Abstract
Background
Core needle biopsy (CNB) is commonly used for histological diagnosis and molecular testing in patients with non-small cell lung cancer (NSCLC). However, the adequacy of CNB specimens is often compromised by low tumor cell content or insufficient nucleic acid quantity, which can negatively impact the success of downstream analyses. Moreover, variations in specimen collection techniques may further contribute to inconsistency in sample quality. This retrospective study aimed to assess and optimize CNB specimen collection methods to improve sample adequacy for molecular testing in NSCLC.
Methods
A total of 546 CNB specimens from NSCLC patients were collected using three different techniques: (I) steam sterilization indicator cards; (II) polypropylene microporous membranes (PPMM) pieces for tissue adherence; and (III) direct rinsing of biopsy needles with sterilized buffer. Cell pellets were harvested from the residual fixative medium after CNB processing. Tumor cellularity was evaluated in formalin-fixed paraffin-embedded (FFPE) tissues and cell pellet samples from each method.
Results
Adequacy rates for molecular testing were 86.4% in FFPE samples and 92.3% in cell pellet samples. Incorporating cell pellet analysis increased overall molecular testing adequacy to 95.2%. Among the evaluated techniques, the steam sterilization indicator cards cohort yielded the lowest adequacy rates for both FFPE and cell pellet samples. The PPMM technique achieved the highest adequacy for FFPE tissues, while the direct needle rinsing technique provided the highest DNA yields in cell pellet samples. Further, cell pellets demonstrated increased sensitivity for detecting actionable mutations compared to plasma-based liquid biopsies.
Conclusions
The use of PPMMs significantly enhances CNB specimen quality and molecular testing adequacy in NSCLC. Routine collection of cell pellets from the residual fixative medium is recommended to maximize diagnostic effectiveness and improve clinical decision-making.
Keywords: Non-small cell lung cancer (NSCLC), core needle biopsy (CNB), cell pellet, fixative medium, molecular testing
Highlight box.
Key findings
• Cell pellets obtained from the residual fixative medium of core needle biopsy (CNB) specimens improved molecular testing adequacy.
• The use of steam sterilization indicator cards to adhere core needle-acquired tissues is not recommended.
• Using microporous polypropylene membranes (PPMMs) to adhere tissue improved the sample adequacy rate.
What is known and what is new?
• Our previous study demonstrated that the residual fixative medium from transbronchial lung biopsy (TBLB) specimens, which is typically discarded as medical waste, often contains exfoliated tumor cells. Harvesting cell pellets from the residual medium significantly improved the molecular testing adequacy.
• This study compared three CNB specimen collection techniques and demonstrates that using steam sterilization indicator cards to adhere tissues from biopsy needles resulted in the shedding of coarse paper fibers into the fixative medium, leading to uneven staining and obscured cellular details in the recovered cell pellets. In contrast, the application of PPMMs reduced fiber contamination and improved staining quality and cellular morphology.
What is the implication, and what should change now?
• This research highlights the utility off cell pellet samples derived from CNB specimens in ancillary molecular testing. The use of steam sterilization indicator cards as adhesion materials should be avoided, as they compromise the adequacy of both FFPE tissues and cell pellet recovery. Pre-cut PPMMs are recommended as a more reliable alternative.
Introduction
Core needle biopsy (CNB), guided by various imaging modalities, is a safe and effective technique for obtaining tumor tissues for histological diagnosis and molecular analysis for non-small cell lung cancer (NSCLC) (1). However, the quality and cellular yield of CNB specimens can vary considerably, depending on whether the biopsy needle penetrates viable, necrotic, or fibrous tissues. Current pathology protocols typically embed only visible portions of the CNB specimens into formalin-fixed paraffin-embedded (FFPE) tissue blocks, discarding the residual fixative medium that may contain valuable cellular materials. Approximately 10–20% of CNB-derived FFPE samples exhibit insufficient cellularity or nucleic acids, creating substantial challenges for molecular testing on NSCLC patients (2-4). Although liquid biopsy has been proposed as a complementary method, its clinical utility remains limited due to a relatively high false-negative rate of approximately 30% (5,6). Therefore, alternative strategies are urgently required to enhance the adequacy of CNB samples for molecular testing.
Our previous study demonstrated that the fixative medium from transbronchial lung biopsy (TBLB) specimens, which is typically discarded as medical waste, frequently contains exfoliated tumor cells due to the fragile nature of lung cancer tissues (7). Centrifugation of the residual fixative medium after tissue block preparation allows the recovery of these tumor cells, providing an important supplementary source for molecular testing. The study demonstrated that harvesting cell pellets from the residual fixative medium of TBLB specimens significantly increased the molecular testing adequacy rate from 78.4% to 94.8%. Building upon these promising results, this study aimed to assess whether a similar approach could enhance the adequacy of CNB samples. A significant limitation of current CNB procedures is the lack of standardized materials for tissue collection from biopsy needles. Clinicians commonly rely on readily available materials such as gauze or steam sterilization indicator cards included in sterilization packages to verify adequate sterilization temperatures, potentially compromising specimen integrity due to tissue embedding and contamination caused by material disintegration.
To overcome these limitations, two alternative CNB specimen collection techniques were introduced: (I) using sterilized, pre-cut polypropylene microporous membranes (PPMMs) instead of steam sterilization indicator cards, which provide enhanced durability, minimal fiber shedding, and improved tissue recovery; and (II) direct rinsing of biopsy needles in sterilized neutral buffer solution without adherence materials. This study systematically evaluates these specimen collection methods to determine the optimal approach for improving sample adequacy in molecular testing. We present this article in accordance with the STROBE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-521/rc).
Methods
Specimen selection and data collection
This retrospective study included 546 CNB specimens from NSCLC patients at the First Affiliated Hospital of Guangzhou Medical University between July 2021 and December 2023. Specimens were allocated into three groups based on different collection techniques: steam sterilization indicator cards (CNB1, n=174), PPMMs (CNB2, n=213), and direct needle rinsing (CNB3, n=159). The procedures for these three techniques are illustrated in Figure 1. Clinicopathological data, including age, gender, histological subtype, and tumor-node-metastasis (TNM) stage, were retrieved from electronic medical records. The study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (approval No. 2021-70, issued on August 16, 2021) and individual consent for this retrospective analysis was waived. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.
Figure 1.
Schematic illustration of the three core needle biopsy specimen collection techniques. (A) Steam sterilization indicator cards were used to adhere tissue from the biopsy needle. (B) Polypropylene microporous membranes pieces were used to adhere tissue from the biopsy needle. (C) The biopsy needle was rinsed in a sterilized neutral buffer to facilitate the release of the tissue strips. (D) Procedure for collecting and preparing cell pellet smears from the residual fixative medium. PBS, phosphate-buffered saline.
Details of three different specimen collection techniques
In the CNB1 group, steam sterilization indicator cards were used to adhere tissue (2–4 needle cores per biopsy), which were subsequently immersed in specimen bottles containing 10% formalin. Steam sterilization indicator cards are typically included in sterilization packages to verify adequate sterilization temperatures. In the pathology laboratory, visible tissues were retrieved carefully, and wrapped in embedding paper for FFPE block preparation, while the residual fixative medium was retained for further processing. The procedures for CNB1 are illustrated in Figure 1A.
For the CNB2 group, sterilized PPMMs were used as an alternative adherence material. Large PPMMs (27 cm in diameter) were first trimmed into smaller pieces (1.5 cm × 2 cm), packaged in single packs (six pieces per pack), and high-pressure sterilized. These PPMMs facilitated tissue retrieval and were processed similarly to CNB1. The procedures for CNB2 are illustrated in Figure 1B.
For the CNB3 group, no adherence materials were used. A specimen bottle containing 9 mL of sterilized phosphate-buffered saline (PBS) and a separate tube containing 1 mL of 100% formalin solution were prepared initially. During the CNB procedure, biopsy needles were directly rinsed into sterilized PBS buffer to release the adhered tissue, followed by the addition of 1 mL of 100% formalin solution to achieve a final formalin concentration of 10%. Subsequent laboratory processing steps matched those of CNB1 and CNB2. The procedures for CNB3 are illustrated in Figure 1C.
Cell pellet preparation and DNA extraction
After histological confirmation of NSCLC, the residual fixative medium was centrifuged and washed twice with PBS, yielding a 250 µL cell pellet suspension. From this, 50 µL was used for cytological smear preparation, stained with hematoxylin and eosin (H&E) for cellularity assessment. The remaining 200 µL was centrifuged, the supernatant discarded, and the pellet stored at −80 ℃ for DNA extraction. Details of the processing steps are illustrated in Figure 1D. DNA extraction was performed using the QIAGEN DNeasy Blood and Tissue Kit (Qiagen, Düssel dorf, Germany), quantified with a Qubit fluorometer (Life Technologies, CA, USA), and adequacy thresholds were set as >10 ng for polymerase chain reaction (PCR) and >30 ng for next-generation sequencing (NGS).
Tumor cellularity assessment
Tumor cellularity was evaluated microscopically in H&E-stained FFPE sections and cell pellet smears by two independent pathologists (J.J. and Y.L.), with discrepancies exceeding 10% resolved by consensus. FFPE tissue and cell pellets were classified into three cellularity levels: (I) low cellularity, <200 tumor cells or <10%, inadequate for molecular analysis; (II) moderate cellularity, 200–1,000 tumor cells and ≥10%, adequate for molecular analysis; and (III) high cellularity, >1,000 tumor cells and ≥10%, highly suitable for molecular analysis. Cell pellet smears were evaluated using one-fifth of the total pellet volume.
Biomarker detection
To evaluate the effectiveness of detecting guideline-recommended biomarkers, a total of 60 cell pellet samples were subjected to NGS analysis. These included 15 cases with known positive NGS results from FFPE tissue and 45 cases that required plasma-based NGS due to insufficient FFPE material. NGS was performed in a laboratory certified under the Clinical Laboratory Improvement Amendments and accredited by the College of American Pathologists, using a 1,021-gene panel covering key oncogenic drivers and actionable targets commonly implicated in NSCLC.
Statistical analysis
Pearson’s Chi-squared test was used for pairwise comparisons among the three groups. Bonferroni correction was applied to adjust for multiple comparisons, setting the significance threshold at P<0.0167. Statistical analyses were conducted using SPSS version 25.0 (IBM Corp.).
Results
Clinicopathological characteristics of patients
This study included 546 NSCLC patients who underwent CNB between July 2021 and December 2023. The detailed clinicopathological characteristics of patients across the three groups are summarized in Table 1.
Table 1. Clinicopathological characteristics of 546 cases.
| Patient characteristics | CNB1 (n=174) | CNB2 (n=213) | CNB3 (n=159) |
|---|---|---|---|
| Gender | |||
| Male | 102 (58.6) | 111 (52.1) | 94 (59.1) |
| Female | 72 (41.4) | 102 (47.9) | 65 (40.9) |
| Age (years) | 66 [32–90] | 62 [26–84] | 66 [35–85] |
| Histology | |||
| Adenocarcinoma | 106 (60.9) | 156 (73.2) | 107 (67.3) |
| Squamous cell carcinoma | 41 (23.6) | 33 (15.5) | 30 (18.9) |
| NSCLC not otherwise specified | 27 (15.5) | 24 (11.3) | 22 (13.8) |
| Lesion size (cm) | 3.4±1.8 | 3.2±1.7 | 3.5±2.0 |
| TNM stage | |||
| I | 4 (2.3) | 9 (4.2) | 2 (1.3) |
| II | 5 (2.9) | 13 (6.1) | 7 (4.4) |
| III | 44 (25.3) | 49 (23.0) | 38 (23.9) |
| IV | 121 (69.5) | 142 (66.7) | 112 (70.4) |
Data were presented as n (%), median [range], or mean ± standard deviation. CNB1: samples collected with steam sterilization indicator cards. CNB2: samples collected with polypropylene microporous membranes. CNB3: samples collected by rinsing the biopsy needle. CNB, core needle biopsy; NSCLC, non-small cell lung cancer; TNM, tumor-node-metastasis.
Microscopic characteristics of FFPE tissue sections and cell pellet smears
Microscopic evaluations of FFPE tissue sections and cell pellet smears revealed that, in most cases, cell pellet smears exhibited excellent tumor cellularity, indicating successful tumor cell recovery from the residual fixative medium. Generally, cell pellet smears reflected the cellularity of FFPE tissue sections. However, in cases with fragile tumor tissues or necrosis, notable differences in cellularity were observed between FFPE sections and cell pellet smears. While FFPE sections in these cases contained limited tumor cells (fewer than 100 cells or <1%), cell pellet smears from the same sample often exhibited abundant tumor cells. Representative cases demonstrating these differences are shown in Figure 2.
Figure 2.
Histologic characterization of FFPE tissue sections and cytomorphology of cell pellet smears obtained from the residual fixative medium. (A, upper panel) A biopsy case with extensive necrosis, resulting in small tissue fragments (original magnification: left, 40×; right, 400×). (B, upper panel) A biopsy case with prominent desmoplastic stroma and sparse tumor cells in the FFPE section (original magnification: left, 40×; right, 400×). (A,B, lower panel) H&E-stained direct smears of cell pellets derived from the same cases, showing tumor cell enrichment on the representative smeared from a 50-µL cell suspension (original magnification: left, 40×; right, 400×). FFPE, formalin-fixed paraffin-embedded; H&E, hematoxylin and eosin.
Furthermore, in the CNB1 group, where steam sterilization indicator cards were used, cell pellet smears contained numerous thick and long paper fibers, leading to uneven staining and obscured cellular details. However, the CNB2 group, which used PPMMs, exhibited minimal fiber contamination, allowing for better visualization and easier assessment of cellular characteristics. The CNB3 group, utilizing direct needle rinsing, yielded smears with a clean background and excellent staining quality, facilitating optimal cellular evaluation. Representative cases of cell pellet smears obtained from different groups are shown in Figure 3.
Figure 3.
Microscopic characteristics of cell pellet smears from the residual fixative medium using three different specimen collection techniques (hematoxylin and eosin staining). (A) The sample obtained using steam sterilization indicator cards for tissue adherence. (B) The sample obtained using polypropylene microporous membranes pieces for tissue adherence. (C) The sample obtained by rinsing the biopsy needle with a sterilized neutral buffer.
Tumor cellularity and sample adequacy rates
Tumor cellularity was assessed in matched FFPE tissue sections and cell pellet samples across the three groups (Table 2). The overall adequacy rates for molecular testing were 86.4% (472/546) for FFPE tissue and 92.3% (504/546) for cell pellet samples. Among the 74 cases with inadequate FFPE tissue, 48 had adequate cell pellet samples, leading to an overall increased adequacy rate of 95.2% when cell pellets were included (Table 3). Pairwise comparisons showed significant differences in combined adequacy rates between CNB1 and CNB2 (adjusted P=0.003) as well as CNB1 and CNB3 (adjusted P=0.005). CNB1 had the lowest FFPE adequacy rate, while CNB2 had the highest. CNB3 had the highest adequacy rate for cell pellet samples, though its FFPE adequacy rate was comparable to CNB1.
Table 2. The cellularity of paired FFPE tissue and cell pellet samples.
| Group | FFPE tissue | Cell pellet | Total | ||
|---|---|---|---|---|---|
| Low | Moderate | High | |||
| CNB1 (n=174) | Low | 18 (10.3) | 7 (4.0) | 4 (2.3) | 29 (16.7) |
| Moderate | 6 (3.4) | 19 (10.9) | 10 (5.7) | 35 (20.1) | |
| High | 2 (1.1) | 36 (20.7) | 72 (41.4) | 110 (63.2) | |
| Total | 26 (14.9) | 62 (35.6) | 86 (49.4) | 174 (100.0) | |
| CNB2 (n=213) | Low | 5 (2.3) | 10 (4.7) | 6 (2.8) | 21 (9.9) |
| Moderate | 2 (0.9) | 14 (6.6) | 33 (15.5) | 49 (23.0) | |
| High | 6 (2.8) | 45 (21.1) | 92 (43.2) | 143 (67.1) | |
| Total | 13 (6.1) | 69 (32.4) | 131 (61.5) | 213 (100.0) | |
| CNB3 (n=159) | Low | 3 (1.9) | 3 (1.9) | 18 (11.3) | 24 (15.1) |
| Moderate | 0 (0.0) | 9 (5.7) | 24 (15.1) | 33 (20.8) | |
| High | 0 (0.0) | 24 (15.1) | 78 (49.1) | 102 (64.2) | |
| Total | 3 (1.9) | 36 (22.6) | 120 (75.5) | 159 (100.0) | |
CNB1: samples collected with steam sterilization indicator cards. CNB2: samples collected with polypropylene microporous membranes. CNB3: samples collected by rinsing the biopsy needle. CNB, core needle biopsy; FFPE, formalin-fixed paraffin-embedded.
Table 3. The adequacy rate of paired FFPE tissue and cell pellet samples.
| Group | Total | Adequacy rate | Adequacy increase by adding cell pellet | Combined adequacy rate | Adjusted P value |
|
|---|---|---|---|---|---|---|
| FFPE tissue | Cell pellet | |||||
| CNB1 | 174 | 83.3% (145/174) | 85.1% (148/174) | 6.3% (11/174) | 89.7% (156/174) | CNB1 vs. CNB2: 0.003; CNB1 vs. CNB3: 0.005; CNB2 vs. CNB3: >0.99 |
| CNB2 | 213 | 90.1% (192/213) | 93.9% (200/213) | 7.5% (16/213) | 97.7% (208/213) | |
| CNB3 | 159 | 84.9% (135/159) | 98.1% (156/159) | 13.2% (21/159) | 98.1% (156/159) | |
| Total | 546 | 86.4% (472/546) | 92.3% (504/546) | 8.8% (48/546) | 95.2% (520/546) | |
CNB1: samples collected with steam sterilization indicator cards. CNB2: samples collected with polypropylene microporous membranes. CNB3: samples collected by rinsing the biopsy needle. CNB, core needle biopsy; FFPE, formalin-fixed paraffin-embedded.
DNA yield
The median DNA yield from 546 cell pellet samples was 249.0 ng (range, 0.0–2,824.0 ng). A total of 97.3% of samples yielded ≥10 ng of DNA, and 79.9% exceeded the NGS adequacy threshold of 30 ng. DNA yield was lowest in CNB1, moderate in CNB2, and highest in CNB3 (Table 4).
Table 4. Comparison of DNA yields from 546 cell pellet samples across three groups.
| Group | Total | DNA yield | ||
|---|---|---|---|---|
| ≥10 ng | ≥30 ng | Range (ng) | ||
| CNB1 | 174 | 92.5% (161/174) | 56.3% (98/174) | 0.0–1,737.0 |
| CNB2 | 213 | 99.1% (211/213) | 87.3% (186/213) | 5.6–2,288.0 |
| CNB3 | 159 | 100.0% (159/159) | 95.6% (152/159) | 16.0–2,824.0 |
| Total | 546 | 97.3% (531/546) | 79.9% (436/546) | 0.0–2,824.0 |
CNB1: samples collected with steam sterilization indicator cards. CNB2: samples collected with polypropylene microporous membranes. CNB3: samples collected by rinsing the biopsy needle. CNB, core needle biopsy.
Molecular testing effectiveness
Among the 15 cases with known positive NGS results from FFPE tissue, identical somatic mutations were detected in their corresponding cell pellet samples, with similar allele frequencies. In 45 cases requiring plasma-based NGS due to insufficient FFPE tissue, all plasma samples were sequenced successfully; however, eight cases showed no genomic alterations, suggesting a lack of detectable circulating tumor DNA (ctDNA). Of these 45 cases, 18 had negative results in both plasma and cell pellet samples, while 22 had consistent positive results. Four cases had targetable mutations detected only in cell pellet samples, highlighting their increased sensitivity over plasma for molecular testing. The four cases with targetable mutations detected only in cell pellet samples were among the eight cases that lacked ctDNA shedding in plasma. The genetic detection profiles of these cell pellet samples are presented in Tables S1,S2.
Discussion
This study demonstrated that CNB for NSCLC yielded a high adequacy rate of FFPE tissue samples (86.4%), surpassing the 78.4% reported in our previous study for TBLB samples (7). Furthermore, our results confirmed that collecting cell pellets from the residual fixative medium significantly improved the adequacy of molecular testing, achieving an overall adequacy rate of 95.2%. These findings highlight the potential to utilize residual materials to mitigate the limitations inherent in small biopsy procedures, particularly for molecular analyses where tissue sufficiency is critical.
The selection of specimen collection techniques significantly influenced the adequacy rates of both FFPE tissue samples and cell pellets derived from the residual fixative medium. Among the techniques evaluated, the use of steam sterilization indicator cards was associated with the lowest adequacy rates. When wet, these indicator cards became soft and fibrous, tightly adhering to biopsy tissues, leading to substantial tissue loss during the retrieval process. Similarly, the excessive shedding of coarse and elongated paper fibers into the residual fixative medium adversely affected the quality of cell pellet smears, causing uneven staining and obscuring cellular morphology. Furthermore, these paper fibers substantially compromise DNA extraction efficiency, resulting in reduced DNA yield from cell pellet samples (8). Thus, the use of steam sterilization indicator cards is not recommended due to these significant drawbacks.
The use of sterilized PPMMs significantly improved the adequacy rate of FFPE tissue samples by facilitating tissue separation during forceps scraping. Unlike steam sterilization indicator cards, PPMMs maintained structural integrity throughout tissue processing, minimized fiber contamination, and facilitated effective tissue retrieval. This resulted in clearer cell morphology on smears and better overall specimen quality. Considering the critical role of high-quality FFPE tissues in routine histopathological diagnosis and subsequent biomarker analyses performed through immunohistochemistry or fluorescence in situ hybridization, it is highly recommended to adopt PPMMs as the supporting material for CNB specimen collection.
Interestingly, direct needle rinsing with sterilized neutral buffer significantly improved cell pellet adequacy but did not confer similar benefits to FFPE tissue samples. This contrasts with our previous findings from TBLB procedures, where rinsing improved adequacy in both FFPE and cell pellet samples (7). We hypothesize that this discrepancy stems from morphological differences between CNB and TBLB specimens. TBLB typically yields small, cohesive tissue fragments that remain stable during processing without supporting materials. Conversely, CNB specimens often comprise elongated, delicate tissue strips prone to fragmentation or cell dispersal upon agitation. Consequently, while direct rinsing effectively releases tissues from the needle, it may also lead to fragmentation and loss of integrity in CNB specimens. Moreover, the need to maintain needle sharpness by preventing contact with the container’s sidewall or bottom further complicates its clinical implementation. Therefore, employing robust, low-contamination materials like PPMMs may provide a safer and more effective alternative.
Our comparative evaluation of molecular biomarker detection across FFPE, plasma, and cell pellet samples further underscores the clinical utility of cell pellets from CNB specimens. Although plasma-based liquid biopsy is increasingly utilized, it demonstrated lower sensitivity due to inherent limitations in detecting ctDNA (9). Several cases lacked detectable ctDNA, resulting in false-negative outcomes. By contrast, cell pellets exhibited increased sensitivity, detecting actionable mutations missed in plasma samples, thereby enhancing clinical decision-making accuracy. Hence, residual cell pellet samples should be prioritized for molecular analysis when FFPE materials are inadequate.
Our study, which focuses on collecting cell pellets from residual fixative medium, is based on the principle of exfoliative cytology in small biopsy specimens. Previous studies by Rosell et al. and Goyal et al. introduced methods to obtain exfoliative cytology from flexible bronchoscopy biopsies (10,11). In their protocols, bronchial biopsies were initially immersed in a balanced salt solution. Upon completion of the bronchoscopy procedure, visible tissue fragments were removed and transferred into formalin for histopathological evaluation, while the rinse fluid was submitted for cytological assessment. The diagnostic accuracy of cytology from rinse fluid was compared with that of histopathological sections for the detection of pulmonary neoplasms. In the study by Rosell et al., a diagnosis of malignancy was established based solely on rinse fluid cytology in 4.8% of cases, thus increasing the overall diagnostic yield (10). On the other hand, Goyal et al. reported no instances in which malignancy was identified by cytology alone when the histological diagnosis was benign (11). Neither study evaluated the molecular adequacy of the cytological material derived from rinse fluid. L’Imperio V et al. described a liquid-based shaking technique capable of retrieving viable cells suitable for flow cytometry and fluorescence in situ hybridization, as well as free subcellular material such as DNA for molecular analysis (12). In their method, interventional pathologists performed a gentle mechanical disaggregation in saline solution within seconds of completing a CNB. The intact tissue specimen was then processed as a FFPE block for histological and immunohistochemical analysis. However, this study does not involve immediate sample collection (saline rinse fluid) during the biopsy procedure. Instead, it focuses on harvesting the residual fixative medium after the specimen has been transported to the pathology department. Mechanical agitation during transportation and handling is presumed to promote further exfoliation of tumor cells into the fixative medium. It is, therefore, hypothesized that the residual fixative medium may contain a comparable number of exfoliated cells to that recovered using the liquid-based shaking technique and a higher number than in the initial saline rinse fluid without shaking.
Lan et al. proposed an alternative method of collecting rinse fluid during CNB (13). Following placement of CNB specimens in 10% neutral buffered formaldehyde, the biopsy needle was flushed multiple times with a liquid-based preservative to obtain needle rinse fluid samples. These samples were processed for cytological examination. In their study, 3.2% (13/406) of cases were diagnosed as malignant based solely on cytological analysis of the needle rinse fluid. The combined use of cytological evaluation of the needle rinse fluid and histological assessment of the FFPE tissue resulted in improved diagnostic performance, reducing false-negative rates and facilitating the subtyping of non-small cell lung carcinoma. However, as with the previously mentioned studies, the molecular adequacy of the cytological material from the needle rinse fluid was not evaluated. Further investigation is warranted to assess the potential utility of combining needle rinse fluid samples with residual fixative medium to improve the performance of ancillary testing in lung cancer diagnostics.
To optimize CNB specimen processing, we recommend routine collection of cell pellets from residual fixative medium, particularly when biopsy tissues exhibit limited cellularity, require decalcification, or are collected using thinner gauge needles (20 or 22 gauge). Establishing standardized protocols for the systematic handling of residual materials from small biopsies, including thoracic procedures, will enhance tissue utilization efficiency, reduce wastage, and maximize molecular diagnostic yield.
Despite these promising findings, there are limitations in this study. The most notable is that the three specimen collection techniques were not compared using matched samples from the same patients, potentially introducing confounding variables. Furthermore, variability in operator experience and technical differences during biopsy procedures may have influenced specimen adequacy, highlighting the need for additional standardized trials to comprehensively validate these findings.
Conclusions
This study highlights the significant impact of CNB specimen collection techniques on tissue integrity and molecular testing adequacy. The use of high-quality adherence materials, such as PPMMs, substantially enhances sample preservation and improves molecular diagnostic yield compared with steam sterilization indicator cards. Moreover, routine collection of cell pellets from residual fixative medium represents a practical and effective strategy to maximize molecular testing adequacy, particularly in cases where FFPE samples have insufficient cellularity. Implementing standardized protocols for CNB specimen processing, including the adoption of PPMMs and systematic cell pellet collection, can improve molecular testing success and optimize tissue utilization.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (approval No. 2021-70), and individual consent for this retrospective analysis was waived.
Footnotes
Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-521/rc
Funding: This work was supported by the National Natural Sciences Foundation of China (grant No. 81772814), the Open Project of the State Key Laboratory of Respiratory Disease (grant No. SKLRD-OP-202205), and the Science and Technology Program of Guangzhou, China (grant No. 202201020433).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-521/coif). J.H. serves as an Executive Editor-in-Chief of Journal of Thoracic Disease. All authors report funding from the National Natural Sciences Foundation of China (grant No. 81772814), the Open Project of the State Key Laboratory of Respiratory Disease (grant No. SKLRD-OP-202205), and the Science and Technology Program of Guangzhou, China (grant No. 202201020433). The authors have no other conflicts of interest to declare.
Data Sharing Statement
Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-521/dss
References
- 1.Tam AL, Lim HJ, Wistuba II, et al. Image-Guided Biopsy in the Era of Personalized Cancer Care: Proceedings from the Society of Interventional Radiology Research Consensus Panel. J Vasc Interv Radiol 2016;27:8-19. 10.1016/j.jvir.2015.10.019 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Poudel B, Desman J, Aihara G, et al. Adequacy of samples obtained via percutaneous core-needle rebiopsy for EGFR T790M molecular analysis in patients with non-small cell lung cancer following acquired resistance to first-line therapy: A systematic review and meta-analysis. Cancer Treat Res Commun 2021;29:100470. 10.1016/j.ctarc.2021.100470 [DOI] [PubMed] [Google Scholar]
- 3.Nam BD, Yoon SH, Hong H, et al. Tissue Adequacy and Safety of Percutaneous Transthoracic Needle Biopsy for Molecular Analysis in Non-Small Cell Lung Cancer: A Systematic Review and Meta-analysis. Korean J Radiol 2021;22:2082-93. 10.3348/kjr.2021.0244 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Faber E, Grosu H, Sabir S, et al. Adequacy of small biopsy and cytology specimens for comprehensive genomic profiling of patients with non-small-cell lung cancer to determine eligibility for immune checkpoint inhibitor and targeted therapy. J Clin Pathol 2022;75:612-9. 10.1136/jclinpath-2021-207597 [DOI] [PubMed] [Google Scholar]
- 5.Schwartzberg LS, Horinouchi H, Chan D, et al. Liquid biopsy mutation panel for non-small cell lung cancer: analytical validation and clinical concordance. NPJ Precis Oncol 2020;4:15. 10.1038/s41698-020-0118-x [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Lin Z, Li Y, Tang S, et al. Comparative analysis of genomic profiles between tissue-based and plasma-based next-generation sequencing in patients with non-small cell lung cancer. Lung Cancer 2023;182:107282. 10.1016/j.lungcan.2023.107282 [DOI] [PubMed] [Google Scholar]
- 7.Jiang J, Tang C, Li Y, et al. Cell pellet from fixative medium of transbronchial lung biopsy sample improves lung cancer ancillary test. Lung Cancer 2023;175:9-16. 10.1016/j.lungcan.2022.11.012 [DOI] [PubMed] [Google Scholar]
- 8.Ye X, Lei B. Improved DNA extraction on bamboo paper and cotton is tightly correlated with their crystallinity and hygroscopicity. PLoS One 2022;17:e0277138. 10.1371/journal.pone.0277138 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Mack PC, Banks KC, Espenschied CR, et al. Spectrum of driver mutations and clinical impact of circulating tumor DNA analysis in non-small cell lung cancer: Analysis of over 8000 cases. Cancer 2020;126:3219-28. 10.1002/cncr.32876 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Rosell A, Monsó E, Lores L, et al. Cytology of bronchial biopsy rinse fluid to improve the diagnostic yield for lung cancer. Eur Respir J 1998;12:1415-8. 10.1183/09031936.98.12061415 [DOI] [PubMed] [Google Scholar]
- 11.Goyal S, Mohan H, Uma Handa, et al. Rinse fluid and imprint smear cytology of bronchial biopsies in diagnosis of lung tumors. Diagn Cytopathol 2012;40:98-103. 10.1002/dc.21502 [DOI] [PubMed] [Google Scholar]
- 12.L'Imperio V, Corral L, Pagni F. Liquid-based shaking of core needle biopsy samples for the molecular characterisation of tumours: A fallback in cytopathology. Cytopathology 2021;32:283-6. 10.1111/cyt.12925 [DOI] [PubMed] [Google Scholar]
- 13.Lan Z, Zhang X, Ma X, et al. Utility of liquid-based cytology on residual needle rinses collected from core needle biopsy for lung nodule diagnosis. Cancer Med 2021;10:3919-27. 10.1002/cam4.3949 [DOI] [PMC free article] [PubMed] [Google Scholar]