A. Subjects
This study was approved by the ethical committee “Area Vasta Emilia Centro, Regione Emiglia-Romagna CE-AVEC” (protocol ID: P-PPRAI1/2 − 01, CE protocol reference number: 105/2018/OSS/AUSLBO, date of registration: 11/05/2018; ClinicalTrials.gov ID: NCT04709367, date of registration: 12/01/2021) and carried out at the INAIL Prosthetic Center (Bologna, Italy). All experiments were performed in accordance with the World Medical Association’s Code of Ethics and the Declaration of Helsinki. All recruited subjects signed an informed consent.
The inclusion criteria determined the involvement of stabilized (i.e., time since amputation > 18 months) transfemoral amputees between 18 and 65 years old. Subjects with concurrent medical issues or unable to safely perform the physical tasks required in the experimental protocol were excluded.
To identify the target number of subjects needed to obtain a statistical power of 95%, a preliminary study was carried out on 6 transfemoral amputees, to measure residual limb volume fluctuations due to physical activity. Results of this preliminary study are reported in 19. Then, using these data, the following equation was applied for the sample size estimation (paired t-test) 20:
$${n=\left[\frac{\left({z}_{\alpha }+{z}_{\beta }\right) {\sigma } }{{\delta }}\right]}^{2}$$
1
where 𝛽 is the type II error probability (0.05) for the desired statistical power of 95% (power = 1 - 𝛽), α the desired significance level (0.05), 𝑧α and 𝑧β the standard normal scores for confidence level α and 𝛽 respectively, 𝜎 the population standard deviation (0.051 dm3), and 𝛿 the expected difference (0.040 dm3), both found in 19. Thus, the target subjects number, \(n\), resulted equal to 24.
B. Measurement systems for the assessment of residual limb volume
Residual limb volumes can be measured through many techniques, as we widely described in 10. In this section, we will briefly recapitulate them in order to clarify the rationale undergoing the methodological approach used in this study.
The simplest measurement system for the assessment of residual limb volume consists in dipping the residual limb or its cast within a box filled with water, and measure the water displacement 21. However, this technique is susceptible to errors due to subject’s movements and surface tension at the limb-water interface, thus resulting in a low reliability 10.
Anthropometric models can be reconstructed by importing anatomical landmarks distances, measured by tapes or calipers, but these models are not accurate enough to guarantee reliable results 22, 23. Furthermore, as all techniques involving contact with tissues, anthropometric measurements influence the residual limb shape during the evaluation 10.
Magnetic Resonance Imaging, ultrasound and spiral X-ray Computed Tomography can detect changes in volume and internal residual limb structures. Nevertheless, they are costly, invasive, and affected by errors due to subject’s movements. In addition, they are time-consuming and not fast enough to allow for measurements of volume changes due to prosthesis doffing.
More recently, Sanders et al. 24–28 developed a bioimpedance device to measure the conductive tissue extracellular fluid (ECF) volume of transtibial residual limbs while donning the prosthesis. As a drawback, only ECF volume can be acquired, without including the one of bone and adipose tissues.
Measurement strategies comprising the use of a portable 3D scanner are among the most efficient solutions, as demonstrated by de Boer-Wilzing et al. 29. Thanks to the recent developments in 3D scanning, these systems are nowadays reliable, safe, fast and portable. All these features are fundamental for clinical applications 15, 30. Dickinson et al. 31 have evaluated the accuracy of three hand-held 3D scanners: high reliability and accuracy for the VIUScan marker-assisted laser scanner and the Go!SCAN 3D optical scanner were demonstrated (both metrology-grade scanners of Creaform Inc; Canada). Moreover, the Go!SCAN50 scanner allows for a specific body-scanning option (namely semi-rigid positioning), consisting of an algorithm implementation within the acquisition software able to compensate small body tremors associated to the hand holding the scanner and to the scanned object. Furthermore, marker dots are not needed to be applied on the object to be scanned, thanks to the system ability to capture the object natural features. Thus, a 3D scanner based approach including the Go!SCAN50 was selected for the assessment of the volume fluctuations of transfemoral residual limbs in the framework of this study.
C. Experimental setup and Data acquisition
To yield the protocol reliable and acceptable for the enrolled amputees, a dedicated experimental set-up was developed (Fig. 1a). It included a mechanical support, adequately designed to help the enrolled amputees standing on the sound limb in a stable and comfortable way during scanning. A laser level and a laser meter were used to project two perpendicular lines on the anterior surface of the residual limb and a dot on the distal end, respectively. Such tools were positioned at the beginning of the protocol for each amputee and were kept in position until the end. The two lines and the dot, projected on the residual limb, were drawn before starting the tests, to identify the same limb orientation for all the scans. A mirror was placed in front of the amputee to allow for visual feedback, hence helping to maintain the same position. Before starting, four dots were drawn on the residual limb as anatomical landmarks to uniquely identify a scan cutting plane; they were used afterwards in the post-processing of the 3D image data. One dot was drawn on the ischial tuberosity, one on the external surface of the greater trochanter, and the two remaining dots were drawn on a horizontal axis, about 1 cm distally with respect to the great trochanter, and located about 5 cm anteriorly and 5 cm posteriorly on the skin (see Video 1in supplementary material). All dots were drawn by an expert prosthetist which identified the bony prominences by palpation. These body regions were selected since usually featured by minimal volume changes because of the presence of bony structures with a few soft tissues 10.
Scan files were acquired with the VXelements software (Creaform Inc; Canada), that allows for real-time visualization of the 3D image data (Fig. 1b, see Video 1 in supplementary material). Once the acquisition was completed, the mesh optimization was carried out (i.e., filling holes, eliminating bad frames, performing data clean-up, smart decimation). Then, the meshes were imported in the VXmodel software (Creaform Inc; Canada) for post-processing. Three different options can be chosen in the software for aligning scans: (i) Global registration, (ii) Surface Best-Fit alignment and (iii) N-Point alignment. To select the best tool, 3 consecutive scans of a lower limb were performed on 4 not-amputated subjects, resulting in a total of 12 scans. Based on these data, the Surface Best-Fit alignment was selected (Table 1) since it involves the smallest volumetric error, as averaged across subjects.
The Surface Best-Fit tool aligns the meshes using their common surface when they are not in the same referential by considering one mesh fixed. Thanks to the Pre-align option of the tool, it was possible to select at least 3 points on the fixed mesh, and then the same points on the mobile one (Fig. 1c). Thus, the dots drawn on the residual limb as anatomical landmarks - visible in the acquired scan textures - were used (see Video 1 in supplementary material). Once the common points were selected, the Surface Best-Fit alignment was completed (Fig. 1d). Since the software allows for cutting meshes along planes, the point drawn on the ischial tuberosity, and the other two about 1 cm distally with respect to the great trochanter, were used to define the scan cutting planes (Fig. 1e). The resulted holes were filled in a planar way and the volume was computed by the software (Fig. 1f).
Table 1
Mean ± standard deviation of the volumetric error [%] of the 3 consecutive scans of a lower limb of 4 not-amputated subjects, for the three alignment tools of the VXmodel software.
Global registration
|
Surface Best-Fit
|
N-Point
|
0.338 ± 0.097
|
0.313 ± 0.072
|
0.315 ± 0.245
|
D. Experimental Protocol
Results reported in 19 highlighted that each amputee is featured by a specific time of stabilization in volume after doffing the prosthesis. Hence, the experimental protocol was constituted of four test days and defined as follows.
1st session: during this test session (Fig. 2 – Monday week 0), a resting period of 10 min was scheduled upon arriving in order to reach a homeostatic condition of the limb within the prosthesis. Then, the prosthesis was doffed, the amputee was helped to reach the mechanical support of the experimental set-up and 7 scans were acquired at intervals of 10 min in a standing position. This session allowed for the characterization, over time, of the residual limb volume changes due to prosthesis removal and for the identification of the time required to stabilize the residual limb volume for each amputee. More in detail, volume change was calculated starting from minute 20 and until it was lower than the error evaluated for the 3D body scanning method (i.e., 0.313%; Table 1). By that time, volume was considered stabilized.
2nd session, 3rd session, 4th session: further three sessions of tests were performed in three different days, a week apart from each other (Fig. 2 – Tuesday week 0, Tuesday week 1, Tuesday week 2). Each session was featured by two testing times, one in the morning and one in the afternoon. During both (morning and afternoon), upon arrival, the amputee rested for 10 minutes with the prosthesis donned. Then, 2 consecutive scans were performed immediately after prosthesis doffing. Other 2 consecutive scans were carried out after the amputee’s stabilization time (evaluated in the 1st session). Then, the amputee donned the prosthesis and 15 minutes of physical activity were performed (i.e., walking at a self-selected speed on a treadmill) and the same scanning sequence (i.e., 2 scans just after doffing the prosthesis and 2 scans after the residual limb volume stabilization) was repeated. This resulted in 48 scans for each amputee.
E. Statistical analyses
All statistical tests were carried out in IBM SPSS Statistics environment and the significance level was set equal to 0.05.
1st session: the normality of the volume data acquired in this test session was verified (Kolmogorov-Smirnov’s test and Shapiro-Wilk’s test), while the assumption of sphericity was violated (Mauchly's test) (\(p = 0.05\)). Accordingly, the 1-way ANOVA with repeated measures and the Greenhouse-Geisser correctional adjustment was used to investigate the effects of the factor time (7 levels; i.e., time points at 10 min interval) on the measured volume (H0: no difference among sample means at different time-points). Then, Bonferroni post-hoc comparisons were carried out.
The mean and the standard deviation of the post-doffing volume changes over time were calculated, using the first scan (\(t=0\), Fig. 2 – Monday week 0) as the reference. Then, the curve trend of the measured data was fitted in Matlab R2018a.
2nd session, 3rd session, 4th session: during each session, volumes were computed and averaged between the 2 consecutive scans resulting at each time-point (Fig. 2). This resulted in 8 volume values per day for each amputee. Afterward, these volume values were averaged over the three different test days, resulting in 8 values for each amputee at the specific time-points of the day. The normality (Kolmogorov-Smirnov’s test and Shapiro-Wilk’s test) and the sphericity (Mauchly's test) of data distribution were preliminarily verified. Then, the 3-way ANOVA with repeated measures was performed to investigate the effects of factors: testing time (2 levels: morning vs afternoon), physical activity (2 levels: before vs after physical activity), prosthesis removal (2 levels: immediately after prosthesis doffing vs after the stabilization time), and their interactions on measured volume.