Feasibility of direct cryobiopsy specimen retrieval through the working channel of an ultrathin bronchoscope—a retrospective study of single institute

Introduction

The widespread adoption of endobronchial ultrasound-guided ultrathin bronchoscopy (EBUS-UT) has advanced the diagnostic approach to peripheral pulmonary lesions (PPLs) (1,2). The ultrathin bronchoscope (UTB) with an outer diameter of 3.0-mm and a 1.7-mm working channel (BF-MP290F, Olympus, Japan) improves access to distal airways and enables positioning closer to peripheral lesions that are difficult to diagnose (3). However, UTB-guided transbronchial forceps biopsy using small forceps often yields limited specimen sizes. The addition of ultrathin cryobiopsy using a 1.1-mm cryoprobe may improve diagnostic yield by enabling the acquisition of larger tissue samples (4,5).

For peripheral lesions, removal of the bronchoscope from the airway with each cryobiopsy after reaching the target necessitates re-localization of the lesion using radial EBUS and fluoroscopic guidance for each biopsy and requires intubation. The cryobiopsy technique reported by Nakai et al. (6) involves retrieving specimens through a 2.0-mm working channel using a short freezing time, without conventional hemostatic methods. In addition, Ariza-Prota et al. recently reported the feasibility of direct mediastinal cryobiopsy specimens retrieval through the working channel using a linear EBUS bronchoscope (7). While the concept of in-channel cryobiopsy specimen retrieval has been previously described, our study demonstrates the feasibility of applying this technique to the substantially narrower 1.7-mm working channel of a UTB in PPLs. We retrospectively reviewed cases in which these techniques performed using a UTB at our institution and evaluated sample size, diagnostic yield, and safety profile. We present this article in accordance with the STROBE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0916/rc).

Methods

We conducted a retrospective study between November 2022 and December 2025 at National Hospital Organization (NHO) Okayama Medical Center, Japan. Patients with PPLs requiring pathological diagnosis who underwent EBUS-UT with additional cryobiopsy involving direct specimen retrieval through the working channel were included. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The Institutional Review Board of NHO Okayama Medical Center approved the study (identifier No. RINKEN 2025-055; approval date: March 6, 2026). Individual consent for this retrospective analysis was waived. Nine patients in this retrospective cohort were also enrolled in a previously published prospective study (8); however, the objectives of the present study differ.

Bronchoscopic procedures

Bronchoscopy was performed under conscious sedation with midazolam and fentanyl. A UTB (1.7-mm working channel; BF-MP290F, Olympus, Tokyo, Japan) was used. After bronchoscope insertion, an endotracheal tube with an inner diameter of 8 mm was intubated under bronchoscopic guidance (to facilitate hemostasis using the two-scope technique). A radial EBUS probe (UM-S20-17; Olympus) was used to localize target lesions under fluoroscopic guidance. Fluoroscopy and radial probe EBUS guided bronchoscope positioning close-to-the-lesion (9), ensuring a reproducible biopsy-oriented approach. The bronchoscope was advanced to the peripheral airways, and a 1.5-mm small forceps biopsy (FB-433D; Olympus, Japan) was performed before cryobiopsy. The forceps biopsy specimens obtained were assessed using rapid on-site evaluation (ROSE). The specimens obtained by forceps biopsy were gently touch-imprinted onto glass slides and stained using CytoQuick (Muto Pure Chemicals Co., Tokyo, Japan). ROSE results were classified as either positive or negative. When forceps biopsy specimen showed inadequate tissue based on ROSE, additional ultrathin cryobiopsy was performed. During the cryobiopsy procedure, a 1.1-mm cryoprobe was inserted through the working channel and frozen for 2–4 seconds. The cryobiopsy specimen was then retrieved through the working channel as done with standard forceps biopsy. A bronchoscope stabilization system was not used. The operator manually maintained the wedged position by advancing and stabilizing the bronchoscope at the patient’s mouth. To avoid loss of bronchoscope fixation, the assistant withdrew the cryoprobe while the operator maintained the bronchoscope position. After cryobiopsy, the bronchoscope was kept wedged in the responsible bronchus for two minutes (Figure 1). Retrieval failure was defined as a situation in which the cryobiopsy specimen became stuck at the tip of the bronchoscope during retrieval after peripheral cryobiopsy and could not be pulled through into the working channel.

Figure 1 Bronchoscopic technique for direct cryobiopsy specimen retrieval through the working channel. Ultrathin bronchoscope positioned close-to-the-lesion, ensuring a reproducible biopsy-oriented approach. The bronchoscope was advanced to the peripheral airways as wedging. During the cryobiopsy procedure, a 1.1-mm cryoprobe was inserted through the 1.7-mm working channel and frozen for 2–4 seconds. The cryobiopsy specimen was then retrieved through the working channel as done with standard forceps biopsy. After cryobiopsy, the bronchoscope was kept wedged in the responsible bronchus for two minutes.

Study outcomes

We evaluated sample size for specimens obtained using small forceps and cryobiopsy. Specimen size was measured microscopically using the OLYMPUS cellSens Standard (Olympus, Tokyo, Japan). We also assessed procedural pathological diagnostic yield and safety. Diagnostic yield and bleeding severity were defined according to the Delphi consensus statement (10,11).

Statistical analysis

Sample sizes were analyzed using a two-tailed Mann-Whitney U test. Diagnostic yield was analyzed using the McNemar’s test. A P value <0.05 was considered statistically significant. All statistical analyses were performed using EZR software (version 1.61; Jichi Medical University Saitama Medical Center, Japan).

Results

Patient characteristics

A total of 18 patients were included in the study (shown in Figure 2). The median age was 80 years (range, 68–90 years), and the study included eight men and 10 women. The median diameter of the PPLs was 13.5 mm (range, 10–28 mm); 15 lesions measured less than 20 mm, and three measured 20–30 mm. On computed tomography, nine lesions were classified as solid, five as part-solid, and four as ground-glass nodules. Lesion location included eight in the right upper lobe, one in the right middle lobe, three in the right lower lobe, five in the left upper lobe, and one in the left lower lobe. The detailed patient characteristics are shown in Table 1.

Figure 2 Study flow diagram. Between November 2022 and December 2025, a total of 153 cryobiopsy procedures using a 1.1-mm cryoprobe were performed at our institution. Most procedures were conducted using therapeutic or thin bronchoscopes (n=126). Among the 27 procedures performed using the ultrathin bronchoscope BF-MP290F, nine procedures were performed using conventional en bloc bronchoscope removal techniques with the two-scope or tube wedge method for bleeding control. The remaining 18 procedures underwent direct cryobiopsy specimen retrieval through the working channel of the ultrathin bronchoscope and were included in the present study.

Bronchoscopic procedures

All bronchoscopic procedures were performed under conscious sedation using midazolam and fentanyl. The median dose of midazolam was 5 mg (range, 2–7 mg), and the median dose of fentanyl was 0.07 mg (range, 0.05–0.10 mg). Radial EBUS findings demonstrated a within pattern in eight cases, an adjacent-to pattern in six cases, and a blizzard pattern in four cases. The median procedure time was 34.5 minutes (range, 14–90 minutes). The median numbers of specimens obtained by forceps biopsy and cryobiopsy were 5 [5–10] and 3 [1–4], respectively. Cryobiopsy was performed for a total of 57 times. Freezing times of 2, 3, and 4 seconds were applied in 23, 26, and four passes, respectively. Retrieval failure at the bronchoscope tip occurred in four passes and was observed exclusively when the freezing time was 4 seconds. No bronchoscope dislocation from the target bronchus was observed during the procedures.

Size of specimens

We evaluated 18 specimens obtained using small forceps and 53 specimens obtained using cryobiopsy. For forceps biopsy, the largest specimen per patient was selected for analysis. For cryobiopsy specimens, the size of all successfully obtained specimens was measured according to each freezing time. The comparison of specimen sizes is presented in Figure 3. Overall cryobiopsy specimens (n=53) were significantly larger than forceps specimens (1.97 vs. 1.00 mm², P<0.001, Mann-Whitney U test). The median sample size for each freezing time was 1.75 mm² at 2 seconds (n=23), 2.75 mm² at 3 seconds (n=26), and 1.71 mm² at 4 seconds (n=4), respectively (Figure 3). Even at the shortest freezing time (2 seconds), the sample size was significantly larger than that obtained with forceps biopsy (P=0.02, Mann-Whitney U test). There was no significant difference between the 2-second and 3-second freezing times (P=0.052, Mann-Whitney U test).

Figure 3 Comparison of specimen sizes obtained by forceps biopsy and cryobiopsy. (A) Comparison of specimen size between small forceps biopsy and cryobiopsy specimens obtained using a 1.1-mm cryoprobe. Cryobiopsy specimens were significantly larger than forceps biopsy specimens (median, 1.97 vs. 1.00 mm²; P<0.001, Mann-Whitney U test). (B) Comparison of cryobiopsy specimen size according to freezing time. The median specimen area was 1.75 mm² for 2-second freezing (n=23), 2.75 mm² for 3-second freezing (n=26), and 1.71 mm² for 4-second freezing (n=4).

Diagnostic yield

The overall specific diagnostic yield was 72.2% [95% confidence interval (CI): 46.5–90.3%]. Forceps biopsy and cryobiopsy yielded 33.3% (95% CI: 13.3–59%) and 67% (95% CI: 31–86.7%), respectively (Table 2). A Venn diagram is presented in Figure 4. McNemar’s test demonstrated a trend toward a higher diagnostic yield for cryobiopsy, with 7 cryobiopsy-only diagnostic cases versus 1 forceps-only diagnostic case (P=0.07). Among cases with specific findings on cryobiopsy, radial EBUS patterns included within-lesion in 100% (8 of 8), adjacent-to-lesion in 50% (3 of 6), and blizzard in 25% (1 of 4). Among the cases diagnosed solely by cryobiopsy, three demonstrated a within-lesion pattern and four showed a non-within-lesion pattern on radial EBUS. A representative case is shown in Figure 5.

Figure 4 Venn diagram of diagnostic overlap between forceps biopsy and cryobiopsy. The Venn diagram demonstrates the overlap in diagnostic yield between forceps biopsy and cryobiopsy. Five cases were diagnosed by both modalities, one case was diagnosed only by forceps biopsy, and seven cases were diagnosed only by cryobiopsy. Among the cryobiopsy-only diagnostic cases, three showed a within-lesion pattern and four showed a non-within-lesion pattern on radial endobronchial ultrasound. McNemar’s test demonstrated a trend toward a higher diagnostic yield for cryobiopsy compared with forceps biopsy, although the difference did not reach statistical significance (P=0.07).

Figure 5 Representative case of cryobiopsy contributing to diagnosis. (A) Chest computed tomography of a 60-year-old woman showing a ground-glass nodule in the right upper lobe (yellow arrow). (B) Radial endobronchial ultrasound demonstrated an adjacent-to-lesion pattern. (C) Small forceps biopsy specimen, which was nondiagnostic. (D) Cryobiopsy specimen obtained using a 1.1-mm cryoprobe through the working channel of an ultrathin bronchoscope. The cryobiopsy specimen provided the diagnosis of lung adenocarcinoma. Staining method: hematoxylin and eosin staining.

Safety

Procedure-related complications are summarized in Table 3. Bleeding was graded after two minutes of bronchoscopic wedging according to the specified classification. Bleeding was mild in most cases: Grade 0 bleeding occurred in 15 patients, Grade 1 in three patients, and no cases of Grade ≥2 bleeding were observed. Hemostasis was achieved in all cases using suction alone with the UTB. A pinhole defect in the working channel was identified during post-procedure maintenance. The lesion was a peripheral nodule in the left upper lobe (Lt B¹⁺²ciβ), and cryobiopsy was performed with a freezing time of 4 seconds.

Discussion

Previous studies have reported the utility of UTB-guided cryobiopsy using hemostatic techniques such as the tube-wedge method (12), two-scope technique (13), and balloon occlusion method (14). However, these approaches generally require bronchoscope removal for each cryobiopsy, necessitating repeated lesion localization using EBUS and often requiring endotracheal intubation. A 1.1-mm ultrathin cryoprobe can be used through a guide sheath, which may enable repeated biopsy from the same location and reduce bleeding complications while maintaining the bronchoscope within the bronchus (15). The large guide sheath (SG-401C, Olympus, Tokyo, Japan) has a maximum outer diameter of 2.54 mm and is therefore compatible only with therapeutic bronchoscopes with a working channel larger than 2.54 mm. However, this approach is limited to therapeutic bronchoscopes that accommodate a large guide sheath, restricting accessibility to the peripheral. A therapeutic bronchoscope with an outer diameter of approximately 6.0-mm typically reaches the segmental or subsegmental bronchi, whereas UTBs can be advanced further into more peripheral (16).

Cryobiopsy using a UTB, which provides improved access to peripheral lesions, may simplify the procedure. In addition, direct retrieval of cryobiopsy specimens through the working channel of the bronchoscope represents a promising alternative approach. This technique, performed with the bronchoscope positioned close-to-the-lesion (9), may allow continuous sampling without bronchoscope removal. Although the limited freezing time resulted in relatively small cryobiopsy specimens, these specimens remained significantly larger than those obtained using small forceps biopsy. Cryobiopsy also showed a trend toward improved diagnostic yield, suggesting that this approach may represent a reasonable option in selected cases. However, as previously reported by Oki et al. (4), small forceps biopsy and cryobiopsy currently appear to play complementary roles in diagnostic yield, and the authors do not recommend omitting forceps biopsy. In the present study, additional cryobiopsy was performed based on the ROSE results of forceps biopsy; nevertheless, in some cases, a diagnosis was achieved by forceps biopsy alone.

Direct retrieval of cryobiopsy specimens through a 1.7-mm working channel raises concerns regarding potential bronchoscope damage, particularly when the freezing time exceeds 4 seconds. The safety of this technique in terms of bronchoscope integrity remains uncertain. No bronchoscope damage was observed when the freezing time was limited to 2–3 seconds, indicating that this duration may be appropriate. In general, longer freezing times are expected to produce larger cryobiopsy specimens. However, in the present study, four cases of retrieval failure occurred with a 4-second freezing time, likely because the specimens became too large to pass through the working channel. In addition, even when large specimens are successfully obtained at the peripheral region, the specimens may be partially shaved or compressed during passage through the narrow working channel. Therefore, the relatively smaller specimen size observed in the 4-second group compared with the 2- and 3-second groups may have been influenced by both the limited sample size and these technical factors. Prolonged freezing time may increase the risk of retrieval failure within the working channel; therefore, freezing time should be limited to a maximum of 3 seconds, with approximately 2 seconds representing an optimal standard approach. Particularly during the introductory phase of this technique, it may be reasonable to begin with a freezing time of 2 seconds and extend to 3 seconds only when the obtained specimen appears macroscopically small. For ground-glass nodules presenting with a blizzard sign, conventional cryobiopsy involving en bloc removal of the bronchoscope together with the cryoprobe and attached specimen (17) may be preferable because it enables the acquisition of larger tissue specimens. Shorter freezing times may reduce diagnostic yield in lesions with ground-glass.

In cryobiopsy procedures, hemostasis is achieved by successful wedging of the bronchoscope. Therefore, dislocation of the hemostatic device is an important concern and may occur even with balloon occlusion techniques (18) or the tube wedge method (12). In the present study, we speculate that bronchoscope wedging was maintained because the cryobiopsy specimens were not excessively large, thereby reducing the pressure applied to the bronchoscope during specimen retrieval. However, as the number of cases increases, the possibility of bronchoscope dislocation, particularly in upper lobe lesions, cannot be excluded, similar to other cryobiopsy techniques. Thus, we believe that backup availability of a second bronchoscope, especially a therapeutic bronchoscope, during the introductory phase of this technique may be desirable to allow prompt conversion to the two-scope method if needed.

This study has several limitations. First, our institution routinely employs various hemostatic techniques for cryobiopsy (19,20). Direct retrieval of cryobiopsy specimens through the working channel using a UTB should therefore be considered one option, while conventional ultrathin cryobiopsy remains the established standard technique. Further prospective studies with larger cohorts are required to clarify the safety profile of this approach, particularly regarding the risk of bronchoscope damage.

Conclusions

Retrieval of frozen specimens obtained using a 1.1-mm cryoprobe through a UTB enables the acquisition of significantly larger samples than small forceps biopsy. Freezing time should be minimized to reduce the risk of bronchoscope damage.

Acknowledgments

We would like to thank Editage (www.editage.com) for the English language editing.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0916/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0916/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0916/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0916/coif). Y.T. received speaker fees from AMCO Inc. Ken Sato received speaker fees from Olympus Marketing, Inc., and AMCO Inc. The other authors have no conflicts of interest to declare.

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 Institutional Review Board of NHO Okayama Medical Center (approval number: RINKEN 2025-055; approval date: March 6, 2026) and individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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