The Application Value and Safety of Ultrafine Bronchoscopy Combined with Frozen Lung Biopsy in the Diagnosis of Peripheral Pulmonary Nodules

Summary

This study aims to evaluate the diagnostic yield, specimen quality, and safety of ultrathin bronchoscopy-guided transbronchial cryobiopsy in diagnosing peripheral pulmonary nodules, compared with conventional forceps biopsy.

Abstract

Accurate diagnosis of peripheral pulmonary nodules (PPNS) remains challenging due to their small size and location. Conventional transbronchial forceps biopsy often yields suboptimal tissue samples. Cryobiopsy has emerged as a promising technique to improve diagnostic accuracy.

The objective of this study is to evaluate the diagnostic yield, specimen quality, and safety profile of ultrathin bronchoscopy-guided transbronchial cryobiopsy in patients with PPNS, and to compare it with conventional forceps biopsy.

In this retrospective, observational study conducted from January 2023 to December 2024 at the department of pulmonary medicine, 60 adult patients with CT-confirmed PPNS (8-30 mm) underwent bronchoscopy evaluation using ultrathin bronchoscopy, radial endobronchial ultrasound (R-EBUS), and virtual navigation. Cryobiopsies were obtained using a 1.1 mm cryoprobe, with forceps biopsy performed in 75% of patients for comparison. The primary outcome was diagnostic yield; secondary outcomes included complication rates, specimen size, tissue quality, and procedural metrics.

A definitive histopathological diagnosis was achieved in 48 of 60 cases, yielding an overall diagnostic rate of 80%. Cryobiopsy demonstrated a significantly higher diagnostic yield (85%) compared to forceps biopsy (62%, p < 0.05). Cryobiopsy specimens were significantly larger (5.2 ± 1.1 mm vs 2.1 ± 0.7 mm) with p < 0.05 and had less crush artifact (92% vs 58%). Malignancy was diagnosed in 34 patients (56.7%), with adenocarcinoma being the most common. Complication rates were low; pneumothorax occurred in 2 cases (3.3%) and moderate bleeding in 5 cases (8.3%), all managed conservatively.

Transbronchial cryobiopsy is a safe and superior diagnostic modality for evaluating PPNS, offering higher diagnostic yield and better specimen quality than forceps biopsy. It should be considered an effective, minimally invasive option for tissue acquisition in patients with indeterminate pulmonary nodules. However, the study's single-center design and limited sample size may restrict generalizability.

Introduction

Lung cancer continues to be the leading cause of cancer-related deaths globally, accounting for approximately 1.8 million deaths each year¹. A major challenge in reducing lung cancer mortality is the fact that many patients are diagnosed at an advanced stage, where treatment options are limited and the prognosis is poor. Early detection of lung cancer, particularly when the disease is still localized and potentially curable, significantly improves survival rates². In this context, the implementation of low-dose computed tomography (LDCT) screening in high-risk populations, especially long-term smokers, has marked a significant advancement in the early detection of lung malignancies³. The major outcomes of widespread LDCT screening are increasing the identification of peripheral pulmonary nodules, small, often asymptomatic lesions usually located in the outer regions of the lung parenchyma⁴. Most of the research studies have shown high-risk, up to 33% of high-risk individuals are undergoing LDCT screening, which may present with pulmonary nodules. Most of the pulmonary nodules are benign, but a sizeable percentage are early-stage lung malignancies, underscoring the significance of prompt and precise identification to facilitate curative therapy^5,6. However, there are significant difficulties in diagnosing peripheral nodules (PPNS), whereas the excellent sensitivity of CT-guided transthoracic needle aspiration (TTNA) frequently surpasses 90% for detecting cancer⁷. Despite its diagnostic precision, CT-guided core needle biopsy (CT-CNB) has a risk of complications, specifically pneumothorax, which can happen in around 27% of cases, with some patients requiring chest tube insertion⁸. While it tends to be less effective for small and marginally situated nodules, conventional flexible bronchoscopy offers a safer alternative option for nodules measuring under 20 mm in diameter, with its diagnostic yield varying between 10% to 50%⁹. In recent years, significant progress has been made in bronchoscopy tools and techniques aimed at improving the diagnostic precision of PPNS to address these limitations. A significant advancement is the creation of ultrathin bronchoscopes (UTBS)¹⁰. Featuring outer diameters as small as 3 mm, UTBS allow for navigation deep into peripheral airways, accessing lesions that conventional bronchoscopes could not previously reach. This enhanced accessibility could boost diagnostic yield for small and distant nodules. Nonetheless, a significant obstacle persists: the biopsy tools suitable for UTBS, like mini forceps, frequently obtain limited and compressed tissue samples. These less-than-ideal samples may be insufficient for an accurate histopathological diagnosis or for performing essential molecular analyses, which are becoming increasingly vital for directing personalized therapy¹¹.

After the research on lung cancer treatment, transbronchial cryobiopsy (TBLC) has arisen as a promising substitute to address the limitations of the traditional biopsy techniques. This method uses a cryoprobe to extract and freeze tissue, enabling the collection of larger, more intact, and better-preserved samples than those obtained with conventional forceps¹². The augmented tissue volume and improved retention of structural characteristics boost histopathological assessment and aid in sophisticated molecular and immunohistochemical evaluation, essential elements of the age of precision oncology for lung cancer¹³. In reaction to the increasing demand for enhanced diagnostic yield and sample quality, TBLC has attracted interest for its possible use beyond interstitial lung diseases, especially in assessing PPNS. When combined with UTBS and state-of-the-art navigation systems, TBLC can reach and sample lesions that were once deemed difficult or unreachable with conventional bronchoscopic instruments¹⁴. Although TBLC has mainly been used for diagnosing interstitial lung diseases, its use in sampling peripheral nodules, particularly in conjunction with UTBS and advanced navigation systems, is currently under investigation¹⁵.

The current study aimed to assess the diagnostic accuracy and safety profile of combining utb with TBLC in patients presenting with radiologically detected PPNS. A standardized procedural workflow was implemented, incorporating pre-procedural imaging review, virtual bronchoscopy planning, and guidance using radial endobronchial ultrasound (R-EBUS). Utb-guided cryobiopsies were performed on a cohort of patients with peripheral lung nodules varying in size and anatomical location¹⁶. For comparative analysis, traditional forceps biopsies were simultaneously conducted during the same procedure. Initial results demonstrated that the integrated UTB-TBLC approach significantly enhanced diagnostic yield, particularly for nodules less than 20 mm in size and those located in the outer third of the lung parenchyma. Cryobiopsy specimens were consistently larger and exhibited superior architectural preservation compared to those obtained with forceps¹⁷. This allowed for more accurate histological subtyping and improved success rates for molecular profiling. Notably, the procedure was well tolerated, with a low incidence of adverse events such as bleeding and pneumothorax¹⁸.

These findings suggest that the combination of UTB and TBLC offers a safe and highly effective strategy for diagnosing peripheral lung lesions, potentially reducing the reliance on more invasive techniques like CT-guided needle aspiration. If validated by larger, multicentre prospective studies, this minimally invasive approach could redefine the standard bronchoscopic diagnostic pathway for early-stage lung cancer¹⁹. Recent advancements in bronchoscopic techniques have notably improved the detection of PPNS, especially those under 20 mm and located in peripheral lung regions, traditionally challenging for conventional flexible bronchoscopes due to limited reach and suboptimal tissue sampling²⁰. Ultrathin bronchoscopes, with diameters as small as 3 mm, enable deeper navigation into peripheral airways, significantly increasing access to distal lesions and elevating diagnostic yields to approximately 66-70%, surpassing those of conventional bronchoscopes²¹.

The advent of UTBS has significantly improved access to small, peripherally located pulmonary lesions. However, the narrow working channels of UTBS limit tissue acquisition tools to mini forceps, which often yield suboptimal specimens characterized by limited volume and significant crush artifacts²². These limitations hinder comprehensive histopathological evaluation and molecular profiling, critical components of personalized cancer therapy^23,24. TBLC uses a cryoprobe to extract larger and well-preserved tissue samples by rapidly freezing the lesion, thereby enhancing diagnostic accuracy and enabling robust molecular analysis^14,25. The integration of UTB and TBLC-facilitated by the development of ultrathin cryoprobes (1.1-1.7 mm in diameter) compatible with UTB working channels-has led to a marked improvement in diagnostic performance¹³. When supported by advanced navigational tools such as radial endobronchial ultrasound (R-EBUS) and virtual bronchoscopy, this approach has demonstrated superior outcomes in accessing and sampling PPNS^26,27.

Recent studies have reported diagnostic yields ranging from 84.4% to 97.2% for cryobiopsy, substantially higher than those achieved by conventional forceps biopsy (63.8-77.8%)²⁸. In particular, a 2024 propensity-matched study demonstrated that TBLC improved diagnostic yield for ground-glass opacity (GGO) lesions by approximately 25 percentage points compared to forceps biopsy (88.8% vs. 63.8%)²⁶.

In terms of specimen quality, cryobiopsy has been shown to retrieve samples that are up to 26 times larger than those obtained via forceps, with better-preserved cellular architecture and minimal crush artifact²⁹. These features significantly improve the feasibility and accuracy of both histological subtyping and molecular characterization³⁰.

The development of thinner cryoprobes (1.1-1.7 mm) has been instrumental in enabling access to more distal airway segments. When combined with robotic bronchoscopy, the use of a 1.1-mm cryoprobe achieved a diagnostic yield of 90%, with 18% of diagnoses made exclusively via cryobiopsy³¹. Additionally, cryobiopsy offers 360° tissue sampling, enhancing the likelihood of capturing extraluminal or adjacent lesion components, a known limitation of forceps biopsies³².

Cryobiopsy samples have demonstrated superior performance in providing sufficient tissue for downstream molecular analyses³³. In comparative studies, TBLC achieved 100% molecular adequacy for next-generation sequencing (NGS) and immunohistochemistry (IHC), significantly outperforming forceps biopsy (89.5%)³⁴.

While TBLC is associated with a higher rate of bleeding than forceps biopsy, grade 2-3 bleeding in 40.5% of cases vs. 8.6% severe bleeding events (grade 4) remain rare. Importantly, the overall risk of pneumothorax remains low, with large studies reporting an incidence of approximately 1.3%³⁵.

Given its enhanced diagnostic yield, superior tissue quality, and molecular testing capability, cryobiopsy is increasingly recognized as a first-line diagnostic tool for the evaluation of PPNS, especially for GGO lesions. When integrated with navigational modalities such as R-EBUS and virtual bronchoscopy, TBLC offers a comprehensive and minimally invasive approach that rivals the diagnostic performance of transthoracic needle aspiration (TTNA), while offering lower complication rates and the added advantage of concurrent mediastinal lymph node assessment³⁶. Although preliminary results are promising, further validation through large-scale, multicenter prospective trials is necessary to standardize protocols, define patient selection criteria, and confirm long-term safety outcomes³⁷. Nevertheless, the UTB-TBLC combination has the potential to significantly advance bronchoscopic diagnosis in early-stage lung cancer, aligning with the goals of precision medicine and minimally invasive oncology.

Protocol

This is a reprospective, observational clinical study conducted at the Department of Pulmonary Medicine, Jiangxi Provincial People's Hospital(The First Affiliated Hospital of Nanchang Medical College), from January 2023 to December 2024. The study was approved by the Institutional Ethics Committee of Jiangxi Provincial People's Hospital(The First Affiliated Hospital of Nanchang Medical College) and adhered to the Declaration of Helsinki. Written informed consent was obtained from all participants before inclusion.

NOTE: A total of 60 adult patients with radiologically confirmed peripheral pulmonary nodules (PPNs), ranging from 8 to 30 mm in diameter, were consecutively enrolled in the study. Cryobiopsies were obtained using a 1.1 mm cryoprobe, with forceps biopsy performed in 75% of patients for comparison. The primary outcome was diagnostic yield; secondary outcomes included complication rates, specimen size, tissue quality, and procedural metrics. All participants were referred to the Department of Pulmonary Medicine for diagnostic bronchoscopy due to indeterminate pulmonary nodules identified on chest CT scans. The following criteria were used to select eligible participants: (i) adults (≥18 years) with one or more peripheral pulmonary nodules (defined as lesions located beyond the segmental bronchi and not visible by conventional bronchoscopy) detected on chest computed tomography (CT)^38,39, (ii) nodule size between 8 mm and 30 mm in greatest diameter, and (iii) No contraindications to bronchoscopy or general anaesthesia. The study excluded participants who met any of the following criteria: (i) patients with bleeding diathesis or uncorrectable coagulopathy, (ii) severe hypoxemia (PaO₂ < 60 mmHg on room air), (iii) nodules with endobronchial extension visible on conventional bronchoscopy, and (iv) pregnancy. All participants underwent a comprehensive clinical assessment including: (i) medical history and physical examination, (ii) pulmonary function testing, (iii) coagulation profile, (iv) HRCT review to assess nodule characteristics (size, location, attenuation, and presence of bronchus sign), and (v) a multidisciplinary team reviewed each case before the procedure to assess eligibility and procedural feasibility.

1. Anesthesia and monitoring

1. Sedation/analgesia
   1. Determine sedation strategy based on patient comorbidity and procedural complexity:
      1. Moderate sedation: administer intravenous midazolam (0.02-0.05 mg/kg) and fentanyl (1-2 µg/kg) 3-5 min prior to bronchoscope insertion.
      2. General anesthesia (if indicated): follow institutional anesthesia standard operating procedure (SOP), including induction agents (propofol, 1-2 mg/kg IV), a muscle relaxant if required, and maintenance with inhalational or IV anesthetics.
   2. Ensure airway access and readiness for conversion to general anesthesia if desaturation or airway compromise occurs.
2. Physiologic monitoring
   1. Continuously monitor ECG, non-invasive blood pressure, respiratory rate, and SpO₂ throughout the procedure³⁸.
   2. Maintain supplemental oxygen to keep SpO₂≥ 92%.
   3. Record baseline vital signs and repeat measurements at 5-min intervals or after any intervention.

2. Bronchoscopic navigation and localization

1. Equipment setup
   1. Select an ultrathin bronchoscope with an outer diameter ≤3.0 mm.
   2. Prepare virtual bronchoscopy navigation software (e.g., lung point), R-EBUS probe (20 MHz, 1.4-1.7 mm), and fluoroscopy unit.
   3. Choose a guide sheath (GS) compatible with the bronchoscope (typically 1.95-2.6 mm inner diameter) for lesion stabilization.
2. Lesion navigation
   1. Advance the bronchoscope to the target bronchial segment.
   2. Use virtual bronchoscopy to map the route to the peripheral pulmonary nodule (PPN).
   3. Confirm lesion position using R-EBUS. Identify concentric pattern for central location within the bronchus or eccentric pattern for peribronchial lesions.
   4. Verify lesion location fluoroscopically in two planes (anteroposterior and lateral) to ensure accurate targeting.
   5. Insert the guide sheath through the bronchoscope when required, and secure the position for repeated biopsies⁴⁰.
3. Safety note
   1. Avoid advancing the bronchoscope beyond the segmental bronchi without clear visualization.
   2. Stop procedure if patient develops SpO₂ < 88%, significant arrhythmia, or hemodynamic instability.

3. Cryobiopsy technique

1. Cryoprobe preparation
   1. Use a 1.1 mm flexible cryoprobe.
   2. Pre-cool cryoprobe to operating temperature per manufacturer's instructions.
   3. Prepare liquid nitrogen or compressed CO₂ as per standard protocol for activation.
2. Biopsy execution
   1. Advance the cryoprobe through the bronchoscope working channel to the lesion under R-EBUS and fluoroscopic guidance.
   2. Activate the cryoprobe for 3-5 s, adjusting freezing time according to nodule size, density, and location:
      Small nodules (≤15 mm): 3 s
      Medium nodules (16-25 mm): 4 s
      Large nodules (>25 mm): 5 s
   3. Withdraw the cryoprobe along with the bronchoscope to retrieve frozen tissue en bloc.
   4. Repeat to obtain 2-3 cryobiopsy specimens per lesion.
   5. For comparison, perform a forceps biopsy in selected patients⁴¹. Use standard forceps (2 mm cup) and collect 2-3 samples from the same lesion.
3. Sample handling
   1. Immediately place specimens in formalin for histopathology.
   2. Maintain cold chain if required for molecular studies (4 °C for short-term, snap-freeze in liquid nitrogen for RNA/DNA analysis).
4. Safety note
   1. Minimize probe activation time in eccentric lesions to prevent bronchial wall injury.
   2. Stop biopsy immediately if resistance is felt during withdrawal, or if significant bleeding occurs.

4. Bleeding management

1. Prophylactic measures
   1. Place a Fogarty balloon catheter (4-6 Fr, 1.5-2 mL inflation volume) or bronchial blocker in the segmental bronchus prior to biopsy.
   2. Confirm balloon position fluoroscopically.
2. Bleeding assessment
   1. Evaluate using the Nashville Bleeding Scale:
      Grade 0: no bleeding
      Grade 1: mild, self-limited
      Grade 2: moderate, requiring suction or instillation of cold saline (4 °C)
      Grade 3: severe, requiring balloon tamponade or surgical intervention
3. Hemostasis protocol
   1. For Grade 1-2 bleeding, perform suction of blood using the bronchoscope. Instill 5-10 mL of ice-cold saline at 4 °C.
   2. For Grade 3 bleeding, inflate the Fogarty balloon to tamponade⁴² and maintain the position until hemostasis is achieved.
   3. Prepare for emergency airway management and surgical backup for uncontrolled bleeding.

5. Post-procedural monitoring

1. Immediate observation
   1. Monitor patients for at least 4 h post-procedure for oxygen desaturation, respiratory distress, bleeding, and hemodynamic instability.
   2. Obtain a chest x-ray within 1 h to detect pneumothorax.
2. Follow-up
   1. Record all adverse events in the case report form.
   2. Schedule clinical or radiologic follow-up at 1 week, 1 month, and 3 months.
   3. Confirm diagnosis via histology, imaging, or additional biopsy as needed⁴³.

6. Statistical analysis

1. Data management
   1. Enter categorical variables as counts and percentages.
   2. Record continuous variables as mean ± standard deviation or median (interquartile range [IQR]), depending on distribution.
2. Comparative analysis
   1. Compare proportions using the chi-square test or Fisher's exact test.
   2. Compare continuous variables using Student's t-test or Mann-Whitney U test.
3. Significance threshold
   1. Set alpha at 0.05 for all comparisons.
   2. Use the statistical software menus or syntax:
      Frequencies for categorical variables
      Descriptives for continuous variables
      Crosstabs with chi-square
      ​Nonparametric tests > independent samples for the Mann-Whitney U test.
4. Safety note
   1. Conduct an interim safety review if pneumothorax or severe bleeding exceeds 10% of cases.
      NOTE: Cases with incomplete pathology reports or lost follow-up were excluded from analysis to maintain data integrity.

Results

Participant characteristics
A total of 60 patients were enrolled in the study between January 2023 and December 2024, all of whom presented with peripheral pulmonary nodules. The mean age of participants was 62.4 ± 10.7 years, with an age range spanning from early middle age to elderly patients. The cohort included 34 males (57%) and 26 females (43%), reflecting a slight male predominance. The average nodule size was 18.2 ± 5.7 mm, ranging from 9 mm to 30 mm, suggesting that the study population prim...

Discussion

PPNS are increasingly identified owing to the widespread application of high-resolution imaging modalities. Despite these advances, obtaining an accurate tissue diagnosis, particularly for small, deep, or peripherally located nodules, continues to present a significant clinical challenge. This study demonstrates that ultrathin bronchoscopy (UTB) combined with transbronchial cryobiopsy (TBLC) provides a higher diagnostic yield and a favorable safety profile compared with conventional forceps biopsy.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biopsy forceps	Olympus or equivalent	https://olympusmedical.co.in/products/all-products/endotherapy-devices/pulmonaly-devices/biopsy-forceps/index.html	Standard transbronchial biopsy forceps
Bronchial blocker	Fuji or equivalent	N/A	Alternative to balloon catheter for hemorrhage control
Cryoprobe (1.1 mm)	Erbe Elektromedizin	https://us.erbe-med.com/us-en/products/cryosurgery/cryoprobes-for-erbecryor-2/flexible-cryoprobe-single-use-oe-11-mm/	1.1 mm flexible cryoprobe; used for tissue freezing and biopsy
ECG Monitor	Philips/GE/Schiller	https://www.philips.co.in/healthcare/ambulatory-monitoring-and-diagnostics/ecg-monitoring	For cardiac monitoring during procedures
Fentanyl	Any pharmaceutical company	N/A	Opioid analgesic used during bronchoscopy
Fluoroscope	GE Healthcare or similar	https://www.gehealthcare.com/products/fluoroscopy-systems	Real-time imaging for needle guidance
Fogarty balloon catheter	Edwards Lifesciences	https://www.edwards.com/healthcare-professionals/products-services/vascular-solutions/clot-management	Used for bleeding control during biopsy
Guide sheath (GS)	Olympus	https://olympusmedical.co.in/products/pulmonology/bronchoscopy/endotherapy-devices/guidesheath-systems/index.html	Aids in stabilizing and guiding the bronchoscope
High-resolution CT scanner	Siemens/GE/Philips	https://www.siemens-healthineers.com/computed-tomography/somatom/somatom-force	Imaging for identifying peripheral pulmonary nodules
Midazolam	Any pharmaceutical company	N/A	Sedative used for moderate sedation
Non-invasive BP Monitor	Philips/GE	N/A	For monitoring blood pressure
Pulse oximeter	Masimo/Nellcor	https://www.masimo.com/technology/pulse-oximetry/see-the-difference/	Continuous oxygen saturation monitoring
Radial Endobronchial Ultrasound (r-EBUS)	Olympus	https://medical.olympusamerica.com/products/probes/radial-ebus-probes	Ultrasound probe used to localize lesions
SPSS Software	IBM	v26	Used for statistical analysis
Ultrathin bronchoscope	Olympus or equivalent	https://www.olympus-europa.com/medical/en/Products-and-Solutions/Products/Product/BF-MP190F.html	≤3.0 mm outer diameter; enables access to peripheral nodules
Virtual Bronchoscopy Navigation (LungPoint)	Broncus Medical	https://www.broncus.com/	Pre-procedure navigation and planning system