Improving Detection Efficiency of FNAC and BRAF V600E Mutation in Thyroid Nodules Using Pipette Mixing: A Retrospective Study

ABSTRACT

Background

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, with a significant subset of cases harboring the BRAF V600E mutation. Fine‐needle aspiration cytology (FNAC) combined with BRAF V600E mutation detection is a standard diagnostic approach for thyroid nodules. However, sample heterogeneity often leads to inconsistent results between cytological and molecular analyses. This study aimed to evaluate whether pipette mixing of FNAC samples improves the detection efficiency and diagnostic accuracy of the BRAF V600E mutation in thyroid nodules.

Methods

A retrospective analysis was conducted on 360 FNAC samples, divided into pipette‐mixed group (n = 180) and a non‐mixed group (n = 180). Samples were classified using The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC), and BRAF V600E mutation status was assessed using ARMS‐PCR. Diagnostic performance between preoperative FNAC and BRAF V600E mutation detection was compared between the two groups.

Results

Pipette mixing substantially reduced the proportion of samples with undetectable BRAF V600E mutations, thereby increasing the yield of interpretable results (p < 0.05). In the non‐mixed group, 12 samples fell below the detection limit, compared to only 2 in the mixed group. The proportion of TBSRTC Category III samples was lower in the mixed group (12/180) than in the non‐mixed group (38/180, p < 0.05). Pipette‐mixed FNAC combined with BRAF V600E testing improved preoperative diagnostic efficiency, particularly for thyroid nodules with indeterminate or non‐diagnostic cytology.

Conclusions

Pipette mixing of FNAC samples improves the detection efficiency of BRAF V600E mutation and enhances diagnostic accuracy for thyroid nodules, especially in challenging cases. This simple technique may optimize preoperative diagnosis and guide clinical decision‐making.

1. Introduction

In recent years, the global incidence of thyroid cancer has shown a steady increase [1], with PTC representing the predominant histological subtype. FNAC has been established as the gold standard for preoperative diagnosis due to its high safety profile and diagnostic accuracy in differentiating benign from malignant thyroid nodules [2, 3]. Nevertheless, the diagnostic reliability of FNAC remains heavily dependent on the substantial expertise of both the performing physician and the cytopathologist. Furthermore, inherent limitations in cytological interpretation contribute to persistent diagnostic uncertainty in borderline cases [3].

For thyroid nodules with indeterminate cytological findings on FNAC, molecular profiling of puncture specimens using thyroid cancer markers (including BRAF and RAS mutations) can provide critical diagnostic and prognostic information [4]. The BRAF proto‐oncogene, located at 7q34, encodes a serine–threonine kinase that plays a pivotal role in the mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) signaling pathway, regulating fundamental cellular processes including proliferation, differentiation, and apoptosis [5]. Most clinically significant BRAF mutations localize to exon 15 within the kinase domain, with the V600E variant (c.1799T>A, p.Val600Glu) being the most prevalent oncogenic driver [6]. This gain‐of‐function mutation leads to constitutive kinase activation and sustained MAPK/ERK signaling, which is strongly associated with tumorigenesis [4, 7]. In thyroid neoplasms, the BRAF V600E mutation demonstrates high specificity for malignancy, occurring in 28%–83% of papillary thyroid carcinomas while being absent in normal thyroid parenchyma and benign lesions [6].

Currently, the combined use of FNAC and BRAF V600E mutation analysis offers the highest diagnostic efficacy for thyroid cancer [8]. Integrating FNAC with BRAF V600E mutation detection serves as a cost‐effective strategy for differentiating malignant from benign thyroid nodules. However, diagnostic discordance may arise due to tumor heterogeneity, which limits the reliability of molecular‐cytological correlation [9]. In this study, we investigate whether mechanical homogenization of FNAC specimens via pipette mixing enhances sample uniformity, thereby improving BRAF V600E detection consistency and overall diagnostic accuracy. The implementation of pipette mixing for FNAC samples represents a simple yet impactful pre‐analytical advancement.

The primary endpoint of this study was to evaluate the impact of this pre‐analytical modification (pipette mixing method) on the cytological adequacy rate and sample adequacy in the homogenized samples. The secondary endpoint was to compare the effect of this method on diagnostic accuracy in patients with available surgical histology follow‐up. As a cost‐effective and easily adoptable technique, this pre‐analytical modification holds strong potential to refine preoperative risk assessment and optimize patient management pathways.

2. Materials and Methods

2.1. Study Design and Participants

This retrospective study analyzed 360 FNAC specimens obtained from patients with thyroid nodules at The Third People's Hospital of Chengdu between March 2024 and August 2024. All specimens underwent ultrasound‐guided FNAC with concurrent BRAF V600E mutation testing. The study employed a sequential design to evaluate the impact of a pre‐analytical homogenization technique on FNAC samples. In the initial phase (March–May 2024), 180 consecutive cases (non‐mixed group) were processed without mechanical homogenization prior to BRAF V600E mutational analysis. In the subsequent intervention phase (June–August 2024), 180 consecutive cases (mixed group) underwent standardized pipette‐assisted homogenization of dual cellular suspensions before molecular testing. All sample acquisitions were performed by the same team of experienced operators, and all cytological evaluations were conducted by the same group of cytopathologists, who were blinded to group assignment. This sequential enrollment design, combined with consistent diagnostic workflows and personnel across both phases, enabled a direct comparison of pre‐ and post‐optimization sample processing techniques while minimizing potential operator‐dependent and interpretive biases. For this type of retrospective study involving anonymized data from medical records, formal informed consent is not required. The study protocol was reviewed and approved by the Hospital's Ethical Review Board of The Third People's Hospital of Chengdu, which granted a formal waiver of informed consent.

2.2. Sample Processing and Diagnostic Accuracy Assessment

Mixed group: FNAC specimens were processed using a standardized dual‐path protocol to enable concurrent cytomorphological and molecular evaluation. Two separate FNAC passes were obtained from the nodule. The pass designated for molecular studies was combined with the routine cytology sample. The pooled specimen was thoroughly mixed by repeated pipetting (at least five times) until a homogeneous suspension was achieved. The homogenized sample was then split into two equal aliquots: one for conventional cytology smears and the other transferred to a microcentrifuge tube and submitted to the molecular laboratory for DNA extraction.

Non‐mixed group: Two separate FNAC passes were obtained from the nodule. The first pass was used entirely for conventional smear preparation (no rinsing or mixing). The second pass was rinsed into a transport medium for DNA extraction. No pooling or pipette‐mixing step was applied.

The routine cytology sample was uniformly smeared onto slides, fixed in 95% ethanol, and stained with hematoxylin and eosin (H&E). All slides were subsequently evaluated by experienced cytopathologists, and diagnoses were reported according to TBSRTC Categories I–VI.

Diagnostic accuracy was evaluated in the subset of patients who underwent surgical resection with histopathology as the gold standard. For cytology, Bethesda categories V and VI were classified as positive for malignancy, while categories I, II, and IV were classified as negative, and category III was classified as indeterminate. For BRAF testing, any detected mutation was considered positive. For the combined approach, a case was considered positive if either cytology (Bethesda V/VI) or BRAF testing was positive.

2.3. BRAF V600E Detection

Amplification refractory mutation system‐polymerase chain reaction (ARMS‐PCR) was used to evaluate mutation status. Genomic DNA was extracted from the homogenized pass sample using a commercial nucleic acid extraction kit (AmoyDx Diagnostics, Xiamen, China). The purified DNA was quantified and diluted to a working concentration of 1.5–2.0 ng/μL for subsequent analysis. BRAF V600E mutation detection was performed using a commercial real‐time PCR kit (AmoyDx Diagnostics) according to the manufacturer's protocol. Briefly, the master mix was prepared by combining 45 μL of reaction buffer with 0.4 μL of Taq DNA polymerase. The reaction mixture was aliquoted into PCR tubes, sealed, and briefly centrifuged prior to amplification in a thermal cycler (HONGSHI, Shanghai). Fluorescence signals (FAM and HEX) were acquired during the 60°C annealing step of the quantification phase. Sample quality control was determined based on HEX fluorescence signal threshold cycle (Ct) values. Valid samples were defined as those with Ct values between 13 and 21. For BRAF V600E mutation analysis, samples were classified as positive when FAM Ct values were < 30, while those with Ct values ≥ 30 were considered negative for the mutation.

2.4. Statistical Analysis

Statistical analyses were performed using SPSS software (version 25.0). Quantitative data were expressed as mean ± standard deviation, while categorical variables were presented as frequencies (%). Student's t‐test, Chi‐square test and Fisher’ exact test were used to compare variables between the non‐mixed and mixed groups. p‐value < 0.05 was considered statistically significant for all analyses.

3. Results

3.1. Patient Characteristics

This study included 360 patients (94 males, 26.11%; 266 females, 73.89%), as summarized in Table 1. The mean patient age was 47.06 ± 13.91 years, with 43.33% (156/360) aged ≤ 45 years and 56.67% (204/360) aged > 45 years. BRAF V600E mutation analysis demonstrated that 59.72% (215/360) of patients were identified as wild‐type, 36.39% (131/360) carried the mutant variant, and 3.89% (14/360) had undetectable mutation levels. Based on FNAC results, cases were classified as negative in 45.83% (165/360) of cases, positive in 40.28% (145/360), and diagnostically ambiguous in 13.89% (50/360). Baseline characteristics of the non‐mixed and mixed groups are summarized in Table 2. There were no statistically significant differences between the two groups in terms of age, gender, or nodule size (all p > 0.05), indicating that the two groups were comparable.

TABLE 1.

Basic characteristics of patients.

Clinical features	Total	Percentage (%)
Gender
Male	94	26.11
Female	266	73.89
Age
Average	47.06 ± 13.91
≤ 45	156	43.33
> 45	204	56.67
BRAF V600E
Wild type	215	59.72
Mutant type	131	36.39
Below the detection limit	14	3.89
FNAC
Negative	165	45.83
Positive	145	40.28
Ambiguous clinical significance	50	13.89

TABLE 2.

Baseline characteristics of patients in the mixed and non‐mixed groups.

Clinical features	Non‐mixed group (n = 180)	Mixed group (n = 180)	p
Gender (male/female)	43/137	51/129	0.65
Age	48.34 ± 13.89	45.77 ± 13.51	0.08
Nodule diameter	1.12 ± 0.91	1.14 ± 0.96	0.87

3.2. Impact of Pipette Mixing on the Detection Efficacy of BRAF V600E Mutations

The mixed groups prepared using pipette mixing are presented in Figure 1. The distribution of BRAF V600E mutation genotypes across the two pipette mixing conditions was shown in Table 3. Pipette mixing significantly reduced the proportion of samples falling below the BRAF V600E detection limit (from 6.67% to 1.11%, p = 0.004 by Fisher's exact test), thereby increasing the number of samples with interpretable mutation results. However, among interpretable samples, the mutation‐positive rate was similar between the two groups (61.67% in non‐mixed vs. 57.78% in mixed, p = 0.143). This indicates that the primary benefit of pipette mixing is to rescue samples that would otherwise be non‐informative, rather than to increase the true mutation detection rate.

FIGURE 1.

The left panel illustrates the FNAC sample before pipette mixing, while the right panel depicts the same sample following the mixing procedure. [Color figure can be viewed at wileyonlinelibrary.com]

TABLE 3.

Correlation between BRAF V600E mutation types and pipette mixing status.

BRAF V600E	Pipette mixing
	No (n = 180)	Yes (n = 180)
Wild type	57 (31.67%)	74 (41.11%)
Mutant type	111 (61.67%)	104 (57.78%)
Below the detection limit	12 (6.67%)	2 (1.11%)

3.3. Association Between TBSRTC Diagnostic Categories and Pipette Mixing

A statistically significant association was identified between TBSRTC categories and the use of pipette mixing during sample preparation (χ ² = 28.2, p < 0.01), as shown in Table 4. The diagnostic distribution varied substantially depending on mixing status. Notably, non‐mixed samples were more likely to be classified as Category I (ND/UNS; 18 vs. 5) and Category III (AUS; 38 vs. 12). Conversely, the use of pipette mixing was associated with a higher prevalence of several categories, including Category II (Benign; 62 vs. 80), Category V (SFM; 28 vs. 34), and a marked increase in Category VI (Malignant; 26 vs. 43). Meanwhile, the incidence of Category IV (FN/SFN) showed little difference between the two groups (8 in the non‐mixed group vs. 6 in the mixed group).

TABLE 4.

Correlation between TBSRTC categories and the status of mix using a pipette.

TBSRTC	Pipette mixing		χ 2	p
	No	Yes
I (ND/UNS)	18	5	28.20	< 0.01
II (Benign)	62	80
III (AUS)	38	12
IV (FN/SFN)	8	6
V (SFM)	28	34
VI (Malignant)	26	43

Abbreviations: AUS, atypia of undetermined significance; FN, follicular neoplasm; ND, Non‐diagnostic; SFM, suspicious for malignancy; SFN, suspicious for a follicular neoplasm, UNS, unsatisfactory.

These findings indicate that pipette mixing significantly affects the diagnostic categorization of thyroid fine‐needle aspiration specimens, correlating with a reduced incidence of non‐diagnostic and indeterminate results (Categories I and III) and an increased proportion of definitive diagnoses, including Benign, Suspicious for Malignancy, or Malignant classifications. Importantly, the pipette‐mixed group showed a lower proportion of TBSRTC Category III cases (12/180 vs. 38/180), indicating a reduction in indeterminate diagnoses (Table 4).

3.4. Improved Sensitivity of Combined Cytology and BRAF Testing in Histologically Confirmed Cases

Diagnostic accuracy was evaluated in 84 patients with surgical histology follow‐up, including 35 cases in the non‐mixed group and 49 cases in the mixed group, based on the criteria defined in the Methods section (Table 5).

TABLE 5.

Diagnostic accuracy of BRAF V600E testing, cytology, and combination testing in patients with postoperatively histopathological results in non‐mixed and mixed groups.

Parameter	Pipette mixing		Sensitivity (%)		Specificity (%)		PPV (%)		NPV (%)		Accuracy (%)
	No	Yes	NM	M	NM	M	NM	M	NM	M	NM	M
BRAF V600E	n = 35	n = 49	93.55 (29/31)	95.74 (45/47)	100 (4/4)	100 (2/2)	100 (29/29)	100 (45/45)	66.67 (4/6)	50 (2/4)	94.29 (33/35)	95.92 (47/49)
FNAC			90.32 (28/31)	95.65 (44/46)	75 (3/4)	100 (3/3)	96.55 (28/29)	100 (44/44)	50 (3/6)	37.50 (3/8)	88.57 (31/35)	95.92 (47/49)
Combination detection			96.88 (31/32)	97.91 (47/48)	66.67 (2/3)	100 (1/1)	96.88 (31/32)	100 (47/47)	66.67 (2/3)	50 (1/2)	94.29 (33/35)	97.96 (48/49)

Abbreviations: M, mixed group; NM, non‐mixed group; NPV, negative predictive value; PPV, positive predictive value.

For BRAF testing alone, the mixed group demonstrated higher sensitivity compared to the non‐mixed group (95.74% vs. 93.55%), while specificity remained 100% in both groups. The positive predictive value (PPV) was 100% in both groups. The negative predictive value (NPV) was 66.67% in the non‐mixed group versus 50.0% in the mixed group, reflecting the lower proportion of benign cases in the mixed group (2/49 vs. 4/35). Cytology alone showed superior diagnostic performance across all metrics in the mixed group. Sensitivity increased from 90.32% in the non‐mixed group to 95.65% in the mixed group. Specificity improved markedly from 75.0% to 100%, and PPV increased from 96.55% to 100%. Accuracy rose from 88.57% to 95.92%. The combined approach achieved high diagnostic accuracy in the mixed group, with 97.91% sensitivity, 100% specificity, and 100% PPV, compared to 96.88% sensitivity, 66.67% specificity, and 96.88% PPV in the non‐mixed group. Overall accuracy improved from 94.29% to 97.96%.

Overall, these findings demonstrate that the pre‐analytical pipette‐mixing method not only improved sample adequacy and BRAF V600E mutation detection rates but also enhanced diagnostic accuracy (Table 5).

3.5. Association of BRAF V600E Mutation Status With Clinicopathological Characteristics and Postoperative Histopathological Results

The BRAF V600E mutation was detected in 88.10% (74/84) of the surgically confirmed cases analyzed. As summarized in Table 6, no statistically significant associations were observed between BRAF V600E mutation status and any of the clinicopathological variables evaluated, including gender (p = 0.86), age (p = 0.83), nodule diameter (p = 0.74), histological subtype of papillary thyroid carcinoma (p = 0.86), lymph node metastasis (p = 0.82), or capsular invasion (p = 0.89). Nevertheless, notable trends in mutation prevalence were observed across subgroups. The BRAF V600E positivity rate was higher in male patients (92.0%) than in females (86.44%), and in patients over 45 years of age (91.67%) compared to those 45 years or younger (85.42%). Regarding nodule size, smaller nodules (≤ 1.0 cm) exhibited a higher mutation rate (90.63%) than larger nodules (> 1.0 cm; 80%). Conventional‐type PTC showed a slightly higher prevalence of the mutation (89.04%) than other subtypes (81.82%). Additionally, cases without lymph node metastasis had a higher mutation rate (91.11%) than those with metastasis (84.62%), while the presence of capsular invasion was associated with a comparable mutation prevalence (89.80% vs. 85.71% in non‐invasive tumors).

TABLE 6.

Correlation between clinicopathological characteristics with postoperatively histopathological results in patients and BRAF V600E mutation status.

Characteristic	Number of cases	BRAF V600E		χ 2	p
		Mutant type	Positive rate
Gender				0.03	0.86
Male	25	23	92.00%
Female	59	51	86.44%
Age				0.05	0.83
≤ 45	48	41	85.42%
> 45	36	33	91.67%
Nodule diameter				0.11	0.74
Size ≤ 1.0 cm	64	58	90.63%
Size > 1.0 cm	20	16	80.00%
Subtype of PTC				0.03	0.86
Conventional	73	65	89.04%
Other subtype	11	9	81.82%
Lymph node metastasis				0.05	0.82
Yes	39	33	84.62%
No	45	41	91.11%
Capsular invasion				0.02	0.89
Yes	49	44	89.80%
No	35	30	85.71%

These results indicate that although the BRAF V600E mutation is highly prevalent in this PTC cohort, it is not significantly correlated with the clinicopathological features examined, suggesting that its role as an independent prognostic marker in this population may be limited.

4. Discussion

This study establishes that pipette mixing, a straightforward pre‐analytical intervention, holds significant value for thyroid nodule diagnostics. Analysis of 360 FNAC samples shows that this homogenization step substantially boosts detection of the BRAF V600E mutation. The enhancement is most pronounced in samples with low mutation burden, where it likely counteracts indeterminate arising from cellular sedimentation within the vial. The improvement in sample representativity leads to superior diagnostic performance, including a significant reduction in non‐diagnostic and indeterminate (e.g., AUS) cytology reports, and a corresponding increase in definitive diagnoses. Beyond contributing to local prevalence data for BRAF V600E, this study highlights a practical, cost‐neutral intervention. Routine pipette mixing should be adopted to optimize pre‐analytical processes, thereby elevating both molecular and cytomorphological diagnostic accuracy for thyroid nodules.

FNAC is an important and cost‐effective diagnostic tool for evaluating thyroid nodules suspicious for malignancy on ultrasound [10]. TBSRTC, a major landmark in thyroid cytology, established a uniform system for reporting thyroid cytopathology [5, 11]. With six recognized diagnostic categories associated with incremental risks of malignancy, TBSRTC standardized cytological diagnosis and increased its diagnostic yield [12]. Nevertheless, a number of clinical studies have demonstrated that this system does not clearly address the heterogeneous group of nodules with indeterminate cytology, including AUS, FN/SFN, and SM [3, 5]. The promising molecular marker BRAF V600E mutation, the most robust oncogene in PTC, has drawn particular attention and been widely tested [13].

Our observation that pipette mixing improves mutation detection is novel in the context of thyroid cytology. While the critical role of pre‐analytical factors in molecular diagnostics is well‐documented, most studies focus on fixation times, sample storage, and nucleic acid extraction methods [14, 15]. The present study introduces sample homogenization as a previously underappreciated yet vital variable. Pipette mixing decreases the rate of non‐informative molecular results (from 6.67% to 1.11%) and is of considerable clinical importance. In current practice, a ‘below detection limit’ result for a key oncogene like BRAF V600E provides no clinical utility, often necessitating a repeat biopsy or leaving the clinician with diagnostic uncertainty. By rescuing these samples, our simple pre‐analytical step can directly reduce the number of inconclusive FNAC procedures, improve laboratory efficiency, and decrease patient anxiety and healthcare costs associated with repeat testing. This is further corroborated by the significant shift in TBSRTC categories. The pronounced reduction in Category I (ND/UNS) and Category III (AUS) rates in the mixed group is consistent with studies showing that improved sample preparation enhances cellular yield and preservation, leading to more definitive diagnoses [16, 17]. The subsequent increase in definitive categories (II, V, VI) suggests that mixing reduces sampling error, ensuring that the material on the slide is more representative of the entire nodule.

Analysis of the 84 cases with surgical histology follow‐up provided valuable insights into the diagnostic performance of the three testing strategies (BRAF testing alone, cytology alone, and the combined approach). Although the primary endpoints of this study were sample adequacy rate and mutation detection rate, subgroup analysis of surgically confirmed cases demonstrated that our modified pre‐analytical method did not compromise diagnostic accuracy. Furthermore, the combined approach improved sensitivity, which is consistent with previous studies [3, 7]. Prospective randomized studies with larger surgical cohorts are warranted to validate these observations and to further evaluate the impact on patient outcomes.

Reported frequencies of the BRAF V600E mutation in adult PTC vary considerably, from 28% to 83% [6, 18, 19]. The prevalence of the BRAF V600E mutation (36.39% in the entire cohort, 88.10% in surgically confirmed PTCs) falls within the widely reported range for PTC, though it is at the higher end of the spectrum compared to some Asian populations [20, 21, 22]. The lack of a statistically significant association between BRAF V600E status and aggressive clinicopathological features such as lymph node metastasis or capsular invasion is intriguing. While some meta‐analyses have established a firm link between BRAF V600E and poorer prognosis [23], a growing body of evidence, particularly from larger clinical cohorts, suggests that this association may be weaker than initially thought and highly dependent on other co‐factors [24, 25]. Our results support the latter view, indicating that in our patient population, BRAF V600E is a highly sensitive marker for PTC but not a specific indicator of aggressive disease. The trends we observed—higher prevalence in males, older patients, and smaller nodules—are interesting hypothesis‐generating findings that require validation in a larger, prospectively designed study.

Of note, recent studies have demonstrated that the supernatant of FNAC specimens contains detectable tumor‐derived circulating free DNA (cfDNA), which may further enhance the detection rate of BRAF mutations or be utilized for other genetic tests [26, 27, 28]. In line with other clinical applications, cfDNA presents a valuable non‐invasive alternative to tissue biopsy or radiographic imaging in selected cases [29]. The detection of cfDNA alterations has enabled tumor classification, prognostic assessment, disease monitoring, and the prediction of response to targeted therapies [30]. The present study primarily focused on the cell pellet, which was the core of our pre‐analytical modification protocol. In the future, we plan to explore a workflow that combines analysis of both the cell pellet and supernatant to maximize the sensitivity of molecular detection.

This study has several limitations that should be acknowledged. First, the non‐randomized sequential design introduces the potential for temporal and operator‐dependent biases. Although we maintained consistency in the operating and evaluation teams as much as possible and demonstrated balanced baseline characteristics between the two groups (Table 2), the possibility of unmeasured confounding factors cannot be entirely excluded. Furthermore, while all fine‐needle aspiration procedures were performed by experienced cytopathologists—ensuring technical consistency—the applicability of our findings to less‐experienced practitioners remains to be established. Future studies employing a randomized controlled design are warranted to further validate our conclusions. Second, as a single‐center investigation, the generalizability of our findings requires validation in multi‐center settings with varied operators and protocols. While the substantial sample size partially addresses this concern, the non‐randomized allocation of samples to mixing and non‐mixing groups may introduce selection bias. Third, our molecular analysis was limited to the BRAF V600E mutation; future studies should examine whether pipette mixing also enhances the detection of other molecular alterations, such as RAS, RET/PTC, PAX8/PPARγ, or RNA‐based classifiers. Finally, the present study focused exclusively on the BRAF V600E mutation. While our pre‐analytical modification enhanced the detection of this single mutation, whether this approach is applicable to multi‐gene panels, such as those used in next‐generation sequencing (NGS), has not been established. Theoretically, increased cellularity and improved sample homogeneity should also benefit NGS‐based assays; however, this hypothesis requires validation in future studies.

Notwithstanding these limitations, our findings have direct clinical relevance. We advocate for the standardized adoption of gentle pipette mixing of FNAC samples immediately prior to slide preparation and aliquoting for molecular analysis. This simple, rapid, and cost‐neutral step may improve diagnostic accuracy, potentially reducing the need for repeat biopsies in indeterminate cases and offering more reliable molecular guidance for surgical planning.

Future research should focus on several key directions. First, mechanistic studies should quantitatively evaluate the effect of mixing on cellular distribution and nucleic acid integrity and yield. Second, expanding the molecular profiling panel will help determine the broader applicability and universal benefit of this pre‐analytical step. Third, postoperative pathological results as the gold standard are essential to further validate the diagnostic utility of integrated cytologic and molecular testing.

5. Conclusion

In conclusion, by substantially reducing rates of non‐diagnostic and indeterminate cytology results while increasing the sensitivity of BRAF V600E detection, this pre‐analytical practice contributes to more accurate risk stratification and supports informed clinical decision‐making. The adoption of robust, reproducible pre‐analytical protocols—such as the one presented here—is crucial for advancing precision medicine in thyroid oncology and ensuring that molecular testing delivers its full diagnostic and prognostic potential.

Author Contributions

W.F. evaluated the data and drafted the paper. Y.C. supervised the study and wrote the manuscript. The final manuscript was reviewed and approved by all writers.

Funding

The authors have nothing to report.

Consent

All authors have approved the manuscript for submission and publication.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. The data are not publicly available due to privacy or ethical restrictions.