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AJR 2005; 184:1821-1828
© American Roentgen Ray Society


Original Report

Gadolinium-Enhanced MDCT Angiography of the Abdomen: Feasibility and Limitations

Christopher Chicoskie1 and Richard Tello1,2

1 Department of Radiology, Boston University, 33 Pleasant St., Wellesley, MA 02482.
2 Deceased.

Received April 24, 2004; accepted after revision September 7, 2004.

 
Address correspondence to C. Chicoskie.


Abstract
Top
Abstract
Introduction
Subjects and Methods
Results
Discussion
References
 
OBJECTIVE. The purpose of this study was to evaluate a protocol for gadolinium-enhanced MDCT angiography of the abdomen and to identify technical parameters that optimize image quality.

CONCLUSION. The degree of enhancement and image quality achieved using this gadolinium-enhanced MDCT angiography appear adequate for angiographic evaluation of the abdominal aorta and its major branches.


Introduction
Top
Abstract
Introduction
Subjects and Methods
Results
Discussion
References
 
Since the initial description of CT angiography (CTA) of the abdomen using helical acquisition and maximum intensity projection by Costello et al. [1] and studies at Stanford [2, 3], CTA techniques have been described for numerous clinical applications in the head and neck [4], thorax [5], abdomen and pelvis [6], and extremities [7, 8]. In particular, abundant work has emphasized CTA evaluation of the renal arteries for renal artery stenosis [9-11] and renal vascular anatomy in potential kidney donors [12].

All these advances in CTA have been established using IV iodinated contrast agents. Iodine is the prototypical IV radiographic contrast agent because of its high attenuation, low cost, and relatively low incidence of adverse physiologic effects [13]. Nonetheless, significant risks exist for the IV use of iodine, including acute allergic-type reactions [14] and nephrotoxicity [15]. Some patients are at high risk for these adverse effects because of allergies, severe asthma, diabetes, and chronic renal insufficiency [15].

MR angiography is an imaging option in patients who have contraindications to iodinated contrast material. Time-of-flight [16] and phase contrast [17] techniques have been well established for use in evaluating the aorta and renal arteries, and contrast-enhanced MR angiography has approached the specificity of conventional angiography [18, 19]. Despite the success of these techniques, some patients are not candidates for MRI because of contraindications.

Gadolinium has several qualities that make it a potentially excellent radiographic contrast agent. It offers greater photon attenuation than iodine at similar concentrations and has a proven better safety profile [20, 21]. The potential use of gadolinium as an alternative contrast agent for radiographic studies was suggested in 1989, when the first case report on gadolinium use for CT was published [22]. In the years that followed, gadolinium gained acceptance as a reasonable contrast agent for conventional angiographic procedures [23-25].

More recently, case reports have been published of studies using gadolinium as a CT contrast agent. These reports have suggested that gadolinium offers sufficient intraluminal vascular enhancement for some clinical applications [26-28], although parenchymal enhancement of most visceral organs has been disappointing [29]. The limited clinical use of gadolinium-enhanced CT to date has included exclusion of aortic dissection and aortic rupture [30]. In a more recent case series, Wagar [31] used CT with dilute gadolinium to help exclude complications of aortic stent-graft repairs. To date, no protocol for dedicated gadolinium-enhanced MDCTA has been described.

The purpose of this study was to evaluate a protocol for gadolinium-enhanced MDCTA of the abdomen and to identify those technical parameters that optimize image quality.


Subjects and Methods
Top
Abstract
Introduction
Subjects and Methods
Results
Discussion
References
 
Phantom Study
Dilutions of both gadopentetate dimeglumine (Magnevist, Berlex) and ioversol 64% (Optiray 300, Mallinckrodt) were made at concentrations of 1:10 and 1:20, secured in individual plastic tubes, and placed in a water phantom. Imaging of the tubes was performed with a 4-MDCT scanner (MX-8000 Quad, Picker Medical Systems) at 100, 120, and 140 kVp. Hounsfield unit measurements were performed in each tube.

Patients
Institutional review board approval was obtained before the initiation of this prospective study. Subjects were randomly selected from a population of patients referred for nonemergent multiphase abdominal CT of potential tumors of the liver or kidneys at a tertiary care hospital. Daily department schedules were screened to identify patients who were scheduled for an abdominal CT that required arterial phase imaging, who had a normal creatinine level (< 1.3 mg/dL [< 114.9 µmol/L]), and who were not participating in any other study.

Of 30 patients who met these criteria and were approached for participation, eight signed the institutional review board approved consent form. One patient withdrew from the study before the gadolinium scanning.

Seven patients (all men) with a mean age of 56 years (range, 42-70 years) and mean body weight of 80 kg (range, 64-100 kg) completed the entire study protocol. Six of these patients had been scheduled for renal mass CT and one patient for dynamic CT of the liver. No patients were diabetic. All patients underwent routine CT with iodinated contrast material followed by the experimental scanning using IV gadolinium.

Imaging Technique
An 18-gauge IV catheter (Angiocath, Deseret Medical) was placed in an antecubital vein or forearm vein in all patients. Each patient also received a standard oral preparation of 800 mL of barium suspension (Medescan, 2.3% w/v, 2.2% w/w, Lafayette Pharmaceuticals). The patient was placed on the table of the CT unit (MX-8000 Quad) in the supine position with the arms extended above the head. Preliminary scanning of the abdomen was performed without IV contrast material at 120 kVp; detector collimation, 2.5 mm; 1 sec per rotation; table feed, 12.5 mm/sec (pitch 5); and 3.2-mm reconstructed slice thickness.

The catheter was connected to a power injector (Mark IV, Medrad Systems) that was preloaded with 120 mL of ioversol 64% (Optiray 300). A timing bolus was first performed with 15 mL of contrast material at a rate of 4 mL/sec. During the injection, a single-level multiple-scan acquisition was performed. The specific time delay in each patient was calculated as reported previously [32].

Routine CTA was then performed with iodinated contrast material according to standard departmental protocol for the patient's given indication. Each patient received a bolus IV injection of 100 mL of ioversol 64% at a rate of 3 mL/sec, with image acquisition beginning after the calculated time delay. Images were obtained with 120 kVp; detector collimation, 2.5 mm; 1 sec per rotation; table feed, 12.5 mm/sec (pitch 5); and 3.2-mm reconstructed slice thickness. Before the patient was removed from the CT table, the level of the renal arteries was marked on his abdominal wall.

Hydration Protocol
After the routine CTA, the patients were removed from the CT unit and asked to void. All patients were hydrated with at least 400 mL of normal saline (range, 400-500 mL) between studies. Patients were asked to void again just before the gadolinium-enhanced MDCTA. After a short interval (range, 40-90 min), the patients were scanned using the gadolinium-enhanced MDCTA protocol.

Gadolinium-Enhanced MDCTA Technique
Each patient was again placed supine on the table of the same CT unit. The study was planned for a total of 15 cm, centered at the level of the renal arteries, as had been previously marked.

The power injector was loaded for the experimental protocol (Fig. 1). The injector was positioned with the tip angled downward. One hundred milliliters of normal saline was back-filled into the injector with negative pressure, and gas bubbles were eliminated. Then 40 mL of gadopentetate dimeglumine was slowly back-filled into the injector so that it layered completely beneath the saline [33] (Fig. 1). The injector tubing was then attached to the IV catheter in the usual fashion.



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Fig. 1. Diagram shows injector loaded for gadolinium-enhanced MDCT angiography.

 
The patient was then scanned for a total length of 15 cm centered at the level of the renal arteries after a bolus injection of gadolinium at a rate of 4 mL/sec using the previously determined time delay, followed by a continuous injection of 100 mL of normal saline at the same rate. All scanning was performed with detector collimation of 2.5 mm, 1 sec per rotation, table feed of 17.5 mm/sec (pitch 7), and a reconstruction interval of 2.5 mm. Three patients were scanned with 120 kVp, and four were scanned with 90 kVp.

Quantitative Image Analysis
Four attenuation measurements were recorded for each patient: before any contrast material was administered; at peak enhancement during the arterial phase iodine scanning; immediately before the gadolinium scanning, after IV hydration; and at peak enhancement during the arterial phase gadolinium scanning. An oval or circular region with an area of at least 3.0 cm2 located within the confines of the lumen of the juxtarenal aorta was selected for each measurement. Mean change in attenuation was calculated for each scan for both the iodine and the gadolinium scans. Differences in the change in attenuation achieved with gadolinium and with iodine were compared using a paired t test and linear regression using statistical software (Stata version 5.0).

Subjective Image Analysis
The standard axial images of both the routine CTA and the gadolinium-enhanced MDCTA were reviewed independently by each of the investigators and directly compared. Multiplanar reconstructions and maximum intensity projection (MIP) images of the abdominal aorta, including the celiac axis, superior mesenteric artery, and renal arteries, were generated at the workstation. MIP images were viewed using a volume that excluded the spine and oral contrast material in the bowel. All images were viewed at a window of 350 H and level of 60 H.

Two investigators who were unaware of each other's interpretation subjectively reviewed the axial images from both the routine CTA and the gadolinium-enhanced MDCTA. Image quality was classified using a 3-point ordinal score. Image evaluation was determined according to the following scale: 1, poor; 2, adequate; and 3, excellent [34].


Results
Top
Abstract
Introduction
Subjects and Methods
Results
Discussion
References
 
Phantom Study
The absolute attenuation measurements of the 1:10 and 1:20 dilutions of ioversol and gadolinium are summarized in Figure 2. The relative decrease in attenuation as peak kilovoltage increases is displayed in Figure 3, where relative attenuation is defined as the normalized ratio compared with that of an arbitrary peak kilovoltage of 100. For example, the attenuation of 1:10 gadolinium at 100 kVp was 335 H, and the attenuation of 1:10 gadolinium at 140 kVp was 266 H. Thus, the relative attenuation of 1:10 gadolinium at 140 kVp is 0.8 relative to that at 100 kVp.



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Fig. 2. Graph shows absolute attenuation of iodine and gadolinium in Hounsfield units, from top to bottom: 1:10 gadolinium solution, 1:20 iodine solution, 1:10 iodine solution, and 1:20 gadolinium solution.

 


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Fig. 3. Graph shows relative density of iodine and gadolinium with varying peak kilovoltage, from top to bottom: 1:20 gadolinium solution, 1:10 gadolinium solution, 1:20 iodine solution, 1:10 iodine solution.

 
For both iodine and gadolinium, the absolute attenuation decreased at higher peak kilovoltage. Furthermore, as peak kilovoltage was increased from 100 to 140, the relative attenuation of iodine decreased 50% more than the decrease in relative attenuation of gadolinium.

Quantitative Assessment of Intraluminal Enhancement
Gadolinium-enhanced MDCTA was successfully completed for evaluation in six of the seven patients. In one patient, the infrarenal aorta was imaged because of an error in table-position calculation. For that scan, attenuation measurements were made in the infrarenal aorta. Multiplanar reconstructions of the major aortic braches were thus not performed in that patient.

The attenuation measurements in the aortic lumen before and after each scanning sequence are recorded in Table 1. The mean unenhanced attenuation before routine CTA was 42 H (range, 36-48 H). The mean peak attenuation during routine CTA was 184 H (range, 153-227 H). The mean change in attenuation from unenhanced attenuation to peak attenuation was 141 H (range, 110-179 H; SD, 25 H).


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TABLE 1 Intraluminal Aortic Attenuation in Each Patient

 

The mean attenuation before gadolinium-enhanced MDCTA, after IV hydration, was 70 H (range, 54-81 H). The mean peak attenuation during gadolinium-enhanced MDCTA was 152 H (range, 103-186 H). The mean change in attenuation from unenhanced attenuation to peak attenuation was 83 H (range, 41-105 H; SD, 25 H).

The mean change in lumen attenuation for gadolinium-enhanced MDCTA was 83 H (SD, 25 H) compared with 141 H (25 H) mean change achieved with routine CTA (p = 0.0003, paired t test). The ratio of change in attenuation for gadolinium-enhanced MDCTA per change in density for routine CTA was 0.58 (0.16) (Fig. 4).



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Fig. 4. Graph shows change in density: linear regression for gadolinium versus iodine.

 
Subjective Evaluation of Image Quality
The gadolinium-enhanced MDCTA axial and multiplanar reconstruction images showed adequate visualization of the abdominal aorta, celiac axis, superior mesenteric artery, and renal arteries in five (83%) of the six cases and poor visualization in one case (17%), as rated by both reviewers. Both reviewers rated the routine CTA axial and multiplanar reconstruction images excellent in all cases. Because of the small sample size, statistical analysis of the subjective data was not performed.

Intraluminal contrast enhancement was visually less on the gadolinium-enhanced MDCTA images than on the routine CTA images (Figs. 5A, 5B, and 5C). The gadolinium-enhanced MDCTA images obtained with 90 kVp were also noisier than both the routine CTA images and the gadolinium-enhanced MDCTA images obtained at 120 kVp (Figs. 6A, 6B, and 6C). However, in the five studies rated adequate by the reviewers, image quality and level of enhancement allowed complete visualization of the renal arteries in their entirety (Figs. 7A, 7B, 7C, and 7D).



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Fig. 5A. 48-year-old man referred for evaluation of a possible renal mass. Axial CT images unenhanced (A), from iodine-enhanced CT angiography (B), and from gadolinium-enhanced MDCT angiography (C) (120 kVp) show origin of right renal artery (long arrow) and cross-section of superior mesenteric artery (short arrow). Note also left renal vein for comparison of intraluminal density (arrowhead). (Window width, 350 H; window level, 60 H)

 


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Fig. 5B. 48-year-old man referred for evaluation of a possible renal mass. Axial CT images unenhanced (A), from iodine-enhanced CT angiography (B), and from gadolinium-enhanced MDCT angiography (C) (120 kVp) show origin of right renal artery (long arrow) and cross-section of superior mesenteric artery (short arrow). Note also left renal vein for comparison of intraluminal density (arrowhead). (Window width, 350 H; window level, 60 H)

 


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Fig. 5C. 48-year-old man referred for evaluation of a possible renal mass. Axial CT images unenhanced (A), from iodine-enhanced CT angiography (B), and from gadolinium-enhanced MDCT angiography (C) (120 kVp) show origin of right renal artery (long arrow) and cross-section of superior mesenteric artery (short arrow). Note also left renal vein for comparison of intraluminal density (arrowhead). (Window width, 350 H; window level, 60 H)

 


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Fig. 6A. 55-year-old man referred for evaluation of a possible renal mass. Axial CT images unenhanced (A), from iodine-enhanced CT angiography (B), and from gadolinium-enhanced MDCT angiography (C) (90 kVp) show celiac axis (arrow). Gadolinium-enhanced MDCT angiography image is noisier. (Window width, 350 H; window level, 60 H)

 


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Fig. 6B. 55-year-old man referred for evaluation of a possible renal mass. Axial CT images unenhanced (A), from iodine-enhanced CT angiography (B), and from gadolinium-enhanced MDCT angiography (C) (90 kVp) show celiac axis (arrow). Gadolinium-enhanced MDCT angiography image is noisier. (Window width, 350 H; window level, 60 H)

 


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Fig. 6C. 55-year-old man referred for evaluation of a possible renal mass. Axial CT images unenhanced (A), from iodine-enhanced CT angiography (B), and from gadolinium-enhanced MDCT angiography (C) (90 kVp) show celiac axis (arrow). Gadolinium-enhanced MDCT angiography image is noisier. (Window width, 350 H; window level, 60 H)

 


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Fig. 7A. Same 55-year-old man as in Figures 6A, 6B, and 6C. Four contiguous axial images from gadolinium-enhanced MDCT angiography show left renal artery in its entirety.

 


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Fig. 7B. Same 55-year-old man as in Figures 6A, 6B, and 6C. Single images of left renal artery for comparison are unenhanced (B), from iodine-enhanced CT angiography (C), and from gadolinium-enhanced MDCT angiography (D). (Window width, 350 H; window level, 60 H)

 


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Fig. 7C. Same 55-year-old man as in Figures 6A, 6B, and 6C. Single images of left renal artery for comparison are unenhanced (B), from iodine-enhanced CT angiography (C), and from gadolinium-enhanced MDCT angiography (D). (Window width, 350 H; window level, 60 H)

 


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Fig. 7D. Same 55-year-old man as in Figures 6A, 6B, and 6C. Single images of left renal artery for comparison are unenhanced (B), from iodine-enhanced CT angiography (C), and from gadolinium-enhanced MDCT angiography (D). (Window width, 350 H; window level, 60 H)

 
Targeted MIP reconstructions from the gadolinium-enhanced MDCTA images showed sufficient enhancement without venous overlap (Figs. 8A, and 8B), although the presence of oral contrast material precluded the generation of adequate MIP images in all cases, especially the mesenteric branches.



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Fig. 8A. Same 55-year-old man as in Figures 6A, 6B, 6C, 7A, 7B, 7C, and 7D. Targeted coronal (A) and axial (B) maximum-intensity-projection images from gadolinium-enhanced MDCT angiography generated from a volume excluding bowel and spine show renal arteries.

 


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Fig. 8B. Same 55-year-old man as in Figures 6A, 6B, 6C, 7A, 7B, 7C, and 7D. Targeted coronal (A) and axial (B) maximum-intensity-projection images from gadolinium-enhanced MDCT angiography generated from a volume excluding bowel and spine show renal arteries.

 

Discussion
Top
Abstract
Introduction
Subjects and Methods
Results
Discussion
References
 
Gadolinium chelates are widely used as contrast agents in MRI because of their ferromagnetic properties. Gadolinium has also been successfully used as an alternative contrast agent for digital subtraction angiography. The potential use of gadolinium as a vascular and parenchymal contrast agent for CT has been suggested in previous works [22, 26-31]. Gadolinium is an attractive radiographic contrast agent because of its lower incidence of allergic reactions, nephrotoxicity, and extravasation injury compared with iodinated contrast agents [21]. The subset of patients who have relative or absolute contraindications to both MRI and iodinated contrasts agents is a small but not insignificant group. It is this group of patients who would benefit from development of gadolinium-enhanced MDCTA.

In vitro studies have shown that, at typically used photon energies, gadolinium has greater X-ray attenuation than iodine at equimolar concentrations [27]. There are two primary reasons for this. Gadolinium has an atomic number of 64, higher than the 53 of iodine. Allowing for the greater atomic number alone, the absorption of gadolinium would be roughly 1.76 times that of iodine (643/533). However, gadolinium also has a k-edge of 50.2 keV, higher than the 33 keV k-edge of iodine. The k-edge is the photon energy at which the contribution of the photoelectric effect to photon absorption is greatest. Absorption drops precipitously below the k-edge [35]. Thus, in general, at higher photon energies the attenuation of photons by gadolinium increases relative to that of iodine, at least in the range of photon energies typically used for diagnostic imaging. The average photon energy produced by an X-ray tube is approximately one third to one half the peak photon energy, or peak kilovoltage. So a peak kilovoltage between 100 and 150 will be optimal. This effect is further increased in vivo, when beam hardening from body tissues increases the average photon energy [27].

Despite these striking advantages of gadolinium as a potential contrast agent, it has several notable disadvantages. The usefulness of a material as a contrast agent depends on the attenuation characteristics of the material, and the concentration of the agent in the organ or structure being imaged. Commercially available gadolinium agents have a concentration of only 0.5 mmol/mL, far lower than the 2.4 mmol/mL concentration of a 300 mg I/mL iodinated contrast agent. Even when injected at full concentration, the greatest achievable in vivo intraluminal molar concentration of gadolinium is only one fifth that obtainable with iodinated agents injected at the same rate. This is the chief limitation of gadolinium as a contrast agent for CTA.

The total dose of gadolinium used for clinical MRI studies is often based on body weight. The usual dose of gadolinium used for many clinical MRI applications is 0.1 mmol/kg, the so-called single gadolinium dose. The safe use of up to 0.3 mmol/kg of body weight, the maximum total approved dose of gadolinium for IV use, has been documented [36]. For a person weighing 70 kg, the maximum approved total gadolinium dose is about 20 mmol, or about 40 mL of currently available agents. Such doses are often used for brain MRI and MR angiography studies. However, this low volume of gadolinium is only a fraction of that used for the typical CTA examination. Finally, despite the lower total dose used, gadolinium is much more expensive than iodine per use.

The protocol described in this article shows that diagnostic-quality gadolinium-enhanced MDCTA is possible, giving appropriate attention to several important parameters. The rate of contrast agent injection and the delay time to imaging are important features of any dynamic contrast-enhanced imaging study [37, 38]. In the case of gadolinium, the available concentration of the contrast agent is also a critical limiting factor. Other parameters, such as slice thickness and pitch, are important for multiplanar reconstructions and to minimize potential artifacts such as blurring and partial volume effects [39].

In this gadolinium-enhanced MDCTA protocol, 40 mL of gadolinium was injected at a rate of 4 mL/sec. The duration of the gadolinium bolus was thus 10 sec. Although tailored time delays for routine CTA of the abdominal aorta do not improve the extent or uniformity of aortic enhancement over fixed empiric time delays [40], timing is essential for the much shorter injection bolus of gadolinium-enhanced MDCTA. Using the calculated timing bolus, the region of interest was adequately scanned during the peak enhancement of the gadolinium bolus in all but one case. Although the duration and timing of peak luminal enhancement do not precisely correspond to the duration of the injection bolus and time delay [41, 42], the reproducibility of peak enhancement would likely be more reliable with automated timing software [40].

The average change in lumen attenuation using the gadolinium protocol was 83 H, compared with that of the iodinated contrast technique of 141 H. The ratio between average change in attenuation using gadolinium-enhanced MDCTA and conventional CTA was 0.59. Clearly, the level of enhancement produced by gadolinium is of the same order of magnitude as that produced by iodine, and can be reasonably expected to produce diagnostic quality images. Using a gadolinium preparation with a higher concentration could directly increase the maximum achievable peak attenuation. But at currently available concentrations of gadolinium, the only means of increasing peak attenuation is to increase the injection rate. Therefore, gadolinium-enhanced MDCTA performed at a higher injection rate, such as 6.5 mL/sec, will produce even greater potential peak density that may approximate that of a typical CTA protocol. Conversely, a higher concentration of gadolinium will also allow a slower but longer bolus injection. If the duration of the injection were increased, other factors such as nominal collimation width and pitch could be reduced to improve the quality of multiplanar reconstructions [39, 43, 44].

Although none of the patients scanned in this study had evidence of significant vascular disease or renal artery stenosis, the level of enhancement and image quality produced by this imaging protocol appears potentially adequate to exclude significant large vessel stenoses of the aortic branches.

Our study has several limitations. The primary limitation is the small number of subjects, which is insufficient for statistical analysis of image quality.

Another important limitation is the residual iodine in the circulation at the time of the gadolinium studies. The alternative would have been to bring the patients back for gadolinium-enhanced MDCTA on another day. However, the expected reproducible peak enhancement achievable with gadolinium is predicted from the unenhanced-to-peak change in density. The average change in density during gadolinium-enhanced MDCTA was 83 H, accounting for the increased baseline density. Furthermore, although the presence of residual iodine in the circulation increases the intraluminal density, it also increases the background density of the soft tissues. Therefore, dedicated gadolinium-enhanced MDCTA performed without recent iodinated contrast administration will have less background attenuation and so potentially better MIP reconstructions.

The presence of an oral contrast agent, which was used in all patients as part of standard departmental protocol for their scheduled study, further limited MIP reconstructions. The targeted reconstructions that were generated were created from a volume meant to exclude both the spine and the small bowel. Some optimized CTA protocols include water taken orally rather than dense positive contrast agents that may obscure visualization of vascular structures [45]. Thus, dedicated gadolinium-enhanced MDCTA without oral contrast agents may also allow better MIP reconstructions.

Future works should evaluate a gadolinium-enhanced MDCTA technique ideal for construction of MIP images, and validate its usefulness compared with conventional CTA or MR angiography. Correlation with routine CTA may be preferable because the fundamental technique and imaging variables are similar. Furthermore, the sensitivity and specificity of CTA have been well validated using digital subtraction angiography and MR angiography as standards for comparison. To optimize the gadolinium-enhanced MDCTA protocol for MIP reconstruction, thin slice collimation should be used with increased pitch to cover the necessary region of interest. Collimation width has been shown to be the most important single factor in evaluation of vessel stenosis with MIP reconstructions [39, 43, 44]. Conversely, increasing the total volume of bolus injection to 60 mL while maintaining low pitch could further minimize the effects of edge blurring and partial volume artifacts and still allow thin collimation. Images should be reconstructed in an overlapping fashion to minimize stairstep artifacts in the z-axis [43]. Peak kilovoltage is a less important factor, although theoretically using a peak kilovoltage that is as low as possible while maintaining average photon energy above the k-edge of gadolinium, such as 100-120 kVp, would be optimal.

We believe that gadolinium-enhanced MDCTA is a promising technique, and we encourage further studies to evaluate its potentially useful role in selected patient populations.


References
Top
Abstract
Introduction
Subjects and Methods
Results
Discussion
References
 

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