Name: Ultrasound Tissue Characterization of Human Achilles Tendon by Stability Quantification of Echo Patterns (Video) | JoVE Journal
Uploaded: 2025-09-05T13:20:00.000+00:00
Duration: 8 min 11 s
Description: Learn ultrasound tissue characterization to assess Achilles tendon structure using a standardized quantitative protocol. This method enables reliable diagnosis of tendon injuries and monitoring of treatment response in clinical practice.

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5 minutes

The authors are grateful to Hans van Schie for his critical review of the final version of the manuscript. This study was supported by University of Turin (Fondo per la Ricerca Locale - ex-60%) and was carried out within the &#34;QuAntitative Imaging of VAScular dysregulation as a funCtional basis for aUtoimmune disordErs and tumorS (AI-VASCUES)&#34; project funded by European Union - Next Generation EU within the PRIN 2022 program (D.D. 104 - 02/02/2022 Ministero dell&#39;Universit&#224; e della Ricerca). This manuscript reflects only the authors&#39; views and opinions, and the Ministry cannot be considered responsible for them.

The protocol follows the research integrity guidelines of the Polytechnic of Turin28. The acquisition of ultrasound images was performed within a research study approved by the local ethics committee ("Comitato Etico Interaziendale": protocol n. 0001612/30/12/2021), and the investigated subjects gave their written consent (for study participation and publication of anonymized images).
1. System preparation
NOTE: The system is made of four components (see Table of Materials): Smart System with the ultrasound scanner software; analysis software; multi-frequency (5-16 MHz) 192-element linear array transducer covering 4 cm imaging width, with a nominal central frequency of 11 MHz; tracker: frame (premounted) and stand-off (premounted).
<ol>
	<li>Transducer preparation 
	NOTE: The transducer must be mounted on the tracker in transverse position, that comes with frame and stand-off mounted in place (Supplemental Figure S1A).
	<ol>
		<li>Release the stand-off from the tracker by unscrewing the two metal screws under the tracker and then cover the superior surface of the stand-off with a thick layer of acoustic gel. Put the two components back together.</li>
		<li>Unscrew the two black screws of the transducer strap and the locking screw of the transducer holder base. Insert the transducer into the transducer holder base and fasten the locking screw. Lower the strap fully onto the neck of the transducer and fasten the two screws.</li>
		<li>Remove the two black screws that hold the transducer holder in place and adjust the transducer holder's height so that it runs (half a millimeter to one millimeter) free of the frame and stand-off, in contact with acoustic gel.</li>
		<li>Connect both the tracker (with the USB cable) and the transducer to the Smart System. Connect the power-brick of the Smart System.</li>
	</ol>
	</li>
	<li>System power up
	<ol>
		<li>Switch on the Smart System by pressing the power button on the top left edge of the unit, until the blue light is visible in the knob. 
		NOTE: A few beeps will be heard.</li>
		<li>From the desktop, double-click on the ultrasound software icon <img alt="Static equilibrium; ΣFx=0, ΣFy=0, torque=0; diagram; represents force vectors and balance principles." src="/files/ftp_upload/68543/68543eq01.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 1"> and then minimize the ultrasound screen on the Smart System toolbar.</li>
		<li>Switch on the tracker by pressing the ON/OFF button. 
		NOTE: A double beep will be heard, confirming Smart System connection with the tracker: the system is now ready for data acquisition.</li>
	</ol>
	</li>
</ol>
2. Subject preparation
<ol>
	<li>Ask the subject to lie down in a comfortable prone position, with the bare foot relaxed dangling over the end of the table.</li>
	<li>Cover the skin of the distal part of the posterior leg with acoustic gel, forming a thick layer to ensure optimal contact between the skin and the transducer.</li>
	<li>Passively dorsiflex the ankle of the subject (until maximum dorsiflexion is reached) by pushing the knee against the metatarsal heads of the subject (Supplemental Figure S1B).</li>
</ol>
3. Image acquisition
<ol>
	<li>On the Smart System, double-click on the analysis software icon <img alt="Static equilibrium; ΣFx=0; diagram; physics; force vectors; balance analysis; educational illustration." src="/files/ftp_upload/68543/68543eq02.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 2">. Click the red button <img alt="Spectroscopy setup diagram for optical absorption measurement and data analysis results." src="/files/ftp_upload/68543/68543eq03.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 3"> (Acquisition) on the upper toolbar. On the message Tracker initialization OK, click OK.</li>
	<li>When the acquisition control window comes up, click on START US to display the ultrasound image from the ultrasound software. 
	NOTE: Use the following acquisitions preset settings: a penetration depth of 30 mm, 4 focal points, and the central beam focus at 20 mm. Keep the lateral and axial pixel sizes equal to 0.0925926 mm. During the acquisition, set the step size (i.e., distance between one slice and the next) to 0.2 mm.</li>
	<li>Click ACQUISITION on the acquisition control window. When the Press the Start-button on the tracker to start the acquisition message appears, click OK.</li>
	<li>Place the tracker on the calcaneal region perpendicular to the long axis of the Achilles tendon. 
	NOTE: Ensure that the calcaneus is observed on the ultrasound scanner screen as a bony landmark served as a reference point.</li>
	<li>Press the START button on the tracker to start the acquisition. Follow the entire scanning procedure through the ultrasound scanner screen on the Smart System. 
	NOTE: The transducer begins to move automatically along the tracker over the length of the tendon from distal to proximal. Once the proximal position is reached, after a few seconds of stop, the transducer goes back to the distal position capturing transverse grey-scale images of the tendon (one image every 0.2 mm over 12 cm, for a total of 600 images, in approximately 45 seconds).</li>
	<li>At the end of the data collection cycle, click OK on the acquisition control window and then EXIT.</li>
	<li>Click on the transverse cross-section (top-left) of the screen in the analysis software menu to refresh the screen and display the acquired images. Save the images in the appropriate folder using the SAVE SCAN line from the FILE dropdown menu.</li>
</ol>
4. Image analysis
NOTE: Either start analysis on the acquired images or open previously saved images by clicking OPEN SCAN from the drop-down menu. Opening the file will take a few seconds and images will open in all four views, which can be scrolled through for three-dimensional tomographic visualization (Supplemental Figure S2).
<ol>
	<li>Contouring
	<ol>
		<li>On the analysis software click on the <img alt="Chromatography system diagram; static equilibrium, ΣFx=0; data analysis for substance separation." src="/files/ftp_upload/68543/68543eq04.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 4"> button on the upper toolbar, to switch from qualitative to quantitative parameter set.</li>
		<li>Click on the <img alt="Equilibrium diagram; ΣFx=0 equation; static concept; educational analysis; research use." src="/files/ftp_upload/68543/68543eq05.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 5"> button on the upper toolbar to set the analysis parameters: the window opens.</li>
		<li>Flag CUSTOMIZED 1 and set the sample window at 25, save by clicking on the SAVE ALL SETTINGS button. 
		NOTE: Sample window 25 is ideal for drawing contours.</li>
		<li>Close the window and launch analysis by clicking on the <img alt="Spectroscopy setup; DNA separation method, chromatography system diagram, spectral fitting." src="/files/ftp_upload/68543/68543eq06.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 6"> button on the upper toolbar.</li>
		<li>Press <img alt="Solid-state NMR diagram, showing spin dynamics, with chemical shift and spectral vectors." src="/files/ftp_upload/68543/68543eq07.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 7"> on the upper toolbar to select the contour draw/edit mode and select CONTOUR from the window (Supplemental Figure S3 and Supplemental Figure S4). Starting from the edge of calcaneus bone (by sliding through scans in the sagittal plane), move the cursor and click the mouse at regular intervals to draw a contour surrounding the tendon in the transverse cross section.</li>
		<li>Draw a contour every 25 images, moving in the sagittal section to the next position, switching from analysis to ultrasound mode.</li>
		<li>When all contours are laid over the area of interest, press the <img alt="Static equilibrium; ΣFx=0, ΣFy=0 diagram; force vectors, fulcrum, lever, mechanical balance." src="/files/ftp_upload/68543/68543eq08.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 8">  button again to close the window.</li>
		<li>Save contours by clicking the SAVE CONTOUR line from the FILE drop down menu.</li>
	</ol>
	</li>
	<li>Statistics
	<ol>
		<li>On the analysis software, launch a new analysis by clicking on the <img alt="Static equilibrium ΣFx=0, diagram, force vectors, mechanical balance illustration, educational physics concept." src="/files/ftp_upload/68543/68543eq09.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 9"> button on the upper toolbar. 
		​NOTE: <img alt="Static equilibrium diagram with ΣFx=0; includes force vectors and balance analysis for educational use." src="/files/ftp_upload/68543/68543eq10.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 10">  button corresponds to the sample window ideal for general analysis.</li>
		<li>Click on the <img alt="Electrophoresis graph showing DNA separation; results analysis with migration distance." src="/files/ftp_upload/68543/68543eq11.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 11">  button on the upper toolbar to display the graph with statistical data: a window will open. Click on CONTOUR to display the statistics graph (Supplemental Figure S5) of all the drawn and interpolated contours.</li>
	</ol>
	</li>
</ol>
5. Reporting
NOTE: The program is equipped with a reporting function. To prepare data for reporting, some steps must be taken.
<ol>
	<li>On the analysis software, select the appropriate positions in the sagittal cross section and click on the STAR button on the upper toolbar to select the positions for the reference images.</li>
	<li>Click on <img alt="Chromatography diagram; chemical separation method with retention factor analysis." src="/files/ftp_upload/68543/68543eq12.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 12">  button on the upper toolbar to import images and statistical data into a PDF editor. Edit the report, entering patient information, evaluation date, operator, selected reference images, and interpretation of the results in the relevant sections (Supplemental File 1).</li>
</ol>

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<li>Salvi, M., Chelli, A., Maffulli, N., Tiberto, G., Cacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transverse+muscle+ultrasound+analysis+%28TRAMA%29%3A+robust+and+accurate+segmentation+of+muscle+crosssectional+area%5BTitle%5D)+AND+%22Ultrasound+Med+Biol%22%5BJournal%5D)">Transverse muscle ultrasound analysis (TRAMA): robust and accurate segmentation of muscle crosssectional area.</a> Ultrasound Med Biol. 45 (3), 672-683 (2019).</li>
<li>Järvinen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neovascularization+in+tendinopathy%3A+from+eradication+to+stabilization%5BTitle%5D)+AND+%22Br+J+Sports+Med%22%5BJournal%5D)">Neovascularization in tendinopathy: from eradication to stabilization.</a> Br J Sports Med. 54 (1), 1-2 (2020).</li>
<li>Wezenbeek, E., Witvrouw, E., Molenaers, G., Mahieu, N., De Clercq, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+does+normal+tendon+structure+look+like%3F+New+insights+into+tissue+characterization+in+the+Achilles+tendon%5BTitle%5D)+AND+%22Scand+J+Med+Sci+Sports%22%5BJournal%5D)">What does normal tendon structure look like? New insights into tissue characterization in the Achilles tendon.</a> Scand J Med Sci Sports. 27 (7), 746-753 (2017).</li>
<li>Elgart, B., Pietrosimone, L. S., Lucero, A., Stafford, H. C., Berkoff, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identifying+Achilles+tendon+structure+differences+by+ultrasound+tissue+characterization+in+asymptomatic+individuals%5BTitle%5D)+AND+%22Scand+J+Med+Sci+Sports%22%5BJournal%5D)">Identifying Achilles tendon structure differences by ultrasound tissue characterization in asymptomatic individuals.</a> Scand J Med Sci Sports. 31 (10), 1914-1920 (2021).</li>
</ol>

The authors declare no potential conflicts of interest.

The procedure presented in this article can be used to evaluate size and structure of the Achilles tendon through a commercially available solution that has been previously developed13,14 and validated in both healthy and pathological tendons14,15,16,17,18. This solution offers several advantages: it i...

The Achilles tendon is a common site of tendinopathy, causing pain, physical impairment, and disability1,2. Achilles tendinopathy is a common disorder arising from overuse, especially among athletes, but it is also prevalent in sedentary people and patients with rheumatologic and endocrine diseases3. Two distinct forms of Achilles tendinopathy can be diagnosed: midportion tendinopathy that affects the tendon 2-7 cm above its calcaneal insertion and insertional tendinopathy that involves the distal 2 cm of tendon proximal to the calcaneus4. Midportion Achilles tendinopathy is more frequently observed than insertional tendinopathy5,6, which generally occurs in rheumatologic7 and endocrine8 patients and in patients with Haglund deformity9.
Among diagnostic tests for the evaluation of the Achilles tendon, ultrasound is currently the most commonly adopted approach, but its use in clinical practice is typically limited to the quantitative measurements of tendon size (i.e., thickness and cross-sectional area) and qualitative assessment of the tendon structure10,11. Moreover, ultrasound poses problems such as inter/intra-operator variability and lack of standardization in acquisition protocols12. Therefore, many challenges arise when attempting to compare or analyze data acquired with different devices and with different acquisition protocols.
Ultrasound Tissue Characterization (UTC) is a new technique proposed to quantify the structural integrity of the tendon and overcome some limitations of the conventional ultrasound approach13. This method enables the quantification of the ultra-structural arrangement of the Achilles tendon&#160;by analyzing the three-dimensional stability of echo patterns (intensity and distribution) in contiguous transverse ultrasound images13. UTC provides a quantitative assessment of tendon integrity, expressed in terms of four different echo types. Echo type I reflects intact and aligned tendon bundles; echo type II corresponds to mildly disrupted and waving tendon bundles; echo type III results from mainly fibrillary tissue; and echo type IV represents areas composed mainly of amorphous matrix with a marked loss of fibrils, cellular elements, or fluid accumulation13,14.
Recent studies have shown that UTC has good to excellent reproducibility15,16,17,18 and can be used to monitor Achilles tendon changes in elite athletes after single exercise bouts19 and chronic exercise load20, in healthy21 and tendinopathic22 subjects after shockwave therapy, and in patients with midportion tendinopathy23,24. Moreover, UTC has been shown to detect and quantify mild tissue disorganisation, where conventional ultrasound fails to detect these changes. In fact, Docking et al.25 showed in patients with unilateral Achilles tendinopathy (midportion or insertional) that both tendons were structurally compromised, supporting the evidence for the bilateral nature of tendinopathy. We also recently found, in psoriatic arthritis patients with insertional tendinopathy, that UTC analysis was able to detect bilateral structural tendon alterations even when symptoms were unilateral26. Consistently, a recent systematic review27 concluded that UTC can also provide prognostic information in patients with Achilles tendinopathy: the decrease of echo type I and II percentages may precede and predict the occurrence of clinical symptoms. The purpose of the following protocol is to describe the procedure for assessing the Achilles tendon through the UTC approach.

<table><tbody><tr><td>5 &amp;ndash; 16 MHz Linear Array Probe&amp;nbsp;</td><td>Terason</td><td>16L5</td><td>&quot;Transducer&quot; in the manuscript</td></tr><tr><td>uSmart 3200T ultrasound scanner</td><td>Terason</td><td>Terason TAB3</td><td>&quot;Smart system&quot; in the manuscript</td></tr><tr><td>UTC Software</td><td>UTC Imaging</td><td>Version 1.0.0</td><td>&quot;Analysis software&quot; in the manuscript</td></tr><tr><td>UTC Tracker</td><td>UTC Imaging</td><td></td><td>&quot;Tracker&quot; in the manuscript</td></tr></tbody></table>

ultrasound tissue characterization of human achilles tendon by stability quantification of echo patterns

Ultrasound Tissue Characterization of Human Achilles Tendon by Stability Quantification of Echo Patterns

The assessment of tendon size and structure is commonly included in the clinical management of healthy athletes and different types of patients to personalize their training or rehabilitation. The quantitative analysis of the Achilles tendon structure described here can be performed with a recently introduced system that is non-invasive, relatively inexpensive, portable, radiation-free, and enables the rapid collection of accurate and reproducible data. It uses a standard ultrasound probe that is automatically displaced by a tracker in precise steps of 0.2 mm across the region of interest to collect successive two-dimensional ultrasound images that are then compiled into three-dimensional data-block. The measurement procedure is simple: a subject can be tested in 10-15 min, and a comprehensive report is automatically generated. The quantification of four different echo types enables the evaluation of the tissue integrity: this quantification can be useful for the diagnostic and prognostic assessment of patients with Achilles tendinopathy.

Figure 1 shows representative images of a normal Achilles tendon. The qualitative assessment of tendon structure (in both longitudinal and transversal scans) showed a homogeneous echogenicity and the quantitative assessment of tendon size (the target for the thickness measurement was taken at position #2 that corresponded to the thickest part of the tendon in the transversal view) documented a normal thickness (4 mm).
The UTC analysis reported a normal structure o...

Ultrasound is the approach most commonly adopted in the clinical setting for the assessment of the size and structure of the Achilles tendon. To expand the Achilles tendon assessment in a more comprehensive manner, the ultrasound tissue characterization (UTC) approach has recently been proposed and validated in clinical studies.

The assessment of tendon size and structure is commonly included in the clinical management of healthy athletes and different types of patients to ...

We aim to describe a standardized protocol using ultrasound tissue characterization to assess Achilles tendon structure and to support its application, both in research and clinical practice for diagnosing injuries and monitoring treatment response. Many approaches for the assessment of tendon disorders have been proposed, the most recent advance is inside the area of ultrasound imaging, which provides an accessible approach for evaluating tendon size and structure. We address the need for a standardized operator independent method to quantitatively assess the tendon structure, overcoming key limitation of traditional ultrasound imaging.Among the many new questions to the method that we described will put forward to the questions of what echo types are associated with various disease states. Do echo type changes precede and predict the occurrence of clinical symptoms? To begin, unscrew the two metal screws underneath the tracker to release the standoff.Cover the superior surface of the standoff with a thick layer of acoustic gel. Then, reattach the standoff to the tracker. Unscrew the two black screws of the transducer strap and the locking screw of the transducer holder base.Insert the transducer into the transducer holder base and fasten the locking screw and the two strap screws. Next, unscrew the two black screws securing the transducer holder. Adjust the holder height so the transducer runs half a millimeter to one millimeter above the standoff, remaining in contact with the acoustic gel.Connect the tracker using the USB cable and attach the transducer to the smart system. Plug in the power brick to supply power. Press the power button on the top left edge of the smart system until the blue indicator lights up.A few beeps will be heard. Double-click on the Ultrasound software icon on the desktop to launch it. Then, minimize the ultrasound screen from the smart system toolbar.Press the on/off button to power on the tracker. Position the subject in a comfortable prone position with the bare foot relaxed and dangling off the end of the table. Apply a thick layer of acoustic gel to the skin on the distal posterior part of the leg to ensure good contact with the transducer.Then, push the knee against the subject's metatarsal heads to passively dorsiflex the ankle until it reaches maximum dorsiflexion. Next, double-click on the analysis software icon on the smart system. Then, click the red acquisition button on the upper toolbar.When the message Tracker initialization ok"appears, click OK.When the Acquisition Control window opens, click Start US to display the ultrasound image from the ultrasound software. In the Acquisition Control window, click Acquisition. When prompted with Press the start button on the tracker to start the acquisition, click OK.Place the tracker on the calcaneal region so that it is perpendicular to the long axis of the Achilles tendon.Then, press the start button on the tracker to begin the acquisition. The transducer begins to move automatically along the tracker over the length of the tendon. Monitor the full scanning procedure on the ultrasound scanner screen of the smart system.At the end of data collection, click OK on the Acquisition Control window. Then, click Exit to close the window. Click on the transverse cross section in the top left of the Analysis software screen to refresh and display acquired images.From the file dropdown menu, select Save Scan, and save images into the designated folder. Click the appropriate button on the upper toolbar of the analysis software to switch from qualitative to quantitative parameter set. Then, press the UTC Quantitative Analysis Properties setting button on the toolbar to open the window.Now, flag Customize Set 1 from the Settings window. Set the sample window to 25. Click Save All Settings to save changes, and then click OK to close the window.Next, press the Show or Edit Contour button on the upper toolbar to open the Contour Mode window and Flag Contour. Click the Analysis 1 button on the toolbar to start contouring. Slide through sagittal scans and click at intervals to draw a contour around the tendon in the transverse cross section, starting from the edge of the calcaneus bone, switching from analysis to ultrasound mode to properly identify tendon margins.Continue drawing a contour every 25 images while moving through the sagittal section. Once all contours are completed, select Save UTC Contour from the file dropdown menu to save all drawn contours. Click the specific Analysis Launch button, which corresponds to the sample window ideal for general analysis for statistics on the toolbar to start a new analysis.Then, press the statistics button on the toolbar to open the statistical graph window. Click Contour to display graphs for all drawn and interpolated contours. For reporting, navigate to the sagittal cross section of the tendon and click the star button on the toolbar to select reference image at significant positions.Press the Export PDF button on the toolbar to import images and statistics into the PDF editor. The normal Achilles tendon showed homogeneous echogenicity in both longitudinal and transverse scans with the thickest region measured at 4 millimeters. at position number 2.UTC Analysis of the normal tendon revealed a high percentage of echo type 1 indicating intact and aligned tendon bundles across all three positions. The pathological Achilles tendon showed moderate hypoechogenic regions proximally and extensive hypoechogenic areas in both the mid portion and distal positions. UTC analysis of the pathological tendon showed a reduced percentage of echo type 1 and a high presence of echo types 3 and 4, especially in the central and distal portions.The echo type 1 percentage in the normal tendon was consistently high, ranging from 86.9%to 90.3%at all positions and remained elevated in the total tendon volume at 83.8%In contrast, the pathological tendon exhibited a much lower echo type 1 percentage at all positions while echo types 3 and 4 were significantly elevated, comprising 50.7%of the total volume.

ultrasound-tissue-characterization-human-achilles-tendon-stability

Research

JoVE Journal

Medicine

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Learn ultrasound tissue characterization to assess Achilles tendon structure using a standardized quantitative protocol. This method enables reliable diagnosis of tendon injuries and monitoring of treatment response in clinical practice.

Ultrasound Tissue Characterization of Human Achilles Tendon by Stability Quantification of Echo Patterns

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