Keywords: Achilles tendon, Tendinopathy, Ultrasound, CEUS

Purpose: To compare morphological and power Doppler features of the Achilles tendon and Contrast-Enhanced Ultrasound (CEUS) behaviour between asymptomatic athletes and athletes who had undergone surgery for repair of a previous Achilles tendon rupture.

Methods: 24 athletes were divided in two groups (A and B). Group A included 14 patients with a median age of 32 years (range 27 to 47 years) who had undergone surgical repair for unilateral Achilles tendon rupture. Group B (control group) included 10 subjects with a median of 34 years (range 27 to 40 years) with no previous or present history of tendinopathy. All patients were evaluated with ultrasound (US), power-Doppler and CEUS with second-generation contrast agent. We studied the uninjured Achilles tendon in athletes who had suffered a previous rupture (Group A) and either the left or right Achilles tendon of the athletes in Group B.

Results: CEUS showed a significantly greater ability to detect a greater number of vascular spots within the tendon of Group A compared to Group B (< 0.05).

INTRODUCTION

Many authors described a direct relationship between tendinopathy and increased vascularisation in Achilles tendons, even though this relationship is controversial [1][2][3]. Contrast-Enhanced Ultrasound (CEUS) with second-generation contrast agent demonstrated a high sensitivity to evaluate vascularity of different tissues [4][5][6][START_REF] Thorelius | Contrast-enhanced ultrasound: beyond the liver[END_REF]. Recently, Rudzki and co-authors have used a contrast-enhanced ultrasound method to demonstrate regional variations in the vascularity of the supraspinatus tendon, with an age-dependent decrease in asymptomatic individuals with intact rotator cuffs [8]. Patients who suffer from an acute Achilles tendon rupture have a nearly 200-fold increased risk of a contralateral tendon rupture [9] and many exhibit intratendinous changes in the contralateral Achilles tendon. This observational study compared the sensitivity of power Doppler US and CEUS to assess the vascularisation of the Achilles tendon in two different populations. The first one was composed by subject who had sustained an Achilles tendon rupture and in which the contralateral tendon was assessed, and the second one in which either the right or left helthy tendon was studied. We wanted to verify whether CEUS F o r P e e r R e v i e w has a higher sensitivity than power Doppler US in detecting vasculature and that vascularity is increased in patients who suffered from a previous rupture.

MATERIALS AND METHODS

All the procedures described in the present study were approved by our Institutional Review Board, and all participants gave written informed consent to participate in the study.

Twenty-four amateur runners were included in the study; all athletes were running at least three times in the week for at least 15 km. They were divided in two groups. Group A was composed of 14 patients, 12 males and 2 females, with a median age of 32 years (range 27 to 47 years). All had undergone surgery for a unilateral Achilles tendon rupture in the past two years from the present study. None of them had symptoms from the controlateral tendon. Group B was composed of 10 asymptomatic subjects (7 males and 3 females, with median age of 34 years (range 27 to 40 years) with no previous tendon pathology.

The non affected Achilles tendon of group A subjects, and either the left or the right Achilles tendon (selected by the subject tossing a coin immediately before undergoing the ultrasound examination) of group B subjects were studied with gray-scale US, power Doppler and CEUS.

All subjects were asked to abstain from sports activities for two days before the evaluation. The exams were conducted with a Technos MPX US (Esaote, Genova, Italy) by the senior radiologist with the patient prone and the ankle at 90°; he was blinded to knowing whether (AUC), calculated as the integral of the same curve; RBF (Regional Blood Flow): blood flow, calculated as the ratio between RBV and MTT (medium transit time). Data were entered in a commercially available software. Descriptive statistics were calculated. All analyses were performed using the SPSS 13.0 statistical software package (SPSS Science Inc., Chicago, IL). A Mann-Whitney's test for independent samples was used, considering significative a p value < 0.05.

RESULTS

The gray-scale analysis did not show any pathological thickening at the middle third and the region of insertion on the calcaneus site of all the tendons evaluated. The anterior-posterior mean diameter was 4.6 ± 3.10 mm in group A and 4.55 ± 3.02 mm in the group B. The tendon structure was homogeneous without any focal hypo-or hyper-echoic lesions. We did not detect oedema in the tendon. Power Doppler exam did not show any intra-tendinous neovascularisation in either group.

The results of CEUS are summarized in Tables I and II. The enhancement curves were drawn considering the data of the second videoclip because the presence of the contrast agent was appreciable in all subjects only at 40-60 seconds from the injection. In two male patients of group A, it was not possible to calculate the enhancement curve due to movement artefacts. Group A subjects showed a values of peak (mean 3,9; SD 0,07), TTP (mean 40,2; SD 5,1), RBV (mean 267; SD 142) significant higher than Group B subjects (peak mean

DISCUSSION

Midsubstance ruptures of the Achilles tendon typically occur in 30-50 year old male recreational athletes [9,10]. The cause of Achilles tendon rupture is unknown, but is thought to be based on mechanical and biological factors [11. Gray-scale US is routinely performed to evaluate tendinopathic Achilles tendon, and has been used to assess the appearance of the Achilles tendon following surgical repair [12][13][14]. Intratendinous Doppler activity has been interpreted as an equivalent of neovessels [15]. Colour-and power-Doppler highlight the increase of intra-and peritendinous vascularisation in chronic tendon pathology [16]. Other studies have shown similar results, and conclude that color/power Doppler US is optimal to visualize soft-tissue hyperemia as a sign of tendinopathy [17,18].

Morphological studies showed increased microvascularisation and failed healing response processes associated with Achilles tendon rupture [19][20][21]. Zanetti et al. [3] observed a correlation between pain and neovascularisation at power Doppler, but they did not demonstrate an increased tendon rupture risk in symptomatic patients. Richards et al. demonstrated with power-Doppler analysis a correlation between increase of Achilles tendon vascularisation and chronic pain [22], and showed that power Doppler is more sensitive than colour-Doppler to study vascularity in tendon pathologies.

The presence of Doppler signal in the Achilles tendon does not necessarily indicate disease [15]. At rest, healthy tendon do not demonstrated increased blood flow at colour and power Doppler US.

Experimental studies with laser Doppler flowmetry (LDF) have showed a minimum flux in healthy tendons [START_REF] Fornage | Achilles tendon: US examination[END_REF]. Although the amount of Doppler activity in non-symptomatic, healthy subjects was minute compared with that of patient with chronic tendinopathy, a threshold would be useful to distinguish normal from pathological intratendinous Doppler activity. This threshold is machine-dependent [20]. The vascular response to exercise in normal tendons evaluated with colour Doppler has not been studied in detail [15]. However, although colour Doppler flow in tendons is considered by some as a sign of abnormality [13,17], colour Doppler flow is part of the physiological response to exercise in Achilles tendons of healthy normal subjects [15]. The tendon should not be evaluated immediately after strenuous activity or eccentric exercise, because such an increased flow could be present and produce a false positive reading [15]. For this reason, we asked our subjects not to perform any sports activity for at least two days before the US scan. However, one issue is to be able to distinguish between physiological CD flow and possible pathological flow, and cut off values must be defined.

CEUS is a new procedure that allows to evaluate intra-and peritendinous microcirculation, being able to show vessels up to 40 µm in diameter. To the best of our knowledge, only one study details the results of CEUS of the Achilles tendon in asymptomatic patients [START_REF] Maffulli | Ultrasonographic findings in subcutaneous rupture of Achilles tendon[END_REF]. This approach allowed to detect in 5 healthy tendons the presence of vessels in the central portion.

In our study, CEUS was more sensitive than power Doppler in demonstrating tendon vascularisation. Indeed, we did not observe in either group of subjects any Achilles tendon vascularisation with power Doppler, while this became evident at CEUS from the echogenicity of the micro bubbles of the contrast agents in the small vessels, and Group A showed significantly higher vascularisation than Group B. This suggested that, in high risk subjects for tendon abnormalities with no clinical signs of tendinopathy and no US changes, vascularisation is higher, although still within physiologic limits (where physiologic should be considered no detectable signal at power Doppler).

The design of our study and the size of our cohorts do not allow to speculate whether a rupture of the contralateral Achilles tendon will occur in the subjects in group A, and whether the subjects of group B will develop tendon pathology. Several variables can influence these events, including the type and level of sports activity practiced.

Limits of our study include the lack of a reference method to verify microvascularisation such as histological analysis or a LDF. In fact, macroscopic and/or standard ultrasound evaluation might be unable to detect tiny tendon changes visible only at histology [START_REF] Maffulli | Surgical repair of ruptured Achilles tendon in sportsmen and sedentary patients: a longitudinal ultrasound assessment[END_REF]. Many factors could have influenced our results, including cardiac output, the technique of injection of the contrast medium, type and level of sport activity practised. However, given the significant differences between the two groups at CEUS, we suspect that a tendon vascularisation threshold value could be predictive of a tissue degeneration. If you could establish this value by using a reliable method, we would be able to provide useful information, above all in professional athletes, in order to identify initial tendinopathy in asymptomatic athletes with no pathological changes observed at standard US. CEUS is at present the only minimally-invasive method that allows to perform, through enhancement curves, an accurate quantitative analysis of vascularisation of the Achilles tendon. It is relatively easy from a technical point of view, and is a quick and highly sensible method to visualise tendon microcirculation in both healthy and symptomatic subjects. There are no reported complications related to CEUS. . A real clinical significance is still controversial, however at the moment it could be reserved to professional athletes to identify a chronic tendinopathy clinicaly silent. Further studies are needed to verify the prognostic implications of the increase of microvascularisation detected by CEUS to manage Achilles tendinopathy. PEAK: maximum signal intensity value; TTP (Time to Peak): time that the contrast agent takes to reach its higher concentration; RBV (Regional Blood Volume): blood volume proportional to the area under the curve (AUC), calculated as the integral of the same curve; RBF (Regional Blood Flow): blood flow, calculated as the ratio between RBV and MTT (medium transit time). Results: CEUS showed a significantly greater ability to detect a greater number of vascular spots within the tendon of Group A compared to Group B (< 0.05).

Conclusions:

In athletes who had suffered a tear of an Achilles tendon, CEUS detected small vessels that were not identified by power-Doppler US in the uninjured controlateral Achilles tendon.

CEUS is useful to evaluate microvascularisation vascularity not detected by other imaging techniques. Vascularity seems to be increased in patients who had suffered from a previous rupture. The first one was composed by subject who had sustained an Achilles tendon rupture and in which the contralateral tendon was assessed, and the second one was a healthy control group in which either the right or left helthy tendon was studied. We wanted to verify whether CEUS has a higher sensitivity than power Doppler US in detecting vasculature and that vascularity is increased in patients who suffered from a previous rupture.

INTRODUCTION

MATERIALS AND METHODS

All the procedures described in the present study were approved by our Institutional Review Board, and all participants gave written informed consent to participate in the study.

Twenty-four amateur runners were included in the study; all athletes were running at least three times in the week for at least 15 km. They were divided in two groups. Group A was composed of 14 patients, 12 TTP (Time to Peak): time that the contrast agent takes to reach its higher concentration; RBV (Regional Blood Volume): blood volume proportional to the area under the curve (AUC), calculated as the integral of the same curve; RBF (Regional Blood Flow): blood flow, calculated as the ratio between RBV and MTT (medium transit time).

Data were entered in a commercially available database software.

Descriptive statistics were calculated. All analyses were performed using the SPSS 13.0 statistical software package (SPSS Science Inc., Chicago, IL). A Mann-Whitney's test for independent samples was used, considering significative a p value < 0.05. The results of CEUS are summarized in Tables I andII. The enhancement curves were drawn considering the data of the second videoclip because the presence of the contrast agent was appreciable in all subjects only at 40-60 seconds from the injection. In two male patients of group A, it was not possible to calculate the enhancement curve due to movement artefacts. Group A subjects showed a values of peak (mean 3,9; SD 0,07), TTP (mean 40,2; SD 5,1), RBV (mean 267; SD 142) significant higher than Group B subjects (peak mean 2,8; SD 1,07; TTP mean 31,9; SD 5,9; RBV mean 150; SD 56,8) with a p<0.0). No difference was observed for the RBF parameter (mean 4,1; SD 1,5 in Group A and mean 3 with SD of 1,1 in Group B).

DISCUSSION

Midsubstance ruptures of the Achilles tendon typically occur in 30-50 year old male recreational athletes [15,17 9,10]. The cause of Achilles tendon rupture is unknown, but is thought to be based on mechanical and biological factors [18,19 11]. However, the aetiology and pathogenesis of failed healing response evident at histological examination of specimens from these patients remains to be clarified [20]. Overuse and ripetitive loading appear to play a significant role [21]. The classical failedhealing response at the basis of tendinopathy lesions, however, is also seen in inactive individuals [22].

Gray-scale US is routinely performed to evaluate tendinopathic Achilles tendon, and has been used to assess the appearance of the Achilles tendon following surgical repair [23-28 12-14].

Intratendinous Doppler activity has been interpreted as an equivalent of neovessels [20 15]. Colour-and power-Doppler highlight the increase of intra-and peritendinous vascularisation in chronic tendon pathology [1, 29 16]. Other studies have shown similar results, and conclude that color/power Doppler US is optimal to visualize softtissue hyperemia as a sign of tendinopathy [20,30,31 17,18] and that it could be used to quantify disease activity during tretment [20].. The neovascularisation appeared to be more likely related to the size of the tendon rather than the symptoms. They speculated that the progression to tendinopathy begins with the increase in tendon size followed by detection of blood flow at power Doppler evaluation, and then by pain [START_REF] Richards | Achilles tendon (TA) size and power Doppler ultrasound (PD) changes compared to MRI: a preliminary observational study[END_REF]. Whether pain in Achilles tendon and intratendinous Doppler flow are predisposing signs of partial or complete rupture, or part of a healing phase is not known [18].

The presence of Doppler signal in the Achilles tendon does not necessarily indicate disease [20 15]. At rest, healthy tendon do not demonstrated increased blood flow at colour and power Doppler US.

Experimental studies with laser Doppler flowmetry (LDF) have showed a minimum flux in healthy tendons [36, 37 23]. Although the amount of Doppler activity in non-symptomatic, healthy subjects was tiny compared with that of patient with chronic tendinopathy, a threshold would be useful to distinguish normal from pathological intratendinous Doppler activity. This threshold is machine-dependent [20]. The vascular response to exercise in normal tendons evaluated with colour Doppler has not been studied in detail [18 15]. However, although colour Doppler flow in tendons is considered by some as a sign of abnormality [27, 30 13,17], colour Doppler flow is part of the physiological response to exercise in Achilles tendons of healthy normal subjects [18, 20 15]. The tendon should not be evaluated immediately after strenuous activity or eccentric exercise, because such an increased flow could be present and produce a false positive reading [20 15]. For this reason, we asked our subjects not to perform any sports activity for at least two days before the US scan. However, one issue is to be able to distinguish between physiological CD flow and possible pathological flow, and cut off values must be defined.

CEUS is a new procedure that allows to evaluate intra-and peritendinous microcirculation, being able to show vessels up to 40 µm in diameter. To the best of our knowledge, only one study details the results of CEUS of the Achilles tendon in asymptomatic patients [38 24]. This approach allowed to detect in 5 healthy tendons the presence of vessels in the central portion, where the velocimetric spectrum did not include a diastolic component a resistance index equal to 1.00.

In our study, CEUS was more sensitive than power Doppler in demonstrating tendon vascularisation. Indeed, we did not observe in either group of subjects any Achilles tendon vascularisation with power Doppler, while this became evident at CEUS from the echogenicity of the micro bubbles of the contrast agents in the small vessels, and Group A showed significantly higher vascularisation than Group B. This suggested that, in high risk subjects for tendon abnormalities with no clinical signs of tendinopathy and no US changes, vascularisation is higher, although still within physiologic limits (where physiologic should be considered no detectable signal at power Doppler). Increased vascularisation can be considered as a risk factor of tendinopathy [2], or can be a normal physiological response to physical activity [START_REF] Malliaras | Achilles tendon Doppler flow may be associated with mechanical load among active athletes[END_REF].

The design of our study and the size of our cohorts do not allow to speculate whether a rupture of the contralateral Achilles tendon will Reply to Rewiever 1 1) Was the senior radiologist blinded to knowing whether a particular patient was group A or group B? This should be indicated.

The senior radiologist was blinded whether a patient belonged to group A or B.

2) In group A patients, we should be told how many (if any) had symptoms related to the contralateral Achilles tendon, if any. It would be interesting to subdivide group A patients into two groups; those with symptoms in the contralateral tendon and those who do not, and look for differences.

None of the patient of group A had symptoms related to the controlateral tendon.

3) On page 9, line 53, contrast-enhanced US is referred to as a 'noninvasive method'. I don't consider the procedure non-invasive. Most would consider an intravenous injection to be at least minimally invasive. This relates to the next point as well.

At least a brief comment should be made as to the potential clinical utility of this technique in the future should be made. This is alluded to at the beginning of page 9 (increased ability to detect microvascularization in asymptomatic patient), but should be more explicitly addressed given that it an invasive technique and that it cannot be done without patient preparation (no physical activity for 48 hours prior to scanning).

I agree that CEUS could be considered as a minimally invasive procedure.

Our results are stll very preliminary and need to be conformed on larger series. A possible field of application could be in professional athletes who are at high rsik of injuries in order to detect asymptomatic degenerative lesion. We tried to better explain this point in the text.

Introduction 6-7) It was changed as asked.

Materials and Methods 8, 9) Details required were added.

12) The correct frequency transducer was written (it was a typing mistake). 10,11,13,14) The sonographic terminology was cheched. Illustrations were eliminated.

  to group A or B. A gray-scale US and power-Doppler with a 13-4 MHz linear probe, with longitudinal and transversal scans, were used to detect any structural tendon abnormalities. Anteroposterior thickness, US-echotexture (homogeneous / dishomogeneous), and signs of peritendinopathy were evaluated at the middle one third and insertion of the Achilles tendon on the calcaneus. Patients who showed clinical signs of tendinopathy were excluded from the study. For power Doppler evaluation, the gain was set to cancel any noise signal. CEUS was then performed using longitudinal scans, with a linear 9-3 MHz probe and dedicated CnTi software (Esaote, Genova, Italy). The radiologist administered a 5 ml volume injection of second-generation contrast agent (Sonovue, Bracco, Milano, Italy) followed by 10 ml saline solution in an antecubital vein of the forearm of all the subjects involved in the present study. Two videoclips of 60 seconds each were recorded, beginning at the end of the injection of the contrast agent. The level of contrast diffusion in the tendon was evaluated in a quantitative fashion drawing time intensity enhancement curves, using QontrastTM software (Bracco, Milano, Italy). The intensity of contrast diffusion in the tendon was calculated on longitudinal tendon scan. The variables analyzed on the curves were the following: PEAK: maximum signal intensity value; TTP (Time to Peak): time that the contrast agent takes to reach its higher concentration; RBV (Regional Blood Volume): blood volume proportional to the area under the curve

3 .

 3 Zanetti M, Metzdorf A, Kundert HP, et al. Achilles tendons: clinical relevance of neovascularization diagnosed with power Doppler US. Radiology 2003;227(2):556-60. 4. Albrecht T, Blomley M, Bolondi L, et al. Guidelines for the use of contrast agents in ultrasound. January 2004. Ultraschall Med 2004;25(4):249-56. 5. Genovese EA, Callegari L, Combi F, et al. Contrast enhanced ultrasound with second generation contrast agent for the follow-up of lower-extremity muscle-strain-repairing processes in professional athletes. Radiol Med (Torino) 2007;112(5):740-50. 6. Harvey CJ, Blomley MJ, Eckersley RJ et al. Developments in ultrasound contrast media. Eur Radiol 2001;11(4):675-89. 7. Thorelius L. Contrast-enhanced ultrasound: beyond the liver. Eur Radiol 2003;13 Suppl 3:N91-108. 8. Rudzki JR, Adler RS, Warren RF, et al. Contrast-enhanced ultrasound characterization of the vascularity of the rotator cuff tendon: age-and activity-related changes in the intact asymptomatic rotator cuff. J Shoulder Elbow Surg 2008;17(1 Suppl):96S-100S. 9. Aroen A, Helgo D, Granlund OG et al. Contralateral tendon rupture risk is increased in individuals with a previous Achilles tendon rupture. Scand J Med Sci Sports 2004;14(1):30-3. 10. Maffulli N, Kenward MG, Testa V et al. Clinical diagnosis of Achilles tendinopathy with tendinosis. Clin J Sport Med 2003;13(1):11-5.

  KM, Cook JL, Bonar F et al. Histopathology of common tendinopathies. Update and implications for clinical management. Sports Med 1999;27(6):393-408. T, Gad A, Reinholt FP et al. Tendon pathology in longstanding achillodynia. Biopsy findings in 40 patients. Acta Orthop Scand 1997;68(2):170-5. 21. Tallon C, Maffulli N, Ewen SW. Ruptured Achilles tendons are significantly more degenerated than tendinopathic tendons. Med Sci Sports Exerc 2001;33(12):1983-90. 22. Richards PJ, Win T, Jones PW. The distribution of microvascular response in Achilles tendonopathy assessed by colour and power Doppler. Skeletal Radiol 2005;34(6):336-42.23. Astrom M, Westlin N. Blood flow in the human Achilles tendon assessed by laser Doppler flowmetry. J Orthop Res 1994;12(2):246-52. 24. Koenig MJ, Torp-Pedersen S, Holmich P, et al. Ultrasound Doppler of the Achilles tendon before and after injection of an ultrasound contrast agent--findings in asymptomatic subjects. Ultraschall Med 2007;28(1):52-6. 25. Alfredson H, Lorentzon M, Backman S et al. cDNA-arrays and real-time quantitative PCR techniques in the investigation of chronic Achilles tendinosis. J Orthop Res 2003;21(6):970-5.

  To compare morphological and power Doppler features of the Achilles tendon and Contrast-Enhanced Ultrasound (CEUS) behaviour between asymptomatic athletes and athletes who had undergone surgery for repair of a previous Achilles tendon rupture. Methods: 24 athletes were divided in two groups (A and B). Group A included 14 patients with a median age of 32 years (range 27 to 47 years) who had undergone surgical repair for unilateral Achilles tendon rupture. Group B (control group) included 10 subjects with a median of 34 years (range 27 to 40 years) with no previous or present history of tendinopathy. All patients were evaluated with ultrasound (US), power-Doppler and CEUS with second-generation contrast agent. We studied the uninjured Achilles tendon in athletes who had suffered a previous rupture (Group A) and either the left or right Achilles tendon of the athletes in Group B. Time-Intensity enhancement curves were calculated.

B

  -mode Ultrasound (US) with colour and power Doppler is considered the only investigation able to evaluate microvascularisation. Many authors described a direct relationship between tendinopathy and increased vascularisation in Achilles tendons, even though this relationship is controversial [1-3]. Contrast-Enhanced Ultrasound (CEUS) with second-generation contrast agent demonstrated a high sensitivity to evaluate vascularity of different tissues [4-13 4-7].Recently, Rudzki and co-authors have used a contrast-enhanced ultrasound method to demonstrate regional variations in the vascularity of the supraspinatus tendon, with an age-dependent decrease in asymptomatic individuals with intact rotator cuffs [14 8].Patients who suffer from an acute Achilles tendon rupture have a nearly 200-fold increased risk of a contralateral tendon rupture[15 9] and many exhibit intratendinous changes in the contralateral Achilles observational study compared the sensitivity of power Doppler US and CEUS to assess the vascularisation of the Achilles tendon in two different populations. The first one was composed by subject who had sustained an Achilles tendon rupture and the contralateral tendon was assessed, and the second one was a healthy control group the tendon of the same side has been studied. We hypothesised that CEUS has a higher sensitivity than power Doppler US.

males and 2

 2 females, with a median age of 32 years (range 27 to 47 years). All had undergone surgery for a unilateral Achilles tendon rupture in the past two years from the present study. None of them had controlateral tendon. Group B was composed of 10 asymptomatic subjects (7 males and 3 females, with median age of 34 years (range 27 to 40 years) with no previous tendon pathology. The non affected Achilles tendon of group A subjects, and either the left or the right Achilles tendon (selected by the subject tossing a coin immediately before undergoing the ultrasound examination) of group B subjects were studied with B-mode gray-scale US, power Doppler and CEUS.All subjects were asked to abstain from sports activities for two days before the evaluation. The exams were conducted with a Technos MPX US (Esaote, Genova, Italy) by the senior radiologist with the patient prone and the ankle at 90°; he was blinded to knowing whether a patient belonged to group A or B. A gray-scale US and power-Doppler with a 13-14 13-4 MHz linear probe, with longitudinal and transversal scansions scans, were used to detect any structural tendon abnormalities. Anteroposterior thickness, US-structure echotexture (homogeneous / dishomogeneous), and signs of peritendinopathy were evaluated at the middle one third and insertion of the Achilles tendon on the calcaneus. Patients who showed clinical signs of tendinopathy were excluded from the study. For power Doppler evaluation, the gain was set to cancel any deep flow signals at the level of the calcaneal tuberosity. For power Doppler evaluation, the gain was set to cancel any noise signal. CEUS was then performed using longitudinal scans, with a linear 9-13 9-3 MHz probe and dedicated CnTi software (Esaote, Genova, Italy). The radiologist administered a 5 ml volume injection of second-generation contrast agent (Sonovue, Bracco, Milano, Italy) followed by 10 ml saline solution in an antecubital vein of the forearm of all the subjects involved in the present study. Two videoclips of 60 seconds each were recorded, beginning at the end of the injection of the contrast agent. The level of contrast diffusion in the tendon was evaluated in a quantitative fashion drawing intensitytime time intensity enhancement curves, using QontrastTM software (Bracco, Milano, Italy). The intensity of contrast diffusion in the tendon was calculated on longitudinal tendon scan. The variables analyzed on the curves were the following: PEAK: maximum signal intensity value;

RESULTS

  The gray-scale analysis did not show any pathological thickening at the middle third and the region of insertion on the calcaneus site of all the tendons evaluated. The anterior-posterior mean diameter was 4.6 ± 3.10 mm in group A and 4.55 ± 3.02 mm in the group B. The tendon structure was homogeneous without any focal hypo-or hyper-echoic lesions. We did not detect oedema in the tendon. Power Doppler exam did not show any intra-tendinous neovascularisation in either group.

  Morphological studies showed increased microvascularisation and failed healing response processes associated with Achilles tendon rupture[32-34 19-21]. Zanetti et al.[3] observed a correlation between pain and neovascularisation at power Doppler, but they did not demonstrate an increased tendon rupture risk in symptomatic patients. Richards et al. demonstrated with power-Doppler analysis a correlation between increase of Achilles tendon vascularisation and chronic pain[35 22], and showed that power Doppler is more sensitive than colour-Doppler to study vascularity in tendon pathologies.

sentence was reworded 16 )

 16 We recorded two clips because it was easier to elaborate data on the software. 17, 18) it was changed as suggested Results[19][20][21][22] We wrote the resuls in the section and eliminate the tables. 23) NS was written for mistake.[START_REF] Maffulli | Surgical repair of ruptured Achilles tendon in sportsmen and sedentary patients: a longitudinal ultrasound assessment[END_REF][START_REF] Maffulli | Ultrasound diagnosis of Achilles tendon pathology in runners[END_REF] We observed that in all patients contrast enhancement in the Achilles tendon was visible only after 40-60 seconds; so the first 40 seconds of the clip are useless to calculate the data. Discussion Discussion was rewritten.

Table I : CEUS results of Group A calculated from enhancement curves

 I

	N, gender, age (yrs)	[Peak] [TTP]	[RBV]	[RBF]
	1.	M	27	4.9	47.9	379.7	5.1
	2.	M	28	4.4	44.7	588.7	4.8
	3.	M	29		Not assessable
	4.	F	29	6.8	40.9	393.9	6.5
	5.	M	30	2.9	31.9	131.6	2.9
	6.	M	30	5.00	39.6	288.2	5.1
	7.	M	31		Not assessable
	8.	F	32	3.9	37.7	254.7	4.2
	9.	M	32	3.5	40.7	191.6	3.5
	10. M	33	2.9	46.5	208.8	3.0
	11. M	34	6.0	38.1	346.1	6.2
	12. M	36	3.4	40.2	212.1	3.5
	13. M 14. M	37 47	1.9 1.6 F 38.8 30.7 o r	133.9 76. 8	2.1 1.9
					P
					e
						e r
						R
						e
						v i e
							w

PEAK: maximum signal intensity value; TTP (Time to Peak): time that the contrast agent takes to reach its higher concentration (seconds); RBV (Regional Blood Volume): blood volume proportional to the area under the curve (AUC), calculated as the integral of the same curve (ml); RBF (Regional Blood Flow): blood flow, calculated as the ratio between RBV and MTT (medium transit time) (ml/seconds)

Table II : CEUS results of Group B calculated from enhancement curves

 II

	N, gender, age (yrs)	[Peak]	[TTP]	[RBV]	[RBF]
	1. M	27	4.0	36.1	223.4	4.30
	2.	F	28	4.1	20.2	155.4	4.6
	3.	F	29	4.1	28.3	229.7	4.4
	4. M	30	2.8	40.1	197.6	2.9
	5. M	31	2.9	31.9	131.6	2.9
	6.	F	34	1.5	29.8	72.6	1.8
	7. M	35	1.6	31.0	75.7	1.7
	8. M	36	1.9	33.8	125.4	2
	9. M	37	3.1	39.7	183.6	3.0
	10. M	40	2.5	28.5	111.6	2.5

  23. Astrom M, Westlin N. Blood flow in the human Achilles tendon assessed by laser Doppler flowmetry. J Orthop Res 1994;12(2):246-52. 24. Koenig MJ, Torp-Pedersen S, Holmich P, et al. Ultrasound Doppler of the Achilles tendon before and after injection of an ultrasound contrast agent--findings in asymptomatic subjects. Ultraschall Med 2007;28(1):52-6. 25. Alfredson H, Lorentzon M, Backman S et al. cDNA-arrays and real-time quantitative PCR techniques in the investigation of chronic Achilles tendinosis. J Orthop Res 2003;21(6):970-5.

	will develop tendon pathology. Several variables can influence these could be reserved to professional athletes to identify a chronic 7. Carrafiello G, Laganà D, Recaldini C, et al. Comparison of
	events, including the type and level of sports activity practiced. tendinopathy clinicaly silent. Further studies are needed to verify the contrast-enhanced ultrasounf and computed tomography in classifying
	Limits of our study include the lack of a reference method to verify prognostic implications of the increase of microvascularisation endoleaks after endovascular treatment of abdominal aorta aneurysms:
	microvascularisation such as histological analysis or a LDF. In fact, detected by CEUS to manage Achilles tendinopathy. preliminary experience. Cardiovasc Intervent Radiol 2006; 29(6):969-
	macroscopic and/or standard ultrasound evaluation might be unable to
	detect tiny tendon changes visible only at histology [40, 41 25]. Many
	factors could have influenced our results, including cardiac output, the
	F o r technique of injection of the contrast medium, type and level of sport activity practised. However, given the significant differences between the two groups at CEUS, we suspect that a tendon vascularisation F o r F o r F o r F o r F o r F o r F o r F o r F o r F o r
	P limit value could be predictive of a tissue lesion. A reliable method to P P P P P P P P P P
	e detect this value can be useful to identify initial tendinopathy in e e e e e e e e e e
	e r asymptomatic athletes with no pathological changes observed at e r e r e r e r e r e r e r e r e r e r
	R standard US. we suspect that a tendon vascularisation threshold value could be predictive of a tissue degeneration. If you could establish R R R R R R R R R R
	e v i e w e e e e e e e e e e this value by using a reliable method, we would be able to provide useful information, above all in professional athletes, in order to v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e identify initial tendinopathy in asymptomatic athletes with no pathological changes observed at standard US. CEUS is at present the w w w w w w w w w w
	only non minimally-invasive method that allows to perform, through
	enhancement curves, an accurate quantitative analysis of
	vascularisation of the Achilles tendon. It is relatively easy from a
	technical point of view, and is a quick and highly sensible method to
	visualise tendon microcirculation in both healthy and symptomatic
	subjects. There are no reported complications related to CEUS. A real
	clinical significance is still controversial, however at the moment it

occur in the subjects in group A, and whether the subjects of group B

John Wiley & SonsJournal of Clinical Ultrasound


Michael Skovdal Rathleff

Carsten Møller Mølgaard

Jens Lykkegaard Olesen

Keywords: foot, musculoskeletal, plantar fascia, Plantar fasciitis, sonography foot, musculoskeletal, plantar fascia, plantar fasciitis, reliability, sonography

HAL is a

Introduction

Ultrasound imaging (US) is a well-established, non-invasive method for examining soft tissue structures of the foot, including the plantar fascia (PF). 1,2 It is a valuable tool in clinical practice to evaluate the integrity of musculoskeletal structures qualitatively 3,4 . Healthy tendons are known to have a well-organized, uniform, hyperechoic pattern of collagen. 5 Conversely, tendons with pathology is often thicker and have a more disorganized, diffuse or hypoechoic appearance on US. 1, 4 In essence, tendon health can be clinically evaluated by recording a grayscale sonogram of the tendon.

Diagnosis of plantar fasciitis is often made due to a combination of patient complaints, localization of pain and measurable thickening of the PF. This has been reported in case control studies where patients with plantar fasciitis was compared to healthy subjects. 2,7 Interestingly a correlation has been seen between a reduction in the thickness of the PF and a reduction in pain. 8 Ultrasonography is often used to support the diagnosis of plantar fasciitis by measuring a thickening of the PF. During rehabilitation US may also be used to secure that rehabilitation is proceeding as expected. The goal during rehabilitation is to observe a reduction in the thickness of the PF as well as a reduction in pain. The main problem with US is that it is a real-time and operator-dependent technique, and factors such as transducer-handling and machine-settings might influence size and appearance of the PF. If patients are examined several times by different examiners, it is essential to know intraobserver and interobserver reliability and precision of the method. This will allow the examiner to know if the changes observed are actual changes in thickness or a result of measurement error. To our knowledge, inter-and intraobserver reliability of US assessment of PF thickness and intra-day variation in the thickness of the PF are not yet known. The purpose of the study is to determine intra-and interobserver reliability and measurement precision of US assessment of PF thickness when using one or the average of multiple scans. We expected that intraobserver reliability would be higher than interobserver reliability and that both inter-and intraobserver reliability would increase if the mean of three measurements was used.

Overall we expected that through a standardized protocol in which one examiner records and measures three sonograms, US will be reliable (ICC>0.75) and precise (Limits of agreement <20%).

Methods

Participants

Twenty healthy volunteers, aged 20 to 31 years, without lower extremity symptoms, were recruited for this study. Exclusion criteria were prior history of surgery to the foot and current or prior pain in PF. Participants provided consent on forms approved by the local Ethics Committee in Denmark (N-2009-0050).

Examiners

Two experienced observers were recruited for the study. Both had used musculoskeletal US as part of daily examinations for 2-5 years. In order to minimize learning effect from customization to the specific US machine used in the study, both examiners were allowed a recommendation period of 4 sessions with the US machine.

Test

Measurements were carried out using the set of recommendations from the ESSR (European Society of Musculoskeletal Radiology), figure 1. [START_REF] Beggs | Musculoskeletal Ultrasound Technical Guidelines: VI. Ankle[END_REF] Subjects were positioned in a prone position. The toes were dorsally flexed, and the talo-crural joint was positioned in 0 degrees. As the PF is Three successive scans were made of each foot to resemble a feasible clinical setting. Between each scan the transducer was moved away from the foot and then repositioned. It was decided at random whether observer 1 or 2 did the first scan and whether the scans were initiated on the left or the right foot. To investigate whether the two examiners would increase reliability during the day of testing, all subjects received a consecutive identification number. This allowed us to ensure that no learning effect would influence the results.

Retest

After a period of approximately 60 minutes, the subjects underwent a second ultrasound examination. During the retest, three additional sonograms of the PF were recorded. Care was taken to maintain the same standardized foot position, to keep the ultrasound machine settings constant, and to replicate the exact measurement. As physical activity and time-of-day might influence on the actual thickness of the PF, we chose a period of 60 minutes between test and retest. [START_REF] Beggs | Musculoskeletal Ultrasound Technical Guidelines: VI. Ankle[END_REF][START_REF] Grigg | Incidental walking activity is sufficient to induce time-dependent conditioning of the Achilles tendon[END_REF]

Sonogram Analysis

All sonograms were measured using the built-in software in the scanner. PF thickness measurements were made at the insertion onto the calcaneus. A third person randomly loaded sonograms onto the screen for examiner 1 and 2 to measure, thereby blinding the observers to which subject they were measuring.

Data Analysis

Statistical analysis was performed using SPSS version 15.0. The dependant measure of PF thickness was calculated using the built-in software in the scanner. Each sequential measurement was arranged in SPSS as a separate variable, and intraexaminer reliability with 95% confidence intervals (CI) was estimated using two-way mixed-model, consistency-type intraclass correlation coefficients (ICC).

Both examiners were blinded during measurement of the current and previous thickness values. The reliability when using a single measurement was estimated using the first 2 measurement variables and the "single measures" output from SPSS. The reliability when using a mean of 2 measurements was estimated using the first 2 measurement variables and the "average measures" output from SPSS. The reliability when using a mean of 3 measurements was similarly estimated using the first 3 measurement variables and the "average measures" output from SPSS. To determine interobserver reliability, a parallel model (two-way random effect, total agreement) with single measurement and mean values of 2 and 3 measurements was calculated.

Measurement precision was assessed by limits of agreement (LOA) and LOA as a percentage of the mean value (LOA%).

Results

The sample included in the study consisted of 4 men and 16 women. Mean age was 24.1 (SD 2.6).

Mean body mass index (BMI) was 22.2 (SD 2.1). Paired t-test shows no significant difference between the right and left PF, (p>0.05), table 1. Paired t-test shows no systematic difference in thickness between test and retest for observer 1 or observer 2 (p>0.05). ICC values show no significant difference in reliability between right and left PF which is the reason why the right and left plantar fascias are pooled in the reliability analysis.

Table 1

Intraobserver

Intraobserver reliability measured by ICC for observer 1 using one sonogram was 0.50 (CI 0.23-0.70) and increased to 0.77 (CI 0.56-0.87) when using the average of three sonograms.

Intraobserver reliability for observer 2 using one sonogram was 0.52 (CI 0.09-0.75) and increased to 0.67 (CI 0.39-0.82) when using the average of three sonograms. When using one sonogram, LOA was 0.8mm for observer 1 and 0.9 for observer 2. LOA decreased to 0.6mm (LOA% of 17.5%) and 0.8mm (LOA% 20.8) when using the mean of three sonograms, table 2.

Table 2

Interobserver

Interobserver reliability when measuring different sonograms measured by ICC was 0.62 (CI 0.37-0.78) and increased to 0.82 (CI 0.65-0.91) when using the mean of three sonograms, table 3. When the observers measured the same set of sonograms, reliability measured by ICC was 0.71 (CI 0.50-0.84) and 0.73 (CI 0.55-0.85) and increased to 0.89 (CI 0.78-0.95) and 0.91 (CI 0.82-0.95) Doing a visual inspection of the consecutive ID number plotted against the difference between test and retest, there are no sign of learning effect with decreased difference with increasing ID number. Furthermore a Bland-Altman plot with the difference between test and retest plotted against the thickness of the PF shows no sign of increased error with increased thickness of the PF.

Discussion

The purpose of this study was to determine the intra-and interobserver reliability of US assessment of PF thickness and to investigate improvements in measurement precision when using the mean value of three measurements compared to one. Intraobserver reliability using one sonogram was 0.50 and 0.52 and using the mean of three sonograms intraobserver reliability increased to 0.67 and 0.77. Interobserver reliability when using one sonogram was 0.62 and increased to 0.82 when using the average of three sonograms. LOA based on intratester reliability shows that changes in thickness that are larger than 0.8mm can be considered actual changes in thickness and not a result of measurement error.

A study by Liang et al [START_REF] Liang | Thinner plantar fascia predicts decreased pain after extracorporeal shock wave therapy[END_REF] examined the intraobserver reliability of US to assess the thickness of fascia plantaris in 14 patients diagnosed with plantar fasciitis. They showed ICC values of 0.90 (CI 0.70-0.97). Craig et al [START_REF] Craig | Plantar fascia thickness, a measure of tissue glycation, predicts the development of complications in adolescents with type 1 diabetes[END_REF] did a similar study on an unknown number of patients with diabetes and found ICC of 0.89. Neither Liang et al [START_REF] Liang | Thinner plantar fascia predicts decreased pain after extracorporeal shock wave therapy[END_REF] or Craig et al [START_REF] Craig | Plantar fascia thickness, a measure of tissue glycation, predicts the development of complications in adolescents with type 1 diabetes[END_REF] Koppenhaver 14 examined the intraobserver reliability US to assess the thickness of transverses abdominis (TrA) and multifidi (MU) with a study design similar to this. They found ICC values of 0.83 (CI 0.67-0.91) and 0.79 (CI 0.61-0.81), corresponding to 15.1% and 19.1% Standard Error of the Estimate (SEM), which approximately corresponds to LOA of 0.7 mm and 0.9 mm. SEM and LOA are comparable if study designs are similar. [START_REF] Atkinson | Typical error versus limits of agreement[END_REF] Springer et al 16 did a similar study where they found a intraobserver SEM of 17% when measuring the thickness of TrA, corresponding to measurements precision of 1.3 mm. Bjordal 17 examined intraobserver reliability when measuring the thickness of the tendon from m supraspinatus. They found LOA of 0.4 mm and a mean value of tendon thickness of 6.6 mm.

Interobserver reliability

O`Connor 18 did an interobserver study of US measurement of Achilles tendon thickness. They found a LOA% of 29%. Ying 19 did a similar study and found interobserver measurement error between 5 observers of 0.9-1.1 mm, which corresponds to a LOA% of approximately 20%.

It appears that intraobserver reliability of US assessments of human tendon and muscle thickness are between 0.4 mm and 1.3 mm when measuring healthy tendons. LOA% is between 15 and 19%.

Interobserver reliability is slightly lower with LOA% of approximately 20%. We found intraobserver reliability of ICC 0.77 and 0.67 and LOA 0.6 mm and 0.8 mm, corresponding to 17.5% and 20.8%. Interobserver reliability was ICC 0.82, LOA 0.57, corresponding to 15.8%. Both intra-and interobserver reliability found in the literature correspond to the findings in this study. Genc et al 26 showed a significant correlation between a decrease in thickness of the PF and reduction in pain. This was present at both 1 and 6 months after treatment. They found that PF thickness decreased 1.7 mm from initial treatment until follow-up at 1 month. At the next follow-up after 6 months, the PF thickness had decreased another 0.6mm. Put into context of the current study, the change in PF thickness that they observed in the first month of treatment reflects actual changes in the thickness. However the change in thickness between 1 and 6 months of follow-up might be caused by measurement error as they only recorded one sonogram. The results of this study showed that if only one sonogram is measured, then the minimal detectable change in PF thickness is 0.8mm while it decreases to 0.6mm if three sonograms are used.

Clinical Relevance

Limitations of ICC

In order to analyze intra-and interobserver reliability, we used ICC and LOA. ICC is a very common method for describing reliability in different settings. However ICC has some limitations.

The size of ICC depends on the ratio of the variability between subjects to the total variability. 18 This means that the size of ICC may be affected by factors related to the study sample itself. 18 If two separate test-retest studies are performed on two study samples where one study sample consists of a homogenous group and the other consists of a very heterogeneous groups, the results will be different. This is not because the methods being used have different reliability in the two groups, but because ICC is dependent on the ratio of the variability between subjects to the total variability. 18 The higher ICC in a heterogeneous group is due to the calculations that ICC is based on and not due to the fact that the methods in the two study samples disagree. The consequence is that it is easier to disguise measurement error in a heterogeneous population. Liang et al [START_REF] Liang | Thinner plantar fascia predicts decreased pain after extracorporeal shock wave therapy[END_REF] and Craig et al [START_REF] Craig | Plantar fascia thickness, a measure of tissue glycation, predicts the development of complications in adolescents with type 1 diabetes[END_REF] both examined pathological groups that may consist of a more heterogeneous sample. This

Comparison of mean thickness of the PF

The earliest study where the thickness of the Pf was quantified by US was published in 1993. 21 Studies that examine healthy subjects find mean values of the thickness between 2.6 and 3.9 mm. 7,[21][22][23][24][25][26][27] The two largest studies, which included 154 and 110 subjects, found mean thickness of the PF of 3.2 mm (women and men combined) and 3.3 mm(±0.3 mm) for women and 3.9 mm (±0.5 mm) for men. The average values in these studies are very similar to the ones found in the present study.

Discrepancy in the positioning of subjects, different reference point of measurement and body mass index may to some extent explain the variation from 2.6 to 3.9 mm.

Largest measurement error from scan or measurement

The results in table 2 and 3 show that the interobserver reliability when both observers measure the same scans is higher than when they measure separate scans. This indicates that scan is a source of error rather than measurement of the scan. The thickness of the PF is not identical across the entire insertion onto the calcaneus. 28 This means that movement of the transducer might affect the recorded thickness. Furthermore it indicates that some of the variance across the different scan is not only due to measurement error, but reflects actual changes in thickness.

Conclusion

Measurement of PF thickness by US is a reliable method. If the same observer measures the same person twice, the difference will be maximum 0.6 -0.8 mm (ICC 0.67 -0.77) in 95 % of the examinations when using the mean of three sonograms. If two different observers examine the same

ABSTRACT Purpose

To determine intra-and interobserver reliability and measurements precision of Ultrasound Imaging (US) assessment of plantar fascia thickness when using one, the mean of two or three measurements..

Methods

Two experienced observers scanned 20 healthy subjects twice with 60 minutes between test and retest. A GE LOGIQe ultrasound scanner was used in the study. The built-in software in the scanner was used to measure the thickness of the plantar fascia (PF). Reliability was calculated using intraclass correlation coefficient (ICC) and limits of agreement (LOA).

Results

Intraobserver reliability using one sonogram was ICC 0.50 and 0.52, and using the mean of three sonograms intraobserver reliability increased up to 0.77. Interobserver reliability when measuring one sonogram was ICC 0.62 and increased to 0.82 when using the average of three sonograms.

LOA showed that when using the average of 3 sonograms, LOA decreased to 0.6 mm, corresponding to 17.5% of the mean thickness of the PF.

Conclusions

The results showed that reliability increases when using the mean of three sonograms compared to one. Limits of agreement based on intratester reliability shows that changes in thickness that are larger than 0.6 mm can be considered actual changes in thickness and not a result of measurement error.

Keywords

Introduction

Ultrasound imaging (US) is a well-established, non-invasive method for examining soft tissue structures of the foot, including the plantar fascia (PF). 1,2 It is a valuable tool in clinical practice to evaluate the integrity of musculoskeletal structures qualitatively 3,4 . Healthy tendons are known to have a well-organized, uniform, hyperechoic pattern of collagen. 5 Conversely, tendons with pathology is often thicker and have a more disorganized, diffuse or hypoechoic appearance on US. 1, 4 In essence, tendon health can be clinically evaluated by recording a grayscale sonogram of the tendon.

Diagnosis of plantar fasciitis is often made due to a combination of patient complaints, localization of pain and measurable thickening of the PF. This has been reported in case control studies where patients with plantar fasciitis was compared to healthy subjects. 2,7 Interestingly a correlation has been seen between a reduction in the thickness of the PF and a reduction in pain. 8 Ultrasonography is often used to support the diagnosis of plantar fasciitis by measuring a thickening of the PF. During rehabilitation US may also be used to secure that rehabilitation is proceeding as expected. The goal during rehabilitation is to observe a reduction in the thickness of the PF as well as a reduction in pain. The main problem with US is that it is a real-time and operator-dependent technique, and factors such as transducer-handling and machine-settings might influence size and appearance of the PF. If patients are examined several times by different examiners, it is essential to know intraobserver and interobserver reliability and precision of the method. This will allow the examiner to know if the changes observed are actual changes in thickness or a result of measurement error. To our knowledge, inter-and intraobserver reliability of US assessment of PF thickness and intra-day variation in the thickness of the PF are not yet known. The purpose of the study is to determine intra-and interobserver reliability and measurement precision of US assessment of PF thickness when using one or the average of multiple scans. We expected that intraobserver reliability would be higher than interobserver reliability and that both inter-and intraobserver reliability would increase if the mean of three measurements was used.

Overall we expected that through a standardized protocol in which one examiner records and measures three sonograms, US will be reliable (ICC>0.75) and precise (Limits of agreement <20%). attached at the plantar surface at the toes, dorsal flexion of the toes creates tension in the PF and makes the borders of the fascia more clearly defined. The transducer was placed over the plantar aspect of the hindfoot to examine the PF at the insertion onto the calcaneus. Long-axis sonograms were obtained approximately 0.5cm medial to the midline of the plantar surface of the foot, figure 2. Sonograms of both the right and the left foot were used in the study. The scanner was from General Electric Healthcare (3135 Easton Tpke, Fairfield, CT, USA), model LOGIQ e. The transducer that was used was a 12L RS, 13 MHz linear transducer. Scan depth was set to 2 cm. Three successive scans were made of each foot to resemble a feasible clinical setting. Between each scan the transducer was moved away from the foot and then repositioned. It was decided at random whether observer 1 or 2 did the first scan and whether the scans were initiated on the left or the right foot. To investigate whether the two examiners would increase reliability during the day of testing, all subjects received a consecutive identification number. This allowed us to ensure that no learning effect would influence the results.

Methods

Participants

Retest

After a period of approximately 60 minutes, the subjects underwent a second ultrasound examination. During the retest, three additional sonograms of the PF were recorded. Care was taken to maintain the same standardized foot position, to keep the ultrasound machine settings constant, and to replicate the exact measurement. As physical activity and time-of-day might influence on the actual thickness of the PF, we chose a period of 60 minutes between test and retest. [START_REF] Beggs | Musculoskeletal Ultrasound Technical Guidelines: VI. Ankle[END_REF][START_REF] Grigg | Incidental walking activity is sufficient to induce time-dependent conditioning of the Achilles tendon[END_REF] All subjects where instructed to rest between test and retest.

Sonogram Analysis

All sonograms were measured using the built-in software in the scanner. PF thickness measurements were made at the insertion onto the calcaneus. A third person randomly loaded sonograms onto the screen for examiner 1 and 2 to measure, thereby blinding the observers to which subject they were measuring.

Data Analysis

Statistical analysis was performed using SPSS version 15.0. The dependant measure of PF thickness was calculated using the built-in software in the scanner. Each sequential measurement was arranged in SPSS as a separate variable, and intraexaminer reliability with 95% confidence intervals (CI) was estimated using two-way mixed-model, consistency-type intraclass correlation coefficients (ICC).

Both examiners were blinded during measurement of the current and previous thickness values. The reliability when using a single measurement was estimated using the first 2 measurement variables and the "single measures" output from SPSS. The reliability when using a mean of 2 measurements was estimated using the first 2 measurement variables and the "average measures" output from SPSS. The reliability when using a mean of 3 measurements was similarly estimated using the first 3 measurement variables and the "average measures" output from SPSS. To determine interobserver reliability, a parallel model (two-way random effect, total agreement) with single measurement and mean values of 2 and 3 measurements was calculated.

Measurement precision was assessed by limits of agreement (LOA) and LOA as a percentage of the mean value (LOA%).

Results

The sample included in the study consisted of 4 men and 16 women. Mean age was 24.1 (SD 2.6).

Mean body mass index (BMI) was 22.2 (SD 2.1). Paired t-test shows no significant difference between the right and left PF, (p>0.05), table 1. Paired t-test shows no systematic difference in thickness between test and retest for observer 1 or observer 2 (p>0.05). ICC values show no significant difference in reliability between right and left PF which is the reason why the right and left plantar fascias are pooled in the reliability analysis.

Table 1

Intraobserver

Intraobserver reliability measured by ICC for observer 1 using one sonogram was 0.50 (CI 0.23-0.70) and increased to 0.77 (CI 0.56-0.87) when using the average of three sonograms.

Intraobserver reliability for observer 2 using one sonogram was 0.52 (CI 0.09-0.75) and increased to 0.67 (CI 0.39-0.82) when using the average of three sonograms. When using one sonogram, LOA was 0.8mm for observer 1 and 0.9 for observer 2. LOA decreased to 0.6mm (LOA% of 17.5%) and 0.8mm (LOA% 20.8) when using the mean of three sonograms, table 2.

Table 2

Interobserver

Interobserver reliability when measuring different sonograms measured by ICC was 0.62 (CI 0.37-0.78) and increased to 0.82 (CI 0.65-0.91) when using the mean of three sonograms, table 3. When the observers measured the same set of sonograms, reliability measured by ICC was 0.71 (CI 0.50-0.84) and 0.73 (CI 0.55-0.85) and increased to 0.89 (CI 0.78-0.95) and 0.91 (CI 0.82-0.95) Doing a visual inspection of the consecutive ID number plotted against the difference between test and retest, there are no sign of learning effect with decreased difference with increasing ID number. Furthermore a Bland-Altman plot with the difference between test and retest plotted against the thickness of the PF shows no sign of increased error with increased thickness of the PF.

Discussion

The purpose of this study was to determine the intra-and interobserver reliability of US assessment of PF thickness and to investigate improvements in measurement precision when using the mean value of three measurements compared to one. Intraobserver reliability using one sonogram was 0.50 and 0.52 and using the mean of three sonograms intraobserver reliability increased to 0.67 and 0.77. Interobserver reliability when using one sonogram was 0.62 and increased to 0.82 when using the average of three sonograms. LOA based on intratester reliability shows that changes in thickness that are larger than 0.8mm can be considered actual changes in thickness and not a result of measurement error.

A study by Liang et al [START_REF] Liang | Thinner plantar fascia predicts decreased pain after extracorporeal shock wave therapy[END_REF] examined the intraobserver reliability of US to assess the thickness of fascia plantaris in 14 patients diagnosed with plantar fasciitis. They showed ICC values of 0.90 (CI 0.70-0.97). Craig et al [START_REF] Craig | Plantar fascia thickness, a measure of tissue glycation, predicts the development of complications in adolescents with type 1 diabetes[END_REF] did a similar study on an unknown number of patients with diabetes and found ICC of 0.89. Neither Liang et al [START_REF] Liang | Thinner plantar fascia predicts decreased pain after extracorporeal shock wave therapy[END_REF] or Craig et al [START_REF] Craig | Plantar fascia thickness, a measure of tissue glycation, predicts the development of complications in adolescents with type 1 diabetes[END_REF] reported whether the measurements were done by a blinded observer, how long time there was between test and retest or which ICC model was used. This makes a direct comparison difficult. Koppenhaver [START_REF] Koppenhaver | The effect of averaging multiple trials on measurement error during ultrasound imaging of transversus abdominis and lumbar multifidus muscles in individuals with low back pain[END_REF] examined the intraobserver reliability US to assess the thickness of transverses abdominis (TrA) and multifidi (MU) with a study design similar to this. They found ICC values of 0.83 (CI 0.67-0.91) and 0.79 (CI 0.61-0.81), corresponding to 15.1% and 19.1% Standard Error of the Estimate (SEM), which approximately corresponds to LOA of 0.7 mm and 0.9 mm. SEM and LOA are comparable if study designs are similar. [START_REF] Atkinson | Typical error versus limits of agreement[END_REF] Springer et al 16 did a similar study where they found a intraobserver SEM of 17% when measuring the thickness of TrA, corresponding to measurements precision of 1.3 mm. Bjordal 17 examined intraobserver reliability when measuring the thickness of the tendon from m supraspinatus. They found LOA of 0.4 mm and a mean value of tendon thickness of 6.6 mm.

Intraobserver reliability

Interobserver reliability

O`Connor 18 did an interobserver study of US measurement of Achilles tendon thickness. They found a LOA% of 29%. Ying 19 did a similar study and found interobserver measurement error between 5 observers of 0.9-1.1 mm, which corresponds to a LOA% of approximately 20%.

It appears that intraobserver reliability of US assessments of human tendon and muscle thickness are between 0.4 mm and 1.3 mm when measuring healthy tendons. LOA% is between 15 and 19%.

Interobserver reliability is slightly lower with LOA% of approximately 20%. We found intraobserver reliability of ICC 0.77 and 0.67 and LOA 0.6 mm and 0.8 mm, corresponding to 17.5% and 20.8%. Interobserver reliability was ICC 0.82, LOA 0.57, corresponding to 15.8%. Both intra-and interobserver reliability found in the literature correspond to the findings in this study. Genc et al 26 showed a significant correlation between a decrease in thickness of the PF and reduction in pain. This was present at both 1 and 6 months after treatment. They found that PF thickness decreased 1.7 mm from initial treatment until follow-up at 1 month. At the next follow-up after 6 months, the PF thickness had decreased another 0.6mm. Put into context of the current study, the change in PF thickness that they observed in the first month of treatment reflects actual changes in the thickness. However the change in thickness between 1 and 6 months of follow-up might be caused by measurement error as they only recorded one sonogram. The results of this study showed that if only one sonogram is measured, then the minimal detectable change in PF thickness is 0.8mm while it decreases to 0.6mm if three sonograms are used.

Clinical Relevance

Limitations of ICC

In order to analyze intra-and interobserver reliability, we used ICC and LOA. ICC is a very common method for describing reliability in different settings. However ICC has some limitations.

The size of ICC depends on the ratio of the variability between subjects to the total variability. 18 This means that the size of ICC may be affected by factors related to the study sample itself. 18 If two separate test-retest studies are performed on two study samples where one study sample consists of a homogenous group and the other consists of a very heterogeneous groups, the results will be different. This is not because the methods being used have different reliability in the two groups, but because ICC is dependent on the ratio of the variability between subjects to the total variability. 18 The higher ICC in a heterogeneous group is due to the calculations that ICC is based on and not due to the fact that the methods in the two study samples disagree. The consequence is that it is easier to disguise measurement error in a heterogeneous population. Liang et al [START_REF] Liang | Thinner plantar fascia predicts decreased pain after extracorporeal shock wave therapy[END_REF] and Craig et al [START_REF] Craig | Plantar fascia thickness, a measure of tissue glycation, predicts the development of complications in adolescents with type 1 diabetes[END_REF] both examined pathological groups that may consist of a more heterogeneous sample. This

Comparison of mean thickness of the PF

The earliest study where the thickness of the Pf was quantified by US was published in 1993. 21 Studies that examine healthy subjects find mean values of the thickness between 2.6 and 3.9 mm. 7,[21][22][23][24][25][26][27] The two largest studies, which included 154 and 110 subjects, found mean thickness of the PF of 3.2 mm (women and men combined) and 3.3 mm(±0.3 mm) for women and 3.9 mm (±0.5 mm) for men. The average values in these studies are very similar to the ones found in the present study.

Discrepancy in the positioning of subjects, different reference point of measurement and body mass index may to some extent explain the variation from 2.6 to 3.9 mm.

Largest measurement error from scan or measurement

The results in table 2 and 3 show that the interobserver reliability when both observers measure the same scans is higher than when they measure separate scans. This indicates that scan is a source of error rather than measurement of the scan. The thickness of the PF is not identical across the entire insertion onto the calcaneus. 28 This means that movement of the transducer might affect the recorded thickness. Furthermore it indicates that some of the variance across the different scan is not only due to measurement error, but reflects actual changes in thickness.

Conclusion

Measurement of PF thickness by US is a reliable method. If the same observer measures the same person twice, the difference will be maximum 0.6 -0.8 mm (ICC 0.67 -0.77) in 95 % of the examinations when using the mean of three sonograms. If two different observers examine the same

  the plantar fascia attached at the plantar surface at the toes, dorsal flexion of the toes creates tension in the PF and makes the borders of the fascia more clearly defined. The transducer was placed over the plantar aspect of the hindfoot to examine the PF at the insertion onto the calcaneus. Long-axis sonograms were obtained approximately 0.5cm medial to the midline of the plantar surface of the foot, figure2. Sonograms of both the right and the left foot were used in the study. The scanner was from General Electric Healthcare (3135 Easton Tpke, Fairfield, CT, USA), model LOGIQ e. The transducer that was used was a 12L RS, 13 MHz linear transducer. Scan depth was set to 2 cm.

Figure 1

 1 Figure 1 about here.

Figure 2

 2 Figure 2 about here.

  of the plantar fascia may explain why the LOA found in the current study corresponds to other studies, but ICC in the current study is lower than what was reported by Liang et al[START_REF] Liang | Thinner plantar fascia predicts decreased pain after extracorporeal shock wave therapy[END_REF] and Craig et al.[START_REF] Craig | Plantar fascia thickness, a measure of tissue glycation, predicts the development of complications in adolescents with type 1 diabetes[END_REF]

  of the plantar fascia subject twice, the measurement error will be maximum 0.6 mm (ICC 0.82) in 95 % of the examinations. of the plantar fascia 9. Beggs, I, Bianchi, S, Bueno, A, and et al. Musculoskeletal Ultrasound Technical Guidelines: VI. Ankle. [First edition]. 2010. European Society of Musculoskeletal Radiology. Accesssed 19-8-2010 on www.essr.org/html/img/pool/ankle.pdf 10. Grigg NL, Stevenson NJ, Wearing SC, Smeathers JE Incidental walking activity is sufficient to induce time-dependent conditioning of the Achilles tendon. Gait Posture 2009 2010: 31(1):64-7. 11 Grigg NL, Wearing SC, Smeathers JE Eccentric calf muscle exercise produces a greater acute reduction in Achilles tendon thickness than concentric exercise. Br J Sports Med 2009; 43: 280-283 12. Liang HW, Wang TG, Chen WS, et al. Thinner plantar fascia predicts decreased pain after extracorporeal shock wave therapy. Clin Orthop Relat Res 2007; 460: 219-225 13. Craig ME, Duffin AC, Gallego PH, et al. Plantar fascia thickness, a measure of tissue glycation, predicts the development of complications in adolescents with type 1 diabetes. Diabetes Care 2008; 31: 1201-1206 14. Koppenhaver SL, Parent EC, Teyhen DS, et al. The effect of averaging multiple trials on measurement error during ultrasound imaging of transversus abdominis and lumbar multifidus muscles in individuals with low back pain. J Orthop Sports Phys Ther 2009; 39: 604-611 15. Atkinson G, Nevill A Typical error versus limits of agreement. Sports Med 2000; 30: 375-

  : foot; musculoskeletal; plantar fascia; plantar fasciitis; reliability;sonography

  Deleted: plantar fasciaDeleted: plantar fascia Deleted: As UI is an integrated part of the diagnosis and evaluation of treatment success, patients are often examined several times to control if rehabilitation is proceeding as expected.

  Twenty healthy volunteers, aged 20 to 31 years, without lower extremity symptoms, were recruited for this study. Exclusion criteria were prior history of surgery to the foot and current or prior pain in PF. Participants provided consent on forms approved by the local Ethics Committee in Denmark (N-2009-0050).ExaminersTwo experienced observers were recruited for the study. Both had used musculoskeletal US as part of daily examinations for 2-5 years. In order to minimize learning effect from customization to the specific US machine used in the study, both examiners were allowed a recommendation period of 4 sessions with the US machine.TestMeasurements were carried out using the set of recommendations from the ESSR (European Society of Musculoskeletal Radiology), figure 1. 9 Subjects were positioned in a prone position. The toes were dorsally flexed, and the talo-crural joint was positioned in 0 degrees. As the PF is

Figure 1

 1 Figure 1 about here.

Figure 2

 2 Figure 2 about here.

  No significant change in the quantitative ultrasound outcomes were expected during the initial and final tests, because subjects were instructed to rest between these tests.

  the plantar fasciaRELIABILITY OF SHOULDER TENDON ULTRASOUND

  the plantar fascia may explain why the LOA found in the current study corresponds to other studies, but ICC in the current study is lower than what was reported by Liang et al[START_REF] Liang | Thinner plantar fascia predicts decreased pain after extracorporeal shock wave therapy[END_REF] and Craig et al.[START_REF] Craig | Plantar fascia thickness, a measure of tissue glycation, predicts the development of complications in adolescents with type 1 diabetes[END_REF]

  the plantar fascia subject twice, the measurement error will be maximum 0.6 mm (ICC 0.82) in 95 % of the examinations.Deleted: then in 95 % of the examinations Deleted: imally Deleted: 57 Deleted: .Deleted: Clinical Relevance ¶The results showed that reliability increases when using the mean of three images compared to one. Limits of agreement based on intratester reliability shows that changes in diameter that are larger than 0.63 mm can be considered actual changes in diameter and not a result of measurement error. ¶

Figure 1 Figure 2

 12 Figure 1 Long-axis sonograms of the plantar fascia at the insertion onto the calcaneus. 254x172mm (150 x 150 DPI)

  All subjectswhere instructed to rest between test and retest.

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	RELIABILITY OF SHOULDER TENDON ULTRASOUND
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Table 1

 1 Diameter of fascia plantaris [mm]

			Men	Women	All
		Mean (SD)	Mean (SD)	Mean (SD)
		Right	Left	Right	Left	Right	Left
	Diameter observer 1	3.93 (0.14)	3.82 (0.16)	3.44 (0.36)	3.45 (0.41)	3.55 (0.37) 3.49 (0.31)
	Diameter observer 2	4.03 (0.39)	3.93 (0.39)	3.54 (0.37)	3.58 (0.40)	3.63 (0.38) 3.67 (0.29)

Table 2

 2

	Limits of	1 image	% of	Mean of 2	% of	Mean of 3 images.	% of
	agreement (LOA)		observers	images.	observers		observers
			average		average		average
	Observer 1 [mm]	0.76	21.1 %	0.67	18.6 %	0.63	17.5 %
	Observer 2 [mm]	0.91	25.3 %	0.82	22.8 %	0.75	20.8 %
	Interobserver [mm]	0.67	18.6 %	0.62	17.2 %	0.57	15.8 %
	Interobserver, both	0.59	16.3 %	0.52	14.4 %	0.47	13.0 %
	observers measure
	observer 1's scans.
	[mm]
	Interobserver, both	0.55	15.6%	0.49	13.9 %	0.43	12.2 %
	observers measure
	observer 2's scans.
	[mm]

Table 3

 3

	Intraclass correlation coefficient.	1 image	Mean of 2 images	Mean of 3 images.
	Intraobserver, observer 1 [ICC 95 % CI]	0.50 (0.23-0.70),	0.73 (0.48-0.85)	0.77 (0.56-0.87)
	Intraobserver, observer 2 [ICC 95 % CI]	0.52 (0.09-0.75)	0.64 (0.32-0.81)	0.67 (0.39-0.82)
	Interobserver [ICC 95 % CI]	0.62 (0.37-0.78)	0.80 (0.60-0.90)	0.82 (0.65-0.91)
	Interobserver [ICC 95 % CI]. Both	0.71 (0.50-0.84)	0.86 (0.71-0.93)	0.89 (0.78-0.95)
	observers measure observer one`s scans.
	Interobserver [ICC 95 % CI]. Both	0.73 (0.55-0.85)	0.89 (0.79-0.94)	0.91 (0.82-0.95)
	observers measure observer two`s scans.

Table legends Table 1 :

 legends1 Diameter of the plantar fascia sub grouped by gender as well as combined, shown for observer 1 and 2.

Table 2 :

 2 Intra-and interobserver reliability presented as Limits of Agreement (LOA), and LOA as percentage of observers' average diameter based on all measurements.

Table 3 :

 3 Intra-and interobserver reliability. Values are presented as ICC with 95 % confidence interval.

John Wiley & SonsJournal of Clinical Ultrasound


Paolo Falsetti

Caterina Acciai

Lucia Lenzi

Keywords: power Doppler ultrasonography, neurogenic heterotopic ossification, myositis ossificans, acquired brain injury

Ultrasonographic diagnosis of neurogenic heterotopic ossification in patients with severe acquired brain injury

admitted to a neurorehabilitation unit.

Results: the incidence of clinical NHO was 7% (one patient with multifocal involvement). In NHO of hip US demonstrated the classical pattern of zone phenomenon, and PDUS demonstrated vascular signal within mineralized NHO and in outer hypoechoic area. No vascular signal was observed in the central hypoechoic core. In NHO of knee and elbow only a heterogeneously hypoechoic mass or hyperechoic mineralized mass were respectively evident, with vascular signals within the lesions. Spectral wave analysis (SWA) demonstrates low resistance vessels in NHO.

Conclusions: bedside US is a useful and cheap tool in diagnosis of NHO. PDUS adds data about neoangiogenetic activity of NHO.

Introduction

Heterotopic ossification is the development of mature lamellar bone (by endochondral ossification) in periarticular soft tissues. This process is frequently associated with neurological disorders (spinal cord injury, hemiplegia due to stroke or traumatic brain injury, encephalitis) and it is defined neurogenic heterotopic ossification (NHO) [START_REF] Van Kuijk | Neurogenic heterotopic ossification in spinal cord injury[END_REF][START_REF] Youssefian | Nodular osteochondrogenic activity in soft tissue surrounding osteoma in neurogenic para osteo-arthropathy: morphological and immunohistochemical study[END_REF][START_REF] Taly | Neurogenic heterotopic ossification: a diagnostic and therapeutic challenge in neurorehabilitation[END_REF] . However heterotopic ossification has been described with the same features in burns, trauma and joint arthroplasty [START_REF] Mccarthy | Heterotopic ossification: a review[END_REF][START_REF] Lane | Idiopatic heterotopic ossification in the intensive care setting[END_REF] . Other terms are used to describe this phenomenon, such as paraosteoarthropathies, myositis ossificans, heterotopic bone formation.

NHO can lead to severe limitation of the range of motion (ROM) and even complete ankylosis of peripheral joints [START_REF] Van Kuijk | Neurogenic heterotopic ossification in spinal cord injury[END_REF][START_REF] Mccarthy | Heterotopic ossification: a review[END_REF] . As not-surgical treatment of NHO, even if controversial, should prevent endochondral ossification, diagnosis should be made before calcification of involved soft tissue [START_REF] Van Kuijk | Neurogenic heterotopic ossification in spinal cord injury[END_REF][START_REF] Taly | Neurogenic heterotopic ossification: a diagnostic and therapeutic challenge in neurorehabilitation[END_REF][START_REF] Jamil | Management of immature heterotopic ossification (HO) of the hip[END_REF][START_REF] Banovac | Prevention and treatment of heterotopic ossification after spinal cord injury[END_REF] .

Though NHO could begin within few weeks after neurological injury, diagnosis is often delayed. Early clinical signs include decreased ROM, local pain and firm swelling, erythema and warmth. The clinical features of the early inflammatory phase mimic tumour, infection, deep venous thrombosis (DVT) and acute arthritis [START_REF] Van Kuijk | Neurogenic heterotopic ossification in spinal cord injury[END_REF][START_REF] Youssefian | Nodular osteochondrogenic activity in soft tissue surrounding osteoma in neurogenic para osteo-arthropathy: morphological and immunohistochemical study[END_REF][START_REF] Taly | Neurogenic heterotopic ossification: a diagnostic and therapeutic challenge in neurorehabilitation[END_REF][START_REF] Mccarthy | Heterotopic ossification: a review[END_REF][START_REF] Scola | Ultrasonographic diagnosis of heterotopic ossification mimicking deep vein thrombosis[END_REF] . Laboratory findings may not be helpful in early diagnosis. In particular, a rise of serum alkaline phosphatase (AP), hydroxyprolinuria and erytrocite sedimentation rate (ESR) is reported but it is very aspecific [START_REF] Van Kuijk | Neurogenic heterotopic ossification in spinal cord injury[END_REF][START_REF] Taly | Neurogenic heterotopic ossification: a diagnostic and therapeutic challenge in neurorehabilitation[END_REF] . Diagnostic radiographic or computed tomography (CT) findings may not appear for one month. Three-phase 99m Technetium bone scanning is a sensitive diagnostic tool for NHO, but its low specificity leads to difficulty in discriminating NHO from other inflammatory, traumatic or degenerative process of the skeleton. Also magnetic resonance imaging (MRI) has proved limited role in the early diagnosis of NHO because of its moderate specificity, high cost and necessity of contrast Gray-scale ultrasonography (US) is proved to be a sensitive imaging method in soft tissues lesions, calcifications and bone-related diseases [START_REF] Kiris | Power Doppler assessment of overall disease activity in patients with rheumatoid arthritis[END_REF][START_REF] Cho | Sonography of bone and bone-related diseases of the extremities[END_REF] . Moreover US has high specificity in early diagnosis and follow-up of NHO, allows differentiation from muscle tears, haematoma, infection, DVT and soft tissues tumour [START_REF] Van Kuijk | Neurogenic heterotopic ossification in spinal cord injury[END_REF][START_REF] Youssefian | Nodular osteochondrogenic activity in soft tissue surrounding osteoma in neurogenic para osteo-arthropathy: morphological and immunohistochemical study[END_REF][START_REF] Scola | Ultrasonographic diagnosis of heterotopic ossification mimicking deep vein thrombosis[END_REF][START_REF] Peck | Early myositis ossificans: a new echographic sign[END_REF][START_REF] Thomas | The role of ultrasound in the early diagnosis and management of heterotopic bone formation[END_REF][START_REF] Alouini-Mekki | La myosite ossifiante circonscrite: apport de l'imagerie[END_REF][START_REF] Bodley | Ultrasound in the early diagnosis of heterotopic ossification in patients with spinal injuries[END_REF][START_REF] Cassar-Pullicino | Sonographic diagnosis of heterotopic bone formation in spinal cord injury patients[END_REF][START_REF] Kramer | Ultrasound appearance of myositis ossificans[END_REF][START_REF] Kirkpatrick | The role of ultrasound in the early diagnosis of myositis ossificans. A case report[END_REF] . US has the advantage of bedside application, it is easy to perform, repeatable and inexpensive and requires no radiation. Doppler US has proved to be a reliable tool for semiquantitative assessment of the vascularity of the soft tissue [START_REF] Kiris | Power Doppler assessment of overall disease activity in patients with rheumatoid arthritis[END_REF] . As demonstrated in a recent work Doppler US can detect hypervascularization inside and outside HO [START_REF] Youssefian | Nodular osteochondrogenic activity in soft tissue surrounding osteoma in neurogenic para osteo-arthropathy: morphological and immunohistochemical study[END_REF] .

The aim of this study was to evaluate the role of bedside US in early diagnosis of NHO, to define the incidence of clinical NHO in this setting and to describe US and power Doppler US (PDUS) features of NHO. or vascular tree or diffuse blush with vessel boundaries not distinguishable [START_REF] Falsetti | Ultrasonography and magnetic resonance imaging of heel fat pad inflammatory-oedematous lesions in rheumatoid arthritis[END_REF] . A spectral Doppler tracing was obtained to confirm that each color signal represented true arterial flow and spectral wave analysis (SWA) was performed in order to define two indices: Pulsatility Index (PI) and Resistance index (RI). In particular the latter, defined as (maximum systolic velocity-end diastolic velocity)/ maximum velocity, was recorded as it correlates with increasing peripheral resistance.

Materials and Methods

Ninety

Results

Six patients over 92 (7 %) with severe acquired brain injury had clinical and US evidence of NHO. The demographic and clinical characteristics of patients with NHO are shown in Table 1 (Tab. 1). In one patient NHO was multifocal involving both hips and left elbow.

US examination showed a focal disorganization of muscle in the involved area, with loss of longitudinal muscular striation and presence of a heterogeneously hypoechoic mass with the characteristics of the classical "zone phenomenon" previously described in literature, in particular in the hip [START_REF] Thomas | The role of ultrasound in the early diagnosis and management of heterotopic bone formation[END_REF][START_REF] Cassar-Pullicino | Sonographic diagnosis of heterotopic bone formation in spinal cord injury patients[END_REF] . The zone phenomenon was always evident in NHO of the hip (2 patients) and appeared as a inner hypoechoic core surrounded by a ring of hyperechoic mineralized islands, with an outer hypoechoic zone adjacent to normal muscle (Fig. 1). PDUS demonstrates vascular signal (grade 2 in both patients) within mineralizing area and in outer hypoechoic area and SWA of vessels within NHO always showed RI<1. No vascular signal was observed in the central hypoechoic core. No synovitic intracapsular vascular signal was observed.

In NHO of knee (4 patients) the classical zone phenomenon was less evident. A heterogeneously hypoechoic mass was evident within insertional area of vastus medialis muscle and proximal enthesis of medial collateral ligament (Fig. 2a). In a case mild hyperechoic deposits were observed also within distal enthesis of medial collateral ligament.

PDUS showed intense vascularisation inside the lesion (grade 3 in 3 patients, grade 2 in one) (Fig. 2b) and SWA of vessels within NHO always showed RI<1 (Fig. 2c).

In NHO of elbow (1 patient) only mild hyperechoic deposits in olecranic recess of the joint were observed and the zone phenomenon was not evident (Fig. 3). PDUS demonstrates vascular signal within mineralizing area (grade 1). In all the site SWA of vessels within NHO always showed RI<1.

Only hip and elbow NHO were associated with significant joint effusion (Fig. 1) but no synovitic intracapsular vascular signal was observed. In knee NHO joint effusion was always small.

The dynamic examination always showed a low compressibility of the hypoechoic areas during the application of a pressure with the probe.

In our case study only a NHO of the hip evolved within one year in complete ankylosis, while in the other cases an acceptable ROM was maintained. NHO is a complication of brain and spinal injury and its prevalence varies from 16% to 53% in traumatic patients. Scanty data are available about incidence of NHO in not-traumatic neurological disorders [START_REF] Lane | Idiopatic heterotopic ossification in the intensive care setting[END_REF][START_REF] Vanden Bossche | Heterotopic ossification: a review[END_REF] . A recent work showed 4% of incidence in a neurorehabilitation unit [START_REF] Taly | Neurogenic heterotopic ossification: a diagnostic and therapeutic challenge in neurorehabilitation[END_REF] . Our data shows comparable results with a similar diagnostic algorhythm.

Discussions

According with literature hip, knee and elbow were the most frequently affected joints [START_REF] Van Kuijk | Neurogenic heterotopic ossification in spinal cord injury[END_REF][START_REF] Taly | Neurogenic heterotopic ossification: a diagnostic and therapeutic challenge in neurorehabilitation[END_REF][START_REF] Vanden Bossche | Heterotopic ossification: a review[END_REF] .

Severe and rapid limitation of range of motion was the most common sign of NHO in our patients, while pain was not always detectable because of sensory deficits and/or concomitant severe aphasia. In knee and elbow a firm swelling was observed in the site on NHO within few days from diagnosis. In NHO of hip palpable mass was not appreciable because of the depth of joint.

In 4 patients AP, ESR and CRP were elevated in early stages of NHO and decreased after beginning of therapy. In 2 cases with small NHO of knee only ESR and CRP was elevated.

Our data confirm the usefulness but incomplete sensitivity of these inexpensive investigation in diagnosis of NHO. In fact, as confirmed in previous works, if NHO formation is small, ALP levels may remain unchanged [START_REF] Vanden Bossche | Heterotopic ossification: a review[END_REF] . Moreover, low specificity of these laboratory findings should be considered, in particular in critical ill patients (concomitant infection, tracheostomy, recent neurosurgery, pressure sores).

In all the cases plegia and muscular hypertonia or spasticity was present in the affected limbs. This risk factor is often reported in literature as cause of muscle hypoxia and increased risk of muscle tears from mobilization [START_REF] Van Kuijk | Neurogenic heterotopic ossification in spinal cord injury[END_REF][START_REF] Youssefian | Nodular osteochondrogenic activity in soft tissue surrounding osteoma in neurogenic para osteo-arthropathy: morphological and immunohistochemical study[END_REF][START_REF] Taly | Neurogenic heterotopic ossification: a diagnostic and therapeutic challenge in neurorehabilitation[END_REF][START_REF] Mccarthy | Heterotopic ossification: a review[END_REF][START_REF] Lane | Idiopatic heterotopic ossification in the intensive care setting[END_REF][START_REF] Vanden Bossche | Heterotopic ossification: a review[END_REF][START_REF] Tsur | Relationship between muscular tone, movement and periarticular new bone formation in postcoma-unaware (PC-U) patients[END_REF] .

Our gray-scale US data support the highly specific pattern of "zone phenomenon" in NHO of the hip, while in other localizations differential diagnosis with arthritis, tumour and abscess could be more difficult.

In hips NHO were localized in a plane deep to the rectus femoris and superior to the coxofemural capsule, as recognized in previous MRI works [START_REF] Ledermann | Pelvic heterotopic ossification: MR imaging characteristics[END_REF] . Iliopsoas muscle or iliopsoas bursa This effusion could be secondary to the adjacent intense inflammatory process.

In knees NHO involved vastus medialis fibers in its entheseal area and proximal fibrocartilaginous enthesis of medial collateral ligament. In our cases knee effusion was minimal, whereas a previous work reported joint effusion with synovial hypertrophy [START_REF] Peck | Early myositis ossificans: a new echographic sign[END_REF] .

In elbow NHO was evident in the olecranic recess, probably in medial head of triceps muscle or in olecranic bursa.

In our experience the low compressibility of lesions underneath the probe is a useful sign to diagnose NHO.

In our knowledge this is the first work in which PDUS was applied to the study of early NHO.

Our data confirm that hypervascularization of mineralizing areas is a relevant diagnostic aspect of NHO lesions, as previously observed with angiographic studies [START_REF] Van Kuijk | Neurogenic heterotopic ossification in spinal cord injury[END_REF][START_REF] Jamil | Management of immature heterotopic ossification (HO) of the hip[END_REF] . Moreover the flow pattern indicate the presence of low resistance vessel as reported in inflammatory neoangiogenesis. The inner core of early NHO appears as a relatively avascular area. Previous observations with MRI confirmed the strong ring enhancement of early HO after administration of Gadolinium-based contrast [START_REF] Wick | Magnetic resonance signal alterations in the acute onset of heterotopic ossification in patients with spinal cord injury[END_REF][START_REF] Ledermann | Pelvic heterotopic ossification: MR imaging characteristics[END_REF] .

No flow signal was observed in synovial layer within adjacent joints. This can strengthen the hypothesis of a reactive and secondary joint effusion [START_REF] Kun | Heterotopic ossification presenting as acute arthritis[END_REF] .

Medical management of NHO is controversial and supported with limited evidence [START_REF] Vanden Bossche | Heterotopic ossification: a review[END_REF][START_REF] Cullen | Management of heterotopic ossification and venous thromboembolism following acquired brain injury[END_REF] . In our case study only a NHO of the hip evolved within one year in complete ankylosis. In our multifocal case therapy has been relatively effective in elbow but not in hips. Previous works reported different outcome after standardized therapy in various involved sites of the same patient [START_REF] Jamil | Management of immature heterotopic ossification (HO) of the hip[END_REF] . NHO in knee seems to have better evolution with respect to other sites. This study has some limitations. The incidence of NHO in brain injured patients could be underestimated because we studied by imaging only sites with clinical signs or symptoms, so 8 subclinical NHO could be missed. Moreover, the time period of follow-up has been limited to about one year. However this diagnostic protocol has been used in previous studies and avoids the high costs of a screening imaging protocol [START_REF] Taly | Neurogenic heterotopic ossification: a diagnostic and therapeutic challenge in neurorehabilitation[END_REF] .

Another limitation is the heterogeneity of the diagnosis of case series, but it is related to the neurorehabilitative setting of the study.

In conclusions, NHO is a frequent cause of reduction of ROM and secondary disability following severe acquired brain injury. As early detection of NHO and prompt starting of therapy are essential to contain related disability, bedside US seems a useful first-step diagnostic tool when clinical and/or laboratory findings induce suspicion of NHO.

  -two consecutive inpatients transferred from Neurosurgery or other intensive care unit and referred to our neurological rehabilitation unit from September 2007 to October 2008 with severe acquired brain injury and clinical suspect of NHO underwent bedside US and PDUS examination. Clinical suspect of NHO derived from reduction of ROM of a joint, with or without local swelling, rising of AP and/or ESR and C-reactive protein (CRP). Location, etiology and severity of the neurological lesion and risk factors for NHO (like DVT, spasticity, pressure sores, and infection) was recorded. In all US positive cases for NHO, diagnosis was confirmed with radiography or CT, and three-phase scintigraphy.In all NHO patients a therapy was initiated with: indometacin 50 mg bid for 15 days, disodium etidronate 300 mg bid per 6 months, 6-metylprednisolone 8 mg until normalization of ESR, and cautious joint mobilization.

4 Bedside

 4 US examination were carried out using a SonoSite (Bothell, USA) Titan with a 5-10 MHz linear transducer (frequency variations depending on site and depth of the lesion). Each US examination was carried out with both longitudinal and transverse scans by the same operator, with the same machine setting. The technical parameters of power Doppler US were: lowest pulse repetition frequency (PFR) avoiding flash artifact, highest gain level without background noise and low filter. The intensity of vascularity at PDUS analysis of the NHO lesions was rated on a semi-quantitative 4-point scale as follows: grade 0= no flow signal; grade 1= 1-2 flow signals (only as coloured spot); grade 2= 2-5 flow signals (both coloured spot and/or definite vessels without secondary branching); grade 3= >5 flow signals

  involved sites. However in coxo-femural joint an effusion was always observed.

Figure 1 .Figure 2c .Figure 3 .

 12c3 Figure 1. Sagittal longitudinal US scan of left hip (patient 1). The classical aspect of the "zone phenomenon" of HO was evident in iliopsoas muscle, with a inner hypoechoic core (curved arrow) surrounded by a ring of hyperechoic mineralized islands (arrowhead), and an outer hypoechoic zone adjacent to normal muscle. Hypoechoic effusion (*) is visible within coxo-femural joint capsule. 170x102mm (75 x 75 DPI)

  1,9-11 .

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