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Vascular and Interventional Radiology |
1 From the Departments of Radiology (M.E.A.P.M.A., M.C.J.M.K., P.M.T.P., M.G.M.H.) and Epidemiology and Biostatistics (M.E.A.P.M.A., M.C.J.M.K., T.S., M.G.M.H.) and Division of Vascular Surgery (M.R.H.M.v.S., H.v.U.), Erasmus MC, Rm EE2140a, Dr Molewaterplein 50, 3015 GE Rotterdam, the Netherlands; and Department of Health Policy and Management, Harvard School of Public Health, Boston, Mass (M.G.M.H.). From the 2002 RSNA scientific assembly. Received October 1, 2003; revision requested December 18; revision received January 19, 2004; accepted February 17. Supported by a Health Care Efficiency Grant from the Health Care Insurance Board (00112) and a Program Grant from the Netherlands Organization for Scientific Research (90466-091). Address correspondence to M.G.M.H. (e-mail: m.hunink@erasmusmc.nl).
| ABSTRACT |
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MATERIALS AND METHODS: In a randomized controlled trial, 73 patients were assigned to CT angiography, and 72 were assigned to DSA. Physician confidence in the treatment decision was measured as a continuous outcome on a scale of 010 (uncertain to certain) and as a dichotomous outcome (further imaging recommended, yes or no). Mean confidence scores and additional imaging recommendations were compared between CT and DSA groups in an intention-to-diagnose-and-treat analysis. To detect trends in confidence, confidence scores were plotted over time, and multiple linear regression analysis was performed. To detect trends in additional imaging recommendations, logistic regression analysis was used. Data from eligible nonrandomized patients were analyzed separately.
RESULTS: No statistically significant difference in baseline characteristics between randomized groups was found. CT had a lower confidence score than did DSA (7.2 vs 8.2, P < .001). Further imaging was recommended more often after CT (25 of 71 patients, 35%) than after DSA (nine of 66 patients, 14%; P = .003). Analysis of trends demonstrated increasing (but not statistically significant) confidence in CT and stable confidence in DSA. No significant difference was found in baseline characteristics between randomized and nonrandomized patients. Among nonrandomized patients, no significant difference in mean confidence score (8.2 vs 8.3, P = .26) was found between CT (n = 24) and DSA (n = 26).
CONCLUSION: With CT angiography, physician confidence decreases with an associated increase in additional imaging prior to revascularization in patients with symptomatic peripheral arterial disease. Given that CT is less invasive than DSA, results suggest that CT may replace DSA in selected cases.
© RSNA, 2004
Index terms: Arteries, peripheral Computed tomography (CT), angiography Digital subtraction angiography, comparative studies
| INTRODUCTION |
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Multidetector row computed tomographic (CT) technology has made a number of new CT applications possible (4). One of these, multidetector row CT angiography, is a minimally invasive method of visualizing the vascular system as an alternative to DSA. CT angiography does not require catheterization and intraarterial injection of contrast medium and therefore causes less patient morbidity than does DSA. It is also quicker because it requires no postprocedural monitoring. If CT angiography can provide all the information necessary to determine which revascularization procedure to perform in a patient with symptomatic peripheral arterial disease, CT could replace DSA in the diagnostic work-up of these patients. CT angiography is a relatively new procedure, however, and physicians may thus be less experienced with it, have less confidence in it, and recommend additional imaging more frequently than they would with DSA.
The hierarchical approach to assessment of new diagnostic imaging technology entails assessment of technical performance, diagnostic accuracy, effect on clinical decision making, and effectiveness and cost-effectiveness of the new technology (5,6). Technical performance and diagnostic accuracy of CT angiography for peripheral arterial disease have been reported (713). Prior to implementation of CT angiography for assessment of peripheral arterial disease on a wide scale, knowledge is also required about its effect on clinical decision makingthat is, confidence in diagnosis and therapeutic decision making (5,1419)and the effect on patient outcomes and costs (5,6).
We performed a randomized controlled trial to evaluate how CT angiography used in the assessment of peripheral arterial disease affects clinical decision making, quality of life, and cost. In this study, our purpose was to compare CT angiography and DSA with regard to recommendations for additional imaging and physician confidence ratings for the chosen therapy. To report our findings, we applied the Standards for Reporting of Diagnostic Accuracy, or STARD, and the revised Consolidated Standards Of Reporting Trials, or CONSORT, recommendations, as appropriate (20,21).
| MATERIALS AND METHODS |
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Those eligible for the trial were adult patients with symptomatic peripheral arterial disease who presented to the Department of Vascular Surgery of our medical center from April 2000 until August 2001. The patients were candidates for percutaneous or surgical intervention and normally would have undergone DSA. Our medical center is an academic hospital and a secondary and tertiary referral center.
After patients gave informed consent, they were randomly allocated to one of two diagnostic strategies. The diagnostic strategies were defined by the initial imaging modality, either CT angiography or DSA. After the initial imaging examination, any additional imaging methods desired could be used with both strategies. Patients were block randomized with a block size of six. An independent statistician prepared the randomization list. The allocation sequence was concealed by means of sealed and numbered opaque envelopes. Research nurses who were not involved in patient care and who were unaware of the allocation sequence enrolled the patients.
Imaging Modalities
Intraarterial DSA.DSA was performed by using a standardized protocol with equipment from two manufacturers (Integris V3000, Philips Medical Systems, Best, the Netherlands; or Angiostar Plus, Siemens Medical Systems, Forchheim, Germany). The procedures were performed by radiology residents in training with the supervision of one of three interventional radiologists (including P.M.T.P.) with at least 3 years of postresidency experience.
Aortic flush series were obtained by using a 4-F pigtail catheter inserted through a 5-F introducer sheath in the left or right femoral artery. DSA series were obtained at contiguous anatomic levels from the abdominal aorta (at the level of the renal arteries) down to the level of the ankles. Images were obtained in the anteroposterior projection and were supplemented with additional oblique views if considered necessary.
Injection rates for nonionic contrast material (Iomeron 300; Altana Pharma, Hoofddorp, the Netherlands) (iodine concentration of 300 g/L) were 1015 mL/sec for a total of 1020 mL per series, depending on cardiac output and the position of the catheter tip. Typically, 150200 mL was used per patient. DSA images were obtained by using an image intensifier 38 cm in diameter with a 512 x 512 acquisition matrix. In all patients, the invasive arterial pressure gradients over the left and right iliac arteries were measured by comparing pressure measurements obtained in the aorta (at the catheter tip) with simultaneous measurements obtained in the femoral introducer sheath and in an additional 4-F sheath inserted in the contralateral femoral artery. Pressure measurements were obtained at rest and during pharmacologic vasodilation induced with 60 mg of intraarterial papaverine (Papaverinesulfaat CF [50 mg/mL]; Centrafarm, Etten-Leur, the Netherlands). A pressure gradient of more than 10 mm Hg during either rest or vasodilation was considered indicative of a hemodynamically significant stenosis.
Multidetector row CT angiography.CT angiography was performed with a multi detector row CT scanner (Somatom Plus4 Volume Zoom; Siemens). The procedures were performed by dedicated CT technologists. A total volume of 120 mL of nonionic contrast material (Visipaque [320 mg/mL]; Amersham Health, Eindhoven, the Netherlands) with an iodine concentration of 320 g/L was injected in an antecubital vein by using a power injector at a monophasic flow rate of 3 mL/sec. Spiral acquisitions in a single examination began at the level of the celiac trunk and ended at the ankles. The scan parameters were pitch of 7, tube current of 110 mAs (120 kV), and 3-mm sections acquired with 4 x 2.5-mm collimation.
Scanning was started 25 seconds after the start of contrast material injection (fixed delay time). The acquisition time was 35 seconds, on average. In patients known to have impaired cardiac output, a longer delay time was used. The images were reconstructed with an increment of 1.5 mm and an effective section width of 3 mm by using the smooth algorithm (B20; Siemens), which resulted in three overlapping data sets of approximately 250 images each. The images were transferred to an online workstation (Easy-Vision, Philips; or Volume Wizard, Siemens) for the preparation of reconstructions.
Sliding maximum intensity projections were obtained with transverse, coronal, and sagittal projections. Whole-volume maximum intensity projection reconstructions with segmentation of bone were obtained. If the aortoiliac arteries were highly calcified, we made additional volume maximum intensity projection reconstructions of these arteries with segmentation of both bone and calcified plaque. Finally, central lumen line reconstructions were obtained in the aortoiliac arteries.
Data Collection and Analysis
We analyzed the data according to the intention-to-diagnose-and-treat principle, which implies that patients belong to the group to which they were assigned by means of randomization, regardless of the actual (sequence of) events surrounding diagnosis and treatment.
Baseline patient characteristics and statistical analysis.At the time of patient inclusion, research nurses obtained baseline characteristics (ie, age, sex, and risk factors) of each patient from the patients themselves, from medical records, and from the hospital information system. To verify that randomization resulted in comparable groups, we compared baseline characteristics of the CT angiography group with those of the DSA group. For dichotomous variables we used the
2 test, and for continuous variables we used the two-sample t test.
For regression analyses, we calculated one comprehensive variable for the risk factor profile of a patient by counting his or her total number of risk factors. Risk factors were male sex, current smoking status, diabetes mellitus (ie, noninsulin dependent diabetes mellitus or insulin-dependent diabetes mellitus), hypertension (ie, diastolic blood pressure > 90 mm Hg and/or use of antihypertensive medication), use of anticoagulants (ie, coumadin or aspirin), coexisting cardiac disease (ie, myocardial infarction, angina pectoris, chronic heart failure and/or decompensatio cordis, percutaneous transluminal coronary angioplasty, coronary artery bypass graft), coexisting cerebrovascular disease (ie, transient ischemic attack, reversible ischemic neurologic deficit or cerebral infarction), hyperlipidemia (ie, cholesterol level > 5.0 mmol/L and/or use of lipid-lowering agents), renal insufficiency (serum creatine level > 130 µmol/L and/or dialysis), and previous surgical and/or radiologic intervention for peripheral arterial disease.
Therapeutic confidence and statistical analysis.We measured the therapeutic confidence of staff physicians during a weekly meeting of vascular surgeons and radiologists (hereby referred to as "the vascular conference"). All DSA and CT angiographic images were interpreted by experienced vascular or interventional radiologists (usually P.M.T.P. and M.G.M.H., each with more than 10 years of experience). At the vascular conference, physicians were shown volume maximum intensity projections with bone segmentation and, if necessary, with segmentation of calcified plaque, sliding of maximum intensity projections in several projections, and central lumen line reconstructions. Presentation of DSA images at the vascular conference included hemodynamic information. After reviewing the information and images, each physician was asked to complete a confidence form independently. The therapeutic confidence was measured with the following verbal rating scale (translated from Dutch): "How sure are you that you can make an accountable therapeutic choice with the diagnostic information available now? Give a number on a scale from 0 (absolutely uncertain) to 10 (absolutely certain)."
Three radiologists (including P.M.T.P. and M.G.M.H.) and four vascular surgeons (including M.R.H.M.v.S. and H.v.U.) completed the confidence forms during the vascular conferences. Their years of experience with diagnosis and treatment of patients with symptomatic peripheral arterial disease varied from 4 to 30 years. The number of physicians who scored a patients initial images varied from one to five, with a mean of 2.5.
Each score given by a physician with regard to a single patient was considered a separate data point. Raters tend to use scales differently as a result of different attitudes toward numeric data. Furthermore, not all raters evaluated all cases.
To adjust for variability in interpretation, we therefore normalized scores for each physician by subtracting that physicians mean score from the individual score and then dividing by that physicians standard deviation. To make it conceptually easier, we transformed the normalized scores back to the 010 scale. Through visual inspection of the scatter plot, we detected outliers in the confidence score that were all more than 3 standard deviations away from the mean and were excluded from the main analysis.
We compared physician confidence in CT angiography and DSA at several levels: the mean scores of all physicians per patient, the mean scores per patient per specialty (radiologists vs vascular surgeons), and the individual physician scores. We compared the mean score in the CT angiography group with that in the DSA group by using the two-sample t test.
To detect trends in confidence, we plotted the confidence physicians had in CT and DSA over time, and we made separate graphs for the radiologists, the vascular surgeons, and each physician who scored more than one-third of all diagnostic tests. We expressed time by ranking the dates of the vascular conferences at which a confidence form was completed. To detect trends in confidence over time, we also used multiple linear regression analysis. The outcome of interest (ie, the dependent variable) was the confidence score.
Two different models were fitted. In the first model, the independent variables were the diagnostic test, time (ranking), and the interaction term between them. In the second model, we included patient characteristics (ie, age, risk factor profile, and symptoms at presentation [critical ischemia vs claudication]) with the variables in the first model.
Recommendations for additional imaging and statistical analysis.Recommendations for additional imaging were measured with the second part of the confidence form, on which we listed possibilities for further diagnostic testing or treatment and requested that the physician indicate the best next step for the patient.
We analyzed the recommendations for additional imaging as a dichotomous outcomenamely, additional diagnostic imaging recommended, yes or no. Because each physician completed the confidence form independently and individual physicians did not always agree on the best next step for a particular patient, all physicians had to recommend additional imaging (strict criterion) before we counted this as a recommendation for additional imaging in the analysis. We used this strict criterion before including a recommendation for additional imaging in the analysis because if all physicians wanted more diagnostic imaging to be performed, then the patient would surely undergo another imaging test.
To compare recommendations for additional imaging between the CT angiography group and the DSA group, we used the
2 test. To detect trends in recommendations for additional imaging, we used logistic regression analysis. The outcome of interest (the dependent variable) was additional imaging recommended versus not recommended. As with the analysis of the confidence scores, we fitted two different hierarchical models with the same independent variables.
Sensitivity analyses.To check the internal consistency of our results, we analyzed the association of the dichotomous outcome (additional imaging recommended, yes or no) with the continuous outcome (confidence score). We calculated the mean confidence scores for all cases in which additional imaging was recommended and for all cases in which additional imaging was not recommended. To compare the mean confidence scores (ie, additional imaging recommended vs not recommended), we used the two-sample t test.
To investigate how robust our results were, we performed a per-protocol analysis. In this analysis, the CT angiography group and the DSA group were defined by the initial diagnostic test the patients actually underwent. We also repeated the intention-to-diagnose-and-treat analysis, including the outliers. Furthermore, we documented how many patients actually underwent additional imaging within 60 days after initial imaging.
To detect selection bias, we collected data from all eligible patientsthat is, randomized and nonrandomized patients. We performed similar analyses on the group of nonrandomized patients, and we documented why they were not randomized.
Analyses were performed with statistical software (SPSS for Windows, version 10.0; SPSS, Chicago, Ill). Two-sided P values of .05 or less were considered to indicate a statistically significant difference.
| RESULTS |
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Therapeutic Confidence
Seven physicians (three radiologists [P.M.T.P., M.G.M.H.] and four vascular surgeons [M.R.H.M.v.S., H.v.U.]) completed confidence forms during the vascular conferences. Each individual physician scored a similar number of CT angiographic images and DSA images. The median physician confidence score ranged from 6 to 8, and the mean score ranged from 6.2 to 8.3. Three outliers were detected: two in the DSA group in the beginning of the trial (confidence scores of 3.3 and 3.2 compared with a mean of 8.1 ± 1.4 [standard deviation]) and one in the CT angiography group when more than half of the patients were already included (confidence score of 2.3 compared with a mean of 7.2 ± 1.6). These outliers were excluded from the main analysis.
Analysis of the mean physician confidence scores showed that there was less confidence in CT than in DSA (7.2 vs 8.2, P < .001; Table 2). When analyzed according to specialty or individual physician, CT also resulted in a lower confidence score than that of DSA. This difference was statistically significant in four of the seven physicians.
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Sensitivity Analyses
We analyzed the association between the continuous outcome (confidence score) and the dichotomous outcome (additional imaging recommended, yes or no). When further diagnostic imaging was recommended, the mean confidence score was lower than it was when no further diagnostic imaging was recommended (6.1 and 8.2, respectively; P < .001), which indicated internal consistency of our results. This difference was statistically significant in all groups (all randomized patients together, the CT group, and the DSA group).
The per-protocol analysis in which groups were compared according to whether patients actually underwent CT or DSA produced results similar to those of the intention-to-diagnose-and-treat analysis. Similarly, inclusion of the outliers in the intention-to-diagnose-and-treat analysis did not significantly change the results (mean confidence score, 7.2 for CT and 8.1 for DSA; P < .05). Within 60 days after initial imaging, 22 patients in the CT group and 11 patients in the DSA group actually underwent additional imaging (30% [22 of 73] and 15% [11 of 72], respectively; P = .03).
For the 50 patients who were not randomized, patients who underwent CT had significantly higher baseline cardiac morbidity (53% [10 of 19]) than that in patients who underwent DSA (20% [five of 25]; P = .02). No statistically significant difference in the other characteristics between patients undergoing CT versus DSA was found. Furthermore, no statistically significant difference was found in baseline characteristics of patients who were randomized and patients who were not randomized. Within the nonrandomized patient group, no statistically significant difference in mean confidence scores was found between CT and DSA (8.2 vs 8.3, P = .73). Analysis of trend showed increasing confidence in both CT and DSA. The difference in confidence between CT and DSA for the nonrandomized patients was small at the beginning of the study and absent at the end.
| DISCUSSION |
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In the Dutch grading system, a score of 5.5 or higher on a 10-point scale is a passing score. A 7.2 and an 8.2 on a 10-point scale are considered good scores. Although CT resulted in a significantly lower confidence score than that of DSA, the confidence scores of both imaging tests are far above the threshold of a 5.5. Given the less invasive nature of CT when compared with DSA, we feel that the slight decrease in physician confidence and the associated increase in additional imaging after initial CT would probably be acceptable in selected cases.
Furthermore, if DSA is still required after CT, it can often be combined with percutaneous intervention. Thus, CT will generally provide enough information to guide planning of the procedure in the sense of reserving room time and choosing the means of arterial access. As such, we believe that CT can replace DSA as the initial imaging test in selected patients with symptomatic peripheral arterial disease prior to revascularization. To justify this approach, analysis of patient outcomes and costs are still required. Furthermore, delineation of patient selection for which this approach is appropriate would also be required.
So far, to our knowledge, investigators who have reported intermediate outcomes for CT angiography and DSA have measured only diagnostic accuracy (713). With DSA as the reference standard, the values of sensitivity and specificity for CT angiography varied from 80% to 100% and from 94% to 100%, respectively (712). To our knowledge, studies have not yet been reported with the regard to (a) the effect of each on diagnosis and therapy choice and (b) cost-effectiveness analyses of CT angiography compared with DSA for peripheral arterial disease.
Previous studies outside the field of peripheral arterial disease have involved measurement of diagnostic and therapeutic effects in a before-after settingthat is, one measurement was performed before the diagnostic test and one after (5,14,15,17,18,2328). In our study, however, the diagnosis was already known to be symptomatic peripheral arterial disease, and patients were undergoing diagnostic imaging work-up prior to revascularization. Thus, the question was not whether the diagnosis or the decision to perform revascularization would change but whether CT provided sufficient information in daily practice to determine which revascularization procedure should be used.
CT angiography is not the only less invasive imaging modality that could possibly replace DSA in the assessment of peripheral arterial diseaseothers include gadolinium-enhanced MR imaging and duplex US. Results of a meta-analysis (29) showed that MR imaging had better discriminatory power than that of US and that MR imaging was a highly sensitive and specific test when compared with DSA. The recent development of multidetector row CT angiography for the evaluation of peripheral arterial disease has shown promising results, which is why we chose to use CT angiography as the less invasive imaging modality.
We performed our measurements in the setting of a randomized controlled trial. A randomized controlled trial is the most reliable setting because, in theory, known and unknown confounders are distributed equally between the two groups (3032). To minimize selection bias, we included patients consecutively and kept records of all eligible patients. Indeed, no statistically significant difference in baseline characteristics between the CT group and the DSA group was present. However, a slight imbalance was present in the number of male patients, patients with diabetes, and patients with cerebrovascular comorbiditymore were present in the CT angiography group. Within the nonrandomized patients, there was a tendency to perform CT angiography, which is less invasive, in case of cardiac morbidity. No statistically significant difference in baseline characteristics was found between randomized and nonrandomized patients. This implies that no obvious selection bias was present and suggests that our results are generalizable to all patients with symptomatic peripheral arterial disease.
The principal limitation of our study design was that physicians were not blinded to the diagnostic test performed. At the same time, however, this was one of its strengths, since we wanted to determine whether CT could provide physicians with sufficient diagnostic information to replace DSA in daily clinical practice. Blinding by means of transferring the information to a schematic drawing, for example, would have introduced an extra step that could have hampered interpretation and confidence and would probably never be used in daily clinical practice. As a result of our pragmatic study design, however, a physicians enthusiasm for CT or DSA could have influenced his or her rating (26).
Furthermore, physicians could have distorted their ratings deliberately. Because they knew the purpose of the study and may have preferred one test to the other, it is possible that they tried to manipulate the direction of the study results. Inspection of the graphs of the physician confidence scores at the individual level, however, suggested that this did not occur. None of the physicians rated one diagnostic imaging test consistently higher or lower than the other.
Another limitation of our study design is that although a physician can be confident about the interpretation of the images, he or she can still be wrong. Technical performance and diagnostic accuracy of CT angiography for peripheral arterial disease have already been reported, however, and were found to be excellent (712). The purpose of the present study was, therefore, not that of a standard diagnostic study. Instead, we set out to evaluate the effect on clinical decision making.
All physicians did not attend all vascular conferences. Therefore, the number of physicians who scored a patients imaging test varied. Furthermore, the scoring behavior between physicians varied. Some scored consistently higher than others for both tests, and some used a broad range, while others used a narrow range. To make the scores of different physicians comparable and to adjust for differing attitudes toward uncertainty, we normalized the scores by assuming that every physician saw a mix of high- and low-quality images. Inspection of the minimum, maximum, median, and mean confidence scores per physician suggested that every physician did indeed see a representative mix of images.
This trial started immediately after the introduction of CT angiography for peripheral arterial disease at our hospital. Therefore, we expected to find a learning curve for CT angiography (ie, the confidence physicians had in CT would increase over time) (33). To detect such a trend, we plotted the confidence physicians had in CT angiography and DSA over time and analyzed this with multiple linear regression (33). We expected to find stable confidence in DSA over time, since this diagnostic imaging test has already been used for many yearsand indeed, this was the case. We also expected to find a decreasing difference in confidence between CT and DSA over time, as the physicians became more comfortable with CT. In our multiple linear regression model, the difference in confidence between CT and DSA decreased over time, but this never reached the point of statistical significance.
Fifty eligible patients were not randomized but did undergo initial diagnostic imaging (CT angiography or DSA). In at least 42 of these 50 patients, the physician decided which test was most suitable on the basis of clinical characteristics and practical considerations. The mean confidence scores were 8.2 in patients who were not randomized and underwent CT, 8.3 in patients who were not randomized and underwent DSA, and 8.2 in patients randomized to DSA. This suggests that in daily practice, physicians learned very quickly how to select patients with symptomatic peripheral arterial disease for whom CT would provide sufficient diagnostic information and thus for whom CT is a feasible alternative to DSA in the diagnostic imaging work-up prior to revascularization.
Additional imaging was recommended after both CT and DSA. Diagnostic imaging tests recommended after initial CT angiography were duplex US, selective DSA, and DSA. Imaging tests recommended after initial DSA were duplex US, CT, MR imaging, and selective DSA. This demonstrates that both DSA and CT have a place in the diagnostic imaging work-up of patients prior to revascularization. Identification of patients in whom CT angiography can be expected to provide sufficient information should help with patient selection in the future.
In conclusion, initial CT angiography results in a decrease in physician confidence and an associated increase in additional imaging in the diagnostic work-up prior to revascularization in patients with symptomatic peripheral arterial disease. Given the less invasive nature of CT angiography than that of DSA, our results suggest that initial CT angiography may replace initial DSA in selected cases.
| ACKNOWLEDGMENTS |
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| FOOTNOTES |
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Abbreviation: DSA = digital subtraction angiography
Authors stated no financial relationship to disclose.
Author contributions: Guarantor of integrity of entire study, M.G.M.H.; study concepts, M.E.A.P.M.A., M.G.M.H.; study design, M.E.A.P.M.A., T.S., M.G.M.H.; literature research, M.E.A.P.M.A.; clinical studies, M.E.A.P.M.A., M.C.J.M.K., M.R.H.M.v.S., H.v.U., P.M.T.P., M.G.M.H.; data acquisition, M.E.A.P.M.A., M.C.J.M.K., M.R.H.M.v.S., H.v.U., P.M.T.P., M.G.M.H.; data analysis/interpretation, M.E.A.P.M.A., T.S., M.G.M.H.; statistical analysis, M.E.A.P.M.A., T.S.; manuscript preparation, M.E.A.P.M.A.; manuscript definition of intellectual content, all authors; manuscript editing, M.E.A.P.M.A.; manuscript revision/review and final version approval, all authors
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