A protocol was approved by the local Institutional Review Board (IRB). Participants provide signed informed consent prior to completing the protocol.
1.&#160;Pre-procedure
<ol>
	<li>Ensure that the candidates for the electromagnetic navigational transthoracic needle aspiration have a peripheral pulmonary nodule. Determine the pulmonary nodule use of imagining studies. 
	NOTE: The most common one and needed for this protocol is by evaluating previously obtained chest CT.</li>
	<li>On the day of the procedure attach&#160;the electronic reference point&#160;pads to the contralateral hemithorax of the target lesion to minimize pad overlap with the TTNA biopsy entrance point. Obtain a baseline inspiratory and expiratory non-contrast chest tomography with 0.5 mm intervals and 0.67 - 0.75 mm thickness.</li>
	<li>Transfer the digital imaging and communication in medicine (DICOM) data from the CT scanner to the planning software. If DICOM is unavailable, a compact disk (CD) copy of the CT can be directly loaded onto the system for planning. Digitally select the target lesion and reconstruct a three-dimensional virtual airway map.</li>
	<li>Transport the patient by stretcher from the CT scanner to the bronchoscopy suite with electronic reference points in place.</li>
	<li>Evaluate the patient for anesthesia, establish I.V. access and attach standard anesthetic monitoring.</li>
	</ol>
 class="jove_title"2. Procedure (Bronchoscopic Phase)
<ol>
	<li>Transfer the patient to the bronchoscopy suite. Administer deep sedation&#160;anesthesia using a propofol infusion (100 - 200 mcg/kg/min). An airway is established with laryngeal mask airway (LMA) or endotracheal tracheal tube (ETT). 
	NOTE: Anesthesia can only be administered by a certified provider.</li>
	<li>Perform a white light bronchoscopy (WLB) inspection in the standard fashion examining the tracheobronchial tree to the segmental level.</li>
	<li>Perform an EBUS-TBNA following the International Association for the Study of Lung Cancer (IASLC) lymph node map for lung cancer staging. If available, fine needle aspiration (FNA) specimens are cytological analyzed on-site. If not, the sample is place in formalin for later evaluation.</li>
	<li>Use the WLB bronchoscope with the EMN bronchoscopy program phase following the previously computer recognized or manually planed endobronchial pathway to the target lesion with an instrument of choice at the proceduralists discretion (Cytology brush, jaw biopsy forces, FNA Needle).</li>
	<li>Advance the R-EBUS through the working channel and attempt to confirm the location of the lesion.</li>
	<li>Once in the target, sample the lesion with the operator selected trackable instrument used to navigate. (Cytology brush, jaw biopsy forces, FNA Needle).</li>
	<li>If the target is unable to be verified by radial EBUS or the airway anatomy prohibits endobronchial navigation remove the bronchoscope and prepare the patient for a transthoracic approach. &#160;If the lesion cannot be diagnosed bronchoscopically prepare the patient for a transthoracic approach.</li>
	</ol>
3. Procedure (Electromagnetic Transthoracic Needle Aspiration)
<ol>
	<li>Allocate all supplies needed for the TTNA. (see Material&#160;Table)</li>
	<li>Position the patient supine in a similar fashion used for chest CT scan imaging acquisition.</li>
	<li>Using a test electromagnetic navigational needle select the entrance point to the chest cavity and mark it on the skin surface. Entrance point should be superior to the surface of the nearest rib and avoid osseous structures and any vascular structure.</li>
	<li>Prepare the skin with the 2% chlorhexidine solution, drape the field using sterile technique and anesthetize locally (subcutaneous infiltration of about 1 to 2 cc of 1% lidocaine).</li>
	<li>Place the sterile electromagnetic navigational needle on top of the entrance point and select the angle to the target based on observation of the transverse and coronal views seen on the electromagnetic system screens. (Crosshair marks in at least two different planes.)</li>
	<li>Stabilize&#160;the needle and firmly advanced through the chest wall into the target lesion.</li>
	<li>Remove the guidance needle stylet from the needle and take care to avoid needle movement. At this point extreme care should be use to prevent inadvertent needle displacement. Cover the needle hub with your finger.&#160;Insert the 20 G FNA needle through the 19 G needle. Sample the lesion and provide the specimen for ROSE.&#160;</li>
	<li>Program the automatic needle biopsy to the desired distance or throw based on the size of the lesion, and then insert it through the 19 G needle. Stabilize both needle and biopsy gun, trigger the needle biopsy gun mechanism to perform the biopsy and remove from the inside of the electromagnetic navigational guide needle making sure it remains in appropriate position.</li>
	<li>Gently advance the guidance probe back into the lumen of the percutaneous needle and keep it stable.</li>
	<li>While the needle is continuously stabilized, the assistant uses the scalpel blade to gently remove the specimen from the inner cannula of the core biopsy needle and place it on the 1/2 x 1/2 inch previously cut non adherent pad. Immerse the pad in a formalin solution.</li>
	<li>Reconfirm appropriate placement of the needle within the lesion and then repeat the operation 4 to 5 times making sure the core needle is rotated clockwise or counterclockwise to sample different areas.</li>
	<li>Once satisfied with the specimens remove the needle, apply pressure and place a small bandage over the puncture site.</li>
	</ol>
4. Post Procedure
<ol>
	<li>If available, perform&#160;bedside ultrasonography to assess for the presence of lung sliding (absence of pneumothorax) or use any other imaging modality (fluoroscopy,etc) .</li>
	<li>Transfer the patient to the recovery area and observe&#160;under standard ambulatory surgical unit protocols until fully awake.</li>
	<li>Obtain a chest radiograph to rule out complications.</li>
	<li>Discharge patient home after education is provided regarding the potential post procedure complications including bleeding and pneumothorax.</li>
	</ol>

<ol>
	<li>Govert, J. A., Dodd, L. G., Kussin, P. S., Samuelson, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+comparison+of+fiberoptic+transbronchial+needle+aspiration+and+bronchial+biopsy+for+bronchoscopically+visible+lung+carcinoma.">A prospective comparison of fiberoptic transbronchial needle aspiration and bronchial biopsy for bronchoscopically visible lung carcinoma.</a> Cancer. 87, 129-134 (1999).</li><li>Gasparini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchoscopic+biopsy+techniques+in+the+diagnosis+and+staging+of+lung+cancer.">Bronchoscopic biopsy techniques in the diagnosis and staging of lung cancer.</a> Monaldi Arch Chest Dis. 52, 392-398 (1997).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=United+States+cancer+statistics:+1999-2010+incidence+and+mortality+web-based+report.">United States cancer statistics: 1999-2010 incidence and mortality web-based report.</a> US Department of Health and Human Services Centers for Disease Control and Prevention and National Cancer Institute. , (2014).</li><li>Silvestri, G. 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=Methods+for+staging+non-small+cell+lung+cancer:+Diagnosis+and+management+of+lung+cancer,+3rd+ed:+American+College+of+Chest+Physicians+evidence-based+clinical+practice+guidelines.">Methods for staging non-small cell lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines.</a> Chest. 143, e211S-e250S (2013).</li><li>Steinfort, D. P., Khor, Y. H., Manser, R. L., Irving, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+probe+endobronchial+ultrasound+for+the+diagnosis+of+peripheral+lung+cancer:+systematic+review+and+meta-analysis.">Radial probe endobronchial ultrasound for the diagnosis of peripheral lung cancer: systematic review and meta-analysis.</a> The European respiratory journal. 37, 902-910 (2011).</li><li>Gex, 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=Diagnostic+yield+and+safety+of+electromagnetic+navigation+bronchoscopy+for+lung+nodules:+a+systematic+review+and+meta-analysis.">Diagnostic yield and safety of electromagnetic navigation bronchoscopy for lung nodules: a systematic review and meta-analysis.</a> Respiration; international review of thoracic diseases. 87, 165-176 (2014).</li><li>Wang Memoli, J. S., Nietert, P. J., Silvestri, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+of+guided+bronchoscopy+for+the+evaluation+of+the+pulmonary+nodule.">Meta-analysis of guided bronchoscopy for the evaluation of the pulmonary nodule.</a> Chest. 142, 385-393 (2012).</li><li>Lee, K. A., Raval, A. A., Amir, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effectiveness+of+endobronchial+percutaneous+biopsy+compared+with+transthoracic+biopsy+for+diagnosis+of+peripheral+lung+lesions.">Cost-effectiveness of endobronchial percutaneous biopsy compared with transthoracic biopsy for diagnosis of peripheral lung lesions.</a> Lung Cancer Management. 3 (2), 135-148 (2014).</li><li>Rivera, M. P., 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=American+College+of+Chest,+P.+Initial+diagnosis+of+lung+cancer:+ACCP+evidence-based+clinical+practice+guidelines+(2nd+edition).">American College of Chest, P. Initial diagnosis of lung cancer: ACCP evidence-based clinical practice guidelines (2nd edition).</a> Chest. 132, 131S-148S (2007).</li><li>Wiener, R. S., Schwartz, L. M., Woloshin, S., Welch, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-based+risk+for+complications+after+transthoracic+needle+lung+biopsy+of+a+pulmonary+nodule:+an+analysis+of+discharge+records.">Population-based risk for complications after transthoracic needle lung biopsy of a pulmonary nodule: an analysis of discharge records.</a> Annals of internal medicine. 155, 137-144 (2011).</li></ol>

The authors received research funding that was provided by Veran Medical Technologies Inc., which produces the EM and TTNA instruments used in this publication.

Electromagnetic guidance facilitates the percutaneous transthoracic biopsy technique. It is critical during the procedure to verify adequate equipment calibration. During needle insertion maintaining a steady angle and keeping visual alignment using at least two different planes is essential to effectively reach the target lesion.
Needle insertion depth can be modified between biopsies to maximize the cover area. Preferably,&#160;the procedure could be assisted by a second operator, especially at the time of biopsy gun operation, but a single operator procedure is feasible.
Familiarization with the instruments and technique takes approximately 2 to 3 needle insertions. If available training on phantom models or cadaveric models under direct supervision of an experienced operator is advisable. Electromagnetic navigational transthoracic needle aspiration (E-TTNA) is a novel technology that supplements the current armamentarium for the diagnosis of peripheral pulmonary nodules. Potential limitations of the procedure are inherent to the electromagnetic navigational technology, like CT to body divergence and related to the dynamic nature of the respiratory system. We recommend anticipation for the management of complications like pneumothorax and bleeding should be taken during any percutaneous procedure.
In this article we describe how the E-TTNA is performed by interventional pulmonologist and the incorporation of the technique to the current available bronchoscopic methods for the evaluation of lung nodules.
E-TTNA potentially can reduce the need for other image guided percutaneous transthoracic procedures providing full integration of bronchoscopic and non-bronchoscopic techniques in one single procedure potentially increasing diagnostic yield, improving safety and decreasing the overall number of procedures. Further studies are needed to determine clinical utility of this modern biopsy technique.

A solitary pulmonary nodule (SPN) is a frequent clinical scenario that is increasing due to the number of radiographic chest studies and the implementation of lung cancer screening programs. A lung nodule by definition is less than 3 cm in diameter and &#160;one that typically lies beyond the bronchoscopically visualized segmental bronchi. A lesion larger than 3 cm is considered a lung mass. The etiologies of the majority of lung nodules are benign conditions (infections, inflammatory, vascular) and in a smaller percentage are caused by lung cancer and other malignancies. Evaluation of a SPN starts with assessment of risk factors, radiographic characteristics, size, growth, location, surgical risk, etc. Risk stratification helps to select the most appropriate individual management, which ranges from radiologic surveillance to primary surgical resection. Diagnostic modalities include bronchoscopic techniques: ( bronchoalveolar lavage (BAL), cytology brush, transbronchial biopsy (TBBx), transbronchial needle aspiration (TBNA), radial probe endobronchial ultrasound (R-EBUS), electromagnetic navigation (EMN), ultrathin bronchoscopy, virtual bronchoscopy navigation) and non-bronchoscopic: image guided transthoracic needle biopsy and surgical resection.
The bronchoscopic yield using simple conventional techniques for lesions less &#60;3 cm ranges from 14 &#8211; 50%. 1,2 Lung cancer remains the leading cause of cancer deaths. It represents 14% of all invasive cancers diagnosed each year and 28% of all cancer deaths in the United States.3 Prognosis and treatment management are determined by the stage. The staging of non-small cell lung cancer (NSCLC) is defined by the TNM system. Determination of the N or nodal status requires strict assessment of the mediastinal and hilar lymph nodes.
Endobronchial ultrasound (EBUS) is a minimally invasive bronchoscopic technique that uses ultrasound to identify structures contiguous to the airways facilitating transbronchial needle aspiration (TBNA). For lung cancer staging a recent meta-analysis reported a median sensitivity of 89% with values ranging from 46% to 97% and a median negative predictive value PV of 91% for EBUS-TBNA. 4 Radial EBUS is another bronchoscopic tool that can be used to localize parenchymal lung lesions. It is a 1.4 mm diameter instrument with a 20-MHz ultrasound probe at the tip that generates 360 degree images with a penetration of 5 cm. Recently reported data of this technique showed a sensitivity of identifying neoplasia of 73% (95% CI, 0.70 - 0.76) for nodules of any size and 71% (95% CI, 0.66 - 0.75) for lesions smaller than 2.5 cm 5 .
Electromagnetic Navigational Bronchoscopy (ENB) is a technology that creates a magnetic field around the patient allowing determination of the spatial location of a sensor device within the magnetic field. This information is superimposed on previously obtained computed tomography (CT) images, which permit a display of the sensor location in relationship to the anatomy. A virtual bronchoscopy reconstruction of the patient&#39;s airways facilitates navigation to the target lesion. Once in the target, samples are taken directly using one of the two available technologies; trackable bronchoscopic instruments or through an extended working channel. Overall diagnostic accuracy was 73.9% (95% CI 68.0%&#160;- 79.2%) and negative predictive value 52.1% (95% CI 43.5% - 60.6%) and when using at least one of the other technologies (virtual bronchoscopy, ENB, Radial-EBUS, guide sheath, ultrathin bronchoscope) the diagnostic yield was 70.0% with a 95% CI of 67.1% to 72.9% 67 One of the commercially available systems has the added feature to complement&#160;bronchoscopic navigation with the ability to convert from a bronchoscopic approach to a transthoracic needle approach in cases where the lesion is unable to be accessed bronchoscopically. This system uses an electromagnetic navigation guided 19 G tracking needle that allows for the sampling of core lung biopsies using a 20 G automatic biopsy device.
Transthoracic needle aspiration (TTNA) is a non bronchoscopic diagnostic approach to lung nodules and masses. TTNA of a peripheral lung lesion can be performed under ultrasonography, fluoroscopy, CT scan or electromagnetic guidance. In an updated meta-analysis by Rivera et al&#160;the pooled sensitivity of TTNA for the diagnosis of peripheral bronchogenic carcinoma was 0.90 (95% CI,&#160;0.88 to 0.91).8 In a study by Wiener RS that included 15,865 patients with a reported risk of pneumothorax after TTNA of 15% (95% CI, 14% - 16%) with 7% (95% CI, 6% - 7.2%) requiring management with a chest tube. 9 Clinical trials investigating the use of E-TTNA are currently underway.
CASE PRESENTATION
An 83 year old female former smoker with oxygen dependent chronic obstructive pulmonary disease was incidentally found with a PET fluorodeoxyglucose avid 1.6 x 1.3 cm spiculated right upper lobe (RUL) nodule (Figure 1). Although her probability for malignancy was high, the patient refused surgical interventions and opted for a diagnostic procedure before considering other therapies. The typical scenario that may prompt consideration for an evaluation for biopsy would be a case of intermediate probability for malignancy or with a high probability for malignancy with a contraindication for surgical resection.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>20 G x 15 cm Programmable automatic biopsy needle</td>
			<td>SuperCore Argon Medical devices</td>
			<td align="right">701120150</td>
			<td></td>
		</tr>
		<tr>
			<td>Non Adherent Pads</td>
			<td>Telfa&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2% chlorhexidine gluconate pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blade #11</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical drape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1% lidocaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gowns</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mask</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scrub hair cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic reference points&#160;</td>
			<td>vPAD2 (Veran Medical Technologies)</td>
			<td>INS-0049</td>
			<td></td>
		</tr>
		<tr>
			<td>Planning software&#160;</td>
			<td>SPiNDrive&#160;2.0 (Veran Medical Technologies)&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eelectromagnetic navigation platform</td>
			<td>SPiNView (Veran Medical Technologies)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>19 G x 105 mm Electromagnetic navigational needle</td>
			<td>SPiNPerc&#160; Veran Medical Technologies</td>
			<td>INS-0029</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard diagnostic Fiberoptic Bronchoscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EBUS Bronchoscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Radial EBUS probe UM-S20-17S</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde-based fixative solution.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol based fixative</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of electromagnetic navigational transthoracic needle aspiration (e-ttna) for sampling of lung nodules

Lung nodule evaluation represents a clinical challenge especially in patients with intermediate risk for malignancy. Multiple technologies are presently available to sample nodules for pathological diagnosis. Those technologies can be divided into bronchoscopic and non-bronchoscopic interventions. Electromagnetic navigational bronchoscopy is being extensively used for the endobronchial approach to peripheral lung nodules but has been hindered by anatomic challenges resulting in a 70% diagnostic yield. Electromagnetic navigational guided transthoracic needle lung biopsy is novel non-bronchoscopic method that uses a percutaneous electromagnetic tip tracked needle to obtain core biopsy specimens. Electromagnetic navigational transthoracic needle aspiration complements bronchoscopic techniques potentially allowing the provider to maximize the diagnostic yield during one single procedure. This article describes a novel integrated diagnostic approach to pulmonary lung nodules. We propose the use of endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) for mediastinal staging; radial EBUS, navigational bronchoscopy and E-TTNA during one single procedure to maximize diagnostic yield and minimize the number of invasive procedures needed to obtain a diagnosis. This manuscript describes in detail how the navigation transthoracic procedure is performed. Additional clinical studies are needed to determine the clinical utility of this novel technology.

All tissue samples obtained by EBUS-TBNA and E-TTNA are processed by the central pathology laboratory for further analysis. Pathological evaluation of the samples includes cytological and histological assessment. Rapid on-site evaluation (ROSE) uses a modified Romanowsky stain that allows adequacy evaluation and a cytomorphological diagnostic assessment of the pathological specimens. A preliminary intra procedure assessment using ROSE of the EBUS-TBNA biopsies taken from the mediastinal lymph node stations 4L,4R and 7 was unrevealing of obvious neoplasm. The navigational bronchoscopic phase, supported with radial EBUS, failed to localize the right upper lobe lesion due to anatomical limitations. The procedure was rapidly converted to E-TTNA. E-TTNA was successfully performed and the obtained tissue was sufficient for further pathological and genetic analysis. The final pathology of the E-TTNA core sample biopsies was diagnostic and consistent with adenocarcinoma. No immediate complications were noted.
<img alt="Figure 1" src="/files/ftp_upload/52723/52723fig1.jpg" /> 
Figure 1. Chest Computed Tomography (Panel A) and Positron Emission Tomography (PET) (Panel B). Right upper lobe nodule with a PET negative mediastinum. Scale in cm.

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Use of Electromagnetic Navigational Transthoracic Needle Aspiration E-TTNA for Sampling of Lung Nodules

We describe the novel use of electromagnetic navigational guided transthoracic needle aspiration for the pathologic assessment of human lung nodules.

Use of Electromagnetic Navigational Transthoracic Needle Aspiration (E-TTNA) for Sampling of Lung Nodules

Lung nodule evaluation represents a clinical challenge especially in patients with intermediate risk for malignancy. Multiple technologies are presently ...

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22.2K Views.  Johns Hopkins University. We describe the novel use of electromagnetic navigational guided transthoracic needle aspiration for the pathologic assessment of human lung nodules. 

Video: Use of Electromagnetic Navigational Transthoracic Needle Aspiration E-TTNA for Sampling of Lung Nodules

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

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 ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the lack of a standardized definition for STG. Biopsy is often required to differentiate benign from malignant lesions. Unlike cervical thyroid, the overlying sternum precludes ultrasound-guided percutaneous fine needle aspiration of STG. Consequently, surgical mediastinoscopy is performed in the majority of cases, causing significant procedure related morbidity and cost to healthcare. Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) is a frequently used procedure for diagnosis and staging of non-small cell lung cancer (NSCLC). Minimally invasive needle biopsy for lesions adjacent to the airways can be performed under real-time ultrasound guidance using EBUS. Its safety and efficacy is well established with over 90% sensitivity and specificity. The ability to perform EBUS as an outpatient procedure with same-day discharges offers distinct morbidity and financial advantages over surgery. As physicians performing EBUS gained procedural expertise, they have attempted to diversify its role in the diagnosis of non-lymph node thoracic pathologies. We propose here a role for EBUS-TBNA in the diagnosis of substernal thyroid lesions, along with a step-by-step protocol for the procedure.

Substernal thyroid lesions are common, and need to be differentiated from malignancy. Obtaining percutaneous fine needle biopsy is not possible due to its retrosternal location. This article proposes a protocol for biopsy of substernal thyroid lesions using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA).

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the examiners&#39; expertise. Speckle tracking Echocardiography (STE) is a promising but technically challenging method that can be used to quantitatively assess regional and global systolic and diastolic myocardial performance. Myocardial strain and strain rate can be measured in all three dimensions&#160;&#8212; radial, circumferential, longitudinal &#8212; of myocardial deformation. Standard cross-sectional two-dimensional B-mode images are recorded and subsequently postprocessed by automated continuous frame-by-frame tracking and motion analysis of speckles within the myocardium. Images are recorded as digital loops and synchronized to a 3-lead EKG for timing purposes. Longitudinal deformation is assessed in the apical 4-, 3-, and 2-chamber views. Circumferential and radial deformation are measured in the parasternal short axis plane.
Optimal image quality and accurate tissue tracking are paramount for the correct determination of myocardial performance parameters. Utilizing transthoracic STE in a healthy volunteer, the present article is a detailed outline of the essential steps and potential pitfalls of quantitative echocardiographic myocardial deformation analysis.

Speckle tracking echocardiography is an emerging diagnostic imaging technique for the quantitative assessment of global and regional myocardial performance. Standard view echocardiographic motion images are recorded and deformation parameters are subsequently measured by automated continuous frame-by-frame tracking and motion analysis of speckles within the B-mode images of the myocardium.

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Use of Electromagnetic Navigational Transthoracic Needle Aspiration (E-TTNA) for Sampling of Lung Nodules

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Femoral Bone Marrow Aspiration in Live Mice

Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling