This research was approved by the Ethics Application Committee of Hokkaido University, Faculty of Health Sciences (approval number: 18–59). A sufficient explanation was given to the participants, and all experiments were carried out after obtaining informed consent from them. The participants were divided into two groups, that is, the control group and the ankle instability (AI) group. For the control group, we recruited eight healthy volunteers (16 feet, mean age: 22.1 years) with the following conditions: (1) no history of fracture, (2) no history of ankle sprain within three years, and (3) no history of repeated ankle sprains. The participants had self-evaluated Cumberland Ankle Instability Tool (CAIT) scores, which is currently the internationally recommended questionnaire for self-evaluation of ankle instability . The CAIT scores were ≥ 25 points for the control group. For the AI group (five volunteers, eight feet, mean age: 23.2 years), the CAIT scores were less than 25 poin[4,30] group were recruited without a clinical diagnosis, but they were required to have a subjective feeling of pain and instability, giving way, or recurring sprains. The exclusion criteria included a history of fractures, a sprain within the previous year, and hospital visits within one year . The participants were also evaluated using the Karlsson Ankle Functional Score (KAFS) to measure ankle joint function .
Anterior drawer stress test
The stress was loaded using a Telos stress device (GAIII/E; Telos Arztund Krankenhausbedarf GmbH, Hungen, Germany). The front pressure cushion of the Telos device was situated 5 cm above the medial malleolus.
The position of the participants during both ultrasound and X-ray imaging was the standard ankle lateral radiograph, as follows: the popliteal fossa of the examinee’s side was tightly applied to the pole of the Telos, and the knee joint was bent to 60° with the patella facing anteriorly and the tibia lying parallel to the table; the medial malleolus and lateral malleolus were aligned and kept perpendicular to the examination table. The heel was pushed against the Telos, and the angle of the ankle joint was 0° (Fig. 1).
Ultrasound stress imaging tests
All ultrasound images were acquired using an Ascendus (Hitachi ALOKA Medical, Ltd. Tokyo, Japan) with EUP -L75 probe (38 mm linear, 4-18MHz) and scanning at a fixed depth of 40 mm. The probe was placed slightly inside the Achilles tendon from the rear of the ankle along the direction of the major axis. The examiner adjusted the focus at the posterior process of the talus. The tibia and the medial tubercle of the posterior process of the talus could be clearly visualized so that the posterior tibia was as long as possible (Fig. 2). The distance between the tibia and the talus was measured as the shortest distance from the tibia backward of the cortical bone border posterior extension line to the medial tubercle of the posterior process of the talus (Figs 2 and 3). In the control group, ultrasound images were obtained one image sequentially every 10 N from 0 to 150 N. In the AI group, one ultrasound image was obtained at 0, 120, and 150 N each. The anterior displacement of the talus in response to the stress exerted by the Telos device was then measured. Data were expressed as the difference in tibiotalar distance at the respective stress load. The distances between the tibia and the talus were measured using Image J ver. 1.48v (National Institutes of Health, Bethesda, MD, USA).
Stress X-ray tests
All participants underwent stress X-rays. Stress X-ray tests were conducted at 0, 120, and 150 N and images were taken sequentially. We evaluated the distance between the tibia and the talus via X-ray by measuring the shortest distance between the distal portion of the rear tibia (lip) to the talar border (Fig. 4). The amount of anterior talar displacement at 120 and 150 N was calculated with reference to the position at 0 N (Figs 3 and 4). The measurements were made using medical image viewer software, EV Insite R. ver.184.108.40.206 (PSP Co., Tokyo, Japan).
Examiners and measurers
The examiners were two radiology technologists with more than three years of clinical experience each in ultrasound imaging, and the measurers were two experts in ultrasound imaging who assessed the tibiotalar distance from the images taken by the examiners. The examiners physically applied probes to the participants’ ankle joints and then captured images of the tibial cortical bone trailing edge and the posterior talar bone inner nodule. Using these images, the measurers determined the shortest distances in millimeters between the tibial cortical bone trailing extensions and the posterior talar process inner nodules. Each examiner and measurer performed the inspection and measurement once, and the same examiners and measurers were always used. The role of the examiner and the measurer was separated, that is, the examiners only performed the ultrasound examination and the measurers only measured the distances from the resulting images. The two examiners performed the ultrasound on the same day for the same participant. The two measurers analyzed the images separately the day after the stress test without any information about the participants. The X-ray examination was performed by an experienced radiology technologist. The examiners and the meas
urers were not informed about the X-ray images and the self-evaluation scores.
All statistical analyses except Bland–Altman analysis were performed using SPSS Statistics v.18 (IBM Corp., Armonk, NY, USA). Comparisons between two groups were performed using the Mann–Whitney U test. Differences in the amounts of anterior talar displacement under each load were analyzed using analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) multiple comparisons test. Error bars represent standard deviation (SD). A p-value of less than 0.05 was considered statistically significant. Inter-examiner and inter-measurer reliability were calculated using intraclass correlation coefficients (ICCs) [i.e., ICC(2,1) and ICC(2,1), respectively]. ICCs were classified according to the evaluation criteria by Koo et al., where a value above 0.9 was classified as excellent reliability, those between 0.75 and 0.9 as good reliability, those between 0.5 and 0.75 as moderate reliability, and those less than 0.5 as poor reliability . The standard error of the mean (SEM) was calculated by dividing the standard deviation by the square root of the sample size. The coefficient of variation (CV) was calculated by dividing the standard deviation by the average, and the minimum detectable change at the 95% confidence level (MDC95) was calculated using the following formula: MDC95 = SEM × 1.96 × √2 (the value 1.96 is the z score associated with the 95% confidence level). Correlations between the measurements of the anterior talar displacement obtained from ultrasound or X-ray images were estimated using Pearson’s correlation analysis. Probability values p < 0.05 were considered statistically significant. R values were classified as follows: a value above 0.9 was very high, a value between 0.70 and 0.9 was high, a value between 0.40 and 0.70 was moderate, a value between 0.20 and 0.40 was weak, and a value below 0.20 was very weak (Rowntree, 1981; Overholser and Sowinski, 2008).