Patient population
This was an observational study where patients were recruited on a prospective and consecutive pattern from the Radiology and Neurology Department.
Institutional ethics review board approval and written informed consent from every patient were obtained.
Patients candidates for inclusion in the study were those referred for carotid conventional US,
either being symptomatic or asymptomatic based on clinical examination and brain imaging.
The cause of referral could either be occurrence of transient ischemic attack or stroke (symptomatic patients) or other unrelated conditions (such as pre-operative work-up in asymptomatic patients).
Both carotid systems of every patient were included in the study and analyzed separately.
A plaque was considered symptomatic if ipsilateral to a stroke occurring during the last six-month period or asymptomatic if located on the contralateral side or ipsilateral to the side of stroke but after the six-month period.
For a plaque to be considered symptomatic,
a stroke needed to be diagnosed on brain imaging with magnetic resonance imaging (MRI).
The primary inclusion criterion was the identification of an internal carotid artery plaque with moderate (50-69%) or severe (70-99%) stenosis.
Exclusion criteria used in this study included any contra-indication to the use of US or CTA contrast agents like history of allergy,
as well as the presence of other diseases mimicking stroke or comorbidities that could cause stroke (including arrhythmias,
cardiac anatomic abnormalities,
thrombophilia and immunologic diseases like antiphospholipid syndrome).
These comorbidities were excluded in an attempt to accurately associate the stroke with carotid disease.
Extensively calcified plaques with acoustic shadowing were also excluded from analysis,
if considered unsuitable for quantification.
Once a patient fulfilled the inclusion criteria,
a CEUS and a CTA examination of the carotid arteries was performed within seven days of the conventional US examination.
Imaging technique
All US examinations were done by a physician radiologist with 9 years of experience on carotid disease and on a GE Logiq S8 (GE Healthcare) with XDclear technology device.
A linear probe (9L) with a 3-10 MHz bandwidth and the carotid arteries preset was used for the purpose of this study.
B-mode and CDU were used for the initial evaluation of carotid plaque echogenicity and surface morphology.
For the CEUS examination an intravenous catheter was placed,
the US device was set to the contrast specific mode (pulse inversion technique) and 2.4 mL of SonoVue (Bracco) followed by 10 mL of saline were administered.
The Mechanical Index (MI) was adjusted to less than 0.1 in order to achieve optimal microbubbles visualization and lower their destruction.
MDCTA examinations were performed on a 128-slice multi-detector CT system (GE Optima CT660,
GE Healthcare),
both with an unenhanced scan and an angiographic scan.
The scan range covered the area from the ascending aorta and up to the intracranial arteries,
at the level of the frontal sinuses.
Eighty milliliters of contrast agent (37% iodine,
iopromide,
Ultravist,
Bayer) was intravenously administered in a bolus followed by 50 ml of saline bolus chaser,
both administered at an injection rate of 4 ml/s.
Real-time bolus tracking was performed at the level of the ascending aorta and used in order to synchronize contrast passage with the angiographic data acquisition.
The slice thickness of the images reviewed was 0.625 mm for optimal isotropic imaging.
Image analysis
The radiologist who performed the carotid US examination recorded video clips of the affected internal carotid artery in long axis plane,
both with CDU and CEUS.
Subsequently,
the clips were reviewed by two different radiologists – observers (A and B),
both with more than ten years of experience with carotid US and who had been previously trained for this study on the use of the software used.
Both observers were blinded to the patient’s history and the findings of CTA.
Both observers chose a frame of the CDU and CEUS video clip,
optimally visualizing carotid plaque surface and then used the surface irregularities software developed for this study in order to quantify surface irregularities,
being blinded to each other’s results.
As a result,
two SII indexes were calculated; one based on the CDU image (SII-CDU) and one derived from CEUS image (SII-CEUS).
CTA was used as the reference method for diagnosing ulceration,
with the latter being defined as the presence of an extension of the contrast agent column beyond the lumen of the vessel and within a plaque measuring at least 1 mm and confirmed in at least two projections.
Images before and after the administration of contrast agent were compared to accurately differentiate ulcerations from calcifications,
with the former being visualized only in the angiographic phase while the latter also existing on the unenhanced acquisition.
The software used in this study for the quantification of carotid plaque surface irregularities was developed based on MATLAB (Mathworks) and offers the potential to quantify the plaque’s surface irregularities.
The physician using this software manually places successive points on the plaque surface and on the outer vascular wall,
for both the upper and lower vessel wall,
based on the plaque’s distribution.
As previously described by Kanber et al [14,
15],
a plaque surface irregularity index (SII) is calculated by the computational summation of the angular deviations of the luminal plaque surface from the straight line,
divided by the physical length of the plaque surface.
The plaque’s surface can be delineated by colour Doppler blood flow signals on CDU and the border of microbubble column on CEUS images.
Given the fact that the US images used by the software are in DICOM format,
pixel spacing and length information were available.
Consequently,
the software can measure the required angles using the cosine rule for the triangles formed by the consecutive points placed by the physician (Fig. 1).
The SII of the carotid wall with the greater plaque surface irregularities was recorded for statistical analysis as this was considered to be more clinically relevant for the detection of ulceration.
Statistical analysis
The IBM SPSS Statistics version 23.0 was used for statistical analysis purposes.
Descriptive statistics included mean and standard deviation (SD) for normally distributed variables and median and inter-quartile range (IQR) for non-normally distributed variables.
The Kolmogorov-Smirnov test was used to test the normal distribution of variables.
Mann-Whitney U test and t-test were used to compare means between groups depending on the normality of distribution.
Receiver Operating Characteristic (ROC) analysis was used for diagnostic accuracy analysis for the detection of ulceration.
Statistical significance level was set at 0.05.