Proposal for a standardized methodology for performing endobronchial ultrasound-guided mediastinal cryobiopsy: a four-step approach

Introduction

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is the preferred method for examining mediastinal and hilar lesions (1). However, inadequate biopsy samples can impact its effectiveness and lower its diagnostic accuracy for certain conditions such as lymphoproliferative and granulomatous disorders (2). In such cases, additional diagnostic procedures like re-biopsies or mediastinoscopy may be necessary, especially when there is a high likelihood of malignancy. It is important to note that there are potential complications associated with both intra and post-operative procedures of video-assisted mediastinoscopy (VAM) and video-assisted mediastinoscopic lymphadenectomy (VAMLA). These complications, including wound infection, mediastinal hematoma and seroma, mediastinitis, pleural effusion, pneumothorax, chylothorax, and left-sided recurrent nerve paralysis, occur in approximately 5–8% of cases (3). In recent years, it has become increasingly important to accurately characterize tissue samples for the treatment of thoracic malignancies, particularly in cases of non-small cell lung cancer (NSCLC). While EBUS-TBNA is effective in diagnosing primary lung cancers, it may not always provide enough tissue for a definitive diagnosis of uncommon tumors, some benign mediastinal conditions or when immunohistochemical and molecular analyses are required. In such cases, a histopathological evaluation and assessment of the surrounding tissue structure is often necessary (4,5). Therefore, mediastinal cryobiopsy seems to play a crucial role in these challenging scenarios. However, there is ongoing debate in the field of interventional pulmonology regarding the best approach for obtaining a mediastinal cryobiopsy. Some interventional pulmonologists use a high-frequency needle knife to create an incision in the tracheobronchial wall adjacent to the mediastinal lesion before inserting the cryoprobe, while others use a needle to create a pathway to the target area. There are also variations in the use of endoscopic or ultrasound imaging for guidance.

In this article, we aim to review the current literature on different methods of performing mediastinal cryobiopsy and share our own clinical experience and methodology in a systematic way for its implementation in a safe, fast, and effective way.

Literature review

Botana-Rial et al. conducted a systematic review of 555 patients, concluding that genetic studies and immunohistochemical determination of programmed death-ligand 1 (PD-L1) were feasible in almost all (97%) of the samples obtained by Cryo-EBUS, while this was only possible in 79% of those obtained by EBUS-TBNA (6). In 2020, Zhang et al. reported the first case of transbronchial mediastinal cryobiopsy (TMC) (7). They described a young male with a mediastinal mass of more than 4 cm who was diagnosed with seminoma. The procedure was performed under local anesthesia and conscious sedation with intravenous midazolam. It involved four passes of TBNA using a 22-gauge needle (Olympus NA-201SX-4022; Olympus, Tokyo, Japan), followed by the insertion of a high-frequency needle knife (Olympus KD-31C-1; Olympus) through the EBUS scope into the airway wall near the location of needle aspiration. After applying cautery, the needle knife was inserted into the mediastinal lesion and then withdrawn. Next, a 1.1 mm cryoprobe (Erbe 20402-401; Erbe, Tübingen, Germany) was inserted into the target area and cooled for 15 seconds before being extracted. Two cryobiopsies were obtained in total. In 2021, Gonuguntla et al. published a case series on four patients who underwent mediastinal cryobiopsy in lymph nodes greater than 1 cm (8). The procedure was carried out as follows: all four patients underwent EBUS-TBNA under general anesthesia with laryngeal mask airway. Initially, a 19-gauge needle was used for puncture, followed by the insertion of a 1.1 mm cryoprobe (Erbe 20402-401; Erbe) into the working channel of the EBUS bronchoscope. The cryoprobe was then guided under endoscopic vision to the puncture site and cooled down for 3 seconds before being removed, resulting in one cryobiopsy for one patient and two for the others.

In 2021, Zhang et al. published a randomized trial involving 197 patients who underwent TMC in lymph nodes greater than 1 cm (9). In their study, all patients underwent four TBNAs and three cryobiopsies. The team again used a high-frequency needle knife to create an incision in the tracheobronchial wall before introducing the cryoprobe into the lesion. The probe was cooled down for 7 seconds. The most common adverse event reported was minor bleeding, which resolved without intervention. Additionally, there were two cases of pneumothorax and one case of pneumomediastinum, both of which resolved spontaneously without the need for drainage. Genova et al. presented a series of five patients who underwent both EBUS-TBNA and TMC for diagnostic and staging purposes (10). Patients with mediastinal lymph nodes with greater diameter ≥2 cm were included. The procedure was carried out under deep sedation using propofol and midazolam. Once suitable lymph nodes were identified through EBUS, the operator performed TBNA with 19-gauge needle at the targeted stations of interest (3 samplings for each station). Afterwards, the puncture hole left by the needle was located through endoscopic vision, and the cryoprobe was inserted into de lymph node. The probe was cooled for 4 seconds, and no major complications were reported.

In 2022, Ariza-Prota et al. published a case series of four patients who underwent mediastinal cryobiopsy (11). The study included patients with mediastinal lymph nodes greater than 1 cm. The procedures were performed under conscious sedation using midazolam and fentanyl. Once the targeted lymph node station was identified, the operator conducted four TBNAs with 22-gauge cytological needle (SonoTip EBUS Pro Flex GUS-45-18-022; Medi-Globe, Rohrdorf, Germany). Following the TBNA puncture, a 1.1 mm cryoprobe (Erbe 20402-401) was inserted into the working channel of the EBUS bronchoscope (EB19-J10U; Pentax Medical) and gently advanced towards the puncture site. The cryoprobe was then introduced gently through the previous puncture site created by the needle. The EBUS image confirmed the cryoprobe’s correct placement within the lymph node. After cooling the cryoprobe for 3 seconds, it was retracted with the bronchoscope along with the frozen biopsy tissue attached to its tip.

In 2023, Fan et al. conducted an open-label, randomized trial at three hospital sites in Europe and Asia to evaluate the safety and added value of combining TMC with standard EBUS-TBNA for diagnosing mediastinal diseases (12). Eligible participants had at least one mediastinal lesion measuring 1 cm or longer in the short axis that required diagnostic bronchoscopy. A total of 271 patients were randomly assigned in a 1:1 ratio to either the combined group, which received both EBUS-TBNA and TMC, or the control group, which received EBUS-TBNA alone. They used a high-frequency needle knife to create an incision in the tracheobronchial wall before introducing the cryoprobe into the lesion. The study found that adding cryobiopsy to standard sampling significantly increased the overall diagnostic yield for mediastinal lesions, with 126 out of 136 participants (93%) in the combined group and 109 out of 135 participants (81%) in the control group achieving successful diagnoses. Subgroup analyses also showed that the combined approach was more sensitive than standard needle aspiration for benign disorders (94% vs. 67%), and it improved the suitability of tissue samples for molecular and immunological analyses of NSCLC. The incidence of adverse events related to the biopsy procedure did not differ between the two trial groups. Their trial concluded that the addition of mediastinal cryobiopsy to standard EBUS-TBNA resulted in a significant improvement in diagnostic yield for mediastinal lesions, with a good safety profile.

In 2023, Ariza-Prota et al. published a prospective study of 50 patients who underwent EBUS-TBNA and TMC using a 22-gauge needle (SonoTip TopGain GUB-42-18-022; Medi-Globe) (13). The authors aimed to simplify the procedure and introduce the Ariza-Pallarés method for its implementation. This was the first time that the mediastinal cryobiopsy procedure was described as completely ultrasound-guided. Patients with mediastinal lesions >1 cm were recruited in the study. All procedures were performed under conscious sedation with midazolam and fentanyl, and with a short oral biter. After identifying the suitable lymph node station with EBUS, three passes of TBNAs were performed. During the last puncture, a “tunnel” was created using the unique crown-cut tip of this needle. The 1.1 mm cryoprobe was then inserted under ultrasound guidance, without the need for endoscopic vision. By focusing only on the ultrasound imaging, the authors were able to identify the trace left by the TBNA puncture and the broken capsule of the lymph node. The position of the cryoprobe within the lymph node was confirmed with the EBUS image. The cryoprobe was cooled down for 4 seconds and retracted with the EBUS scope, with the frozen biopsy tissue attached to its tip. The cryobiopsy site was immediately examined and no complications were reported. The authors state that in numerous cases, the site from the previous puncture could not be located using endoscopic vision. However, with the use of ultrasound imaging, the trace left by the needle inside the lesion and the broken capsule could be identified in all cases, making the process simpler and reproducible in all lymph node stations. All patients received post-procedural chest radiographs or pleural echography to confirm that a pneumothorax had not been produced and the patient was discharged 2 h after verifying that there had been no complications. Follow-up was conducted on all patients at 24 h via phone call and 2 weeks after the procedure to check that there were no delayed complications.

When to do it? Possible indications

• Suspected lymphoproliferative disorders (both for de novo diagnosis and for recurrence /relapse diagnosis);
• Suspicion of benign granulomatous processes (sarcoidosis, silicosis, rare mediastinal infections and ganglionar tuberculosis);
• Metastases from other non-pulmonary or infrequent tumors (seminoma, thymoma, thymic carcinoma, etc.);
• Non-diagnostic EBUS-TBNA;
• Necrotic lymph nodes/lesions;
• NSCLC stages III–IV (immunohistochemical and molecular analysis);
• Restaging the mediastinum after induction chemotherapy and/or radiotherapy for locally advanced NSCLC.

After careful analysis, these are the possible indications that our group determined for considering performing a mediastinal cryobiopsy. It should be noted that these indications are not set in stone and may differ based on the resources of each interventional pulmonology unit, including technology, EBUS-TBNA diagnostic yield, expert cytopathologists, rapid on-site evaluation (ROSE), etc. Therefore, it is crucial to take these individual factors into account when determining the optimal utilization of Cryo-EBUS and delivering top-quality care to your patients. One scenario where Cryo-EBUS is particularly advantageous is in restaging the mediastinum after receiving chemotherapy and/or radiotherapy for locally advanced NSCLC. In these cases, the lymph nodes can become necrotic or stiff, making it challenging to obtain a viable sample through traditional TBNA methods for immunohistochemical and molecular testing. However, since incorporating Cryo-EBUS into our diagnostic approach, we have observed a significant increase in our diagnostic yield from 62% to 94%. Each case should be approached individually as every situation is unique. Regarding the risk of mediastinitis, there is limited data directly linking the procedure to this rare complication. When a necrotic lymph node or lesion is accessible by EBUS, we always opt for TBNA, and we see no reason for this to be any different for Cryo-EBUS. However, if our suspicion is a cystic lesion, we do not perform EBUS-TBNA, and therefore would not perform a mediastinal cryobiopsy in that scenario. One crucial factor to consider is the presence of ROSE, which allows for real-time assessment of sample quality. If ROSE indicates that the sample is too necrotic for accurate immunohistochemical and molecular analysis, or contains very few viable cells, we have seen excellent results with mediastinal cryobiopsy.

When not to do it?

The contraindications of a Cryo-EBUS are the same as for EBUS-TBNA in our experience.

• Current or recent myocardial ischemia;
• Severe hypoxemia;
• Hemodynamic instability;
• Severe pulmonary hypertension;
• Poorly controlled heart failure;
• Chronic obstructive pulmonary disease (COPD)/asthma exacerbation;
• Life-threatening dysrhythmias;
• Patient on anticoagulation/dual antiplatelet therapy;
• Clotting abnormalities;
• Intolerance to sedation/anesthesia;
• Vascular image patterns of grade III–IV on the ultrasound (14):
  • Grade 0: no blood flow or small amounts of flow;
  • Grade I: a few main vessels running toward the center of the lymph node from the hilum;
  • Grade II: a few punctiform or rod-shaped flow signals, a few small vessels found as a long strip of a curve;
  • Grade III: rich flow, more than four vessels found with different diameters and twist or helical flow signal;
  • Grade IV [bronchial artery (BA) inflow sign]: the blood flow from the BA toward the lymph node that was visualized as blue signals on EBUS color Doppler-mode image.

Four-step technique

The Ariza-Pallarés method outlines four crucial steps for effectively performing a mediastinal cryobiopsy, applicable to both the transbronchial (EBUS) and transesophageal [endoscopic ultrasound with bronchoscope (EUS-B)] approaches (15).

Step 1: the planning (select the best place, choose it wisely)

It is crucial to thoroughly evaluate the chest computed tomography (CT) scan or fluorodeoxyglucose positron emission tomography (FDG-PET) to determine the complexity of the procedure. Factors such as location, size, and vascularity of the lesion or lymph node must be taken in consideration to determine the feasibility of the technique. If the planning reveals that the EBUS-TBNA procedure will be challenging, it is likely that the cryobiopsy will also be. However, if the needle is inserted successfully into the lymph node, the 1.1 mm cryoprobe can also be inserted. The initial step in the planning process is to determine whether the lymph node or lesion is in contact with the tracheobronchial wall, regardless of the station. If there is no contact and the space between them is more than 1 cm (Figure 1A), the closest space on the ultrasound image should be chosen for the first TBNA. Here, we present different CT levels from the same patient, showing how the space between the 4R station and the right paratracheal wall decreases as we move down the trachea (Figure 1B), this is a crucial aspect to consider then in the ultrasound imaging, suggesting that the nearer the lesion is to the TBNA entry point, the simpler the procedure will be. Based on our experience, mediastinal cryobiopsy can be performed in all lymph node stations. We have ranked the lymph node stations accessibility from easiest to most challenging as follows: 11L, 11Ri, 7, 11Rs, 4R, 2L, 2R, 10R, 10L, 3p, and 4L.

Figure 1 Thorax CT from different levels for the same patient. (A) Asterisk showing the space between 4R station and the tracheobronchial wall. (B) CT showing how the space between the 4R station and the right paratracheal wall decreases as we move down the trachea. CT, computed tomography.

Step 2: the puncture (the first puncture will guide the rest of the process)

Based on our experience, we recommend that this procedure be performed by two operators. While a high skill bronchoscopist in EBUS-TBNA may be able to perform the technique after mastering the learning curve, we can ensure that having two operators makes the process easier, quicker, and safer. Sedation was performed with midazolam (0.07 mg·kg⁻¹) and fentanyl citrate (0.5–2 µg·kg⁻¹), starting with boluses of 1–3 mg of midazolam and 0.1 µg of fentanyl citrate. Sedation was maintained with intermittent boluses of 1.2 mg midazolam and 0.1 µg fentanyl citrate according to the clinical judgment of the pulmonologist. When viewing the ultrasound image, it is important to always use the Doppler mode to avoid any vessels (Figure 2A), carefully choose the location with the thinnest mucosa and lymph node capsule (Figure 2B), and steer clear of any cartilages before performing the TBNA (Figure 2C). The use of Doppler is essential in this procedure. Here we show a significant distance between the TBNA entry point and the lymph node capsule. This distance is due to the enlarged mucosa and cartilage, which should be avoided during the procedure (Figure 3A,3B).

Figure 2 Lymph node stations and Doppler-mode on the ultrasound image. (A) Arrow showing a vessel in the proximal zone of the lymph node. (B) Arrow showing the location with the thinnest mucosa and lymph node capsule. (C) Needle sheath avoiding cartilage before performing the puncture. ADP, adenopathy.

Figure 3 Distance between the mucosa and the lymph node capsule. (A) Arrow showing significant distance between the TBNA entry point and the lymph node capsule in station 7. (B) Arrow showing the distance between the TBNA entry point and the lymph node capsule in station 4R. ADP, adenopathy; TBNA, transbronchial needle aspiration.

Should we use suction during this step? As with all EBUS-TBNA procedures, we rely on the ultrasound features of the lymph node/lesion and its vascularization in Doppler mode to make this decision. If we observe grade 2 or higher vascularization, suction is typically not used. The use of suction and the specific characteristics of the 22-gauge 3-point needle tip with crown cut (Figure 4), also aid in creating a more distinct trace mark within the lesion, which will serve as a guide for the tunneling step and insertion of the cryoprobe. How many passes are typically performed during the puncture step? This step involves a conventional EBUS-TBNA. Our standard practice is to make 8–12 passes during each TBNA, which is also applicable in this step.

Figure 4 22-gauge SonoTip TopGain crown cut tip needle.

Step 3: “the tunnel”

Once the first TBNA has been performed in the optimal location, we will proceed with the second TBNA while implementing tunneling techniques. First, we must locate the trace left by the TBNA in the lymph node from the previous puncture. To identify this trace, the EBUS operator must be in the same position and angle as the first TBNA, carefully scanning for a subtle line or small white dots within the lymph node. This trace will guide us in making the second TBNA and creating a “tunnel” (Figure 5). Once the trace is localized, we can move on to the second TBNA. During the tunneling process, we do not use aspiration as it may cause further trauma or bleeding in the proximal area of the lymph node. Tunneling involves creating a pathway through the mucosa, submucosa, and capsule of the lymph node to allow for easy insertion of the cryoprobe. If the trace is not clearly visible, we can identify the broken capsule by observing a discontinuity and loss of echogenicity in the usually homogenous and hyperechoic lymph node capsule (Figure 6). To create the “tunnel”, we will first perform 8–12 conventional TBNA passes without aspiration, then shorten the needle length by 1 cm to focus on the proximal part of the lymph node. This TBNA sample is also sent for further analysis. When the needle can easily enter and exit the proximal area without resistance, we can confirm that the capsule has been successfully broken and we can proceed to step 4.

Figure 5 Arrows on the ultrasound image showing the trace left by the TBNA needle inside the lymph node. TBNA, transbronchial needle aspiration.

Figure 6 Arrow pointing at the broken lymph node capsule.

Step 4: the cryobiopsy

Now that the “tunnel” has been made, the next step is to obtain the cryobiopsy sample. This is an ultrasound-guided procedure, and it is crucial to identify the broken capsule and the trace from the previous TBNA, as shown in step 3. The EBUS operator must be in the same position and angle as when performing the second TBNA. The Doppler mode should be used again to ensure that there are no vessels in the cryoprobe’s path (Figure 7). Once the needle trace and the broken capsule are identified, the 1.1 mm cryoprobe is introduced into the working channel of the EBUS bronchoscope (Figure 8A). There is no need to visually locate the puncture site since this is an ultrasound-guided procedure. In this case, it would have been impossible to do so because the puncture site is not visible (Figure 8B). The needle trace can be seen inside the lymph node (Figure 8C). Using ultrasound imaging and Doppler mode, the cryoprobe is gently advanced into the lymph node to confirm its correct position (Figure 9). As for where to take the cryobiopsy samples, we recommend performing three cryobiopsies per station, one distally, one medially, and one more proximal to the capsule (Figure 10A-10C). This allows to obtain samples from different areas of the lymph node. The “fanning” technique can also be performed by adjusting the lever of the EBUS scope to access different zones of the lesion (16). During this process, it is advisable to hold the cryoprobe firmly with the fourth and fifth fingers, and once it is fixed, put the lever of the EBUS scope in a neutral position and press the pedal for 3–5 seconds, and then retracted with the EBUS scope and the frozen biopsy tissue attached to the tip of the probe. The pedal should be pressed until the sample is secured outside the airway (Figure 11). After retrieving the cryobiopsy in saline and fixed formalin (Figure 12), the cryobiopsy site should be examined. Key points in this step: (I) lever of the EBUS bronchoscope in neutral position before taking the scope out with the cryobiopsy sample; (II) always keep the tip of the cryoprobe image on the ultrasound; and (III) keep the pedal close to your foot and keep it pressed until the sample is secured (Video 1).

Figure 7 Doppler mode to ensure that the 1.1 mm cryoprobe path is free of vessels.

Figure 8 Introduction of the cryoprobe. (A) 1.1 mm cryoprobe being introduced gently into the working channel of the EBUS bronchoscope. (B) No endoscopic vision for letting us locate the previous puncture site. (C) Ultrasound image showing the needle trace inside the lymph node. EBUS, endobronchial ultrasound.

Figure 9 Arrow in the ultrasound image and Doppler mode pointing the tip of the cryoprobe in the correct and desired position.

Figure 10 Ultrasound image of the cryoprobe. (A) Ultrasound image confirms the cryoprobe tip positioned distally within the lymph node. (B) Cryoprobe’s tip located in the lymph node’s medial area. (C) Cryoprobe’s tip positioned in the proximal zone of the lymph node.

Figure 11 The Cryoprobe firmly grasped with the fourth and fifth fingers, once fixed, the lever of the EBUS scope should be in neutral position. The pedal needs to be pressed for 3–5 seconds until the sample is secured outside the airway. EBUS, endobronchial ultrasound.

Figure 12 Cryobiopsy attached to the tip of the 1.1 mm cryoprobe.

Conclusions

The Ariza-Pallarés method for performing EBUS-guided mediastinal cryobiopsy is a minimally invasive, feasible, and safe technique that can be performed in a bronchoscopy suite under moderate sedation by a highly experienced interventional pulmonologist in performing EBUS. This novel approach has shown to have a significantly higher diagnostic yield compared to EBUS-TBNA, particularly in the diagnosis of lymphoproliferative disorders, non-pulmonary uncommon tumors, and NSCLC that require further molecular and immunohistochemical testing. We have applied this method using various types of TBNA needles, each of different diameters (19- and 22-gauge) and characteristics (standard needle tip and 3-point needle tip). It is worth noting that the 22-gauge crown cut tip needle only requires a single TBNA to create the “tunnel”, while other conventional needles may require between three and four TBNA’s before the 1.1 mm cryoprobe can be inserted in the desired target. This can significantly prolong the procedure, increase the need for sedation, and potentially increase the risk of complications. Regarding mediastinal cryobiopsy and its potential complications, our experience has shown that we have not encountered any cases of pneumothorax, pneumomediastinum, mediastinitis, or major bleeding. We believe that the complications associated with this technique are no different from those of EBUS-TBNA. However, in studies where pneumomediastinum and pneumothorax have been reported (9,12), the cryoprobe was frozen for more than 6 seconds. It is important to mention that the sample size does not increase after freezing for more than 6 seconds. Additionally, it should be noted that the proximal part of the cryoprobe begins to freeze at this point. This issue is crucial, as freezing for more than 6 seconds can result in obtaining samples not only from the lymph node, but also from the pleura and mucosa, which may contribute to the reported complications in previous studies. Therefore, we recommend freezing for 3–5 seconds, which we believe is both safe and sufficient to obtain high-quality samples for accurate diagnosis. A crucial aspect to consider is the learning curve associated with this technique. Based on our experience, we highly recommend performing a minimum of 30 procedures within a 3-month timeframe and obtaining samples from different lymph node stations rather than solely on one. This approach will greatly aid in mastering the learning curve. The Ariza-Pallarés method demonstrates that the use of a high-frequency needle knife is not necessary for performing mediastinal cryobiopsy; we have eliminated this step of the process by directly introducing the 1.1 mm cryo-probe under ultrasound guidance, therefore, simplifying the procedure and making it more accessible and reproducible. Our objective is to share the Ariza-Pallarés method, so that numerous interventional pulmonology colleagues can employ this technique efficiently and safely in their daily clinical practice. Standardization of the methodology for conducting mediastinal cryobiopsy is crucial for future published trials to be easily comparable.

Acknowledgments

Funding: None.

Footnote

Peer Review File: Available at https://med.amegroups.com/article/view/10.21037/med-23-65/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://med.amegroups.com/article/view/10.21037/med-23-65/coif). The 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. All clinical procedures described in this study were performed in accordance with the ethical standards of the institutional research committee and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patients for publication of this article and accompanying images and video. A copy of the written consent is available for review by the editorial office of this journal.

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