Published online before print March 29, 2002, 10.1148/radiol.2232011024
(Radiology 2002;223:403.)
A more recent version of this article appeared on May 1, 2002
Anterior Cruciate Ligament Tears and Associated Meniscal Lesions: Assessment at Dual-Detector Spiral CT Arthrography1
Bruno C. Vande Berg, MD, PhD,
Frederic E. Lecouvet, MD, PhD,
Pascal Poilvache, MD,
Jean-Emile Dubuc, MD,
Baudouin Maldague, MD and
Jacques Malghem, MD
1 From the Departments of Radiology (B.C.V.B., F.E.L., B.M., J.M.) and Orthopedic Surgery (P.P., J.E.D.), Cliniques Universitaires St Luc, Université Catholique de Louvain, 10 avenue Hippocrate, 1200 Brussels, Belgium. Received June 11, 2001; revision requested July 12; revision received August 28; accepted September 28. Address correspondence to B.C.V.B. (e-mail: vandeberg@rdgn.ucl.ac.be).
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ABSTRACT
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PURPOSE: To assess dual-detector spiral computed tomographic (CT) arthrography of the knee in the evaluation of anterior cruciate ligament (ACL) tears and associated meniscal lesions.
MATERIALS AND METHODS: ACL and meniscal abnormalities in 125 consecutive patients who underwent dual-detector spiral CT arthrography of the knee were evaluated on the basis of both initial interpretations and retrospective review of CT images and were compared with arthroscopic findings. The sensitivity and specificity of CT arthrography for the detection of ACL tears and meniscal lesions in knees with abnormal ACLs were determined.
RESULTS: The sensitivities and specificities for the detection of ACL tears were 90% and 96%, respectively, at initial interpretation and 95% and 99%, respectively, at retrospective interpretation. The sensitivities and specificities for the detection of meniscal tears in knees with abnormal ACLs were 92% and 88%, respectively, at initial interpretation and 96% and 94%, respectively, at retrospective interpretation.
CONCLUSION: Dual-detector spiral CT arthrography of the knee is an accurate method for detecting ACL tears and associated meniscal lesions.
© RSNA, 2002
Index terms: Knee, arthrography, 452.122, 452.12119 Knee, CT, 452.12112, 452.12115, 452.12119 Knee, ligaments, menisci, and cartilage, 452.4852, 452.4857
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INTRODUCTION
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Magnetic resonance (MR) imaging is the dominant imaging technique for evaluating internal derangements of the knee because it is noninvasive and has clinically acceptable accuracy for the detection of meniscal (13) and anterior cruciate ligament (ACL) lesions (2,46). In our institution, MR imaging and dual-detector spiral computed tomography (CT) arthrography with iodinated contrast material are routinely used in the assessment of internal derangements of the knee. Dual-detector spiral CT arthrography is used mainly because of the limited availability of MR imaging and a subsequent shorter waiting list for spiral CT arthrography. The choice of the imaging technique depends primarily on the clinical findings. MR imaging is preferentially performed in injured knees with suspected ligamentous lesions, in patients with known allergic reaction to iodinated products, and in children. Dual-detector spiral CT arthrography is preferentially performed in knees with suspected meniscal or hyaline cartilage lesions and has been found to be accurate in the detection of meniscal lesions (7). In the current study, our purpose was to assess dual-detector spiral CT arthrography of the knee for the evaluation of ACL tears and associated meniscal lesions.
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MATERIALS AND METHODS
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Patients
Between February 1, 1998, and December 30, 2000, 1,193 spiral CT arthrography procedures and 2,326 MR imaging studies of the knee were performed in our institution. Our study population consisted of 125 of the 1,193 patients who underwent spiral CT arthrography of the knee in whom subsequent arthroscopy was performed at our institution. We excluded patients who had undergone knee arthroscopy prior to knee arthrography, patients who were referred by physicians outside of the institution, and patients who did not undergo arthroscopy. The findings in the first 50 patients in the current study group were retrospectively analyzed in a previous study designed to evaluate spiral CT arthrography in the assessment of meniscal tear (7). These patients were also included in the current study to avoid selection bias in our planned retrospective evaluation of the initial interpretations.
The 97 men and the 28 women in the study group had a mean age ± SD of 44.19 years ± 14 (range, 1677 years; median, 42 years). All patients were referred by orthopedic surgeons from our institution for spiral CT arthrography in the right (n = 76) or left (n = 49) knee because of clinical suspicion of a meniscal lesion. Questionnaires were administered to the patients before spiral CT arthrography to record localization and duration of pain and history of trauma or knee surgery.
Review of each patients charts and questionnaire responses was conducted to classify ACL tears, when present, according to the time interval between injury to the knee (as indicated by the patient) and the date of the spiral CT arthrography examination. Acute ACL lesions were classified as those 6 weeks old or less, subacute lesions were those between 7 and 12 weeks old, and chronic lesions were those more than 12 weeks old (8). Fifty-three patients had a history of knee trauma that had occurred 1 week to 5 years before CT arthrography (median delay, 8 weeks; mean delay, 21 weeks). Symptoms lasted a mean of 28 weeks (range, 1200 weeks; median, 8 weeks) before CT arthrography.
Informed consent was obtained from patients before the arthrographic procedure. Ethical committee approval from our institution was not solicited because CT arthrography is routinely performed at our institution to assess internal derangement of the knee (7). Our institutional review board does not require its approval or informed consent for review of patient records and imaging findings.
Arthrography
A volume of 10 mL of ionic contrast material (ioxaglate meglumine and ioxaglate sodium [320 mg of iodine per milliliter], Hexabrix 320; Guerbet, Aulnay-sous-Bois, France) mixed with 1 mL of a 0.1% solution of epinephrine was injected with fluoroscopic control through a 20-gauge needle placed in the suprapatellar pouch of the knee joint after sterile skin preparation (9). Knee effusion, if present, was drained before injection of the contrast material. After intraarticular injection, patients were asked to walk around the room and to perform full-range knee flexions. The radiologist performing the procedure used fluoroscopy to evaluate for homogeneous coating of the menisci and articular surfaces by contrast material. No complications were encountered. Patients walked to the nearby CT scanner.
Spiral CT Scanning
CT studies were performed with a dual-detector helical real-time scanner (Twin RTS; Marconi, Cleveland, Ohio) with the spiral scanning mode. All patients were in a supine position, with 15° knee flexion. After a lateral projection scout image was obtained, scanning was performed for 65-85 seconds to obtain images in the area between the upper pole of the patella and the tibial plateaus. Spiral scanning was performed at 140 kVp and 135 mAs with a focus of 0.75 mm. A dynamic oscillating focus was used (10). The field of view at acquisition was 430 mm. The table speed was 0.75 mm/sec (effective pitch, 0.75), and the effective section thickness was 0.55 mm. The collimation beam was 1 mm.
For image reconstruction, a 360° linear interpolation algorithm, a high-frequency kernel with spatial definition of 14 line pairs per centimeter, an increment of 0.3 mm (40% section overlap), and a zoom factor of 1.94 were used. Images were reconstructed with a 512 x 512 matrix, and in-plane resolution was 0.43 mm. Longitudinal resolution was 0.3 mm, because, with a pitch of 0.75 and a reconstruction increment of 60% of the nominal section width, longitudinal resolution equaled the reconstruction increment of 0.3 mm (11,12). All reconstructed images were prospectively stored on erasable optical disks.
Image Analysis
First, a musculoskeletal radiologist with 11 years of experience (B.C.V.B.) retrospectively reviewed the 125 spiral CT arthrograms in the study population at a workstation (Omnipro; Silicon Graphics, Mountain View, Calif). Sagittal, transverse, and coronal reformations with a 0.45-mm section thickness and a 2-mm interval were viewed with a zoom factor of 2.04.5 at window width and level settings suitable for viewing bone (window width, 1,900 HU; window level, 450 HU). Images of the ACL were also viewed at soft-tissue settings (window width, 1,000 HU; window level, 200 HU). Coronal and sagittal oblique reformations in the plane of the ACL were also obtained. Printed images were not used for retrospective interpretation. The radiologist was blinded to the clinical history of the patients and to the initial interpretation of the findings at the CT arthrographic examinations and the arthroscopic examinations. The radiologist retrospectively determined the status of the ACL according to criteria noted later in this article. He also noted the presence of meniscal abnormalities, including contour irregularity, peripheral separation, and tear (7).
Second, the initial reports were reviewed by a resident to determine the reported status of the ACL and of both menisci. In routine clinical practice, this initial interpretation was performed in consensus by two of the three musculoskeletal staff radiologists, including, for 69 of the 125 patients in the present study, the radiologist who performed the retrospective interpretations. All initial reports included information related to ACL status. In one report, the status of the lateral meniscus was not mentioned; this lateral meniscus was arbitrarily considered to be normal during retrospective review of the initial reports. The radiologists who performed the initial interpretations were aware of the patients clinical history but were not aware whether the patient would undergo arthroscopy. Initial interpretations had been performed with printed films that contained coronal, sagittal, and transverse 0.43-mm-thick reformations, as described in a previous study (7). In equivocal cases, additional information had been obtained at the workstation by means of analysis of reformatted images in sagittal and coronal oblique planes viewed at bone and soft-tissue settings. Specifically, in the retrospective review of the initial interpretations, the following information was collected: the presence of a normal or abnormal ACL and the presence of normal or abnormal medial and lateral menisci. The frequency of the different signs that suggest an abnormal ACL was not determined because these signs were not systematically assessed in the initial reports.
The ACL was considered to be normal if it appeared as a continuous tubular structure on contiguous coronal and sagittal reformations. Its attenuation was intermediatehigher than that of fat, equivalent to that of the patellar tendon, and lower than that of iodinated contrast material (1315). A straight or slightly concave anterior contour and a slightly concave medial contour on sagittal and coronal reformations, respectively, were considered to be normal. On sagittal and coronal reformations, linear streaks of contrast material parallel to the long axis of the ACL were frequently observed within otherwise normal-appearing ACLs, mainly at their periphery. These longitudinal streaks were considered to be normal; their frequency within the proximal, middle, and distal thirds of the ACL was determined at retrospective interpretation. On transverse reformations, the ACL was identified as an oval structure of intermediate attenuation adjacent to the lateral femoral condyle. Presence of the infrapatellar plica anterior to the ACL was considered to be a normal variant (1618).
Signs of ACL tear at spiral CT arthrography were adapted from those observed at MR imaging (2,46,8,19,20). Direct signs of ACL tear at spiral CT arthrography included ligament discontinuity, abnormal contours, and abnormal course. Ligament discontinuity was defined by lack of visualization of the ACL with fatty tissue where the ACL was expected to be or by the presence of intraligamentous contrast material that appeared either as a cleavage plane perpendicular or oblique to the long axis of the ACL or as a fleck of contrast material. Abnormal contour was defined as a bowing or undulating anterior contour with anterior convexity in the lower half of the ACL. Abnormal course was defined as a situation in which the course of an ACL ran almost parallel to the tibial plateaus. Indirect signs of ACL tears at spiral CT arthrography included anterior translocation of the lateral tibial plateau (4), abnormal depression of the lateral femoral condyle notch (21), and fracture of the posterior margin of the lateral tibial plateau (22). These signs were noted at retrospective interpretation but were not systematically noted at initial interpretation.
Arthroscopy
All arthroscopic examinations of the knee were performed by two orthopedic surgeons from our institution (P.P., J.E.D.) at a mean delay of 9.2 weeks (range, 132 weeks; median delay, 8 weeks) after spiral CT arthrography. Sagittal, coronal, and transverse reformatted spiral CT arthrographic images were available to surgeons on film. Original reports included descriptions of meniscal and ligamentous lesions, with notation of the presence of displaced meniscal fragments. Initial reports did not mention whether the ACL lesions were considered to be partial or complete or acute, subacute, or chronic.
At the time of arthroscopy, the appearance of the ACL and of any meniscal lesion patterns was noted on the charts. Surgical notes and drawings were used as the standard of reference in the determination of meniscal and ACL status. Sensitivity, specificity, and positive and negative predictive values with 95% CIs were calculated for the detection of ACL tears and meniscal abnormalities in knees with abnormal ACLs and knees with intact ACLs at both initial and retrospective interpretations. To determine the value of spiral CT arthrography for detection of meniscal lesions in knees with abnormal ACLs and knees with intact ACLs, the ACL status was derived based on the findings at arthroscopy. The
2 test with the Yates correction for continuity was used to assess the statistical significance of the difference in sensitivity and specificity for the detection of meniscal lesions between knees with abnormal ACLs and knees with intact ACLs. A P value of less than .05 was considered statistically significant.
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RESULTS
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ACL Tears
At arthroscopy, 104 ACLs were normal and 21 ACLs were torn. At initial interpretation of spiral CT arthrograms, 19 of the 21 ACLs that proved to be torn at arthroscopy were found to be abnormal (Figs 1, 2). Two ACL lesions that were revealed at arthroscopy were overlooked at spiral CT arthrography. On the basis of the time interval between trauma and spiral CT arthrography, eight tears were considered to be acute, six subacute, and seven chronic. Of the 104 ACLs that were normal at arthroscopy, 100 were classified as normal and four as abnormal at spiral CT arthrography. At initial interpretation of the spiral CT arthrography results, the sensitivity and specificity of spiral CT arthrography for the detection of ACL tears were 90% and 96%, respectively (Table 1).

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Figure 1a. (a) Sagittal reformation (window width, 1,000 HU; window level, 200 HU) obtained after spiral CT arthrography of the left knee in a 42-year-old man who had sustained trauma to the knee 8 weeks previously shows that the anterior contour of the ACL is convex (small arrows). The presence of intraligamentous contrast material (large arrow) running perpendicular to the long axis of the ACL suggests ligament disruption. (b) Coronal oblique reformation (window width, 1,000 HU; window level, 200 HU) shows contrast material (large solid arrow) delineating a cleavage plane within the ACL. The body of the medial meniscus (small solid arrow) is abnormally small, and a meniscal fragment (open arrow) is present in the intercondylar space. At arthroscopy, an ACL tear and a displaced bucket-handle tear of the medial meniscus were revealed.
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Figure 1b. (a) Sagittal reformation (window width, 1,000 HU; window level, 200 HU) obtained after spiral CT arthrography of the left knee in a 42-year-old man who had sustained trauma to the knee 8 weeks previously shows that the anterior contour of the ACL is convex (small arrows). The presence of intraligamentous contrast material (large arrow) running perpendicular to the long axis of the ACL suggests ligament disruption. (b) Coronal oblique reformation (window width, 1,000 HU; window level, 200 HU) shows contrast material (large solid arrow) delineating a cleavage plane within the ACL. The body of the medial meniscus (small solid arrow) is abnormally small, and a meniscal fragment (open arrow) is present in the intercondylar space. At arthroscopy, an ACL tear and a displaced bucket-handle tear of the medial meniscus were revealed.
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Figure 2a. (a) Sagittal reformation (window width, 1,000 HU; window level, 200 HU) of spiral CT arthrography data obtained 55 weeks after trauma to the left knee in a 31-year-old man shows accumulation of contrast material (arrows) in an area where the ACL is expected to be present. (b) Sagittal reformation (window width, 1,000 HU; window level, 200 HU) shows a complex tear (black arrow) of the posterior horn of the lateral meniscus and anterior translocation of the tibia (white arrow). P = posterior. (c) On a coronal reformation (window width, 1,000 HU; window level, 200 HU), contrast material (large black arrow in center of image) is present between the medial aspect of the lateral condyle and the posterior cruciate ligament (small arrows) in an area where the ACL is expected to be found. A complex tear (large black arrow on right side of image) is visible in the body of the medial meniscus. Tears of the ACL and of both menisci were revealed at arthroscopy. L = left.
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Figure 2b. (a) Sagittal reformation (window width, 1,000 HU; window level, 200 HU) of spiral CT arthrography data obtained 55 weeks after trauma to the left knee in a 31-year-old man shows accumulation of contrast material (arrows) in an area where the ACL is expected to be present. (b) Sagittal reformation (window width, 1,000 HU; window level, 200 HU) shows a complex tear (black arrow) of the posterior horn of the lateral meniscus and anterior translocation of the tibia (white arrow). P = posterior. (c) On a coronal reformation (window width, 1,000 HU; window level, 200 HU), contrast material (large black arrow in center of image) is present between the medial aspect of the lateral condyle and the posterior cruciate ligament (small arrows) in an area where the ACL is expected to be found. A complex tear (large black arrow on right side of image) is visible in the body of the medial meniscus. Tears of the ACL and of both menisci were revealed at arthroscopy. L = left.
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Figure 2c. (a) Sagittal reformation (window width, 1,000 HU; window level, 200 HU) of spiral CT arthrography data obtained 55 weeks after trauma to the left knee in a 31-year-old man shows accumulation of contrast material (arrows) in an area where the ACL is expected to be present. (b) Sagittal reformation (window width, 1,000 HU; window level, 200 HU) shows a complex tear (black arrow) of the posterior horn of the lateral meniscus and anterior translocation of the tibia (white arrow). P = posterior. (c) On a coronal reformation (window width, 1,000 HU; window level, 200 HU), contrast material (large black arrow in center of image) is present between the medial aspect of the lateral condyle and the posterior cruciate ligament (small arrows) in an area where the ACL is expected to be found. A complex tear (large black arrow on right side of image) is visible in the body of the medial meniscus. Tears of the ACL and of both menisci were revealed at arthroscopy. L = left.
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At retrospective interpretation, the sensitivity and specificity for the detection of ACL tears were 95% and 99%, respectively (Table 1). Ligamentous discontinuity was observed in 20 of 21 torn ACLs, with accumulation of contrast material in 17 cases and presence of fat in five cases (Table 2). The pattern of contrast material in abnormal ACLs was typically not linear or linear and oblique or perpendicular to the ligament. Thirty-nine (37%) of the 104 ACLs that were revealed to be normal at arthroscopy showed linear streaks of contrast material parallel to the ligament fascicles that involved the distal (n = 29), middle (n = 12), and/or proximal third (n = 4) of the ACL (Fig 3).
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TABLE 2. Direct and Indirect Signs of ACL Tear at Retrospective Interpretation of Spiral CT Arthrograms in 21 Abnormal Knees
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Figure 3a. (a) Sagittal reformation (window width, 2,000 HU; window level, 400 HU) obtained after spiral CT arthrography of the left knee in a 41-year-old man shows a normal ACL (small arrows) with a straight anterior contour. A linear streak of contrast material (large arrow) that courses parallel to the ACL is present in the distal third of the ACL. (b) Coronal oblique reformation (window width, 2,000 HU; window level, 400 HU) along the plane of the ACL also shows contrast material (arrow) in the distal third of the ACL. At arthroscopy, the ACL was found to be normal. Most likely, the contrast material is accumulated between fascicles of the ACL.
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Figure 3b. (a) Sagittal reformation (window width, 2,000 HU; window level, 400 HU) obtained after spiral CT arthrography of the left knee in a 41-year-old man shows a normal ACL (small arrows) with a straight anterior contour. A linear streak of contrast material (large arrow) that courses parallel to the ACL is present in the distal third of the ACL. (b) Coronal oblique reformation (window width, 2,000 HU; window level, 400 HU) along the plane of the ACL also shows contrast material (arrow) in the distal third of the ACL. At arthroscopy, the ACL was found to be normal. Most likely, the contrast material is accumulated between fascicles of the ACL.
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Meniscal Abnormalities
In knees revealed to have an abnormal ACL at arthroscopy, 23 of 25 menisci that proved to be abnormal at arthroscopy were also categorized as abnormal at the initial interpretation of spiral CT arthrography results (Figs 1, 2). Among the 17 normal menisci at arthroscopy, 15 were categorized as normal at spiral CT arthrography. The sensitivities and specificities of spiral CT arthrography for the detection of meniscal abnormalities in knees with abnormal ACLs were 92% and 88%, respectively, at initial interpretation and 96% and 94%, respectively, at retrospective interpretation (Table 3).
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TABLE 3. Detection of Meniscal Abnormalities at Initial and Retrospective Interpretation of Spiral CT Arthrograms in Knees with Abnormal ACL and Knees with Intact ACL
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In knees revealed to have an intact ACL at arthroscopy, the sensitivities and specificities of spiral CT arthrography for the detection of meniscal abnormalities were both 95% at initial interpretation and were 95% and 98%, respectively, at retrospective interpretation (Table 3). Differences in sensitivity and specificity for the detection of meniscal abnormalities in knees with abnormal ACLs and knees with intact ACLs were not statistically significant either at initial (P = .85 and P = .60, respectively) or retrospective (P = .68 and P = .92, respectively) interpretation.
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DISCUSSION
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The findings of this study demonstrated that dual-detector spiral CT arthrography of the knee enabled clinically valuable detection of ACL tears and associated meniscal lesions. First, ACL abnormalities were detected with sensitivities and specificities of 90% and 96%, respectively, at initial interpretation and 95% and 99%, respectively, at retrospective interpretation. These results are equivalent to those obtained at MR imaging (2,46) and are well in line with observations that conventional CT (1315) and tomography after arthrography (14,23,24) also enable recognition of ACL tears. At dual-detector spiral CT arthrography, 20 of 21 ACLs that proved to be torn at arthroscopy showed discontinuity, with either accumulation of contrast material or presence of fat in an area where the ACL was expected to be found. Presence of contrast material within the ACL should be interpreted cautiously because intraligamentous contrast material was found in 39 of 104 ACLs that proved to be normal at arthroscopy. However, in torn ACLs, contrast material accumulated in large ligamentous defects or formed a cleavage plane perpendicular or oblique to the long axis of the ACL. In normal ACLs, intraligamentous contrast material appeared as linear streaks parallel to the long axis of the ligament, mainly at the periphery of the distal third of the ACL. The latter finding deserves further investigation because it contradicts the finding of Lee et al (25), who reported a lack of leakage of contrast material from the synovial space within the ACL at MR arthrography of cadaveric knees.
Meniscal lesions in knees with abnormal ACLs were detected with sensitivities and specificities of 92% and 88%, respectively, at initial interpretation and 96% and 94%, respectively, at retrospective interpretation. These sensitivity values for the detection of meniscal lesions in knees with abnormal ACLs at spiral CT arthrography could be superior to those obtained at conventional MR imaging, which have been reported to range between 69% and 88% (2629), but this observation remains to be assessed in a comparative study. The location and configuration of meniscal lesions observed in knees with abnormal ACLs could partially account for the decreased sensitivity of MR imaging. The meniscal separation and peripheral tears that are associated with ACL tears (30,31) can be missed at MR imaging (5,2629,3234) and could be better detected at spiral CT arthrography. However, we also observed lower sensitivity and specificity values in the detection of meniscal tears in knees with abnormal ACLs than in knees with intact ACLs. The fact that this difference did not reach statistical significance could be related to the relatively small number of patients.
Our study had some limitations. First, results obtained with dual-detector spiral CT arthrography and submillimeter spatial resolution may not be extrapolated to CT arthrography performed with standard spatial resolution. The use of submillimeter collimation in multidetector row systems, however, contributes to an increase in radiation dose compared with the use of more conventional scanning techniques with 1- or 2-mm section thicknesses. Second, the relatively small proportion of patients with ACL tears in our study is related to our selection criteria, because MR imaging rather than spiral CT arthrography was performed in patients suspected of having ligament lesions. Third, patients were selected by the orthopedic surgeons, who decided first that spiral CT arthrography should be performed and second that surgery should be performed. The fact that patients with major knee trauma who were suspected of having ligament lesions were examined with MR imaging rather than with spiral CT arthrography could have influenced the pattern of observed ACL lesions in the current series. However, a spectrum of ligament changes is likely to have been observed because our series included eight acute, six subacute, and seven chronic ACL tears among 53 patients with a history of knee trauma. The fact that the decision to perform arthroscopy was based not only on the clinical findings but also on the imaging findings introduced a verification bias, with a possible increase in the value of CT arthrography signs of ACL lesions. Fourth, the fact that the radiologist who performed the retrospective interpretation was involved in the initial interpretation of the spiral CT arthrography results could have introduced recall bias as an additional limitation. Fifth, inter- and intraobserver reproducibility in the detection of ACL tears was not determined in the current study. Finally, no partial ACL tear was noted at surgery. The value of spiral CT arthrography in detecting partial ACL tears is unknown.
Spiral CT arthrography is more invasive than conventional MR imaging. It uses ionizing radiation and is subject to the potential complications inherent in intraarticular injection of iodinated contrast material (35). Comparative studies between spiral CT arthrography and MR imaging of the knee are needed to define conditions in which spiral CT arthrography could benefit patients despite its increased invasiveness.
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FOOTNOTES
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Abbreviation: ACL = anterior cruciate ligament
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., F.E.L., J.E.D.; literature research, B.C.V.B., P.P., F.E.L.; clinical studies, B.C.V.B., F.E.L., J.M., P.P.; data acquisition, B.C.V.B., F.E.L., B.M., P.P., J.E.D.; data analysis/interpretation, B.C.V.B., F.E.L., J.M.; statistical analysis, B.C.V.B.; manuscript preparation, B.C.V.B.; manuscript definition of intellectual content, B.C.V.B., P.P., B.M.; manuscript editing, B.C.V.B., J.M., B.M.; manuscript revision/review, all authors; manuscript final version approval, B.C.V.B.
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