Validity and reliability of a novel 3D ultrasound approach to assess static lengths and the lengthening behavior of the gastrocnemius medialis muscle and the Achilles tendon in vivo

The study was approved by the Ethics Committee of the University of Graz, Austria (registration number: 39/20/63 ex 2020/21). All participants (see the “Results” section) were informed about the study procedure, its purpose, and MRI safety. Written informed consent was obtained beforehand.

Experimental design

To test the validity of the approach, ultrasound data, which were collected during an initial ultrasound measurement session, were compared with the MRI data captured 24.4 ± 8.1 h after the ultrasound assessment. Reliability analyses were conducted based on the ultrasound data captured during the initial and a second ultrasound measurement sessions (separated by 2.1 ± 0.6 days).

Validity assessment

To test the validity of the 3D ultrasound approach, the static lengths of the GM MTU, the GM muscle belly, and the Achilles tendon were determined using both the proposed ultrasound procedure (see the “Reliability assessment” section) and MRI. Owing to contraindications (use of the contraceptive coil) and technical issues, six participants were excluded from the validity assessment.

The MRI data were acquired using a 3 T Magnetom Vida system with syngo MR XA20A software (Siemens Medical Systems, Erlangen, Germany) at the MRI-Lab at Graz University. The T1 measurements were obtained using a 3D space sequence derived from the commercial Numaris/X VA20A package. The full sequence protocol is provided in the Appendix.

Before scanning, two spherical markers (6-mm diameter, MR-PinPoint®, No. 187, Beekley Medical®) were placed with their center at the level of marks indicating the origin and insertion of the GM MTU determined during the first ultrasound measurement session, therefore, corresponding to the ultrasound scanning position. Two custom-made splints (Ortho-Aktiv; Graz, Austria) were then placed on the participant’s ankle joints to ensure the same joint position during all measurements. The right ankle joint was stabilized at 90° and the left ankle joint at 80° (i.e., 10° dorsiflexion).

The participant was positioned supine with the knees slightly flexed. The participant’s feet were placed in a Head/Neck-20 coil with two Body-18 coils covering the upper part of the measurement volume in an overlapping setup and a Spine-32 coil covering the lower part, ensuring a signal-to-noise ratio (SNR) optimized volume coverage from the soles of the feet to the middle of the thighs.

Reliability assessment

To examine the intra- and inter-rater reliability, a randomization of the starting order for both investigators and the test legs was performed (www.randomizer.org) and a standardized protocol was used. The participant was instructed to lie prone on an examination bench. First, the static length measurements were performed, followed by the dynamic examinations.

Static length assessment

The ankle joint angle (Fig. 1A) was controlled with a goniometer (Ka We V01, Medizintechnik).

Fig. 1figure 1

Measurement set-up for the static (A) and dynamic (B) trials. Placement of reflective markers, ultrasound transducer, and electromyographic sensors to assess gastrocnemius medialis muscle belly, tendon, and muscle–tendon unit lengthening behavior, and muscle activity throughout dorsiflexion rotations, respectively. Marker placement locations: 1 = medial and lateral condyle; 2 = most superficial point of the medial condyle; 3 = four-marker cluster; 4 = medial and lateral malleolus; 5 = proximal insertion of the Achilles tendon onto the calcaneus; 6 = four markers attached to footplate; US1 − US4 = markers placed on the ultrasound probe. B shows the procedure used to calculate the location of the muscle–tendon junction in vivo. Starting from marker US1, this point is corrected by the distance xUS along the horizontal direction, which is formed by vector US1US2, and then by the distance hprobe + yUS along the vertical direction, which is formed by the cross product of markers US1, US2, and US4

First, a 5-cm linear array ultrasound transducer (LA523, MyLab 60; Esaote S.p.A., Genova, Italy) was used to detect the anatomical landmarks needed for the tissue length assessments (Fig. 2). The landmarks were the most superficial point of the medial epicondyle detected at the popliteal fossa, the GM muscle–tendon junction (MTJ), and the proximal tendinous insertion at the calcaneus (Fig. 2). The MTJ was determined by following the path of the muscle belly and visualizing its most distal point in the transverse plane. The anatomical sites were marked, and two ultrasound images were recorded showing the landmarks (Fig. 2). To avoid bias, the skin marks of the first investigator were removed before the second investigator started.

Fig. 2figure 2

Determination of the anatomical landmarks. A The ultrasound transducer was placed in the longitudinal direction onto the heel to locate the proximal tendon attachment point at the calcaneus. The most superficial point of the femoral epicondyle was marked on the skin by localizing with the ultrasound probe in both the longitudinal (B) and transverse (C) planes. The reflective markers were placed collinear with the vertical axis above the determined landmarks. These markers were corrected along the vertical axis according to the distance from the center of the marker to the most superficial point of the medial condyle visible in the ultrasound images (hmarker + lcond). Accordingly, a correction was made according to the distance from the center of the marker to the proximal attachment point of the Achilles tendon at the calcaneus (hmarker + lcalc)

To control for possible muscle activation during the passive measurements, surface electromyographic (EMG) signals of the gastrocnemius lateralis were recorded (Fig. 1). Skin preparation and electrode placement (Blue Sensor N, Ambu A/S, Ballerup, Denmark) were carried out according to the SENIAM guidelines [17].

The reflective markers associated with the 3D motion capture system (10 cameras, Miqus M3, Qualisys AB, Gothenburg, Sweden) were placed on to predefined sites displayed in Fig. 1A, B. Moreover, a 59-mm linear array transducer (LogicScan 128; Telemed, Vilnius, Lithuania) fitted with a rigid cluster of four reflective ultrasound markers was fixed over the GM MTJ (Fig. 1A, B).

The marker positions and EMG data, as well as the ultrasound videos, were simultaneously recorded during two trials at 2000 Hz and 60 Hz, respectively. Afterwards, the second leg was prepared and measured as described above.

Dynamic lengthening assessment

After finishing the static measurements, the lengthening behavior of the GM MTU of the starting leg was assessed. The knee was placed at  ~ 20° flexion using a custom-made cushion (Fig. 1B) [6], and a custom-made footplate was applied to the foot (Fig. 1B) [18, 42]. Furthermore, an inclino-dynamometer [7, 18] was attached to the footplate, and the ultrasound transducer was placed onto the GM MTJ.

Altogether, two rotations were performed using the inclino-dynamometer to move the foot sole into dorsiflexion. The externally applied torque was simultaneously measured [42]. The displacement of the MTJ was recorded.

Data analysesUltrasound examination

The MTJ displacement was manually tracked in the ultrasound videos [23]. The analysis of the tissue length and lengthening behavior was conducted using custom software programmed in MATLAB. To determine the absolute GM MTU, muscle belly, and tendon lengths, the origin and heel markers (Fig. 1) were corrected to determine the locations of the anatomical landmarks in vivo in 3D (Fig. 2A, C).

For the dynamic trials, the heel marker was corrected along the direction defined by the medially fixated footplate markers (Fig. 1B). Finally, the location of the MTJ was determined in 3D as displayed in Fig. 1B. The location of the MTJ was then determined using the calculated vectors, the ultrasound probe height (hprobe), and the vertical (yUS) and horizontal (xUS) coordinates of the MTJ obtained from the tracking procedure.

Muscle belly length was computed as the linear distance between the corrected origin and the MTJ, and tendon length was calculated as the distance between the MTJ and the corrected heel marker position by applying the Pythagorean theorem. The MTU length was calculated as the sum of both lengths.

To assess the lengthening behavior, the individual GM muscle belly, tendon, and MTU lengths were calculated over the range from 0 Nm (i.e., resting length) to the maximum applied common torque (5.5 Nm) for all participants.

Magnetic resonance imaging examination

Two T1 space measurements were performed with overlapping FoVs and later combined into a single 3D volume, using the Numaris/X VA20A angio-compose algorithm. MRI evaluations of the combined 3D volumes were performed with Siemens View&GO in Siemens reference space.

The origin of the GM MTU was determined by locating the longitudinal and transverse slices with the largest marker diameter and 3D referencing the position in the transverse plane. The most superficial point of the medial epicondyle was determined and marked in the three spatial planes by scrolling through the referenced coronal slices. The proximal tendinous insertion of the GM MTU and the GM MTJ were also detected in the three spatial planes. Based on the marker positions, the muscle belly, tendon, and MTU lengths were directly measured in a single 3D volume for both legs (slice thickness of 1 mm).

Statistical analyses

All the statistical analyses were performed using SPSS (version 22.0, SPSS Inc., Chicago, IL, USA). The level of significance was set to α = 0.05.

Sample size calculation

The sample size was based on the ICC estimates of Walter et al. [41]. To achieve an ICC of 0.9, a minimum acceptable ICC of 0.7, a power of 80%, and a significance level of 5%, the sample size calculation resulted in 18 subjects or legs. To account for any possible dropout, 16 healthy subjects (32 legs) were included, as in previous studies [3, 24, 33].

Validity

A dependent t test was used to compare the first ultrasound measurements (day 1) of both investigators, which were compared with the MRI analyses. Since the t test showed no significant difference (n.s.) between the raters, the collapsed ultrasound data (rater 1 + rater 2) were used. To assess the absolute agreement between the 3D ultrasound approach and MRI for the tissue lengths of both legs, Bland–Altman plots were utilized [8].

Reliability

For the reliability analyses, the coefficient of variation (CV) and intraclass correlation coefficient were used [21, 22, 24, 33, 34].

Moreover, the absolute reliability of the tissue lengths was evaluated by further calculating the standard error of measurement , where SD is the mean standard deviation of the respective test–retest length pair across the 15 participants [24]. The minimal detectable change (MDC) with a 95% CI was then derived [MDC95 = 1.96 × sqrt(2) × SEM [16]].

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