The detection of a new mass or growth, whether self-detected or identified through imaging, often causes significant patient anxiety. Achieving a prompt, accurate diagnosis while minimizing discomfort is clinically imperative. FNAC is a widely accepted, minimally invasive, cost-effective technique for evaluating palpable and deep-seated masses, performed across specialties including pathology, radiology, surgery, and endocrinology.^1-7

Despite its advantages, FNAC inadequacy rates remain a challenge, leading to repeat procedures, delayed diagnosis, and increased costs. ROSE addresses this by providing immediate on-site assessment of sample adequacy and preliminary diagnosis, improving diagnostic yield and reducing non-diagnostic rates.^8-12

Toluidine blue, a basic thiazine metachromatic dye with high affinity for nucleic acids, enables rapid staining (within seconds to minutes) and excellent visualization of nuclear and cytoplasmic details. It has been used effectively in various cytopathological and histopathological contexts, including oral, cervical, and gastric lesions, and shows promise for ROSE in FNAC.^13-16

 This study evaluates the effectiveness and feasibility of toluidine blue-stained ROSE in FNAC across multiple organ sites, comparing it to conventional methods.^17-20

Study Design: Prospective randomized observational study. Duration and Setting: 18 months (June 2024-December 2025) at the Department of Pathology, Santosh Medical College and Hospital, Ghaziabad. Sample Size: 150 participants (75 per group), calculated using standard formula (z=1.96, p=0.5, E=0.08). Inclusion Criteria: Adults 18 years and above scheduled for FNAC based on clinical or radiological suspicion, willing to consent. Exclusion Criteria: Bleeding disorders, anticoagulation precluding FNAC, inability to consent. Randomization: Computer-generated sequence; consecutive recruitment. Procedure: Ultrasound or CT-guided FNAC under sterile conditions. In the ROSE group, smears were immediately stained with toluidine blue for on-site evaluation by cytopathologist or cytotechnologist for adequacy and preliminary diagnosis. Control group used standard Papanicolaou/Giemsa staining without ROSE. Outcomes: Sample adequacy, number of passes required, diagnostic accuracy (vs. final cytology/histopathology), staining quality (nuclear/cytoplasmic detail), turnaround time, repeat procedure rates, operator feedback (1-5 Likert scale), and cost analysis. Statistical Analysis: Chi-square test for categorical variables, t-test for continuous; p<0.05 significant. Data analyzed using appropriate software. Ethical Considerations: Informed consent obtained; study approved by institutional ethics committee.

Table 1: Baseline Demographic and Clinical Characteristics of the Study Population (N=150)

Baseline characteristics were comparable between groups.

Table 2: Comparison of Sample Adequacy Between the Two Groups

ROSE showed significantly higher adequacy.

Table 3: Number of FNAC Passes Required to Achieve Adequacy

Fewer passes required with ROSE.

Table 4: Diagnostic Accuracy of ROSE with Toluidine Blue vs. Final Diagnosis (Histopathology)

Sensitivity: 96.4%, Specificity: 100%, PPV: 100%, NPV: 97.8%.

Table 5: Comparison of Diagnostic Accuracy Metrics Between Groups

Higher overall accuracy with ROSE.

Table 6: Comparison of Staining Quality - Cytoplasmic Detail

Superior cytoplasmic detail with toluidine blue.

Graph Descriptions (Data Trends from Thesis):

1. Bar chart of sample adequacy: ROSE 96% vs standard 84%.
2. Bar chart of passes: Higher single-pass in ROSE.
3. Pie charts of diagnosis distribution: Fewer inadequate cases in ROSE.
4. Bar chart of diagnostic metrics: Superior accuracy in ROSE.
5. Grouped bar chart of site-wise adequacy: ROSE superior, especially liver/gallbladder (100% vs 50%).

The present study demonstrates that incorporation of toluidine blue-based Rapid On-Site Evaluation (ROSE) in FNAC significantly enhances sample adequacy, reduces the number of needle passes, improves diagnostic accuracy, and decreases the need for repeat procedures compared to conventional Papanicolaou/Giemsa staining. These findings align closely with and extend the existing body of literature on ROSE in cytopathology.

In this randomized comparative study involving 150 patients, ROSE with toluidine blue achieved an adequacy rate of 96.0% versus 84.0% in the standard group (p=0.011), with single-pass adequacy in 77.3% of cases. This substantial improvement mirrors the results of Ammanagi et al. (2012)¹, who reported that on-site toluidine blue staining allowed decisions on further management in 98% of thyroid, breast, and lymph node FNAC cases on the same day, reducing unsatisfactory samples from 14% initially.

Similarly, Selhi et al. (2018)³ observed an increase in diagnostic yield for hepatic FNACs from 86.25% to 95.8% with ROSE utilizing toluidine blue. The current study’s adequacy improvement and site-specific benefits (100% adequacy in thyroid and liver/gallbladder lesions with ROSE) corroborate these observations.

Kothari et al. (2019)⁶ evaluated ROSE with toluidine blue in 1500 FNAC samples and reported sensitivity of 98%, specificity of 100%, and diagnostic concordance of 99.2%. Our results are highly consistent, with sensitivity of 96.4%, specificity of 100%, and overall diagnostic accuracy of 98.6% (p=0.045).

Recent research further validates these benefits. Kumar et al. (2024)¹⁵ reported 100% correlation between ROSE and cytological diagnosis in pulmonary lesions. Tummidi et al. (2024) demonstrated that ROSE lowered non-diagnostic rates from 7.7% to 1.3%.

Chowdhury et al. (2023)¹¹ found sensitivity of 97.9% for toluidine blue in lung lesions. Kimambo et al. (2023) reported high sufficiency in breast FNAB with ROSE.

The staining quality assessment revealed toluidine blue’s strength in cytoplasmic detail (69.3% excellent vs. 54.7% with Giemsa, p=0.038). Mendoza and Afify (2019)⁷ found 20% concentration optimal. Hewer et al. (2020)⁹ described ultrafast toluidine blue staining.

Operator feedback was positive (ease 4.6/5, speed 4.8/5). Michael et al. (2020)¹⁰ noted benefits in reducing non-diagnostic rates.

Post et al. (2023)¹³ showed ROSE improved adequacy. De Mesa Alvarez et al. (2023)¹⁴ confirmed high agreement.

Verma and Gupta (2018)¹⁵ reported 93% accuracy with supravital staining. Anila et al. (2018)⁵ converted 31% of inadequate samples.

Our study showed fewer repeat procedures (2.7% vs 12.0%, p=0.026). These outcomes align with broader evidence.

Limitations: Single-center study. Future Directions: Multi-center validation and telecytology integration.

ROSE with toluidine blue is an effective, feasible, and cost-efficient adjunct to FNAC. It significantly improves sample adequacy, reduces passes and repeats, enhances diagnostic accuracy, and supports timely clinical decisions. Routine adoption is recommended to optimize cytopathology services and patient outcomes.

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