European Journal of Cardiovascular Medicine

Thyroid nodules are among the most common endocrine abnormalities encountered in clinical practice, with a prevalence that has significantly increased over the past few decades due to the widespread use of high-resolution ultrasonography. Palpable nodules are detected in approximately 4-7% of adults, whereas ultrasonography can reveal nodules in up to 30-70% of otherwise normal individuals. Although the majority of these nodules are benign, about 5-15% harbor malignancy, necessitating careful evaluation to distinguish benign from malignant lesions and thereby optimize management strategies. The clinical challenge lies in identifying nodules that require surgical intervention while minimizing unnecessary procedures for benign conditions.^[1]

The diagnostic evaluation of thyroid nodules involves a combination of clinical assessment, imaging, laboratory studies, and cytopathology. Fine-needle aspiration cytology (FNAC) is considered the gold standard for initial evaluation and remains an essential tool for triaging patients. However, FNAC has inherent limitations, including a non-diagnostic or indeterminate rate that may range between 10-25%. These limitations have driven the integration of imaging and biochemical markers to improve diagnostic accuracy.^[2]

The Thyroid Imaging Reporting and Data System (TIRADS), introduced by the American College of Radiology (ACR) in 2015, provides a standardized approach for risk stratification of thyroid nodules based on ultrasonographic features. It assigns points according to composition, echogenicity, shape, margin, and echogenic foci, generating a cumulative score that correlates with the risk of malignancy. By introducing uniform reporting and management recommendations, TIRADS aids clinicians in identifying high-risk lesions for biopsy and in avoiding unnecessary procedures for benign nodules. Studies have demonstrated that the malignancy rate progressively increases from TIRADS 2 to TIRADS 5 categories, thereby reinforcing its utility as a non-invasive, reproducible diagnostic tool.^[3]

Despite its advantages, imaging alone cannot provide definitive histopathologic confirmation. This is where cytological evaluation through FNAC plays a crucial role. The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) classifies thyroid lesions into six diagnostic categories, each with a specific risk of malignancy and management recommendation. However, discrepancies between imaging and cytology are not uncommon, especially in follicular-patterned lesions where cytologic features alone may not reliably predict malignancy. Therefore, adjunctive biochemical markers such as serum thyroglobulin (Tg) have been increasingly studied for their diagnostic and prognostic significance.^[4]

Thyroglobulin is a large glycoprotein produced exclusively by the follicular cells of the thyroid gland, serving as a precursor in thyroid hormone synthesis. Elevated serum thyroglobulin levels may be observed in benign hyperplastic nodules, multinodular goiter, thyroiditis, and differentiated thyroid carcinomas, particularly papillary and follicular types. Measurement of serum thyroglobulin has been routinely used in the follow-up of patients post-thyroidectomy to detect residual, recurrent, or metastatic disease. However, recent research has explored its potential role as a preoperative marker for differentiating benign from malignant thyroid nodules.^[5]

Aim

To compare the TIRADS scoring system with serum thyroglobulin levels in the cytological diagnosis of thyroid lesions.

Objectives

1. To correlate the cytological diagnosis and TIRADS scoring system with serum thyroglobulin levels in thyroid lesions.
2. To study serum thyroglobulin levels in differentiating benign and malignant thyroid lesions.
3. To evaluate the diagnostic utility of combining TIRADS and thyroglobulin levels with cytological findings in predicting malignancy.

Source of Data: The study was conducted on patients presenting with thyroid swellings referred to the Department of Pathology, Shri Atal Bihari Vajpayee Medical College and Research Institute, Bowring and Lady Curzon Hospital, Bengaluru. The data were collected from outpatient subjects attending the FNAC clinic and radiology department.

Study Design: This was a hospital-based cross-sectional comparative study conducted among patients with clinically or sonographically detected thyroid nodules.

Study Location: Department of Pathology, Bowring and Lady Curzon Medical College and Research Institute, Bengaluru, Karnataka, India.

Study Duration: The study was Jun 2022 to Dec 2023 following Institutional Ethics Committee (IEC) approval.

Sample Size: A total of 50 cases were included in the study based on convenience sampling.

Inclusion Criteria:

• Adult patients presenting with palpable thyroid nodules or sonographically detected thyroid lesions.
• Patients providing informed written consent for participation and blood sampling.

Exclusion Criteria:

• Pediatric patients.
• Patients unwilling to provide consent.
• Patients with incomplete clinical or laboratory data.

Procedure and Methodology: All consenting patients with thyroid swellings were evaluated clinically and subjected to ultrasound examination of the neck. Based on sonographic features, thyroid nodules were categorized according to the American College of Radiology (ACR) TIRADS scoring system, which assigns points for nodule composition, echogenicity, margins, shape, and echogenic foci to classify nodules from TIRADS-1 (benign) to TIRADS-5 (highly suspicious for malignancy).

Subsequently, Fine-Needle Aspiration Cytology (FNAC) was performed under aseptic precautions using a 23-gauge needle, either freehand or under ultrasound guidance. Smears were prepared, air-dried, and stained with Romanowsky (Giemsa) and Papanicolaou stains. Cytological interpretation was carried out as per the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC), and each case was categorized accordingly.

For biochemical analysis, 3 mL of venous blood was collected in plain tubes from each patient after informed consent. Serum was separated after clotting and centrifugation. Serum thyroglobulin (Tg) and thyroid-stimulating hormone (TSH) levels were estimated in the Central Laboratory of Bowring and Lady Curzon Hospital using the Beckman Coulter Access 2 Chemiluminescent Immunoassay System, which employs a two-site immunoenzymatic (“sandwich”) methodology. Quality control was ensured by running internal standards for every batch.

The cytological diagnosis, corresponding TIRADS category, and serum thyroglobulin values were recorded for each patient. Cases were classified as benign or malignant based on cytology, and correlations were drawn between TIRADS scores and thyroglobulin levels.

Sample Processing: All slides were reviewed by two independent pathologists to ensure consistency. Discordant cases were re-evaluated jointly to reach consensus. Laboratory assays were carried out following manufacturer protocols, and calibration curves were verified periodically to maintain assay accuracy.

Statistical Methods: Data were entered into Microsoft Excel and analyzed using SPSS version 21. Descriptive statistics (mean, standard deviation, percentages) were used to summarize the data. Comparative analysis was performed using Student’s t-test for continuous variables and Chi-square test for categorical variables. Pearson correlation and Receiver Operating Characteristic (ROC) curve analysis were applied to evaluate the diagnostic performance of thyroglobulin levels relative to cytological and TIRADS findings. A p-value < 0.05 was considered statistically significant.

IEC Approval: The study was conducted after obtaining approval from the Institutional Ethics Committee (IEC) of Bowring and Lady Curzon Medical College and Research Institute, Bengaluru.

IEC Number: BLCMCRI/IEC/RP/057/2021-22, dated 23.08.2021

Data Collection: All data, including demographic details, clinical findings, radiological reports, cytological categories, and laboratory results, were systematically recorded in pre-designed proformas. Data confidentiality was maintained throughout, and the records were securely stored in the Department of Pathology for five years post-study completion.

Table 1: Comparison of TIRADS with serum thyroglobulin (Tg) in cytological diagnosis (N = 50)

Table 1 summarizes the overall distribution of demographic, sonographic, biochemical, and cytological characteristics in 50 subjects. The mean age of participants was 42.7 ± 13.4 years (95 % CI 38.9-46.5). Females predominated (66 %) and showed a statistically significant preponderance (χ² = 5.12, p = 0.024). The mean nodule size was 2.47 ± 1.08 cm, suggesting most lesions were small-to-moderate in dimension. TIRADS categorization revealed 12 % TR2, 34 % TR3, 26 % TR4, and 28 % TR5 nodules, indicating that over half were in the intermediate-to-high-risk strata. Mean serum thyroglobulin (Tg) levels rose steadily with increasing TIRADS grade-from 24.7 ng/mL in TR2 to 142.6 ng/mL in TR5-a highly significant trend (ANOVA F = 18.73, η² = 0.55, p < 0.001). Correlation analysis confirmed a strong positive association between TIRADS score and Tg (Spearman ρ = 0.62, 95 % CI 0.41-0.76). Cytological findings (Bethesda system) were benign in 58 %, AUS/FLUS 18 %, follicular neoplasm 12 %, and suspicious/malignant 12 %. The relationship between Bethesda categories and TIRADS levels was statistically significant (χ² = 16.04, Cramer V = 0.40, p = 0.001), supporting good concordance between imaging-based risk and cytopathology.

Table 2: Correlation of cytology & TIRADS with serum Tg

Table 2 further examines how serum Tg values varied across Bethesda categories and their association with TIRADS. Mean Tg levels increased from 41.3 ± 22.8 ng/mL in benign (II) to 139.4 ± 66.8 ng/mL in malignant (V/VI) cytology, showing a highly significant difference (ANOVA F = 13.91, p < 0.001). Pairwise contrasts demonstrated progressive elevation of Tg from benign to indeterminate to malignant lesions, with large standardized mean differences (Cohen d = 0.78-2.42). The ordinal correlation between TIRADS and Bethesda categories was strong (Goodman-Kruskal γ = 0.61, p < 0.001), and TIRADS correlated positively with Tg (Spearman ρ = 0.62, p < 0.001). These findings indicate that as ultrasound suspicion increases, cytological atypia and biochemical activity (Tg output) also rise in parallel.

Table 3: Serum thyroglobulin for differentiating benign vs malignant cytology

Table 3 compares Tg concentrations between benign (Bethesda II-IV) and malignant (Bethesda V/VI) lesions to test its discriminative performance. Malignant nodules exhibited markedly higher mean Tg (138.9 ± 70.8 ng/mL) compared with benign ones (45.3 ± 27.1 ng/mL) with a mean difference of -93.6 ng/mL (95 % CI -131.2 to -56.0; p < 0.001). The standardized effect size was very large (Hedges g = 1.90). ROC analysis yielded an AUC = 0.84 (95 % CI 0.73-0.95), confirming good discriminatory power of Tg for malignancy. A Youden-optimized cut-off of 72 ng/mL achieved 83 % sensitivity and 82 % specificity (LR⁺ 4.56, LR⁻ 0.21). Model calibration was satisfactory (Brier score 0.12). Thus, serum Tg effectively distinguished malignant from benign thyroid cytology in this cohort.

Table 4: Diagnostic utility of combining TIRADS + Tg + Cytology to predict malignancy

Table 4 evaluates the diagnostic utility of combining modalities. Individually, cytology, TIRADS, and Tg achieved AUCs of 0.82, 0.79, and 0.84, respectively. When integrated in a logistic model (Cytology + TIRADS + Tg), the AUC rose to 0.91 (95 % CI 0.83-0.98), indicating excellent discrimination. This combined approach improved accuracy by ΔAUC = +0.09 (p = 0.028) and produced a significant net reclassification improvement (NRI = 0.21, 95 % CI 0.04-0.38; p = 0.014) compared with cytology alone. The model demonstrated balanced sensitivity (83 %) and specificity (88 %) with acceptable calibration (Hosmer-Lemeshow p = 0.61).

Figure: ROC curve with AUC

In cohort (N=50), higher TIRADS strata were common (TR4-TR5 = 54%) and showed a graded rise in serum thyroglobulin (Tg) from TR2 (24.7 ± 11.9 ng/mL) to TR5 (142.6 ± 64.2 ng/mL), with a large between-group effect (ANOVA η² = 0.55; Jonckheere trend p<0.001) and a strong monotonic association (Spearman ρ=0.62). This “dose-response” mirrors the original and validation work on ultrasound risk stratification wherein malignancy probability increases stepwise across categories, supporting the biological plausibility that increasingly suspicious sonographic phenotypes reflect greater follicular activity/tumor burden captured by Tg. Li HJ et al.(2023)^[6] first demonstrated a clear escalation of malignancy rates across their TIRADS tiers, while the ACR TI-RADS multi-institutional analysis by Grani G et al.(2024)^[7] confirmed robust risk discrimination in contemporary practice. Tg-TIRADS gradient is therefore consistent with the expectation that higher TIRADS bins are enriched for malignant histotypes (or aggressive biology), even when cytology is the primary reference.

Cytology (Bethesda) correlated significantly with TIRADS (χ²=16.04; Cramer V=0.40) and with Tg (ANOVA F=13.91; pairwise d up to 2.42). Eissa MS et al.(2024)^[4] also report concordance between sonographic suspicion and cytopathology, particularly for features embedded in risk systems (composition, echogenicity, margins, calcifications). The ACR TI-RADS study emphasizes that standardized scoring reduces subjectivity and aligns biopsy decisions with malignant yield-features that likely drive the association we observed between TIRADS levels and Bethesda categories. Calcifications-one of the ACR TI-RADS point-earning features-have repeatedly emerged as malignancy predictors, especially in follicular-patterned nodules where cytology can be indeterminate.

When dichotomized by cytology (Benign II-IV vs Malignant V/VI), Tg showed a very large separation (g=1.90) and good standalone discrimination (AUC 0.84; 95% CI 0.73-0.95).  Youden-optimized cut-off of ~72 ng/mL (Se 0.83; Sp 0.82) aligns closely with external thresholds. Hussein MA et al.(2024)^[8] prospectively proposed ~53 ng/mL with Se 72% and Sp 73%, while Kyrilli A et al.(2023)^[9] reported that Tg ≥70 ng/mL (particularly in nodules >1.7 cm) associated with increased cancer risk. Hussein IH et al.(2024)^[10] and Ito Y et al.(2025)^[11] further reinforced that elevated preoperative Tg is an independent predictor of malignancy in follicular/Hürthle neoplasms, often alongside specific US features (e.g., calcification). Cut-off sits between the 53-75 ng/mL band repeatedly cited across cohorts, likely reflecting differences in inclusion criteria, nodule size mix, and assay platforms.

Most importantly, combining modalities improved performance: the logistic model with Cytology + TIRADS + Tg achieved AUC 0.91 (ΔAUC +0.09 vs cytology alone; p=0.028) and a significant NRI (0.21). This “triple-test” approach is in line with the direction of recent evidence syntheses that recommend integrating biochemical markers with structured US risk and cytology to refine preoperative decisions, particularly when molecular testing is unavailable or cytology is indeterminate.  Mahmoud SA et al.(2024)^[12] concluded study that preoperative Tg “shows promise” for differentiating benign from malignant nodules in indeterminate cytology, while cautioning about heterogeneity in assays and thresholds-considerations that also apply to setting.

The present study, demonstrated a strong and statistically significant correlation between TIRADS categories, cytological findings, and serum thyroglobulin (Tg) levels. Serum Tg values increased progressively with higher TIRADS grades, reflecting escalating malignant potential. Likewise, Tg levels showed a clear distinction between benign and malignant cytological groups, establishing its diagnostic value as a complementary biomarker. When combined, TIRADS scoring, cytology, and serum Tg estimation achieved superior diagnostic accuracy (AUC = 0.91) compared to any single modality. These findings affirm that integrating biochemical and imaging parameters with cytology enhances the precision of thyroid lesion evaluation, particularly in indeterminate cases, facilitating more informed clinical decision-making and potentially reducing unnecessary surgical interventions.

LIMITATIONS OF THE STUDY

1. Small sample size (N = 50) limits the generalizability of findings and may underrepresent less common thyroid malignancies.
2. The study was single-center and hospital-based, possibly introducing referral bias.
3. Histopathological confirmation was unavailable for all cytologically benign cases, restricting complete sensitivity-specificity validation.
4. Serum thyroglobulin is a nonspecific biomarker and can be influenced by benign goiter, thyroiditis, or gland volume, which were not fully controlled.
5. The study did not include antithyroglobulin antibody (TgAb) levels, which may interfere with Tg assay accuracy.
6. Operator dependency in ultrasonographic interpretation could introduce inter-observer variability in TIRADS scoring.
7. The cross-sectional design precludes assessment of longitudinal Tg trends or post-therapeutic changes.
8. Advanced molecular and immunohistochemical markers were not incorporated, which might further improve diagnostic precision.

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