Clinical utility of rapid on-site evaluation of brush cytology during bronchoscopy using endobronchial ultrasound with a guide sheath

Clinical utility of rapid on-site evaluation of brush cytology during bronchoscopy using endobronchial ultrasound with a guide sheath | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Clinical utility of rapid on-site evaluation of brush cytology during bronchoscopy using endobronchial ultrasound with a guide sheath Kazuhiro Nishiyama, Kei Morikawa, Shotaro Kaneko, Makoto Nishida, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4497598/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Previous studies have shown that rapid on-site evaluation (ROSE) improves the diagnostic yield of bronchoscopy using endobronchial ultrasonography with guided sheath (EBUS-GS) for peripheral pulmonary lesions (PPL). There are many reports on the ROSE of imprint cytology of forceps biopsy; however, there are few reports on the ROSE of brush cytology. We investigated the utility of ROSE in brushing PPL specimens during bronchoscopy. We retrospectively analyzed 214 patients who underwent bronchoscopy with EBUS-GS for PPL. Sensitivity to bronchoscopy was significantly higher in the ROSE group than in the non-ROSE group (96.8% vs. 83.3%, P = 0.002), wherein the sensitivity to brushing also significantly improved (92.9% vs. 75.0%, P < 0.001). Conversion of ROSE results from negative to positive was observed in 79.5% of first-time ROSE-negative cases. We also found that brush specimens containing high tumor cell counts increase with repeated ROSE (42.1–69.0%). This study suggests that ROSE of brush cytology improves bronchoscopy sensitivity and increases the collected tumor cell counts. Biological sciences/Cancer/Lung cancer Health sciences/Medical research Health sciences/Diseases/Respiratory tract diseases Rapid on-site evaluation endobronchial ultrasound with a guided sheath peripheral lung lesions brushing Figures Figure 1 Figure 2 Introduction Bronchoscopy is a pathological diagnostic method for peripheral pulmonary lesions (PPL). In bronchoscopic specimen collection from PPL, it is important to know the approach to the lesion and the specimen collection method. Various techniques for approaching lesions have been examined, including electromagnetic navigation bronchoscopy, virtual bronchoscopic navigation, radial endobronchial ultrasound (R-EBUS), ultrathin bronchoscopy, and guide sheath (GS) [ 1 – 6 ]. Bronchoscopy using endobronchial ultrasonography with a GS (EBUS-GS) is commonly performed for PPL, while forceps biopsy is a common specimen collection method in bronchoscopy for PPL. Other methods include brushing, washing, and cryobiopsy, which are often combined with forceps biopsies [ 7 ]. In recent years, with the advent of molecular-targeted drugs and immune checkpoint inhibitors for lung cancer therapy, genetic mutation and programmed cell death ligand-1 (PD-L1) testing of bronchoscopic specimens has become increasingly important. Next-generation sequencing (NGS), which can detect multiple genetic mutations simultaneously, is often used in genetic mutation testing [ 8 ]. Conventional NGS testing requires a certain tissue size and tumor content [ 9 ]. Against this background, obtaining large tissue specimens with high tumor cell content for bronchoscopy has been considered with the spread of cryobiopsy [ 10 – 12 ]. However, it is now possible to perform NGS on cytological specimens [ 13 – 15 ]. Furthermore, it has been suggested that the level of PD-L1 in cytological specimens correlates strongly with that in tissue specimens [ 16 – 18 ]. Therefore, cytological specimens used for bronchoscopy are becoming increasingly important. Brushing has been reported to have a high diagnostic accuracy [ 19 ]; hence, brushing has received renewed attention in recent years. Rapid on-site evaluation (ROSE) is used to confirm tumor cells in cytological specimens, and its utility in bronchoscopy has been studied using endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), which suggested that ROSE improves the diagnostic yield of EBUS-TBNA and decreases the number of biopsies required [ 20 – 22 ]. The same has been reported for bronchoscopy of PPL [ 23 – 28 ]. When performing ROSE during bronchoscopy using EBUS-GS for PPL, two types of cytology specimens must be evaluated: imprint cytology of forceps biopsy and brush cytology. There are many reports on ROSE of imprint cytology of forceps biopsy [ 23 – 26 ], but there are few reports on brush cytology [ 27 , 28 ]. We investigated the improvement in diagnostic yield and changes in ROSE results when ROSE was performed on brush cytology during bronchoscopy using EBUS-GS for PPL. Methods Patients This retrospective study was approved by the Ethics Committee of the St. Marianna University School of Medicine on October 26, 2023, with approval number 6224. The data used in this study were anonymized; therefore, the requirement for informed consent was waived by the Ethics Committee of the St. Marianna University School of Medicine and the study was compliant with The Declaration of Helsinki. We analyzed patients who underwent bronchoscopy for PPL at St. Marianna University Hospital between October 2020 and October 2022. PPL was defined as a nodule surrounded by the pulmonary parenchyma. Patients who underwent endobronchial biopsy or did not undergo EBUS-GS were excluded. After a one-year study of medical records following bronchoscopy, the final diagnosis was defined as malignant for cases with a pathologic diagnosis of malignancy and benign for other cases. Biopsy procedure Prior to bronchoscopy, a high-resolution chest computed tomography (CT) scanner was used to identify the bronchi that led to lesions. The order of bronchoscopy was randomly determined. ROSE was performed for all bronchoscopies where cytologists could participate. ROSE was not performed when cytologists were unavailable. Bronchoscopy was performed using a combination of a bronchoscope (BF-1T260 or BF-P260F; Olympus, Tokyo, Japan), an R-EBUS probe (20 MHz mechanical radial type, UM-S20-17S; Olympus), and a GS kit (K-201 or K-203). During bronchoscopy, patients received intravenous midazolam and fentanyl for mild sedation and analgesia, and the bronchoscope was inserted orally or nasally. We inserted the GS with an R-EBUS probe through the working channel of the bronchoscope and guided it to the lesion site. EBUS images were classified into three categories: "within," "adjacent to," and "invisible." After reviewing the fluoroscopic and EBUS images, we removed the R-EBUS probe and performed a brushing and forceps biopsy. In the ROSE group, the ROSE results of the glass slides produced by brushing were immediately communicated to the examiner. If the EBUS image was "adjacent to" or "invisible," we adjusted the position of the R-EBUS probe or changed the bronchus into which it was inserted to be as "within" as possible. The number of brushings, forceps biopsies, and changes in procedure were determined at the discretion of the examiner. Processing of specimens In the ROSE group, brushes were smeared onto four glass slides. One slide was rapidly air-dried and stained with Cyto-Quick stain (Cyto-Quick; Muto Pure Chemicals, Tokyo, Japan), whereas the other three slides were fixed in 95% alcohol. The Cyto-Quick-stained slide specimens were microscopically evaluated by a cytologist in the same bronchoscopy laboratory. Alcohol-fixed slides were Papanicolaou-stained after bronchoscopy and microscopically evaluated by a cytologist. In the non-ROSE group, the materials from the brush were smeared onto two glass slides and fixed in 95% alcohol. Similar to the ROSE group, they were Papanicolaou-stained and evaluated after bronchoscopy. In both groups, forceps biopsy specimens were placed in 10% formalin. The sections were stained with hematoxylin and eosin after bronchoscopy for histological evaluation. Cytological specimen diagnosis Cytological diagnosis was performed according to the World Health Organization reporting system for lung cytopathology [ 29 , 30 ]. Lung cytopathology specimens were classified into five categories: insufficient/inadequate/non-diagnostic, benign, atypical, suspicious for malignancy, malignant. Among the five categories of lung cytopathological specimen types, those suspicious for malignancy and diagnosed as malignant were defined as positive, while all others were defined as negative. The cytological diagnosis and evaluation were confirmed by several pathologists and cytologists at our hospital. To evaluate the tumor cell counts, the ROSE results were classified into four categories: no malignant findings (class 1), atypical cells or suspected malignant cells (class 2), sufficient malignant cells (class 3), and many malignant cells (class 4). Sufficient malignant cells were based on the Bethesda system in thyroid cytopathology, with a minimum of 6–7 clusters [ 31 ]. Classes 1 and 2 were defined as ROSE-negative, while classes 3 and 4 were ROSE-positive. The ROSE classification was performed for each brushing. The highest class for each brushing was designated as the ROSE diagnosis. Statistical analysis Statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) [ 32 ]. Continuous variables were expressed as mean ± standard deviation (SD), and dichotomous variables were analyzed using Student's t-test. Categorical variables were expressed as percentages, and differences were compared using Fisher's exact test. When comparing the sensitivity between the two groups, we used multivariable logistic regression to control for the potentially confounding roles of lesion size, lesion location, number of specimens, and EBUS images. Statistical significance was set at P < 0.05. Results In total, 239 patients underwent bronchoscopy for PPL between October 2020 and October 2022. Of the 239 patients, 20 who underwent endobronchial biopsy and five who did not undergo EBUS-GS were excluded. Ultimately, 214 patients were included in the study. Among them, 129 patients with ROSE were enrolled in the ROSE group, and 85 patients without ROSE were included in the non-ROSE group (Fig. 1). Baseline characteristics of the patients and details of bronchoscopy using EBUS-GS are summarized in Table 1. The mean ± SD of lesion size was 38.34 ± 19.62 mm in the ROSE group and 33.58 ± 16.65 mm in the non-ROSE group, with no statistically significant difference. The lesion locations were divided into three groups: (1) right upper lobe and left upper lobe; (2) right middle lobe and lingula; and (3) right lower lobe and left lower lobe. The number of cases in each of these groups were 67 (51.9%), 14 (10.9%), and 48 (37.2%), respectively, in the ROSE group and 50 (58.8%), 7 (8.2%), and 28 (32.9%), respectively, in the non-ROSE group. Brushing was performed on all patients, and forceps biopsy was performed on approximately 90% of patients in both groups. The number of cases with "within" EBUS images was significantly more in the ROSE group than in the non-ROSE group (89.9% vs. 78.8%, P = 0.029). The final diagnosis of malignancy was significantly more in the ROSE group than in the non-ROSE group (89.9% vs. 84.7%, P < 0.001). Table 2 shows the sensitivity of bronchoscopy in both groups. The sensitivity of the bronchoscopy results was significantly higher in the ROSE group than in the non-ROSE group (96.8% vs. 83.3%, P = 0.002). The multivariate analysis also showed a statistically significant difference (P = 0.004). Using the sampling method, the sensitivity of brushing was significantly higher in the ROSE group than in the non-ROSE group (92.9% vs. 75.0%, P < 0.001), and the sensitivity of forceps biopsy was also higher in the ROSE group than in the non-ROSE group (87.6% vs. 78.8%, P = 0.137). Figure 2 shows the change in ROSE results for the 126 patients in the ROSE group whose final diagnosis was malignancy. Forty-four patients (34.9%) initially had ROSE- negative results. Of these, 35 (79.5%) converted to a ROSE-positive final diagnosis (Fig. 2b). Of the 35 patients whose final diagnosis was ROSE-positive, 20 (57.1%) had repeated cell sampling at the same site, 9 (25.7%) changed bronchial branches, and 6 (17.1%) changed the depth of the brushing position. On the other hand, 82 patients (65.1%) initially had ROSE-positive results. The initial ROSE result was class 3 in 29 patients (35.3%). In 16 (55.2%) of these patients, the final ROSE diagnosis improved to class 4 (Fig. 2c). The number of patients with a class 4 ROSE result increased with each successive ROSE, from 53 patients (42.1%) at the initial ROSE to 87 patients (69.0%) at the sixth ROSE. In addition, 30 patients (23.8%) were class 1 at the initial ROSE, but this number decreased to two patients (1.6%) at the sixth ROSE (Fig. 2d). Discussion In this study, the ROSE of brush cytology improved the sensitivity of bronchoscopy using EBUS-GS for PPL. Multiple ROSE procedures tended to change the ROSE diagnosis from negative to positive in some cases and increased tumor cell counts. The ROSE of brush cytology is useful in bronchoscopy for PPL. In addition, ROSE increased the sensitivity of brushing. The ROSE of a portion of a brushing specimen allows for the real-time evaluation of the brushing specimens. We believe that this real-time evaluation may have allowed the examiner to repeat the procedure until sufficient specimens were collected, thereby improving the brushing sensitivity. The rationale for this was that there were many cases wherein ROSE diagnosis changed from negative to positive during bronchoscopy. Of the 44 initially ROSE-negative cases, 35 (79.5%) converted to a ROSE-positive diagnosis. Of these, 20 tested positive after repeated brushing of the same site. Fifteen of 20 patients had adenocarcinomas. Kurimoto et al. reported that tumors for which specimen collection is not possible even with EBUS imaging are often adenocarcinomas [ 33 ]. Because adenocarcinomas are often covered by tracheal epithelium, specimen collection may not be possible without repeated brushing and destruction of the tracheal epithelium. Therefore, if adenocarcinoma is suspected based on chest CT imaging or tumor markers before bronchoscopy, it may be important to repeat the specimen collection at the same site. In contrast, the brushing position changed during bronchoscopy in 15 patients. Of these, nine changed the bronchial branches, and six changed the depth of the brushing position. Patients in whom the bronchial branch changed had another bronchial branch, which was a candidate for specimen collection based on the chest CT imaging prior to bronchoscopy. All patients for whom the depth of the brushing position was changed had lesions measuring > 30 mm. We believe that it is important to identify the lesion in detail prior to bronchoscopy, such as through chest CT imaging, and decide how the procedure should be modified according to the ROSE results. Tumor cell counts collected by brushing increased with multiple ROSE. As a result of multiple ROSE, more than half of the patients whose initial ROSE diagnosis was class 3 were reclassified as class 4 at the final ROSE diagnosis. In addition, the percentage of class 4 ROSE diagnoses increased and the percentage of class 1 ROSE diagnoses decreased with each repeated ROSE; thus, repeated ROSE allows the collection of more tumor cells. When performing NGS or PD-L1 testing on specimens collected during bronchoscopy, the higher the tumor cell count collected, the higher the success rate of the tests. Therefore, when NGS or PD-L1 testing is performed on cytology specimens, additional brushing may be necessary even if tumor cells are present in the cytology specimen. Further studies are needed to determine the tumor cell counts required for NGS or PD-L1 testing of cytology specimens. This study has some potential limitations. This was a single-center, retrospective study, which may have influenced the judgment of the examiner regarding the presence or absence of ROSE, and selection bias may exist. However, the ROSE group did not have significantly more small lesions or pulmonary apex cases that were difficult to diagnose using bronchoscopy; hence, the possible effect was likely to be small. Prospective randomized multicenter trials are required to avoid possible bias. In conclusion, bronchoscopy sensitivity was improved by performing ROSE on brush cytology during bronchoscopy using EBUS-GS for PPL. In some cases, the ROSE diagnosis changed from negative to positive during bronchoscopy, and the tumor cell counts increased. Declarations Competing interests The authors declare no competing interests. Author Contribution K.N. conceived the research concept and strategy, designed and supervised the study, analyzed the data and wrote the main part of manuscript. K.M. contributed the concept and revised the final manuscript. S.K., M.N., A.M., Y.N., Y.N., Y.S., and H.T. performed the bronchoscopic procedures at the participating center. N.S., C.O. processed and evaluated cytology specimens. N.O., J.K. performed the histological examination. H.K. reviewed titles, abstracts and full text articles. H.H., M.M. reviewed titles and abstracts and provided critical revision of the manuscript. All the authors discussed the results and implications, commented on the manuscript at all stages, and reviewed the final version prior to submission. All authors read and approved the final manuscript. Acknowledgement The authors thank Kenichiro Tanabe from St. Marianna University Graduate School of Medicine, Pathophysiology, and Bioregulation for statistical advice. Data Availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. References Kurimoto, N. et al . Endobronchial ultrasonography using a guide sheath increases the ability to diagnose peripheral pulmonary lesions endoscopically. Chest. 126, 959–965 (2004). Asahina, H. et al . Transbronchial biopsy using endobronchial ultrasonography with a guide sheath and virtual bronchoscopic navigation. Chest. 128, 1761–1765 (2005). Ishida, T. et al . 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Endobronchial ultrasonography using a guide sheath increases the ability to diagnose peripheral pulmonary lesions endoscopically. Chest. 126, 959–965 (2004). Tables Tables 1-2 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table12.docx Cite Share Download PDF Status: Published Journal Publication published 12 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 16 Jul, 2024 Reviews received at journal 23 Jun, 2024 Reviews received at journal 22 Jun, 2024 Reviewers agreed at journal 20 Jun, 2024 Reviewers agreed at journal 04 Jun, 2024 Reviewers invited by journal 04 Jun, 2024 Editor assigned by journal 04 Jun, 2024 Editor invited by journal 04 Jun, 2024 Submission checks completed at journal 01 Jun, 2024 First submitted to journal 29 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4497598","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":312668771,"identity":"b8d92f7c-e19c-4a53-b487-0bc3977cf9d9","order_by":0,"name":"Kazuhiro 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Nobuyuki","middleName":"","lastName":"Oike","suffix":""},{"id":312668785,"identity":"dacd7881-23bb-4465-8b74-cfdaa576918a","order_by":14,"name":"Junki Koike","email":"","orcid":"","institution":"Department of Pathology, St. Marianna University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Junki","middleName":"","lastName":"Koike","suffix":""},{"id":312668786,"identity":"8acd42e9-a0ff-4443-b2c4-359808f2bc55","order_by":15,"name":"Masamichi Mineshita","email":"","orcid":"","institution":"Department of Respiratory Medicine, St. Marianna University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Masamichi","middleName":"","lastName":"Mineshita","suffix":""}],"badges":[],"createdAt":"2024-05-29 14:06:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4497598/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4497598/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-72138-z","type":"published","date":"2024-09-12T15:57:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58515609,"identity":"4c772a3b-16c2-45c0-b57c-88af1e486483","added_by":"auto","created_at":"2024-06-17 16:41:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68819,"visible":true,"origin":"","legend":"\u003cp\u003eFlow chart of patients enrolled in the study. PPL, peripheral pulmonary lesions; EBUS-GS, endobronchial ultrasound with a guide sheath; ROSE, rapid on-site evaluation.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4497598/v1/65dd0099aded95a0a00c1b13.png"},{"id":58515610,"identity":"2878d802-bafa-43d2-83b7-33f087e96929","added_by":"auto","created_at":"2024-06-17 16:41:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":457273,"visible":true,"origin":"","legend":"\u003cp\u003eThe change from the initial ROSE to the final ROSE diagnosis. (a) All cases and cases with an initial (b) ROSE-negative or (c) ROSE-positive results are shown. (d) The change in the ROSE class arranged by the number of brushing. ROSE, rapid on-site evaluation.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4497598/v1/b95f307269483bbc563a2ea9.png"},{"id":64619998,"identity":"14fe9e77-d445-49a1-bb16-e1cdcc7dc67f","added_by":"auto","created_at":"2024-09-16 16:17:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":780951,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4497598/v1/243de913-ee71-4fdd-8343-3852d7a11af5.pdf"},{"id":58515608,"identity":"0f005461-c5c5-433e-b5cb-14fc5b1fba74","added_by":"auto","created_at":"2024-06-17 16:41:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":464397,"visible":true,"origin":"","legend":"","description":"","filename":"Table12.docx","url":"https://assets-eu.researchsquare.com/files/rs-4497598/v1/919a1cf3ef4c670466a6c15d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clinical utility of rapid on-site evaluation of brush cytology during bronchoscopy using endobronchial ultrasound with a guide sheath","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBronchoscopy is a pathological diagnostic method for peripheral pulmonary lesions (PPL). In bronchoscopic specimen collection from PPL, it is important to know the approach to the lesion and the specimen collection method. Various techniques for approaching lesions have been examined, including electromagnetic navigation bronchoscopy, virtual bronchoscopic navigation, radial endobronchial ultrasound (R-EBUS), ultrathin bronchoscopy, and guide sheath (GS) [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Bronchoscopy using endobronchial ultrasonography with a GS (EBUS-GS) is commonly performed for PPL, while forceps biopsy is a common specimen collection method in bronchoscopy for PPL. Other methods include brushing, washing, and cryobiopsy, which are often combined with forceps biopsies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, with the advent of molecular-targeted drugs and immune checkpoint inhibitors for lung cancer therapy, genetic mutation and programmed cell death ligand-1 (PD-L1) testing of bronchoscopic specimens has become increasingly important. Next-generation sequencing (NGS), which can detect multiple genetic mutations simultaneously, is often used in genetic mutation testing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Conventional NGS testing requires a certain tissue size and tumor content [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Against this background, obtaining large tissue specimens with high tumor cell content for bronchoscopy has been considered with the spread of cryobiopsy [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, it is now possible to perform NGS on cytological specimens [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, it has been suggested that the level of PD-L1 in cytological specimens correlates strongly with that in tissue specimens [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, cytological specimens used for bronchoscopy are becoming increasingly important. Brushing has been reported to have a high diagnostic accuracy [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]; hence, brushing has received renewed attention in recent years.\u003c/p\u003e \u003cp\u003eRapid on-site evaluation (ROSE) is used to confirm tumor cells in cytological specimens, and its utility in bronchoscopy has been studied using endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), which suggested that ROSE improves the diagnostic yield of EBUS-TBNA and decreases the number of biopsies required [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The same has been reported for bronchoscopy of PPL [\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. When performing ROSE during bronchoscopy using EBUS-GS for PPL, two types of cytology specimens must be evaluated: imprint cytology of forceps biopsy and brush cytology. There are many reports on ROSE of imprint cytology of forceps biopsy [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], but there are few reports on brush cytology [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. We investigated the improvement in diagnostic yield and changes in ROSE results when ROSE was performed on brush cytology during bronchoscopy using EBUS-GS for PPL.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003e This retrospective study was approved by the Ethics Committee of the St. Marianna University School of Medicine on October 26, 2023, with approval number 6224. The data used in this study were anonymized; therefore, the requirement for informed consent was waived by the Ethics Committee of the St. Marianna University School of Medicine and the study was compliant with The Declaration of Helsinki.\u003c/p\u003e \u003cp\u003eWe analyzed patients who underwent bronchoscopy for PPL at St. Marianna University Hospital between October 2020 and October 2022. PPL was defined as a nodule surrounded by the pulmonary parenchyma. Patients who underwent endobronchial biopsy or did not undergo EBUS-GS were excluded.\u003c/p\u003e \u003cp\u003eAfter a one-year study of medical records following bronchoscopy, the final diagnosis was defined as malignant for cases with a pathologic diagnosis of malignancy and benign for other cases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBiopsy procedure\u003c/h2\u003e \u003cp\u003ePrior to bronchoscopy, a high-resolution chest computed tomography (CT) scanner was used to identify the bronchi that led to lesions. The order of bronchoscopy was randomly determined. ROSE was performed for all bronchoscopies where cytologists could participate. ROSE was not performed when cytologists were unavailable.\u003c/p\u003e \u003cp\u003eBronchoscopy was performed using a combination of a bronchoscope (BF-1T260 or BF-P260F; Olympus, Tokyo, Japan), an R-EBUS probe (20 MHz mechanical radial type, UM-S20-17S; Olympus), and a GS kit (K-201 or K-203). During bronchoscopy, patients received intravenous midazolam and fentanyl for mild sedation and analgesia, and the bronchoscope was inserted orally or nasally. We inserted the GS with an R-EBUS probe through the working channel of the bronchoscope and guided it to the lesion site. EBUS images were classified into three categories: \"within,\" \"adjacent to,\" and \"invisible.\" After reviewing the fluoroscopic and EBUS images, we removed the R-EBUS probe and performed a brushing and forceps biopsy. In the ROSE group, the ROSE results of the glass slides produced by brushing were immediately communicated to the examiner. If the EBUS image was \"adjacent to\" or \"invisible,\" we adjusted the position of the R-EBUS probe or changed the bronchus into which it was inserted to be as \"within\" as possible. The number of brushings, forceps biopsies, and changes in procedure were determined at the discretion of the examiner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProcessing of specimens\u003c/h2\u003e \u003cp\u003eIn the ROSE group, brushes were smeared onto four glass slides. One slide was rapidly air-dried and stained with Cyto-Quick stain (Cyto-Quick; Muto Pure Chemicals, Tokyo, Japan), whereas the other three slides were fixed in 95% alcohol. The Cyto-Quick-stained slide specimens were microscopically evaluated by a cytologist in the same bronchoscopy laboratory. Alcohol-fixed slides were Papanicolaou-stained after bronchoscopy and microscopically evaluated by a cytologist. In the non-ROSE group, the materials from the brush were smeared onto two glass slides and fixed in 95% alcohol. Similar to the ROSE group, they were Papanicolaou-stained and evaluated after bronchoscopy. In both groups, forceps biopsy specimens were placed in 10% formalin. The sections were stained with hematoxylin and eosin after bronchoscopy for histological evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCytological specimen diagnosis\u003c/h2\u003e \u003cp\u003eCytological diagnosis was performed according to the World Health Organization reporting system for lung cytopathology [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Lung cytopathology specimens were classified into five categories: insufficient/inadequate/non-diagnostic, benign, atypical, suspicious for malignancy, malignant. Among the five categories of lung cytopathological specimen types, those suspicious for malignancy and diagnosed as malignant were defined as positive, while all others were defined as negative. The cytological diagnosis and evaluation were confirmed by several pathologists and cytologists at our hospital.\u003c/p\u003e \u003cp\u003eTo evaluate the tumor cell counts, the ROSE results were classified into four categories: no malignant findings (class 1), atypical cells or suspected malignant cells (class 2), sufficient malignant cells (class 3), and many malignant cells (class 4). Sufficient malignant cells were based on the Bethesda system in thyroid cytopathology, with a minimum of 6\u0026ndash;7 clusters [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Classes 1 and 2 were defined as ROSE-negative, while classes 3 and 4 were ROSE-positive. The ROSE classification was performed for each brushing. The highest class for each brushing was designated as the ROSE diagnosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Continuous variables were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), and dichotomous variables were analyzed using Student's t-test. Categorical variables were expressed as percentages, and differences were compared using Fisher's exact test. When comparing the sensitivity between the two groups, we used multivariable logistic regression to control for the potentially confounding roles of lesion size, lesion location, number of specimens, and EBUS images. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u0026nbsp;In total, 239 patients underwent bronchoscopy for PPL between October 2020 and October 2022. Of the 239 patients, 20 who underwent endobronchial biopsy and five who did not undergo EBUS-GS were excluded. Ultimately, 214 patients were included in the study. Among them, 129 patients with ROSE were enrolled in the ROSE group, and 85 patients without ROSE were included in the non-ROSE group (Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Baseline characteristics of\u0026nbsp;the patients and details of\u0026nbsp;bronchoscopy using EBUS-GS are summarized in Table 1. The mean \u0026plusmn; SD of lesion size was 38.34 \u0026plusmn; 19.62 mm in the ROSE group and 33.58 \u0026plusmn; 16.65 mm in the non-ROSE group, with no statistically significant difference. The lesion locations\u0026nbsp;were divided into three groups: (1) right upper lobe and left upper lobe; (2) right middle lobe and lingula; and (3) right lower lobe\u0026nbsp;and left lower lobe. The number of cases in each of these groups\u0026nbsp;were 67 (51.9%), 14 (10.9%), and 48 (37.2%), respectively, in the ROSE group and 50 (58.8%), 7 (8.2%), and 28 (32.9%), respectively, in the non-ROSE group. Brushing was performed on all patients, and forceps biopsy was performed on approximately 90% of patients in both groups. The number of cases with \u0026quot;within\u0026quot; EBUS images was significantly more in the ROSE group than in the non-ROSE group (89.9% vs. 78.8%, P = 0.029). The final diagnosis of malignancy was significantly more in the ROSE group than in the non-ROSE group (89.9% vs. 84.7%, P \u0026lt; 0.001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Table 2 shows the sensitivity of bronchoscopy in both\u0026nbsp;groups.\u0026nbsp;The sensitivity of the bronchoscopy results was significantly higher in the ROSE group than in the non-ROSE group (96.8% vs. 83.3%, P = 0.002). The multivariate analysis also showed a statistically significant difference (P = 0.004). Using the sampling method, the sensitivity of brushing was significantly higher in the ROSE group than in the non-ROSE group (92.9% vs. 75.0%, P \u0026lt; 0.001), and the sensitivity of forceps biopsy was also higher in the ROSE group than in the non-ROSE group (87.6% vs. 78.8%, P = 0.137).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Figure 2 shows the change in ROSE results for the 126 patients in the ROSE group whose final diagnosis was malignancy. Forty-four patients (34.9%) initially had ROSE- negative results. Of these, 35 (79.5%) converted to a ROSE-positive final diagnosis (Fig. 2b). Of the 35 patients whose final diagnosis was ROSE-positive, 20 (57.1%) had repeated cell sampling at the same site, 9 (25.7%) changed bronchial branches, and 6 (17.1%) changed the depth of the brushing position. On the other hand, 82 patients (65.1%) initially had ROSE-positive results. The initial ROSE result was\u0026nbsp;class 3 in 29 patients (35.3%). In 16 (55.2%) of these patients, the final ROSE diagnosis improved to class 4 (Fig. 2c). The number of patients with a class 4 ROSE result increased with each successive ROSE, from 53 patients (42.1%) at the initial ROSE to 87 patients (69.0%) at the sixth ROSE. In addition, 30 patients (23.8%) were class 1 at the initial ROSE, but this number decreased to two patients (1.6%) at the sixth ROSE (Fig. 2d).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, the ROSE of brush cytology improved the sensitivity of bronchoscopy using EBUS-GS for PPL. Multiple ROSE procedures tended to change the ROSE diagnosis from negative to positive in some cases and increased tumor cell counts.\u003c/p\u003e \u003cp\u003eThe ROSE of brush cytology is useful in bronchoscopy for PPL. In addition, ROSE increased the sensitivity of brushing. The ROSE of a portion of a brushing specimen allows for the real-time evaluation of the brushing specimens. We believe that this real-time evaluation may have allowed the examiner to repeat the procedure until sufficient specimens were collected, thereby improving the brushing sensitivity. The rationale for this was that there were many cases wherein ROSE diagnosis changed from negative to positive during bronchoscopy. Of the 44 initially ROSE-negative cases, 35 (79.5%) converted to a ROSE-positive diagnosis. Of these, 20 tested positive after repeated brushing of the same site. Fifteen of 20 patients had adenocarcinomas. Kurimoto et al. reported that tumors for which specimen collection is not possible even with EBUS imaging are often adenocarcinomas [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Because adenocarcinomas are often covered by tracheal epithelium, specimen collection may not be possible without repeated brushing and destruction of the tracheal epithelium. Therefore, if adenocarcinoma is suspected based on chest CT imaging or tumor markers before bronchoscopy, it may be important to repeat the specimen collection at the same site. In contrast, the brushing position changed during bronchoscopy in 15 patients. Of these, nine changed the bronchial branches, and six changed the depth of the brushing position. Patients in whom the bronchial branch changed had another bronchial branch, which was a candidate for specimen collection based on the chest CT imaging prior to bronchoscopy. All patients for whom the depth of the brushing position was changed had lesions measuring\u0026thinsp;\u0026gt;\u0026thinsp;30 mm. We believe that it is important to identify the lesion in detail prior to bronchoscopy, such as through chest CT imaging, and decide how the procedure should be modified according to the ROSE results.\u003c/p\u003e \u003cp\u003eTumor cell counts collected by brushing increased with multiple ROSE. As a result of multiple ROSE, more than half of the patients whose initial ROSE diagnosis was class 3 were reclassified as class 4 at the final ROSE diagnosis. In addition, the percentage of class 4 ROSE diagnoses increased and the percentage of class 1 ROSE diagnoses decreased with each repeated ROSE; thus, repeated ROSE allows the collection of more tumor cells. When performing NGS or PD-L1 testing on specimens collected during bronchoscopy, the higher the tumor cell count collected, the higher the success rate of the tests. Therefore, when NGS or PD-L1 testing is performed on cytology specimens, additional brushing may be necessary even if tumor cells are present in the cytology specimen. Further studies are needed to determine the tumor cell counts required for NGS or PD-L1 testing of cytology specimens.\u003c/p\u003e \u003cp\u003eThis study has some potential limitations. This was a single-center, retrospective study, which may have influenced the judgment of the examiner regarding the presence or absence of ROSE, and selection bias may exist. However, the ROSE group did not have significantly more small lesions or pulmonary apex cases that were difficult to diagnose using bronchoscopy; hence, the possible effect was likely to be small. Prospective randomized multicenter trials are required to avoid possible bias.\u003c/p\u003e \u003cp\u003eIn conclusion, bronchoscopy sensitivity was improved by performing ROSE on brush cytology during bronchoscopy using EBUS-GS for PPL. In some cases, the ROSE diagnosis changed from negative to positive during bronchoscopy, and the tumor cell counts increased.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eK.N. conceived the research concept and strategy, designed and supervised the study, analyzed the data and wrote the main part of manuscript. K.M. contributed the concept and revised the final manuscript. S.K., M.N., A.M., Y.N., Y.N., Y.S., and H.T. performed the bronchoscopic procedures at the participating center. N.S., C.O. processed and evaluated cytology specimens. N.O., J.K. performed the histological examination. H.K. reviewed titles, abstracts and full text articles. H.H., M.M. reviewed titles and abstracts and provided critical revision of the manuscript. All the authors discussed the results and implications, commented on the manuscript at all stages, and reviewed the final version prior to submission. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Kenichiro Tanabe from St. Marianna University Graduate School of Medicine, Pathophysiology, and Bioregulation for statistical advice.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKurimoto, N. \u003cem\u003eet al\u003c/em\u003e. Endobronchial ultrasonography using a guide sheath increases the ability to diagnose peripheral pulmonary lesions endoscopically. Chest. 126, 959\u0026ndash;965 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsahina, H. \u003cem\u003eet al\u003c/em\u003e. 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Chest. 126, 959\u0026ndash;965 (2004).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1-2 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rapid on-site evaluation, endobronchial ultrasound with a guided sheath, peripheral lung lesions, brushing","lastPublishedDoi":"10.21203/rs.3.rs-4497598/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4497598/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePrevious studies have shown that rapid on-site evaluation (ROSE) improves the diagnostic yield of bronchoscopy using endobronchial ultrasonography with guided sheath (EBUS-GS) for peripheral pulmonary lesions (PPL). There are many reports on the ROSE of imprint cytology of forceps biopsy; however, there are few reports on the ROSE of brush cytology. We investigated the utility of ROSE in brushing PPL specimens during bronchoscopy. We retrospectively analyzed 214 patients who underwent bronchoscopy with EBUS-GS for PPL. Sensitivity to bronchoscopy was significantly higher in the ROSE group than in the non-ROSE group (96.8% vs. 83.3%, P\u0026thinsp;=\u0026thinsp;0.002), wherein the sensitivity to brushing also significantly improved (92.9% vs. 75.0%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Conversion of ROSE results from negative to positive was observed in 79.5% of first-time ROSE-negative cases. We also found that brush specimens containing high tumor cell counts increase with repeated ROSE (42.1\u0026ndash;69.0%). This study suggests that ROSE of brush cytology improves bronchoscopy sensitivity and increases the collected tumor cell counts.\u003c/p\u003e","manuscriptTitle":"Clinical utility of rapid on-site evaluation of brush cytology during bronchoscopy using endobronchial ultrasound with a guide sheath","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-17 16:41:09","doi":"10.21203/rs.3.rs-4497598/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-16T05:46:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-24T00:56:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-22T21:55:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100949147030037539581265585261606402427","date":"2024-06-20T12:08:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266068088946034327891766944676368899320","date":"2024-06-04T21:42:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-04T19:15:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-04T19:13:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-04T18:15:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-01T08:30:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-29T14:02:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c421fd37-bfcb-4a6d-ad39-5d14d9ba031f","owner":[],"postedDate":"June 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33050755,"name":"Biological sciences/Cancer/Lung cancer"},{"id":33050756,"name":"Health sciences/Medical research"},{"id":33050757,"name":"Health sciences/Diseases/Respiratory tract diseases"}],"tags":[],"updatedAt":"2024-09-16T16:13:34+00:00","versionOfRecord":{"articleIdentity":"rs-4497598","link":"https://doi.org/10.1038/s41598-024-72138-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-09-12 15:57:54","publishedOnDateReadable":"September 12th, 2024"},"versionCreatedAt":"2024-06-17 16:41:09","video":"","vorDoi":"10.1038/s41598-024-72138-z","vorDoiUrl":"https://doi.org/10.1038/s41598-024-72138-z","workflowStages":[]},"version":"v1","identity":"rs-4497598","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4497598","identity":"rs-4497598","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}