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Musculoskeletal Imaging |
1 From the Departments of Radiology (B.C.V.B., J.M., B.M., F.E.L.) and Orthopedic Surgery (P.P.), Cliniques Universitaires St Luc, Université Catholique de Louvain, 10 av Hippocrate, 1200 Brussels, Belgium. Received October 2, 2003; revision requested December 19; final revision received May 17, 2004; accepted June 17. Address correspondence to B.C.V.B. (e-mail: vandeberg@rdgn.ucl.ac.be).
| ABSTRACT |
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MATERIALS AND METHODS: The institutional review board required neither its approval nor informed patient consent for the retrospective review of patient data; however, informed patient consent had been obtained before the MR imaging examinations were performed. The presence of meniscal tears with notch and recess fragments was determined at MR imaging and at subsequent arthroscopy in 101 consecutive knees to determine the value of MR imaging for the depiction of these lesions. Initial reports were reviewed to evaluate results of initial interpretations. MR images were retrospectively analyzed to determine the value of several MR image signs for the detection of displaced tears with notch or recess fragments.
RESULTS: At arthroscopy, 37 (41%) of 91 torn menisci had partially detached fragments. Twenty-six torn menisci had notch fragments, and 14 had recess fragments; three torn menisci had one notch and one recess fragment each. At initial MR image analysis, 38 (36%) of 105 torn menisci had partially detached fragments. Twenty-eight torn menisci had notch fragments, and 13 had recess fragments; one torn meniscus had two recess fragments, and three torn menisci had one notch and one recess fragment each. At initial analysis, sensitivities and specificities were, respectively, 69% and 94% for detection of tears with notch fragments and 71% and 98% for detection of tears with recess fragments. At retrospective analysis of sagittal MR images, the presence of at least one sign indicative of meniscal tear with a notch fragment had sensitivities and specificities, respectively, of 65% and 78% for observer 1 and 77% and 73% for observer 2. The presence of at least one sign indicative of a meniscal tear with a recess fragment had sensitivities and specificities, respectively, of 64% and 77% for observer 1 and 64% and 76% for observer 2.
CONCLUSION: Meniscal tears with notch and recess fragments are frequently seen at arthroscopy and can be depicted at knee MR imaging with moderate sensitivity and high specificity.
© RSNA, 2005
| INTRODUCTION |
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Meniscal fragments may migrate inwardly toward the intercondylar notch (5), and several magnetic resonance (MR) image signs have been validated for their detection (718). Meniscal fragments may also migrate outwardly into the meniscal recesses between the capsule and the femoral condyles (superior meniscal recesses) or the meniscal recesses between the capsule and the tibial plateaus (inferior meniscal recesses) (5). Recess fragments are frequently observed at arthroscopy (1,19), but descriptions of their MR imaging appearance remain limited (11,2022). Thus, the purpose of our study was to retrospectively evaluate MR imaging for the depiction of meniscal tears with partially detached fragments displaced in the intercondylar notch or in the meniscal recesses of the knee.
| MATERIALS AND METHODS |
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Informed consent was obtained from the patients before they underwent MR imaging, and no complications occurred during the examinations. Our institutional review board does not require its approval or informed consent for the review of patients records and imaging findings. We excluded patients who either did not undergo arthroscopy or did undergo arthroscopy but at other institutions. During the specified period, a total of 1560 MR imaging examinations of the knee were performed at our institution.
MR Imaging
All MR imaging examinations were performed by using a 1.5-T unit (Intera; Philips Medical Systems, Best, the Netherlands) with a dedicated phased-array quadrature knee coil provided by the manufacturer. Patients were imaged while in the conventional supine position, with a slight degree of knee flexion. Coronal conventional spin-echo T1-weighted (509/16 [repetition time msec/echo time msec], without fat suppression) and fast spin-echo intermediate-weighted (2227/18 [effective], echo train length of five, with fat suppression) sequences, sagittal fast spin-echo intermediate-weighted (2325/18 [effective], echo train length of four, without fat suppression) and T2-weighted (4532/90 [effective], echo train length of nine, without fat suppression) sequences, and a transverse fast spin-echo intermediate-weighted sequence (2660/45 [effective], echo train length of eight, with fat suppression) were performed.
For all sequences, the section thickness was 3 mm with 0.3-mm intervals. The fields of view were 180 mm for the sagittal sequences and 160 mm for the coronal and transverse sequences. The matrix size was 512 x 346. Two signals were acquired for the coronal T1-weighted spin-echo and sagittal fast spin-echo sequences, and three signals were acquired for the coronal and transverse fast spin-echo intermediate-weighted sequences. For each coronal sequence, 16 sections were obtained in a plane parallel to the posterior bicondylar line of the femur on transverse scout views. For each sagittal sequence, 24 sections were obtained in a plane perpendicular to the bicondylar line with 10° of internal rotation. Twenty transverse sections were obtained in a plane parallel to the medial tibial plateau on the sagittal and coronal images. In general, transverse sections were obtained from the proximal aspect of the patella to the fibula.
Coronal and sagittal images were obtained in all knees. Transverse images of the menisci in nine knees were not available: They were not obtained at all in two knees, and the transverse images encompassed only the patella in seven knees. All MR images were stored in a local archiving system (Amserver; Kodak, Rochester, Minn).
Initial Analyses
The initial analyses of the 101 MR images obtained in the 101 patients had been performed by one of three staff musculoskeletal radiologistsJ.M., B.C.V.B., or F.E.L.who had 25, 11, and 6 years experience, respectively. Film hard copies of all coronal, sagittal, and (available) transverse MR images were viewed as part of typical workday caseloads. The radiologists were aware of the patients clinical histories.
The radiologists determined the presence or absence of meniscal lesions, including contour irregularity, meniscocapsular separation, and tears. They also determined the presence or absence of partially detached meniscal fragments. Contour irregularity was defined as an altered meniscus shape with truncation, blunting, flattening, and/or rounding of the inner borders on coronal or sagittal MR images. Meniscal tear was defined as an area of abnormal intrameniscal signal intensity that extended to the articulating surface of the meniscus on more than one contiguous coronal or sagittal MR image. A questionable extension of abnormal signal intensity to the free meniscus surface viewed on only one MR image was not considered to be a tear (23,24) and was not mentioned in the reports. The identified meniscal tears were found to involve the medial or lateral meniscus.
The presence or absence of a notch or recess fragment was assessed as follows: A partially detached, displaced meniscal fragment was defined as a piece of intermediate- to low-signal-intensity meniscus that was continuous with the adjacent torn meniscus and located near the torn meniscus. Contiguous MR images obtained in the same planes were analyzed to demonstrate the continuity of the partially detached, displaced fragment with the adjacent meniscus and the fragments lack of continuity with adjacent capsular, ligamentous, osseous, or tendinous structures.
Notch fragments were defined as meniscal fragments that were located in the intercondylar notch. When a notch fragment was detected, it was classified as large or small. A large notch fragment corresponded to a notch fragment that extended from the anterior to the posterior horns of the corresponding meniscus (718). A small notch fragment corresponded to a notch fragment that did not extend from the anterior to the posterior horns and that involved either the anterior or the posterior aspect of the intercondylar notch. Recess fragments were defined as meniscal fragments that were located in the articular space between the capsule and the femoral condyles (superior meniscal recess) or in the articular space between the capsule and the tibial plateaus (inferior meniscal recess) (25,26).
Retrospective Analyses
In July 2002, all MR images were retrieved from the archiving database, renumbered after identification data had been deleted, and burned onto CD-ROMs. Two musculoskeletal radiologists (B.C.V.B., F.E.L.) independently reviewed all of the available intermediate-weighted fast spin-echo MR images (sagittal, coronal fat saturated, and transverse fat saturated), with 1-month intervals between the reviews of each series of imaging planes. Coronal T1-weighted spin-echo and sagittal T2-weighted fast spin-echo MR images were not included in this retrospective analysis because the analysis of these images alone for the detection of meniscal tears can be difficult.
The MR images were viewed at optimal window widths and levels and with a zoom factor of 1.5 on a viewing console (Easy Vision; Philips Medical Systems). The readers were aware that the patients had undergone knee arthroscopy after MR imaging. However, they were blinded to the patients names, ages, sex, and medical histories and to the number and topographic features of displaced meniscal fragments observed at initial MR image analysis and arthroscopy. No images were lost because of failure of the archiving system.
The two radiologists (B.C.V.B., F.E.L.) used several MR imaging findings for the detection of meniscal tears with partially detached, displaced meniscal fragments on each corresponding imaging plane to identify the torn menisci with partially detached fragments. The MR imaging findings indicative of the presence of notch fragments included a notch fragment, the double posterior cruciate ligament sign, the flipped-meniscus sign, the absence of the bow-tie sign, the too-tall anterior horn sign, and the disproportionate posterior horn sign. The findings indicative of the presence of recess fragments included a recess fragment and absence of the bow-tie sign.
The finding of a notch fragment was positive when a bandlike meniscal fragment was visible in the intercondylar notch in any of the three imaging planes (10,13). The readers were aware that the fibers of a torn anterior cruciate ligament lying parallel to the tibial surface could create an appearance similar to that of a notch fragment. In general, ligament fibers remain attached to the tibia, whereas meniscal tissue inserts medially or laterally into the intercondylar notch.
The double posterior cruciate ligament sign was positive for the presence of a notch fragment when a bandlike meniscal fragment was visible under the posterior cruciate ligament and created the appearance of a double posterior cruciate ligament on sagittal intermediate-weighted MR images (8). The flipped-meniscus sign was positive when an anteriorly displaced triangular meniscal fragment was located posterior to the anterior horn of the same meniscus on sagittal intermediate-weighted images (9). The too-tall anterior horn sign was positive when the anterior horn of the meniscus was too tall or was at least 6 mm in diameter on sagittal intermediate-weighted images (9). The disproportionate posterior horn sign was positive when the inner portion of the posterior horn was larger than the outer portion on sagittal intermediate-weighted images (14).
The absent bow-tie sign was considered to be positive when only one or no meniscal body segment was visible on two consecutive peripheral sagittal sections (12,16,17). The MR imaging appearance of the normal meniscal body segment is continuous across the anterior and posterior horns of the meniscus, and each depicted body segment is termed a "bow tie" because of its appearance (12). The absent bow-tie sign was considered to be negative for the presence of a notch fragment when a meniscal body segment was visible on two consecutive image sections.
The presence of a recess fragment was based on the visualization of a low- to intermediate-signal-intensity bandlike meniscal fragment within the meniscal recess in any of the three MR imaging planes. The absent bow-tie sign was also evaluated as an MR image sign potentially indicative of a recess fragment.
At retrospective analysis, the readers did not report on meniscal fragments that were not in the notch or recess, including nondisplaced meniscal fragments, fragments displaced between the femoral and tibial articular surfaces, and completely detached meniscal fragments.
After the retrospective analyses of the MR images were completed, one radiologist (B.C.V.B.) reviewed the initial reports to determine the presence or absence of meniscal tears and the presence or absence of notch or recess fragments as observed at the initial interpretation. Initial reports that were printed out and renumbered according to the numbers on the CD-ROM reports after the identification data had been deleted were retrospectively reviewed. All initial reports included information on the meniscus status and on the presence or absence of partially detached, displaced meniscal fragments. Data on the other meniscal fragments that were described in the reports as partially detached but not displaced or as partly detached and displaced between the femoral and tibial articular surfaces were excluded from this study. These fragments move easily, depending on the knees position and distention, and, thus, such classification is not relevant.
Arthroscopy
All arthroscopic examinations of the knee were performed by the same orthopedic surgeon (P.P.), who had 14 years of experience at our institution, at a mean of 12.4 weeks (median delay, 12 weeks; range, 132 weeks) after MR imaging. All MR images were available to the surgeon on film hard copies. The original reports made by the radiologists included descriptions of the meniscal and ligamentous lesions, with notations regarding the presence or absence of displaced meniscal fragments, which included the size of the notch fragments and the topographic features of the recess fragments.
Arthroscopy was performed, with spinal anesthesia (bupivacaine, Marcaine 0.5%; Astra Zeneca, Brussels, Belgium) induced in the patient, by using a 30° whole-angle arthroscope (Karl Storz, Tuttlingen, Germany) that was 4 mm in outer diameter and a one-chip high-resolution camera (Linvatec; Tampere, Finland). High anterolateral and anteromedial portals were routinely used to introduce the arthroscope; accessory portals, including posteromedial, suprapatellar, or high medial portals, were used when necessary. During arthroscopy, the entire joint, including the suprapatellar pouch, the patellofemoral joint, the medial compartment, the medial recesses, the lateral compartment, and the lateral recesses, was carefully and systematically examined. The menisci were carefully probed by using a 4-mm probing hook (Linvatec). Upper and lower meniscal recesses were carefully examined and probed to exclude meniscal fragments.
The arthroscopic images were either recorded on a video printer or digitized on a computer. At arthroscopy, the presence of meniscal lesions, as well as the presence and topographic features of displaced meniscal fragments, was noted in the charts. No patient data were excluded from the study because of insufficient inspection of the articular space or insufficient palpation of all menisci.
Statistical Analyses
The orthopedic surgeon reviewed the surgical notes and drawings after the retrospective analysis of the MR images was completed, and arthroscopic surgical findings were used as the standard of reference for determining the meniscal status and the presence or absence of partially detached meniscal fragments. Sensitivity, specificity, and positive and negative predictive values for the detection of meniscal tears and partially detached notch or recess fragments at initial image interpretation, with corresponding 95% confidence intervals, were calculated. Statistical values were calculated for the medial and lateral menisci together (without separate analysis for these menisci).
The findings at the retrospective interpretations were used to calculate the sensitivities, specificities, and positive and negative predictive values of each MR image sign in each corresponding imaging plane for the detection of partially detached notch or recess fragments. The retrospective interpretations were also used to calculate the sensitivities, specificities, and positive and negative predictive values for the detection of partially detached notch or recess fragments observed in each of the three imaging planes.
In the sagittal plane, the direct visualization of a fragment or the presence of a finding that was considered positive for the detection of a fragment on sagittal MR images was considered to indicate the presence of a displaced meniscal notch or recess fragment. No direct visualization of a notch or recess fragment and the presence of only those signs that were considered negative for the detection of a fragment on sagittal MR images were considered to indicate the absence of displaced meniscal notch or recess fragments.
In the coronal and transverse planes, the findings for visualization of notch or recess fragments were considered to indicate the findings in the corresponding imaging planes because no other finding or sign was addressed on the coronal and transverse images. Interobserver agreement between the two readers regarding each MR imaging finding and for each imaging plane was assessed by using
coefficients (27). A
value of between less than or equal to 0.41 and 0.60 represented poor agreement; a
value of 0.610.80, substantial agreement; and a
value of 0.811.00, almost perfect agreement.
| RESULTS |
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= 0.693). The presence of any one sign that was indicative of a tear related to a notch fragment on sagittal MR images had sensitivities of 65% and 77% and specificities of 78% and 73% for readers 1 and 2, respectively, with substantial interobserver agreement (
= 0.699).
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= 0.742). On transverse fat-saturated intermediate-weighted fast spin-echo MR images, visualization of a notch fragment had sensitivities of 62% and 62% and specificities of 94% and 93% for readers 1 and 2, respectively, with substantial interobserver agreement (
= 0.752).
Meniscal tear with recess fragment.On sagittal intermediate-weighted fast spin-echo MR images, direct visualization of a recess fragment had a sensitivity of 21% and a specificity of 98% for both readers, with poor interobserver agreement (
= 0.485) (Table 4). The absent bow-tie sign had sensitivities of 64% and 64% and specificities of 77% and 76% for readers 1 and 2, respectively, with substantial interobserver agreement (
= 0.693) (Table 4). The presence of any one sign that was indicative of a tear related to a recess fragment on sagittal intermediate-weighted fast spin-echo MR images had a sensitivity of 64% for both readers and specificities of 77% and 76% for readers 1 and 2, respectively, with substantial interobserver agreement (
= 0.682).
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= 0.713). On transverse fat-saturated intermediate-weighted fast spin-echo MR images, direct visualization of a recess fragment had sensitivities of 46% and 46% and specificities of 96% and 94% for readers 1 and 2, respectively, with poor interobserver agreement (
= 0.460). | DISCUSSION |
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In the current study of 37 meniscal tears with arthroscopically proved notch or recess fragments, the sensitivity and specificity at initial MR image analysis were, respectively, 69% and 94% for the detection of tears with notch fragments and 71% and 98% for the detection of tears with recess fragments. More specific findings in the current study are as follows:
First, 37 (41%) of 91 torn menisci had partially detached fragments displaced in the intercondylar notch or in meniscal recesses at arthroscopy. Twenty-six (29%) torn menisci had notch fragments, 15 of which were large and 11 of which were small. The frequency of 29% for tears with notch fragments that we observed is similar to that observed by Dandy (19), who noted a frequency of 28.4% for tears with notch fragments out of 1000 symptomatic meniscal tears, including 96 large bucket-handle tears (type 1 locked tear) and 188 small notch fragments (detached anterior and posterior tears). The frequency of displaced bucket-handle tears that we observed is also similar to that observed in most arthroscopy series (10,19,32,33). In our series, 14 (15%) torn menisci with displaced recess fragments were found at knee arthroscopy; nine fragments were inferior, and five were superior. Dandy (19) observed inferior recess fragments in 5.3% of the examined meniscal lesions (inverted tears) but did not mention the frequency of superior recess fragments.
Second, the sensitivity and specificity of initial MR image analysis for the detection of tears with notch fragments were 69% and 94%, respectively. Numerous studies (715,17,18) have addressed the value of MR imaging for the depiction of notch fragments and generally yielded more favorable sensitivity values than those calculated in the present study. Differences in study design may partially account for this discrepancy because, to our knowledge, all but one study performed before this one were retrospective, but they were performed with only those patients who had surgically proved large bucket-handle tears (715,17,18). In a prospective MR imaging study, Watt et al (16) calculated a sensitivity of 44% for the MR imaging detection of displaced bucket-handle tears.
Third, at initial analysis, MR imaging had a sensitivity and a specificity of 71% and 98%, respectively, for the detection of meniscal tears with recess fragments. We cannot compare these results with those in prior studies: Ruff et al (11) observed three superior recess fragments during a 4-year period, and Lecas et al (20) observed 11 inferior meniscal fragments in 3686 knee MR imaging examinations. We can only speculate as to why tears with recess fragments depicted at MR imaging are rarely reported (815,21). Recess fragments can be smaller, have less biomechanical impact, and be less visible than notch fragments (1,2,5). Even at arthroscopy, recess fragments may be obscured at standard inspection because they can be entrapped in articular recesses owing to synovial hypertrophy (5,20,22,34). Therefore, recess fragments may not be consistently mentioned in the charts (19,20), and the evaluation of these fragments is impossible in retrospective MR studies.
Fourth, the sensitivity of several MR image signs appeared to range from 8% to 69% for the detection of tears with notch fragments and from 21% to 64% for the detection of tears with recess fragments. None of these MR signs was extremely sensitive, but their appearance may prompt the reader to search for a fragment more carefully. The absent bow-tie sign has been found to be the most reliable indirect sign of a notch fragment (17,18,34). In the current study, the absent bow-tie sign had sensitivities of 62% and 58% for readers 1 and 2, respectively, in the detection of tears with notch fragments; these values are lower than previously reported sensitivities, which range from 71% to 97% (12,13,16,17). Our study results indicate that the absent bow-tie sign had specificities of 80% and 78% for readers 1 and 2, respectively, in the detection of tears with notch fragments; these values are higher than those reported by Watt et al (62%) (16) and Dorsay and Helms (64%) (17).
Moreover, the absent bow-tie sign was found to enable the detection of tears with recess fragments with a sensitivity of 64% for both readers and with specificities of 77% and 76% for readers 1 and 2, respectively. Actually, the absent bow-tie sign is an indication of substance loss from the free edge of the body of the meniscus. The fact that the absence of the bow-tie sign does not depend on the migration pattern of the meniscal fragment may explain the value of this sign for the detection of tears with either notch or recess fragments.
All studies that have addressed the value of MR imaging in the detection of meniscal tears with displaced fragments have yielded some false-positive and false-negative observations. In the current study, there were 10 false-positive and eight false-negative findings of tears with notch fragments and three false-positive and four false-negative findings of tears with recess fragments. All of the false-positive findings at MR imaging corresponded to meniscal tears without displaced fragments at arthroscopy, but none was considered to be normal at arthroscopy. All of the false-negative findings at MR imaging that corresponded to tears with displaced fragments at arthroscopy were considered to be torn menisci without displaced fragments at MR imaging.
The dynamics of partially detached, displaced meniscal fragments could partially account for these interpretation errors. Actually, a displaced meniscal tear fragment is probably not static, and fragments may move in and out of the central or peripheral articular recesses. Difficulty in differentiating meniscal fragments from osteophytes, ligament fragments, and loose bodies may also cause false-positive findings, and the differential diagnosis of meniscal fragments must be further assessed.
Our study had several limitations. First, because of ethical reasons, the referring surgeon could not be blinded to the findings of preoperative MR imaging. Knowledge of such findings may have influenced the clinical decision to proceed or not to proceed to arthroscopy, and, thus, it may have created selection bias in favor of a positive meniscal pathologic entity in those patients who underwent arthroscopy and possibly bias in the arthroscopic evaluation of the meniscus.
In addition, patients with substantial clinical signs underwent surgery. As a result, a selection bias toward the clinically important meniscal lesions that were associated with clinically important symptoms was possibly created. Therefore, the finding that 41% of the torn menisci had displaced fragments might have been skewed because many meniscal tears without displaced fragments might be less symptomatic and/or treated conservatively without surgery. It is also possible that clinically occult displaced meniscal tear fragments may have been missed at preoperative MR imaging and thus were not selected for surgery.
Second, the use of arthroscopy as the standard of reference for the evaluation of displaced meniscal tear fragments has limitations intrinsic to the assessment of the menisci. Mainly in the posterior horns, menisci may be difficult to evaluate (1,5,19,35,36). No posterior arthroscopic portal was used, and some posterior fragments that were suspected at MR imaging to exist may have remained undetected at arthroscopy (36). In addition, only one surgeon was involved in the assessment of meniscal status, and the degree of interobserver agreement for the detection of displaced meniscal tear fragments at arthroscopy was not determined.
Third, we focused on notch and recess fragments. Consequently, nondisplaced meniscal fragments and fragments displaced in the articular space were not taken into account. Indirect MR image signs, including the absent bow-tie sign, could be positive for these fragments also. Therefore, the values for these signs reported herein apply only to the depiction of tears with notch and recess fragments. The fact that detached fragments and fragments displaced between the femoral and tibial articular surfaces were not taken into account may have contributed to the false-negative results in the current study.
Fourth, the limited number of knees in the current series made it necessary for us to study the imaging data for both the medial menisci and the lateral menisci together. We observed a ratio of medial-to-lateral large notch fragments of 1.5:1.0, as in most previous studies (10,12,16,37). The observed ratio of medial-to-lateral recess fragments of 7:1 should be further assessed but is reminiscent of the observation made by Dandy (19), who noted that recess fragments originated more frequently from the medial than from the lateral meniscus. The limited number of cases also made it necessary for us to study large and small notch fragments and superior and inferior notch fragments together. The distribution of displaced meniscal fragments definitely should be further assessed with a larger series of patients and correlated with the various clinical situations and/or the associated ligamentous lesions.
Fifth, clustering was another potential limitation: In three of the 101 patients, more than one fragment was observed at arthroscopy, so a total of 40 meniscal fragments were observed in 37 tears with displaced meniscal fragments. (Three patients had one notch and one recess fragment each.) The potential effect of three clustered measurements on our finding of 40 fragments was small, however, and an assessment of the actual effect of clustering with a random-effects model was not feasible without a larger set of clustered data.
Sixth, during the retrospective assessment of the MR images to determine the value for each sign of tear with a displaced fragment, the intercondylar notch and the meniscal recesses were not physically masked on the images. Therefore, the visualization of displaced meniscal fragments or the presence or absence of other MR image signs could have influenced interpretations of other signs and thus led to cautious interpretations of the value of each sign. Finally, because the radiologists who performed the retrospective interpretations were involved in the initial MR image interpretations, recall bias may have been introduced and been an additional limitation. However, there was an interval of at least 12 months between the initial analysis of the MR images and the retrospective blinded analysis of the images.
In conclusion, in the current study, 41% of torn menisci had partially detached fragments displaced in the intercondylar notch or in the meniscal recesses at arthroscopy. The sensitivity and specificity of MR imaging at initial analysis were, respectively, 69% and 94% for the detection of tears with notch fragments and 71% and 98% for the detection of tears with recess fragments.
| FOOTNOTES |
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Author contributions: Guarantor of integrity of entire study, B.C.V.B.; study concepts, B.C.V.B., P.P., J.M.; study design, B.C.V.B., P.P., F.E.L., J.M.; literature research, B.C.V.B., P.P.; clinical studies, B.C.V.B., P.P., F.E.L., J.M.; data acquisition and analysis/interpretation, B.C.V.B., P.P., F.E.L., J.M.; statistical analysis, B.C.V.B.; manuscript preparation, B.C.V.B., B.M.; manuscript definition of intellectual content, B.C.V.B., F.E.L., J.M.; manuscript editing, B.C.V.B., F.E.L., J.M., P.P.; manuscript revision/review and final version approval, all authors
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