Bronchoscopic Cytology and Metagenomic Sequencing to Differentiate Cancer Treatment-related and Infectious Lung Injury

Summary

Here, we present a protocol to evaluate bronchoalveolar lavage fluid (BALF) through integrated bronchoscopy-guided collection, cytological analysis, and metagenomic next-generation sequencing (mNGS). This comprehensive workflow accurately distinguishes cancer treatment-related lung injury from pulmonary infections, facilitating precise and timely clinical decisions.

Abstract

Patients with cancer treatment-related lung injury (CTLI) frequently present with non-specific respiratory symptoms and radiological changes that closely mimic infectious pneumonia or tumor progression, presenting a significant challenge for a definitive diagnosis. Traditional diagnostic processes, mainly evaluated through blood biomarkers and standard microbial cultures, usually cannot make a clear diagnosis and take too much time. Here, we present a comprehensive protocol to diagnose CTLI by combining bronchoalveolar lavage fluid (BALF) cytological analysis with metagenomic next-generation sequencing (mNGS). The procedural workflow consists of three primary stages. First, standardized bronchoscopy is performed to obtain high-quality BALF samples. Second, conducting cytological analysis of the obtained BALF samples provides a snapshot of the lung microenvironment. This allows identification of inflammatory features and screening for malignant cells to exclude tumor progression. Finally, mNGS is utilized to identify or exclude active infectious etiologies. This advanced genomic technique achieves rapid, highly sensitive, and unbiased pathogen detection, successfully overcoming the limitations of traditional cultures. Representative results using this method demonstrate that this approach can effectively distinguish immune-related pneumonitis from active pulmonary infections or tumor progression. Compared with traditional diagnostic methods, this protocol has the advantage of quickly and accurately distinguishing CTLI from infectious etiologies and occult malignancies. Ultimately, this standardized workflow clarifies clinical diagnoses, guides critical treatment decisions, and improves patient outcomes.

Introduction

Advances in antitumor treatments, such as immune checkpoint inhibitors and targeted therapies, have significantly improved patient survival but also increased the incidence of non-infectious pulmonary toxicities, collectively known as cancer treatment-related lung injury (CTLI)^{1,2,3,4,5,6,7}. Notably, checkpoint inhibitor pneumonitis (CIP) has emerged as a fatal immune-related adverse event, with overall incidence rates of 2–19% and mortality rates reaching up to 35%^8,9,10. Diagnosing CTLI presents a major clinical challenge because its non-specific respiratory symptoms and radiological manifestations—such as ground-glass opacities, consolidation, or reticular opacities—closely mimic those of infectious pneumonia or tumor progression^11,12,13,14.

Currently, a CTLI diagnosis must be determined by carefully excluding other etiologies¹⁵. Traditional diagnostic workflows heavily rely on blood biomarkers and standard microbial cultures, which are time-consuming and frequently fail to yield a definitive diagnosis^{16,17,18,19,20,21,22,23}. While surgical lung biopsy provides clear histopathological evidence, its invasiveness makes it unsuitable for critically ill cancer patients. Consequently, there is a critical need for an effective, minimally invasive diagnostic approach. In clinical practice, the protocol described herein should be applied to cancer patients who have previously received or are currently receiving anti-tumor treatments (e.g., immunotherapy or targeted therapy) and present with new respiratory symptoms and lung imaging changes. It is particularly recommended when CTLI is clinically suspected, standard non-invasive tests fail to provide a clear diagnosis, and the patient cannot tolerate invasive surgical examinations.

Bronchoscopy combined with bronchoalveolar lavage (BAL) serves as a minimally invasive "liquid biopsy" of the lower respiratory tract²⁴. By integrating BAL fluid (BALF) cytological analysis with metagenomic next-generation sequencing (mNGS), this approach significantly improves upon existing diagnostic workflows. First, BALF cytology provides a direct snapshot of the pulmonary microenvironment; it can identify characteristic inflammatory features²⁵, such as lymphocytosis and an inverted CD4/CD8 ratio in CIP, while simultaneously screening for malignant cells to rule out tumor progression^{26,27,28,29,30}. Second, mNGS achieves rapid, highly sensitive, and unbiased pathogen detection, overcoming the low detection rates and delays of traditional cultures, particularly for fastidious bacteria or atypical pathogens^{31,32,33,34,35}. The combined use of these modalities has been shown to significantly improve the detection of mixed infections and guide precise treatments, drastically reducing patient mortality^4,5.

Despite this clinical potential, there is currently no standardized protocol for comprehensive BALF collection and analysis in the context of CTLI. Here, we introduce a standardized protocol for obtaining and processing BALF via bronchoscopy, explicitly combining cytological evaluation with mNGS. The methodological novelty of this workflow lies in its dual-pronged diagnostic capability: it leverages cytology to map the immune microenvironment and exclude malignancy, alongside mNGS to definitively rule in or rule out active infections. Compared to traditional diagnostic methods, this integrated protocol offers the distinct advantage of rapidly and accurately differentiating CTLI from infectious etiologies and occult tumor progression. Ultimately, this standardized workflow accelerates the diagnostic timeline and guides timely, targeted clinical interventions (Figure 1).

Protocol

This protocol adheres to the principles of the Declaration of Helsinki and was approved by the institutional review board of our hospital. Informed consent was obtained from all participants prior to the procedure.

1. Preprocedure preparation

1. Patient evaluation (within 1 week of bronchoscopy)
   1. Obtain recent laboratory tests, including a complete blood count and coagulation profile, to ensure safety for an invasive procedure (Supplemental File 1).
   2. Perform a 12-lead electrocardiogram.
   3. Assess vital signs and ensure hemodynamic stability.
   4. Measure the patient's resting oxygen saturation on room air to evaluate pulmonary function.
      NOTE: If the SpO₂ is low or the patient has respiratory compromise, consider an arterial blood gas analysis for baseline PaO₂/PaCO₂.
   5. Screen for all contraindications.
   6. Instruct the patient to fast for at least 6 h and avoid drinking water for 2 h before the procedure to reduce aspiration risk.
   7. Perform oral and nasal suction and cleansing immediately prior to the procedure to minimize upper airway contamination.
2. Equipment and personnel preparation
   1. Prepare all required bronchoscopy apparatus and monitoring equipment. Select an appropriate bronchoscope size based on the patient's airway anatomy. Verify the suction functionality of the bronchoscope.
      NOTE: Ensure emergency resuscitation equipment, including supplemental oxygen delivery devices and suction apparatus, is immediately available in the procedure room to manage potential airway complications. Any board-certified bronchoscopist with standard training can execute this procedure independently without specialized certifications.
   2. Warm the sterile physiological saline (0.9% NaCl) intended for lavage to 37 °C.
   3. Draw up 100–150 mL of the warmed sterile saline into multiple 20 mL syringes to facilitate rapid instillation.
3. Patient positioning and monitoring setup
   1. Place the patient supine on the bronchoscopy table.
   2. Install continuous monitoring equipment for the patient before starting the procedure.
      1. Apply electrocardiogram (ECG) leads directly to the patient's chest for continuous cardiac activity monitoring.
      2. Connect a pulse oximeter probe to the patient's finger to continuously monitor blood oxygen saturation and pulse.
      3. Affix a non-invasive blood pressure cuff to the patient's upper arm to continuously detect venous blood pressure.
4. Oxygen support device setup and postprocedure weaning
   1. Prepare and configure the appropriate oxygen support device based on the patient's baseline respiratory status. Complete this configuration prior to initiating the bronchoscopy.
   2. High-flow nasal cannula (HFNC) setup
      1. Select an HFNC system for patients with an SpO₂ ≥ 90% on room air and no hypercapnia. Connect the cannula to the patient and set the initial gas flow rate to ≥60 L/min with an FiO₂ of 1.0. Titrate the FiO₂ continuously during the procedure to maintain SpO₂ ≥ 90%.
   3. Transition strategy for non-invasive ventilation(NIV) patients
      1. Remove the NIV mask and transition the patient to an HFNC system immediately prior to bronchoscope insertion. Set the HFNC gas flow rate to ≥60 L/min with an FiO₂ of 1.0. Perform active airway suctioning during the unmasked period to clear secretions and optimize airway patency.
      2. Closely monitor the patient for desaturation during the temporary transition from NIV to HFNC. If the SpO₂ drops below 90% and remains refractory to suctioning and maximum HFNC settings, abort the bronchoscopy procedure immediately. Promptly reapply the NIV mask to restore positive pressure or escalate to invasive endotracheal intubation if the patient's respiratory status deteriorates.
   4. Invasive mechanical ventilation (IMV) setup
      1. Set the tidal volume to 6–8 mL/kg of predicted body weight, FiO₂to 1.0, positive end-expiratory pressure (PEEP) to 5–8 cmH₂O, and respiratory rate to 12–16 breaths/min. Insert the bronchoscope directly through the established endotracheal tube.
   5. Maintain continuous vital sign monitoring upon completion of the bronchoalveolar lavage (BAL). Titrate supplemental oxygen to sustain SpO₂ between 92% and 96% (or 88% to 92% for patients with chronic hypercapnia).
   6. Verify the patient meets all of the following criteria before reducing oxygen support: complete emergence from sedation, restoration of protective airway reflexes, hemodynamic stability, and the absence of active bleeding or severe bronchospasm.
   7. For HFNC weaning, de-escalate the concentration first, followed by the flow rate. Decrease the FiO₂ in 5% to 10% increments until reaching ≤40%. Subsequently, decrease the flow rate in 5% to 10% increments. Discontinue the HFNC completely when the patient maintains the target SpO₂ with an FiO₂ ≤ 35% and a flow rate ≤ 20 L/min.
   8. For NIV weaning, ensure hypoxemia is improved and vital signs are stable, then proceed with one of two methods:
      1. Decrease the IPAP by 2–3 cmH₂O per adjustment, reduce PEEP to 4–5 cmH₂O, and decrease FiO₂ to ≤40%.
      2. For intermittent weaning, transition the patient to HFNC to facilitate and gradually extend the duration of spontaneous breathing. Reinitiate full NIV support immediately if the patient exhibits tachypnea (>30 breaths/min), tachycardia (>140 beats/min), respiratory muscle fatigue, altered mental status, or SpO₂ < 90%.
   9. For IMV weaning, perform a spontaneous breathing trial or a cuff leak test. Proceed to extubation only after the patient successfully passes the trial, demonstrates adequate spontaneous cough strength, and exhibits minimal airway secretions.
5. Sedation and anesthesia
   1. Inject 15 mL of 2% lidocaine into the nasal mucosa and oropharynx, divided into 2–5 doses.
   2. Instill 10 mL of 2% lidocaine in divided doses through the working channel of the bronchoscope. Apply these doses step-by-step while advancing the bronchoscope into the airway to cover the tracheal mucosa, carina, and bronchial tree.
      NOTE: The absolute maximum safe dose of topical lidocaine is uncertain. Toxicity usually occurs when the serum concentration exceeds 5 µg/mL, but subjective side effects may appear at lower concentrations. Carefully track the total volume of anesthetic administered.
   3. Administer systemic sedation according to patient preference, anxiety degree, and expected procedure tolerance to ensure adequate anesthesia, maximize patient comfort, and reduce coughing. Administer midazolam 2–2.5 mg via slow intravenous injection, or reduce the dose to 0.5–1 mg for frail or elderly patients³⁶.
   4. Monitor SpO₂, ECG, and non-invasive blood pressure (NIBP) continuously is adopted³⁷.
      NOTE: Systemic sedation significantly increases the risks of refractory hypoxemia, airway collapse, and emergency tracheal intubation. Closely monitor the patient throughout the procedure.

2. BAL procedure

NOTE: Perform the BALF collection procedure strictly in accordance with standard protocols and clinical practice guidelines.

1. Target lung segment selection (Figure 2)
   1. Review the patient's most recent high-resolution chest computed tomography (HRCT) scan prior to the procedure to determine the precise anatomical distribution of the pulmonary disease.
   2. Select the target lung segment for the BAL based on the specific disease distribution identified on the HRCT scan.
   3. Direct the bronchoscope to perform the lavage in the most radiographically affected segment or the segment demonstrating the most rapid progression for localized or markedly asymmetric disease.
   4. Direct the bronchoscope to lavage either the right middle lobe or the lingula of the left upper lobe for diffuse or symmetrical disease, as these anatomical sites typically yield an adequate fluid return.
2. Prelavage and sample discard
   1. Instill 20–30 mL of warmed saline through the bronchoscope into the wedged segment. Wait 20–30 s.
   2. Apply gentle suction (negative pressure 50–100 mmHg) to aspirate the fluid back. Collect this first aliquot into a trap and discard it without sending it for analysis, as it may contain mucus and bronchial debris.
3. Main lavage procedure
   1. Connect a sterile syringe containing 0.9% normal saline firmly to the working channel port of the bronchoscope.
   2. Inject 20 mL of saline through the working channel over 3–5 s. Pause immediately for 5–10 s after injection, allowing the liquid to fully disperse and settle into the distal alveolar spaces by gravity.
   3. Apply gentle suction to recover the instilled fluid into a sterile collection trap. Maintain a suction pressure below 100 mmHg to avoid significant airway collapse or damage to the airway mucosa.
   4. Repeat the instillation, pause, and suction sequence strictly until the total infused volume is between 100 and 150 mL to ensure the lower respiratory tract sample is adequate and representative.
      NOTE: Continuously monitor the patient's vital signs, oxygen saturation, and comfort throughout the procedure. Pause the procedure immediately if severe coughing or desaturation occurs and resume only after the patient stabilizes.
4. Fluid recovery technique
   1. Use low suction pressure (≤100 mmHg) to avoid airway collapse.
   2. Employ a “pulsed suction” technique instead of continuous suction: intermittently occlude and release the suction tubing to allow air to mix in, mobilizing the fluid and preventing the bronchus from collapsing against the scope.
   3. Target a retrieval of at least 40–60 mL of BALF.
      ​NOTE: Cease the lavage if the patient develops complications (e.g., bronchospasm, intolerable hypoxemia), even if the full volume is not recovered. Keep the total procedure time for BAL instillation and aspiration under 2–3 min to minimize hypoxia risk.
5. Postlavage care
   1. Withdraw the bronchoscope carefully upon completing the BAL.
   2. Suction any remaining secretions from the central airways.
      ​NOTE: Continue HFNC or NIV support if utilized until the patient is stable in recovery. Monitor the patient for post-procedural complications, such as bleeding or pneumothorax.

3. Sample handling and quality control

1. Measurement, transport, and initial evaluation of the BALF
   1. Record the volume of fluid returned versus the volume instilled immediately during the procedure.
   2. Calculate the recovery percentage using the following formula:
      Recovery percentage = (volume recovered / volume instilled) × 100%
      ​NOTE: A recovery of ≥30% is generally considered a good BALF sample.
   3. Place the collected BALF on ice immediately. Transport the BALF promptly to the laboratory.
      CAUTION: Human BALF samples are potentially infectious. Handle all samples with appropriate standard precautions.
   4. Observe and record the macroscopic color and turbidity of the BALF sample prior to processing.
      ​NOTE: Clear or pale yellow fluid with no visible turbidity is typical for non-infectious states. A cloudy or milky appearance is associated with pulmonary alveolar proteinosis (PAP)³⁸. Purulent fluid indicates a potential bacterial infection. Blood-tinged fluid indicates potential bleeding or diffuse alveolar hemorrhage³⁹.
2. Cell viability assessment
   1. Dilute the BALF to a concentration of approximately 1 × 10⁶ cells/mL using sterile physiological saline.
   2. Mix the diluted BALF with Trypan blue at a 1:1 volume ratio to yield a 20 µL reaction mixture.
      CAUTION: Trypan blue is a toxic chemical and a suspected carcinogen.
   3. Incubate the reaction mixture for 3 min at room temperature (approximately 25 °C).
   4. Place the mixture on a hemocytometer. Examine the cells under a low-power light microscope.
   5. Count a minimum of 200 cells to systematically determine the cell viability.
      ​NOTE: Viable cells remain unstained, transparent, and morphologically intact, whereas non-viable cells take up the blue dye. An initial cell viability of >90%–95% is required to ensure reliable downstream cytology counts.
3. Aliquot distribution
   1. Divide the BALF into designated tubes for different analyses during the lavage operation.
   2. Designate Tube 1 as "discarded" for the first returned aliquot.
   3. Designate Tube 2 for microbiology and allocate 10–20 mL of BALF for the pathogen work-up (e.g., mNGS).
      NOTE: Perform routine bacterial Gram stain/culture, fungal stain/culture, acid-fast bacillus (AFB) smear, and PCR simultaneously in clinical practice.
   4. Designate Tube 3 for cytology and reserve at least 10 mL for total and differential cell counts.
   5. Designate Tube 4 as an optional reserve and store any remaining fluid (at 4 °C for short-term or -80 °C for longer-term) for additional tests.
      ​NOTE: Freeze the mNGS sample at -80 °C immediately if testing cannot start right away. Process cytology samples fresh; if a delay is anticipated, refrigerate the sample at 4 °C for up to 24 h to preserve cell morphology.

4. Laboratory processing of BALF

1. Total cell count
   1. Invert the tube to gently mix the BALF and resuspend the cells.
   2. Dilute the BALF with an appropriate volume of sterile physiological saline if the sample contains macroscopic mucus plugs and debris.
   3. Prewet a piece of sterile medical gauze thoroughly with sterile saline prior to filtration. Filter the diluted sample through two layers of the prewetted sterile medical gauze.
   4. Collect the filtrate into a sterile collection tube. Count the total nucleated cells using a hematology analyzer.
      ​NOTE: Low epithelial cell contamination (<5% of cells) and a minimal proportion of red blood cells (<10%) indicate a high-quality alveolar sample.
2. Differential cell count
   1. Transfer 1 mL of the well-mixed BALF sample into a 1.5 mL microtube. Centrifuge the microtube at 211 × g for 5 min to concentrate the cells.
   2. Remove the supernatant, leaving exactly 50–100 µL of fluid in the microtube. Resuspend the cell pellet thoroughly in the remaining supernatant.
   3. Pipette 10–20 µL of the resuspended cell suspension onto the surface of a clean glass slide, near the frosted edge.
   4. Hold a second clean glass slide at a 30–45° angle to the first slide. Push the second slide forward using a gentle wedge-smear technique to create a thin, uniform cell smear.
   5. Repeat steps 4.2.3 to 4.2.4 to prepare 2–3 identical slides.
   6. Allow the smeared slides to air-dry completely at room temperature.
   7. Stain the slides using the Wright-Giemsa method.
   8. Observe the stained slides under a light microscope using a high-power oil immersion objective (1,000× magnification).
   9. Identify and categorize at least 400 cells to determine the differential cell count.
      NOTE: Maintain any BALF specimens with a high clinical suspicion of malignancy at 4 °C and submit them to the central pathology laboratory for standardized cytocentrifugation within 2 h of sample collection.
3. mNGS pipeline
   1. Inactivate the bronchoalveolar lavage fluid (BALF) sample by incubating it at 65 °C for 30 min.
   2. Transfer 0.5 mL of the inactivated BALF sample into a 1.5 mL microcentrifuge tube containing 1 g of 0.5 mm glass beads.
   3. Secure the microcentrifuge tube to a horizontal platform on a vortex mixer. Agitate the mixture at 2,800–3,200 rpm for 30 min to facilitate mechanical microbial cell wall disruption and subsequent DNA release.
   4. Centrifuge the tube briefly to settle the beads, and carefully transfer 0.3 mL of the supernatant into a new 1.5 mL microcentrifuge tube.
   5. Extract total nucleic acids from the supernatant using a micro-volume DNA extraction kit, strictly following the manufacturer’s instructions.
   6. Measure the concentration of the extracted DNA using a fluorometer.
   7. [PAUSE POINT] Store the extracted DNA at -20 °C for short-term use or -80 °C for long-term storage if library preparation is not performed immediately.
   8. Construct the metagenomics libraries from the extracted DNA utilizing an ultra-low input library preparation kit.
   9. Inspect the constructed libraries to ensure appropriate fragment sizes and sufficient concentration.
   10. Sequence the qualified libraries on a high-throughput sequencing platform.
4. Analysis of sequencing data
   1. Eliminate low-quality reads and adapter sequences from the raw sequencing data using sequence processing software.
   2. Discard reads with a length shorter than 35 base pairs (bp), reads exhibiting low complexity, and reads with a Phred quality score (Q score) below 20.
   3. Align the high-quality reads to the human reference genome (version hg38) using a Burrows-Wheeler Alignment algorithm to identify human DNA sequences.
   4. Discard the aligned human host sequences to isolate the non-host sequencing data.
   5. Blast the remaining non-host sequencing reads against a classification reference database to obtain microbial information at the species level. Construct the database based on published microbial genome databases, such as the reference sequence database at the National Center for Biotechnology Information (NCBI). Ensure it contains comprehensive pathogen data (e.g., 25,863 pathogens comprising 12,142 bacteria, 2,680 fungi, 10,061 viruses, 654 parasites, 206 mycobacteria, and 120 mycoplasma/chlamydia).
   6. Establish negative ('No template' control, NTC) and positive controls by processing them through the identical extraction, library preparation, and bioinformatics analysis procedures.
   7. Examine the strictly mapped read number (SMRN) and genomic coverage for diagnostic assessment.
      NOTE: SMRN denotes the quantity of sequences that are precisely aligned with the microorganism at the species level.
   8. Classify a bacterial, fungal, mycoplasma, or chlamydia mNGS result as positive if the microorganism is not detected in the NTC AND its genomic coverage ranks in the top 10 among microbes in the same genus, OR if the ratio of SMRN_sample to SMRN_NTC is > 10 (when SMRN_NTC ≠ 0).
   9. Confirm that the SMRN_sample meets the following minimum thresholds for a positive result: ≥3 for bacteria, fungi, mycoplasmas, and chlamydiae; ≥100 for parasites; and ≥1 for Mycobacterium tuberculosis.
   10. Classify a viral mNGS result as positive if the virus is not detected in the NTC, OR if the ratio of SMRN_sample to SMRN_NTC is >5 (when SMRN_NTC ≠ 0).
   11. Confirm that the SMRN_sample for viruses is ≥1.
   12. Compile a list of candidate pathogens along with their read counts or relative abundances.
   13. Record any antibiotic resistance genes or virulence factors identified by the bioinformatics pipeline.
   14. Define a significant hit by combining the number of reads, statistical comparison with control groups, and clinical relevance.
   15. Assess whether each identified pathogen can account for the clinical situation.

Results

By following the above standardized protocol, clinicians can reproducibly obtain high-quality BALF samples and leverage both cellular and genomic analyses to distinguish CTLI from its mimics. Below, we present a representative result from our case to illustrate typical findings. A 68-year-old female with stage IV poorly differentiated lung cancer (right pleural region primary tumor) on pembrolizumab immunotherapy is presented to illustrate the BALF cytology + mNGS approach (Figure 1<...

Discussion

In the current era of cancer therapy, treatment-related organ injuries, such as those induced by immune checkpoint inhibitors (ICIs), remain a major clinical concern that can limit treatment progression or even be life-threatening^3,4. Among ICI-induced immune-related adverse events (irAEs), pneumonitis has garnered significant attention as a primary cause of fatal outcomes¹⁰, accounting for 79% of irAE-related deaths in ICI-treated patient...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2% lidocaine	Hunan Kelun	N/A	Local anesthetic for airway mucosa.
0.5 mm glass beads	Sigma	N/A	Used for mechanical cell wall disruption of microbes.
BF-260 electronic bronchoscope	Olympus	N/A	The bronchoscope is used to obtain alveolar lavage fluid.
Burrows-Wheeler Alignment	Open Source	0.7.17(RRID:SCR_010910)	Algorithm for mapping sequences to the human genome.
High-flow humidified oxygen therapy instrument	BMC Medical	N/A	Provide a certain flow, heated and humidified breathing gas for patients with spontaneous breathing.
Vortex mixer	Scientific Industries	N/A	Scientific Industries
Midazolam	Jiangsu Enhua	N/A	Systemic sedative for patient comfort.
Nextseq 550 platform	Illumina	N/A	The gene sequence to sequence the genome in BALF
QIAseq Ultralow Input Library Kit	Tiangen	N/A	Kit for constructing metagenomic library
Qubit 4.0	Thermo Fisher Scientific	N/A	Instrument for detecting BALF nucleic acid content
TIANamp Micro DNA Kit DP316	Tiangen	N/A	The DNA extraction kit to extract total nucleic acids from BALF
Trypan blue	Biosharp	N/A	Dye used for cell viability assessment.
Water bath	Joan	N/A	Used for BALF sample inactivation at 65 °C.
Wright-Giemsa stain	yuanmu	N/A	Used for differential cell staining.
XN-9000 Hematology analyzer	Sysmex	SN-13699	Hematology analyzer to count total nucleated cells from BALF sample