Reliability and methodology of quantitative assessment of harvested and unharvested patellar tendons of ACL injured athletes using ultrasound tissue characterization

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Reliability and methodology of quantitative assessment of harvested and unharvested patellar tendons of ACL injured athletes using ultrasound tissue characterization © The Author(s), 2019. Published version Pereira, Carla S.; Santos, Rafael C. G.; Whiteley, Rod; Finni, Taija Pereira, C. S., Santos, R. C. G., Whiteley, R., & Finni, T. (2019). Reliability and methodology of quantitative assessment of harvested and unharvested patellar tendons of ACL injured athletes using ultrasound tissue characterization. BMC Sports Science, Medicine and Rehabilitation, 11, Article 12. https://doi.org/10.1186/s13102-019-0124-x 2019 RESEARCH ARTICLE Open Access Reliability and methodology of quantitative assessment of harvested and unharvested patellar tendons of ACL injured athletes using ultrasound tissue characterization Carla S. Pereira 1,2* , Rafael C. G. Santos 1 , Rod Whiteley 1 and Taija Finni 2 Abstract Background: Ultrasound tissue characterization (UTC) imaging has been previously used to describe the characteristics of patellar and Achilles tendons. UTC imaging compares and correlates successive ultrasonographic transverse tendon images to calculate the distribution of four color-coded echo-types that represent different tendon tissue types. However, UTC has not been used to describe the characteristics of patellar tendons after anterior cruciate ligament reconstruction (ACLR). The aim of this cross-sectional study was to assess the intra and inter-rater reliability of the UTC in unharvested and harvested patellar tendons of patients undergoing ACLR. Methods: Intra and inter-rater reliability of both UTC data collection and analysis were assessed. Ten harvested and twenty unharvested patellar tendons from eighteen participants were scanned twice by the same examiner. Eleven harvested and ten unharvested patellar tendons from sixteen participants were scanned and analyzed twice by two different examiners. Twenty harvested and nineteen unharvested patellar tendons from twenty-three participants were analyzed twice by two examiners. Results: Quantification of the proportion of echo-types I, II, III and IV in the areas of interest: (1) patella apex, (2) proximal tendon, (3) mid tendon, (4) distal tendon, and overall tendon of harvested and unharvested patellar tendons all displayed excellent intra-rater reliability (ICC 2,1 : 0.94 to 0.99), excellent inter-rater reliability for harvested and unharvested patellar tendon scanning and analysis (ICC 2,1 : 0.89 to 0.98), and excellent inter-rater reliability for analysis (ICC 2,1 : 0.95 to 0.99). Intra-rater reliability for the measure of volume was good (ICC 2,1 : 0.69 harvested, 0.67 unharvested), whilst mixed results were observed for the measure of mid tendon thickness (ICC 2,1 : 0.88 harvested, 0.57 unharvested). Inter-rater reliability for scanning and analysis was good for volume (ICC 2,1 : 0.67) and excellent for thickness (ICC 2,1 : 0.97), while the inter-rater reliability for analysis was fair to poor for volume (ICC 2,1 : 0.59 harvested, 0.30 unharvested), and excellent to poor for mid tendon thickness (ICC 2,1 : 0.85 harvested, 0.24 unharvested). Conclusion: UTC imaging is a reliable tool to characterize the quality of most aspects of unharvested and harvested patellar tendons in subjects undergoing ACLR. Keywords: Anterior cruciate ligament, Echo-types distribution, Graft, Tendon quality, UTC © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: [email protected] 1 ASPETAR Orthopaedic and Sports Medicine Hospital, Sports City Street, Inside Aspire Zone, Al Buwairda St, DohaPO Box 29222Qatar 2 Faculty of Sport and Health Sciences, Biology of Physical Activity, Neuromuscular Research Center, University of Jyväskylä, Jyväskylä, Finland Pereira et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:12 https://doi.org/10.1186/s13102-019-0124-x Background Ultrasound tissue characterization (UTC) has been used to assess the integrity of tendon structure in animals and humans. [1–8] UTC captures contiguous transverse ultrasound images over the length of the tendon and semiquantifies the stability of the echotexture over successive transverse ultrasonographic images. [1–3] Four different echo-types have been proposed to discriminate the underlying tendon tissue types; type I = intact and aligned collagen bundles; type II = discontinuous, swollen and wavy collagen bundles; type III = loose matrix; and type IV = amorphous matrix. [2] The validation of this method to date has originally been based on histopathologic studies of the superficial digital flexor tendons of horses, [1,2,9], and subsequently the use of UTC has expanded to human tendons. [3,5,6] Reliability of UTC imaging in both healthy and pathological tendons has demonstrated high intra- and inter-observer reproducibility for both acquisition and analysis. [3,10] Studies using UTC imaging have documented alterations in tendon appearance in the presence of clinically diagnosed Achilles tendinopathy, [3,7,11–18] patellar tendinopathy, [11,18] systemic disease such as diabetes, [19] after platelet-rich plasma (PRP) injection, [4]and after different therapeutic exercise programs. [5,6,12,14] Anterior cruciate ligament (ACL) injuries are one of the most devastating injuries encountered in sports medicine due to the likely requirement of surgery, and the extended recovery and rehabilitation period following the injury. Where surgical reconstruction of the injured ligament is decided (ACL reconstruction - ACLR), surgeons may choose from a range of possible grafts to repair the torn ligament, including allograft –from cadavers or synthetic, and autograft –when either a portion of the quadriceps tendon, hamstrings tendons (Hst), or frequently, the patellar tendon (BTB) is harvested. [20] BTB autograft has garnered increased attention and popularity in recent decades, which has been attributed to the hypothesis that BTB grafts provide superior post-operative stability via its bone-to- bone attachments [21,22]. However, BTB grafts have been associated with increased donor site morbidity, particularly anterior knee pain and quadriceps weakness have been reported [23–25]. Potentially increased understanding of the effects of BTB grafts on tendon structure may help negate the potential side effects of this surgical approach. While UTC has documented reliability and normative data for typical anterior knee pain populations (echo-type I (%) 58 ± 7; echo-type II (%) 34 ± 5; echo-types III (%) 6 ± 4; echo-type IV (%) 3 ± 2) [10], there are no normative or reliability data for those undergoing ACLR using a BTB graft. To establish the utility of interventions for these populations and to understand meaningful changes of the tendon tissue characteristics as they relate to symptoms, reliability and normative data need to be documented in this population. Therefore, the aim of the current study was to assess the intra- and inter-rater reliability of UTC imaging in harvested patellar tendons after ACLR and to provide normative values for this population. Methods Participants The patellar tendons assessed in this study were from participants who sought conservative or surgical treatment for an ACL injury at Aspetar, Orthopaedic and Sports Medicine Hospital, Doha, Qatar. Thirty-seven male athletes registered within Qatar’s sporting federations regularly attending Aspetar Orthopaedic and Sports Medicine Hospital for rehabilitation following ACL injury and/or ACLR during the period of February to August 2018 were invited to participate in the study. Patients were deemed suitable to participate in the study if they were: male, had a diagnosed ACL tear confirmed by magnetic resonance imaging or a previously performed ACLR, and agreed to take part in one or more phases of this study and to be assessed by different examiners and/or on different days (Table 1). Written informed consent was obtained from each participant or legal guardian. Ethical approval was obtained by ethical committee of the Anti-Doping Laboratory Qatar Research Office (2017000227). Ultrasound tissue characterization (UTC) UTC imaging utilizes a 5–12 MHz ultrasound (US) transducer (SmartProbe 12 L5, Terason 2000, Teratech, USA) fixed in a transverse position into a 12 cm tracking device (UTC Tracker, UTC imaging, Netherlands), allowing the capture and storage of a sequence of transverse images of the tendon at regular intervals of 0.02 cm (Fig. 1). Participants lay supine with their knees flexed at approximately 100° and their feet parallel resting on the plinth. Coupling gel was applied between the US probe and the stand-off pad, and between the standoff pad and the skin to optimize contact. The examiners held the UTC tracker device resting with full contact on participant’s anterior knee, parallel to the long axis of the patellar tendon (Fig. 2). The US transducer was placed initially over the apex of the patella and manually moved down to ensure the patellar tendon was centrally located on the transverse view in the UTC acquisition software. Once a good position was visually affirmed, the data acquisition was initiated. The US transducer then moved down the track driven by a motor, from proximal to distal, resulting in a total of 598 sequential transverse images acquired in 45 s. With these scans the UTC algorithm creates a 3D block of the scanned area allowing additional reconstructed coronal and sagittal views (Fig. 3). A scan was considered satisfactory and included for analysis when the upper surface of the patella and Pereira et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:12 Page 2 of 13 tibial tuberosity were at the same level with the patellar tendon horizontal and taut on the sagittal view of the UTC acquisition software, and the patella and tibial tuberosity were aligned longitudinally with the patellar tendon vertically displayed in the coronal view of the UTC acquisition software (Fig. 3). The patellar tendons of participants whose UTC scans did not meet the above criteria (mostly due to painful limited knee flexion post- Table 1 Participants’graft type, sport, patellar tendon investigated and participation time in the different analysis Participant’s number Graft Sports Intra-rater Harvested Intra-rater Unharvested Inter-rater Acquisition & Analysis Inter-rater Harvested Inter-rater Unharvested Involved Involved Uninvolved Involved Uninvolved Involved Involved Uninvolved 1 Hst Football 10.5 M 10.5 M 2 BTB Revision Football 3 M 3 M 3 BTB Table tennis 6 W 6 W preop 4 BTB Futsal preop preop 5 BTB Football 1Y 1Y 6 BTB Football preop preop 7 Hst Volleyball preop 8 BTB Football 6 M 6 W, 6 M preop 9 BTB Football 6 M 10 Allograft Basketball 6 M 11 BTB Handball 6 W, 6 M preop 12 BTB Revision Football 6 W 6 W 13 BTB Revision Football 3 M, 4.5 M 4.5 M 14 Conservative Football preop 15 BTB Handball 6 M 16 Hst Football 6 M 6 M 6 M 6 M 17 BTB Football 9 M 9 M 9 M 9 M 18 BTB Football preop preop 19 BTB Football 9 M 10.5 M 9 M 10.5 M 20 BTB Football 7.5 M 7.5 M 21 Hst Handball 4.5 M 4.5 M 4.5 M 4.5 M 22 Hst Football 6 M 6 M 6 M 23 BTB Hockey 4.5 M 4.5 M 24 BTB Football 4.5 M 4.5 M 25 BTB Football 3 M 3 M 3 M 3 M 26 BTB Football 6 W 6 W 27 Hst Cycling 6 W 6 W 28 BTB Handball 6 M 6 M 6 W, 6 M preop 29 BTB Basketball 6 W 6 W 6 W 6 W 30 BTB Football 6 W 6 W 6 W 31 BTB Rugby preop 32 Hst Handball 3 M 33 BTB Sky diving 6 W 34 BTB Football 6 W 6 W 6 W 35 BTB Football 3 M 3 M 3 M 36 Conservative Football preop preop 37 BTB Football 3 M 3 M “preop”: pre-operation. “W”,“M”, and “Y”denote weeks, months, and years post-operative respectively BTB Bone patellar tendon bone graft, Hst Hamstring graft Pereira et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:12 Page 3 of 13 operatively) were excluded. Due to the presence of swelling and thickness of the harvested patellar tendon we adopted the factory preset of the UTC imaging software for patellar tendons (PT_UTC_VH4028) for medium size participants, with US parameters standardized as: 12 MHz, focus at 2.8 cm, and depth of 4 cm. For these settings each pixel unit can be considered as equivalent to 1.0 mm. In all cases the right knee was scanned first. UTC data analysis and processing All analyses were performed on the UTC analyzer v.2.0.2 using a window size 17. Two examiners scanned the same patellar tendons on the same day. Only one examiner scanned the same patellar tendons twice, 1 day apart. Subsequently software analysis of the same patellar tendons was performed on different days to avoid any possibility of bias in this phase. For the analysis, the margin of the patellar tendon (contour) was manually traced in the transverse images of the tendon creating at least 10 sections along the patellar tendon length to quantify the whole tendon structure (Fig. 4). The first contour of each tendon was drawn from the notch of the tibia. This contour determines the last (most distal) transverse image included in the patellar tendon characterization analysis. The examiner ensured longitudinal alignment between the notch of the tibia and the patellar apex to draw this contour. The second contour was drawn from the first transverse image immediately distal to the patellar apex. This is the first area of interest, set as reference mark 1 in the UTC acquisition software, and is the first transverse image included in the characterization analysis which defines the beginning of the patellar tendon length measurement. Twenty-six images distal to reference mark 1, another contour was drawn (2nd area of interest = reference mark 2) representing the proximal area of the patellar tendon (0.52 cm distal from patellar apex). The 3rd area of interest or mid tendon (reference mark 3) was drawn 51 images distal from reference mark 2 (1.54 cm distal from patellar apex). [8,26] Additionally, at 75% of the distance between the reference mark 1 and the notch of the tibia (last contour), a fourth contour was drawn (4th area of interest = reference mark 4) to characterize the distal part of the patellar tendon (Fig. 5a). Between reference marks 2 and 3, another two contours were drawn approximately 0.5 cm apart. Between reference marks 3 and 4, additional contours were drawn at approximately 0.5 cm intervals, and between reference mark 4 and the notch of the tibia another contour was drawn. (Note that each additional contour provided to the software reduces the amount of interpolation required to depict the patellar tendon.) Only the transverse images between the Fig. 1 Superior view of UTC transducer transversely fixed into tracking device for scanning a right patellar tendon Fig. 2 Lateral view of UTC tracking device showing silicone pad in contact with left patellar tendon Pereira et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:12 Page 4 of 13 patellar apex and the notch of the tibia were considered in the characterization analysis of the patellar tendon. Measurement of thickness of the mid tendon was done manually using the measuring tool of the UTC imaging software (Fig. 5a). The distance in centimeters between the first and last contours represents the length of the patellar tendon (Fig. 5b). The UTC algorithm quantifies the proportion of echotypes in each specific area of interest, (1) patellar apex, (2) proximal tendon, (3) mid tendon, (4) distal tendon, and (5) overall tendon (all the tendon information between the first and last contours, patellar apex and notch of the tibia, respectively). Four sub-types of tendon are classified according to 4 primary tendon features appearing on grayscale ultrasound images: continuity, integrity and alignment of the collagen tendon bundles, and brightness [2]. In essence, alignment is measured by the degree of variation from a true, straight line of a series of pixels within the window being examined. Variation in brightness is estimated by comparing adjacent pixels on their grayscale value –i.e. the representation of the pixel on a scale from complete black through to bright white. The echo-type I (green) is generated by intact and aligned collagen bundles. These collagen bundles appear Fig. 3 Transverse (a), sagittal (b), and coronal (c&d) views of a harvested patellar tendon. The cross-hair is placed in the center of the harvested region (3A) at the distal pole of the patella (3B). Horizontal line ensures that patella and tibia tuberosity are at the same level (3B). The vertical lines in (3C) and (3D) allow confirmation that patella apex and tibia tuberosity are aligned. Horizontal and vertical alignment are requirements for a scan to be considered of a satisfactory quality to be saved and included for analysis Fig. 4 Example of contours drawn in cross-sectional view in harvested (a) and unharvested (b) patellar tendons. Echo-types I are shown as green, echotype 2 as blue, echo-type III as red, and echo-type 4 as black. Note that only the area inside the marked yellow circumference is quantified as patellar tendon, and it is in this area that all calculations regarding relative percentages of different echo-types are made Pereira et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:12 Page 5 of 13 linear within the window, with little to no variation in their grayscale “whiteness”value. The echo-type II (blue) is reported in the presence of discontinuous, swollen, and wavy collagen bundles. It is defined by pixels that are aligned but display variation of about 10% of the gray levels. The echo-type III (red) is generated by a loose matrix consisting mainly of smaller fibrils. It is represented by much less aligned pixels with gray level variation of more than 10%. The echo-type IV (black) is generated by mainly amorphous matrix with loose fibrils, cells and fluid (hematoma and exudate). It is represented by echoes with a severe lack of stability and no pixels alignment over sequential transverse images. [2,3] In addition to echo-type characterization, the UTC algorithm also quantifies the area within the contour (volume) drawn in four selected areas of interested (reference marks 1–4). After running the UTC software analyses, a range of raw data was exported for analysis. The following variables were assessed: length of the patellar tendon; thickness of the mid tendon; percentage of echo-types I, II, III, and IV; tendon volume at patellar apex, proximal, mid, and distal tendon; and percentage of echo-types I, II, III and IV in the whole tendon. Repeated measures - intra-rater and inter-rater reliabilities Twenty unharvested and ten harvested patellar tendons from 18 participants were scanned and analyzed twice, 1 day apart, by the same examiner (C.S.P., physiotherapist –3 years of experience with UTC imaging acquisition and analysis) to test the intra-rater reliability of acquisition and analysis. Ten unharvested and eleven harvested (ACLR) patellar tendons from 16 participants were scanned, on the same day, by two different examiners (C.S.P. and R.C.G.S., sports physician –6 months of experience with UTC imaging acquisition and analysis) to investigate inter-rater reliability. For these tendons, each examiner analyzed their own scans to test the inter-rater reliability of the acquisition and the analysis. Additionally, nineteen unharvested and twenty harvested patellar tendons from 23 participants were analyzed by the two (A) (B) Fig. 5 Example of sagittal images of the patellar tendon after acquisition with UTC imaging. aThe four areas of interest (1–4) are depicted. The vertical black bar shows the measurement of tendon thickness. The first area of interest (patella apex) was defined as the first image distal to the patella apex.The second area of interest (proximal tendon)started0.52cmdistaltomark1,andthethird(midtendon) 1.54 cm further distally. The fourth area of interest (distal tendon) was set at 75% of tendon length. bThe length of the tendon is depicted by the horizontal white bar. In this case, 15 contours were drawn as shown. In all subjects a minimum of 10 of such contours were created. Each of these contours were individually marked in a transverse view to outline the extent of the tendon (see Fig. 4) for that area of interest Pereira et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:12 Page 6 of 13 examiners (C.S.P. and R.C.G.S) to describe the interrater reliability of the analysis (Fig. 6). Statistical analysis Descriptive statistics (mean ± standard deviation) were calculated for participants’demographics and all UTC variables. Data was tested for normality through visual inspection of histograms and Q-Q plots as well as calculation of Shapiro-Wilk statistics. The majority of the studied variables in harvested and unharvested tendons were normally distributed with exception of the following 9 variables: tendon thickness, percentage of echo-types III at proximal tendon, and percentage of echo-types III and IV at the distal tendon in harvested tendons, and the variables of percentage of echo-types I and II at mid-tendon, and percentage of echo-types III and IV in all areas of unharvested tendons. [27] Test –retest reliability of both UTC data collection and analysis were assessed for harvested and unharvested patellar tendons. Two-way mixed single measures intra-class correlation for absolute agreement between repeated scans (ICC 2,1 ) was calculated to yield the standard error of the measurement (SEM = SD (Day 1) × [√(1-ICC)]), [17,28] standard error of measurement as percentage of the grand mean (SEM % GrM = SEM/ Average Acquisitions 1&2 × 100), and the minimal detectable change of all UTC parameters (MDC = 1.96 × SEM × √2). [7,14,16,18,29,30] MDC for the variables of harvested and unharvested patellar tendons were calculated based on the intra-rater reliability analysis, when tendon scanning and contour drawing were performed two times by the same examiner (C.S.P.). ICC values were considered poor when less than 0.40, fair between 0.40 and 0.59, good between 0.60 and 0.74, and excellent when above 0.75. [3,31] 95% confidence intervals (CI) are reported parenthetically after the group estimator where applicable. SPSS version 21 was used for all statistical analyses (SPSS Inc., Chicago, Illinois, USA). Results The mean age of the participants at the time of data acquisition was 23 years (range: 16 to 36 years), body mass of 75.9 ± 15 kg, and height of 177 ± 11 cm. The sport, the time of the data acquisition, and the type of graft used for the ACLR for each participant are detailed in Table 1. Repeated measures –intra-rater reliability Analysis to quantify the proportion of each of the echotypes (I, II, III and IV) in each of the areas of interest Fig. 6 Description of the number of patellar tendons and participants included, and the analyses performed Pereira et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:12 Page 7 of 13 (patellar apex, proximal tendon, mid tendon, distal tendon, and overall tendon) of harvested and unharvested patellar tendons displayed excellent intra-rater reliability (ICC 2,1 : 0.95–0.99 harvested, 0.89–0.98 unharvested) (Table 2). Intra-rater reliability for the measure of volume in the four areas of interest of the tendon was good (ICC 2,1 : 0.69 harvested, 0.67 unharvested), and the intra-rater reliability for the measure of thickness of the mid tendon was excellent for harvested (ICC 2,1 : 0.88) but fair for unharvested (ICC 2,1 : 0.57) tendons (Table 3). The measurement of tendon length displayed excellent intra-rater reliability (4.5 ± 0.6 cm, ICC 2,1 = 0.79, SEM = 0.3 cm, SEM % GrM = 7.4%, MDC = 0.9 cm) for harvested tendons, and (4.9 ± 0.7 cm, ICC 2,1 = 0.94, SEM = 0.2 cm, SEM % GrM = 3.6%, MDC = 0.5 cm) unharvested tendons. The minimal detectable change for harvested tendons was 7.5% for echo-type I, 6.9% for echo-type II, 4.8% for echo-type III and 2% for echo-type IV. For unharvested tendons, the MDC was 14.1% for echo-type I, 10.6% for echo-type II, 6.3% for echo-type III and 1.2% for echo-type IV. Repeated measures –inter-rater reliability –acquisition and analysis Analysis of the amount of echo-types I, II, III and IV in the four areas of interest and in the overall tendon when two examiners acquired and analyzed their own scans of mixed harvested and unharvested patellar tendons demonstrated excellent inter-rater reliability (ICC 2,1 : 0.89– 0.98) (Table 4). Volume of the tendon in the areas of interest, and thickness of the mid tendon showed good (ICC 2,1 : 0.67) and excellent (ICC 2,1 : 0.97) inter-rater reliability, respectively (Table 5). The tendon length of mixed harvested and unharvested tendons appeared to have good inter-rater reliability (4.5 ± 0.5 cm, ICC 2,1 = 0.63, SEM = 0.2 cm, SEM % GrM = 7.3%). Repeated measures –inter-rater reliability - analysis When two examiners analyzed the same scan of harvested and unharvested tendons separately, the interrater reliability was excellent for the echo-type variables in the different areas of interest (ICC 2,1 : 0.95–0.99) (Table 6) and mid tendon thickness of harvested tendons (ICC 2,1 : 0.85) (Table 7). The inter-rater reliability of the volume in different levels of the tendon was fair for harvested (ICC 2,1 : 0.59) and poor for unharvested (ICC 2,1 : 0.30) tendons (Table 7). Moreover, the mid tendon thickness of unharvested tendons also displayed poor interrater reliability (ICC 2,1 : 0.24) when two examiners analyzed the same scan (Table 7). On the other hand, tendon length displayed excellent reliability for both harvested and unharvested tendons (4.7 ± 0.7 cm, ICC 2,1 = 0.86, SEM = 0.3 cm, SEM % GrM = 5.4%, and 4.8 ± 0.6 cm, ICC 2,1 = 0.79, SEM = 0.3 cm, SEM %GrM = 6.6% respectively). Discussion This is the first study to investigate the reliability of UTC in the measurement of tendon structure following ACLR. Results of the current study suggests that the UTC imaging displays excellent reliability for quantifying the proportion of each of the echo-types (I, II, III and IV) in each of the areas of interest (patellar apex, Table 2 Echo-types values of harvested and unharvested tendons for the two acquisition days, done by one examiner Intra-rater reliability for acquisition and analysis –1 examiner / 2 acquisitions/ 2 different days/ 2 analysis per patellar tendon Examiner C.S.P. DAY 1 DAY 2 Area/ Echo-types Type I (%) Type II (%) Type III (%) Type IV (%) Type I (%) Type II (%) Type III (%) Type IV (%) ICC (95% CI) SEM (ICC) % GrM (%) SEM (ICC) (%) Harvested patellar tendons of 10 participants (n= 10) Patellar Apex 56.0 ± 10.8 33.5 ± 5.9 7.3 ± 4.8 2.9 ± 1.6 54.7 ± 9.8 31.6 ± 6.9 8.9 ± 4.2 4.8 ± 2.6 0.95 (0.89–0.97) 21.1 5.3 Proximal tendon 60.0 ± 6.6 32.1 ± 5.1 5.8 ± 3.9 2.2 ± 1.5 60.5 ± 8.8 29.4 ± 6.3 6.6 ± 4.1 3.6 ± 2.8 0.97 (0.94–0.98) 16.1 4.0 Mid Tendon 62.8 ± 7.0 29.4 ± 4.0 5.4 ± 3.5 2.5 ± 1.7 64.8 ± 8.8 27.2 ± 6.8 5.2 ± 3.3 3.1 ± 2.6 0.98 (0.95–0.98) 15.1 3.8 Distal Tendon 46.9 ± 7.9 35.3 ± 6.3 13.2 ± 8.4 4.5 ± 2.6 50.0 ± 12.6 34.0 ± 7.6 11.4 ± 5.8 4.6 ± 2.8 0.95 (0.89–0.97) 17.2 4.3 Overall Tendon 53.6 ± 5.4 33.3 ± 4.1 9.5 ± 4.7 3.7 ± 1.7 54.9 ± 7.0 31.6 ± 4.5 9.2 ± 4.3 4.1 ± 2.4 0.99 (0.97–0.99) 9.0 2.2 Unharvested patellar tendons of 14 participants (n= 20) Patellar Apex 57.5 ± 12.3 39.0 ± 10.6 2.5 ± 5.2 0.9 ± 1.9 56.1 ± 13.3 40.5 ± 12.0 2.6 ± 4.7 0.8 ± 1.6 0.94 (0.90–0.96) 25.5 6.4 Proximal tendon 65.7 ± 10.3 33.0 ± 8.6 0.8 ± 2.3 0.3 ± 0.9 67.4 ± 8.5 31.9 ± 8.0 0.5 ± 0.9 0.2 ± 0.4 0.97 (0.94–0.97) 21.0 5.2 Mid Tendon 68.6 ± 10.2 30.1 ± 8.7 0.9 ± 1.9 0.3 ± 0.7 70.2 ± 9.3 29.0 ± 8.6 0.4 ± 1.2 0.2 ± 0.5 0.98 (0.97–0.98) 14.6 3.7 Distal Tendon 53.3 ± 10.1 41.3 ± 7.8 4.3 ± 5.6 1.2 ± 2.0 52.9 ± 17.7 42.2 ± 12.9 4.1 ± 9.3 0.7 ± 1.7 0.89 (0.83–0.92) 31.6 7.9 Overall Tendon 60.4 ± 7.8 35.2 ± 6.3 3.2 ± 3.3 0.8 ± 1.2 61.6 ± 9.8 33.9 ± 6.9 3.6 ± 4.3 0.8 ± 1.2 0.98 (0.96–0.98) 15.1 3.8 n= number of tendons assessed. Mean ± standard deviation ICC (95% CI) Intra-class coefficient of reliability (95% confidence interval), SEM % GrM Standard error of measurement as percentage of the grand mean, SEM Standard error of measurement Pereira et al. BMC Sports Science, Medicine and Rehabilitation (2019) 11:12 Page 8 of 13