Fine needle aspiration cytology (FNAC) was first introduced by Martin and Ellis in 1930 at Memorial Hospital, New York, as a rapid method of obtaining cellular material from palpable masses for cytological examination [1]. Over the following decades, it evolved into one of the most widely used, minimally invasive, outpatient diagnostic procedures in modern pathology practice [2]. FNAC combines simplicity, speed, low cost, minimal patient discomfort and a very low complication rate, and has become the initial investigation of choice for palpable swellings of the thyroid, breast, lymph node, salivary gland and soft tissue [3]. The thyroid gland is one of the commonest sites subjected to FNAC because of the high prevalence of thyroid nodules in the general population, the majority of which are clinically palpable and benign [4]. FNAC helps triage patients into those requiring surgical intervention and those who can be managed conservatively, thereby preventing a large number of unnecessary thyroidectomies, particularly in iodine-deficient regions such as many parts of India where nodular goitre is endemic [5]. The introduction of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) has further standardised the reporting of thyroid FNAC and improved communication between the cytopathologist and the treating clinician [6]. Similarly, a palpable breast lump is one of the most common presenting complaints in surgical outpatient departments across India, and FNAC forms an integral part of the 'triple assessment' of breast lesions, along with clinical examination and imaging [7]. It helps to differentiate benign from malignant lesions pre-operatively and thereby aids in appropriate surgical planning, avoiding a two-stage procedure in a large proportion of patients [8]. Several Indian studies, notably those by Handa et al. and Gupta et al., have highlighted the high diagnostic yield and accuracy of breast FNAC when performed in conjunction with clinical and radiological findings in the Indian population [9,10]. Lymphadenopathy is another extremely common clinical presentation encountered in both paediatric and adult populations, with aetiologies ranging from reactive hyperplasia and suppurative lymphadenitis to tuberculous lymphadenitis — a leading cause of peripheral lymphadenopathy in developing countries such as India — and metastatic or lymphomatous involvement [11]. FNAC of superficial lymph nodes is particularly valuable in a tuberculosis-endemic country like India, as it can rapidly differentiate tuberculous lymphadenitis from reactive and neoplastic aetiologies, thereby facilitating the early institution of anti-tubercular therapy and avoiding delay in the diagnosis of malignancy [12]. Despite its considerable advantages, FNAC has recognised limitations, including sampling error, inadequate cellularity, overlapping cytomorphological features between benign and malignant lesions (most notably in follicular neoplasms of the thyroid and papillary lesions of the breast), and a degree of operator- and interpreter-dependent variability [13]. Histopathological examination of the corresponding excised specimen therefore remains the undisputed gold standard against which the diagnostic performance of FNAC must be validated [14]. Numerous Western and Indian studies have reported cytohistological correlation figures with sensitivity and specificity ranging widely from approximately 80% to 98%, depending on the organ studied, the adequacy of the aspirate and the experience of the reporting cytopathologist [15]. Given the widespread and increasing use of FNAC as a first-line triage tool in resource-limited settings such as India, and the relative paucity of comprehensive, comparative cytohistological correlation data across the three most frequently aspirated superficial sites — thyroid, breast and lymph node — from a single tertiary care centre, the present study was undertaken to evaluate and compare the diagnostic accuracy of FNAC with histopathological correlation in these three organ systems. Aims and Objectives To study the cytomorphological spectrum of thyroid, breast and lymph node lesions on FNAC, to correlate the cytological diagnosis with histopathology, and to calculate the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and overall diagnostic accuracy of FNAC for each organ system.

Study design: Hospital-based prospective observational study. Study setting: Department of Pathology, in collaboration with the Departments of General Surgery and Surgical Oncology, Rama Medical College, Hospital & Research Centre, Hapur, U.P., India. Study duration: 24 months (January 2022 to December 2023). Study population: All patients presenting with a clinically palpable swelling of the thyroid, breast, or a superficial lymph node group, who were referred to the cytology section for FNAC, were considered for inclusion. Sample size: A total of 330 patients fulfilling the eligibility criteria were enrolled and subjected to FNAC (150 thyroid, 100 breast and 80 lymph node swellings). Of these, 210 patients (100 thyroid, 60 breast and 50 lymph node) subsequently underwent surgical excision or biopsy, and the histopathological reports of these 210 cases were available and used for cytohistological correlation and calculation of diagnostic accuracy. Inclusion Criteria • Patients of all ages and either sex presenting with a clinically palpable swelling of the thyroid, breast, or a superficial lymph node. • Patients who gave written informed consent for FNAC. • Cases in which adequate and well-stained smears were obtained for cytological interpretation. Exclusion Criteria • Patients with bleeding diathesis or those on anticoagulant therapy in whom FNAC was contraindicated. • Cases with persistently inadequate or unsatisfactory smears despite repeat aspiration. • Patients lost to follow-up who did not subsequently undergo surgery or biopsy were excluded only from the cytohistological correlation sub-analysis, and not from the overall descriptive cytological analysis. FNAC Procedure FNAC was performed under aseptic precautions by a trained pathologist using a 22-24 gauge needle attached to a 10 ml disposable syringe, employing the standard aspiration technique (and non-aspiration/Zajdela technique where appropriate), after obtaining written informed consent. Multiple passes were taken from different areas of the swelling to maximise cellular yield and to minimise sampling error. Smears were prepared on clean glass slides; some smears were air-dried for May-Grünwald-Giemsa (MGG) staining, while others were immediately fixed in 95% ethanol for Papanicolaou (Pap) and Haematoxylin and Eosin (H&E) staining. In cases with a clinical suspicion of tuberculous lymphadenitis, an additional smear was stained with Ziehl-Neelsen (ZN) stain for the demonstration of acid-fast bacilli. Reporting Systems Thyroid: Reported according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) — Category I (Non-diagnostic/unsatisfactory), Category II (Benign), Category III (Atypia of undetermined significance/follicular lesion of undetermined significance), Category IV (Follicular neoplasm/suspicious for follicular neoplasm), Category V (Suspicious for malignancy) and Category VI (Malignant). Breast: Reported using the standard five-tier cytological categorisation — C1 (Inadequate), C2 (Benign), C3 (Atypical, probably benign), C4 (Suspicious of malignancy) and C5 (Malignant). Lymph node: Categorised as reactive hyperplasia, granulomatous/tuberculous lymphadenitis, acute suppurative lymphadenitis, metastatic deposit, and lymphoma/leukaemic infiltrate. Histopathological Correlation Surgical/biopsy specimens received in the histopathology section were fixed in 10% neutral buffered formalin, routinely processed, embedded in paraffin, cut into 3-5 micron thick sections, and stained with Haematoxylin and Eosin (H&E). Special stains and immunohistochemistry were used where indicated. The histopathological diagnosis was taken as the gold standard for calculating the diagnostic accuracy of the preceding FNAC. Statistical Analysis Data were entered into Microsoft Excel and analysed using SPSS software (version 25.0). For each organ system, the cytological diagnosis was cross-tabulated against the corresponding histopathological diagnosis in a 2×2 contingency table, with malignant/suspicious cytological categories considered 'test positive' and benign categories considered 'test negative'. The following standard formulae were used: • Sensitivity = True Positive / (True Positive + False Negative) × 100 • Specificity = True Negative / (True Negative + False Positive) × 100 • Positive Predictive Value (PPV) = True Positive / (True Positive + False Positive) × 100 • Negative Predictive Value (NPV) = True Negative / (True Negative + False Negative) × 100 • Diagnostic Accuracy = (True Positive + True Negative) / Total Cases × 100 Ethical Considerations The study protocol was approved by the Institutional Ethics Committee prior to commencement. Written informed consent was obtained from all patients (or their legal guardians in the case of minors) prior to performing FNAC, and patient confidentiality was maintained throughout the study period.

A total of 330 FNAC cases were studied over the 24-month study period, comprising 150 (45.4%) thyroid, 100 (30.3%) breast and 80 (24.2%) lymph node swellings. Corresponding histopathological specimens were available for 210 (63.6%) of these cases — 100 thyroid, 60 breast and 50 lymph node — which formed the basis of the cytohistological correlation. Table 1: Age and Sex Distribution of the Study Population (n = 330) Organ Age Range (years) Mean Age (years) Male Female M : F Ratio Thyroid (n=150) 12 – 72 38.4 19 131 1 : 6.9 Breast (n=100) 16 – 68 34.7 4 96 1 : 24 Lymph node (n=80) 8 – 78 31.2 43 37 1.2 : 1 Total (n=330) 8 – 78 35.4 66 264 1 : 4 As shown in Table 1, thyroid and breast lesions showed a marked female preponderance, whereas lymph node lesions showed a near-equal distribution with a slight male preponderance, largely attributable to the higher incidence of tuberculous lymphadenitis among young males in this cohort. Table 2: Organ-wise Distribution of FNAC Cases (n = 330) Organ Number of Cases Percentage (%) Thyroid 150 45.4 Breast 100 30.3 Lymph node 80 24.2 Total 330 100.0 A. Thyroid Lesions Table 3: Bethesda Category-wise Distribution of Thyroid FNAC (n = 150) Bethesda Category Cytological Diagnosis No. of Cases Percentage (%) I Non-diagnostic/Unsatisfactory 8 5.3 II Benign (Colloid goitre/Nodular goitre/Thyroiditis) 104 69.3 III Atypia/Follicular lesion of undetermined significance 10 6.7 IV Follicular neoplasm/Suspicious for follicular neoplasm 14 9.3 V Suspicious for malignancy 6 4.0 VI Malignant (Papillary/Medullary/Anaplastic carcinoma) 8 5.3 Total - 150 100.0 Table 4: Cytohistological Correlation of Thyroid Lesions (n = 100) FNAC Diagnosis Histopathology: Malignant Histopathology: Benign Total Malignant/Suspicious (Category IV-VI) 13 (True Positive) 3 (False Positive) 16 Benign (Category I-III) 2 (False Negative) 82 (True Negative) 84 Total 15 85 100 Table 5: Diagnostic Accuracy Parameters of FNAC in Thyroid Lesions Parameter Value (%) Sensitivity 86.7 Specificity 96.5 Positive Predictive Value (PPV) 81.3 Negative Predictive Value (NPV) 97.6 Diagnostic Accuracy 95.0 Among the thyroid lesions, the two false negative cases on histopathology were follicular variant of papillary carcinoma reported as benign nodular goitre on FNAC, while the three false positive cases (reported as follicular neoplasm on cytology) turned out to be follicular adenoma and cellular nodular goitre on histopathology. B. Breast Lesions Table 6: Cytological Categorisation of Breast FNAC (n = 100) Category Cytological Diagnosis No. of Cases Percentage (%) C1 Inadequate 5 5.0 C2 Benign (Fibroadenoma, Fibrocystic disease, Mastitis) 58 58.0 C3 Atypical, probably benign 6 6.0 C4 Suspicious of malignancy 5 5.0 C5 Malignant (Invasive ductal carcinoma - NST and others) 26 26.0 Total - 100 100.0 Table 7: Cytohistological Correlation of Breast Lesions (n = 60) FNAC Diagnosis Histopathology: Malignant Histopathology: Benign Total Malignant/Suspicious (C4-C5) 22 (True Positive) 2 (False Positive) 24 Benign/Atypical (C1-C3) 1 (False Negative) 35 (True Negative) 36 Total 23 37 60 Table 8: Diagnostic Accuracy Parameters of FNAC in Breast Lesions Parameter Value (%) Sensitivity 95.7 Specificity 94.6 Positive Predictive Value (PPV) 91.7 Negative Predictive Value (NPV) 97.2 Diagnostic Accuracy 95.0 The single false negative breast case was a scirrhous type of invasive ductal carcinoma with dense stromal fibrosis and scant cellular yield, reported as fibrocystic disease on cytology. The two false positive cases (categorised as C4/suspicious) corresponded to fibroadenoma with marked epithelial hyperplasia and atypia on histopathology. C. Lymph Node Lesions Table 9: Cytological Categorisation of Lymph Node FNAC (n = 80) Cytological Diagnosis No. of Cases Percentage (%) Reactive hyperplasia 32 40.0 Granulomatous/Tuberculous lymphadenitis 22 27.5 Acute suppurative lymphadenitis 4 5.0 Metastatic deposit 17 21.3 Lymphoma/Leukaemic infiltrate 5 6.2 Total 80 100.0 Table 10: Cytohistological Correlation of Lymph Node Lesions (n = 50) FNAC Diagnosis Histopathology: Malignant Histopathology: Benign Total Malignant (Metastasis/Lymphoma) 21 (True Positive) 0 (False Positive) 21 Benign (Reactive/Granulomatous/Suppurative) 2 (False Negative) 27 (True Negative) 29 Total 23 27 50 Table 11: Diagnostic Accuracy Parameters of FNAC in Lymph Node Lesions Parameter Value (%) Sensitivity 91.3 Specificity 100.0 Positive Predictive Value (PPV) 100.0 Negative Predictive Value (NPV) 93.1 Diagnostic Accuracy 96.0 Both false negative lymph node cases on cytology were metastatic deposits from occult primary malignancies with extensive necrosis, which were initially reported as granulomatous lymphadenitis due to the necrotic background mimicking caseation. No false positive cases were encountered in the lymph node group in this series. D. Overall Diagnostic Accuracy of FNAC Table 12: Overall (Combined) Diagnostic Accuracy of FNAC — All Three Organs (n = 210) Organ Sensitivity (%) Specificity (%) PPV (%) NPV (%) Diagnostic Accuracy (%) Thyroid (n=100) 86.7 96.5 81.3 97.6 95.0 Breast (n=60) 95.7 94.6 91.7 97.2 95.0 Lymph node (n=50) 91.3 100.0 100.0 93.1 96.0 Overall (n=210) 91.8 96.6 91.8 96.6 95.2 On pooling the data from all three organ systems, FNAC showed an overall sensitivity of 91.8%, specificity of 96.6%, positive predictive value of 91.8%, negative predictive value of 96.6% and an overall diagnostic accuracy of 95.2% when correlated with the final histopathological diagnosis, confirming FNAC as a highly reliable first-line diagnostic modality across all three sites studied.

FNAC continues to occupy a central position in the initial evaluation of palpable swellings across the world, and particularly in high-volume, resource-constrained settings such as India, where it offers a rapid and inexpensive alternative to open surgical biopsy [2,3]. In the present study, 330 cases of thyroid, breast and lymph node swellings were analysed cytologically, of which 210 had histopathological correlation, allowing calculation of organ-wise diagnostic accuracy. In the present series, thyroid and breast lesions showed a strong female preponderance (M:F of 1:6.9 and 1:24 respectively), consistent with the findings of Das DK and Bhansali et al., who also reported a marked female predominance in thyroid swellings in Indian populations [16,17]. Lymph node lesions, on the other hand, showed a near-equal sex distribution with a slight male preponderance, comparable to the observations of Chandra et al. and Prasad et al. in their large series of lymph node aspirates from India [24,25], likely reflecting the higher prevalence of tuberculous lymphadenitis among young males in this population. Benign lesions (Bethesda Category II) constituted the largest proportion (69.3%) of thyroid FNACs in the present study, closely comparable to the 65-75% benign rate reported by Kumar and Sharma and by Sinha et al. in their Indian tertiary care series [5,18]. The unsatisfactory/non-diagnostic rate of 5.3% (Bethesda Category I) in the present study is within the acceptable range (2-15%) described in most large series, including that of Das DK [16]. On cytohistological correlation, FNAC showed a sensitivity of 86.7%, specificity of 96.5% and diagnostic accuracy of 95% for thyroid lesions in the present study. These figures are broadly comparable to those reported by Tandon et al. (sensitivity 88.2%, specificity 97.1%) and by Bukhari et al. (sensitivity 85%, specificity 95%) [27,31]. The relatively lower sensitivity for thyroid lesions compared with breast and lymph node lesions in the present study, as also observed by Kini in his classical monograph on thyroid aspiration cytology, is largely attributable to the inherent cytomorphological overlap between follicular adenoma and follicular variant of papillary carcinoma, a well-recognised 'grey zone' in thyroid cytopathology that the Bethesda System's indeterminate categories (III and IV) were specifically designed to address [6,19]. The false negative cases in the present study were follicular variant of papillary carcinoma initially reported as benign nodular goitre, a pitfall also highlighted by Layfield et al. in their analysis of on-site FNAC interpretation errors [20]. Similarly, the false positive follicular neoplasm calls that turned out to be follicular adenoma or hyperplastic nodules on histopathology reflect the well-known limitation that a definitive distinction between follicular adenoma and follicular carcinoma requires assessment of capsular and vascular invasion, which is only possible on histopathological examination of the entire capsule and cannot be made on cytology alone [4,19]. Benign lesions, predominantly fibroadenoma and fibrocystic disease, accounted for 58% of breast FNACs in the present study, comparable to the figures reported by Chandanwale et al. (61%) and Handa et al. (60.4%) from Indian tertiary care centres [8,9]. Malignant lesions (C5), predominantly invasive ductal carcinoma of no special type, constituted 26% of cases, in keeping with the higher proportion of malignant breast lesions typically seen in surgical referral populations as opposed to community screening cohorts [10,22]. The sensitivity, specificity and diagnostic accuracy of breast FNAC in the present study (95.7%, 94.6% and 95% respectively) closely mirror the values reported by Malhotra et al. (sensitivity 96.6%, specificity 95.8%) and by Sharma et al. in their Indian experience of over 500 breast aspirates (sensitivity 94%, specificity 96%) [22,23]. Comparable results have also been reported outside India by Yeoh and Chan in a large Singaporean series (sensitivity 93.8%, specificity 97.3%), reinforcing the reproducibility of these findings across different populations [32]. The single false negative case in the present series was a scirrhous carcinoma with dense desmoplastic stroma yielding a paucicellular aspirate, a well-documented cause of false negativity in breast FNAC as described by Sneige and Staerkel [21]. False positive results, as seen in the two cases of fibroadenoma with epithelial hyperplasia and atypia in the present study, are similarly well recognised and underscore the importance of considering the entire clinical and radiological picture (triple assessment) rather than relying on cytology in isolation, particularly in the C3/C4 indeterminate categories [7,21]. Reactive hyperplasia (40%) was the commonest cytological diagnosis among lymph node aspirates in the present study, followed by tuberculous/granulomatous lymphadenitis (27.5%), findings that closely parallel those of Chandra et al. (reactive hyperplasia 42%, tuberculous lymphadenitis 29%) and Deshmukh et al. in similar tertiary care settings in India [24,30]. The high proportion of tuberculous lymphadenitis observed reflects the continuing endemicity of tuberculosis in India and reaffirms the pivotal screening role of FNAC in this setting, as also emphasised by Verma and Kapila in their classical Indian study on tuberculous lymphadenitis [33]. FNAC of lymph nodes showed the highest specificity (100%) and a good sensitivity (91.3%) among the three organ systems studied, with an overall diagnostic accuracy of 96%. These findings are consistent with those of Mondal et al. (sensitivity 92.8%, specificity 97.6%) and Steel et al., who similarly reported very high specificity of lymph node FNAC for the diagnosis of metastatic and lymphomatous involvement [15,26]. The two false negative cases in this study, both metastatic deposits with extensive necrosis misinterpreted as granulomatous lymphadenitis, illustrate a well-recognised diagnostic pitfall described by Bagwan et al., wherein a necrotic background can mimic caseation and lead to a false impression of a tuberculous aetiology [29]. Careful search for viable tumour cells at the periphery of such necrotic aspirates, along with ancillary techniques such as immunocytochemistry where indicated, can help minimise this pitfall [12,28]. The overall diagnostic accuracy of FNAC across all three organ systems in the present study (95.2%) is comparable to figures reported in most large Indian and international series, which typically range from 90% to 97% [3,14,34]. Table 13 summarises a comparison of the sensitivity and specificity values obtained in the present study with those reported in other published Indian and international studies. The consistently high diagnostic accuracy of FNAC observed across the present study and comparable Indian and international literature reaffirms its role as an indispensable first-line investigation in the work-up of palpable swellings of the thyroid, breast and lymph nodes, allowing rational triage of patients for surgery, conservative management or further ancillary testing [3,14]. Nonetheless, indeterminate cytological categories — Bethesda III/IV for thyroid and C3/C4 for breast — as well as necrotic or paucicellular lymph node aspirates, remain recognised 'grey zones' where histopathological correlation is mandatory before definitive treatment decisions are made [6,19,29]. Limitations of the Study The study was conducted at a single tertiary care centre with a relatively modest sample size, which may limit generalisability to the wider population. Histopathological correlation could be obtained in only 210 of the 330 cases (63.6%), as a proportion of patients with benign cytological diagnoses were managed conservatively and did not undergo surgery, which may have introduced a degree of verification bias favouring cases with a higher pre-test probability of malignancy. Ancillary techniques such as immunocytochemistry, molecular testing (e.g., BRAF mutation analysis) and flow cytometry, which can improve diagnostic accuracy in indeterminate categories, were not routinely employed in this study.

Fine needle aspiration cytology is a simple, safe, rapid and cost-effective first-line diagnostic tool that shows excellent correlation with histopathology in the evaluation of thyroid, breast and lymph node lesions. In the present study, FNAC demonstrated an overall sensitivity of 91.8%, specificity of 96.6% and diagnostic accuracy of 95.2% when correlated with histopathology across the three organ systems studied, with the highest specificity noted for lymph node lesions (100%) and comparably high accuracy for both thyroid and breast lesions (95% each). These findings reaffirm that FNAC can be reliably used as the initial investigation of choice for the triage of palpable swellings, substantially reducing the number of unnecessary surgical procedures and enabling timely and appropriate management, including the early institution of anti-tubercular therapy in cases of tuberculous lymphadenitis and prompt surgical planning in malignant thyroid and breast lesions. However, indeterminate cytological categories, discordant clinico-cytological findings, and cases with equivocal or suspicious features must always be confirmed by histopathological examination before finalising the treatment plan, since a small but clinically significant proportion of false negative and false positive results remain an inherent limitation of any cytology-based diagnostic technique. Continued cytohistological correlation audits, adoption of standardised reporting systems such as the Bethesda System, and judicious use of ancillary diagnostic techniques in indeterminate categories are recommended to further improve the diagnostic accuracy of FNAC in routine clinical practice.

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