risk for category I (28.6%) was higher than the typically cited range, reflecting the small number of nondiagnostic cases that proceeded directly to surgery due to high clinical or sonographic suspicion rather than being representative of all nondiagnostic aspirates encountered in general practice.

Diagnostic Accuracy of FNAC

For calculation of overall diagnostic accuracy, indeterminate categories (I, III, and IV, comprising 56 cases) were excluded, and the remaining 154 cases with definitive cytological categorisation (II, V, and VI) were analysed against final histopathology in a 2x2 contingency table. Within this subset, FNAC correctly identified 115 of 117 cytologically benign cases as benign on histopathology, and 34 of 37 cytologically malignant cases as malignant on histopathology. Table 3 presents the 2x2 contingency table, and Table 4 presents the resulting diagnostic performance indices.

Table 3. Contingency table of FNAC versus histopathological diagnosis (definitive categories, n=154)

TP, true positive; FP, false positive; FN, false negative; TN, true negative.

Table 4. Diagnostic performance indices of FNAC against histopathology

CI, confidence interval, calculated using the Wilson score method. Indices calculated from Table 3 (definitive Bethesda categories II, V, and VI only; n=154).

When the three false-negative cases (Bethesda category II reported as benign but malignant on histopathology) were reviewed, all corresponded to papillary thyroid microcarcinomas measuring less than 1 cm, an established cause of sampling-related false negatives due to the small target lesion size relative to needle placement accuracy. The two false-positive cases (Bethesda category VI reported as malignant but benign on histopathology, in this dataset attributed to one category V and one category VI case being reclassified as benign hyperplastic nodules with reactive atypia on final histopathology) similarly reflect a recognised, if uncommon, pitfall of cytological overcall in the presence of marked reactive or regenerative atypia.

This study demonstrates that FNAC, when reported using the Bethesda system, provides excellent diagnostic accuracy for thyroid nodules, with a sensitivity of 91.9%, specificity of 98.3%, and overall diagnostic accuracy of 96.8% for definitive cytological categories. These findings are broadly concordant with published meta-analyses and large institutional series, which have generally reported sensitivity in the range of 83-97% and specificity in the range of 90-99% for thyroid FNAC against histopathological correlation, supporting the continued role of FNAC as the first-line investigation for thyroid nodule evaluation (14,15,16).

The distribution of cases across Bethesda categories in our cohort, with the majority falling into category II (56.2%), is consistent with the expected pattern in populations undergoing surgery, although it should be noted that our cohort, derived exclusively from patients who proceeded to surgery, is subject to selection bias and does not reflect the true population-level distribution of Bethesda categories, in which category II would be expected to constitute a substantially larger proportion, often 60-70%, of all FNAC performed, with comparatively fewer patients in this category proceeding to surgical excision (17). The progressive increase in risk of malignancy observed across categories II through VI in our study mirrors the risk stratification implicit in the Bethesda system's design and corroborates its clinical utility in guiding management decisions (9,18).

The risk of malignancy observed for indeterminate categories III (18.2%) and IV (40.0%) in our cohort falls within, albeit toward the higher end of, the range reported in the literature, where pooled estimates from meta-analyses generally place the risk of malignancy for category III between 10% and 30% and for category IV between 25% and 40% (10,19). This elevated risk likely reflects, at least in part, the referral bias inherent to a surgical cohort, in which patients with indeterminate cytology who were referred for surgery may have done so partly on the basis of additional risk factors, such as suspicious sonographic features or family history, that were not captured in the cytological diagnosis alone. This observation reinforces the value of incorporating sonographic risk stratification systems, such as the American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS), alongside cytology to refine the pretest probability of malignancy in indeterminate nodules (20).

The false-negative cases identified in our study, all corresponding to papillary microcarcinomas, highlight a well-recognised limitation of FNAC: the inherent risk of sampling error in small lesions, where the needle may preferentially sample adjacent benign parenchyma rather than the diagnostic focus itself (21). This underscores the importance of meticulous ultrasound-guided targeting, particularly for nodules under 1 cm, and supports continued sonographic surveillance even after benign cytology in nodules with high-risk sonographic features. Similarly, the false-positive cases observed, attributable to reactive or regenerative atypia mimicking malignant features, illustrate a recognised cytological pitfall that may be mitigated through cytopathologist experience and, increasingly, through ancillary immunocytochemical or molecular markers (22).

This study has several limitations. As a single-institution study restricted to patients who ultimately underwent surgery, it is subject to referral and selection bias, which may inflate the apparent risk of malignancy within indeterminate categories relative to the general FNAC population. The relatively small number of cases within categories I, IV, and V limited the precision of category-specific risk estimates, as reflected in the wide confidence intervals for these subgroups. Molecular testing, which has been shown in several studies to improve risk stratification for indeterminate nodules, was not performed in this cohort and represents an important area for future prospective evaluation (12,22).

Fine needle aspiration cytology, when reported using the Bethesda System for Reporting Thyroid Cytopathology, demonstrates excellent diagnostic accuracy for the preoperative evaluation of thyroid nodules, with high sensitivity, specificity, and overall concordance with histopathology for definitively benign and malignant categories. The progressive increase in risk of malignancy observed across Bethesda categories in this study supports the clinical validity of the system in guiding management decisions. However, indeterminate categories, particularly AUS/FLUS and follicular neoplasm, continue to pose a substantial diagnostic challenge, with a risk of malignancy too high to safely defer surgery yet too low to justify routine total thyroidectomy without further risk stratification. Incorporation of sonographic risk stratification systems and, where available, molecular testing alongside cytology may help refine management decisions for this challenging subset of patients and reduce the rate of diagnostic surgery for ultimately benign disease.

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