The authors want to thank the interventional pulmonology team, endoscopy staff, anesthesia team, cytopathology team, and hybrid operating room radiology technicians at UT Southwestern Medical Center.

The protocol described in this article outlines standard clinical practice. The University of Texas Southwestern Medical Center Institutional Review Board approved the prospective data collection of patients undergoing standard-of-care bronchoscopy with ssRAB (STU-2021-0346), and individual consent is waived for inclusion in our database. Routine procedure consent is obtained from the patient prior to the procedure. Patients who have DPLD radiographically and are acceptable candidates for bronchoscopic biopsy are referred for this procedure5,27. Patients over 18 years of age are deemed able to undergo the procedure by the referring and performing physicians. Exclusion criteria include bleeding disorders (elevated INR &#62;1.3, thrombocytopenia &#60;100,000/&#181;L), hypoxia with pulse oximetry &#60;90% on 2 L/min supplemental oxygen, pulmonary hypertension (echocardiographically measured systemic pulmonary artery pressure &#62;50 mmHg), or severe cardiac disease. The details of the equipment used in this study are listed in the Table of Materials.
1. Pre-procedural planning
<ol>
	<li>Upload the patient&#8217;s thin slice CT chest to the planning software. The software will automatically create a 3-dimensional reconstruction of the airways and lungs.</li>
	<li>Select targets in the lungs for proposed sampling approximately 10 mm from the pleural border. NOTE: This may be an area of ground glass, infiltrate, nodularity, or fibrosis after discussion with the referring physician, radiologist, or clinical judgment. Prior reported literature for peripheral lung lesions shows an increased diagnostic yield if the targeted area is&#160; &#62;2 cm28.</li>
	<li>Plan a pathway to each target site. 
	NOTE: In the event that the pathway is not feasible during the procedure, consider planning a secondary pathway.</li>
	<li>Review the plan in all three CT views (axial, coronal, and sagittal) and virtual bronchoscopy (Figure 1).</li>
	<li>Export the plan to the controller console.&#160;</li>
</ol>
2. Patient preparation
<ol>
	<li>Induce and maintain the patient under general anesthesia with a minimum size 8.0 single-lumen endotracheal tube. Use neuromuscular blockade and total intravenous anesthesia with ventilator protocols to reduce the development of atelectasis29. 
	NOTE: Monitoring of paralysis is performed using a train-of-four test with a peripheral nerve stimulator29.</li>
	<li>Tuck the patient&#8217;s arms to allow for full rotation of the c-arm during CBCT spin.</li>
	<li>Position the patient and c-arm so that the targeted area for TBLC is isocentered on fluoroscopy.</li>
</ol>
3. Conventional bronchoscopy
<ol>
	<li>Insert the diagnostic or therapeutic bronchoscope via a bronchoscope adapter into the endotracheal tube.</li>
	<li>Perform an airway examination and minimize suction to reduce the development of atelectasis30.</li>
	<li>Remove the bronchoscope.</li>
</ol>
4. Robotic-assisted bronchoscopy
<ol>
	<li>Docking
	<ol>
		<li>Move the robotic bronchoscope to a position adjacent to the patient.</li>
		<li>Dock the robotic arm with the magnetic bronchoscope adapter. Insert the catheter and vision probe into the endotracheal tube.</li>
	</ol>
	</li>
	<li>Registration
	<ol>
		<li>Position the catheter so that direct vision matches the virtual bronchoscope image at the carina.</li>
		<li>Maneuver the robotic catheter via a scroll wheel and track ball on the controller console into both mainstem airways, then bilateral upper and lower airways to collect airway data.</li>
		<li>Compare virtual versus actual bronchoscope images after registration is complete. If significant mismatching or divergence is noted, perform re-registration; otherwise, accept the registration.</li>
	</ol>
	</li>
	<li>Navigation
	<ol>
		<li>Maneuver the catheter using the scroll wheel and track ball on the controller console through the airways to the target lesion following the planned pathway.</li>
		<li>Use the &#8220;Preview Path&#8221; feature to follow images of the airways if divergence (discrepancy between the virtual and actual airways) is noted.</li>
	</ol>
	</li>
	<li>Use fluoroscopy, r-EBUS, and CBCT to confirm location.
	<ol>
		<li>Remove the vision probe when the catheter is within 5&#8211;10 mm of the target lesion.</li>
		<li>Advance the r-EBUS probe with probe rotation under fluoroscopy. Advance to the pleural border (Figure 2A).</li>
		<li>Retract the r-EBUS probe under fluoroscopy approximately 10 mm from the pleural border to the anticipated biopsy target site. Use the r-EBUS probe to visualize the target area and assess the surrounding parenchyma and any vasculature in the potential biopsy area. Remove the r-EBUS probe.</li>
		<li>Insert the 1.1 mm touch cryoprobe via the catheter and extend under fluoroscopy to the pre-determined target area for biopsy (Figure 2B).</li>
		<li>Perform cone beam CT spin per system-specific protocol. The ventilation may be continued or held per provider preference using an end-inspiratory breath hold with the ventilator&#8217;s adjustable pressure-limiting valve set to match the positive end-expiratory pressure (PEEP) or vital capacity maneuver. 
		NOTE: The CBCT spin may be performed with the extension of the r-EBUS probe without rotation or 1.1 mm cryoprobe in the anticipated biopsy site.</li>
		<li>Interpret and compare the intra-procedure imaging to the pre-procedure CT chest and plan to ensure the catheter is at the target. If augmented fluoroscopy is available on the CBCT, segment the target for visualization with 2-D fluoroscopy during biopsy (Figure 3).</li>
		<li>Adjust the catheter based on fluoroscopy, CBCT, and r-EBUS to ensure that sampling occurs in the appropriate location.</li>
		<li>Repeat CBCT after the catheter is adjusted if necessary.</li>
	</ol>
	</li>
	<li>Tissue sampling
	<ol>
		<li>Ensure the 1.1 mm touch cryoprobe is in the appropriate biopsy position.</li>
		<li>Press the pedal to activate the freeze cycle from 4 s to 6 s, then retract the probe in one motion while continuing to depress the pedal.</li>
		<li>Release the pedal while placing the probe tip with tissue biopsy in sodium chloride 0.9% or fixative to release the biopsy from the tip.</li>
		<li>Repeat steps 4.5.1&#8211;4.5.3 to perform TBLC. 
		NOTE: The authors typically perform 1&#8211;4 biopsies at each site. During the biopsy process, the catheter may be adjusted slightly prior to each biopsy to ensure adequate tissue procurement; this may require repeating CBCT spins or using r-EBUS to confirm position in step 4.4.</li>
		<li>After the final biopsy, inject 1&#8211;2 mL of normal saline and air in a 10 mL Leuer lock syringe into the catheter to clear any blood or secretions.</li>
		<li>Insert the vision probe to view the sampling site and retract the catheter slowly. If there is evidence of bleeding via direct vision or blushing on fluoroscopy, then instill topical 1:10,000 epinephrine 1 mL, additional cold saline, or 50&#8211;100mg of tranexamic acid via Leuer lock syringe. Then, leave the catheter in place for 3&#8211;5 min for additional tamponade.</li>
		<li>Repeat step 4.5.6. If there is no evidence of bleeding, retract the catheter to the trachea.</li>
		<li>If significant bleeding is noted, then remove the robotic bronchoscope and follow protocols for the management of iatrogenic bleeding after flexible bronchoscopy31.</li>
	</ol>
	</li>
	<li>Additional target sites: If additional target sites are planned for biopsy, then repeat steps 4.3&#8211;4.5.</li>
	<li>After the conclusion of robotic-assisted bronchoscopy, retract the catheter, undock the robotic system, and move out of position.</li>
</ol>
5. Conventional bronchoscopy&#160;
<ol>
	<li>Reinsert the diagnostic or therapeutic bronchoscope through the bronchoscope adapter into the airways for airway examination and suctioning.</li>
	<li>If bronchoalveolar lavage is indicated, advance the bronchoscope into a subsegmental airway where TBLC was not performed to create a wedge. Instill serial aliquots of normal saline and then return via hand suction.&#160;</li>
</ol>
6. Procedure conclusion
<ol>
	<li>Remove the bronchoscope.</li>
	<li>Perform fluoroscopy or focused ultrasound5 to assess for pneumothorax. If pneumothorax is identified, consider the placement of a chest tube versus conservative management with serial observation depending on size and patient&#8217;s clinical status.</li>
	<li>If necessary, transfer the TBLC specimens to the container with a fixative if initially placed in 0.9% sodium chloride.</li>
	<li>Reverse anesthesia, extubate, and awaken the patient.</li>
	<li>Transfer the patient to the post-anesthesia care unit.</li>
	<li>Perform and review post-procedure chest radiograph (Figure 4).</li>
</ol>
7. Follow-up post procedure
<ol>
	<li>Review results from the bronchoscopy at a multi-disciplinary discussion attended by pulmonologists who are experts in interstitial lung disease, thoracic radiologists, and thoracic pathologists to determine an ILD subtype.</li>
	<li>Discuss the results of the bronchoscopy and MDD conference with the patient, as well as plans for further management and follow-up.&#160;</li>
</ol>

Advancements in Pulmonary Lesion Biopsy Through Robotic Bronchoscopy

<ol>
	<li>Jain, P., Hadique, S., Mehta, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transbronchial+lung+biopsy.">Transbronchial lung biopsy.</a> Interventional Bronchoscopy: A Clinical Guide. , 15-44 (2013).</li><li>Maher, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+lung+disease:+A+review.">Interstitial lung disease: A review.</a> JAMA. 331 (19), 1655-1665 (2024).</li><li>Wahidi, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+forceps,+cryoprobe,+and+thoracoscopic+lung+biopsy+for+the+diagnosis+of+interstitial+lung+disease:+The+chill+study.">Comparison of forceps, cryoprobe, and thoracoscopic lung biopsy for the diagnosis of interstitial lung disease: The chill study.</a> Respiration. 101 (4), 394-400 (2022).</li><li>Hutchinson, J. P., Fogarty, A. W., Mckeever, T. M., Hubbard, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-hospital+mortality+after+surgical+lung+biopsy+for+interstitial+lung+disease+in+the+United+States.+2000+to+2011.">In-hospital mortality after surgical lung biopsy for interstitial lung disease in the United States. 2000 to 2011.</a> Am J Respir Crit Care Med. 193 (10), 1161-1167 (2016).</li><li>Davidsen, J. R., Laursen, C. B., Skaarup, S. H., Kronborg-White, S. B., Juul, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transbronchial+lung+cryobiopsy+for+diagnosing+interstitial+lung+diseases+and+peripheral+pulmonary+lesions-a+stepwise+approach.">Transbronchial lung cryobiopsy for diagnosing interstitial lung diseases and peripheral pulmonary lesions-a stepwise approach.</a> J Vis Exp. (197), e65753 (2023).</li><li>Korevaar, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=European+Respiratory+Society+guidelines+on+transbronchial+lung+cryobiopsy+in+the+diagnosis+of+interstitial+lung+diseases.">European Respiratory Society guidelines on transbronchial lung cryobiopsy in the diagnosis of interstitial lung diseases.</a> Eur Respir J. 60 (5), 2200425 (2022).</li><li>Raghu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+pulmonary+fibrosis+(an+update)+and+progressive+pulmonary+fibrosis+in+adults:+An+official+ATS/ERS/JRS/ALAT+clinical+practice+guideline.">Idiopathic pulmonary fibrosis (an update) and progressive pulmonary fibrosis in adults: An official ATS/ERS/JRS/ALAT clinical practice guideline.</a> Am J Respir Crit Care Med. 205 (9), e18-e47 (2022).</li><li>Maldonado, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transbronchial+cryobiopsy+for+the+diagnosis+of+interstitial+lung+diseases:+Chest+guideline+and+expert+panel+report.">Transbronchial cryobiopsy for the diagnosis of interstitial lung diseases: Chest guideline and expert panel report.</a> Chest. 157 (4), 1030-1042 (2020).</li><li>Rodrigues, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+yield+and+safety+of+transbronchial+lung+cryobiopsy+and+surgical+lung+biopsy+in+interstitial+lung+diseases:+A+systematic+review+and+meta-analysis.">Diagnostic yield and safety of transbronchial lung cryobiopsy and surgical lung biopsy in interstitial lung diseases: A systematic review and meta-analysis.</a> Eur Respir Rev. 31 (166), 210280 (2022).</li><li>Tang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transbronchial+lung+cryobiopsy+for+peripheral+pulmonary+lesions.+A+narrative+review.">Transbronchial lung cryobiopsy for peripheral pulmonary lesions. A narrative review.</a> Pulmonology. S2531-o437 (23), 00163 (2023).</li><li>Fielding, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+human+use+of+a+new+robotic-assisted+fiber+optic+sensing+navigation+system+for+small+peripheral+pulmonary+nodules.">First human use of a new robotic-assisted fiber optic sensing navigation system for small peripheral pulmonary nodules.</a> Respiration. 98 (2), 142-150 (2019).</li><li>Benn, B. S., Romero, A. O., Lum, M., Krishna, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robotic-assisted+navigation+bronchoscopy+as+a+paradigm+shift+in+peripheral+lung+access.">Robotic-assisted navigation bronchoscopy as a paradigm shift in peripheral lung access.</a> Lung. 199 (2), 177-186 (2021).</li><li>Kalchiem-Dekel, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-sensing+robotic-assisted+bronchoscopy+in+the+diagnosis+of+pulmonary+parenchymal+lesions.">Shape-sensing robotic-assisted bronchoscopy in the diagnosis of pulmonary parenchymal lesions.</a> Chest. 161 (2), 572-582 (2022).</li><li>Ali, M. S., Ghori, U. K., Wayne, M. T., Shostak, E., De Cardenas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+performance+and+safety+profile+of+robotic-assisted+bronchoscopy:+A+systematic+review+and+meta-analysis.">Diagnostic performance and safety profile of robotic-assisted bronchoscopy: A systematic review and meta-analysis.</a> Ann Am Thorac Soc. 20 (12), 1801-1812 (2023).</li><li>Oberg, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+robotic-assisted+cryobiopsy+for+peripheral+pulmonary+lesions.">Novel robotic-assisted cryobiopsy for peripheral pulmonary lesions.</a> Lung. 200 (6), 737-745 (2022).</li><li>Verhoeven, R. L., F&#252;tterer, J. J., Hoefsloot, W., Van Der Heijden, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone-beam+CT+image+guidance+with+and+without+electromagnetic+navigation+bronchoscopy+for+biopsy+of+peripheral+pulmonary+lesions.">Cone-beam CT image guidance with and without electromagnetic navigation bronchoscopy for biopsy of peripheral pulmonary lesions.</a> J Bronchology Interv Pulmonol. 28 (1), 60 (2021).</li><li>Verhoeven, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone-beam+ct+and+augmented+fluoroscopy&#8211;guided+navigation+bronchoscopy:+Radiation+exposure+and+diagnostic+accuracy+learning+curves.">Cone-beam ct and augmented fluoroscopy&#8211;guided navigation bronchoscopy: Radiation exposure and diagnostic accuracy learning curves.</a> J Bronchology Interv Pulmonol. 28 (4), 262 (2021).</li><li>Kheir, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone-beam+computed+tomography-guided+electromagnetic+navigation+for+peripheral+lung+nodules.">Cone-beam computed tomography-guided electromagnetic navigation for peripheral lung nodules.</a> Respiration. 100 (1), 44-51 (2021).</li><li>Setser, R., Chintalapani, G., Bhadra, K., Casal, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+beam+CT+imaging+for+bronchoscopy:+A+technical+review.">Cone beam CT imaging for bronchoscopy: A technical review.</a> J Thorac Dis. 12 (12), 7416 (2020).</li><li>Wagh, A., Ho, E., Murgu, S., Hogarth, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+diagnostic+yield+of+navigational+bronchoscopy+for+peripheral+pulmonary+lesions:+A+review+of+advancing+technology.">Improving diagnostic yield of navigational bronchoscopy for peripheral pulmonary lesions: A review of advancing technology.</a> J Thorac Dis. 12 (12), 7683 (2020).</li><li>Styrvoky, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-sensing+robotic-assisted+bronchoscopy+with+concurrent+use+of+radial+endobronchial+ultrasound+and+cone+beam+computed+tomography+in+the+evaluation+of+pulmonary+lesions.">Shape-sensing robotic-assisted bronchoscopy with concurrent use of radial endobronchial ultrasound and cone beam computed tomography in the evaluation of pulmonary lesions.</a> Lung. 200 (6), 755-761 (2022).</li><li>Styrvoky, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+dose+of+cone+beam+ct+combined+with+shape+sensing+robotic+assisted+bronchoscopy+for+the+evaluation+of+pulmonary+lesions:+An+observational+single+center+study.">Radiation dose of cone beam ct combined with shape sensing robotic assisted bronchoscopy for the evaluation of pulmonary lesions: An observational single center study.</a> J Thorac Dis. 15 (9), 4836 (2023).</li><li>Inomata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+radial+endobronchial+ultrasonography+combined+with+transbronchial+lung+cryobiopsy+in+patients+with+diffuse+parenchymal+lung+diseases:+A+multicentre+prospective+study.">Utility of radial endobronchial ultrasonography combined with transbronchial lung cryobiopsy in patients with diffuse parenchymal lung diseases: A multicentre prospective study.</a> BMJ Open Respir Res. 8 (1), 000826 (2021).</li><li>Zhou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+diagnostic+efficacy+of+cone+beam+computed+tomography-guided+transbronchial+cryobiopsy+for+interstitial+lung+disease:+A+cohort+study.">Safety and diagnostic efficacy of cone beam computed tomography-guided transbronchial cryobiopsy for interstitial lung disease: A cohort study.</a> Eur Respir J. 56 (2), 2000724 (2020).</li><li>Benn, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+beam+CT+guidance+improves+transbronchial+lung+cryobiopsy+safety.">Cone beam CT guidance improves transbronchial lung cryobiopsy safety.</a> Lung. 199, 485-492 (2021).</li><li>Ali, S. O., Castellani, C., Benn, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transbronchial+lung+cryobiopsy+performed+with+cone+beam+computed+tomography+guidance+versus+fluoroscopy:+A+retrospective+cohort+review.">Transbronchial lung cryobiopsy performed with cone beam computed tomography guidance versus fluoroscopy: A retrospective cohort review.</a> Lung. 202 (1), 1-9 (2023).</li><li>Hackner, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transbronchial+lung+cryobiopsy:+Prospective+safety+evaluation+and+90-day+mortality+after+a+standardized+examination+protocol.">Transbronchial lung cryobiopsy: Prospective safety evaluation and 90-day mortality after a standardized examination protocol.</a> Thera Adv Respir Dis. 16, 17534666221077562 (2022).</li><li>Kops, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+yield+and+safety+of+navigation+bronchoscopy:+A+systematic+review+and+meta-analysis.">Diagnostic yield and safety of navigation bronchoscopy: A systematic review and meta-analysis.</a> Lung Cancer. 180, 107196 (2023).</li><li>Pritchett, M. A., Lau, K., Skibo, S., Phillips, K. A., Bhadra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+considerations+to+reduce+motion+and+atelectasis+during+advanced+guided+bronchoscopy.">Anesthesia considerations to reduce motion and atelectasis during advanced guided bronchoscopy.</a> BMC Pulm Med. 21 (1), 1-10 (2021).</li><li>Paradis, T. J., Dixon, J., Tieu, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+bronchoscopy+in+the+diagnosis+of+airway+disease.">The role of bronchoscopy in the diagnosis of airway disease.</a> J Thorac Dis. 8 (12), 3826 (2016).</li><li>Bernasconi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iatrogenic+bleeding+during+flexible+bronchoscopy:+Risk+factors,+prophylactic+measures+and+management.">Iatrogenic bleeding during flexible bronchoscopy: Risk factors, prophylactic measures and management.</a> ERJ Open Res. 3 (2), 00084 (2017).</li><li>Bian, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnostic+efficiency+and+safety+of+transbronchial+lung+cryobiopsy+using+1.1-mm+cryoprobe+in+diagnosing+interstitial+lung+disease.">The diagnostic efficiency and safety of transbronchial lung cryobiopsy using 1.1-mm cryoprobe in diagnosing interstitial lung disease.</a> Research Square. , (2024).</li><li>Kronborg-White, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pilot+study+on+the+use+of+the+super+dimension+navigation+system+for+optimal+cryobiopsy+location+in+interstitial+lung+disease+diagnostics.">A pilot study on the use of the super dimension navigation system for optimal cryobiopsy location in interstitial lung disease diagnostics.</a> Pulmonology. 29 (2), 119-123 (2023).</li><li>Abdelghani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+modalities+during+navigational+bronchoscopy.">Imaging modalities during navigational bronchoscopy.</a> Expert Rev Respir Med. 18 (3-4), 175-188 (2024).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnostic+value+of+transbronchial+lung+cryobiopsy+combined+with+rapid+on-site+evaluation+in+diffuse+lung+diseases:+A+prospective+and+self-controlled+study.">The diagnostic value of transbronchial lung cryobiopsy combined with rapid on-site evaluation in diffuse lung diseases: A prospective and self-controlled study.</a> BMC Pulm Med. 22 (1), 124 (2022).</li><li>Goorsenberg, A., Kalverda, K. A., Annema, J., Bonta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+optical+coherence+tomography+and+confocal+laser+endomicroscopy+in+pulmonary+diseases.">Advances in optical coherence tomography and confocal laser endomicroscopy in pulmonary diseases.</a> Respiration. 99 (3), 190-205 (2020).</li><li>Kheir, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+bronchoscopic+lung+cryobiopsy+and+a+genomic+classifier+in+the+multidisciplinary+diagnosis+of+diffuse+interstitial+lung+diseases.">Using bronchoscopic lung cryobiopsy and a genomic classifier in the multidisciplinary diagnosis of diffuse interstitial lung diseases.</a> Chest. 158 (5), 2015-2025 (2020).</li><li>Chaudhary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+lung+disease+progression+after+genomic+usual+interstitial+pneumonia+testing.">Interstitial lung disease progression after genomic usual interstitial pneumonia testing.</a> Eur Respir J. 61 (4), (2023).</li><li>Tian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+confocal+laser+endomicroscopy+in+pulmonary+medicine.">The role of confocal laser endomicroscopy in pulmonary medicine.</a> Eur Respir Rev. 32 (167), 2201245 (2023).</li></ol>

DP has no conflicts of interest to declare. KS reports a relationship with Intuitive Surgical Inc. that includes travel reimbursement.

This manuscript provides a stepwise approach for performing RAB with fluoroscopy, r-EBUS, and cone beam CT to obtain targeted TBLC.&#160;
There are several critical steps in this protocol. First, patient selection is imperative to ensure patients are both appropriate candidates (the biopsy procedure may have a direct impact on diagnosis and further care) and medically able to undergo the procedure5,6. Pre-procedure preparation includes...

Flexible bronchoscopy with transbronchial lung biopsy (TBBX) is a diagnostic modality used for the evaluation of abnormal chest imaging, including masses, nodules, non-resolving infiltrates, or parenchymal lung diseases1. Diffuse parenchymal lung diseases (DPLD) can often be characterized by fibrosis and/or inflammation. While some patients can be diagnosed noninvasively with a thorough history, physical examination, relevant serologies, high-resolution computed tomography (HRCT) findings, and multi-disciplinary discussion (MDD), many patients need an invasive procedure to establish a diagnosis2. Conventional transbronchial lung biopsies with forceps are limited due to small biopsy size and crush artifacts; as a result, surgical lung&#160;biopsy has been considered the gold standard, although it has significant morbidity and mortality3,4.
Transbronchial lung cryobiopsy (TBLC) is a technique that can be used to diagnose interstitial lung disease (ILD) or diffuse parenchymal lung disease (DPLD) and might serve as an alternative to surgical lung biopsy (SLB)5. According to the European Respiratory Society guidelines, TBLC is recommended as a substitute for SLB in eligible patients6. Similarly, the American Thoracic Society guidelines offer a conditional recommendation for TBLC as an alternative to SLB in medical centers with the necessary expertise in performing and interpreting TBLC results7. TBLC has historically provided good accuracy in diagnosis compared to SLB but is limited by complications, including bleeding and pneumothorax8. A recent meta-analysis showed an overall diagnostic yield of 77% that improved to 80.7% with MDD, and reported a pneumothorax rate of 9.2% and bleeding rate of 9.9%9. TBLC is also used in the evaluation of PPLs10.&#160;
The development of robotic-assisted bronchoscopy (RAB) allows for targeted sampling in the lung by navigating through the airways under direct vision with easy catheter maneuverability, precise catheter tip articulation, stability, and the ability to maintain a bronchoscopic wedge in distal airways with the catheter using a robotic arm. The Ion endoluminal system utilizes shape-sensing technology for navigation to access specific targeted areas in the lung. Studies using shape-sensing robotic-assisted bronchoscopy (ssRAB) have shown favorable diagnostic outcomes and safety profile, primarily for PPLs suspicious of malignancy11,12,13,14. A 1.1 mm cryoprobe for TBLC combined with ssRAB has been shown to be safe and effective for the diagnosis of pulmonary nodules compared to transbronchial biopsy with forceps15. This technique can be used to obtain targeted lung biopsies larger than conventional transbronchial biopsies using forceps that are relatively free of crush artifacts.&#160;
Radial endobronchial ultrasound (r-EBUS) and cone beam computed tomography are used in conjunction with conventional bronchoscopy, electromagnetic, or robotic navigational systems for real-time confirmation prior to sampling PPLs16,17,18,19,20,21,22. R-EBUS has also been utilized during TBLC for DPLD to increase the pathologic confidence of lung specimens, decrease bleeding, and have a shorter procedure time23. The addition of CBCT has improved the safety profile of TBLC for DPLD by confirming the probe tip is in a safe zone for biopsy, allowing objective measurement of the distance from the pleura with the ability to visualize and avoid vasculature24,25,26.&#160;
This protocol will describe a procedure to obtain targeted TBLC in the setting of parenchymal lung disease for patients who are able to tolerate and benefit from the procedure using the Ion endoluminal system in conjunction with fluoroscopy, r-EBUS, and CBCT in a clinical setting under general anesthesia. This multimodal approach allows for precise sampling of targeted areas of interest.&#160;

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% normal saline, 1000 mL</td>
			<td>Any make</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Leuer lock syringes</td>
			<td>Any make</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL slip tip syringes</td>
			<td>Any make</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bronchoscope</td>
			<td>Intuitive</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bronchoscope processor and video screens</td>
			<td>Intuitive</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide gas tank</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cone beam computed tomography system with c-arm and controller console</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable valve for biopsy channel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable valve for suction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ERBECRYO 2 1-pedal footswitch AP &#38; IP X8 Equipment US</td>
			<td>Erbe</td>
			<td>20402-201</td>
			<td></td>
		</tr>
		<tr>
			<td>ERBECRYO 2 Cart</td>
			<td>Erbe</td>
			<td>20402-300</td>
			<td></td>
		</tr>
		<tr>
			<td>ERBECRYO 2 Cryosurgical unit</td>
			<td>Erbe</td>
			<td>10402-000</td>
			<td></td>
		</tr>
		<tr>
			<td>ERBECRYO 2 System</td>
			<td>Erbe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible Cryoprobe, OD 1.1 mm, L1.15 m with oversheath, OD 2.6 mm, L817 mm</td>
			<td>Erbe</td>
			<td>20402-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible gas hose; L 1m for Erbokryo CA/AE/ERBECRYO 2</td>
			<td>Erbe</td>
			<td>20410-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas bottle adapter H; CO2; Pin index</td>
			<td>Erbe</td>
			<td>20410-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion endoluminal system with robotic arm, controller console</td>
			<td>Intuitive</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ion fully articulating catheter</td>
			<td>Intuitive</td>
			<td>490105</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion instruments and accessories</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ion peripheral vision probe</td>
			<td>Intuitive</td>
			<td>490106</td>
			<td></td>
		</tr>
		<tr>
			<td>Laptop with PlanPoint planning software</td>
			<td>Intuitive</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Probe driving unit</td>
			<td>Olympus</td>
			<td>MAJ-1720</td>
			<td></td>
		</tr>
		<tr>
			<td>Radial EBUS Probe</td>
			<td>Olympus</td>
			<td>UM-S20-17S or UM-S20-20R-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Radial endobronchial ultrasound system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen containers with fixative per institution standards</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile disposable cups</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suction tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Topical 1:10,000 epinephrine, 10 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Topical tranexamic acid 1000mg, 10 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Universal ultrasound processor&#160;</td>
			<td>Olympus</td>
			<td>EU-ME2</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire basket; 339 x 205 x 155 / 100 mm</td>
			<td>Erbe</td>
			<td>20180-010</td>
			<td></td>
		</tr>
	</tbody>
</table>

robotic-assisted bronchoscopy combined with multimodal imaging for targeted lung cryobiopsies

Robotic-assisted bronchoscopy (RAB) allows for targeted bronchoscopic biopsy in the lung. A robotic-assisted bronchoscope is navigated through the airways under direct vision after establishing a pathway to a target lesion based on mapping performed on a 3-dimensional (3D) lung and airway reconstruction obtained from a pre-procedure thin-slice computed tomography chest. RAB has maneuverability to distal airways throughout the lung, precise catheter tip articulation, and stability with the robotic arm. Adjunct imaging tools such as fluoroscopy, radial endobronchial ultrasound (r-EBUS), and cone beam computed tomography (CBCT) can be used with RAB. Studies using shape-sensing robotic-assisted bronchoscopy (ssRAB) have shown favorable diagnostic outcomes and safety profiles in both malignant and non-malignant processes for the biopsy of peripheral pulmonary lesions (PPLs). A 1.1 mm cryoprobe combined with ssRAB has been shown to be safe and effective for the diagnosis of PPLs compared to a traditional bronchoscopy with forceps biopsy. This technique can also be used for targeted lung sampling in benign processes. The aim of this article is to describe a stepwise approach to performing RAB combined with fluoroscopy, r-EBUS, and CBCT to obtain targeted transbronchial lung cryobiopsies (TBLC).

The described technique allows for targeted transbronchial lung cryobiopsies via RAB with fluoroscopy, r-EBUS, and CBCT guidance. Compared to conventional bronchoscopy with random TBLC, this technique allows for targeting specific areas of DPLD or PPLs of interest while assessing surrounding structures prior to biopsy. This technique can be used with r-EBUS and fluoroscopy only or with a combination of CBCT. While this technique had been devised for PPLs, it can be utilized in benign and diffuse parenchymal lung...

Author Spotlight: Expanding Interventional Pulmonology Research with Robotic-Assisted Bronchoscopy

This article aims to describe a stepwise approach to performing robotic-assisted bronchoscopy combined with fluoroscopy, radial endobronchial ultrasound, and cone beam computed tomography to obtain targeted transbronchial lung cryobiopsies.&#160;

Robotic-assisted Bronchoscopy Combined with Multimodal Imaging for Targeted Lung Cryobiopsies

Robotic-assisted bronchoscopy (RAB) allows for targeted bronchoscopic biopsy in the lung. A robotic-assisted bronchoscope is navigated through the airways ...

robotic-assisted-bronchoscopy-combined-with-multimodal-imaging-for

Research

JoVE Journal

Medicine

320 Views.  University of Texas Southwestern Medical Center. This article aims to describe a stepwise approach to performing robotic-assisted bronchoscopy combined with fluoroscopy, radial endobronchial ultrasound, and cone beam computed tomography to obtain targeted transbronchial lung cryobiopsies. 

Video: Author Spotlight: Expanding Interventional Pulmonology Research with Robotic-Assisted Bronchoscopy

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.
The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.
Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

This article describes bronchoscopic techniques in the bovine lung under experimental conditions, i.e. bronchoscopically guided inoculation, bronchoalveolar lavage, bronchial brushing, and transbronchial lung biopsy.

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

Flexible Bronchoscopy for Novice Bronchoscopists

Systematic Bronchoscopy: the Four Landmarks Approach

Flexible bronchoscopy is a technically difficult procedure and has been identified as the most important procedure that should be integrated into a simulation-based training program for pulmonologists. However, more specific guidelines that govern bronchoscopy training are needed to meet this demand. To ensure patients a competent examination, we propose a systematic, stepwise approach, splitting the procedure into four &#34;landmarks&#34; to support novice endoscopists navigating the bronchial maze. The procedure can be evaluated based on three established outcome measures to ensure a thorough and effective inspection of the bronchial tree: diagnostic completeness, structured progress, and procedure time.
The stepwise approach relying on the four landmarks is used at all simulation centers in Denmark and is being implemented in the Netherlands. To provide instant feedback to novice bronchoscopists when training and to relieve time constraints from consultants, we suggest that future studies should implement artificial intelligence&#160;as a feedback and certification tool when training new bronchoscopists.

Author Spotlight: Learning Systematic Bronchoscopy in a Simulation-Base Setting 

Here, we present a protocol to navigate the bronchial maze in a structured manner, splitting the bronchoscopy into a stepwise approach-the four landmarks approach.

Flexible bronchoscopy is a technically difficult procedure and has been identified as the most important procedure that should be integrated into a ...