Measurement of Healthy and Injured Triceps Surae Morphology

The Achilles tendon originates at the myotendinous junctions of the gastrocnemius and soleus muscles and inserts on the posterior calcaneus. The Achilles tendon consists primarily of densely packed, organized collagen tissue arranged in a hierarchical fashion to maximize tensile strength¹. Despite its ability to withstand heavy forces, the Achilles tendon is susceptible to several types of injuries throughout one's lifespan. These injuries, such as Achilles tendinopathy and Achilles tendon ruptures, are often accompanied by changes in the structure of the triceps surae and surrounding tissues. In Achilles tendinopathy, patients often exhibit fusiform tendon thickening, tendinosis, collagen disorganization, and neovascularization, a process of proliferation of vascular and neural tissue into the tendon². Additionally, pathological changes associated with Achilles tendinopathy include paratendinitis, intratendinous, and/or entheseal calcifications, and bursitis^2,3. After Achilles tendon ruptures, structural changes are common occurrences, irrespective of treatment, and include Achilles tendon thickening and increased tendon length^4,5. Furthermore, muscular changes, such as triceps surae muscular atrophy, are also commonly associated with Achilles tendon injuries^5,6.

The ability to assess triceps surae and surrounding tissue structures provides valuable insight into structural integrity, tissue quality, and size, which are known to relate to symptoms, function, and prognosis^4,7,8,9. Ultrasound imaging is a reliable and valid assessment tool of these structures, including, but not limited to, Achilles tendon length¹⁰, thickness^10,11, cross-sectional area (CSA)¹², gastrocnemius anatomical CSA¹³, and neovascularization^14,15. Assessment of these measures provides valuable insight into the understanding of healthy triceps surae tissue, as well as quantification of structural alterations to evaluate injury risk, severity, and recovery, as well as understanding healthy tissue qualities¹⁶.

Despite the clinical and research utility of ultrasound imaging in assessing triceps surae structure, there are often differences in imaging techniques and measurement parameters among clinical and research studies^17,18. As a result, comparisons between studies are difficult. Therefore, the purpose of this methods paper is to describe a valid and reliable protocol for efficiently evaluating triceps surae muscle and tendon structure using musculoskeletal ultrasound imaging. This protocol aims to demonstrate the feasibility of incorporating this tool into research and clinical settings through its entirety or as specific parts in healthy and injured individuals. In addition, representative values for healthy and injured triceps surae are provided.

The following protocol follows the guidelines set forth by and has been approved by the University Institutional Review Board to ensure safe and ethical human subject research. All subjects provided informed consent for participation in the research studies and dissemination of data. The full protocol takes approximately 20 minutes to perform by a trained sonographer. However, individual measurements may be taken per assessment need.

1. Marking of the skin

1. Ask the participant to be seated at the end of a plinth with their legs hanging off the table.
2. Palpate and mark the medial tibial plateau and most distal aspect of the medial malleolus bilaterally. Measure the distance between the ipsilateral markings to determine tibial length.
3. Calculate 25% of the tibial length. Measure and mark this distance, distal from the medial tibial plateau, to indicate the location for gastrocnemius measurements (Figure 1).
4. Calculate 30% of the tibial length. Measure and mark this distance, proximal from the most distal aspect of the medial malleolus, to indicate the location for soleus measurements (Figure 1).
5. Record the 25% and 30% tibial length for longitudinally measuring the same location during future evaluations.
6. Ask the participant to move into a prone position on the plinth with the knees fully extended and their feet hanging relaxed off the edge of the plinth with ankles in a resting position (Figure 2).
7. Transfer the gastrocnemius and soleus markings to the posterior aspect of the lower leg. Palpate and mark the midpoint of the gastrocnemius (the distal most point between the two muscle bellies). Ask the participant to gently plantarflex against manual resistance to palpate this location.

Figure 1: Skin measurement markings. Tibial length is measured as the distance from the medial tibial plateau to the most distal aspect of the medial malleolus. Soleus muscle thickness is measured at 30% of the tibial length proximal to the most distal aspect of the medial malleolus (blue mark). Gastrocnemius muscle CSA is measured at 25% of the tibial length distal to the medial tibial plateau (red mark). Please click here to view a larger version of this figure.

Figure 2: Participant positioning for ultrasound imaging. The participant is prone with knees fully extended and feet hanging relaxed in a resting ankle position off the edge of the plinth. Please click here to view a larger version of this figure.

2. Ultrasound imaging

NOTE: All images will be taken on both legs. All images should be taken along the midline of the Achilles tendon. Use palpation of the medial and lateral borders of the Achilles tendon to determine midline location.

1. Use B-mode ultrasound with a 5 cm linear array transducer with parameters set for visualization of the triceps surae. Set frequency to 10 MHz and depth of measurement to 3.5 cm for visualization. Adjust the foci between 0.75 cm and 1.75 cm with gain adjusted to 49 for optimal visualization of tissue.
   NOTE: Depth, foci, and gain may be adjusted as appropriate for visualization.
2. Place ultrasound gel on the posterior calcaneus. Holding the probe on the long axis, visualize the proximal calcaneal notch and align the notch with the midpoint of the ultrasound probe. Mark this location on the skin (Figure 3).
3. Marking the skin for measurement of Achilles tendon thickness and CSA will vary depending on the participant. Use the following guidelines to determine this position.
   1. For healthy individuals, measure 2 cm proximal from the proximal calcaneal notch and mark this location on the skin.
   2. For individuals with Achilles tendinopathy, visualize the thickest portion of the Achilles tendon with the ultrasound probe on the long axis and mark this location on the skin. Record the distance of this marking from the proximal calcaneal notch forlongitudinally measuring the same location during future evaluations.
   3. For individuals with Achilles tendinopathy without visible fusiform thickening, mark the skin at the location of most tendon pain upon palpation. Record the distance of this marking from the proximal calcaneal notch for longitudinally measuring the same location during future evaluations.
   4. For individuals post Achilles tendon rupture, visualize the location of rupture with the ultrasound probe in the long axis and mark this location on the skin. Record the distance of this marking from the proximal calcaneal notch for longitudinally measuring the same location during future evaluations.
   5. For the uninvolved limb, make the skin marking at the same distance from the proximal calcaneal notch as the involved limb.
4. Gastrocnemius tendon length (Full tendon length): Using an extended field-of-view and holding the probe on the long axis, begin the image at the calcaneus by visualizing the Achilles tendon insertion. Glide the probe proximally along the midline of the Achilles tendon towards the mark at the gastrocnemius midpoint until the myotendinous junction is visualized. This will end the image. Take three images of the Achilles' full tendon length (Figure 4).
5. Soleus tendon length (Free tendon length): Using an extended field-of-view and holding the probe on the long axis, begin the image at the calcaneus by visualizing the Achilles tendon insertion. Glide the probe proximally along the midline of the Achilles tendon towards the mark at the soleus until the soleus myotendinous junction is visualized. Take three images of the Achilles' free tendon length.
6. Achilles tendon thickness: Using the B-mode view, place the probe on the long axis on the skin marking made for Achilles tendon thickness and CSA (step 2.3). Take three images of Achilles tendon thickness.
7. Achilles tendon cross-sectional area: Using the B-mode view, place a gel standoff pad on the skin marking made for Achilles tendon thickness and CSA (step 2.3). Holding the probe on the short axis, take three images of the Achilles tendon cross-section.
   NOTE: Tendon is of an anisotropic quality; depending on the angle of the sound waves, it will reflect back, making it look bright, or reflect away, making it look dark (Figure 5). Therefore, toggling the probe back and forth will help with visualizing the border of the tendon.
8. Soleus thickness: Using the B-mode view, place the probe on the long axis on the skin marking made for the soleus (30% of the tibial length from the distal aspect of the medial malleolus). Visualize the anterior border of the soleus muscle. Take three images for soleus thickness¹⁹.
   1. To distinguish between the soleus and flexor hallucis longus, have the participant actively flex and extend the great toe. This will cause movement of the fibers of the flexor hallucis longus without moving the soleus fibers.
9. Gastrocnemius cross-sectional area: Using an extended field-of-view, hold the ultrasound probe on the short axis and in line with the mark for the gastrocnemius (25% of the tibial length from the medial tibial plateau). Visualize the medial border of the medial gastrocnemius and begin the image by gliding the probe from medial to lateral until the lateral border of the lateral gastrocnemius is visualized. Take three images for gastrocnemius CSA¹³.
   1. To capture the most medial aspect of the medial gastrocnemius it may be needed to ask the participant to abduct their legs and internally rotate the hip slightly. If this is required, ensure the participant is not plantarflexing their foot, as this will impact measurements.
10. Using the B-mode view, determine the presence or absence of calcifications within the Achilles tendon (Figure 6D) and/or at the Achilles tendon insertion (enthesis; Figure 6C). These calcifications are unattached from the calcaneus and appear as hyperechoic areas with a shadow below, present in multiple planes of view. Take an image to confirm the findings. Note whether the calcifications occur within the Achilles tendon or at the enthesis.
11. Using the B-mode view, determine the presence or absence of bursitis. This is visualized by a hypoechoic area deep to the Achilles tendon at the proximal calcaneus (retrocalcaneal bursitis) and/or superficial to the Achilles tendon insertion (subcutaneous calcaneal bursitis). Take an image to confirm the findings (Figure 6B).
12. Using the Doppler setting on the ultrasound, assess for the presence or absence of neovascularization within the Achilles tendon.
    1. Position the Doppler box (i.e., region of interest) on the tendon, hold the ultrasound probe still, and avoid pressing down or distending the tissue as this may occlude and prevent visualization of blood flow.
    2. Scan the length of the Achilles free tendon, being sure to assess each portion of the tendon without movement of the probe to prevent artifact. If there is vasculature visible within or in contact with the tendon, take three, 3 s videos in the region with maximal blood flow (Figure 6A).

Figure 3: Achilles tendon thickness and CSA measurement locations. Extended field of view ultrasound images of (A) a healthy and (B) tendinopathic Achilles tendon. Dotted lines indicate tendon borders. Red arrows indicate measurement locations. Yellow lines indicate the distance proximal to the proximal calcaneal notch (white arrow). In the case of thickening at the 2 cm mark (B), healthy tendon thickness should be taken at a healthy area in the free tendon (blue arrow). Please click here to view a larger version of this figure.

Figure 4: Achilles tendon length measurements. Extended field of view ultrasound image of the Achilles tendon. Free tendon length (yellow line) is measured from the proximal Achilles tendon insertion on the proximal calcaneal notch to the soleus myotendinous junction (MTJ). Full tendon length (red line) is measured from the proximal Achilles tendon insertion on the proximal calcaneal notch to the gastrocnemius myotendinous junction (MTJ)¹⁰. Please click here to view a larger version of this figure.

Figure 5: Tendon anisotropy. Achilles tendon anisotropy occurs when the ultrasound waves are not perpendicular to the structure. Tilting the ultrasound probe will cause the tendon structure to appear (A) hyperechoic or (B) hypoechoic depending on the relationship of ultrasound waves to the tendon. Dotted lines indicate tendon borders. Please click here to view a larger version of this figure.

Figure 6: Additional ultrasound imaging findings. (A) Neovascularization, (B) retrocalcaneal bursitis, (C) entheseal calcifications, (D) intratendinous calcifications. Areas colored red and yellow indicate blood flow within the tendon region of interest (yellow box). Dotted lines indicate bursa borders. Arrows indicate calcifications²⁰. Please click here to view a larger version of this figure.

3. Ultrasound measurements

NOTE: Ultrasound measurements can be made on the machine based on the settings and tools for the respective ultrasound machine. Images can also be exported for taking measurements on software such as Osirix DICOM viewer. The average of the three trials for each measure will be used for analysis.

1. Gastrocnemius tendon length (full tendon length): Open the longer extended field-of-view tendon image (taken in step 2.4). Measure from the most proximal point of the tendon insertion (proximal calcaneal notch) to the gastrocnemius myotendinous junction (Figure 4).
2. Soleus tendon length (free tendon length): Open the shorter extended field-of-view tendon image (taken in step 2.5). Measure from the most proximal point of the tendon insertion (proximal calcaneal notch) to the soleus myotendinous junction (Figure 4).
3. Tendon thickness at 2 cm: Open the shoter extended field-of-view tendon image. Measure from the most proximal point of the tendon insertion (proximal calcaneal notch) to 2 cm, with the end of the measurement on the deep border of the tendon. Measure from this point on the deep border of the tendon to the direct superficial aspect of the tendon to get the 2 cm tendon thickness. This measure should represent healthy tendon thickness (Figure 7A).
   1. In the case of Achilles tendinopathy, if the tendon is thickened at this 2 cm distance, locate a healthy area in the free tendon to take this measurement and document the distance from the proximal calcaneal notch (Figure 7B).
      NOTE: Thickness is used to describe the raw thickness of the tendon from the most superficial to deep.
4. Tendon cross-sectional area: Open image taken in step 2.7. Visually identify the borders of the Achilles tendon and outline the circumference of the tendon (Figure 8A).
5. Tendon thickness: Open image taken in step 2.6. At the center of the image, measure the Achilles tendon from the superficial border to the deep border of the tendon (Figure 8B).
6. Soleus thickness: Open image taken in step 2.8. At the center of the image, measure from the superficial border to the deep border of the soleus muscle (Figure 9).
7. Gastrocnemius cross-sectional area: Open image taken in step 2.9. Visualize the borders of both the medial and lateral heads of the gastrocnemius and outline the medial gastrocnemius and then the lateral gastrocnemius heads (Figure 10).
8. Achilles tendon thickening: Use this calculation to determine the degree of thickening of the tendon in Achilles tendinopathy. Subtract thickness at 2 cm (or adjusted location; Figure 7A) from tendon thickness (Figure 7B) to obtain tendon thickening (measurement in step 3.5 minus measurement in step 3.3).

Figure 7: Achilles tendon thickening. Achilles tendon thickening is calculated by subtracting (A) the thickness of the healthy portion of the tendon from (B) the thickness of the thickest portion of the tendon³¹. Dotted lines indicate tendon borders. Red lines indicate tendon thickness. Yellow lines indicated a 2 cm distance proximal to the proximal calcaneal notch. Please click here to view a larger version of this figure.

Figure 8: Achilles tendon cross-sectional area and thickness. Ultrasound images of the Achilles tendon in (A) cross-section and (B) longitudinal view at the thickest portion. Dotted lines indicate tendon borders. The red line indicates tendon thickness. A cross-section of the tendon is shown with and without a border for clarity. Please click here to view a larger version of this figure.

Figure 9: Soleus muscle thickness. Ultrasound image of the soleus muscle. Dotted lines indicate muscle borders. The red line indicates muscle thickness. Please click here to view a larger version of this figure.

Figure 10: Gastrocnemius muscle cross-sectional area. Extended field of view ultrasound image of medial and lateral gastrocnemius muscles in cross-section. Dotted lines indicate tendon borders. Please click here to view a larger version of this figure.

The measures in this protocol have been demonstrated to be reliable and valid^10,20. Excellent reliability has been reported for extended field-of-view Achilles tendon length to gastrocnemius (interclass correlation coefficient (ICC): 0.944) and Achilles tendon length to soleus (ICC: 0.898)¹⁰. The minimal detectable change at the group level (MDC_group) for Achilles tendon length measures is 0.43 cm for length to gastrocnemius and 0.41 cm for length to soleus. The MDC at the individual level (MDC_individual) is 1.83 cm for length to gastrocnemius and 1.73 cm for length to soleus¹⁰. Further, comparison between Achilles tendon length measures assessed via extended field-of-view ultrasound imaging and physical measurements on cadaver specimens reveals excellent validity for length to gastrocnemius (ICC: 0.895) and moderate to good validity for length to soleus (ICC:0.744)¹⁰. Excellent reliability has also been established for Achilles tendon thickness (ICC: 0.898)¹⁰ and Achilles tendon CSA measures (ICC: 0.986)²⁰ with a MDC_group of 0.007 cm for thickness (MDC_individual=0.03 cm) and 0.009 cm² for CSA²¹.

Previously, Park et al. established the reliability and validity of anatomical CSA of the gastrocnemius muscles using ultrasound imaging¹³. Triceps surae muscle ultrasound imaging measures have also been established as reliable. In a sample of 10 health participants, excellent reliability was calculated for medial (ICC: 0.988, MDC_group: 0.305 cm², MDC_individual: 0.964 cm²) and lateral (ICC: 0.838, MDC_group: 0.605 cm², MDC_individual: 1.91 cm²) gastrocnemius CSA. In the same healthy sample, soleus thickness was found to have good reliability (ICC: 0.787, MDC_group: 0.093 cm, MDC_individual: 0.293 cm).

Representative results are presented with data from healthy participants (Delaware, USA), participants with Achilles tendinopathy (Delaware, USA), and participants 6 months post Achilles tendon rupture (Delaware, USA and Stockholm, Sweden; Table 1). It is also important to note the demographic differences between groups (Table 2). Consistent with published literature, the sample of individuals post Achilles tendon rupture has a greater proportion of males to females, whereas the individuals with Achilles tendinopathy are closer to an even distribution of males and females^22,23. Additionally, in this data set, the healthy participants are younger than the participants with Achilles tendinopathy and Achilles tendon rupture.

Key findings for healthy participants include an Achilles tendon mean thickness of 0.47 cm and CSA of 0.58 cm², consistent with findings by Jackson et al.²⁴ and Schmidt et al.²⁵. While there are no significant differences between limbs in Achilles tendon length measures for healthy individuals¹⁰, there is a notably large range for all morphological values. Tendon thickness and CSA vary between individuals as a result of body size, sex, age, exercise, and comorbidities^26,27,28,29. Therefore, when evaluating patients with injuries, it is important to use the uninvolved side for comparison. Further, when comparing individuals, using limb symmetry helps to account for body size, physical activity level, and other factors.

For individuals with Achilles tendinopathy, these findings, consistent with the current literature, show thicker tendons with larger CSA compared to asymptomatic limbs³⁰, as well as tendon thickening in the symptomatic region. While CSA and tendon thickness are different within regions, the results also show that individuals post Achilles tendon rupture have Achilles tendon elongation, larger Achilles tendon CSA, and lower triceps surae muscle thickness/CSA in the involved compared to the uninvolved limb.

Table 1: Representative results of healthy participants, participants with unilateral Achilles tendinopathy, and participants 6 months post Achilles tendon rupture. Abbreviations: AT = Achilles tendon; CSA = cross-sectional area; MG = medial gastrocnemius; LG = lateral gastrocnemius. Group data merged for presentation, 6 weeks n=54, 6 months n=56⁵.

Table 2: Demographics of each set of representative data. Abbreviations: ATR = Achilles tendon rupture. Subject data published in⁴ is a partial sample of subject data reported in²⁰. Group data merged for presentation⁵.

Critical steps in the protocol to ensure the validity and reliability of the methods include the use of skin markings on participants to guide the ultrasound exam and the required training of individuals performing the measurements used in research studies. Skin markings at specific documented locations assist in the ability to assess variables of interest consistently and accurately at the same locations in the same individual over time. Furthermore, applying skin markings to different individuals in the same systematic way allows for the comparison of the same variable between individuals. Required reliability training is another critical step in this protocol, allowing for comparison of findings across evaluators. The measurements used as reference values for the reliability training are from a single person who is an expert in ultrasound imaging with over 14 years of experience. Prior to performing the measurements with research participants, researchers should demonstrate the reliability of at least 0.800 (ICC_2,k) on every ultrasound variable in the study population of interest. Reliability training results in a high volume of practice, leading to better assessment and execution of measurements.

Modification and troubleshooting of these methods are required when certain situations arise. Common occurrences include performing measurements on growing children and adolescents, varying body compositions, and open wounds. Musculoskeletal growth of children and adolescents requires additional markings on the skin to account for bone and tendon length changes over time. Tibial length should be measured at all follow-up time points to account for differences in triceps surae muscle locations. Achilles tendon CSA should be assessed at several points along the tendon (e.g., 10%, 20%, and 30% of free tendon length) to account for non-uniform growth in tendon size. Body composition (i.e., muscle size, presence of excess adipose tissue, etc.) may require adjustments in the ultrasound settings utilized. Typically, increased muscle size or excess adipose tissue will require increasing the depth utilized so that the whole structure is visualized in the image³². The gain and focus location should also be adjusted to optimize visualization. Lastly, modifications to the protocol must be made if there is an opening in the skin (i.e., small and open cuts) to ensure a sanitary environment for the participant and the equipment. In these circumstances, a sterile probe cover is placed over the ultrasound transducer, and sterile ultrasound gel is used to protect the participant from infections.

There are some limitations to using ultrasound imaging. As with other imaging modalities, ultrasound imaging requires practice to become proficient with the resultant image quality limited by the skill of the imager¹⁰. While practice and proficiency with the device are important, equally critical is knowledge of the anatomy of the structures of interest. Both practice and familiarization with the body regions being examined will be key in optimizing image quality and ensuring efficiency with the protocol. Additionally, ultrasound imaging is limited by structure depth. When visualizing a deeper tissue, it will result in lower image quality when compared to more superficial tissues, even with the appropriate settings and transducer selection³³. Acoustic shadowing, where the image below a hyperechoic region appears anechoic, is an artifact present when imaging calcifications in the tendon^34,35. The shadowing caused by calcifications makes it difficult or impossible to visualize the structure beneath them. Limitations also arise with equipment. This protocol utilizes the extended field-of-view setting to capture structures that are larger/longer than the length of the probe. Some ultrasound imaging devices do not have this setting, resulting in adjustments to assess Achilles tendon length and gastrocnemius CSA.

Magnetic resonance imaging (MRI) is often used to visualize and quantify structural properties of soft tissue such as tendons and muscles. However, MRI comes with several drawbacks to use, such as its large size, lack of portability, and expense¹⁶. Further, as a static imaging tool, it does not allow for the evaluation of dynamic properties of soft tissues. This protocol using ultrasound imaging offers an alternative method to other imaging modalities as a dynamic, comfortable, compact, and cheaper means to visualize and measure triceps surae structure and morphology¹⁶.

Ultrasound imaging offers real-time imaging, enabling users to observe structures during movements. With this protocol, the toggling of the ultrasound probe is used to distinguish between tissues of differing echogenicity. Additionally, having the participant actively flex the first toe allows for distinguishing between the soleus and the deeper flexor hallucis longus. For optimal accuracy, measurements should be performed soon after image capture to visualize structure borders. Another benefit to ultrasound imaging is patients are not limited by positioning constraints such as with other imaging modalities such as MRI, which require patients to stay unmoving in a fixed position for long periods of time. Finally, with portable ultrasound imaging devices becoming more inexpensive, the use of ultrasound imaging in clinical practice and research studies is increasingly feasible.

Determining recovery of structure is an important feature of recovery from Achilles tendon injuries. Further, structural measures of the triceps surae can provide insight into general Achilles tendon health and can predict the development of symptoms. This protocol has been used clinically as well as in research studies involving the assessment of healthy individuals as well as the evaluation of individuals with Achilles tendinopathy and individuals post Achilles tendon ruptures. Continued use of this method across research studies will allow for a deeper understanding of Achilles tendon morphology as well as structural changes associated with injury, other conditions, and recovery.