The authors thank the echocardiographic study subject for volunteering in the video as well as Witten/Herdecke University and HELIOS Research Center (HRC-ID 000416 assigned to Kai O. Hensel) for funding.

The protocol content has been ethically approved by the Witten/Herdecke University Ethics Committee.
1. Technical Requirements
<ol>
	<li>Utilize an echocardiography device featuring speckle tracking technology equipped with an adequate sector array tissue harmonic imaging transducer.</li>
	<li>During image acquisition, record and connect a standard 3-lead EKG directly to the echocardiography device in order to synchronize echocardiographic motion images to electromechanical activity. This is mandatory for timing purposes during subsequent postprocessing analyses. Connect the study subject to the EKG and unfreeze the ultrasound image to start detecting the EKG signal.</li>
	<li>Record digital loops as explained in detail below (steps 2.1-2.5) and store the data as moving images in DICOM format on an external disk. Subsequently, transfer the files to an off-line workstation.</li>
	<li>Perform post-processing analyses using appropriate software as outlined in detail below (steps 3.1-3.13).</li>
</ol>
2. Recording of Echocardiographic Digital Loops
<ol>
	<li>Examine the patient in the left lateral decubitus position (patient lying on the left side with the left arm stretched above the head). 
	NOTE: This part of the protocol requires the patient/study subject to be present.</li>
	<li>Alternatively, when combining STE with stress echocardiography modalities such as a bicycle ergometer, ensure that the patient is in a 45-degree upright position. In this case, utilize a standard bicycle ergometry device and perform standard stress echocardiography testing as previously described11. During the recording of echocardiographic images, tilt the ergometer to achieve a left lateral body position in order to minimize artifacts by interfering lung tissue.</li>
	<li>Take special care to optimize image quality to guarantee accurate assessment of myocardial deformation. To do so, adjust the frame rate between 60 and 80 frames per second using the &#34;adjust frame rate&#34; option. Furthermore, pay attention to include all aspects of the myocardial structures that shall be analyzed throughout the entirety of the cardiac cycle.</li>
	<li>Obtain cross-sectional two-dimensional greyscale B-mode images in standard apical long axis and parasternal short axis planes as described by the European Association of Cardiovascular Imaging and the American Society of Echocardiography12. Record several consecutive cardiac cycles (actually only one is necessary, recording of at least three cardiac cycles is advised in order to be able to choose the one with the best image quality during subsequent post processing) in each of the following planes:
	<ol>
		<li>For the assessment of longitudinal &#949; and SR, capture standard apical 4-, 3-, and 2-chamber views as previously described12. To do so, position the transducer at the apex of the heart near the apical impulse (usually between the 3rd and 5th intercostal space and between the mid-clavicular and anterior axillary line). Aim towards the right shoulder and angulate the transducer until all anatomical structures of interest become visible.</li>
		<li>Record images in the parasternal short axis view at the level of the mitral valve, the papillary muscles and the apex to detect circumferential &#949; and SR as well as radial &#949; and SR as described elsewhere in detail12. To do so, place the probe at the left parasternal border at the 2nd or 3rd intercostal space and angulate until you obtain a cross-sectional perpendicular view of the LV.</li>
	</ol>
	</li>
	<li>When combining STE with cardiac stress testing such as bicycle ergometry or any other functional testing modality requiring serial measurements (see step 2.2), repeat step 2.4 at each desired time point.</li>
</ol>
3. Postprocessing Analysis
NOTE: This part of the protocol includes the evaluation and interpretation of the recorded echocardiographic images. It does not require the patient to be present and can be performed at any time following the previous part of the procedure.
<ol>
	<li>Utilizing the quantitative echocardiography analysis software, click &#39;File&#39; and &#39;Open&#39; and chose the desired echocardiographic study data. Select a patient/study and pick an echocardiographic plane that shall be analyzed.</li>
	<li>Click the &#39;Q&#39;-icon in the right lower corner of the selected image. Next, press the &#39;aCMQ&#39; button on the left.</li>
	<li>Chose the cardiac cycle of the highest image quality by using the green &#39;QRS&#39; skip keys at the bottom of the screen. Use the keyboard space bar to play and pause the loop.</li>
	<li>Select a region of interest (ROI) to be analyzed by confirming the echocardiographic view on the left side of the screen. Next, have the software automatically detect the timing of end-diastole and suggest a ROI. 
	NOTE: A first speckle tracking analysis is subsequently being computed by the software. Segmental and global &#949; curves are displayed on the bottom of the screen.</li>
	<li>Click &#39;Strain rate&#39; below the graphs to visualize segmental and global SR.</li>
	<li>Visually verify the tracking quality suggested by the software. 
	NOTE: To do so, critically control whether all aspects of the myocardium to be analyzed are completely covered by the ROI during the entirety of the cardiac cycle. Avoid including surrounding non-myocardial tissues into the ROI.</li>
	<li>If necessary, manually reposition the entire ROI or single aspects of it, or even re-draw the ROI completely (see 3.8-3.9) in order to guarantee exact measurements. 
	NOTE: Optionally, set the ROI to be transparent to adjust the ROI coverage to the appropriate position and width of the myocardium.</li>
	<li>In the apical 4-, 3-, and 2-chamber view, have the software automatically determine a possible ROI dividing the myocardium into seven segments.
	<ol>
		<li>In case ROI re-definition is necessary, click &#39;Draw&#39; on the left and start out tagging the endocardial border at three reference points: the two opposing insertion points of the AV valve and the LV wall starting with the basal infero-septal/ basal infero-lateral/basal inferior part of the valve finishing with the center of the apex. Ensure that both endpoints of the tracked-endocardium are on the same level completely excluding the valvular tissue.</li>
		<li>If repositioning is necessary in order to optimize position and width of the ROI, click &#39;Edit&#39; on the left side of the screen. Move each segment margin as well as the endocardial and epicardial borders individually with the cursor. Utilize an orthogonal line pointing towards the apex for navigation/orientation when moving the ROI in its entirety.</li>
		<li>Finally, start the speckle tracking re-analysis by pressing the &#39;Compute&#39; button on the left side of the screen. 
		NOTE: The software now automatically detects &#34;acoustic markers&#34;, which deflect myocardial ultrastructures corresponding to myocardial fiber organization in their movement throughout the contraction and relaxation of the myocardium. These acoustic markers are traced through the entire duration of a complete cardiac cycle. The necessary calculation may take seconds to minutes. &#949; and SR are calculated by the software and presented in a numeric and graphic manner.</li>
	</ol>
	</li>
	<li>In the parasternal view, have the software automatically suggest a predefined ROI. Adjust this ROI manually, dividing the myocardium into six segments. 
	NOTE: The width of the ROI, should exactly match the thickness of the myocardium. Where necessary, optimize position and width of the ROI as described in 3.8.2. A dot in the center of the ROI may be utilized for navigation/orientation when moving the ROI in its entirety.
	<ol>
		<li>Next, start the speckle tracking re-analysis by pressing the &#39;Compute&#39; button on the left side of the screen.</li>
	</ol>
	</li>
	<li>Chose segmental and global &#949; and SR to be displayed in curves or the comprehensive bull&#39;s eye format. To do so, click the &#39;Preferences&#39; button at the bottom left corner of the screen. Different types of waveforms and displaying options can be selected in this menu.</li>
	<li>If manual repositioning of the ROI is not sufficient to achieve appropriate overall speckle tracking quality, start over from 3.1 and redefine the ROI or consider selecting a different cardiac cycle prior to continuing to the next step.</li>
	<li>Save and export the data for subsequent statistical analyses. If desired, cine loops or still frames can be exported as illustrations. To do so, click &#39;Export&#39; at the bottom left corner of the screen and select the desired format and file directory.</li>
</ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain,+strain+rate,+torsion,+and+twist:+echocardiographic+evaluation.">Strain, strain rate, torsion, and twist: echocardiographic evaluation.</a> Curr. Cardiol. Rep. 17, 568 (2015).</li><li>Kukulski, T., 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=Identification+of+acutely+ischemic+myocardium+using+ultrasonic+strain+measurements.+A+clinical+study+in+patients+undergoing+coronary+angioplasty.">Identification of acutely ischemic myocardium using ultrasonic strain measurements. A clinical study in patients undergoing coronary angioplasty.</a> J. Am. Coll. Cardiol. 41, 810-819 (2003).</li><li>Suffoletto, M. 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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=Subclinical+Alterations+of+Cardiac+Mechanics+Present+Early+in+the+Course+of+Pediatric+Type+1+Diabetes+Mellitus:+A+Prospective+Blinded+Speckle+Tracking+Stress+Echocardiography+Study.">Subclinical Alterations of Cardiac Mechanics Present Early in the Course of Pediatric Type 1 Diabetes Mellitus: A Prospective Blinded Speckle Tracking Stress Echocardiography Study.</a> J Diabetes Res. 2016, 2583747 (2016).</li><li>Kalam, K., Otahal, P., Marwick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+implications+of+global+LV+dysfunction:+a+systematic+review+and+meta-analysis+of+global+longitudinal+strain+and+ejection+fraction.">Prognostic implications of global LV dysfunction: a systematic review and meta-analysis of global longitudinal strain and ejection fraction.</a> Heart. 100, 1673-1680 (2014).</li><li>Hensel, K. O., Grimmer, F., Jenke, A. C., Wirth, S., Heusch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+real-time+blood+glucose+levels+on+left+ventricular+myocardial+strain+and+strain+rate+in+pediatric+patients+with+type+1+diabetes+mellitus+-+a+speckle+tracking+echocardiography+study.">The influence of real-time blood glucose levels on left ventricular myocardial strain and strain rate in pediatric patients with type 1 diabetes mellitus - a speckle tracking echocardiography study.</a> BMC Cardiovasc. Disord. 15, 175 (2015).</li><li>Kurt, M., Tanboga, I. H., Aksakal, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Dimensional+Strain+Imaging:+Basic+principles+and+Technical+Consideration.">Two-Dimensional Strain Imaging: Basic principles and Technical Consideration.</a> Eurasian J Med. 46, 126-130 (2014).</li><li>Cameli, M., Lisi, M., Righini, F. M., Mondillo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+echocardiographic+techniques+to+assess+left+atrial+size,+anatomy+and+function.">Novel echocardiographic techniques to assess left atrial size, anatomy and function.</a> Cardiovasc. Ultrasound. 10 (4), (2012).</li><li>Pellikka, P. A., Nagueh, S. F., Elhendy, A. A., Kuehl, C. A., Sawada, S. 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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=Recommendations+for+cardiac+chamber+quantification+by+echocardiography+in+adults:+an+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> J. Am. Soc. Echocardiogr. 28, 1-39 (2015).</li><li>Curtis, J. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Apical+Rotation+in+Severe+Aortic+Stenosis+is+Associated+with+Reduced+Survival:+A+Speckle-Tracking.">Increased Apical Rotation in Severe Aortic Stenosis is Associated with Reduced Survival: A Speckle-Tracking.</a> J. Am. Soc. Echocardiogr. , (2015).</li><li>Auger, D., 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=Effect+of+cardiac+resynchronization+therapy+on+the+sequence+of+mechanical+activation+assessed+by+two-dimensional+radial+strain+imaging.">Effect of cardiac resynchronization therapy on the sequence of mechanical activation assessed by two-dimensional radial strain imaging.</a> Am. J. Cardiol. 113, 982-987 (2014).</li><li>To, A. C., 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=Strain-time+curve+analysis+by+speckle+tracking+echocardiography+in+cardiac+resynchronization+therapy:+Insight+into+the+pathophysiology+of+responders+vs.+non-responders.">Strain-time curve analysis by speckle tracking echocardiography in cardiac resynchronization therapy: Insight into the pathophysiology of responders vs. non-responders.</a> Cardiovasc. Ultrasound. 14 (14), (2016).</li><li>Seo, 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=Three-dimensional+propagation+imaging+of+left+ventricular+activation+by+speckle-tracking+echocardiography+to+predict+responses+to+cardiac+resynchronization+therapy.">Three-dimensional propagation imaging of left ventricular activation by speckle-tracking echocardiography to predict responses to cardiac resynchronization therapy.</a> J. Am. Soc. 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The authors have nothing to disclose.

Significance of the technique with respect to alternative methods
The current gold standard for the echocardiographic assessment of LV systolic function is the LV ejection fraction (EF)13. However, determination of EF is based on a simplistic approach that is closely correlated to the radial component of myocardial contraction but does not take into consideration the important longitudinal and circumferential planes. Hence, EF oversimplifies the three-dimensional complexity of myocardial deformation. As a consequence, EF measurements do not unmask subtle cardiac dysfunctions but only detect LV deterioration at a relatively advanced state14. On the other hand, STE-derived &#949; and SR have been shown to detect subclinical cardiac alterations while EF still remained normal15. STE has emerged to be a robust tool for research and bare important clinical implications providing a significant advance in the management of cardiovascular patients16. Global LV longitudinal &#949; has the highest clinical value among the STE-derived LV deformation parameters and better reflects global myocardial function when compared to circumferential and radial &#949;7.
Longitudinal &#949; and SR are assessed in the apical long axis views and reproducibly assess global and regional LV deformation (Figures 1 and 2). Caution must be paid to maintain highest requirements for image quality and myocardial tracing, since inadequate tissue-tracking yields incorrect deformation parameters that often are not as obvious as demonstrated in Figure 3. Circumferential and radial wall motion can be quantitatively measured in the parasternal short axis (Figures 4 and 5). Even though global LV longitudinal &#949; has been pointed out as the most significant single STE-derived deformation parameter, circumferential and radial wall motion yield important additional information for a variety of clinical scenarios17,18.
Fortunately, novel echocardiography devices provide the possibility to assess global longitudinal &#949; measurements at the patient&#39;s bedside within a few min. This gives the physician the possibility to perform a robust detection of LV myocardial deformation parameters without the time-consuming comprehensive STE-measurements that require the recording and off-line postprocessing of various cross-sectional echocardiographic images. Utilizing the bull&#39;s eye visualization, the physician can review global LV function at a glance (Figure 7).
Furthermore, besides the assessment of global LV function, segmental (regional) myocardial function can be analyzed using STE. This enables physicians to measure myocardial dyssynchrony and the response to cardiac resynchronization therapy19,20.
Limitations, critical steps and troubleshooting
Despite the promising advantages of STE, the technology has important limitations. First and foremost, the dependence of reproducible STE-derived measurements on echocardiographic image quality cannot be overstated. It is therefore crucial to take special care improving image quality as far as possible21. Even subtle artifacts can lead to significant misinterpretations of &#949; or SR, as demonstrated in Figures 3 and 6. Furthermore, the tissue tracking software automatically includes all segments regardless of their image quality. At times it may even suggest unrealistic deformation parameters for ROIs that include non-cardiac tissue. Thus, dedicated verification of accurate ROI size and position and incremental fine-tuning using thorough visual assessment is absolutely essential.
Another approach aimed to avoid artifacts during echocardiographic image acquisition is to advise the patient to hold his/her breath for a few seconds. While this is usually feasible for regular echocardiographic studies in competent adult patients, it is certainly challenging and oftentimes plainly unrealistic to try so in pediatric patients or during cardiac stress testing such as bicycle ergometry.
Moreover, optimal adjustment of frame rate prior to image acquisition is mandatory. Frame rates of less than 30 frames per sec result in overly smooth time-strain curves and lack sufficient temporal resolution. High frame rates over 100 frames per sec oftentimes yield noisy &#949; curves that are only reliable with exceptionally high image quality. A range of 60 to 80 frames per sec has been established to best match the software requirements for optimal tissue tracking in average adult patient populations16. In pediatric cardiology and especially in neonatology, patients naturally have higher heart rates than adult individuals. Based on a recent STE study in premature neonates, the authors suggested to adjust frame rate settings according to the patients&#39; heart rate. A frame rate/heart rate ratio of 0.7 to 0.9 frames per sec per bpm was proposed to achieve optimal myocardial speckle tracking results22. In conclusion, standardization of views, frame or volume rate and image quality should be prerequisites for STE-derived assessment of myocardial performance in order to achieve reliable, well-reproducible results.
In addition, it is noteworthy to mention that STE-derived assessment of myocardial performance currently is a reasonably time-consuming method. Despite the promising value for clinical decision making, the multiple step nature of the procedure including the many necessary quality control and software calculation steps are probably the most relevant limitation hindering STE from being used in day-to-day clinical routine care. Companies should be encouraged to optimize tissue tracking software development with a special focus on feasibility in order to expedite and improve the work-flow, rendering it more user friendly.
Finally, an important limitation of STE is the variance of STE-derived &#949; and SR values between different software packages. Various commercial companies supplying STE analysis software utilize different underlying mathematic algorithms that at times yield non-consistent deformation parameters. Thus, a given myocardial &#949; or SR measured with a device from company A has to be interpreted with caution when reference values are derived from an STE software package of company B.
Future applications
Currently, STE is increasingly used to detect subtle changes of myocardial performance that remain undetected by conventional echocardiography23. While the LV has been assessed in various STE studies, little is still known regarding left atrial, right ventricular and right atrial mechanics for a variety of clinical and scientific scenarios. Novel modifications to the technique even allow the assessment of vessel stiffness for large arteries utilizing speckle tracking technology. Furthermore, STE can be used in experimental animal models to gather valuable information regarding myocardial performance without the need for invasive procedures. 3D-STE is another promising development allowing comprehensive and time-efficient myocardial deformation analyses. Moreover, STE can be combined with pharmacological or ergometer stress testing to better detect wall motion abnormalities when compared to conventional stress echocardiography. In addition, myocardial twist and torsion can be assessed utilizing STE, which could add incremental clinical value to global &#949; and SR imaging. Further studies are needed in order to illuminate both the clinical significance and limitations of these potential implications of STE.

Scientific and clinical scenarios in cardiovascular medicine are more and more addressed by continuous variables and cutoff values rather than simplistic &#34;yes or no&#34; algorithms. Imaging techniques have evolved to be able to assess cardiac function in ever increasing detail. Speckle tracking echocardiography (STE) is an emerging diagnostic tool for the quantitative evaluation of myocardial performance. While conventional echocardiography is limited by subjective image interpretation and a strong dependence on the individual examiner&#39;s expertise, STE has been introduced as a reproducible and more objective method to quantify global and regional systolic and diastolic function1,2.
Left ventricular (LV) myocardial deformation &#8212; longitudinal and circumferential shortening as well as radial thickening in systole and vice versa in diastole &#8212; can be described measuring the parameters strain (&#949;) and strain rate (SR). &#949; is a dimensionless percent change in myocardial length. SR is a time derivate of &#949;3. These important indices of myocardial function have been shown to be able to identify myocardial ischemia4, predict response to cardiac resynchronization therapy5 and to detect subclinical myocardial dysfunction while conventional echocardiographic parameters still remain normal6. In a systematic meta-analysis, global longitudinal &#949;, the most frequently used quantitative LV systolic function parameter, has been shown to have superior prognostic value for the prediction of major adverse cardiac events then LV ejection fraction (EF), the current gold standard for the assessment of LV systolic function7. Even very subtle alterations such as the effect of short term metabolic changes on myocardial mechanics in asymptomatic patients can be detected utilizing STE8.
Technically, STE uses greyscale 2D or 3D B-mode motion images recorded in standard echocardiography views. Several consecutive cardiac cycles are recorded in apical 4-, 3- and 2-chamber views to measure longitudinal deformation and in the parasternal short axis view for circumferential and radial deformation9. Moreover, by capturing the short axis view at the level of the mitral valve, the papillary muscles and the apex, LV torsion can be assessed3. Subsequently to image acquisition and storage as digital loops, myocardial deformation is measured on an off-line work station or on the ultrasound device itself. The software detects unique myocardial pixel patterns in the recorded greyscale images, so-called &#34;speckles&#34; and traces them throughout the analyzed cardiac cycle. Vectors are measured and deformation parameters are subsequently calculated. This way regional and global myocardial deformation can be assessed in systole and diastole for both the left and right ventricle and atrium10.

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		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Phillips iE33 ultrasound system</td>
			<td style="width:155px;">Philips Healthcare</td>
			<td style="width:155px;"></td>
			<td style="width:619px;">http://www.umiultrasound.com/ultrasound-machine/philips/ie33</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;"> 
			S5-1 broadband sector array transducer&#160;</td>
			<td style="width:155px;">Philips Healthcare</td>
			<td style="width:155px;"></td>
			<td>5-1 MHz, http://www.usa.philips.com/healthcare/product/HC989605412081/s5-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QLAB Advanced Quantification Software Version 10.5</td>
			<td style="width:155px;">Philips Healthcare</td>
			<td style="width:155px;"></td>
			<td>Q-App: Automated Cardiac Motion Quantification (aCMQ), www.philips.com/QLAB-cardiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xcelera R3.3L1 (Version 3.3.1.1103)&#160;</td>
			<td style="width:155px;">Philips Healthcare</td>
			<td style="width:155px;"></td>
			<td>http://www.usa.philips.com/healthcare/product/HC830038/xcelera-r41-cardiology-information-management-system</td>
		</tr>
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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.

The principle parameters for the quantitative assessment of myocardial performance are &#949; and SR. Technically, all cardiac chambers can be analyzed using STE. However, since speckle tracking methodology has been mostly used to study the LV, the focus of this article is on LV myocardial mechanics. Generally, longitudinal &#949; and SR are the most commonly assessed LV deformation parameters. Longitudinal &#949; and SR describe systolic shortening (and diastolic lengthening) of the myocardium. Hence, systolic values are annotated as negative numbers. Figures 1 and 2 represent good examples of STE-derived segmental and global &#949; and SR analyses. Optimal image quality and adequate tissue coverage of the ROI are paramount for reliable assessment of myocardial deformation. Suboptimal tissue tracking frequently results in misinterpretation of true &#949; and SR values. An example of poor tissue tracking quality in the apical (purple) and mid-lateral (blue) segments is displayed in Figure 3. Apical and mid-lateral &#949; is largely underestimated in this example of a healthy patient, who &#8212; when correctly assessed &#8212; exhibits normal LV deformation with unremarkable heterogeneous &#949; and SR values in all LV segments.
<img alt="Figure 1" src="/files/ftp_upload/54736/54736fig1.jpg" /> 
Figure 1: Apical 4-chamber view-derived longitudinal strain and strain rate. Apical 4-chamber view (AP4) derived &#949; and SR are presented in the left and right panels, respectively. Each colored time-strain curve (left and right, bottom) corresponds to one of the seven color-coded myocardial segments (left and right, top) and visualizes segmental (= regional) &#949; or SR, respectively. The white dotted line represents global longitudinal LV &#949; or SR, respectively. Note the smooth parallel shape of the segmental time-strain and SR curves. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54736/54736fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54736/54736fig2.jpg" /> 
Figure 2: Apical 2-chamber view-derived longitudinal strain and strain rate. Left and right panels show Apical 2-chamber (AP2) derived LV myocardial &#949; and SR, respectively. Time-strain curves correspond to the color-coded segments as described above for Figure 1. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54736/54736fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54736/54736fig3.jpg" /> 
Figure 3: Inadequate apical tissue tracking: AP2-derived longitudinal strain. This healthy patient actually has normal LV myocardial &#949; values, when properly assessed. As an artifact, in this figure apical (purple) and mid-lateral (blue) segments exhibit poor tissue tracking quality and thus the real segmental &#949; is greatly underestimated. Peak LV global myocardial &#949; values are displayed (Figure 2, bottom right). Note the absence of dark red color-coding, representing myocardial contraction in the color-coded curved M-mode (Figure 2, bottom left). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54736/54736fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Circumferential &#949; and SR describe the circular systolic shortening of the LV myocardium (and its lengthening in diastole) and are therefore denoted as negative numbers in systole. Circumferential LV deformation is assessed in the parasternal short axis view in three different planes: at the level of the mitral annulus, the papillary muscles (Figure 4) and at the apex. Inclusion of all three levels yields LV myocardial torsion &#8211; basal clockwise and apical counterclockwise rotation in systole &#8211; expressed as LV systolic twist and diastolic untwist.
<img alt="Figure 4" src="/files/ftp_upload/54736/54736fig4.jpg" /> 
Figure 4: LV circumferential strain. Parasternal short axis view at the level of the papillary muscles yields circumferential &#949;. The myocardium is subdivided into six segments. Regional peak LV circumferential &#949; values are displayed at the top. Segmental colors correspond to the time-strain curves displayed at the bottom. The white dotted line represents global circumferential &#949;. Note the smooth and parallel shape of the segmental and global time-strain curves. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54736/54736fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
While longitudinal and circumferential &#949; is negative in systole, radial &#949; reflects systolic myocardial thickening and is thus positive in systole. An example of a well-performed LV radial &#949; assessment is demonstrated in Figure 5. In contrast, Figure 6 shows incorrect tissue tracking of the anteroseptal myocar &#8212; when adequately measured &#8212; unremarkable deformation parameters.
<img alt="Figure 5" src="/files/ftp_upload/54736/54736fig5.jpg" /> 
Figure 5: LV radial strain. Parasternal short axis view at the level of the papillary muscles also yields radial &#949;. Myocardial ROI coverage is displayed at the top left side. Segmental peak systolic radial &#949; values are presented (in %) in the bottom on the left. Radial time-strain curves are displayed at the top right aspect of the figure. Radial strain is displayed in % on the y-axis and time corresponding to the ECG (at the bottom) is presented on the x-axis. Colored curved M-mode with dark red representing positive radial &#949; is shown on the right side of the bottom. Note the homogeneous shape of the segmental radial &#949; curves corresponding well to the uniform curved M-mode color-coding. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54736/54736fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54736/54736fig6.jpg" /> 
Figure 6: Inadequate tissue tracking: LV radial strain. The conceptual content and scaling of this figure correspond to Figure 5. In this example poor anteroseptal tissue tracking resulted in inaccurate radial &#949; values and heterogeneous radial time-strain curves in an asymptomatic healthy patient. Note the deviation of the septal (red) and anteroseptal (yellow) segmental radial &#949; curves. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54736/54736fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For a comprehensive illustration, LV myocardial longitudinal &#949; can be displayed in a so-called bull&#39;s eye view (Figure 7). This way, peak systolic longitudinal &#949; values of 17 segments altogether representing the entirety of the LV myocardium can be visualized in a single view. Time-strain curves for AP4-, AP2-, and AP3-derived segmental longitudinal &#949; are also depicted, corresponding to the color-coding as outlined for Figures 1 and 2. This illustration can be of benefit to visualize regionally varying deformation deficits such as in cardiac amyloidosis which is characterized by apical sparring in the bull&#39;s eye view.
<img alt="Figure 7" src="/files/ftp_upload/54736/54736fig7.jpg" /> 
Figure 7: LV global strain: bull&#39;s eye view. Longitudinal time-strain curves are displayed for LV segmental myocardial &#949; derived from the AP4 (top left), AP2 (top right) and the AP3 (bottom left) planes. The differently colored curves correspond to the segmental division as indicated in Figures 1 and 2. Longitudinal &#949; is displayed in % on the y-axes and relative time corresponding to the ECG (at the bottom) is represented on the x-axis. Peak segmental longitudinal &#949; values are depicted in the bull&#39;s eye illustration (bottom right) for 17 segments analyzed in the AP4, AP2 and AP3 views. Red shaded color-coding corresponds to the legend indicated on the right. Note the higher homogeneity in the AP4-derived segmental time-strain curves when compared to AP2 and AP3. This correlates well with the challenging image acquisition of the anterior LV wall aspects oftentimes leading to artifacts and heterogeneous deformation parameters. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54736/54736fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation at JoVE.com

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

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 ...

transthoracic-speckle-tracking-echocardiography-for-quantitative

Nanyang Technological University

Research

JoVE Journal

Medicine

19.4K Views.  Witten/Herdecke University. 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.

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

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 ...

Magnetic Resonance Derived Myocardial Strain Assessment Using Feature Tracking

Purpose: An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that strain is a more sensitive and earlier marker for contractile dysfunction than the frequently used parameter EF. Current technologies for CMR are time consuming and difficult to implement in clinical practice. Feature tracking is a technology that can lead to more automization and robustness of quantitative analysis of medical images with less time consumption than comparable methods.
Methods: An automatic or manual input in a single phase serves as an initialization from which the system starts to track the displacement of individual patterns representing anatomical structures over time. The specialty of this method is that the images do not need to be manipulated in any way beforehand like e.g. tagging of CMR images.
Results: The method is very well suited for tracking muscular tissue and with this allowing quantitative elaboration of myocardium and also blood flow.
Conclusions: This new method offers a robust and time saving procedure to quantify myocardial tissue and blood with displacement, velocity and deformation parameters on regular sequences of CMR imaging. It therefore can be implemented in clinical practice.

An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that strain is a more sensitive and earlier marker for contractile dysfunction than the frequently used parameter EF.

Purpose: An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that ...

Ultrasonic Assessment of Myocardial Microstructure

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. Ultrasonic assessments of cardiac tissue can include analyses of backscatter signal intensity within a given region of interest. Previously established techniques have relied predominantly on the integrated or mean value of backscatter signal intensities, which may be susceptible to variability from aliased data from low frame rates and time delays for algorithms based on cyclic variation. Herein, we describe an ultrasound-based imaging algorithm that extends from previous methods, can be applied to a single image frame&#160;and accounts for the full distribution of signal intensity values derived from a given myocardial sample. When applied to representative mouse and human imaging data, the algorithm distinguishes between subjects with and without exposure to chronic afterload resistance. The algorithm offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, mice undergo RR upon removal of the aortic constriction. In this paper, we describe a step-by-step procedure to perform a minimally invasive surgical aortic debanding in mice. Echocardiography was subsequently used to assess the degree of cardiac hypertrophy and dysfunction during LV remodeling and RR and to determine the best timing for aortic debanding. At the end of the protocol, terminal hemodynamic evaluation of the cardiac function was conducted, and samples were collected for histological studies. We showed that debanding is associated with surgical survival rates of 70-80%. Moreover, two weeks after debanding, the significant reduction of ventricular afterload triggers the regression of ventricular hypertrophy (~20%) and fibrosis (~26%), recovery of diastolic dysfunction as assessed by the normalization of left ventricular filling and end-diastolic pressures (E/e&#39;&#160;and LVEDP). Aortic debanding is a useful experimental model to study LV RR in rodents. The extent of myocardial recovery is variable between subjects, therefore, mimicking the diversity of RR that occurs in the clinical context, such as aortic valve replacement. We conclude that the aortic banding/debanding model represents a valuable tool to unravel novel insights into the mechanisms of RR, namely the regression of cardiac hypertrophy and the recovery of diastolic dysfunction.

Here we describe a step-by-step protocol of surgical aorta debanding in the well-established mice model of aortic-constriction. This procedure not only allows studying the mechanisms underlying the left ventricular reverse remodeling and regression of hypertrophy but also to test novel therapeutic options that might accelerate myocardial recovery.

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, ...

Morphological and Functional Assessment of the Right Ventricle Using 3D Echocardiography

Traditionally, it was believed that the right side of the heart has a minor role in circulation; however, more and more data suggest that right ventricular (RV) function has strong diagnostic and prognostic power in various cardiovascular disorders. Due to its complex morphology and function, assessment of the RV by conventional two-dimensional echocardiography is limited: the everyday clinical practice usually relies on simple linear dimensions and functional measures. Three-dimensional (3D) echocardiography overcame these limitations by providing volumetric quantification of the RV free of geometrical assumptions. Here, we offer a step-by-step guide to obtain and analyze 3D echocardiographic data of the RV using the leading commercially available software. We will quantify 3D RV volumes and ejection fraction. Several technical aspects may help to improve the quality of RV acquisition and analysis as well, which we present in a practical manner. We review the current opportunities and the limiting factors of this method and also highlight the potential applications of 3D RV assessment in current clinical practice.

Here, we provide a step-by-step acquisition and analysis protocol for the 3D volumetric assessment of the right ventricle, mainly focusing on the practical aspects that maximize the feasibility of this technique.