Changes in Muscle Hardness from Resting to Mid-Range Lengthened Positions Detected by Shear Wave Elastography (SWE) with a Novel Protocol of Ultrasound Probe Placement

Round 1

Reviewer 1 Report

The article presents an interest topic related to the shear wave elastography, namely the ultrasound probe placement.

I have several comments.

Reliability test. Why were included only 12 of the 26 subjects?

Results. Table 1. Include the measurement unit for body mass index. BMI should be included in the table caption. Correct years (instead of year).

Discussion. Mention anterior rotation (line 209).

Point out the novelty of the study protocol. The clinical implications of the novel protocol should also be mentioned.

Author Response

Thank you very much for the efforts and expertise invaluable comments to improve this article. This article has been revised accordingly based on your comments. Below please also kindly find the point-to-point response and the detailed revisions to the manuscript.

Reliability test. Why were included only 12 of the 26 subjects?

The subject amount for reliability test was based on the convenient sampling technique. Usually, 10 subjects were good enough to detect the reliability of the measurement tool. A previous work done by Chino K et al. 2012 with similar sample size was also added as reference to support this study design as follows:

on page 5, line 168, stating: “A total of twelve subjects participated the reliability test [24][27].”

Results. Table 1. Include the measurement unit for body mass index. BMI should be included in the table caption. Correct years (instead of year).

As suggested, the years was corrected in Table 1. BMI is an index and has no unit. To avoid the potential future misunderstanding, the full name of BMI was also added to the table caption Please find the revised Table 1 and the caption on page 5, line 170 as follows:

Table 1 Demographic of the subjects (mean ± SD).

Gender (Female : Male)	Age (year)	Height (m)	Weight (kg)	Body mass index
12 : 14	33.5 ± 5.9	1.66 ± 0.10	60.3 ± 10.8	21.4 ± 2.6

 

Discussion. Mention anterior rotation (line 209).

As suggested, more discussion on rotation has been added accordingly on page 7, line 219, stating: “One of the body positions that can affect the measurement is the external and internal rotation of the lower extremity in supine position that can be observed anteriorly”

Point out the novelty of the study protocol. The clinical implications of the novel protocol should also be mentioned.

As suggested, the novelty of the probe placement procedure was emphasized as follows:

On page 7, line 221-223: “…Previous works took consideration of the ultrasound probe location along muscle contraction direction of the lower extremity, however, the probe location between the muscle boarders was usually undermined during experimental procedure (12, 16)…”

On page 7, line 226-228:” This novel probe placement procedure provides more detailed guidance to the SWE operator that can help to eliminate the bias during the SWE measurement.”

Author Response File: Author Response.docx

Reviewer 2 Report

The authors investigate the stiffness of leg muscles. The first finding is, that stiffness is higher in lengthened position compared to rest position. This is in line with the results from previous studies. The second finding is, that the proposed new method to place the probe yield good inter- and intra-rater reliability. However, it is unmentioned (or unknown) how big the improvement in reliability compared to a non-standardized probe placement is. I have some comments.

 

1. Line 55-56: Precisely speaking: SWE is measuring the shear wave speed (SWS). Some vendors convert the SWS to shear modulus (µ). Even if the author stated this in the equation (line 98), it should be mentioned, that the primary measurement value is SWS. In general, I recommend using shear wave speed in the entire document. Especially the averaging of quadratic values (µ ~ SWS^2) yield some bias compared to averaging of the primary values (SWS).
2. Line 56-59: This sentence is referring to Young’s modulus. Again, none of the listed studies in Brandenburg et al. [15] measures the Young’s modulus – their primary measurement value is SWS.
3. Line 56-59: The shear modulus does not necessary assume isotropy. In a transverse isotropic medium, there are two shear moduli: µ_perpendicular and µ_parallel. Witch are measured depends on the orientation of the muscle fibers in relation to the shear wave propagation and the shear wave deflection.
4. Line 63: It should be mentioned that the reported value of 9 kPa correspond to shear modulus.
5. Line 77-78: I totally agree that there is a need to precisely orientate the probe along the fibers. However, the muscle fibers should also be orientated precisely compared to the shear wave propagation direction. Since the SWE use acoustic radiation force impulse (ARFI) to generate shear waves, the shear wave propagation is horizontal (in the image). In figure 1 is a slide (about 15°) mismatch. It should be how big this mismatch was for all other measurements!
6. Line 99: The “3” in kg/m3 should be superscripted.
7. Line 101: Please provide a space between values and units: “10 s”!
8. Line 124-133: The resulting probe position is well visible in figure 2. However, to better understand all steps (i to vi) of the novel probe positioning, a figure will be helpful. Can you please further explain where you choose to draw a transverse line, where the muscle is thickest? Or is it a specific distance from the tendon or bone, or is it the most central position of the whole muscle? In lines 134-139, you explained the position of the probe placement on the RF. However, in figure 2a, the probe is approximately one probe length away from the patella, while in b), it is approximately two probe lengths.
9. Line 124-133 and figure 1: I agree that a standardized probe positioning is necessary. However, the area you measured is only a small part of the whole muscle (7 mm diameter in figure 1). Are the stiffness values the same in different areas? Is the localized stiffness representative of the stiffness of the whole muscle?
10. Line 165: “A total of 26 subjects (age: 33.5 ± 5.9 years)” This are redundant information which are still mentioned in “subjects” and table 1.
11. Table 2: It should be discussed why GM_RST has such a low ICC compared to the others.
12. Table 3: Please mention the unit (kPa) of the presented values.
13. Line 197-202: This is only a repetition of the methods.
14. 2. Effect of RST vs. MRL position on muscle hardness: The comparison with literature is missing. For example, Brandenburg et al. [15] and Gennisson et al. (Ultrasound Med Biol. 2010 May;36(5):789-801. doi: 10.1016/j.ultrasmedbio.2010.02.013.) could be cited regarding the stiffening due to lengthening.
15. Discussion: I miss the literature comparison regarding the reliability. Did your new probe positioning really improve the ICC compared to non-standardized studies?

Author Response

Thank you very much for the efforts and expertise invaluable comments to improve this article. This article has been revised accordingly based on your comments. Below please also kindly find the point-to-point response and the detailed revisions to the manuscript.

Regarding the improvement in reliability of this study compared to a non-standardized probe placement in previous studies, our novel probe placement procedure has achieved the improvement of the reliability by 4% to 26% comparing with previous work. More detailed information has been added to the manuscript as follows:

• On page 6, line 204-209, stating: “…In a previous study, the intra-rater reliability of GM at MRL condition was reported to be moderate to good (ICC=0.68) [24]. Another study reported moderate to good inter-rater reliability for the muscle measurement (ICC: TA=0.616, RF=0.679, GM=0.728) [27]. Our probe placement procedure has achieved better intra-rater (ICC: GM_RST=0.696, GM_MRL=0.862) and inter-rater reliability (ICC: TA_RST=0.871, TA_MRL=0.851, RF_RST=0.732, RF_MRL=0.711, GM_RST=0.707, GM_MRL=0.895) with the increase ranged from 4% to 26%... ”

1. Line 55-56: Precisely speaking: SWE is measuring the shear wave speed (SWS). Some vendors convert the SWS to shear modulus (µ). Even if the author stated this in the equation (line 98), it should be mentioned, that the primary measurement value is SWS. In general, I recommend using shear wave speed in the entire document. Especially the averaging of quadratic values (µ ~ SWS^2) yield some bias compared to averaging of the primary values (SWS).

This mentioned paragraph is an introduction of the basic theory of SSI, as such the underling technical issue was not involved. To clarify and avoid the unnecessary misleading beyond the focus of the current study, the paragraph has been re-written as follows:

• On page 2, line 54-56, stating: “…The ultrasound probe generated a radiation force followed by tissue deformation, resulting in propagation of a transient shear wave. This propagation was captured by the ultra-fast ultrasound image to calculate the tissue hardness [13] …”

2. Line 56-59: This sentence is referring to Young’s modulus. Again, none of the listed studies in Brandenburg et al. [15] measures the Young’s modulus – their primary measurement value is SWS.

Thank you for pointing this out and suggesting a review paper (Brandenburg et al., 2014) for us to refer to. Upon reading the suggested review, it seems only one study reported SWS with SSI method, while the remaining SSI studies have reported shear modulus (μ) or Young`s modulus (E). Please feel free to correct us if this is not the case.

The table 2 of (Brandenburg et al., 2014) is also copied for your kind reference as below:

 

3. Line 56-59: The shear modulus does not necessary assume isotropy. In a transverse isotropic medium, there are two shear moduli: µ_perpendicular and µ_parallel. Witch are measured depends on the orientation of the muscle fibers in relation to the shear wave propagation and the shear wave deflection.

Thank you for pointing this issue out. Indeed, the muscle hardness calculated from shear wave velocity is influenced by the orientation between muscle fiber direction and the probe direction. To clarify this issue to the research community, a paragraph of limitation has been added in the manuscript based on your comments as follows:

• On page 7, line 260-265, stating: “…There were some limitations of the study. Although our novel probe placement procedure managed to eliminate the influence of the measurement errors caused by the probe location, attention should be paid to the fact that we did not eliminate the orientation between muscle fiber direction and the probe. The shear modulus does not necessary assume isotropy. In a transverse isotropic medium, there are two shear moduli: µ_perpendicular and µ_parallel, which are measured depends on the orientation of the muscle fibers in relation to the shear wave propagation and the shear wave deflection. For the in-vivo materials such as muscle, the mechanical property is not isotropy, resulting in the hardness varies among the different directions of muscle fibers and the probe. The influence of muscle fiber direction has been reported before [14], and future studies should take this issue into consideration…”

4. Line 63: It should be mentioned that the reported value of 9 kPa correspond to shear modulus.

The manuscript was revised accordingly based on your comments as follows: on page 2, line 60-61, stating: “…However, there have been variations in the revealed results in shear modulus, such as 9.24 ± 1.98 kPa in Gastrocnemius Medialis (GM) …”

5. Line 77-78: I totally agree that there is a need to precisely orientate the probe along the fibers. However, the muscle fibers should also be orientated precisely compared to the shear wave propagation direction. Since the SWE use acoustic radiation force impulse (ARFI) to generate shear waves, the shear wave propagation is horizontal (in the image). In figure 1 is a slide (about 15°) mismatch. It should be how big this mismatch was for all other measurements!

We appreciate your pinpoint that the ultrasound probe was not precisely parallel to the muscle fiber direction in Figure 1. This is due to the limitation of the vivo material, an illustration of muscle is attached below for your information. We have further acknowledged this limitation in Discussion based on your comments as follows:

• On page 7, line 260-265, stating: “…For the in-vivo materials such as muscle, the mechanical property is not isotropy, resulting in the hardness varies among the different directions of muscle fibers and the probe. The influence of muscle fiber direction has been reported before [14], and future studies should take this issue into consideration…”

6. Line 99: The “3” in kg/m3 should be superscripted.

As suggested, the “3” in kg/m3 has been superscripted accordingly.

7. Line 101: Please provide a space between values and units: “10 s”!

As suggested, the space has been added between “10” and “s”, and also to the other values of 3.3 s and 6.6 s.

8. Line 124-133: The resulting probe position is well visible in figure 2. However, to better understand all steps (i to vi) of the novel probe positioning, a figure will be helpful. Can you please further explain where you choose to draw a transverse line, where the muscle is thickest? Or is it a specific distance from the tendon or bone, or is it the most central position of the whole muscle? In lines 134-139, you explained the position of the probe placement on the RF. However, in figure 2a, the probe is approximately one probe length away from the patella, while in b), it is approximately two probe lengths.

As suggested, a figure was added to the manuscript as Figure 3 to clearly demonstrate all the steps (Figure 3. Illustration of applying the novel probe placement procedure on tibialis anterior muscle. (a) Mark the body landmarks of the fibular head and lateral malleolus for TA; (b) Drew a transverse line on the upper one-third of the line between landmarks for TA; and (c) Locate the middle point between the muscle boarder, which was confirmed with ultrasound B-mode image.).

Please find the Figure 3 on page 4, line 132. The related description of the methodology has also been revised as follows:

• One page 4, line 124-127: “i) find the body landmarks, … (Figure 3a); ii) … draw a transverse line on it (Figure 3b); iii…; iv) mark the medial and lateral border of the target muscle on the skin (Figure 3c);”

The probe was place on the thickest part of the take muscle. Detailed description has been added as follows:

• On page 5, line 137-138, stating: “For RF muscle, the body landmarks were anterior superior iliac spine and the superior border of the patella, with the transverse line drawn in the middle of the line between landmarks (18).”
• On page 5, line 143-144, stating: “For TA muscle, the body landmarks were fibula head and lateral malleolus, with the transverse line drawn on the upper one-third of the line between landmarks (25).”
• On page 5, line 148-149, stating: “For GM muscle, the body landmarks were the popliteal fossa and the cross point of Achilles tendon and two malleoli, with the transverse line drawn on the upper one-third of the line between landmarks (26).”

The reason of why the distance between the probe and the patella seems different between fig 2a and 2b is that the patella tended to slide lower during leg flexion.

9. Line 124-133 and figure 1: I agree that a standardized probe positioning is necessary. However, the area you measured is only a small part of the whole muscle (7 mm diameter in figure 1). Are the stiffness values the same in different areas? Is the localized stiffness representative of the stiffness of the whole muscle?

We agree that the muscle stiffness values are different at different areas, and this is one potential limitation of the ultrasound elastography. To clarify this issues, we have add more statement to the study limitation section as follows:

On page 7, line 265-269, stating: “…In this study, we have reported the muscle hardness in terms of shear modulus within the region of interest for TA, RF and GM at both RST and MRL conditions. Due to the limitation of the probe size and the nature of the in-vivo materials, it shall be noted that the localized hardness cannot represent the whole muscle. Mapping the muscle hardness along the whole muscle area could be considered as a potential future research direction...”

10. Line 165: “A total of 26 subjects (age: 33.5 ± 5.9 years)” This are redundant information which are still mentioned in “subjects” and table 1.

As suggested, the redundant information was deleted as follows:

On page 5, line 165, stating: “…A total of 26 subjects participated in this study…”

11. Table 2: It should be discussed why GM_RST has such a low ICC compared to the others.

Thank you for noticing this point. The shape of the GM muscle may contribute to the decrease of the ICCs. Discussion about this point was added into the manuscript as follows:

On page 6, line 209-211, stating: “…The reliability of GM was found to be the lowest among three target muscles. This might be because the curved shape of the GM muscle makes the ultrasound probe slides easily on the GM muscle during the muscle contraction. This made it difficult to maintain the same probe location, resulting in the lowest ICC values of the GM muscle…”

12. Table 3: Please mention the unit (kPa) of the presented values.

As suggested, it has been revised accordingly on page 6, line 185, as follows: “Table 3 Shear modulus (kPa) of each muscle at both resting and mid-range lengthened positions (mean ± SD)”

13. Line 197-202: This is only a repetition of the methods.

The above mention sentences were rewritten to decrease the repetition and highlight the novelty of the probe placement procedure as follows:

On page 6, line 200-203, stating: “We have followed the protocol of previous studies regarding the probe placement procedure [16, 23, 24]. Upon identified the targeted muscle, one step forward has been taken by marking the edges of the muscle and mid-point between the edges.”

 

14. Effect of RST vs. MRL position on muscle hardness: The comparison with literature is missing. For example, Brandenburg et al. [15] and Gennisson et al. (Ultrasound Med Biol. 2010 May;36(5):789-801. doi: 10.1016/j.ultrasmedbio.2010.02.013.) could be cited regarding the stiffening due to lengthening.

As suggested, the citation was added to the manuscript and the statement on page 7 line 231 was revised as follows: “This finding is in line with previous works (28, 29), however, our study provides…”

15. Discussion: I miss the literature comparison regarding the reliability. Did your new probe positioning really improve the ICC compared to non-standardized studies?

Thank you for point this out. As suggested, the comparison between the previous works was added, please find the detailed revision on page 6, line 204-209, stating: “…In a previous study, the intra-rater reliability of GM at MRL condition was reported to be moderate to good (ICC=0.68) [24]. Another study reported moderate to good inter-rater reliability for the muscle measurement (ICC: TA=0.616, RF=0.679, GM=0.728) [27]. Our probe placement procedure has achieved better intra-rater (ICC: GM_RST=0.696, GM_MRL=0.862) and inter-rater reliability (ICC: TA_RST=0.871, TA_MRL=0.851, RF_RST=0.732, RF_MRL=0.711, GM_RST=0.707, GM_MRL=0.895) with the increase ranged from 4% to 26%...”

Author Response File: Author Response.docx